Abstract The introduction of this series on method development describes in brief the background on analytical methods and method development. The importance of analytical methodology broader than chromatography is discussed, explaining the basics of method development in analytical chemistry. It identifies how chromatographic separation can be achieved by changing ones parameters. A large number of degrees of freedom enable us to develop chromatographic methods to resolve a wide range of analytical questions.

This chapter has been written by Peter Schoenmakers, Stella Brudin and Peter Pruim.

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Developing (implementing, improving, validating, etc.) adequate chromatographic methods is needed for a chromatographic analysis to take place. The need of methods in analytical chemistry is not reserved strictly to chromatography. For example, there are numerous titration methods, methods for elemental analysis, etc.

The heart of a chromatographic system is formed by the column.

  • In the column the molecules will be retained shorter or longer based on the difference in affinity for the stationary and / or the mobile phase.
  • The separation is also determined by the width of the chromatographic peaks, this is based on physical transport phenomena which are difficult to describe with only theories.

Significant changes in the chromatographic separation can be achieved by changing the:

  • Chemical composition of a column (stationary phase)
  • Chemical composition of the mobile phase (LC),
  • Temperature (GC),
  • Pressure (SFC)
  • Programmed conditions (e.g. temperature program in GC or gradients in LC)

A large number of parameters must be carefully defined. Adequate robustness of chromatographic methods is often desired.  For example, subtle variations between different LC columns (which are nominally identical) may lead to retention-time changes with dramatic consequences. A large number of degrees of freedom enable us to develop chromatographic methods to resolve a wide range of analytical questions. Method development in chromatography is difficult and sometimes slow, which is why (expert) knowledge is required. Taking the results that can be achieved using chromatography into account it is also a rewarding activity.

What is a method?

All activities, settings and conditions for a chromatographic analysis together make up a method. A complete method must allow the experiment to be repeated. The method ensures that measurements can be performed in practice.

When a chromatographic method is described a distinction can be made between:

  • Static (using established data). Static parameters include all instrument settings, as well as hardware parameters, such as the choice of detector and the size of the sample loop. Also, a temperature program or a gradient program can be described by a set of static parameters.
  • Dynamic (acts) (see Table). Dynamic parameters (acts) are defined in the method, but each time the method is applied they need to be conducted again. Typical examples are weighing samples, adding reagents, and preparing buffer solutions.

Chromatographic method
Set parameters
Equipment Sample treatment
(weighing out, dilution)
Scope (analyze components; matrices)
Column Calibration Limit of detection, Limit of quantification
Injection volume Interactive data handling Linear workspace
Flow of pressure Precision
(repeatable unit, reproducibility, robustness)
Temperature Selectivity / specificity
(Matrix effects, interference)
Detector settings
Data acquisition parameters

The number of operations to be performed is related to the degree of automation. Injection often takes place automatically. The only thing left to the analyst is the filling the sample vial. Sometimes integration is automated, but often an interactive process between the analyst and the computer is preferred. In highly automated ('robotized') systems dilution and derivatization of samples can be performed.

Robustness of a method

The static parameters should be clearly displayed in a table and easy to reproduce. The exact values of the parameters, however, are not always accurate or exact. By implementing a method in different places there will be small differences in the actual values of the set parameters.  The results obtained with a method can be affected to a greater or lesser extent by the exact values of the set parameters. Similarly, the dynamic parameters (acts) for an analysis will always affect the results to some extent give. We speak of a robust method if small deviations from the specified parameters do not lead to substantially different results (see Table). 

A method also has a number of properties in the form of restrictions (scope, detection, linearity, specificity, stability) and results (accuracy, precision). The scope of a method is almost always limited and not in all cases clearly marked, neither in terms of analyzed compounds, nor in terms of the matrix.

Comparing a standardized and a robust method
Parameter Standardized method Robust Method
Flow rate (ml / min)  1 0.9 - 1.1
Mobile phase
(Vol% iso-propanol
in cyclohexane)
3 2 - 4
Injection volume (µ l) 20 15 - 25
Column temperature (°C) 30  25 - 35
Sample concentration
1 0.5 - 1.5
Excitation wavelength (nm) 295 285 - 305
Emission wavelength (nm) 335 325 - 345
Limit of detection (mg / kg) 1 10
(Relative Standard deviation,%)
5 10
Demands to the user  High Low
Effort for implementation Great Small
Reliability  Low High
Comparison between a standardized and a robust method for the determination of a preservative at low (ppm) level, using normal-phase LC with fluorescent detection (thanks to Jeroen Oomen, SRTCA, Amsterdam) 

Determining the C3 and C4 alcohols in Scotch whiskey can serve as a model. Quite often, the intended application of the method can be different to a greater or lesser degree. For example if the matrix is changed (C3 and C4 alcohols in imitation Scotch whiskey) or differs somewhat from that for which the method was originally developed for (C3 and C4 alcohols in Irish whiskey or in Schiedam genever), it is tempting to quickly change or extend the scope of a method (Higher alcohols in whiskey). 

It is necessary to recognize how well a method is developed and defined. As a tool, methods are classified into three groups in the next section. These classes are characteristic for different levels of development.

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