CHROMATOGRAPHIC TERMS

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Symbol and Name Recommended Other Symbols and

by the IUPAC* Names in Use

K_c Distribution Constant (for GLC) K_p Partition Coefficient

Distribution Coefficient

k Retention Factor k Capacity Factor

N Plate Number n Theoretical Plate Number; No. of Theoretical Plates

H Plate Height HETP Height Equivalent to One Theoretical Plate

R Retardation Factor (in columns) R_R Retention Ratio

R_S Peak Resolution R Peak Resolution

a Separation Factor a Selectivity; Solvent Efficiency

t_R Retention Time

V_R Retention Volume

V_M Hold-Up Volume V_M Volume of the Mobile Phase

V_G Volume of the Gas Phase

V_O Void Volume, Dead Volume

Chromatography Common Abbreviations

a Selectivity; separation factor

AED Atomic Emission Detector

b Phase ratio

C4, C8, C18 Alkyl chain length of an LC reversed bonded phase

CE, CZE Capillary Electrophoresis, Capillary Zone Electrophoresis

CI Chemical Ionization

DAD, PDA (Photo) Diode Array Detector

dp Particle diameter

df Film thickness

ECD Electron Capture Detector

EI Electron Impact

ELCD Electrolytic Conductivity Detector

EPA Environmental Protection Agency

FAMEs Fatty Acid Methyl Esters

FID Flame Ionization Detector

FPD Flame Photometric Detector

FS Fused Silica

FT-IR Fourier Transform Infrared

GLC Gas Liquid Chromatography

GPC Gel Permeation Chromatography

GSC Gas Solid Chromatography

HETP or H Height Equivalent to one Theoretical Plate

HPLC High Performance Liquid Chromatography

HRGC High Resolution Gas Chromatography

I.D. Internal Diameter

IPC Ion Pair Chromatography

IRD Infrared Detector

IS Internal Standard

k (or k') Capacity factor (syn capacity ratio, partition ratio)

K Partition coeficcient

l Wave length

LSC Liquid Solid Chromatography

LIF Laser Induced Fluorescence (detector)

MAOT Maximum Allowable Operating Temperature

MDQ / MDL Minimum Detectable Quantity / Minimum Detectable Level

MIP Microwave Induced Plasma

MS Mass Spectrometry

MSD Mass Selective Detector

N Theoretical plate (number of)

NP Normal Phase

NPD Nitrogen Phosphorus Detector

O.D. Outer Diameter

ODS Octadecyl silane

PCBs Polychlorobiphenyls

PID Photo Ionization Detector

PLOT Porous-Layer Open Tubular (column)

PMT Photo-Multiplier Tube

PNAs Polynuclear Aromatics

ppb Part per billion

ppm Part per million

ppt Part per trillion

PTFE Polytetrafluoroethylene (Teflon®)

PTGC Programmed Temperature Gas Chromatography

RS Resolution

RP Reversed Phase

RSD Relative Standard Deviation

SCOT Surface Coated Open Tubular (column)

SEC Size (or Steric) Exclusion Chromatography

SFC Supercritical Fluid Chromatography

SFE Supercritical Fluid Extraction

SIM Single Ion Monitoring

S/N Signal to noise

TCD Thermal Conductivity Detector

TID Thermionic Detector (usually known as NPD)

TLC Thin Layer Chromatography

THMs Trihalomethanes

TMS Trimethylsilyl (derivative)

tR Retention time

UV Ultra-Violet (detector)

VR Retention volume

VOC Volatile Organic Compound

W Peak width

WCOT Wall Coated Open Tubular (column)

LIQUID CHROMATOGRAPHY AND GAS CHROMATOGRAPHY

GLOSSARY

absolute calibration - syn. direct calibration or external standard; method relating detector response to sample concentration in order to perform quantitative analysis. Standard solutions of a sample to be quantitated are prepared and equal volumes chromatographed. Peak heights or peak areas are plotted versus concentrations to produce a calibration curve.

absolute retention time - syn. retention time; the time elapsed between the introduction of the sample and the appearance of the peak maximum.

adsorbent - packing used in adsorption chromatography. Silica gel and alumina are the most frequently used adsorbents in HPLC.

adsorption - process which occurs at the surface of a liquid or solid as a result of the attractive forces between the adsorbate and solute; the basis for separation in GSC and LSC.

adsorption chromatography - one of the basic LC modes. elies on the adsorption process to effect the separation. Silica gel and alumina are the most frequently used supports; molecules are retained by the interaction of their polar functional groups with the surface functional group (e.g. silanols of silica).

adsorption isotherm - in adsorption, a plot of ilibrium concentration of sample in the mobile phase per unit volume us. the concentration in the stationary phase per unit weight. The shape of the adsorption isotherm can determine the chromatographic behavior of the solute: tailing, fronting, overload, etc.

affinity chromatrography - a technique in which a biospecific adsorbent is prepared by coupling a specific ligand (such as an enzyme, antigen, or hormone) for the macromolecule of interest to a solid support (or carrier). This immobilized ligand wil interact only with molecules that can selectively bind to it. Molecules that will not bind elute unretained. The retained compound can later be released in a purified state. Affinity chromatography is not a chromatographic technique as such, but is actually selective filtration.

alumina - an adsorbent sometimes used in adsorption chromatography. Aluminum oxide (Al2O3) is a porous adsorbent that is available with a sligtly basic surface. For this reason, it can have advantages over silica, which is considered to have an acidic surface.

analyte - the chemical species being investigated by an analytical method. Usually the identification and/or the amount is to be determined by a separation and estimation technique.

anion-exchange chromatography - the ion-exchange procedure used for the separation of anions. Both resins and bonded phases are available for this mode. The tetralkylammonium group is a typical strong anion-exchange functional group. An amino group on a bonded or coated stationary phase would be the example of a weak anion exchanger.

asymmetry - factor describing the shape of a chromatographic peak. Theory assumes a Gaussian shape and that peaks are symetrical. The peak asymetry factor is the ratio (at 10% of the peak height) of the distance between the peak apex and the back side of the chromatographic curve to the distance between the peak apex and the front side of the chromatographic curve. A value > 1 is a tailing peak, while a value <1 is a fronting peak.

band - mobile phase zone which contains a sample component; band is usually in the column; peak is the band signal on the chromatogram.

baseline - constant signal produced by the background level of the instrument; usually represented by a flat line on the recorder.

biocompatible - term to indicate that the column or instrument component will not irreversibly or strongly adsorb or deactivate biomolecules such as proteins.

bonded columns - silica is traditionally the common "normal" chromatography packing material. When another material is chemically bonded to the surface of the silica, a new column packing with unique separation properties can result. When an alkyl group is chemically joined to a silica surface, a hydrophobic paraffinic packing is obtained. Similarly, amine, sulfonic acid, or nitrile groups could be bonded to silica.

bonded-phase chromatography - (BPC) the most popular LC mode. A stationary phase chemically bonded to a support is used for the separation. The most popular support is microparticulate silica gel, and the most popular type of bonded phase is the organosilane, such as octadecyl (for reversed-phase chromatography). Approximately 70% of all HPLC is carried out on chemically bonded phases.

bleeding - column bleed; loss of the stationary phase due to its own volatility. Every phase has a maximum operating temperature.

C4, C8, C18, etc. - refers to the alkyl chain length of a reversed bonded phase.

capillary tubing - tubing for connecting various parts of the chromatograph. Most capillary tubing used in HPLC is <0.020 in. i.d. The smallest useful i.d. is about 0.004 in.

cartridge column - a type of column that has no endfittings and is held in a cartridge holder. The column is a tube; the packing is contained by frits in each end of the tube. Cartridges are easy to change and are less expensive and more convenient than conventional columns with endfittings.

cation-exchange chromatography - the form of ion-exchange chromatography that uses resins or packing with functional groups that can separate cations. A sulfonic acid would be an example of a strong cation-exchange group; a carboxylic acid would be a weak cation-exchange group.

chain length - the length of carbon chain in the hydrocarbon portion of a reversed-phase packing. It is expressed as the number of carbon atoms (e.g. C8, C18).

channeling - occurs when voids created in the packing material of a column may cause mobile phase and accompanying solutes to move more rapidly that the average flow velocity, resulting in band broadening. The voids are created by poor packing or by erosion of the packed bed.

chemisorption - sorption caused by a chemical reaction with the packing. Most such interactions are irreversible; usually occur on packings with reactive functional groups such as silanol or bonded amino phases.

chiral stationary phases (CSP) - stationary phases that are designed to separate enantiomeric compounds. They can be bonded to solid supports or created in situ on the surface of the solid support, or they can be surface cavities that allow specific interactions with one enantiomeric form.

column chromatography - any form of chromatography that uses a column or tube to hold the stationary phase. Open-column chromatography, HPLC, and open-tubular capillary chromatography are all examples.

corrected retention time (T) - the retention time corrected for the pressure drop developed along the chromatographic column; column inlet and outlet pressures need to be measured.

correction factor - syn. response factor; calculation coefficient which corrects the different response of detectors to different compounds; used to convert peak areas to numbers proportional to weight of sample.

counterion - in an ion-exchange process, the ion in solution used to displace the ion of interest from the ionic site. In ion pairing, it is the ion of opposite charge added to the mobile phase to form a neutral ion pair in solution.

coupled columns - a form of column switching. Uses a primary column connected to two secondary columns via a selector valve. Fractions from the first column can be selectively transferred to the other two columns for additional separation. Term also used to describe two or more columns connected in series to provide increased plate number.

coverage - refers to the amount of bonded phase on a silica support in bonded-phase chromatography. Coverage is usually described in mmol/m2 or in terms of %C.

cross-linking - during the process of copolymerization of resins to form a three-dimensional matrix, a disfunctional monomer is added to form cross-linkages between adjacent polymer chains. The degree of cross-linking is determined by the amount of this monomer added to the reaction. For example, divinylbenzene is a typical cross-linking agent for polystyrene ion-exchange resins. The swelling and diffusion characteristics of a resin are governed by its degree of cross-linking.

cyano phase - a stationary phase that usually consists of cyanopropylsilyl groups; used in both normal and reversed-phase chromatography.

deactivated support - support which has been chemically treated to reduce its surface activity; the most common treatments include acid washing (AW) and silanizing with dimethyl dichlorosilane (DMCS).

dead band - range within which the signal can change without causing a response in a potentiometric recorder.

dead volume (Vo) dead time (To) - syn. gas hold-up; volume of carrier gas required to transport a compound not retained by the stationary phase throughout the column; usually measured by injection of air or methane.

degassing - the process of removing dissolved gas from the mobile phase before or during use. Dissolved gas may come out of solution in the detector cell and cause baseline spikes and noise. Dissolved air can affect electrochemical detectors (by reaction) or fluorescence detectors (by quenching). Degassing is carried out by heating the solvent or by vacuum (in a vacuum flask), or on-line using evacuation of a tube made from a gas-permeable substance such as PTFE, or by helium sparging.

derivative(s) - compound(s) obtained from original sample, usually through chemical reactions which are more easily chromatographed; active protons such as those found in acids, amines, alcohols, and phenols are reacted to form more inert esters, ether, or silyl derivatives.

detection limit - syn. minimum detectable quantity; amount of sample which produces a signal twice the noise level.

detector - part of the gas chromatograph which constantly monitors the composition of the column effluent by measuring some physical property of the carrier gas and eluted compounds.

diaphragm pump - a reciprocating piston pump which acts not directly on the mobile phase, but on nydraulic fluid in a closed system. The pumped hydraulic fluid compresses a flexible steel diaphragm, which in turn pumps mobile phase with a reaction in flow stream pressure pulsation.

dimethyl dichlorosilane (DMCS) - reagent employed to block the silanol groups of the diatomaceous supports through chemical reaction.

displacement - a form of chromatography (like elution and frontal) where stronger solvents are used in series to displace (desorb) sample components; used occasionally in liquid chromatography.

distribution coefficient (K) - syn. partition coefficient; ratio of the concentration of solute or sample in the stationary phase to the concentration of the same in the gas phase.

dual piston pump - see "reciprocating piston pump".

eddy diffusion - phenomena occurring in packed columns due to lack of homogeneity of packing; expressed quantitatively by the first term of the van Deemter equation; produces peak broadening.

effective plate number - number of theoretical plates calculated by using the adjusted retention time instead of the absolute retention time; is considered to be a better measure of the efficiency of capillary columns.

efficiency of a column - column characteristic expressed quantitatively by the number of theoretical plates; efficient columns have many theoretical plates and show only limited band broadening.

effluent splitter - device which divides the column effluent in two or more streams; useful when two detectors measure the same sample simultaneously, or when part of the effluent is collected or passed to an auxiliary instrument.

electrochemical detector - an HPLC detector based upon an electroreductive or electro-oxidative process at a micro electrode in a low volume detector flowcell. The process is similar to polarographic techniques, and such electrochemical methods can be adapted to HPLC.

electronic integrator - instrument which converts the chromatographic signal into a frequency count proportional to peak area.

eluant - syn. carrier gas; general designation of mobile phase in chromatography.

eluate - that which is eluted from the column; designation of the sample when separated and dissolved in the mobile phase.

elution analysis - syn. elution technique; elution chromatography; the most commonly used technique in chromatography; the sample components are transported by the carrier gas and separated according to their partition coefficients.

elutropic series - a relative ranking of HPLC solvents ranging from non-polar to very polar properties. Very useful in choosing solvents for separation of analytes by column-solvent partitioning phenomena when developing an HPLC method. Polarity effects are due, in part, to dielectric constant, dipole moment and hydrophobic-hydrophillic properties.

endcapping - a column is said to be endcapped when a small silylating agent (e.g. trimethylchlorosilane) is used to bond residual silanol groups on a packing surface. Most often used with reversed-phase packings. May cut down on undesirable adsorption of basic or ionic compounds.

endfitting - the fitting at the end of the column that connects it to the injector or detector. Most HPLC endfittings contain a frit to hold the packing and have a low dead volume for minimum band spreading. Usually made of stainless steel.

exclusion limit - in SEC, the upper limit of molecular weight (or size), beyond which molecules will elute at the same retention volume, called the exclusion volume. Many SEC packings are referred to by their exclusion limit. For example, a 105 column of porous silica gel will exclude any compounds with a molecular weight higher than 100,000 based on a polystyrene calibration standard.

exclusion volume (Vc) - the retention volume of a molecule on an AEC packing; all molecules larger than the size of the largest pore are totally excluded and elute at the interstitial volume of the column.

external standard - syn. absolute calibration; quantitative analysis method in chromatography where dilutions of pure standard are compared to unknown samples; the standards are the components of interest.

extracolumn effects - the band-broadening effects of parts of the chromatographic system outside of the column itself. Extracolumn effects must be minimized in order to maintain the efficiency of the column. Areas of band broadening can include the injector, connecting tubing, endfittings, frits, detector cell volume, and internal detector tubing. The variances of all of these contributions are additive.

ferrule - part of fitting (see fittings).

fittings - pieces of plumbing used to connect the column to the instrument by high pressure, high temperature seal.

flow rate - volume of mobile phase per unit time passing through the column, usually reported as milliliters per minute.

flow splitters - (see effluent splitter).

fluorescence detector - a very sensitive and selective HPLC detector, equipped with two monochrometers at right angles to one another. The flow cell is illuminated at one face, and compounds which are excited by that light (can fluoresce) emit light at a different wavelength. This emitted light is measured via the second monochrometer using a photomultiplier tube.

frit - the porous element at either end of a column that serves to contain the column packing. It is placed at the very ends of the column tubs or, more commonly, in the endfitting. Frits are made from stainless steel or other inert metal or plastic, such as porous PTFE or polypropylene.

frontal analysis - a form of chromatography where pure sample flows through the column; each component breaks through at a different time depending on its affinity for the column; not commonly used today.

fronting - peak shape in which the front part of a peak (before the apex) in a chromatogram tapers in advance of the remainder of the peak. There is an asymmetric distribution with a leading edge. The asymmetry factor for a fronting peak has a value <1. The opposite effect is tailing. Fronting is related to the shape of the sorption isotherm.

gas chromatography (GC, GLC) - a form of chromatography where the mobile phase is gas.

Gaussian curve - a standard error curve, based on a mathematical function, that is a symmetrical, bell-shaped band or peak. Most chromatographic theory assumes a Gaussian peak.

gel filtration chromatography (GFC) - size-exclusion chromatography carried out with aqueous mobile phases. Generally refers to separations carried out on soft gels such as polydextrans. Most gel filtration separations involve biopolymers.

gel permeation chromatography (GPC) - SEC carried out with organic mobile phases. Used for the separation and characterization of polymers. SEC with aqueous mobile phases is referred to as a aqueous GPC, or GFC.

ghost-peak - spurious signal due to sample carry over in a syringe or injection valve.

gradient elution - technique for decreasing separation time by increasing mobile phase strength over time during the chromatographic separation. Also known as solvent programming. Gradients can be continuous or stepwise. Binary, ternary, and quaternary solvent gradients have been used routinely in HPLC.

guard column - a small column placed between the injector and the analytical column. Protects the analytical column against contamination by sample particulates and, perhaps, by strongly retained species. The guard column is usually packed with the same material as the analytical column and is often of the same i.d. It is much shorter, costs less, and is usually discarded when it becomes contaminated.

H - Same as HETP.

head pressure - the pressure above gravity at the head of the column. Expressed in psig, bar, atm, or MPa.

headspace analysis - analysis of the vapors above a liquid sample; commonly used for analysis of foods, flavors, fragrances, etc.

heart cutting - in preparative LC, refers to collection of the center of the peak, where purity should be maximum. Also used in column switching.

height equivalent to a theoretical plate (HETP) - value obtained by dividing the column length by the number of theoretical plates; taken as an indication of column quality.

HETP - height equivalent to a theoretical plate. A carryover from distillation theory; a measure of a column's efficiency. For a typical HPLC column well-packed with 5-mm particles, HETP (or H) values are usually between 0.01 and 0.03 mm. HETP = L/N, where L is column length, and N is the number of theoretical plates.

hexamethyl disilazane (HMDS) - sililating reagent employed to deactivate solid supports by blocking of silanol groups.

hydrophilic - "water-loving"; refers both to stationary phases that are compatible with water and to water-soluble molecules in general. Most columns used to separate proteins are hydrophilic in nature and should not sorb or denature protein in the aqueous environment.

hydrophobic - "water-hating"; refers both to stationary phases that are not compatible with water and to molecules in general that have little affinity for water. Hydrophobic molecules have few polar functional groups; most are hydrocarbons or have high hydrocarbon content.

hydrophobic interaction chromatography - a technique in which reversed-phase packings are used to separate molecules by virtue of the interactions between their hydrophobic moieties and the hydrophobic sites on the surface. High salt concentrations are used in the mobile phase; separations are effected by changing the salt concentration. The technique is analogous to "salting out" molecules from solution. Gradients are run by decreasing the salt concentration over time.

internal standards (IS) - substance used as reference in quantitative analysis; the internal standard is first mixed with standard solutions; later it is added to the unknown, and the ratio of peak heights (or areas) of internal standard and analyte is used for quantitative analysis.

interstitial volume - (Vo) - the total value of mobile phase within the length of the column. It is made up of the intraparticle volume (inside the packing itself) and interparticle volume (between the packing particles). Same as void volume. Also abbreviated Vi or Vm.

ion chromatography (IC) - An ion-exchange technique in which low concentrations of anions or cations are determined using low-capacity ion exchangers with weak buffers. Conductivity detectors are often used. Ion chromatography is practiced in two forms. In suppressed IC, a second column is used to remove the buffer ions so that sample ions can be more easily detected; membrane separator is sometimes used. In nonsuppressed IC, weakly conducting buffers at low concentration are carefully selected, and the entire effluent is passed through the detector; ions are detected above the background signal.

ion-exchange chromatography (IEC) - a mode of chromatography in which ionic substances are separated on cationic or anionic sites of the packing. The sample ion (and usually a counterion) will exchange with ions already on the ionogenic group of the packing. Retention is based on the affinity of different ions for the site and on a number of other solution parameters (pH, ionic strength, counterion type, etc.).

ion-exchange capacity - the number of ionic sites on the packing that can take part in the exchange process. Exchange capacity is expressed in mequiv/g; typical strong anion-exchange resin may have 3-5 mequiv/g capacity.

ion-pair chromatography - form of chromatogrpahy in which ions in solution can be "paired" or neutralized and separated as an ion pair on a reversed-phase column. Ion-pairing agents are usually ionic compounds that contain a hydrocarbon chain that imparts a certain hydrophobicity so that the ion pair can be retained on a reversed-phase column. Ion-pairing can also occur in normal-phase chromatography when one part of the pair is loaded onto a sorbent, but this technique is not as popular as the RPC technique.

irregular packing - refers to the shape of a silica gel based packing. Irregular silicas are available in microparticulate sizes. The packings are made by grinding silica gel into small particles and then sizing them into narrow fractions using classification machinery. Spherical packings are now used more often than irregular packings in HPLC, but less-expensive irregular packings are still widely used in prep LC.

irreversible adsoprtion - when a compound that has a very strong affinity for the adsorbent is injected onto a column, it can be adsorbed so strongly that it cannot be eluted from the column. A chemical reaction between the sample and the surface of the adsorbent is an example of irreversible adsorption.

isocratic - use of a constant-composition mobile phase in liquid chromatography.

Kovats index (Kovats retention index) - characterization system of the chromatographic behavior of substances in gas chromatography; normal alkanes are used as reference compounds to establish a scale of retention; widely used as a qualitative tool in Europe.

linear range - extension of the calibration plot (usually expressed in decades of concentration) within which the detector response is clearly linear.

linear velocity (u) - the velocity of the mobile phase moving through the column. Expressed in cm/s. Related to flow rate by the cross-sectional area of the column. Sometimes expressed as v.

linearity - proportionally between detector response and amount of sample; a calibration plot with a slope of 1.0 is the ideal case.

liquid-liquid chromatography (LLC) - same as partition chromatography. The earliest form of HPLC, it gave way to chemically bonded phases in the early 1970s.

liquid-solid chromatography (LSC) - same as adsorption chromatography.

loading - the amount of stationary phase coated or bonded onto a solid support. In liquid-liquid chromatography, the milligram amount of liquid phase per gram of packing. In BPC, the loading may be expressed in mmol/m2 - or in %C. See coverage.

longitudinal diffusion - same as molecular diffusion term/ B-term in van Deemter equation. See van Deemter equation.

mass transfer - band-broadening effect due to the lack of equilibrium between the mobile and stationary phase when partitioning the sample; this effect is expressed by the third term of the van Deemter equation.

mean pore diameter - the average pore diameter of the pore in a porous packing. The pore diameter is important in that it must allow free diffusion of solute molecules into and out of the pore so that the solute can interact with the stationary phase. In SEC, the packings have different pore diameters, and therefore molecules of different sizes can be separated. For a typical adsorbent such as silica gel, 60-Å and 100-Å pore diameters are most popular. For packings used for the separation of biomolecules, pore diameters ≥300 Å are used.

mesh - usual way to characterize particle size; the mesh number indicates the number of wires per inch of the sieve through which the particles are passed.

micellar chromatography - the addition of micelles to the mobile phase to effect separations. The micelles act as displacing or partitioning agents and provide another parameter that can be used to change selectivity.

micro LC - refers collectively to techniques in which a column of smaller-than-usual internal diameter (i.d.) is used for separation. In micor HPLC, columns of <0.5 mm i.d. are used.

minimum plate height - the minimum of the curve that results from a plot of H vs u. This value represents the most theoretical plates that can be obtained for a certain column and mobile phase system. Usually occurs at very low flow rates.

mobile phase - in LC the mobile phase is a solvent mixture such as methanol and water for reversed phase LC, or hexane for adsorption chromatography that flows continuously through the column.

modifier - additive that changes the character of the mobile phase. For example, in reversed phase, water is the weak solvent; methanol, the strong solvent, is sometimes called the modifier.

molecular diffusion - effect that produces band-broadening expressed by the second term of the van Deemter equation; the effect is due to the sample diffusion in the mobile phase.

molecular weight distribution - the distribution of molecular weight of molecules in a polymer sample. Distribution can be defined as weight average and number average.

monomeric phase - refers to a bonded phase in which single molecules are bonded to a support. For silica gel, monomeric phases are prepared by the reaction of an alkyl or aryl monochlorosilane. Polymeric phases are generally prepared from a di- or tri-chlorosilane reactant.

net retention time (Tog) - the adjusted retention time per gram of liquid phase corrected for the pressure drop along the chromatographic column.

noise - random fluctuation of the chromatographic signal; short-term noise (less than 1 sec) is often electrical in nature; long-term noise can be due to flow rate changes, temperature changes, or column "bleed".

normalization - quantitative method commonly employed when the entire sample is eluted from the column; the area percent is taken as weight percent composition; of limited usefulness since most detectors give different responses to different samples.

normal-phase chromatography - a mode of chromatography carried out with a polar stationary phase and a nonpolar mobile phase. Adsorption on silica gel using hexane as a mobile phase would be a typical normal-phase system. Also refers to the use of polar bonded phases, such as CN or NH2 . Sometimes referred to a straight-phase chromatography.

octadecylsilane (ODS) - the most popular reversed phase in HPLC. Octadecylsilane phases are bonded to silica or polymeric packings. Both monomeric and polymeric phases are available.

open tubular column - syn. capillary column, Golay column, WCOT column, SCOT column; a column with a hole down the middle, frequently called a capillary column.

overload - in preparative chromatography, the overload condition is defined as the mass of sample injection onto the column at which efficiency and resolution begin to be affected if the sample size is further increased. See sample capacity.

packing - material contained inside the column; responsible for the separation.

particle size (dp) - the average particle size of the packing in an LC column. A 5-mm column would be packed with particles having definite particle size distribution; packings are never monodisperse. See particle-size distribution.

particle-size distribution - a measure of the distribution of the particles used to pack the LC column. In HPLC, a narrow particle-size distribution is desirable. A particle-size distribution of dp ± 10% would mean that 90% of the particles fall between 9 and 11 mm for an average 10-mm dp packing.

partition - distribution phenomena of the sample between the mobile phase and the stationary phase.

partition chromatography - separation process in which one of the liquid phases is held stationary on a solid support while the other is allowed to flow freely down the column. Solutes partition themselves between the two phases based on their individual partition coefficients. Liquid-liquid chromatography is an example.

partition coefficient (K) - quantitative expression of the partition equilibrium; usually expressed as the ratio of concentration of the sample in the stationary phase and the mobile phase.

partition ratio (k) - syn. capacity ratio, capacity factor; column characteristic expressed as the ratio of the retention volumes to the dead volumes; k' = t'R/to.

phase ratio(b) - column characteristic defined as the ratio of mobile phase to stationary phase.

