Questions and Answers – Chromedia April 2010

Nicholas H. Snow, Seton Hall University

How would you remove natural organic matter (NOM; like humic and fulvic acids) in water samples? I am extracting 500 ml of source water.

Think about the solubility of the matrix components as well as the analytes. If using an SPE-based method, you might consider pH adjustment to deprotonate the acids (high pH) so they stay dissolved in the water as is passes through the SPE cartridge or is mixed with an oragnic solvent. You want to plan an extraction where the analytes are soluble in the extraction phase but the interferences are not. Check application notes and literature from SPE vendors.

Is SPE a good method for extracting proteins, for example samples as milk, vaccines?

Yes. Best next step would be to check application notes at one of the major vendors for SPE materials and supplies.

Are all SPE C18 cartridges the same (different vendors)?

No. Each vendor will use a different source for the particles themselves and a different procedure for preparing the bonded phase. Like HPLC columns, they have subtle differences in properties, so you will need to perform experiments to determine equivalence.

"When do you have a complex soil matrix, what type of extraction of organic pollution is more useful for this purpose?"

There are several possibilities, depending on how strongly you suspect the organic contaminants are bound to the soil – for instance were they spilled today or have they been seeping into the soil for years? Listed in order of simple and inexpensive to more challenging and expensive, I would explore.

1. Try extracting with an appropriate organic solvent. Use an ultrasonic bath to assist if needed.
2. Soxhlet extraction.
3. Accelerated Solvent Extraction
4. Supercritical Fluid Extraction

Definitely check the literature and think about the goals of your analysis before beginning.

What is a matrix? (in slide 12). Can we use this technique for quantitative separation?

The matrix is all that stuff in your sample that is not the compounds of interest. You can use all of the discussed techniques for quantitative analysis. For multi-step techniques such as SPE, SPME, ASE, etc., I suggest you plan to use an internal standard, added to all samples, blanks and standards prior to extraction, to account for variable extraction recovery.

How do you begin to develop an SPE separation method, when there are so many cartridges, solvents, and individual component properties? How do you validate your separation protocol?

I begin by thinking about he solubility of my analytes and major matrix components. We are looking for a sorbent and solvent combination that will initially trap both analytes and matrix on the sorbent, then allow washing of the matrix from the sorbent while retaining the analytes, then allow elution of the analytes. Nearly all of the major vendors of SPE materials have method development guides and examples on their websites; this is a good place to begin.

In your experience, have you found SPE vendors to be variable in their sorbents: both vendor-to-vendor as well as lot-to-lot variability from a single vendor?

Yes. This is one reason that I generally use internal standard quantitation with SPE. Recovery from lot to lot and vendor to vendor may vary significantly. Also there is often analyst to analyst variability as well.

What happens if you have a family of compounds with very similar properties and you need to separate only one of them that is in very low concentration, How can you handle this task?

In general, I would think of extraction as a bulk phase transfer process. Separate my analyte and the similar compounds from all the rest of the matrix (like all the steroids from urine) using the extraction method. Next use high resolution gas or liquid chromatography to separate the compounds in the family from each other. Extraction methods alone generally lack the selectivity to separate closely related compounds.

bulk C18 or prepacked C18 cartridges?

For SPE, premade cartridges will be much easier to use and more reproducible.

How critical is the extraction time in SPME and SBSE? Is the time important to reproducibility?

Extraction time is critical in all extraction methods. During method development, it is important to optimize extraction time be performing several experiments using different extraction times and plotting peak area versus extraction time. As time increases, the plot should increase until it reaches a plateau. Extraction time should be set to a time reflected on the plateau so that small variations in time will not have a major effect on peak area.

I am extracting multi water soluble vitamins from fish oil. What approach do you suggest with SPE. Currently we are using hexane extraction. With no recovery studies done, I would like to move to SPE extraction.


How does the solvent drop stay on the end of the syringe in that last micro-extraction technique?

In single drop micro-extraction a drop of organic solvent is suspended from the tip of a micro-syringe. The drop stays on the tip because the surface tension of the solvent and its repulsion of water will help keep it intact. Of course, with too large a drop or too much agitation, the drop may fall off.

Do you have any suggestions or strategies regarding desorption from adjuvants (e.g. alum salts) of recombinant proteins particularly in multivalent vaccine preparations (e.g. 3 or more proteins)? Thanks very much!


When you use the SPME you are only extracting the analytes and getting rid of solvent. Is it any solvent, for example would it work for dodecane as a solvent ?

Just like liquid-liquid extraction, you should be sure that the sample solvent is not soluble in the fiber material. In the case of dodecane as analyte solvent, dodecane would likely overload the fiber.

Follow up question--- If quantitating with an internal standard with SPE, what are the requirements for internal standard , eg, can we use same internal standards we used with GC?

You can generally use the same internal standards you would use for a GC method. Similar GC properties will most likely lead to similar SPE properties. For GCMS you can often use deuterated analogs of your analytes as internal standards.

Can you address how to obtain a representative sample or sub-sample for analysis?

This is one of the biggest challenges and easiest things to challenge in any analytical method. For a sample to be representative, it must be taken from a homogeneous mixture that is also at equilibrium. Assuring these two conditions is often difficult. When sampling, I note carefully any observations that may lead to doubt about whether the sample is representative.