K = kb

phenyl phase - a nonpolar bonded phase prepared by the reaction of dimethylphenylchlorosilane with silica gel. Claimed to have affinity for aromatic compounds.

photoconductometric detector - an HPLC detector which works on a two fold principle. Firstly, high energy photoexcitation of a neutral molecule raises its energy level to generate a charged, ionized species. Secondly, this new charged species is detected by a conductometric measuring cell. Chloro-aromatics are one of the analytes measured at very low levels by this technique.

pirkle columns - chiral "brush-type" stationary phases, based on 3.5-dinitrobenzoylphenylglycine silica, used in the separation of a wide variety of enantiomers. Named after the developer, Dr. William Pirkle, University of Illinois.

plate height - syn. height equivalent to a theoretical plate, HETP; column length corresponding to a theoretical plate, normally found by dividing the column length by the number of theoretical plates.

plot columns - syn. porous layer open tubular columns; see open tubular column.

polarity - a measure of the separation of charges in a molecule; hydrocarbons are non-polar; alcohols are polar; more polar stationary phases interact more strongly with polar samples and usually provide a better separation.

polystyrene-divinylbenzene resin (PS-DVB) - the most common polymer base for ion-exchange chromatography. Ionic groups are incorporated by various chemical reactions. Neutral PS-DVB beads are used in reversed-phase chromatography. Porosity and mechanical stability can be altered by varying the cross-linking through the variation of the DVB content.

porosity - for a porous adsorbent, the ratio of the volume of the interstices to the volume of the solid particles. The pore volume is also used as a measure of porosity.

preparative chromatography - chromatographic technique in which the purpose is the separation of sizable amounts of pure materials.

programmed temperature gas chromatography (PTGC) - technique employed to speed up the elution of long-retained compounds by gradual heating of the column as the separation occurs.

pulsating flow - flow originating from a reciprocating pump. Normally, the pulses are dampened out by a pulse damper, by an electronic pressure feedback circuit, or by an active damper pump head. Some detectors (e.g. electrochemical) are affected by flow pulsations.

reciprocating piston pump - the most common HPLC pump design which is available in single or multiple-piston arrangements forces mobile phase through the column on a forward piston stroke, and refills the cylinder with mobile phase on the recycle stroke.

recorder - electromechanical instrument which transforms the chromatographic signal into a graphical record.

reduced plate height (h) - Used to measure efficiencies of columns. An HPLC column with an h value ≤2 is considered to be well-packed, h = H/dp.

reduced velocity (v) - along with the reduced plate height, is used to compare different chromatographic columns. It relates the solute diffusion coefficient (Dm) in the mobile phase to the particle size of the column packing (dp). v = dp/Dm.

relative retention time - syn. solvent efficiency; ratio between the net retention time of a substance and that of a standard compound.

residual silanols - the silanol (-Si-OH) groups that remain on the surface of a packing after a phase in chemically bonded onto its surface. These silanol groups may not be accessible to the reacting bulky organosilane (e.g. octadecyldimethylchlorosilane) but may be accessible to small polar compounds. Often they are removed by endcapping with a small organosilane such as trimethylchlorosilane. See endcapping.

resolution - a quantitative measure of the separation of two peaks; it accounts for narrowness of peaks and the separation of peak maxima.

response - detector characteristic which defines the detectable types of compounds by a particular detector.

retention index - see Kovats index.

retention time (adjusted) (t'R = tR- to) - the retention value obtained by subtracting the dead time from the uncorrected retention time.

retention time (uncorrected) (tR) - time elapsed between sample introduction and maximum of response.

retention volume (adjusted) (V) retention volume corrected by subtracting the dead volume.

retention volume (corrected) - retention volume corrected for pressure drop across the column.

retention volume (uncorrected) - volume of mobile phase required to elute the peak maximum of a compound; calculated by multiplying the retention time by the flow rate.

reversed-phase chromatography (RPC) - the most common HPLC mode. Uses hydrophobic packings such as octadecyl- or octylsilane phases bonded to silica or neutral polymeric beads. Mobile phase is usually water and a water-miscible organic solvent such as methanol or acetonitrile. There are many variations of RPC in which various mobile phase additives are used to impart a different selectivity. For example, for the RPC of anions, the addition of a buffer and tetraalkylammonium salt would allow ion pairing to occur and effect separations that rival ion-exchange chromatography.

sample capacity - refers to the amount of sample that can be injected onto a LC column without overload. Often expressed as grams of sample per gram of packing. Overload is defined as the sample mass injected at which the column efficiency falls to 90% of its normal value.

sample loop - a loop of calibrated volume used in a sample injection valve; normal volumes range from 5 to 100 microliters.

sample valve - syn. injection valve; a device used to inject fixed volumes of liquid sample; may be operated manually or automatically; provides very reproducible injection volumes.

SAX - strong anion exchanger. A typical strong anion exchange functional group would be tetraalkylammonium.

SCX - strong cation exchanger. A typical strong cation exchange functional group would be a sulfonic acid.

selectivity (a = (t'R(2)/t'R(1)) - measures the selective solubility of a stationary phase for 2 compounds; high a values mean high selectivity and easy separation.

sensitivity - term which quantitatively describes the signal obtained per amount of sample introduced.

separation factor - ratio of retention times of two peaks; has been replaced by selectivity a which is the ratio of adjusted retention times.

silanol - the Si-OH group found on the surface of silica gel. There are different strengths of silanols, depending on their location and relationship to each other. The strongest silanols are acidic and often lead to undesirable interactions with basic compounds during chromatography.

silica gel - the most commonly used packing in liquid chromatography. It has an amorphous structure, is porous, and consists of siloxane and silanol groups. It is sued as a bare packing for adsorption, as the support in liquid-liquid chromatography or for chemically bonded phases, and, with various pore sizes, as packing in size-exclusion chromatography. Micro-particulate silicas of 5- and 10-mm average particle diameter are used in HPLC.

siloxane - the Si-O-Si bond. A principal bond found in silica gel or for attachment of a silylated compound or bonded phase. Stable except at high pH values.

silylation - a chemical reaction involving a silane reagent such as trimethyl chlorosilane (TMS); used to silanize or deactivate active sites found in samples on solid support surfaces and even on glass tubing.

single-piston pump - see "reciprocating piston pump".

size-exclusion chromatography (SEC) - same as steric exclusion chromatography.

slurry packing - the technique most often used to pack HPLC columns with microparticles. The packing is suspended in a slurry (10% wt/vol) and is rapidly pumped into the empty column. Special high-pressure pumps are used.

solid phase extraction (SPE) - A sample-preparation technique that uses a solid-phase packing contained in a small plastic cartridge. The solid stationary phases are the same as HPLC packings; however, the principle is different from HPLC. Sometimes referred to as digital chromatography. The process as most often practiced requires four steps: conditioning the sorbent, adding the sample, washing away the impurities, and eluting the sample in as small a volume as possible with a strong solvent.

solid support - 5 to 20 mm particles contained in the column whose surface is coated with stationary phase in liquid-liquid chromatography, or derivatized with a bonded phase in BPC.

solute - syn. sample or analyte.

solvent strength - refers to the ability of a solvent to elute a particular solute or compound from a column. Described by Lloyd Snyder for LEAC (LSC) adsorption chromatography on alumina; solvents were quantitatively rated in an elutropic series. No elutropic series exists for other modes.

sorbent - refers to an adsorption packing used in liquid chromatography. A common sorbent is silica gel.

spherical packing - refers to spherical solid packing materials. Spherical packings are generally preferred over irregular particles.

standard addition - a method of quantitative analysis where standard amounts of sample are added to the unknown; from the increase in peak height the concentration in the unknown can be estimated.

stationary phase - syn. liquid phase; liquid covering the surface of the solid support in the column in LLC, or the derivatized particles in BPC.

steric exclusion chromatography (SEC) - a major LC mode in which samples are separated by virtue of their size in solution. Also known as size-exclusion, gel permeation, gel filtration, or gel chromatography.

supercritical fluid chromatography (SFC) - a technique that uses a supercritical fluid as the mobile phase. The technique has been applied to the separation of substances that cannot be handled effectively by liquid chromatography (because of detection problems) or gas chromatography (because of the lack of volatility). Examples are separations of triblycerides, hydrocarbons, and fatty acids. GC detectors and HPLC pumps have been used together in SFC.

support - refers to solid particles. Support can be naked or coated or can have a chemically bonded phase in HPLC.

suppressor column - in ion chromatography, refers to the column placed after the ion-exchange column. Its purpose is to remove or suppress the ionization of buffer ions so that sample ions can be observed in a weakly conducting background with a conductivity detector.

surface area - in an adsorbent, refers to the total area of the solid surface as determined by an accepted measurement technique such as the BET method using nitrogen adsorption. The surface area of a typical porous adsorbent such as silica gel can vary from 100 to 600 m2/g.

surface coverage - usually refers to the mass of stationary phase per unit area of an LC support. Often expressed in mmol/m2 of surface. Sometimes the %C is given as an indicator of surface coverage.

swelling - process in which resins and gels increase their volume because of their solvent environment. Solvent enters ion-exchange resin to dilute ions;; in gels, solvent penetrates pores. If swelling occurs in packed columns, blockage or increased back pressure can occur. In addition, column efficiency can be affected.

syringe pump - a popular and useful pump design for Micro-LC and SFC, consists of a single, large volume cylinder and piston assembly (typically 50 mL in volume), such that uninterrupted mobile phase flow is available without pulsation.

tailing - the phenomenon in which the normal Gaussian peak has an asymmetry factor >1. The peak will have skew in trailing edge. Tailing is caused by sites on the packing that have a stronger-than-normal retention for the solute. A typical example of a tailing phenomenon is the strong adsorption of amines on the residual silanol groups of a low-coverage reversed-phase packing.

theoretical plate - one equilibrium between the mobile and stationary phase; a measure of column efficiency; the more plates, the better is the column efficiency.

tortuosity - a property of a packed column that indicates the degree of unevenness of the path followed by the solute molecule as it passes down the column. Covered in the A-term of the van Deemter equation.

total permeation volume (Vp) - the retention volume on an SEC packing in which all molecules smaller than the smallest pore will elute. In other words, at Vp, all molecules totally permeate all of the pores and elute together.

trimethyl chlorosilane (TMS) - common reagent employed in the formation of derivatives, and in deactivation of packing materials.

uncorrected retention time - see retention time; time from injection to peak maximum.

UV detector - the most popular detector for HPLC, a UV spectrophotometer (for variable-wavelength detection) or photometer (for single-wavelength detection) equipped with a low-volume flow through "curvette", commonly referred to as a flow cell. Detects analytes which readily absorb light at the selected wavelength.

van Deemter equation - theoretical relationship which describes sample band broadening as a function of three phenomena; eddy diffusion, molecular diffusion, and mass transfer.

void - the formation of a space, usually at the head of the column, caused by a settling or dissolution of the packing. A void in the column leads to decreased efficiency and loss of resolution. Even a small void can be disastrous for small microparticlate columns. The void can sometimes be removed by filing it with glass beads or porous packing.

void volume (Vi) - the total volume of mobile phase in the column; the remainder of the column is taken up by packing material. Can be determined by injecting an unretained substance that measures void volume plus extracolumn volume. Also referred to as interstitial volume. Vo or Vm are sometimes used as a symbols.

wall effect - the consequence of the looser packing density near the walls of the rigid HPLC column. Mobile phase as a tendency to flow slightly faster near the wall because of the decreased permeability. The solute molecules that happen to be near the wall are carried along faster than the average of the solute band and, consequently, band spreading results.

WAX - weak anion exchanger. Ionizable groups such as primary, secondary, or tertiary amino groups on a packing are considered to be weak.

WCX - weak cation exchanger. Carboxylic groups on a packing are typical of a weak cation exchanger.

well coated open tubular column - syn. WCOT; capillary column, Golay column.

zwitterions - compounds that carry both positive and negative charges in solution.

Chromatography Common Abbreviations

a Selectivity; separation factor

AED Atomic Emission Detector

b Phase ratio

C4, C8, C18 Alkyl chain length of an LC reversed bonded phase

CE, CZE Capillary Electrophoresis, Capillary Zone Electrophoresis

CI Chemical Ionization

DAD, PDA (Photo) Diode Array Detector

dp Particle diameter

df Film thickness

ECD Electron Capture Detector

EI Electron Impact

ELCD Electrolytic Conductivity Detector

EPA Environmental Protection Agency

FAMEs Fatty Acid Methyl Esters

FID Flame Ionization Detector

FPD Flame Photometric Detector

FS Fused Silica

FT-IR Fourier Transform Infrared

GLC Gas Liquid Chromatography

GPC Gel Permeation Chromatography

GSC Gas Solid Chromatography

HETP or H Height Equivalent to one Theoretical Plate

HPLC High Performance Liquid Chromatography

HRGC High Resolution Gas Chromatography

I.D. Internal Diameter

IPC Ion Pair Chromatography

IRD Infrared Detector

IS Internal Standard

k (or k') Capacity factor (syn capacity ratio, partition ratio)

K Partition coeficcient

l Wave length

LSC Liquid Solid Chromatography

LIF Laser Induced Fluorescence (detector)

MAOT Maximum Allowable Operating Temperature

MDQ / MDL Minimum Detectable Quantity / Minimum Detectable Level

MIP Microwave Induced Plasma

MS Mass Spectrometry

MSD Mass Selective Detector

N Theoretical plate (number of)

NP Normal Phase

NPD Nitrogen Phosphorus Detector

O.D. Outer Diameter

ODS Octadecyl silane

PCBs Polychlorobiphenyls

PID Photo Ionization Detector

PLOT Porous-Layer Open Tubular (column)

PMT Photo-Multiplier Tube

PNAs Polynuclear Aromatics

ppb Part per billion

ppm Part per million

ppt Part per trillion

PTFE Polytetrafluoroethylene (Teflon®)

PTGC Programmed Temperature Gas Chromatography

RS Resolution

RP Reversed Phase

RSD Relative Standard Deviation

SCOT Surface Coated Open Tubular (column)

SEC Size (or Steric) Exclusion Chromatography

SFC Supercritical Fluid Chromatography

SFE Supercritical Fluid Extraction

SIM Single Ion Monitoring

S/N Signal to noise

TCD Thermal Conductivity Detector

TID Thermionic Detector (usually known as NPD)

TLC Thin Layer Chromatography

THMs Trihalomethanes

TMS Trimethylsilyl (derivative)

tR Retention time

UV Ultra-Violet (detector)

VR Retention volume

VOC Volatile Organic Compound

W Peak width

WCOT Wall Coated Open Tubular (column)

Structure-related sample treatment

(Bio-)Analytical Chemistry: From B(egin) to E(nd)

Hubertus Irth, Henk Lingeman

BioMolecular Separations Group, Faculty of Sciences, VU University Amsterdam, Amsterdam, the Netherlands

The start
Introduction

Sample treatment (ST) is the bottleneck in most chromatographic/electrophoretic separations. As a result the primary goal of the present module on “Structure related sample treatment” techniques and approaches that can be used for this purpose.

The main ST objectives discussed in this module are:

- Learn technologies and techniques:

o How do they work,

o What are the applications,

o What are the advantages and limitations,

- Possibilities to improve laboratory operations:

o Cost (labour intensive),

o Errors (accuracy, precision),

o Time,

- Productivity issues:

o Automation,

o Parallel versus serial processing,

o On-line versus off-line,

- Help to make the transition from SOP’s to (new) techniques a successful one:

o Criteria for choosing sample treatment techniques.

This means that the problems are addressed that are related to: time, labour, automation, enrichment, selectivity, organic solvents, inorganic salts, contamination, etc.

The resulting ST goals are:

1. Removal of interferences (high and low molecular weight compounds, organic and inorganic compounds);
2. Enrichment of the analyte(s);
3. Phase transfer of the solutes(s).

The main focus will be on the development of total (bio-)analytical procedures an in particular on the ST steps needed to determine these compounds with either gas chromatography (GC), liquid chromatography (LC), ion chromatography (IC), affinity chromatography (AC) or capillary electrophoresis (CE), with the emphasis on automated/hyphenated systems including high-throughput and bio-specific assays. This includes a discussion on the theoretical backgrounds, advantages and disadvantages of pharmaceutical, bio-analytical, food, clinical environmental and process analytical methods and procedures.

Figure 1.1: Relative speed of different steps in (bio-)analytical sample treatment.

The fact that the polarity of the compounds of interest is still increasing and the fact that sampling and sample preparation (SP) are the most time consuming parts of the total analytical scheme, explains the focus on the numerous combinations of sampling and SP procdures in combination with GC / LC and CE separation-detection approaches (Figure 1.1)

The most important options for ST, sampling plus sample preparation, are:

- Sample collection: Taking a representative sample;

- Sample storage and stabilization: Using proper containers and freezing of unstable samples;

- Initial (primary) sample preparation: Reducing the sample size;

- Weighing or volumetric dilution: Taking precautions for unstable and reactive samples;

- Alternative sample processing methods: Introducing solvent replacement, desalting, evaporation procedures etc.;

- Removal of particulates: Applying filtration and centrifugation steps;

- Selective (secondary) sample preparation: Introduction of liquid-liquid extraction (LLE) and solid-phase extraction (SPE) approaches;

- Derivatization: Enhancing detection and improving separation.

In this module various definitions will be used:

- Sample treatment: Total process of analyzing a sample including sampling, initial (primary) and selective (secondary) sample preparation, analysis, detection, quantitation and validation.

- Sampling: Process of taking a reliable and representative sample.

- Initial sample preparation: Process of storing, stabilizing and preparing a sample for selective clean-up and analysis.

- Selective sample preparation: Process of sample concentration, selective sample clean-up and/or phase transfer of the analyte(s).

- Analysis: Process of selective (qualitative or quantitative) determination of the analyte(s).

- Detection: Process of identifying or detection of the analyte(s).

- Quantitation: Process of data acquisition, data reduction and data interpretation.

- Validation: Process of guaranteeing the repeatability / reproducibility, robustness / ruggedness of the overall sample treatment process.

These steps are summarized in the overall analytical procedure as given in Figure 1.2.

Figure 1.2: Overall analytical procedure.

Another reason that sampling and SP are so important is that looking at the time spend on the analysis, about 60% is related to sample processing. In addition to this about 30% of the errors during SP/ST is due to sample processing and another 20% to the operator. This means that automation (Figure 1.3) of SP/ST techniques certainly will improve the overall quality of the analytical procedure.

Figure 1.3: Lab-automation in combinatorial chemistry (www.pa.bosch.com).

Analytical chemistry plays an important role in separating, isolating and quantifying various chemical compounds. Virtually every item of commerce has been subjected to analytical testing (Figure 1.4) at one or more stages in its manufacturing process.

Figure 1.4: Analytical testing can be performed using numerous techniques and procedures (www.pennpharm.co.uk).

Apart from the classical methods, such as titrimetric and gravimetric techniques, many instrumental techniques have been developed, for the determination of not only the active ingredient, but also the quantification of related compounds or impurities associated with it (Figure 1.5).

Figure 1.5: Traditional techniques like titrimetric and gravimetric analysis are, nowadays, replaced by modern separation techniques.

The recently developed analytical methods have the advantage of not only using small amounts of sample, reagents and less time, but also produce accurate results. These analytical techniques may be:

- physico-chemical methods,

- electro-analytical methods

- separation-based methods.

The physico-chemical methods like spectroscopy include colorimetry and spectrophotometry covering ultra-violet and visible region or fluorimetry, nephelometry (Figure 1.6) or turbidimetry, and nuclear-magnetic resonance and mass spectrometry. The electro-analytical methods cover potentiometry, amperometry, voltammetry, electrophoresis and polarography.

Figure 1.6: Nephelometry is based on the principle that a dilute suspension of small particles will scatter light (usually a laser) passed through it rather than simply absorbing it. The amount of scatter is determined by collecting the light at an angle (www.lib.mcg.edu).

The separation-based methods involve (high-performance) liquid chromatography (LC), (high-performance) thin-layer chromatography (HPTLC / TLC), capillary electrochromatography (CEC), supercritical-fluid chromatography (SFC) and gas chromatography (GC). The combination of GC and LC with mass spectrometry (MS), nowadays, are the most powerful tools employed for the quantification and identification of analytes in pure as well as in associated forms. In addition to chromatographic separation methods, electrophoretic techniques, like capillary electrophoresis (CE), isotachophoresis (ITP) (Figure 1.7) gel electrophoresis (GE) and isoelectric focusing (IEF), are relatively popular for the separation and quantitation of (charged) organic compounds.

Figure 1.7: Isotachophoresis is one of the electrophoretic techniques used for the separation of charged molecules (www.bd.com).

The still increasing interest on the determination of drugs has forced the analytical chemists to develop methods for their trace analysis in the presence of biological matrices. Since every year a large number of drug candidates are synthesized it means that also a large number of methods/procedures must be developed that are applicable for the routine analysis of these drug candidates. These methods should be rapid, precise, accurate and cost effective. Although costly sophisticated instruments like LC, HPTLC, GC, GC-MS and LC-MS are available, the spectrophotometer is being preferred by ordinary laboratories for its simple, economical and easy handling techniques (Table 1.1).

Table 0.1: Bio-analytical techniques.

In summary: The philosophy and concepts of sampling, sample preparation in analytical chemistry will be presented in a practical way to provide the analytical chemist with the necessary tools to determine low- and high-molecular-weights compounds in a variety of matrices using chromatographic / electrophoretic, spectroscopic or immunological methods.

In addition, the philosophy and concepts for the method development procedures will be based on the physico-chemical properties of the analyte(s) and the matrices in which these compounds are present (Figure 1.8).

Figure 1.7: Physico-chemical properties.
Physico-chemical properties

(& Structure-activity relationships of drugs)

In order to develop analytical procedures for low-molecular weight (LMW) compounds in pharmaceutical formulations and biological samples the starting point always must the physico-chemical properties of both the compounds of interest and the sample matrix. Therefore, the focus will be on these physico-chemical properties and the structure-activity relationships between the solutes and matrix components such as the interaction between drugs and biological matrix components (e.g., proteins, enzymes, receptors) or pesticides and humic substances.

Pharmaceutical chemistry Bio-analysis Pharmacochemistry

The intention is not to discus all the basic principles of analytical chemistry, physical chemistry, organic chemistry, thermodynamics and kinetics in detail, but just to provide the information necessary to develop new or to modify existing analytical methods that can be used in pharmaceutical chemistry, bio-analysis and pharmacochemistry.

For example, during the development of new drug entities (NCE) it is of utmost importance to optimize the structure of a new compound (drug candidate) for a certain target. This can be done by changing the 3-dimensional (3-D) structure, or in other words the way the functional groups are arranged around the basic molecule. In addition the physico-chemical and bio-pharmaceutical properties of a compound are determined by its structure (Figure 1.8).

Figure 1.8: Fit of NCE in receptor pocket (www.tripos.com).

In addition the physico-chemical and bio-pharmaceutical properties of a compound are determined by its structure. These properties, on its turn, are related to processes like solubility, absorption, protein binding, partitioning, elimination and metabolism (Figure 1.9).

Figure 1.9: Summary of metabolism process (www.elmhurst.edu).

These properties, on its turn, are related to processes like solubility, absorption, protein binding, partitioning, elimination and metabolism. In (bio)-analytical chemistry (BAC) these properties determine which analytical techniques can be used and what the actual conditions must be. It will be obvious that there is a strong correlation between the various physico-chemical properties (Figure 1.10).

Figure 1.10: Relation between physico-chemical properties.

In the case of drugs it can be stated that when the molecular weight is over 500, the number of H-bridge donors is over 5, the number of H-bridge acceptors is over 10 and the calculated partitioning coefficient is over 5, the compound will badly orally adsorbed (Figure 1.11). This gain shows the importance of studying the physico-chemical properties of organic compounds.

Figure 1.11: Solubility of chloramphenicol is determined by the presence of hydrophilic and lipophilic groups (www.auburn.edu).

From the example mentioned above it will be clear that the emphasis will be on the relation between the chemical structure, or in other words the physico-chemical properties of a compound, and the determination of these compounds in a (complex) sample matrix. The final objective is that the information provided can be used to develop simple qualitative and quantitative methods for organic compounds in a variety of matrices. In order to obtain this goal the following parameters will be discussed:

- Physico-chemical properties of organic molecules like polarity, acid-base properties, solubility, stability, absorption/adsorption, etc;

- Composition of sample matrices and their effect on the analytical results (e.g., analyte-matrix binding, stability);

- ST/SP procedures (e.g., filtration, centrifugation, extraction).

There are two magic words that will be discussed throughout the whole monograph: pH and pKa (Figure 1.12).

Figure 1.12: Questions to be answered!

After studying the module on “Structure-related sample treatment” it should be possible to answer the following starting questions (Figure 1.12) and the final question (Figure 1.13):

Figure 1.13: Final analytical question to be answered!

With respect to environmental, pharmaceutical and bio-analysis this means that the following topics features are of importance:

- Insight in structures of chemical compounds and physico-chemical properties like pK_a, polarity, stability, solubility, chromophores, etc;

- Composition of biological materials and, to lesser extent, pharmaceutical formulations and environmental samples, and their influence on the analytical results (e.g. stability, matrix binding);

- Initial (primary) sample treatment procedures (e.g., filtration, precipitation, extraction);

- Selective (secondary) sample preparation procedures (e.g. solid-phase extraction, immunological approaches, hyphenated and automated system

In order to do deal with these features a number of sub-questions should be answered. These sub-questions are related to the following physico-chemical properties of the analyte(s) or the matrix molecules:

- Name, molecular weight, structure;

- Appearance, colour, smell;

- Acid-base properties;

- Functional groups;

- Polarity (partitioning coefficients);

- Solubility;

- (Chemical) stability;

- Spectral properties;

- Bio-pharmaceutical properties;

- Sample treatment / sample preparation;

- Separation / detection;

- Quantitative aspects (validation).

Except for the relatively straightforward analytical applications in most cases an overall sample analysis scheme should be constructed (Figure 1.14). This because direct sample injection into a chromatographic/electrophoretic system, normally is not possible because unwanted matrix components can disturb the separation and/or detection or can clog the analytical device. In other cases the concentration of the analyte(s) or the degradation products or metabolites may be too low to allow a direct injection procedure. The result is that ST/SP is an important aspect of the total analytical procedure and that in order to provide an adequate ST/SP procedure accurate knowledge about the properties of the compounds to be separated and the sample matrix is a prerequisite.

Figure 1.14: Sample analysis scheme.

Bio-analytical chemistry

Bio-analytical chemistry (BAC) involves a number of different disciplines such as therapeutic drug monitoring (TDM), toxicology (clinical, forensic, post-mortem), drugs-of abuse, pharmaceutical analysis, food analysis and environmental analysis.

One of the most important disciplines is therapeutic drug monitoring (TDM) (Figure 1.15). TDM can be defined as:

‘The measurement and the clinical use of blood (serum/plasma) drug levels (concentrations) to adjust each patient’s individual drug dosage and schedule to each patient’s individual therapeutic requirement’.

Figure 1.15: Therapeutic drug monitoring in epilepsy (www.e-epilepsy.org.uk).

In order to perform TDM, quantitative methods for the determination of these drugs, their metabolites and their degradation products should be available.

A number of variables complicate bio-analytical procedures. For example, after a drug has been administered to an individual, it is metabolized into products that can be excreted more easily. On its way through the body multiple metabolites are formed meaning that depending on the bioactivity and relative concentrations, the parent drug and/or the metabolites should be determined.

Biomonitoring outshines the indirect assessment of exposure in determining which pollutants enter the body, and whether they cause disease or disability (Figure 1.16). Non-persistent toxicants move quickly out of the blood as they are metabolized to water-soluble compounds that can be extracted in urine. Some chemicals or metabolites may bind to proteins or to DNA and persist in the body for a longer period of time.

Figure 1.16: Biomonitoring of pollutants (www.nature.com).

Postmortem toxicology, for example, is used to determine if alcohol, drugs or other poisons have contributed to the death of a person (Figure 1.17). Fundamentally it is different from TDM and clinical toxicology. The reason is that it is far more difficult to interpret post-mortem results. Clinical toxicology is dealing with patients with suspected poisoning and the primary objective is to assist in the treatment of such a patient.

Figure 1.17: Postmortem toxicology (www.answers.com).

Drugs-of-Abuse can be defined as: ‘Any substance that, because of some desirable effect, is used for some purpose other than that intended’ (Figure 1:18).

Figure 1.18: Mechanism of action of high concentration of amphetamine (www.cnsforum.com).

The questions in pharmaceutical analysis are again quite different. Questions that are relevant are dealing with the identity, concentration and stability of the drug in the formulation (Figure 1.19). In addition information on the impurities, shelf-life and release-rate of the drug from the formulation are essential. The connection between pharmaceutical and bio-analysis is that after administration of the drug the concentration in tissue or a biological fluid is a key factor. While during drug development parameters like acid/base properties, polarity (log P values), solubility and stability are important features.

Figure 1.19: Pharmaceutical analysis (www.chem.agilent.com).

In food analysis the properties of foods and their constituents are characterized. Analytical procedures are used in food analysis to provide information about a wide variety of characteristics such as composition (e.g., lipids, proteins, water, carbohydrates, and minerals), structure, physico-chemical properties and sensory attributes. The data obtained are used to produce on an economical, safe and nutritious way.

Environmental system analysis is an important tool to analyze sustainability, e.g. environmental impact, optimal resource management and how outside factors affect farm management (Figure 1.20).

Figure 1.20: Environmental system analysis (www-mat21.slu.se).

Trace environmental quantitative analysis (TEQA) is not only dealing with the determination of organic and inorganic compounds in e.g., air, ground, water, plant effluent, leachate, soil, sediment), by also with the determination of these compounds in body fluids and tissues because the intake of environmental pollutants by humans, and the potential of biohazards is a key issue in environmental chemistry. This means that bioterrorism and biomonitoring are important issues in this respect.

Environmental monitoring is completely different from TDM (Figure 1.21).

Figure 1.21: Environmental monitoring (B.J. Alloway, D.C. Ayres, Chemical Principles of Environmental Pollution, Blackie Academic & Professional, London 1997).

Environmental analysis can be:

- Analytes entering the environment, amounts involved, original sources and spatial distribution;

- Effects of analytes on humans, crops, livestock and eco-systems;

- Trends in concentration of analytes over time and the reason for this;

- Extent inputs, concentrations, effects and trends can be modified;

- Establish baseline concentrations for comparison;

- Assess need for legislative controls;

- Activate emergency procedures;

- Determine suitability of resources for proposed uses.

In addition to in TDM BAC is important in Pharmacochemistry. The strategy in pharmcochemistry is to find and to document structure-activity relationships (SAR), with the goal to develop new drug candidates (Figure 1.22).

Figure 1.22: Structure-activity relationships of taxol (www.ch.ic.ac.uk).

Using the term activity means for example:

- Dynamic interactions with biological targets like receptors and enzymes;

- Kinetic parameters like membrane penetration and metabolic conversion;

- Toxicological properties like mutagenesis, cell and organ toxicity.

Quantitative-structure activity relationships (QSAR) play an important role in drug development (Figure 1.23).

Figure 1.23: Quantitative SAR plays an important role during drug development (www.goldenhelix.com).

` Pharmacochemistry can be divided a number of (sub) disciplines like:

- (Bio)organic chemistry (synthesis and combinatorial chemistry) (Figure 1.23);

- Physical chemistry and structural chemistry (SAR, computational chemistry);

- (Bio-)analytical chemistry;

- Molecular and structural biology (including bio-informatics);

- Pharmacology (including bio-transformation and partitioning);

- Molecular toxicology.

Figure 1.23: Electron density in the amino acid cystein calculated using a quantum-chemistry computer program (Computational Chemistry). The picture shows the surface where the electron density is 0.002 electrons/Å3 (meaning that nearly all electrons are inside the surface). The grey scale shows the electrostatic potential at this surface, darker portions representing negative potential (http://nobelprize.org).

The popularity of LC can be explained by the fact that in bio-analytical research analyte stability, metabolism (biotransformation), distribution as well as excretion are important and that the strength of LC is that qualitative as well as accurate quantitative information can be obtained of the parent compounds as well as of polar and extremely polar metabolites. LC also has some disadvantages: relatively time-consuming sample preparation techniques are normally needed and relatively large sample volumes (0.5 -1.0 mL) are normally required.

In contrast, immunological techniques require no sample preparation and only 50 -100-mL volumes are needed, but these techniques lack the selectivity of LC methods (Figure 1.24).

Figure 1.24: Immunoassay formats (www.piercenet.com).

For a considerable number of analytes radio-immunoassays (RIA) are more frequently used than LC methods, but although this is the case for, for example, cyclosporins, the RIA procedures overestimate the cyclosporin concentration because the polyclonal antibodies used react with cyclosporin as well as its metabolites (Figure 1.25).

Figure 1.25: Format of radio-immunoassay (http://users.rcn.com).

However, although cross-reaction is one of the major limitations of immunoassays (IA), the production of highly specific monoclonal antibodies can overcome this problem which means that especially enzyme-linked immunosorbent assay (ELISA) (Figure 1.26) will be an important bio-analytical tool next to LC.

Figure 1.26: Principle of enzyme-linked immunosorbent assay (www.biosystemdevelopment.com).

Since both techniques, LC and IA, have their own advantages and limitations, the combination of these two techniques, the so-called bio-specific detection, seems to be rather promising.

There are many reasons why an analytical procedure should be performed. There are an increasing number of substances that, for legal reasons must be monitored to ensure the safety of the individual as their presence may be detrimental to both humans and animals. There is an increased consumer awareness concerning the quality and safety of manufactured products and this makes manufacturer’s test their products as quality ensures that financial losses from law suits are reduced. The increased or decreased concentrations of the natural constituents of the body can be used to diagnose diseases and to monitor its treatment.

An analytical procedure is a means to an end; it provides information which can concern the composition of a sample or the status of a chemical process. This analytical information is then used as a basis for a decision: is a sample within specifications, is this water sample safe to drink or is the process under control?

The analytical laboratory is often on the critical path in many organizations as the assay results are needed before a decision can be taken. Therefore, the analytical laboratory is under increasing pressure to improve its efficiency and improve turnaround times while maintaining the quality and consistency of the data reported. Analytical data and information must not be confined within the laboratory, it is a resource that should be used and disseminated throughout the laboratory’s organization.

Contents

Structure-related sample treatment

The start

Introduction

Physico-chemical properties

Bio-analytical chemistry

Contents

Introduction

Summary

Introduction

Rationale sample treatment

Introduction

Choice sample treatment

Categorization techniques

Sensitivity & selectivity requirements

Quality & validation

Introduction

Quality & validation

Validation

Method validation (Why, how, when)

Metabolism & stability

Introduction

Metabolism

Degradation

Stability in solution

Stability in matrix

Method development

Introduction

Separation-detection systems

Liquid chromatography

Separation modes

Detection

Gas chromatography

Introduction

Stationary phase

Detection

Gas chromatography – mass spectrometry

Capillary electrophoresis

Introduction

System theoretical background

Conclusions

Sample treatment/preparation

Sample treatment

Sample preparation

Conclusions

Problems & questions

Molecular Properties (Non-covalent interactions)

Organic structures

Hetero atoms

Stereochemistry

Reaction kinetics

Reaction rate

Reaction constants

Non-covalent interactions

Electrostatic interactions

Dipole interactions

Hydrophobic interactions

Solubility parameters

Drug-protein interactions

Protein binding

Disruption of protein binding

Receptor-ligand binding

Chromatographic partitioning

Partitioning isotherms

Secondary chemical equilibria

Molecular Properties (Molecular effects)

Introduction

Acid, bases, buffers

Dissociation & pH

Ionization & Activity

Electronic effects

Electronegativity

Inductive & resonance effects

Hammett equation

Taft equation

Ionization constants

Microscopic constants

Macroscopic constants

Molecular Properties (Partition Related Processes)

Calculations of log P

Hansch method

Substituent additivity

Limitation of substituent additivity

Application of p-values

Rekker method

Relation p- and ¦-values

Collander equation

Correlation log P and protein binding

Solubility and dissolvation

Determination of solubility

Calculation of solubility

Solubility and log p

Solubility of liquids

Solubility of solids

Distribution coefficient

Partition coefficients & reversed-phase chromatography

Use of liquid chromatography

Relation log P/log D and pH

Partition coefficient and transport

Equilibrium models

Relation rate constants and log P

Bilinear model

Molecular Properties (Structure-Stability Aspects)

General stability features

Stability in solution

Matrix stability

Storage and stability

Degradation and stabilization

Hydrolysis

Minimizing hydrolysis

Racemization & epimerization

Oxidation

Minimizing oxidation

Photochemical reactions

Light absorption by the skin

Processes on molecular level

Photobiochemistry

Therapeutic applications

pH profiles

Calculation pH minimum

Calculations rate constants

Infuence ionic strength

Influence ionization

Bio-pharmaceutical determination of drugs

(Bio-)analytical strategy

How to start?

Appearance of solute(s)

Acid/base properties

Functional groups

Polarity

Solubility

Chemical stability

Spectral properties

Bio-pharmaceutical aspects

Sample preparation – chromatography

Sampling: Techniques & precautions

Sampling statistics

Sample size & number of samples

Sampling efficiency

Modes of sampling / analysis

Off-line sampling

On-line sampling

In-line sampling

Contact-less sampling

Sampling methods

Sampling of gases & liquids

Sampling of solids

Biological samples

Volatile compounds

Direct analysis of macromo-lecule-containing samples

Direct-sample introduction

Column-switching systems

Micellar systems

Wide-pore columns

Restricted-access materials

Protein-coated columns

Sample preparation: Introduction

Introduction

Division sample treatment

Summary procedures

Physico-chemical properties

Physico-chemical properties analytes

Physico-chemical properties matrix

Choice of sample

Choice of environmental sample

Choice of biological sample

Sample preparation: Basic techniques

Handling of liquid samples

Chemicals, reagents & vials

Equipment & procedures

Sampling: Techniques and precautions

Requirements for sampling

Preparation of biological samples

Initial sample preparation: Digestion / solubilization

Solubilization of samples

Release of analyte from biological matrix

Removal of endogenous compounds

Initial sample preparation: Techniques

Microwave irradiation

Principles

Applications

Filtration

Off-line methods

Materials

Selective materials

Influence materials on protein binding

Automation

Bio-analytical applications

Precipitation

Acid precipitation

Organic solvent precipitation

Inorganic salt precipitation

Precipitation efficiency

Precipitation of pigments / bile salts

Lyophilization

Equipment and procedures

Dissolution after lyophilization

Applications

Ultrafiltration

Comparison ultrafiltration – dialysis

Principles

Applications

Various techniques

Freezing & thawing

Homogenization

Saponification

Dilution

Dehydration

Ultrasonic techniques

In-vivo microdialysis

Sample preparation: Off-line procedures (Liquid extractions)

Introduction

Rationale off-line procedures

Extraction techniques

Liquid-liquid extractions

Soxhlet extractions

Accelerated extractions

Miniaturized extractions

Solid-liquid extractions

Supercritical-fluid extractions

Headspace extractions

Chemical modification procedures

Miscellaneous off-line techniques

Sample prepararion: Off-line procedures (Solid-phase extraction)

Principles

Non-selective systems

Sorbents

Applications

Selective systems

Sorbents

Applications

Membrane-based extractions

Miniaturized extractions

Special extraction modes

Solid-phase microextraction

Stir bar sorption extraction

Matrix solid phase dispersion

Molecular imprinted polymers

Restricted access materials

Sample preparation: Automated methods (Continuous-flow)

Rationale for automation

Continuous-flow systems

Introduction

Principles

Dispersion

Detection\

Applications

Membrane-based systems

Dialysis

Electrodialysis

Microdialysis

Liquid membranes

Ultrafiltration

Solid-phase extraction techniques

Non-selective systems

Selective systems

Sample preparation: Automated methods (Discrete systems)

Sample processor (dedicated automation)

Robotics (flexible automation)

Coupled column systems

Introduction

Principle columns switching

Column-switching systems

Coupling LC and GC

Coupling LC and CE

Coupling LC and TLC

Monitoring systems

Comparison of automated systems

Separation: Principles & applications

Liquid chromatography

Objectives

Developing new methods

Modes of chromatography

Chromatographic parameters

Principles

LC instrumentation

Method validation

Treatment of chromatographic data

Gas chromatography

Capillary electrophoresis

Various separation techniques

Detection: Principles & applications

Detection in LC

General aspects

Refractive-index detection

Absorbance detection

Amperometric detection

Fluorescence detection

Chemiluminescence detection

Phosphorescence detection

Mass-spectrometric detection

Radiochemical detection

Detectors in GC

Detection in GC

Detection in electrophoresis

Various detection techniques

Reaction-detection systems

Introduction

Rationale derivatizations

Types of derivatizations

Properties of derivatization reagents

Optimization derivatizations

Pre-column techniques and applications

Derivatization of amines

Derivatization of thiols

Derivatization of carboxylic acids

Derivatization of alcohols & phenols

Derivatization of carbonyls

Derivatization of other functionalities

Post-column techniques and applications

Principles

Reactors

Special applications

On-column techniques & applications

Solid-state derivatizations

Immobilized enzyme reactors

Immobilized enzyme reactors

Electrochemical detection

Optical detection

Molecular spectroscopy

General principles

Absorbance

Luminescence

Vibrational

Nuclear-magnetic resonance

Mass spectrometry

Hyphenated techniques

Mass spectrometry-based techniques

Sample treatment

Matrix effects

Traditional sample preparation

Recent developments

Protein labelling techniques

Isotope coded affinity tag in proteomics

Coupling of ICAT to MS

ICAT in practice

Other labelling techniques

Ion mobility mass spectrometry

IMS-MS for peptides and proteins

Theory

Experimental

Immunological procedures

Introduction

Principles

Standard curve

Modes

Evaluation

Radiolabelled immunoassays

Labelled antigen assays

Labelled antibody assays

Enzyme immunoassays

Homogeneous assays

Heterogeneous assays

Avidin-biotin systems

Fluorescence immunoassays

Homogeneous assays

Heterogeneous assays

Chemiluminescence immunoassays

Direct assays

Indirect assays

Electrochemical immunoassays

Liposome immunoassays

Homogeneous assays

Heterogeneous assays

Protein-based analytical procedures

(On-line ) digestion of proteins

Principles of proteolytic digestion

Digestion methods

Multidimensional systems

Applications

Validation analytical methods

Key issues in validation

Equipment qualification

Transfer of analytical methods

Regulatory validation issues

Computerized analytical systems

Evaluation (Bio-analytical strategies)

Integration of sample treatment

Comparison of sample treatment

Decision trees

Future trends

Structure-related sample treatment

(Bio-)Analytical Chemistry: From B(egin) to E(nd)

Hubertus Irth, Henk Lingeman

BioMolecular Separations Group, Faculty of Sciences, VU University Amsterdam, Amsterdam, the Netherlands

The start
Introduction

Sample treatment (ST) is the bottleneck in most chromatographic/electrophoretic separations. As a result the primary goal of the present module on “Structure related sample treatment” techniques and approaches that can be used for this purpose.

The main ST objectives discussed in this module are:

- Learn technologies and techniques:

o How do they work,

o What are the applications,

o What are the advantages and limitations,

- Possibilities to improve laboratory operations:

o Cost (labour intensive),

o Errors (accuracy, precision),

o Time,

- Productivity issues:

o Automation,

o Parallel versus serial processing,

o On-line versus off-line,

- Help to make the transition from SOP’s to (new) techniques a successful one:

o Criteria for choosing sample treatment techniques.

This means that the problems are addressed that are related to: time, labour, automation, enrichment, selectivity, organic solvents, inorganic salts, contamination, etc.

The resulting ST goals are:

1. Removal of interferences (high and low molecular weight compounds, organic and inorganic compounds);
2. Enrichment of the analyte(s);
3. Phase transfer of the solutes(s).

The main focus will be on the development of total (bio-)analytical procedures an in particular on the ST steps needed to determine these compounds with either gas chromatography (GC), liquid chromatography (LC), ion chromatography (IC), affinity chromatography (AC) or capillary electrophoresis (CE), with the emphasis on automated/hyphenated systems including high-throughput and bio-specific assays. This includes a discussion on the theoretical backgrounds, advantages and disadvantages of pharmaceutical, bio-analytical, food, clinical environmental and process analytical methods and procedures.

Figure 1.1: Relative speed of different steps in (bio-)analytical sample treatment.

The fact that the polarity of the compounds of interest is still increasing and the fact that sampling and sample preparation (SP) are the most time consuming parts of the total analytical scheme, explains the focus on the numerous combinations of sampling and SP procdures in combination with GC / LC and CE separation-detection approaches (Figure 1.1)

The most important options for ST, sampling plus sample preparation, are:

- Sample collection: Taking a representative sample;

- Sample storage and stabilization: Using proper containers and freezing of unstable samples;

- Initial (primary) sample preparation: Reducing the sample size;

- Weighing or volumetric dilution: Taking precautions for unstable and reactive samples;

- Alternative sample processing methods: Introducing solvent replacement, desalting, evaporation procedures etc.;

- Removal of particulates: Applying filtration and centrifugation steps;

- Selective (secondary) sample preparation: Introduction of liquid-liquid extraction (LLE) and solid-phase extraction (SPE) approaches;

- Derivatization: Enhancing detection and improving separation.

In this module various definitions will be used:

- Sample treatment: Total process of analyzing a sample including sampling, initial (primary) and selective (secondary) sample preparation, analysis, detection, quantitation and validation.

- Sampling: Process of taking a reliable and representative sample.

- Initial sample preparation: Process of storing, stabilizing and preparing a sample for selective clean-up and analysis.

- Selective sample preparation: Process of sample concentration, selective sample clean-up and/or phase transfer of the analyte(s).

- Analysis: Process of selective (qualitative or quantitative) determination of the analyte(s).

- Detection: Process of identifying or detection of the analyte(s).

- Quantitation: Process of data acquisition, data reduction and data interpretation.

- Validation: Process of guaranteeing the repeatability / reproducibility, robustness / ruggedness of the overall sample treatment process.

These steps are summarized in the overall analytical procedure as given in Figure 1.2.

Figure 1.2: Overall analytical procedure.

Another reason that sampling and SP are so important is that looking at the time spend on the analysis, about 60% is related to sample processing. In addition to this about 30% of the errors during SP/ST is due to sample processing and another 20% to the operator. This means that automation (Figure 1.3) of SP/ST techniques certainly will improve the overall quality of the analytical procedure.

Figure 1.3: Lab-automation in combinatorial chemistry (www.pa.bosch.com).

Analytical chemistry plays an important role in separating, isolating and quantifying various chemical compounds. Virtually every item of commerce has been subjected to analytical testing (Figure 1.4) at one or more stages in its manufacturing process.