How many samples (or a range) are usually measured to validate a method ?

The number of required samples, blanks and standards required to validate a method will vary among industries and applications. In general, you need enough samples to generate valid statistics for:
Precision – how close repeated measurements of the same sample are to each other
Accuracy – how close a measurement is to the true value
Detection limit – the smallest amount of analyte that can be detected
Sensitivity – how much does the instrument response change if the amount of analyte changes
The specific procedures for measuring these will vary between industries. Guidelines are generally promulgated by regulatory bodies or industry groups such as USP, ICH, ASTM, AOAC, EPA, etc.

What is spme?

SPME stands for solid phase micro-extraction. There is an excellent discussion in the sample preparation section of chromedia.org.

Would you introduce again the book you mentioned earlier?

There are numerous excellent books and introductory materials related to sample preparation. A few of my favorites are:

1. www.Chromedia.org. SPME section.
2. Pawliszyn, Solid Phase Microextraction Theory and Practice, Wiley, 1997.
3. Kolb and Ettre, Static Headspace Gas Chromatography, 2nd. Edition, Wiley, 2006.
4. sample preparation chapters in Grob, Modern Practice of Gas Chromatography, 4th. Edition, Wiley, 2003 and Miller and McNair, Basic Gas Chromatography, 2nd. Edition, Wiley, 2009.

How does work in detail single drop extraction ? Do you need a special type of syringe?

No special syringe needed – a standard pointed needle GC syringe will do. You will want to place your sample into a vial. Then draw your extraction solvent into the syringe and insert the needle containing the solvent into the sample liquid. Depress the plunger slowly to form the solvent drop in the sample. Suspend to complete extraction then withdraw the extraction solvent into the needle and transfer to the GC for injection.

Any experience/thoughts/ futher references on FBE (fluid bed extraction)?

Fluidized bed extraction is simply solid phase extraction without the cartridge. The solid phase material is placed directly into the sample and agitated as in a reaction. The solid phase material and extracted analytes are then recovered and the analytes are desorbed by combining with an elution solvent. The advantages are control over the amount and type of sorbent and it is quite simple. Disadvantages are that it is labor intensive.

On the methods such as stir bar or single drop, what is the accuracy of quantitation?

In SBSE and SPME quantitation is usually very accurate with results generally within a few percent of the true value for standards. Precision may vary from a few percent to +/- 10-20%, depending on extraction conditions and sample matrix. For best precision, extraction time, temperature and agitation rate should be carefully controlled. Generally, I use internal standard quantitation with both techniques.

Any comments on "Disposable Pipette EXtraction (DPX)"? Thanks.

Disposable pipette extraction is exactly as the name implies. Solid phase extraction sorbent is placed in a disposable pipette tip and extraction is formed by drawing sample, followed by wash solvent followed by elution solvent through it. The sorbent is retained in the disposable pipette tip and discarded following extraction. It should work in a similar manner to SPE, except that usually a smaller mass of sorbent is used.

You did not discuss the importance of MAE? How You rate MAE in analysis as compare to other sample prep techniques?

This was an omission on my part (sorry). Microwave Assisted Extraction is also useful for extracting analytes from solids or other difficult matrices. It is very efficient in terms of energy transfer into the sample matrix.

Explain SPME and pesticide extraction slide. You skipped over it. Usually, pest ext involves a lot of organic solvents - bad!

This was simply the extraction of a standard mixture form water. SPME is potentially good for pesticides because it reduces exposure to reactive surfaces such as glassware and filter materials. A literature reference is provided on the slide for all the details.

What do you think about QUECHERS ? What do these kits cost ?

QUECHERS stands for “quick, easy, cheap, effective, rugged and safe” analysis for pesticide residues, especially in combination with GCMS and LCMS. Im short, a homogenized sample is combined with appropriate solvent(s) and passed through a SPE cartridge for cleanup prior to GC and LC analysis. An online search will turn up numerous applications. QUECHERS method is reported to be less expensive than traditional SPE.

Do you have experience with SFE (supercritical fluid extraction)?

Yes. SFE generally involved the use of supercritical carbon dioxide as extraction solvent and is hardware intensive as it operates at elevated temperatures and pressures. For a very basic introduction see: N.H. Snow, M. Dunn and S. Patel, “Determination of Crude Fat in Food Products by Supercritical Fluid Extraction and Gravimetric Analysis,” Journal of Chemical Education, 1997, 74(9), 1108-1111

If so, how can you fractionate what is extracted?

In SFE, the polarity of the fluid can be adjusted by mixing it with an organic modifier. Fractionation can occur by collecting separate fractions as the elute from the extraction cartridge and as the solvent polarity is changed.

When is best to use accelerated solvent extraction?

ASE has its strength in extracting compounds that are readily soluble in traditional solvents from solids.

What is more convenient? Use more times an low volume or less times and more volume during the extraction?

A single large extraction will always be simpler. Multiple extractions using smaller volumes that add up to the larger volume will produce a larger recovery of a more pure extract. Multiple extractions are also favorable in cases where the partition coefficient favors the original (sample) phase. See the sample preparation chapter in Grob, Modern Practice of Gas Chromatography, 4th. Edition, Wiley, 2003, for more details.