Figure 1.4: Analytical testing can be performed using numerous techniques and procedures (www.pennpharm.co.uk).

Apart from the classical methods, such as titrimetric and gravimetric techniques, many instrumental techniques have been developed, for the determination of not only the active ingredient, but also the quantification of related compounds or impurities associated with it (Figure 1.5).

Figure 1.5: Traditional techniques like titrimetric and gravimetric analysis are, nowadays, replaced by modern separation techniques.

The recently developed analytical methods have the advantage of not only using small amounts of sample, reagents and less time, but also produce accurate results. These analytical techniques may be:

- physico-chemical methods,

- electro-analytical methods

- separation-based methods.

The physico-chemical methods like spectroscopy include colorimetry and spectrophotometry covering ultra-violet and visible region or fluorimetry, nephelometry (Figure 1.6) or turbidimetry, and nuclear-magnetic resonance and mass spectrometry. The electro-analytical methods cover potentiometry, amperometry, voltammetry, electrophoresis and polarography.

Figure 1.6: Nephelometry is based on the principle that a dilute suspension of small particles will scatter light (usually a laser) passed through it rather than simply absorbing it. The amount of scatter is determined by collecting the light at an angle (www.lib.mcg.edu).

The separation-based methods involve (high-performance) liquid chromatography (LC), (high-performance) thin-layer chromatography (HPTLC / TLC), capillary electrochromatography (CEC), supercritical-fluid chromatography (SFC) and gas chromatography (GC). The combination of GC and LC with mass spectrometry (MS), nowadays, are the most powerful tools employed for the quantification and identification of analytes in pure as well as in associated forms. In addition to chromatographic separation methods, electrophoretic techniques, like capillary electrophoresis (CE), isotachophoresis (ITP) (Figure 1.7) gel electrophoresis (GE) and isoelectric focusing (IEF), are relatively popular for the separation and quantitation of (charged) organic compounds.

Figure 1.7: Isotachophoresis is one of the electrophoretic techniques used for the separation of charged molecules (www.bd.com).

The still increasing interest on the determination of drugs has forced the analytical chemists to develop methods for their trace analysis in the presence of biological matrices. Since every year a large number of drug candidates are synthesized it means that also a large number of methods/procedures must be developed that are applicable for the routine analysis of these drug candidates. These methods should be rapid, precise, accurate and cost effective. Although costly sophisticated instruments like LC, HPTLC, GC, GC-MS and LC-MS are available, the spectrophotometer is being preferred by ordinary laboratories for its simple, economical and easy handling techniques (Table 1.1).

Table 0.1: Bio-analytical techniques.

In summary: The philosophy and concepts of sampling, sample preparation in analytical chemistry will be presented in a practical way to provide the analytical chemist with the necessary tools to determine low- and high-molecular-weights compounds in a variety of matrices using chromatographic / electrophoretic, spectroscopic or immunological methods.

In addition, the philosophy and concepts for the method development procedures will be based on the physico-chemical properties of the analyte(s) and the matrices in which these compounds are present (Figure 1.8).

Figure 1.7: Physico-chemical properties.
Physico-chemical properties

(& Structure-activity relationships of drugs)

In order to develop analytical procedures for low-molecular weight (LMW) compounds in pharmaceutical formulations and biological samples the starting point always must the physico-chemical properties of both the compounds of interest and the sample matrix. Therefore, the focus will be on these physico-chemical properties and the structure-activity relationships between the solutes and matrix components such as the interaction between drugs and biological matrix components (e.g., proteins, enzymes, receptors) or pesticides and humic substances.

Pharmaceutical chemistry Bio-analysis Pharmacochemistry

The intention is not to discus all the basic principles of analytical chemistry, physical chemistry, organic chemistry, thermodynamics and kinetics in detail, but just to provide the information necessary to develop new or to modify existing analytical methods that can be used in pharmaceutical chemistry, bio-analysis and pharmacochemistry.

For example, during the development of new drug entities (NCE) it is of utmost importance to optimize the structure of a new compound (drug candidate) for a certain target. This can be done by changing the 3-dimensional (3-D) structure, or in other words the way the functional groups are arranged around the basic molecule. In addition the physico-chemical and bio-pharmaceutical properties of a compound are determined by its structure (Figure 1.8).

Figure 1.8: Fit of NCE in receptor pocket (www.tripos.com).

In addition the physico-chemical and bio-pharmaceutical properties of a compound are determined by its structure. These properties, on its turn, are related to processes like solubility, absorption, protein binding, partitioning, elimination and metabolism (Figure 1.9).

Figure 1.9: Summary of metabolism process (www.elmhurst.edu).

These properties, on its turn, are related to processes like solubility, absorption, protein binding, partitioning, elimination and metabolism. In (bio)-analytical chemistry (BAC) these properties determine which analytical techniques can be used and what the actual conditions must be. It will be obvious that there is a strong correlation between the various physico-chemical properties (Figure 1.10).

Figure 1.10: Relation between physico-chemical properties.

In the case of drugs it can be stated that when the molecular weight is over 500, the number of H-bridge donors is over 5, the number of H-bridge acceptors is over 10 and the calculated partitioning coefficient is over 5, the compound will badly orally adsorbed (Figure 1.11). This gain shows the importance of studying the physico-chemical properties of organic compounds.

Figure 1.11: Solubility of chloramphenicol is determined by the presence of hydrophilic and lipophilic groups (www.auburn.edu).

From the example mentioned above it will be clear that the emphasis will be on the relation between the chemical structure, or in other words the physico-chemical properties of a compound, and the determination of these compounds in a (complex) sample matrix. The final objective is that the information provided can be used to develop simple qualitative and quantitative methods for organic compounds in a variety of matrices. In order to obtain this goal the following parameters will be discussed:

- Physico-chemical properties of organic molecules like polarity, acid-base properties, solubility, stability, absorption/adsorption, etc;

- Composition of sample matrices and their effect on the analytical results (e.g., analyte-matrix binding, stability);

- ST/SP procedures (e.g., filtration, centrifugation, extraction).

There are two magic words that will be discussed throughout the whole monograph: pH and pKa (Figure 1.12).

Figure 1.12: Questions to be answered!

After studying the module on “Structure-related sample treatment” it should be possible to answer the following starting questions (Figure 1.12) and the final question (Figure 1.13):

Figure 1.13: Final analytical question to be answered!

With respect to environmental, pharmaceutical and bio-analysis this means that the following topics features are of importance:

- Insight in structures of chemical compounds and physico-chemical properties like pK_a, polarity, stability, solubility, chromophores, etc;

- Composition of biological materials and, to lesser extent, pharmaceutical formulations and environmental samples, and their influence on the analytical results (e.g. stability, matrix binding);

- Initial (primary) sample treatment procedures (e.g., filtration, precipitation, extraction);

- Selective (secondary) sample preparation procedures (e.g. solid-phase extraction, immunological approaches, hyphenated and automated system

In order to do deal with these features a number of sub-questions should be answered. These sub-questions are related to the following physico-chemical properties of the analyte(s) or the matrix molecules:

- Name, molecular weight, structure;

- Appearance, colour, smell;

- Acid-base properties;

- Functional groups;

- Polarity (partitioning coefficients);

- Solubility;

- (Chemical) stability;

- Spectral properties;

- Bio-pharmaceutical properties;

- Sample treatment / sample preparation;

- Separation / detection;

- Quantitative aspects (validation).

Except for the relatively straightforward analytical applications in most cases an overall sample analysis scheme should be constructed (Figure 1.14). This because direct sample injection into a chromatographic/electrophoretic system, normally is not possible because unwanted matrix components can disturb the separation and/or detection or can clog the analytical device. In other cases the concentration of the analyte(s) or the degradation products or metabolites may be too low to allow a direct injection procedure. The result is that ST/SP is an important aspect of the total analytical procedure and that in order to provide an adequate ST/SP procedure accurate knowledge about the properties of the compounds to be separated and the sample matrix is a prerequisite.

Figure 1.14: Sample analysis scheme.

Bio-analytical chemistry

Bio-analytical chemistry (BAC) involves a number of different disciplines such as therapeutic drug monitoring (TDM), toxicology (clinical, forensic, post-mortem), drugs-of abuse, pharmaceutical analysis, food analysis and environmental analysis.

One of the most important disciplines is therapeutic drug monitoring (TDM) (Figure 1.15). TDM can be defined as:

‘The measurement and the clinical use of blood (serum/plasma) drug levels (concentrations) to adjust each patient’s individual drug dosage and schedule to each patient’s individual therapeutic requirement’.

Figure 1.15: Therapeutic drug monitoring in epilepsy (www.e-epilepsy.org.uk).

In order to perform TDM, quantitative methods for the determination of these drugs, their metabolites and their degradation products should be available.

A number of variables complicate bio-analytical procedures. For example, after a drug has been administered to an individual, it is metabolized into products that can be excreted more easily. On its way through the body multiple metabolites are formed meaning that depending on the bioactivity and relative concentrations, the parent drug and/or the metabolites should be determined.

Biomonitoring outshines the indirect assessment of exposure in determining which pollutants enter the body, and whether they cause disease or disability (Figure 1.16). Non-persistent toxicants move quickly out of the blood as they are metabolized to water-soluble compounds that can be extracted in urine. Some chemicals or metabolites may bind to proteins or to DNA and persist in the body for a longer period of time.

Figure 1.16: Biomonitoring of pollutants (www.nature.com).

Postmortem toxicology, for example, is used to determine if alcohol, drugs or other poisons have contributed to the death of a person (Figure 1.17). Fundamentally it is different from TDM and clinical toxicology. The reason is that it is far more difficult to interpret post-mortem results. Clinical toxicology is dealing with patients with suspected poisoning and the primary objective is to assist in the treatment of such a patient.

Figure 1.17: Postmortem toxicology (www.answers.com).

Drugs-of-Abuse can be defined as: ‘Any substance that, because of some desirable effect, is used for some purpose other than that intended’ (Figure 1:18).

Figure 1.18: Mechanism of action of high concentration of amphetamine (www.cnsforum.com).

The questions in pharmaceutical analysis are again quite different. Questions that are relevant are dealing with the identity, concentration and stability of the drug in the formulation (Figure 1.19). In addition information on the impurities, shelf-life and release-rate of the drug from the formulation are essential. The connection between pharmaceutical and bio-analysis is that after administration of the drug the concentration in tissue or a biological fluid is a key factor. While during drug development parameters like acid/base properties, polarity (log P values), solubility and stability are important features.

Figure 1.19: Pharmaceutical analysis (www.chem.agilent.com).

In food analysis the properties of foods and their constituents are characterized. Analytical procedures are used in food analysis to provide information about a wide variety of characteristics such as composition (e.g., lipids, proteins, water, carbohydrates, and minerals), structure, physico-chemical properties and sensory attributes. The data obtained are used to produce on an economical, safe and nutritious way.

Environmental system analysis is an important tool to analyze sustainability, e.g. environmental impact, optimal resource management and how outside factors affect farm management (Figure 1.20).

Figure 1.20: Environmental system analysis (www-mat21.slu.se).

Trace environmental quantitative analysis (TEQA) is not only dealing with the determination of organic and inorganic compounds in e.g., air, ground, water, plant effluent, leachate, soil, sediment), by also with the determination of these compounds in body fluids and tissues because the intake of environmental pollutants by humans, and the potential of biohazards is a key issue in environmental chemistry. This means that bioterrorism and biomonitoring are important issues in this respect.

Environmental monitoring is completely different from TDM (Figure 1.21).

Figure 1.21: Environmental monitoring (B.J. Alloway, D.C. Ayres, Chemical Principles of Environmental Pollution, Blackie Academic & Professional, London 1997).

Environmental analysis can be:

- Analytes entering the environment, amounts involved, original sources and spatial distribution;

- Effects of analytes on humans, crops, livestock and eco-systems;

- Trends in concentration of analytes over time and the reason for this;

- Extent inputs, concentrations, effects and trends can be modified;

- Establish baseline concentrations for comparison;

- Assess need for legislative controls;

- Activate emergency procedures;

- Determine suitability of resources for proposed uses.

In addition to in TDM BAC is important in Pharmacochemistry. The strategy in pharmcochemistry is to find and to document structure-activity relationships (SAR), with the goal to develop new drug candidates (Figure 1.22).

Figure 1.22: Structure-activity relationships of taxol (www.ch.ic.ac.uk).

Using the term activity means for example:

- Dynamic interactions with biological targets like receptors and enzymes;

- Kinetic parameters like membrane penetration and metabolic conversion;

- Toxicological properties like mutagenesis, cell and organ toxicity.

Quantitative-structure activity relationships (QSAR) play an important role in drug development (Figure 1.23).

Figure 1.23: Quantitative SAR plays an important role during drug development (www.goldenhelix.com).

` Pharmacochemistry can be divided a number of (sub) disciplines like:

- (Bio)organic chemistry (synthesis and combinatorial chemistry) (Figure 1.23);

- Physical chemistry and structural chemistry (SAR, computational chemistry);

- (Bio-)analytical chemistry;

- Molecular and structural biology (including bio-informatics);

- Pharmacology (including bio-transformation and partitioning);

- Molecular toxicology.

Figure 1.23: Electron density in the amino acid cystein calculated using a quantum-chemistry computer program (Computational Chemistry). The picture shows the surface where the electron density is 0.002 electrons/Å3 (meaning that nearly all electrons are inside the surface). The grey scale shows the electrostatic potential at this surface, darker portions representing negative potential (http://nobelprize.org).

The popularity of LC can be explained by the fact that in bio-analytical research analyte stability, metabolism (biotransformation), distribution as well as excretion are important and that the strength of LC is that qualitative as well as accurate quantitative information can be obtained of the parent compounds as well as of polar and extremely polar metabolites. LC also has some disadvantages: relatively time-consuming sample preparation techniques are normally needed and relatively large sample volumes (0.5 -1.0 mL) are normally required.

In contrast, immunological techniques require no sample preparation and only 50 -100-mL volumes are needed, but these techniques lack the selectivity of LC methods (Figure 1.24).

Figure 1.24: Immunoassay formats (www.piercenet.com).

For a considerable number of analytes radio-immunoassays (RIA) are more frequently used than LC methods, but although this is the case for, for example, cyclosporins, the RIA procedures overestimate the cyclosporin concentration because the polyclonal antibodies used react with cyclosporin as well as its metabolites (Figure 1.25).

Figure 1.25: Format of radio-immunoassay (http://users.rcn.com).

However, although cross-reaction is one of the major limitations of immunoassays (IA), the production of highly specific monoclonal antibodies can overcome this problem which means that especially enzyme-linked immunosorbent assay (ELISA) (Figure 1.26) will be an important bio-analytical tool next to LC.

Figure 1.26: Principle of enzyme-linked immunosorbent assay (www.biosystemdevelopment.com).

Since both techniques, LC and IA, have their own advantages and limitations, the combination of these two techniques, the so-called bio-specific detection, seems to be rather promising.

There are many reasons why an analytical procedure should be performed. There are an increasing number of substances that, for legal reasons must be monitored to ensure the safety of the individual as their presence may be detrimental to both humans and animals. There is an increased consumer awareness concerning the quality and safety of manufactured products and this makes manufacturer’s test their products as quality ensures that financial losses from law suits are reduced. The increased or decreased concentrations of the natural constituents of the body can be used to diagnose diseases and to monitor its treatment.

An analytical procedure is a means to an end; it provides information which can concern the composition of a sample or the status of a chemical process. This analytical information is then used as a basis for a decision: is a sample within specifications, is this water sample safe to drink or is the process under control?

The analytical laboratory is often on the critical path in many organizations as the assay results are needed before a decision can be taken. Therefore, the analytical laboratory is under increasing pressure to improve its efficiency and improve turnaround times while maintaining the quality and consistency of the data reported. Analytical data and information must not be confined within the laboratory, it is a resource that should be used and disseminated throughout the laboratory’s organization.

Contents

Structure-related sample treatment

The start

Introduction

Physico-chemical properties

Bio-analytical chemistry

Contents

Introduction

Summary

Introduction

Rationale sample treatment

Introduction

Choice sample treatment

Categorization techniques

Sensitivity & selectivity requirements

Quality & validation

Introduction

Quality & validation

Validation

Method validation (Why, how, when)

Metabolism & stability

Introduction

Metabolism

Degradation

Stability in solution

Stability in matrix

Method development

Introduction

Separation-detection systems

Liquid chromatography

Separation modes

Detection

Gas chromatography

Introduction

Stationary phase

Detection

Gas chromatography – mass spectrometry

Capillary electrophoresis

Introduction

System theoretical background

Conclusions

Sample treatment/preparation

Sample treatment

Sample preparation

Conclusions

Problems & questions

Molecular Properties (Non-covalent interactions)

Organic structures

Hetero atoms

Stereochemistry

Reaction kinetics

Reaction rate

Reaction constants

Non-covalent interactions

Electrostatic interactions

Dipole interactions

Hydrophobic interactions

Solubility parameters

Drug-protein interactions

Protein binding

Disruption of protein binding

Receptor-ligand binding

Chromatographic partitioning

Partitioning isotherms

Secondary chemical equilibria

Molecular Properties (Molecular effects)

Introduction

Acid, bases, buffers

Dissociation & pH

Ionization & Activity

Electronic effects

Electronegativity

Inductive & resonance effects

Hammett equation

Taft equation

Ionization constants

Microscopic constants

Macroscopic constants

Molecular Properties (Partition Related Processes)

Calculations of log P

Hansch method

Substituent additivity

Limitation of substituent additivity

Application of p-values

Rekker method

Relation p- and ¦-values

Collander equation

Correlation log P and protein binding

Solubility and dissolvation

Determination of solubility

Calculation of solubility

Solubility and log p

Solubility of liquids

Solubility of solids

Distribution coefficient

Partition coefficients & reversed-phase chromatography

Use of liquid chromatography

Relation log P/log D and pH

Partition coefficient and transport

Equilibrium models

Relation rate constants and log P

Bilinear model

Molecular Properties (Structure-Stability Aspects)

General stability features

Stability in solution

Matrix stability

Storage and stability

Degradation and stabilization

Hydrolysis

Minimizing hydrolysis

Racemization & epimerization

Oxidation

Minimizing oxidation

Photochemical reactions

Light absorption by the skin

Processes on molecular level

Photobiochemistry

Therapeutic applications

pH profiles

Calculation pH minimum

Calculations rate constants

Infuence ionic strength

Influence ionization

Bio-pharmaceutical determination of drugs

(Bio-)analytical strategy

How to start?

Appearance of solute(s)

Acid/base properties

Functional groups

Polarity

Solubility

Chemical stability

Spectral properties

Bio-pharmaceutical aspects

Sample preparation – chromatography

Sampling: Techniques & precautions

Sampling statistics

Sample size & number of samples

Sampling efficiency

Modes of sampling / analysis

Off-line sampling

On-line sampling

In-line sampling

Contact-less sampling

Sampling methods

Sampling of gases & liquids

Sampling of solids

Biological samples

Volatile compounds

Direct analysis of macromo-lecule-containing samples

Direct-sample introduction

Column-switching systems

Micellar systems

Wide-pore columns

Restricted-access materials

Protein-coated columns

Sample preparation: Introduction

Introduction

Division sample treatment

Summary procedures

Physico-chemical properties

Physico-chemical properties analytes

Physico-chemical properties matrix

Choice of sample

Choice of environmental sample

Choice of biological sample

Sample preparation: Basic techniques

Handling of liquid samples

Chemicals, reagents & vials

Equipment & procedures

Sampling: Techniques and precautions

Requirements for sampling

Preparation of biological samples

Initial sample preparation: Digestion / solubilization

Solubilization of samples

Release of analyte from biological matrix

Removal of endogenous compounds

Initial sample preparation: Techniques

Microwave irradiation

Principles

Applications

Filtration

Off-line methods

Materials

Selective materials

Influence materials on protein binding

Automation

Bio-analytical applications

Precipitation

Acid precipitation

Organic solvent precipitation

Inorganic salt precipitation

Precipitation efficiency

Precipitation of pigments / bile salts

Lyophilization

Equipment and procedures

Dissolution after lyophilization

Applications

Ultrafiltration

Comparison ultrafiltration – dialysis

Principles

Applications

Various techniques

Freezing & thawing

Homogenization

Saponification

Dilution

Dehydration

Ultrasonic techniques

In-vivo microdialysis

Sample preparation: Off-line procedures (Liquid extractions)

Introduction

Rationale off-line procedures

Extraction techniques

Liquid-liquid extractions

Soxhlet extractions

Accelerated extractions

Miniaturized extractions

Solid-liquid extractions

Supercritical-fluid extractions

Headspace extractions

Chemical modification procedures

Miscellaneous off-line techniques

Sample prepararion: Off-line procedures (Solid-phase extraction)

Principles

Non-selective systems

Sorbents

Applications

Selective systems

Sorbents

Applications

Membrane-based extractions

Miniaturized extractions

Special extraction modes

Solid-phase microextraction

Stir bar sorption extraction

Matrix solid phase dispersion

Molecular imprinted polymers

Restricted access materials

Sample preparation: Automated methods (Continuous-flow)

Rationale for automation

Continuous-flow systems

Introduction

Principles

Dispersion

Detection\

Applications

Membrane-based systems

Dialysis

Electrodialysis

Microdialysis

Liquid membranes

Ultrafiltration

Solid-phase extraction techniques

Non-selective systems

Selective systems

Sample preparation: Automated methods (Discrete systems)

Sample processor (dedicated automation)

Robotics (flexible automation)

Coupled column systems

Introduction

Principle columns switching

Column-switching systems

Coupling LC and GC

Coupling LC and CE

Coupling LC and TLC

Monitoring systems

Comparison of automated systems

Separation: Principles & applications

Liquid chromatography

Objectives

Developing new methods

Modes of chromatography

Chromatographic parameters

Principles

LC instrumentation

Method validation

Treatment of chromatographic data

Gas chromatography

Capillary electrophoresis

Various separation techniques

Detection: Principles & applications

Detection in LC

General aspects

Refractive-index detection

Absorbance detection

Amperometric detection

Fluorescence detection

Chemiluminescence detection

Phosphorescence detection

Mass-spectrometric detection

Radiochemical detection

Detectors in GC

Detection in GC

Detection in electrophoresis

Various detection techniques

Reaction-detection systems

Introduction

Rationale derivatizations

Types of derivatizations

Properties of derivatization reagents

Optimization derivatizations

Pre-column techniques and applications

Derivatization of amines

Derivatization of thiols

Derivatization of carboxylic acids

Derivatization of alcohols & phenols

Derivatization of carbonyls

Derivatization of other functionalities

Post-column techniques and applications

Principles

Reactors

Special applications

On-column techniques & applications

Solid-state derivatizations

Immobilized enzyme reactors

Immobilized enzyme reactors

Electrochemical detection

Optical detection

Molecular spectroscopy

General principles

Absorbance

Luminescence

Vibrational

Nuclear-magnetic resonance

Mass spectrometry

Hyphenated techniques

Mass spectrometry-based techniques

Sample treatment

Matrix effects

Traditional sample preparation

Recent developments

Protein labelling techniques

Isotope coded affinity tag in proteomics

Coupling of ICAT to MS

ICAT in practice

Other labelling techniques

Ion mobility mass spectrometry

IMS-MS for peptides and proteins

Theory

Experimental

Immunological procedures

Introduction

Principles

Standard curve

Modes

Evaluation

Radiolabelled immunoassays

Labelled antigen assays

Labelled antibody assays

Enzyme immunoassays

Homogeneous assays

Heterogeneous assays

Avidin-biotin systems

Fluorescence immunoassays

Homogeneous assays

Heterogeneous assays

Chemiluminescence immunoassays

Direct assays

Indirect assays

Electrochemical immunoassays

Liposome immunoassays

Homogeneous assays

Heterogeneous assays

Protein-based analytical procedures

(On-line ) digestion of proteins

Principles of proteolytic digestion

Digestion methods

Multidimensional systems

Applications

Validation analytical methods

Key issues in validation

Equipment qualification

Transfer of analytical methods

Regulatory validation issues

Computerized analytical systems

Evaluation (Bio-analytical strategies)

Integration of sample treatment

Comparison of sample treatment

Decision trees

Future trends

Introduction
Summary

The main objective of this monograph is to describe the overall analytical procedure. This means starting with the ‘Object’ and ending with the ‘Information’. These two parameters are connected with each other via the ‘Analytical Method’.

Figure 2.1: Analytical method based on liquid chromatography (www.toxics.usgs.gov).

In summary it can be stated that a (bio-)analytical procedure is used to obtain qualitative and/or quantitative information on a sample (Figure 2.2). In this “Introduction” section an overview will be given on the different stages of such a procedure emphasizing on sampling, sample preparation (SP) and separation/detection of the analytes.

Figure 2.2: Example of a flow diagram for a processing line of canned tuna fish in brine (www.fao.org).

The rationale for sample handling including the choice of the various sample preparation techniques with respect to their sensitivity and selectivity will be discussed. The influence of drug metabolism on the optimum sample treatment techniques will be overviewed. The objectives that will be highlighted are:

- New techniques and methods (i.e. principles, advantages/disadvantages, applications);

- Possibilities to improve laboratory operations (i.e. cost, accuracy, time);

- Throughput aspects (i.e. automation, parallel versus serial processing, off-line versus on-line);

- Strategies in choosing most successful method.

Finally, a short introduction will be given in chromatographic and electrophoretic techniques that are normally used in (bio-) analytical systems (Figure 2.3).

Figure 2.3: Proteomics chart (swehsc.pharmacy.arizona.edu/mass_spec.html).
Introduction

Normally a sample cannot be injected directly into a separation system. In the sample unwanted matrix components can be present that disturb the separation and/or detection of the analyte(s) or even damage the analytical system (Figure 2.4). In trace analysis, the concentration of the analyte frequently is so low that pre-concentration (trace-enrichment) should be performed to improve the detectability of the analyte(s). The result is that a number of sample handling – the combination of sample treatment / sample preparation (ST/SP) - steps should be performed to allow injection of the analyte(s) in the separation system.

Figure 2.4: Composition of milk (www.delaval-us.com).

Figure 2.5: Stage of the analytical process.

In this Figure 2.6 it can be seen that the ST/SP steps can be divided into three parts:

- Stabilizing of the sample (e.g., avoiding degradation);

- Initial (non-selective) sample preparation (e.g., removal of interfering matrix components, dissolution);

- (Selective) sample treatment (e.g., concentration, clean-up).

Figure 2.6: Division of sample treatment technique

The initial SP step is also called sample preservation or sample stabilization (Figure 2.7). This is rather important because there normally is a delay between sample collection and analysis. This means that sample preservation is needed to be sure that both the chemical and physical properties are changing during the analytical process.

Figure 2.7: Sample stability during storage at different temperatures (www.ipj.quintessenz.de).

The importance of proper ST/SP steps can be illustrated by comparing the selectivity, the degrees of freedom and the costs of the various steps of the analytical process (Table 2.1).

Table 2.1: Role of sample preparation / sample treatment in separation process.

From this Table it can be seen that especially optimizing the SP/ST steps can give an extra dimension to the analytical process.

In summary the following statements can be made:

- The isolation and determination of low concentrations of (organic) compounds is a real challenge;

- The lower the concentration of the analytes, the longer method development will take;

- The ST/SP procedure(s) always should be adapted to the final goal of the analytical procedure;

- Always the most simple SP/ST procedure should be chosen, which is in agreement with the goal of the procedure;

- Always the best compromise should be found between the selectivity of the sample ST/SP step and of the separation / detection procedure.

The first stage is sampling (Figure 2.8). Here, a representative sample is taken and submitted for assay.

Figure 2.8: Issues involved in sampling (www.gifted.uconn.edu).

The information required, in this stage, by the analytical chemist are the sampling details, the history of its transport and storage before receipt in the laboratory. It is important to ensure that any sample taken at this, or any subsequent stage, is representative of the original; i.e. that it is homogeneous (Figure 2.9).

Figure 2.9: Taking a representative sample.

In many cases sampling has become an integral part of the instrumental analytical procedure; here the sampling stream is fed directly into the analytical instrument. This concept known as ‘sample-less laboratory’ is, at present, only applicable for process chemistry (Figure 2.10). In this type of set-up the analytical instruments are placed next to the process stream to monitor, and sample feed lines are constructed. However, normally the sampling site is remote from the laboratory and then the traditional scheme as outlines in Figure 2.5 will be used.

Figure 2.10: Environmental monitoring using smart sensor-based modules (www.esmart.com).

Although nowadays there is a trend in instrumentation towards the sampling site this will not obviate the need for SP/ST. On the contrary, in the near future sampling will become an integral part of the total analytical procedure.

Often, obtaining a representative sample is a relatively simple part of the bio-analytical process; the subsequent stages of ST/SP, analysis and interpretation are normally the most time-consuming parts of the bio-analytical process (Figure 2.11).

Figure 2.11: Where to take a representative sample? (C.I. Measures, J.M. Edmond, Anal. Chem., 61 (1989) 544).

In the next step, the ST/SP stage, the sample must be prepared for instrumental analysis. This means that the sample must be treated in such a way that the majority of solutes – matrix constituents as well as dissolved solutes – interfering with the detection or quantitation of the analyte(s) are removed, while at the same time the sample is concentrated. The ST/SP and the instrumental analysis stage are intended to improve specificity and sensitivity of the assay (Figure 2.12).

Figure 2.12: When a medical test is imperfect, scientists try to strike a balance between sensitivity and specificity. To do this, they plot sensitivity and 1-specificity on a graph, called a "ROC curve". (ROC means Receiver-Operator-Characteristic) (www.halls.md).

The initial SP steps can be divided in: labelling, mechanical processing, homogenization, gravimetric or volumetric measurement and fractionation (Figure 2.13). The initial SP steps are used to: preconcentrate the analytes, to get the analytes into solution, get the analyte(s) into a phase which is compatible with mode of separation, reduce the requirements for a and/or N during the separation step, make the analytes compatible with the detection device and/or enhance the sensitivity of the detector.

Figure 2.13: Nuclear protein fractionation procedure (www1.qiagen.com).

The degree of selective SP steps depends upon many parameters – a major one is the objective of the bio-analytical method (Figure 2.14).

Figure 2.14: Example of selective DNA sample preparation (www.millipore.com).

Both initial and selective SP steps are used to (i) remove matrix effects, (ii) to achieve a partial separation, and (iii) to reduce the number of interferences in the separation step. Especially the latter means that the SP steps applied depending on the separation technique involved. For example, using ion chromatography (IC) ionic leachables should be removed, with LC particulate matter must be removed and in case GC is applied non-volatile compounds should be removed. In all cases co-eluting compounds must be separated from the actual analyte(s).

Following the ST/SP step and the sample introduction step is the instrumental analysis stage. As explained in the ‘The Start’ the instrumental separation techniques discussed are mainly chromatography and capillary electrophoresis combined with various detection principles. The detector output from the chromatograph can be linked to a data handling system or a chart recorder for the eventual calculation of the analyte concentration. Results of the analysis are then evaluated and if acceptable validated or the sample is re-assayed to confirm any findings. Once the results are acceptable, interim results can be issued and the final report prepared.

Over the past decade, technological advances have meant that analytical techniques can measure lower quantities of analytes and computer control of instruments has enabled the data produced to manage efficiently. However, until recently these advances were not matched by improved sample preparation procedures meaning that sample clean-up and sample concentration became, in many cases, the rate limiting step during the development of an analytical procedure.

The discussion on ST/SP for low-molecular weight (< 1 kD) (LMW) and high-molecular weight (> 1 kD) (HMW) compounds will be separated. This because the sample preparation of HMW compounds (e.g., peptides, proteins, lipids) requires a quite different approach compared with the LMW solutes.

Figure 2.15: Interactions of large molecules are different from interactions of small molecules.

The actual reason for the analysis can be quite different. There are a number of reasons to perform a separation procedure. The number of compounds that has to be determined in environmental samples, food products, biological and pharmaceutical samples is still increasing. On the other hand the requirements for safety and quality are becoming higher and higher

Another reason is that not only the parent compounds should be determined, but in quite a number of cases also the corresponding degradation products or metabolites. The result is that frequently both polar and non-polar compounds should be determined simultaneously, which means that high quality separation/detection techniques should be used. The latter can be illustrated with the administration of a drug (Figure 2.16).

Figure 2.16: Biotransformation of codeine in the human body.

Shortly after administration, the drug is (partly) metabolized into products (metabolites) that are more water soluble and so can be excreted easier. This means that depending on the bioactivity and the relative concentrations the parent compound and/or the metabolites should be determined.

The information from the analytical procedure can be used to determine the composition of a sample and/or the concentration of an analyte in that sample, or to determine the status of a chemical process. The information obtained in this way can be used to determine if a sample is according to its specification, if the water is safe to drink or if no residues of anti-microbial agents are present anymore in, for example, meat products.

The overall conclusion is that chromatographers are looking for faster, more cost-effective, easy-to-use, convenient, and safer techniques. In other words, automation is one of the key issues in modern analytical chemistry.

Table 2.2: Different automation approaches.

Nowadays there is a suitable automation device for every analytical problem depending on the number of samples to be analyzed and the complexity of the sample an/or the matrix (Table 2.2).

Rationale sample treatment

Introduction

Looking in a complex matrix for traces in the ng/mL to pg/mL range is just like looking for a needle in haystack. Since the samples are usually complex and ‘dirty’, isolation and quantitation of organic compounds – especially those present at low concentrations – in a real-life matrix is an analytical challenge (Figure 2.17).

Figure 2.17: Urine consists of excess water and waste products that have been filtered from the blood by the kidneys (www.allrefer.com).

In order to achieve reliable chromatographic/electrophoretic data, relatively ‘pure’ samples must be analyzed, and therefore, ST/SP is an essential part of the separation procedure. The objectives of the method will indicate how much effort should be put into a ST/SP scheme. Some of the factors to consider are the concentration of the analyte, the matrix involved and the specificity required. A balance should be struck between the specificity that is obtained by the ST/SP scheme with that from the instrumental assay. Insufficient sample clean-up may result in interference with the analysis or a too much effort in ST/SP may result in the under-utilization of the separation/detection method. A ST/SP step can have several objectives:

- Removal of unwanted macromolecular (e.g. proteins) material and other interfering matrix components to improve the selectivity;

- Removal of material if the resolving power of the LC column is insufficient to separate all analytes present in the sample completely or in a reasonable time;

- Removal of material that can effect the chromatographic/electrophoretic resolution or repeatability of the total analytical procedure;

- Solubilization of compounds to enable injection under the initial separation conditions;

- Concentration of the analyte to improve the detection of the analyte with the analytical instrumentation;

- Improvement of the accuracy and precision of the total analytical procedure;

- Dilution of the sample to reduce solvent strength or to avoid solvent incompatibility;

- Removal of materials (e.g. solid contaminants) that can block the instrumental tubing, valves, column/capillaries or frits to increase the system’s lifetime;

- Stabilization of the analyte to avoid hydrolytic, microbiological or enzymatic degradation.

The objectives mentioned are constrained by:

- Physico-chemical properties of analyte (e.g., stability, pKa, Log D, etc.);

- Analytical techniques available;

- Human expertise available;

- Time;

- Matrices available.

Table 2.3: pKa values of functional groups (www.chem.sc.edu).

A special sample treatment technique is derivatization. Fitting a derivatization step into a complete analytical procedure, in a harmonious way, is one of the major analytical challenges. Derivatization procedures are not only used to improve the detectability, but also to change the polarity of the analytes to facilitate either the ST/SP or the chromatographic / electrophoretic separation. An example is the determination of phase II metabolites. These types of compounds normally have a relatively polar character, which means that a liquid-liquid extraction (LLE) or solid-phase extraction (SPE) is problematic. Derivatization can transform these compounds into less polar products which can be more easily extracted.

Aqueous biological matrices such as plasma, serum, urine, tissue homogenates, and saliva contain a multitude of endogenous compounds. Examples are proteins in plasma samples and fatty acids in urine samples. These solutes are often present in much higher concentrations than the analytes or their metabolites. Many of these endogenous compounds have reactive functional groups (e.g. the carboxylic acid function of amino acids and fatty acids) which can participate in the derivatization reactions and can interfere with the analysis of the compounds of interest, if not sufficiently separated by the ensuing separation procedure(s). Furthermore, most derivatization reactions are best performed in non-aqueous solvents and the presence of an anhydrous medium may even be obligatory. The injection of non-aqueous samples into certain separation systems – i.e. reversed-phase (RP) LC or CE – is in some cases not compatible with the applied system and in other systems the injection of (semi)aqueous samples – e.g. normal-phase (NP) LC – may be troublesome.

In summary it can be concluded that the aim of ST/SP procedures is to obtain a sample aliquot that is:

- Relatively free of interferences;

- Will not damage the analytical system;

- Is compatible with the intended separation method.

The latter means, for example, that the sample solvent will be compatible with the mobile phase, of the chosen separation system, without affecting sample retention or resolution. In addition, it may also be necessary to concentrate the analytes and/or label them for improved detection or better separation. The requirements for a ST/SP procedure are:

- To provide a high recovery;

- With a minimum number of steps;

- That automation is relatively easy.

Quantitative, or nearly quantitative, recovery of each of the analytes enhances sensitivity and assay precision, although this does not mean that all of the analyte present in the original sample must be included in finally separated sample. In case the recovery is less than 100%, the ST/SP step must be reproducible. A smaller number of ST/SP steps plus automation reduces the overall time and effort required and decreases the opportunity for imprecision by the analytical chemist. The decision to automate is often based on a cost justification.

In summary this means that:

- The objective of the analytical procedure will indicate the effort required for a sample preparation scheme.

- Insufficient sample preparation will result in interferences during the analytical procedure & too much sample preparation will result in under-utilization of the instrument analysis.

However, the goals for an analytical procedure can vary significantly. Therapeutic-drug monitoring (TDM) is quite different from environmental monitoring and because the final goal is different also the methods of choice will be different. In general environmental monitoring can be performed to determine:

1. Amounts entering the environment;
2. Effects on organisms and (eco)systems;
3. Concentration effects over time;
4. Trend modifications;
5. Establishment of standard concentrations;
6. Need for legislative controls;
7. Activation of emergency procedures;
8. Suitability determination of water and land resources.

This means that monitoring programs can be chosen dealing with:

• Pollution sources (e.g. emission rates);

• Transport mechanisms;
• Target analytes (including speciation).

It will be obvious that TDM and environmental monitoring are completely different from each other; the objectives are different and with that the sampling, SP procedures and the analysis will be different.

It is important to remember that sampling and SP procedures are not linked to an analyte, but linked to a certain problem. This means that for the determination of compound X in matrix Y using LC a completely different procedure can be needed compared with the LC determination of the same compound in human serum. Furthermore, a different procedure will be used for LC, IC or GC. Using IC, charged compound should be removed, while by using LC solid particles should be removed, and by using GC non-volatile material should be removed.

For a successful method development it is important that the objective are clear, that the chemistry of the analyte and any associated reactions is known and that methods chosen are compatible with the technical expertise of the analytical staff and the available information.

Choice sample treatment

Methods and procedures tend to be developed for individual applications as such, and so the sampling and SP step should be tailored to the requirements of the method.

For different types of samples different strategies should be used. In bio-analysis, with sample volumes of 0.1-1 mL, the majority of the procedures are nowadays performed in an off-line mode with the separation using, for example, disposable cartridges. For the treatment of larger sample volumes, i.e. environmental samples with sample volumes of 10-1000 mL, usually on-line procedures are preferred. The sample size strongly depends on the amount of the available sample (e.g. limited in bio-analysis) as well as the combination of analyte concentration in the sample and the limit of determination. Although for the analysis of large samples on-line procedures are to be preferred in terms of repeatability, off-line systems may also be used.

For the treatment of pharmaceutical formulations, the strategy will be quite different compared with the strategy for biological samples. In pharmaceutical preparations (e.g., creams, syrups, ointments, tablets), in principle, one single analyte in a relatively high concentration, should be determined in a matrix consisting of a limited number of components. Contrarily to biological samples, the majority of the sample procedures for pharmaceutical formulations are performed with simple techniques in an off-line mode with the separation. However, the basic principles and techniques for sample clean-up and sample concentration for most of the matrices will be about the same.

In developing a suitable procedure a number of features should be considered such as the:

- Physico-chemical parameters:

o Physico-chemical properties of the analyte(s);

o Chemical and physical composition of the matrix;

o Degree of analyte-matrix binding(e.g., drug-protein binding, binding to humic substances);

o Analyte stability during sampling and SP procedures;

o Presence of interferences from sample or containers used during sampling, storage or transport, or SP.

- Techniques and expertise in the laboratory:

o Available techniques – both with respect to sampling and SP and separation/detection – in a laboratory;

o Expertise available in a laboratory;

o Time available to develop a method.

- Optimization of sample treatment:

o Recovery during the analytical procedure should be as high as possible (preferably over 75%);

o Compatibility of solvent used during the final SP step and the conditions used during the injection of the sample into the separation system;

o Simplicity of the applied techniques,

o Repeatability and reproducibility of the methods used;

o Possibility of concentrating (enrichment) of the analyte(s) during the final SP technique.

Of course, it will be hardly possible to obtain the ideal conditions for the SP. The first five features, listed above, are physico-chemical parameters that cannot be changed during the analytical process, while the other six features are the variables that should be optimized during the sampling and SP procedure to obtain the most suitable conditions for sample preparation and the subsequent separation / detection procedure.

Another parameter that is of importance is the fate of the analyte. This means if the free or the bound fraction of the total amount should be determined. Analytes can be bound to proteins or humic substances. This can be problematic in a number of cases. An example is the sorption of the pesticide 2,4-dichlorophenol to soil with different concentrations (Figure 2.18).

Figure 2.18: Sorption of 2,4-dichlorophenol to lOESS soil with different concentrations (www.chemistrymag.org).

With respect to the stability of the analyte during the sample preparation process it should be taken into account that sample losses – due to the influence of light, dissolved gases, heat or adsorption – are normally more pronounced in dilute than in concentrated solutions. In particular the pH is of major importance for the stability of quite a number of solutes. Problems caused by contaminants, e.g. plasticizers, optical brighteners in paper tissues, fibres from laboratory coats, are well known. Therefore, the use of plastic containers should be avoided whenever possible. In a number of cases, for example, PVC tubing should be replaced by rubber tubing. Since these effects affect the reliability of the assay, optimizing the overall recovery is a necessity in order to obtain a good sensitivity and proper accuracy and precision. Another major problem is the cleaning of glassware. To avoid any problems ultrasonication should be part of every cleaning procedure.

However, until a few years ago no sensational progress has been made in the development of sampling and SP procedures. Most of the applied techniques are still laborious and time-consuming, and lack accuracy and precision. The new developments, in the last years, in sample clean-up methods are closely related to the development of new sorbent materials in LC, which allow the direct injection of, for example, plasma samples, the introduction of automated sample processors, and the application of robotic systems.

One of the present and future trends in chromatography and electrophoresis is miniaturization; although the concentration sensitivity is limited it will be a major topic in analytical chemistry, especially for the separation of HMW compounds. Therefore, automated pre-column switching systems which can be used for the preconcentration and clean-up of aqueous samples in combination with micro bore or narrow bore technology and chip technology (Figure 2.19) will also be discussed in this module.

Figure 2.19: Glass-capillary ink-jet device with an integrated liquid chromatography column (www.microfab.com).

The necessary sampling and SP steps for the determination of analytes in a complex and dirty matrices is one of the most troublesome to perform, and therefore, the degree of SP depends on quite a number of parameters (Table 2.4). The most important ones are the:

- Concentration of the analyte;

- Composition of the matrix;

- Number of samples to be analyzed;

- Chosen separation / detection system.

Categorization techniques

Sample preparation / treatment can be considered as a number of unit operations, each of which capable of a specific task. An overview of SP techniques that can be used is given in Table 2.5. These procedures can be categorized into four groups:

- Initial techniques: sampling, stabilization, storage, solubilization and release of analyte from the biological matrix;

- Procedures sample handling: addition, mixing, separation, or removal of liquids;

- Selective techniques: removal of interfering endogenous solutes;

- Selectivity and/or sensitivity enhancement.

This categorization is an extension of earlier schemes which did not consider the Group 4 methods (Table 2.5) to be part of sample preparation. However, with respect to the integrated approach of sample preparation/treatment and detection, this group of methods is nowadays incorporated into the total analytical scheme.

However, in order to develop an analytical procedure suitable for routine trace analysis, normally a combination of several steps must be chosen. A general approach is presented in Table 2.4. This approach will be extensively discussed in last section of this modules where some extended guidelines are given how to use a strategy for the development of analytical procedures. In principle, an analytical procedure should consist of one or more initial and selective steps, and because normally a procedure should be developed possessing a certain accuracy, precision and repeatability these steps should be incorporated into the total analytical procedure. This can only be achieved if the selectivity and sensitivity of the procedure are guaranteed, which means that relatively clean samples should be analyzed.

Table 2.5: Categorization of sample preparation techniques.

In principle, a combination of a non-selective (initial) step, to stabilize, to concentrate or facilitate storage of the sample (e.g., protein precipitation, dialysis, ultrafiltration, freezing) and a selective step (e.g., LLE, SPE) to isolate the sample will be necessary (Figure 2.20).

Figure 2.20: Protein dilution (www.miramar.sdccd.net).

In actual practice the difference between initial and selective steps is not sharp. LLE, for example, can be used to denaturate proteins (initial procedure) and at the same time to isolate the analyte (selective procedure), and by using switching valves this non-selective and selective techniques can be combined in a single automated system (Figure 2.21).

Figure 2.21: Supercritical extraction as an example of a selective procedure (www.kobelco-co.jp).

The applicability of most of the ST/SP techniques is limited to liquids or dissolved samples. In Table 2.6 a number of frequently used techniques are given and it is indicated if these techniques can be used for gaseous, liquid / dissolved or solid / semi-solid samples.

Table 2.6: Applicability sample preparation/treatment techniques.

It can be seen that the number of techniques available for gaseous samples is limited: dialysis, adsorption chromatography and GC are the most obvious techniques. For the treatment of solid and semi-solid samples (e.g., SPE, SFE, thermal extraction, static headspace, GC, SFC) are the number of options somewhat larger, but far the most possibilities exist for the treatment of solvents and dissolved samples.

A different division of ST/SP techniques in which either non-selective or selective phase-transfers are used is given in Table 2.7. Microwave and SF extraction are listed as non-selective phase-transfer techniques, but by choosing the proper solvent(s) some selectivity can be obtained.

Table 2.7: Sample preparation/treatment based on phase-transfer mechanism.

The relation between sample preparation and analytical instrumentation is presented in Table 2.8. This Table is providing an interesting overview on the techniques that can be used for various types of analytes and the most obvious sample preparation and analytical techniques. However, it should be taken into account that any sample preparation scheme always should be tailored to the objective of the method. In general it can be stated that the method chosen should be the simplest one that is consistent with the analytical objectives.

Table 2.8: Relation sample preparation and analytical instrumentation (Sample Preparation Techniques in Analytical Chemistry, S. Mitra, Wiley, 2003).

Another important parameter is the time needed for the total analytical procedure. The total analysis time is the sum of all the different steps involved in the whole procedure. An example is given in Figure 2.22.

Figure 2.22: Time for chemical analysis.

This means that the overall analysis time strongly depends on the number of steps involved. On its turn this means that the type of steps, or operations, involved is of importance for the overall analytical performance (Figure 2.23). As a result the use of selective procedures certainly will have a positive effect on the overall performance of an analytical procedure and the overall analysis time. On the other hand the way of processing the samples (e.g. parallel or serial) certainly can influence the overall analysis time, but will not necessarily influence the overall system performance.

Figure 2.23: Sample preparation operations.

Sensitivity and selectivity requirements

The techniques mentioned in group 4 of Table 2.5 are mainly focused on derivatization of an analyte to enhance the assay sensitivity and specificity such as pre-column derivatization reactions and post-column derivatization and reaction detectors. The use of specific detection devices such as the electrochemical – in particular amperometric (AMP) detection – conventional-induced fluorescence (CIF), laser-induced fluorescence (LIF) and chemiluminescence (CL) detection systems. Diode-array detectors (DAD) with associated computer interpretation are also part of this group.

However, it is important to explain that there is a significant difference in sensitivity and selectivity, not only with respect to the detectability of the analyte but also regarding the total analytical procedure. Although, in principle, selectivity and sensitivity are separate analytical variables, they are connected in practice. For example, when an assay for an aqueous sample (e.g., urine, surface water) is developed and validated at the ng/mL level, down scaling to the pg/mL level may result in unexpected interferences in the chromatograms electropherograms. So as a result, selectivity should be optimized again.

In addition sensitivity / selectivity issues are different in (Figure 2.24):

- Qualitative: Is the analyte present or not?

- Quantitative: How much of the analyte is present?

- Semi-quantitative: Is the concentration above or below the threshold level?

Figure 2.24: Selectivity versus sensitivity (www.indiana.edu).

Regarding sensitivity of an analytical procedure it is necessary to provide some definitions. Quantitation, using separation techniques, nearly always means calibration versus a standard as can be seen in Figure 2.25.

Figure 2.25: Calibration in separation science using an (internal) standard (Sample Preparation Techniques in Analytical Chemistry, S. Mitra, Wiley, 2003).

The sensitivity of a method is defined as the defined as the ability to distinguish between small difference in analyte concentration at a desired confidence level. This means measuring the slope of the calibration curve.

In practice the limit of detection (LOD) and limit of quantitation (LOQ) are frequently used. The LOD is defined as the minimum detectable concentration or amount in a standard aqueous solution, and the LOQ as the minimum detectable concentration or amount in the real matrix (Figure 2.26).

Figure 2.26: Limit of quantitation (www.antoine.frostburg.edu).

In the literature a number of definitions can be found, but the IUPAC definition is 'the limit of detection, expressed as concentration or amount, is derived from the smallest measure that can be detected with reasonable certainty for a given analytical procedure' (Figure 2.27). In practice this means the concentration where the signal-to-noise ratio reaches an acceptable value.

Figure 2.27: Dose-response curve for Leu-enkephalin showing an LOD of 1 x 10^-9 M (www.senseomics.com).

The LOD is usually defined as the mean blank ± 3s_blank. However, in complex matrices a better value seems to be mean blank ± 10s_blank. The LOQ is usually defined as mean blank ± 6s_blank or mean blank ± 10s_blank.

Another important parameter that can be determined from the calibration plot is the limit of linearity (LOL). This is the point where the calibration curve becomes nonlinear.

The recovery (%) is a frequently under-estimated parameter in chemical analysis. Low recoveries, during sampling and SP, normally means a negative effect on quantitative issues as LOD, LOQ and LOL. The result is that high recoveries (> 80%) and constant recoveries are of utmost importance (Figure 2.28). Discussing recoveries means that absolute and relative recoveries should be distinguished. To evaluate an analytical process only absolute recoveries should be taken into account meaning the overall recovery of the total analytical procedure including all the steps/operations involved in the procedure.

Figure 2.28: Temperature dependence of dynamic dialysis. Absolute recovery of mixture containing the 14C-CBs 122, 126 and 169, as a function of dialysis time; diffusion of lipids though membrane as a function of dialysis time (www.rsc.org).

High recoveries certainly will help to improve the detectability of an analyte. Other possibilities are the use of:

- Selective detection modes (e.g. fluorescence, mass spectrometry);

- Specific detection modes (e.g. bio-specific detection);

- Multidimensional separations (e.g. column switching);

- Derivatization procedures (Figure 2.29).

Figure 2.29: Derivatization of GlcN with 1-naphthyl isothiocyanate (www.ualberta.ca) .

An example of using multidimensional separations in combination with selective detection approaches is given below. In the example the hyphenation of LC with a bio-specific assay and MS is shown (Figure 2.30) to show that fully integrated systems can be constructed that allow the simultaneous determination of biological (e.g., affinity, IC₅₀ values, selectivity) and chemical (e.g., concentration, molecular mass, accurate mass, structural information) data.

Figure 2.30: On-line combination of a reversed-phase LC separation with an acetylcholine-esterase assay and MS detection.

The use of the LC-MS systems provides the possibility to follow precisely what happens with the concentrations of the analyte, the enzyme, substrate and the products formed. This because the MS can be tuned to the exact m/z values of the compounds and their fragments (Figure 2.31).

Figure 2.31: The resulting MS traces of the on-line acetylcholine esterase assay mentioned above.

In Figure 2.31 1 means the addition of the substrate and 2 the addition of the enzyme. The concentration of the analyte (galanthamine) can be measured by evaluating the m/z value of 288 and at the m/z values of 104 and 146 the fragments formed during the reaction can be observed.

Obtaining lower LOD values in increasingly complex samples means that stringent demands are placed on the resolving power of separation systems. Improved resolution can be obtained by changing the:

- Plate number (N);

- Selectivity (a);

- Retention parameter (k’) (Figure 2.32).

Figure 2.32: Retention in separation systems (www.lcresorces.com).

Improving the separation efficiency can be done by improvement of peak capacity which is the “Maximum number of components (n) that can be placed side by side in the available separation space with a given resolution which satisfies the analytical goals”. The Equation to determine the peak capacity is:

In this Equation is r the number of standard deviations taken as equaling the peak width (typically 4) and k’_i the retention parameter of the last peak in a series. For a column LC system the total peak capacity is given by:

where x is the number of identical columns used, yielding a coupled system which is only equivalent to a longer linear system (Figure 2.33).

Figure 2.33: Peak capacity versus capcity ratio (www.chromatography-online.org).

Using orthogonal systems, for this purpose, the amount of cross information is minimized and the amount of component information is maximized (Figure 2.34).

Figure 2.34: Orthogonal separation systems.

Finally the use of derivatization procedures can result in enhancement of the sensitivity / selectivity or specificity by using pre-column derivatization, on-column labeling or post-column tagging procedures (Figure 2.35).

Figure 2.35: Comparison amino acids derivatization procedures (www.jeol.se).

The terms selectivity and specificity are normally the reason for confusing. However, as can be seen in the statement below (www.iupac.org) these terms are not equivalent to each other and scientist should take good care of using the proper terms an definitions during their research and in the corresponding reports.

Quality and validation

Introduction

During the development, optimization and validation of a ST / SP procedure, it is of great importance to test the method under development from the beginning with the most suitable separation / detection combination and that from the beginning the proper quality- and validation parameters are taken into consideration (Figure 2.36).

Figure 2.36: Quality- and validation parameters (www.technet.pnl.gov).

Quality assurance (QA) and quality control will control the systematic and random errors in an analytical system. QA deals with manipulations showing that set quality standards are met, while QC deals with procedures leading to statistical control of the manipulations in the analytical process.

This means that QC is the combination of a number of steps like protocolling and verification, but also of determining the analytical accuracy and precision; in other words validation. QC should be part of all steps of the process: sampling, storage, sample preparation / treatment, analysis, data handling and reporting. In a number of cases, for example for the determination of drugs, the sampling step is preceded by an administration step. The various aspects of QC are summarized in Figure 2.37.

Figure 2.37: Summary of QC aspects.

The difference between GLP and GMP is that in GLP the procedures are involved dealing with the running of a laboratory, while the definition of GMP is the procedures dealing with specific techniques in the analytical process. An SOP is the written description of the procedures and methods used in the analytical process.

During every step problems can occur that will influence the reliability of the results obtained. This means that an important aspect of QC is the use of a SOP. These SOP’s should be available in the form of detailed protocols that can be used by everyone working in the laboratory.

SOP’s are in particular of importance in sample preparation because a strict use op SOP’s will significantly reduce bias and improve precision of an analytical procedure.

In order to obtain reliable results, all steps including the storage procedures, should be carefully controlled and incorporated in protocols. This means that sampling, and ST also should be denoted in the protocols and not only the analytical separation/detection part of the procedure.

Quality control

Quality control (QC) is a term embracing a number of aspects such as: protocolling and audit ability, and others with analytical precision and accuracy. But also drug manufacturing, purity, formulation stability, labeling of clinical trial samples and selection of patients are quality control aspects.

Quality control in analytical chemistry can be divided in several steps:

- Administration;

- Sampling;

- Storage;

- Analysis;

- Data interpretation;

- Rapport.

In every step a number of problems can occur, resulting in unreliable data. A definition of quality control is: development and incorporation of standard operation procedures. These operation procedures should be available in the form of detailed protocols, which should be followed by every one involved in all of the six mentioned stages.

Problems during oral administration of a drug, for example, can be: administration of the drug partly via the trachea instead of the gullet, problems with the proper dose if the drug added to food or drink, and compliance difficulties with patients (Figure 2.38). After intravenous injections, the problem can be that sometimes part of the drug is administered subcutaneously. Another parameter is that during a study or treatment the administration scheme is changed by one of the physicians without notice.

Figure 2.38: Mean trovafloxacin serum concentrations determined following 1 hour intravenous infusions of alatrofloxacin at daily doses of 200 mg (trovafloxacin equivalents) to healthy male volunteers and following daily oral administration of 200 mg trovafloxacin for 7 days to six male and six female healthy young volunteers (www.dailymed.nlm.nih.gov.

With respect to the sampling it is important that large volumes of blood from patients cannot be collected without changing the degree of protein binding, which can strongly influence the pharmacokinetics of the analyte, that samples are taken at fixed time points, and that proper sample containers are used. Problems can be that quantitative collection or urine and faeces is not always possible, which is necessary in bioavailability studies but of minor importance in metabolism studies. Moreover, the sample volume may be limited, because not always the same sample volume is collected.

In order to be sure that all samples are properly stored, all sample containers should be labelled and the labels should contain all the necessary information (e.g., number of the study, date of sampling, location of sampling, amount of sample, initial treatment).

A frequently used storage procedure is freezing of the sample at -20⁰C. However, this can result in a number of problems:

- Concentration of the analytes on air-liquid surfaces;

- Oxidation of the analytes by the oxygen in the air;

- Shifting of the equilibrium between the analytes and matrix components;

- Presence of non-homogeneous samples after defrosting.

A problem related to biological samples, and probably also to environmental samples that contain high concentrations of macromolecules, can be that chemicals can leach from plastic containers and contaminate the sample. The result is that these chemicals can displace analytes from their binding place onto the macromolecules and so change their effective concentration.

This is showing that matrix effects can have a large influence on the accuracy and precision. Matrix spikes can be used to determine matrix effects during sample preparation and subsequent analysis and matrix spiking is performed by adding a known amount of a solute similar to the analyte but which is not present in the sample. The sample is subsequently analyzed for the presence of the added solute to evaluate matrix effects. Matrix spikes can be used to accept or reject an analytical method.

Another issue to be taken into account is contamination. Contamination can occur at any moment during the analytical process. The lower the concentration of the analyte, the more problematic the effect. Contamination can occur during:

- Sampling (e.g., equipment, containers);

- Transport / storage (e.g., containers, cross-contamination);

- Sample preparation (e.g., carryover, dilutions, glassware);

- Separation / detection (e.g., carryover, instrumentation, syringes).

This again means that blanks are essential to determine the influence of contamination sources during the whole sample treatment process. Blanks are samples in which no analyte is present. Different types of blanks must be measured to guarantee the quality on the analytical method. Blanks only identify contaminations but do not account for various errors that may occur. The types of blanks to be used should be given in the SOP for contamination control. An overview of the various blanks that can be measures is:

- System blank: To determine the background signal without the sample;

- Solvent blank: To determine the influence of the solvents/reagents used;

- Method blank: To determine the contamination of all steps in the analytical procedure;

- Matrix blank: To determine contamination during sampling, transport and storage;

- Equipment blank: To determine contamination of the equipment used during the analytical process.

The most important conclusions so far are that all steps of the analytical procedure should be carefully controlled and embedded in protocols. The same is valid for the required sample treated and that no ST procedure may be considered to be without potential problems.

Validation

Quantitation and validation are key parameters in quantitative analytical methods. A definition of validation is: 'Validation is the procedure used to prove that a test method consistently yields what it is expected and requited to do with adequate accuracy and precision'.

Analytical LC methods should be quantitated and/or validated prior to and during use to obtain confidence in the results generated. The fundamental criteria for assessing the reliability and overall performance of an analytical method are:

- Evaluation of analyte stability;

- Selectivity;

- Limits of quantitation and detection;

- Accuracy;

- Precision;

- Linearity and recovery.

The extent to which a method is validated is dependent on its prospective use, the number of samples to be assayed and the use to which data are put.

Specific analytical techniques in LC may require additional validation such as antibody binding characteristics, peak purity determination, and evaluation of matrix effects or structural confirmation of the analyte. Ideally each assay should be cross validated with a method utilizing a highly specific detector such as mass spectrometer. Once in use, the performance of the method should be monitored using quality control standards. If a method is set up in another laboratory, the performance of that assay should be monitored with quality control standards sent from the originating laboratory.

Method validation (why, how, when)

The reason for validating an analytical procedure is to demonstrate the performance and reliability of a method and hence the confidence that can be placed on the results. In addition, it has been stated that all bio-analytical methods must be validated if the results are used to support registration of a new drug or the reformulation of an existing one. It should be noted that the initial validation is only a beginning, as a method should be monitored continuously during its application to ensure that it performs as originally validated.

The aim of this section is to discuss briefly method validation and to provide the basis for a comprehensive framework for validating analytical methods An extensive treatment of the validation process is given in a separate section on (bio)-analytical validation.

With respect to validation the role of the bio-analytical chemist is a rather important one; should analytical data be incorrect, then the efforts of a researcher can be in vain. Dubious analytical data can waste valuable resource, cost money through delays in registration of new compounds or even worse be a cause for wrong diagnosis and treatment. Therefore, the bio-analytical chemist has a vital contribution to make. It is against this background of responsibilities that a bio-analytical chemist validates a method to demonstrate to him self, as well as to other scientists, that the data produced are reliable.

A key parameter in validation nearly always is the concentration of the various compounds analyzed.

In pharmaceutical and industrial analyses the concentrations of the analytes are roughly known in many cases. In bio-analysis the concentration can be strongly dependent of pharmacokinetic parameters such as absorption, partitioning, metabolism, excretion and chemical degradation. In addition, parameters like the way of administration, the formulation of the drug, the sex and age of subject and the time point of administration can play an important role. In environmental samples, the concentrations can either be extremely low or extremely high.

In addition to the concentration the concentration range is an important issue. The results of the effects mentioned above is that the linear dynamic range of most analytical methods should be at least two or three orders of magnitude, which frequently is rather problematic.

Furthermore, the sensitivity of a method is a parameter that determines the ability to distinguish between small differences in analyte concentrations at a desired confidence level. Normally sensitivity is expressed as the slope of the calibration curve and is called the calibration sensitivity.

Both in environmental and in bio-analysis (Table 2.9) a large variety of sample matrices can be used. Every matrix will have its own problems (matrix effects) and the number and the concentration of the analyte(s) can be completely different; this in particular with respect to cell material, macromolecules, metabolites and degradation products.

Table 2.9: Matrices used in bio-analytical procedures.

Dealing living organisms means that the differences between individuals and sex result in differences in absorption, metabolism and elimination. In addition there are matrix differences between the individuals there selves. Dealing with environmental samples, the same differences exist; surface water is not surface water, two soil samples can be completely different in their composition. This means that the interferences are different in many cases (Figure 2.39). For example, dog serum is different compared with human serum.

Figure 2.39: The diagram shows a three sided grid with each side representing the content of a particular particle on a scale from 0% to 100%. The bottom line is the sand content, starting at 0% at the bottom right hand corner, and rising to 100% in the bottom left hand corner (http://web.ukonline.co.uk).

In order to determine if soil sample is polluted with a certain compound, a number of parameters are of importance. The pollution can be heterogeneous divided over the soil and the composition of the soil is not always the same; the percentage of sand, clay and small stones can be different as well as the ionic composition and acidity of the soil. This means that the sampling and SP procedure should be sufficient selective to guarantee a reproducible recovery of the analyte(s) during the clean-up of the various samples.

Both during the final and the initial (during all sampling and SP steps) validation procedure a number of parameters should be validated (Table 2.10). During the validation of a ST/SP procedure the following parameters are of importance:

Table 2.10: Validation parameters.

For quantitative determinations and validations two types of standards can be used: external in internal standards. An external or absolute standard is a solution of the analyte of a known concentration. On the other hand an internal standard is a solution of a structurally related compound with a known concentration, which is treated exactly the same as the analyte solution. For this reason the internal standard solution is frequently called the recovery- or calibration standard. Internal- en external standards have there own advantages and disadvantages (Table 2.11). For quite a number of applications, nowadays certified standards are available and can be used.

Table 2.11: Advantages and disadvantages of internal- and external standards.

In principle the internal standard must be structurally about the same as the analyte and must have similar physico-chemical properties. Baseline resolution between the internal standard and the analyte during the chromatographic/electrophoretic separation is a necessity, the internal standard may not be present in the matrix and the concentration of the internal standard should be about the same as that of the analyte.

The internal standard should be added to the sample directly after sampling and can be used as a qualitative marker to monitor the SP steps, to correct for dilution and injection errors, to control the stability of the analytical system and/or to follow chemical reactions (Figure 2.40).

Figure 2.40: Effect of Ge hydride as internal standard in urine samples: A, calibration graphs in Milli-Q water ( ) and in urine (1 + 4) ( ) without internal standard; B, calibration graphs in Milli-Q water ( ) and in urine (1 + 4) ( ) with Ge internal standard (www.rsc.org).

An external standard is a separate sample with a known concentration of the analyte. No additional compounds are added to the sample. The standard is treated exactly the same as real samples, which means that the recovery of the standards and real samples is the same. The use of an external standard is relatively simple because no internal standard should, which in many cases is rather troublesome. In particular when using automated sample preparation procedures, such as column-switching (CS) systems, in combination with high recoveries the use of an external standard can significantly speed-up the whole analytical procedure.

The smallest amount of an analyte that can be detected, but not quantified, is called the limit of detection (LOD). The smallest amount of the analyte that can be quantified is called the limit of quantitation (LOQ). This means that the LOQ value is depending on the background signal, which is caused by matrix components and/or electronic noise. Both the LOD and the LOQ values can be defined in solutions of standards or in real samples.

During method development it should already be decided if peak height of peak area measurement should be used for quantitation purposes. Both methods have their own advantages and disadvantages, meaning that for every application it should be determined which the most suitable parameter is. A number of these parameters are given in Table 2.12 using a LC separation procedure.

Table 2.12: Peak height versus peak area measurements in LC.

The recovery of the analytes during SP frequently is the most important parameter determining the LOQ of the total analytical procedure. This parameter can be used also to determine the efficiency and as an indicator for the robustness / ruggedness of the system. Recoveries can be determined by spiking standards as well as samples with different concentrations of the analytes. The concentrations should be divided over the entire linear dynamic range of the method. In order to avoid artefact formation an internal standard can be used and is it advisable to spike the sample just before the actual analysis, by comparison of extracted and non-extracted sample, or by using radio-labelled analytes.

The first approach can be used in nearly all cases, but requires the presence of a pure reference compound. During the first stages of method development, a fast indication of the recovery can be obtained by comparing the response of the extracts with the response of a non-extracted standard with in concentration in the mid of the linear dynamic range (Figure 2.41).

Figure 2.41: Structures of single-dye reference compounds ( www.fbi.gov).

The limitation of the second approach is that a labelled analyte must be available, as well as facilities to handle these compounds. The labelled analyte is added to the matrix and is processed in exactly the same way as all the other samples. The main advantage of this procedure is that during all steps of the analytical procedure, it can be seen if sample losses are occurring.

In most cases a linear dynamic range of 2 – 3 orders of magnitude is necessary. During the initial validation (development of the sampling and SP procedure) of a method, procedures should be developed in case the response is outside the calibration range of the method. In principle there are three possibilities that can be considered:

- To which extent is it possible to extrapolate outside the concentration range of the standards;

- Is it possible to repeat the ST/SP procedure with a smaller or larger amount of sample, to be sure that the response is within the linear range of the method? If this is the case, what is the matrix effect if a smaller or larger sample volume is used? How is in the latter case the nominal volume adapted, by adding the matrix or by adding a buffer;

- Is it possible to measure the sample again after dilution? What are the effects of dilution of a sample with a buffer or of the matrix?

The best option depends on the combination of the chosen separation/detection technique. However, before starting the method development the analytical chemist should answer this type of questions.

A next issue is the accuracy and the precision of a method. This means that random and systematic errors have to be discussed. Random errors have a Gaussian distribution and equal probability of being above or below the mean. The individual measurements cluster around the mean value. Systematic errors tend to bias the measurement in one direction and they are determined as the deviation from the true value.

The initial validation of the ST/SP procedures provides an indication on the intra-assay and inter-assay reproducibility of the analytical procedure. The reproducibility can be determined by performing two series of experiments. Every series consists of a six-fold measurement of three sample concentrations divided over the entire calibration curve. The response of the samples is compared with a calibration standard. This normally is sufficient as a first initial validation. In order to obtain more information with respect to the reliability and robustness / ruggedness, a third series of experiments should be performed.

An important question is how many samples should be analyzed in order to obtain reliable results. A relatively simple approach to find the number of samples to be analyzed is to repeat the sample preparation and the analytical procedure to determine an overall standard deviation (s). In this case Student’s t distribution can be used. The number of samples required to achieve a given confidence level is calculated as:

In this Eqn. t is the statistic value selected for a given confidence level and e is the acceptable level of error. The degrees of freedom that determine t can first be chosen arbitrarily and then modified by successive iterations until the number chosen matches the number calculated.

Accuracy should never be confused with precision (Figure 2.42).

Figure 2.42: Accuracy and precision (www.chemistrydaily.com).

Precision is associated with measured values, for example, measurements of the concentration of a chemical in seawater. Precision is a measurement of how closely the analytical results can be duplicated. Replicate samples (Prepared identically from the same sample) are analyzed in order to establish the precision of a measurement. The precision is usually reported as a standard deviation (SD) or average replicate error. The SD value should be as small as possible. In many cases the precision depends on the concentration of the analyte, which means that it should be determined at various concentrations of which one should be close to the limit of detection. In most cases duplicate measurements are sufficient, only in those cases when two values differ more than a SD of each other, a third measurement should be performed.

The reproducibility of an analytical procedure decreases rapidly with decreasing concentrations. In general it can be stated that the uncertainty in trace analysis increases exponentially compared the major and minor component analysis. Furthermore, the uncertainty from ST steps will also increase with decreasing concentrations. In many cases ST/SP accounts for the majority of the overall variability.

Precision is important when sample preparation is involved. The reproducibility decreases disproportionately with decreasing concentrations (Figure 2.43).

Figure 2.43: Reproducibility as a function of concentration during analytical measurements (V. Meyer, LC-GC North Am., 20 (2002) 106).

Accuracy should never be confused with precision. Accuracy measures how close to a true or accepted value a measurement lies. The precision is expressed as the percentage-bias or error and can be both positive and negative. In principle, duplicate measurements are normally are sufficient, only in those cases when two values differ more than a SD of each other, a third measurement should be performed. Measurement of the accuracy is not so easy because an absolute measurement is necessary and chromatographic and electrophoretic methods relative methods.

Another problem is the comparison of a real sample and a spiked sample. After spiking of the sample it is important that sufficient time is taken for equilibration of the sample. Shaking of a sediment sample, during several min, with a solution of the analyte(s) in acetone, can result – after extraction – in a significantly higher recovery as a real sample. In a real sample the analyte(s) can be bound to matrix components in a completely different way. The same problem can occur when analyzing biological tissue samples (e.g., liver, kidney). In the spiked sample the analyte(s) are present in the intracellular solvent, while in the real sample they can be present in the intracellular volume.

In summary the relation between accuracy and precision can be seen in Figure 2.44.

Figure 2.44: Relation between accuracy and precision (www.envisagement.com).

Selectivity is an important parameter during the initial validation procedure in case no absolute but a relative method (e.g. chromatography) is used. Using relative methods, standards must be used to determine the concentrations of the analytes. In principle there are two types of interferences during the development of analytical methods: components which are always present in the matrix and components which are normally not present in the matrix, but are coming from the applied equipments or reagents.

Interferences can have various influences on the analytical experiment. It can be a small but constant deviation of the accuracy at low concentrations and variable problems due to the use different batches of reagents or reagents / chemicals (e.g. SPE cartridges). The result is that the selectivity of the overall procedure should be checked during all steps of the method development.

Changes in the analytical method and re-validation. After changing parts of a validated method, the analytical chemist should decide which part of the method should be re-validated. Changes can be: the use of chemical or reagent with a different purity or of another manufactures or the use of another SP procedure (e.g. SPE instead of LLE). In this case the accuracy, precision, LOD, LOQ and stability should be re-evaluated.

An overview of the important issues in method validation is given in Table 2.13.

Table 2.13: Important issues in method validation (Sample Preparation Techniques in Analytical Chemistry, S. Mitra, Wiley, 2003).

The overall conclusion is that a validation process should involve:

- Determination of the parameters that should be involved in the validation (e.g., accuracy, precision, linear dynamic range);

- Analysis of unknown samples (unknown concentrations) to determine the accuracy and precision;

- Equivalence testing, a comparison with existing methods should be made;

- Collaborative testing, this to determine the ruggedness of the method.

Metabolism and stability

Introduction

Metabolism and degradation of organic compounds are two parameters that can have a major influence on the analytical procedure. In order to understand these parameters first of all a short introduction in the pharmacokinetic processes involved will be given.

Pharmcokinetics involve the kinetics of drug absorption, distribution, metabolism and elimination (ADME):

– Absorption

• Transport of the drug from the site of administration to the bloodstream;

– Distribution

• Movement of the drug from the bloodstream to extravascular tissue (site of action);

– Metabolism

• Biochemical mediated alteration of the drug;

– Elimination

• Removal of the drug and/or metabolites from the body.

These processes are represented in Figure 2.45.

Figure 2.45: Summary of pharmacokinetic processes.

During bio-analysis, in addition to the parent compounds, also metabolites and/or degradation products can provide valuable information on the sample. This means that in many cases, the parent compound should be determined simultaneously with its metabolic and or degradation products. In general it can be stated that degradation products and, to lesser extent, metabolites are interfering compounds and should be removed during the sampling and SP procedure. For an accurate interpretation of the data, and analytical chemist always should be aware of potential metabolites or degradation products (Figure 2.46).

Figure 2.46: Biotransformation reactions.

A definition of metabolism can be the combination of all chemical reactions occurring in living cells. These reactions are allowing cells to grow, reproduce and interact with the environment. Metabolism can be divided in catabolic and anabolic reactions.

The catabolic reactions provide the necessary energy (e.g. breakdown of food) for the organism, while the anabolic reactions are using this energy (e.g. synthesis of proteins).

Macromolecules (e.g., polysaccharides, proteins) cannot be rapidly taken up by cells are degraded into smaller parts before they are used in the cell metabolism For these catabolism reactions several types of enzymes can be used. For example, proteases digest proteins into amino acids and glycoside hydrolases digest polysaccharides into monosaccharides.

Figure 2.47: Catabolism of proteins (www.bact.wisc.edu).

Carbon fixation is an example of an anabolic reaction. Photosynthesis is the synthesis of glucose from sunlight, carbon dioxide and water (oxygen is produced as waste product). ATP and NADPH (co-enzymes) convert carbon dioxide in glycerate-3-phosphate, which is then converted into glucose. Both ATP and NADPH are produced by photosynthetic reactions (Figure 2.48).

Figure 2.48: Photosynthesis (www.eia.doe.gov).

Another process is the detoxification of xenobiotics, including drug molecules. Xenobiotics are detoxified by special enzymes including cytochrome P450 oxidases and glutathione-S-transferases. The metabolizing pathway of polycyclic aromatic hydrocarbons is given in Figure 2.49.

Figure 2.49: Metabolism of benzo(a)pyrene (http://mol-devserver.tara.tsukuba.ac.jp).

Although metabolism and degradation are related there is a distinct difference between these two processes. For example: Bupropion is metabolized in the liver to R,R-hydroxybupropion, S,S- hydroxybupropion, threo-hy drobupropion and erythro-hydrobupropion and further metabolized to inactive metabolites and eliminated via the urine (Figure 2.50).

Figure 2.50: Metabolism of bupropion.

Bupropion was developed to decrease side effects associated with pH induced degradation products. For bupropion a pH dependent degradation pathway is described. The observed degradation products at pH 7 are shown (Figure 2.51).

Figure 2.51: Degradation products of bupropion in pH controlled buffers (www.geocities.com).

To make the distinction between degradation and metabolism clear, degradation can be defined as the chemical breakdown of compounds by physico-chemical effects. However, in some cases the terms metabolism, metabolic degradation and degradation are mixed (Figure 2.52).

Figure 2.52: Amino acid degradation (http://138.192.68.68/bio/Courses/biochem2).

The result of metabolism and degradation is that early metabolite testing using, for example, LC-MS/MS approaches is a must in bio-analytical chemistry (Figure 2.53).

Figure 2.53: Early metabolite testing (www.genpharmtox.com).

Metabolism

Since every exogenous compound entering the body should be eliminated, drug metabolism is an important feature. Therefore, drug metabolism is focused on the conversion of body-foreign solutes into highly water-soluble compounds that can be excreted easily via the urine. Any ’first pass’ effect will influence the ratio of the analyte and its metabolites and so their concentration in the various biological fluids. An example is the fast hydrolysis of esters to free carboxylic acids, which will rapidly conjugate and excreted via the urine (Figure 2.54).

Figure 2.54: The process of metabolism (www.nlm.nih.gov).

Metabolism is an integral part of drug administration and it may affect the pharmacological effect of a drug. The metabolites formed can be pharmacologically active or inactive. Active metabolites may have different modes of action and different potencies and the formation of active metabolites changes the profile of drug action (Figure 255).

Figure 2.55: Summarizing metabolism scheme (www.elmhurst.edu).

Drug metabolism can be divided into two different types of reactions. Phase I mechanisms involve oxidation, reduction and hydrolysis reactions (Figure 2.56), while phase II reactions involve conjugation of these metabolites with polar endogenous solutes (e.g., glucuronic acid, hippuric acid, sulphate) (Figure 2.57).

Figure 2.56: Biotransformation reactions and metabolites.

Conjugation of the analyte can increase the water solubility with a factor of 100. In particular in TDM studies it should be known if either the parent compounds should be determined or if the metabolites should analyzed as well. Furthermore, the extent of metabolism determines the relative concentrations of the parent compounds and corresponding metabolites in the biological matrix.

Figure 2.57: Phase II conjugation reactions.

Phase I reactions include oxidation, hydroxylation, N- and O-dealkylation and sulfoxide formation as well as reduction and hydrolysis reactions (Figure 2.58). Many drugs undergo a combination of phase I and phase II reactions.

Figure 2.58: Metabolism of estrogens (www.breast-cancer-research.com).

Phase II reactions include conjugation reactions, such as with glucuronic acid, as well as acetylation, methylation and conjugation with amino acids and sulfate. Phase II reaction remove or mask functional groups (e.g. amino, carboxyl, hydroxyl, sulfhydryl) on the drug or a Phase I metabolite by the addition of an endogenous substrate (Figure 2.59).

Figure 2.59: Metabolism of farnesol (www.biochemj.org).

An additional problem in developing a bio-analytical procedure is that plasma levels in some patients are relatively low. One of the reasons is that one group of patients can be considered as fast metabolizers, while another group are slow metabolizers which results in increased plasma levels. However, high or low plasma levels are not always due to a slow or fast metabolism (Figure 2.60).

Figure 2.60: A. PM poor metabolizer, absent or greatly reduced ability to clear or activate drugs; B. IM intermediate metabolizer. Heterozygotes for normal and reduced activity genes; C. EM extensive metabolizer. The norm; D. UM Ultra Metabolizer. Greatly increased activity accelerating clearance or activation (www.healthhanddna.com).

A number of features, in addition to the physico-chemical properties of the analyte, such as administration route, concomitant administration, degree of drug-protein binding, dose, formulation, partition volume, solubility, speed and degree of absorption and elimination, time of administration – before, during or after meal – and gastrointestinal disturbance will influence the plasma levels. This means again that selectivity and sensitivity are among the key parameters in bio-analytical procedures. The required selectivity of the bio-analytical method depends on the formed metabolites, the degradation processes, and the presence of interfering compounds in the biological matrix. In urine, normally, significantly more interferences are observed compared with plasma, while saliva is a relatively clean matrix.

Depending on the functional groups present – i.e., amine, carboxyl, ether (nitrogen or oxygen), ester, phenol, and hydroxyl – the parent compounds can be oxygen or nitrogen dealkylated, followed by conjugation of the oxygen/nitrogen atom forming water soluble solutes that can be excreted via the urine As a result the concentration of the parent analyte(s) in plasma or serum can be relatively low, while the concentration of the corresponding metabolites is relatively high in urine. This is the case for analytes which are extensively metabolized. It will be clear that urinary excretion is the most important elimination pathway for exogenous compounds. But in case the renal function is disturbed, plasma concentrations can be increased.

Degradation

Analyte stability is of utmost importance. This means that during all steps of the analytical procedure the stability of the analyte(s) should be checked. In other words the sample studied must be representative of the object under investigation. This means that biological, chemical and physical processes that may influence the composition of the sample are collection should be carefully checked. The biological processes involved are biodegradation and enzymatic reactions, the chemical processes can be hydrolysis, oxidation, precipitation and photo instability, while the physical processes that can cause problems are, for example, adsorption, diffusion and volatilization.

Since samples are normally not stored under exactly the same conditions as the original source it is not possible to guarantee the integrity of a sample for indefinite time. Therefore, samples must be preserved during the time the analytical cycle is completed. This can be done by performing tests how long a sample can be stored without degradation occurring. Analyte stability in solution and analyte stability in the matrix are discussed separately (Figure 2.61).

Figure 2.61: Verification of analyte stability (www.fao.org).

Stability in solution

Normally standard solutions of analyte(s) are made. This means that it should be known during which period and under which conditions these solutions can be stored. In case this type of information is not available, the solutions should be prepared freshly until sufficient data are available, this to avoid the use of standards with an unknown or not precisely known concentration which can result in errors in the quantitation of unknown samples (Figure 2.62).

Figure 2.62: Redox reaction between ascorbic acid (vitamin C) and 2, 6-dichloroindophenol (DCIP) (www.chemlab.truman.edu).

In addition to the stability of standard solutions, also the stability during all steps of the SP/ST should be monitored. For example:

- An effective way of deproteination of plasma samples is by using perchloric acid or trichloroacetic acid. A number of compounds – in particular esters – however, are not stable at pH values of 2 and lower. This means that this approach cannot be used in combination with this type of compounds.

- A number of pesticides are hydrolyzing relatively fast in aqueous solutions (Figure 2.63) or are photochemically not stable. This means that in case these compounds must be determined in surface water, also the hydrolysis products should be determined in order to avoid an underestimation of the concentration of the parent compound.

Figure 2.63: Degradation of atrazine under various conditions (i.e. hydrolysis, biotransformation).

Stability in matrix

In addition to the stability of the analyte in solution also its stability in the matrix is important. In this respect three parameters are of importance:

1. What is the time period that a sample can be stored at room temperature or deep frozen before it is degraded in such a way that it is not acceptable anymore?

These data are important to determine how much time there can be between the collection of the sample (sampling) and the moment the sample is frozen, the time period that samples can be stored and time samples can be stored in an autosampler.

In order to study the stability at room temperature, an experiment can be performed during 24 h using a spiked sample of which an aliquot is analyzed (in triplicate) every hour or every two hours. In the case of homogenous liquid samples (e.g., body fluids, water samples), 24 hours normally is sufficient. Dealing with solid or semi-solid samples (e.g., faces, sediment) the stability should normally be studied during a longer period. An exception is urine; this because the ‘composition’ of this fluid is depending on many parameters the stability frequently is studied for a period of 24–48 h. When the sample seems not to be stable, it should be studied what should be done to avoid degradation. This means that either anti-oxidants, enzyme inhibitors, preservatives or algae inhibitors can be added.

After freezing of the sample the stability should be studied continuously during the whole time period of storage. In this case samples spiked with the analyte(s), at two different concentration levels, are divided in several aliquots. These aliquots are measured, in duplicate or triplicate, at indicated time points to find out the degree of degradation during such conditions. The time period of such a stabilization study depends on the requirements with respect to the stability, although it is advised to determine always the maximum time the sample can be stored.

In case the results indicate that the sample is not completely stable, additional experiments must be performed to indicate the maximum time of storage.

The most usual storage temperature for many samples is -20⁰C. Blood plasma normally is in the solid state at this temperature. However, just around the macromolecules (e.g. glycoproteins) that are present the water molecules still have sufficient potential energy to be ‘mobile’ and to take part in a degradation reaction. Urine and other samples with a high ionic strength (e.g. waste water) even can contain micro-drops of water at -25⁰C. In principle, every laboratory should have the facilities to store samples at -40⁰C and -80⁰C, to avoid the degradations problems sometimes occurring at -20⁰c. A solution of avoid this type of problems is to freeze-dry the samples before they are frozen.

2. What is the effect several freezing – thawing cycles of the sample?

This effect can be studied by spiking the matrix (sample) with known concentrations of the analyte(s) and test aliquots of this solution with several free-thaw cycles. This approach normally provides sufficient information on the possibilities of thawing and freezing the same sample more than once. However, this only is the case when all degradation products and metabolites are known. Unknown compounds can degrade into several parts during this type of procedures.

3. What is the stability of sample extracts?

With a last series of experiments the stability of the extracts, obtained after SP/ST, should be determined. This type of information will provide information on how many samples can be processed simultaneously, what the maximum time can be between the SP/ST and the actual analyses.

Method development

Introduction

There a number of separation systems can be used to determine organic compounds in complex matrices. The most important techniques are LC, GC and CE. In order to develop an adequate analytical method / procedure, the combination of SP/ST, separation and reaction/detection procedure is more critical as the separation mode itself. Due to the many possibilities there is a need for clear guidelines in chosen the optimum approach. The most suitable method can be found by answering the questions given in Table 2.14. These questions are related to the physico-chemical properties of the analyte and of the matrix as well as the objectives of the overall method.

Table 2.14: Guidelines for method choice in analytical chemistry.

Separation-detection systems

In analytical chemistry frequently selectivity, sensitivity, reproducibility, speed and costs are the critical parameters when organic compounds should be determined in complex samples. This means that a combination of techniques should be chosen, to develop a qualitative or quantitative method. An overview of some of the techniques that can be used is given in Table 2.15.

Table 2.15: Overview of analytical techniques.

In order to choose the proper methods and to develop a suitable method first of all, the questions of Table 2.14 should be answered. After answering these questions first of all the separation and detection technique must be chosen, and based on this the sampling and SP procedure can be selected.

From the Tables 2.14 and 2.15 it can be seen that numerous combinations of separation and detection techniques can be chosen for a particular problem. The first choice that should be made is which mode of chromatography/electrophoresis should be applied. In general, the analyte(s) should be determined quantitatively, which means that selectivity and sensitivity are the most critical parameters. The result is that CE, GC and LC will be the most obvious separation techniques. The most suitable detection modes are UV-VIS, DAD, FL and MS in combination with LC, FID, ECD, NPD and MS for GC, and UV-VIS, DAD, LIF and MS in combination wit CE. Especially, the use of MS or MS/MS approaches is still gaining popularity.

The sampling and SP approach will be selected on the basis of the chosen separation-detection system. Normally a combination of initial, non-selective, technique in combination with a selective SP procedure will be applied.

The physico-chemical properties of the analytes and the origin of the sample (e.g. biological, environmental, food) determine which separation technique can be used. Using (capillary) GC mainly volatile solutes, compounds that are thermally sufficiently stable, and have a molecular-weight of less than 500 can be determined without a derivatization procedure. This means that the applicability, in bio-analysis, is limited because only 25-35% of the compounds of interest can be determined with this technique. In environmental analysis it is just the other way around, which explains why GC is still frequently used in these cases. An important feature is that the separation power of GC is about 100 times higher as of LC. This means that if a problem can be solved with both LC and GC, GC normally is the first option. Other differences between GC and LC are that automated sample preparation is LC is more sophisticated than in GC, but in GC a number of selective detection approaches are available, meaning that the requirements for the sample preparation are less critical. Nowadays, both LC-MS(/MS) and GC-MS(/MS) techniques can be used for routine analysis. Another limitation of GC, the relatively small injection volumes, can be circumvented by using PTV (Programmed Temperature Vaporizer) injectors. The result is that volumes up to about 1 mL can be injected, even in a narrow-bore capillary GC system.

CE is in particular developed for the separation of charged compounds. The main advantage of this technique is its high efficiency and the speed of analysis. The most important limitations are the low concentration sensitivity and the fact that silica-based capillaries are used. The fact that the inside wall of the capillary normally is negatively charged means that matrix components (e.g., proteins, humic substances) easily can be sorbed on the wall and disturb the analysis. The result is that CE is mainly suitable for the qualitative and semi-quantitative determination of relatively high concentrations of organic and inorganic compounds in relatively simple matrices.

Liquid chromatography

Separation modes

LC is a technique that can be used for the separation of polar and non-polar compounds, for charged and neutral compounds, and for volatile and non-volatile compounds (Figure 2.64).

Figure 2.64: Block diagram of an LC system (www.protein.iastate.edu).

In particular, when mixtures of compounds with different polarities, i.e., drug and their metabolites, parent compounds and their degradation products, should be measured, LC is the method of choice. An overview on the selection of the phase systems can be found in Table 2.16.

Table 2.16: Various phase systems of liquid chromatography.

The following phase systems can be distinguished (Figure 2.65):

- Adsorption LC: Adsorption LC probably is one of the oldest types of chromatography. It is using a mobile liquid or gaseous phase that is adsorbed onto the surface of a stationary solid phase. The equilibration between the mobile and stationary phase accounts for the separation of different solutes. In this mode a polar sorbent (i.e., silica, alumina) in combination with a non-polar eluent is used. The limited stability and reproducibility, as well as the bad peak shape which is frequently inherent with the analysis of neutral (basic) compounds explains its limited popularity. In most applications adsorption LC can be replaced by normal-phase (NP) partition chromatography.

Figure 2.65: Different mechanisms of retardation in liquid chromatography (J.F. Rubinson, K.A. Rubinson, Contemporary Chemical Analysis, Prentice Hall, Upper Saddle River, 1998).

.

Nowadays, adsorption chromatography has been replaced by partition chromatography (Figure 2.66). This form of chromatography is based on a thin film formed on the surface of a solid support by a liquid stationary phase. The solutes equilibrate between the mobile phase and the stationary liquid. Partition chromatography can be performed in the NP and in the reversed-phase (RP) mode.

Figure 2.66: Liquid partition chromatography (www.liv.ac.uk).

- Normal-phase LC: NP-LC is performed by using silica sorbents which or chemically modified with polar and/or hydrophilic functional groups and in combination with non-polar eluents (Figure 2.67). The most popular sorbents are the amino- and the cyanopropyl sorbent. Both sorbents can be used for the separation of neutral polar compounds. The cyanopropyl sorbent can be used in combination with both polar and non-polar eluents. The use of NP-LC can be advantageous in case of fluorescence detection.

Figure 2.67: Normal-phase interaction on a cyanopropyl sorbent (E.M. Thurman, M.S. Mills, Solid-Phase Extraction: principles and Practice, Wiley, New York, 1998).

- Reversed-phase LC: The most popular mode of LC is RP-LC. In this case a non-polar sorbent is combined with polar eluents. The positive features of RP-LC are a fast equilibration, excellent stability, good reproducibility and peak symmetry, as well as a large application area. For neutral compounds, in principle, RP-LC is the method of choice. The most popular sorbents are the cyanopropyl, octyl and octadecyl phases. In case a more hydrophobic sorbents or an extreme pH value (<2 or >8) must be applied, polymeric (i.e. copolymers of styrene-divinylbenzene) or graphitized carbon can be applied.

- Ion-exchange LC: IEC is performed using either resin (polymeric sorbent) or silica gel material which are chemically modified with charged functionalities (Figure 2.68). These functionalities are covalently attached to the sorbent. Solute ions of the opposite charge in the eluent are attracted to the sorbent by means of electrostatic forces. IEC is applied for the separation of ionized compounds. Depending of the ion-exchanger, weak or strong, the retention is controlled by the ionic strength or the pH, respectively.

Figure 2.68: Principle of ion-exchange chromatograph (www.forumsci.co.il).

- Ion-pair LC: In IP-LC charged counter-ions are used in combination with a neutral, normally hydrophobic, sorbent. IP-LC can be applied as an alternative for IEC, for the separation of charged compounds. The disadvantage of IP-LC is that, depending on the counter-ion used, equilibration is rather time-consuming (Figure 2.69).

Figure 2.69: Ion-pairing mechanism (www.registech.com).

- Ion-suppression LC: IS-LC is a special mode of RP-LC in which the pH of the eluent is adapted in such a way that the ionization of the analytes is suppressed (Figure 2.70). This approach is mainly used for the separation of weak acids.

Figure 2.70: Ion-suppression LC (www.crawfordscientific.com).

Detection

One of the limitations of LC may that at the moment no universal and sufficiently sensitive detectors are available; this in spite of the fact that a wide variety of detection systems varying from selective (amperometric) to universal (refractive index) and from insensitive (conductometry) to sensitive (fluorescence) are available. The limited applicability of selective detectors can be overcome by labeling the analyte with a suitable reagent. A derivatization procedure can be part of the SP procedure, and can be performed before and after the chromatographic / electrophoretic procedure.

Fixed-wavelength absorption detectors (UV-VIS) are in particularly suitable for the quantitation of one or more compounds possessing the same or similar chromophores. The limitation is that identification cannot be performed. Diode-array detectors (DAD) can be used to obtain some structural information, while MS techniques can be used to perform structure confirmation of analyte(s) (Figure 2.71).

Figure 2.71: A, Diode-array detector (www.chromatography-online.org); B, Diode-array spectrum (www.ribbonchem.it).

The choice of the detection system mainly depends on the required limit of determination and the structure of the analyte. For determination limits in the μg/mL – high ng/mL range, UV-VIS detection can be used. Unsaturated compounds can be detected at wavelengths over 250 nm, while aliphatic compounds can be detected between 200 and 250 nm (Figure 2.72). The application of wavelengths below 280 nm is not favourable, in many cases, because of matrix interferences. However, in combination with a proper choice of eluent and SP procedure, wavelengths between 200 and 210 nm can be used to improve the sensitivity.

Figure 2.72: Absorption spectrum of benzydamine (photobiology.com).

The absorption maximum not always is the optimum detection wavelength, because the background signal also can be relatively high at this wavelength. In general it can be stated that the matrix background will be lower at longer wavelengths, but at these wavelengths also the molar absorptivities of the analytes will be lower. This means that always a compromise should be found to determine the maximum signal-to-noise ratio. During method development, in order to find the optimum detection wavelength, the presence of a DAD spectrum can be rather helpful. A proper optimization can result in a gain in sensitivity of 1-2 orders of magnitude. Another way to increase the sensitivity is to apply a proper SP procedure, to remove interfering matrix components.

In case sensitivities in the ng/mL to high pg/mL are required, UV-VIS detection can be used for conjugated and aromatic compounds, while for aliphatic compounds a more sensitive technique like AMP, FL or mass MS should be used (Figure 2.73).

Figure 2.73: Simultaneous detection of SERRS and fluorescence of anticancer drug mitoxantrone within a single living cancer cell (MCF-7) by confocal spectral imaging. Bottom left window shows the reference spectra of a) fluorescence of drug-DNA complex (red); the drug fluorescence in high and low polarity environment (blue and violet, respectively); b) SERRS of the drug (green). Fitting the intracellular spectra with the reference ones provides the respective quantitative maps (monochrome images) that can be superposed (multicolor image) to analyze their colocalisation with cellular compartments: nucleus (red); polar cytosolic regions (blue); cellular membrane and those of organelles (violet). The SERRS map indicates the co-localization of silver colloid aggregates with the membranes (www.jobinyvon.com).

AMP detection only can be used when oxidable or reducible structures are present in the molecule that can be oxidized at an acceptable potential. Electrochemical-active functional groups can be introduced into a molecule, during the SP procedure, via a derivatization procedure (Figure 2.74).

Figure 2.74: Amperometric detection in liquid chromatography (www.epsilon-web.net).

It is preferred to use a relatively low potential, which means that interferences by eluent and matrix components can be avoided. The LOQ values can be in the low pg/mL range.

Using FL detection, determination limits in the low pg/mL range can be obtained (Figure 2.75). A derivatization procedure, using a suitable fluorescence probe, can be used to derivatize which do not possess native fluorescence. The derivatization reaction can be performed either before or after the separation. Applying laser-induced fluorescence (LIF) the detectability can be improved with a factor of 10 – 100. In spite of the improved sensitivity, LIF is still not rather popular because of the limited availability of commercial systems and the fact that only a few lasing wavelengths are available, which means that a derivatization procedure cannot be avoided in many cases. The introduction of relatively cheap diode lasers, certainly will increase the applicability of LIF detection.

Figure 2.75: A, Fluorescence detection (www.boomer.org); B, laser-induced fluorescence detection (www.azonano.com).

MS detection can in principle be applied for all compounds. Determination limits in the ng/mL to high pg/mL level can be obtained, and both qualitative and quantitative (i.e. structure elucidation) data can be obtained.

In a mass spectrometer analytes are determined by measuring the mass-to-charge ratio (m/z). This is done by ionizing the analyte molecules and directing the resulting ions through a series of magnetic and/or electrostatic fields. Four basic types of mass spectrometers are used, in combination with LC, nowadays: magnetic sector, quadrupole, ion-trap and time-of-flight. The magnetic sector instruments use magnetic fields to alter the path of the ionized solute (Figure 2.76).

Figure 2.76: Magnetic-sector instrument (www.geo.uib.no).

Scanning of molecular weights is performed by changing the strength of the magnetic field. The quadrupole instruments are based on the interaction of the ionized solute with an oscillating electric field on four electrodes. By varying the dc and ac potential on the electrodes, only ions of a selected mass will oscillate in a stable path and reach the detector (Figure 2.77).

Figure 2.77: Quadrupole mass analyzer (www.waters.com).

Ion-trap analyzers also an electric field is used, but the ions with stable trajectories are trapped in a cell (Figure 2.78).

Figure 2.78: Ion-trap analyzer (www.zenobi.ethz.com).

The time-of-flight instrument analyses the time required for ions to reach the detector (Figure 2.79).

Figure 2.79: Quadrupole time-of-flight mass spectrometry (www.edoc.hu-berlin.de).

The advantage of the sector instruments is that the molecular weight resolution is relatively high (ca. 0.001 u), the quadrupole instruments are relatively inexpensive, and the time-of-flight instruments have the advantage that there is no upper mass limit of detection. A solute to be analyzed should, therefore, first be vaporized at low pressure, in order to avoid the analyte molecules to react with each other and to facilitate the ionization.

Several ionization modes are in use nowadays, but the two most frequently applied are electron impact (EI) and chemical ionization (CI) (Figure 2.80).

Figure 2.80: Electron-impact ionization (www.phim.unibe.ch).

In EI the analytes in the gas phase are bombarded with a beam of electrons resulting in ionization and strong fragmentation of the formed ions. The limitation of EI-MS is that normally only small ions are formed meaning that no information on the molecular weight can be obtained. CI is a milder ionization technique and as a result fragmentation is minimized resulting in molecular weight information (Figure 2.81). In CI a reagent gas (e.g., methane, ammonia) is ionized by a beam of electrons, and subsequently the analyte is ionized by the transfer of protons from the ionized methane.

Figure 2.81: Niggard spectra (polymer additive), obtained by negative APCI (A), negative APCI with in source fragmentation (B), electron impact ionisation (C) (www.waters.com).

The coupling of LC and the MS allies a highly powerful separation technique with a detector possessing unique sensitivity and selectivity (m/z selection; peak patterns) capability. For about a decade, interest has now been focused on the use of LC-MS for the identification and determination of compounds that cannot easily be handled by GC-MS, i.e., compounds with relatively HMW, thermo labile compounds and compounds of a rather polar nature. Unfortunately, the operating conditions peculiar to LC and MS are somewhat incompatible. Consequently, all LC-MS interfaces designed so far impose some restrictions on the normal operation of the LC, the mass spectrometer, or both.

Gas chromatography

Introduction

Gas chromatography can be used for the separation of volatile organic compounds. However, numerous solutes that should be determined in the bio-analytical or environmental field are not suitable for direct GC determination because of there limited stability at the elevated temperatures used during GC separations.

Derivatization with a suitable reagent can significantly improve the temperature stability of an analyte. A Derivatization step should be considered as an integral part of the total ST/ P procedure. During a derivatization reaction normally a polar function of the analyte is converted into a less polar functionality. Derivatization procedures, both for GC and LC, will be discussed extensively in one of the other Chapters of this Monograph.

SP in GC can be performed nowadays with the traditional techniques like the various modes of extraction, but also multi-dimensional approaches that can be performed either at-line or on-line are widely available. Especially the use of ‘pre-columns’ is gaining in popularity (Figure 2.82).

Figure 2.82: GCxGC system (www.mams.rmit.edu.au).

The popularity of GC in environmental analysis is due to fact that GC, in principle, is a more efficient a faster technique – compared with LC – and can be combined easily with a number of selective and sensitive detectors. Moreover, the analytes in environmental analysis are still relatively non-polar compared with the drugs that should be determined in biological samples (Figure 2.83).

Figure 2.83: Especially for volatile compounds, gas chromatography is a valuable approach (www.resonancepub.com).

Stationary phases

The key parameter in performing a good GC separation is choosing the optimum stationary phase and column and optimum flow rate of the carrier gas and optimum temperature or temperature program belonging to the chosen set of hardware and the physico-chemical properties of the analyte(s) (Fig. 2.84). The fact that the choice of the sorbent in more critical in GC as in LC is due to the fact that in GC no mobile phase is present, the carrier gas mainly act as a transport medium and there are hardly any selective reactions between the analytes and the carrier gas and the carrier gas and the stationary phase.

Figure 2.84: Rapid temperature programming in gas chromatography (www.rvmscientific .com).

The following features are important in choosing the proper separation system:

- The boiling point range of the analytes and the properties of the resulting vapours phase of these analytes;

- The number of components that must be separated;

- The presence of polar and/or non-polar groups in the analytes and the presence or absence of functional groups;

- The combination of stationary phase and column dimensions as well as the film thickness of the stationary phase with respect to the required selectivity and resolution;

- The flow rate of the carrier gas as a parameter to speed up the total analysis, with a minimum loss in resolution of the most critical pair of compounds to be separated;

- The fact that either a temperature program should be used or that the analysis can be performed at a constant temperature.

Stationary phases in GC can be divided based on their polarity or selectivity with respect to one or more physico-chemical properties of the analyte molecules such as molecular mass or molecular shape, boiling point or the presence of certain functional groups (Table 2.17). The chemical composition of some of these phases is given in Figure 2.85. First of all the stationary phases available for packed columns will be discussed and later on, the nowadays far more popular, sorbents for capillary columns will be discussed.

Figure 2.85: Chemical structures of some phases for wall-coated open tubular columns (www.uga.edu).

The separation on the non-polar sorbents like SE-30, OV-1 and Apiezon L, a branched alkane polymer with a melting point of 43⁰C, is primarily based on molecular size and shape. In general the elution order corresponds to the molecular mass of the compounds. In particular for neutral and weakly acidic compounds these phases are suitable.

Table 2.17: Stationary phase for GC (D.A. Skoog, D.M. West, F.J. Holler, S.R. Crouch, Fundamentals of Analytical Chemistry, Thomson, Belmont, 2004).

.

The polyethyleneglycol (PEG) stationary phases are the most frequently used polar sorbents. PEG 400 is a suitable sorbent for the separation of alcohols, ethers, aldehydes and other compounds with low boiling points, while Carbowax 20M can be used for the separation of polar compounds with higher boiling points.

These phases are in particularly useful for the determination of strong basic compounds. Another group of polar stationary phases are the poly(cyanopro-pylphenyldimethyl) siloxanes (Figure 2.85). These moderately polar sorbents are used for the separation of compounds containing several hydroxyl groups (e.g. steroids).

In summary it can be stated by using only a limited number of sorbents, most separations can be performed in case temperature programming is applied:

- Analytes possessing non-polar groups: OV-101, Apiezon L

- Analytes with moderately polar groups: OV-1, OV-1701

- Analytes with polar groups: Carbowax 20 M.

As mentioned before different type of columns are used for GC separations (Figure 2.86).

Figure 2.86: Different column types used for GC separations Different column types used for GC separations (D.C. Harris, Quantitative Chemical Analysis, Freeman, New York, 2007).

In the picture on the left (Figure 2.86) a packed column with an internal diameter of 2-4 mm and a tubular construction of either glass or stainless steel can be seen. The column is packed with particles which either act as the retarding stationary phase or are coated with a thin film of organic material which acts as the stationary phase. The other pictures depict open tubular columns. These columns have a narrow internal diameter between 0.17-0.53 mm. There are two major types of capillary columns: the porous layer open tubular (PLOT) columns in which the inner surface of the column has an embedded layer that contains the stationary phase and the wall coated open tubular (WCOT) in which a thin film of organic stationary phase is bonded directly onto the inner surface of the column. For trace analysis mainly WCOT columns are used, while PLOT columns are applied for the determination of low-molecular weight gases. A comparison between packed and capillary columns is given in Table 2.18.

The chemical structure of some of the stationary phases described in Table 2.18 is pictured in Figure 2.85. The non-polar phases are more stable and chromatographically robust than the polar phases which tend to have a lower temperature tolerance and are more susceptible to oxidative damage if air is introduced into the column.

Table 2.18: Stationary phase for wall coated open tubular columns.

Detection

In addition to the excellent separation power of GC another advantage compared with LC is the large number of sensitive and selective detectors that can be used (Table 2.19).

Table 2.19: Detectors in gas chromatography.

There are a number of differences between the detectors used in LC and those in GC. The most important difference is that in LC mainly concentration sensitive detectors are used, while in GC most detection devices are mass sensitive. Some of the GC detectors (i.e. TCD, FID) can be considered to be nearly universal detectors since they respond to a physical change in the carrier gas. The main advantage of the FID (in GC) compared with the refractive-index (RI) detector (in LC) is that the FID is more sensitive and can be used also in combination with temperature programming. Both detectors are considered to be universal.

In addition to the FID, there is a special FID which is selective for nitrogen and phosphor containing compounds, the NPD. In the NPD-mode this detector is about 50 times more sensitive for nitrogen and 500 times for sensitive for phosphor containing compounds (Figure 2.86).

Figure 2.86: Nitrogen-phosphorus detector (www.chromatography-online.org).

Another selective detector is the ECD. This detector is frequently used in environmental analysis in case trace analysis of chlorinated pesticides or herbicides should be performed. In addition to the halogenides (F < Cl < Br < I) also a number of organometal compounds (e.g. tetraalkyl lead), NO_x and SO_x compounds can be selectively determined with this detector. By means of a derivatization procedure with pentafluorophenyl groups, during the SP, a large number of compounds can be determined via GC-ECD (Figure 2.87).

Figure 2.87: Electron-capture detector (www.chromatography-online.org).

Gas chromatography - mass spectrometry

However, nowadays the most popular detector in combination with a GC separation is the mass spectrometer (GC-MS). This combination allows the separation, identification and quantitation of complex mixtures of compounds. In order for a compound to be determined by GC-MS it should be sufficiently volatile and thermally stable. The procedure is relatively straightforward. The sample is injected into the GC and the analytes are separated on the GC column. The latter part of the column passes through a heated transfer line and ends at the entrance of an ion source (Figure 2.88), where the analyte molecules, eluting from the column, are converted into analyte ions.

Figure 2.88: Ion source for gas chromatography – mass spectrometry systems (www.bris.ac.uk).

In principle, there are two possibilities for production of the ions. The most frequently used one is electron impact (EI) ionization and in a limited number of cases chemical ionization (CI) is used. For EI a beam of electrons ionize the sample molecules resulting in the loss of one electron. The resulting ion is called the molecular ion and is given by M^+. (radical cation). The corresponding peak in the mass spectrum indicates the molecular weight of the compound. Due to the high energy of the electrons, the molecular ions usually fragments into smaller ions with characteristic relative abundances and providing a fingerprint of the molecule. These data can be used to identify analytes and to elucidate the chemical structure of unknown compounds.

The next part is the mass analyzer (Figure 2.89). The mass analyzer is used to separate the positively charged ions according to various mass related properties depending upon the analyzer used.

Figure 2.89: Schematic of quadrupole and ion trap mass analyzer .

There are several types of analyzers in use. The quadrupoles (Figure 2.90) and the ion-traps are the most frequently applied nowadays.

Figure 2.90: Quadrupole mass analyzer (www.ael.gsfc.nasa.gov).

After the ions are separated they enter a detector and after amplification the signal is represented in a mass spectrum in which the intensity of the signal is plotted against the m/z (mass/charge) value.

The power of GC-MS is in the production of mass spectra from each of the analytes detected instead of merely an electronic signal that varies with the amount of analyte. These data can be used to determine the identity as well as the quantity of unknown chromatographic components with an assuredness simple unavailable by other techniques.

Capillary electrophoresis

Introduction

In addition to GC and LC there are a number of other separation techniques that are frequently used. The most popular of these techniques is capillary electrophoresis (CE). Since this technique is in many cases a good alternative for the separation of ionic compounds, a short introduction will be given in this Section.

CE is a series of techniques (capillary-zone electrophoresis – CZE, isoelectric focusing – IEF, capillary-gel electrophoresis – CGE, isotachophoresis – ITP, micellar electrokinetic chromatography – MEKC, capillary electrochromatography - CEC) which are based on the migration van charged particles by an electric field in which capillaries with internal diameters of 20-200 mm are used. Differences in the effective charge / mass ratio of the ions and/or differences in their micro environment will result in changing migration speeds allowing a separation of both low and high-molecular weight compounds. A limitation of the more traditional forms of electrophoresis (e.g. polyacrylamide gel electrophoresis – PAGE) is the limited separation efficiency because of band broadening problems. By performing the separation in a capillary the band broadening - which is due to convections - is significant reduced by the so called wall effect? These convections are caused by the differences in density between the sampling zone and the electrolyte or by heat development the so called Joule heating (Figure 2.91). In a capillary the heat can be dissipated more efficiently because of a better surface / volume ratio. The wall effect and the heat transfer are more pronounced at smaller internal diameters of the capillary. Additional advantages of miniaturization are less dilution of the sample, the possibility of analyzing real small samples (e.g. single-cell analysis) and the use of extremely small amounts of chemicals.

Figure 2.91: Joule heating in capillary electrophoresis (www.chemsoc.org).

Separations in the capillary are performed using a high voltage resulting in an electroosmotic en electrophoretic transport of charged buffer and charged analyte ions (Figure 2.92).

Figure 2.92: Set-up of capillary electrophoresis system (www.answers.com).

In comparison with LC and the traditional electrophoretic approaches, CE has the following properties:

- Electrophoretic separations are performed in a capillary;

- Electric field strengths of 500 V/cm, or higher, are used;

- An electropherogram is similar to a chromatogram;

- Plate number are comparable, or higher, compared with GC;

- Extreme small sample volumes can be handled;

- Automation is relatively easy;

- Only small amounts of reagents are necessary;

- A wide variety of analytes can be determined.

System

In CE the electrophoretic separation is performed in a narrow-bore capillary. In most cases fused-silica capillaries are used with a length of 30-100 cm, an internal diameter of 0.010 to 0.075 mm and an outer diameter of 0.150-0.375 mm. In CE (Figure 2.92) the capillary is filled with buffer and each end is immersed in a vial containing the same buffer. The analyte is injected at one end and an electric field of 100-700 V/cm is applied over the capillary by applying potentials of 20-30 kV. The analytes will migrate will migrate through the capillary due to the applied electric field (electrophoresis). Since most analytes will have different electrophoretic mobilities, the components will be separated in discrete bands. Due to the electroosmotic flow (EOF) (Figure 2.93) all analytes will migrate to the negative electrode. A small volume (ca. 10 nL) is injected at the positive end of the capillary and the separated analytes are detected near the negative end of the capillary.

Figure 2.93: Electroosmotic flow in CE systems(www.answers.com).

Detection in CE is similar to detection in LC. This means that on-column detection performed by using MS, electrochemical and optical techniques. Still the most popular mode of detection is UV absorbance either by using a fixed-wavelength or a DAD detector. Using this detection device part of the capillary is used as the detection cell. The advantage is that no losses in resolution will occur. The disadvantage, however, is that the optical path length of the detection cell (ca. 50 mm) is far less than that of a traditional UV cell (ca. 1 cm). According to Lambert-Beer the sensitivity is proportional to the path length of the cell. This will result in rather unfavourable concentration detection limits. Possibilities to solve this problem are the use of capillary configurations with extended path lengths (e.g., Z-cell, bubble cell) or the use of optical fibres.

Fluorescence detection is another frequently applied mode in CE. This technique can be used both for analytes possessing native fluorescence and compounds that have tagged with a suitable fluorophore. Fluorescence detection offers high sensitivity and improved selectivity (Figure 2.94). The set-up can be quite complicated because a light beam is needed that can be focused on the capillary. This explains why LIF detection is quite popular in CE, especially since detection limits of 10^-18 – 10^-21 mol can be obtained.

Figure 2.94: Schematic diagram of the CE-LS system used for low-temperature spectral characterization of CE-separated analytes (www.rsc.org).

In order to identify sample components (analytes) CE can be directly coupled directly with MS. In most cases the capillary outlet is introduced into an ion source that utilizes ESI. Just like in LC only volatile buffers can be used (Figure 2.95).

Figure 2.95: CE-MS sprayer (www.chemsoc.org).

As mentioned before, the injection volumes in CE are rather small. This means that with respect to overloading the injection plug length is more critical than the volume. Normally the length of the injection plug may not exceed 1% of the total length of the capillary. There are several possibilities to inject a sample; the two most common ones are hydrodynamic and electrokinetic injection.

Hydrodynamic injection is applied by applying some pressure at the injection end of the capillary, vacuum at the exit end of the capillary or by siphoning action. In this case the amount of sample loaded is nearly independent of the sample matrix (Figure 2.96).

Electrokinetic, or electromigration, injection is performed by replacing the injection-end reservoir with the sample vial and applying the voltage. Normally, a field strength 3-5 times lower than that used for the actual separation is applied. In this mode the analyte enters the capillary both by migration and by the EOF. Sample loading is dependent on the EOF, sample concentration and sample mobility, which means that the quantity loaded, is dependent on the electrophoretic mobility of the individual analytes. Due to ionic strength effects electrokinetic injection normally is less reproducible as hydrodynamic injection.

Figure 2.96: Hydrodynamic and electrokinetic injection in CE (www.chemsoc.org).

Theoretical background

The overall electrophoretic mobility of an ion, in a fused silica capillary, for example – is controlled by two parameters: the effective electrophoretic mobility and the electrosmotic mobility.

The effective electrophoretic mobility (m_eff) can be expressed as:

In this Eqn. m_i represents the combined influence of several parameters like solvation, effective ion radius, charge of the ions, dielectric constant and viscosity of the electrophoretic medium. The degree of ionization (a_i) is depending of the pKa of the analyte, the temperature and the pH of the medium. The correction factor for retardation and relaxation effects is given by g_i.

An electrophoretic separation is based on differences in mobility. The mobility of a certain ion q (m_q) can be represented as:

In this Eqn. v is the electrophoretic mobility and E the field strength over the capillary. The electrophoretic mobility is proportional to the charge of the analyte and inversely proportional to the frictional forces in the buffer. As a result the electrophoretic mobility, at a certain pH, can be given as:

In this Eqn. z is the charge of the analyte, h is the viscosity and r is the Stokes radius of the analyte.

Figure 2.97: Electroosmotic flow (www.answers.com).

The second parameter is the electroosmotic (EOF) mobility (Figure 2.97). The EOF normally is larger than the electrophoretic mobility of the analytes in case fused silica capillaries are used. The EOF is the result of a potential gradient between the capillary wall and the layer of electrolytes directly associated with the capillary. Because of the negatively charged silanol groups of the capillary wall and the positively charged cations of the electrolyte solution an ionic double layer exists. The resulting potential gradient can be described by the Stern model. The thickness of the double layer (d) is defined as the distance between the non-mobile layer and the point in the electrolyte solution where the potential equals 0.37 of the potential on the borderline of the non-mobile and the diffusion layer. By applying a potential difference over the capillary, the mobile parts of the adsorbed cations migrate towards the cathode. The remaining part will stay, immobilized, on the capillary wall. During the migration process the remaining liquid will migrate in the capillary resulting in a nearly ideal flat flow profile (Figure 2.98).

Figure 2.98: (a) Laminar flow pattern as usually obtained in LC, (b) elecroosmotic flow pattern as usually obtained in CE (www.answers.com).

The mathematical definition of the electroosmotic flow (v_eo) is given by the Helmholtz-Smulukovski equation:

In this Eqn. is E the field strength, e the dielectric constant of the electrolyte solution, h the viscosity of the electrophoretic medium and z the zèta potential. The zeta-potential is the potential difference on the border of the immobilized and the flowing medium and is depending on the surface of the capillary, the type of counter ions (cations) and the total concentration of the ions. Another way to influence the zeta-potential is by changing the pH of the buffer solution. An increase will result in more deprotonated silanol groups on the surface and, as a result, a larger potential difference over the double layer.

There are several possibilities to determine the EOF. The simplest one is by injecting an electrically neutral marker. Phenol and acetone are frequently used markers for this purpose. However, it is important that a pH is chosen in which the marker solute is completely neutral.

The Efficiency (N), in a situation that only axial diffusion occurs, is given by :

The overall mobility of the ion is given as m_tot, V is the applied potential difference and D the diffusion coefficient. From this Eqn. it can be seen that the efficiency is, more or less, linear with the applied voltage. However, by using high voltages the band broadening due to Joule heating, will not be negligible anymore. As a result the optimum efficiency can only be obtained at a certain potential.

Although equation above is suggesting that N is independent on the capillary length, this is not really the case as can be seen in Eqn.:

In this Eqn. is P the electrical power generate per unit of length (l), K is the molar conductivity and C the molarity of the electrolyte. V and r are respectively, the applied potential difference and the diameter of the capillary. A decrease of l will result in a quadratic increase of P/L (at a constant V), and in more band broadening of the electrophoretic peaks.

Conclusions

The popularity of CE is related to the wide variety of compounds that can be separated if numerous matrices and the fact that these separations can be performed relatively fast with high efficiencies. Additional advantages are that method development can be relatively fast and the costs are relatively low. Limitations of the technique are that the reproducibility (e.g. migration times), accuracy/precision and detectability are not optimal at the moment. The fact that CE is complimentary to GC and LC and that in addition to free flowing zone electrophoresis different modes like capillary electrochromatography (CEC) and micellar electrokinetic chromatography (MEKC) are available, explains the popularity of these techniques for the separation of peptides, proteins and nucleotides, the applicability in pharmaceutical drug quality control and screening techniques in the environmental analysis.

Sample treatment / preparation

Sample treatment

The necessity to remove matrix components from the sample to be processed is the normally the most important reason to build in ST/SP steps in an analytical procedure. In most cases this is the only possibility to ensure sufficient selectivity and sensitivity in combination with an electrophoretic or chromatographic separation. In other words, the required reliability can only be obtained when sufficiently clean samples are analyzed.

The objective of the method and subsequently the chosen separation/detection system determine already which ST/SP procedures can or cannot be applied and if manual or automated procedures should be used.

Questions that are important in choosing the proper ST/SP procedures are:

- Should only the analyte itself, or also the degradation products or metabolites be determined? In this case it should be taken into account that degradation products / metabolites are frequently more polar than the parent compound(s).

- Should the free fraction, the matrix-bound fraction or the total amount of the analyte be determined? It may never be assumed that the applied ST/SP procedure will quantitatively disrupt the analyte-matrix bonds.

- Is the analyte enzymatically and/or chemically stable in the matrix? Is it possible to store the samples, should the sample be stabilized or is an immediate analysis necessary?

During method development of an ST/SP procedure there are a number of features that should be taken into account:

- Stability of the analyte;

- Interference by pollutants from reaction- and reagent containers;

- Recovery of the analyte;

- Compatibility of the solvent used in the final ST/SP step and the solvents used in the subsequent separation system;

- Simplicity of the ST/SP procedure;

- Robustness and reliability of the method;

- Possibilities of sample enrichment.

The majority of the available ST/SP procedures can be performed in a manual and automated way. This means that they can be combined off-line, at-line, on-line or in-line with the separation system. In Table 2.20 an overview is given of frequently applied ST/SP techniques. In addition to the techniques mentioned in this Table a filtration or precipitation step is frequently introduced to avoid clogging of the separation system. In bio-analysis protein precipitation is a more or less standard procedure, while in environmental analysis filtration is normally used to remove solid particles from the sample. The disadvantage of using precipitation techniques is that none of the applied research can be used to precipitate proteins or other macromolecules quantitatively.

Sample clean-up in most cases is the time-limiting step in the analytical procedure. The result is that during the past decade a number of methods/procedures have been developed to completely omit a clean-up step in combination with a chromatographic/electrophoretic separation. In a number of cases this can be done by performing the sample clean-up on-line with the separation on a special precolumn.

Table 2.20 : Sample treatment / separation techniques and their application area.

One of the approaches is the use of the so-called ‘restricted-access surface’ materials (RAM columns). An example is silica sorbent with relatively small pores. The surface outside of the pores is modified with a hydrophilic phase, while the inside of the pores is coated with a hydrophobic phase (Figure 2.99).

Figure 2.99: Pinkerton ‘Internal-Surface Reversed Phase’ (ISRP) material (www.registech.com).

This type of sorbents has been developed for bio-analytical applications. Proteins and other macromolecules are not able to enter the pores and are excluded from the material. Small molecules, on the contrary, will enter the pores and are retained by means of hydrophobic interactions. The advantage of such an approach is that complex samples can be directly injected into a separation system, which can save quite some time (Figure 2.100).