Selection of the LC or HPLC Separation Mode based on the nature of the sample | Chemistry Net

Selection of the LC or HPLC Separation Mode based on the nature of the sample

In previous posts four different LC modes have been discussed with regard to separation mechanism (LSC, LLC, IEC, SEC). Several of the factors which might influence a decision for choice of one mode over another have been presented.

This post presents a general approach for mode selection considering the nature of the sample.

There is a direct relation between several separation techniques and LC / HPLC corresponding modes (Table I.1). These separation techniques can help the analyst to establish an initial set of chromatographic conditions.

Separation Technique
Corresponding LC Mode
Thin layer chromatography
Liquid-liquid extraction
Paper chromatography
Column chromatography

Table I.1: Separation techniques in chemistry related to liquid chromatography (LC). Where, LSC (liquid-solid chromatography), LLC (liquid-liquid chromatography), IEC (ion-exchange chromatography), SEC (steric exclusion chromatography).

Assuming that the analyst has little or even no prior knowledge of the nature of his sample, a general approach for the correct selection of the LC mode can be used. There are several criteria which can be used to decide whether a particular LC mode is more appropriate than another such as:

 What is the molecular weight of the sample?

Compounds of very low molecular weight which are volatile are best separated by gas chromatography.

Compounds in the molecular weight range of 200-2000 are best separated by the normal LC modes such as: LSC, LLC and ion exchange chromatography  (IEC).

Compounds with molecular weight above 2000 are separated by steric exclusion chromatography (SEC).

What is the solubility of the sample in a few common solvents?

Knowledge of the solubility of the sample in a few common solvents can be useful for the selection of the LC mode. A useful statement to remember is that “like dissolves like”.

The following solvents – in the order given below - have been found to give enough  information for the solubility of an unknown sample:

Water, benzene or isooctane, methylene chloride and isopropanol

In general:

Water soluble compounds are best separated by ion-exchange chromatography (IEC) or LLC.

Compounds that are sparingly soluble in water but very soluble in water containing a drop of acid (HCl) or a drop of base (NaOH) are ionizable compounds and they are best separated by ion exchange chromatography.

Compounds that are nonpolar and hydrocarbon-soluble can usually be separated by LSC. Aromatic compounds are more soluble in benzene while aliphatic compounds in isooctane.

Compounds soluble in methylene chloride can be separated by normal LLC and LSC.

Compounds which are water-insoluble but soluble in isopropanol are best separated by reverse phase LLC with water-isopropanol mixtures as mobile phases.

Steric exclusion chromatography can be applied to compounds soluble to any of the above solvents.

What is the structure of the sample?

The presence or absence of certain functional groups can favor one technique over another. A simple infrared spectrum can give information of the functionalities which might be present.

Ionic and ionizable groups (such as organic acids or bases) suggest the use of ion-exchange chromatography.

Aliphatic or aromatic compounds suggest the use of LSC or LLC. LSC works best for the separation of isomeric compounds while LLC for the separation of homologs.

Compounds with functional amino or carboxylic acid groups suggest use of LSC.

Polymers, especially those of high molecular weight are best separated by steric exclusion chromatography


  1. C.A. Dorschel, J.L. Ekmanis, J.E. Oberholtzer  et al. “LC Detectors” Anal. Chem., 61, 951A–968A, 1989 
  2.  Nina Hadden et al., “Basic Liquid Chromatography”, Varian Aerograph, 1971
  3. C. F. Simpson, “Techniques in Liquid Chromatography” Wiley-Hayden: Chichester, England, 1982 
  4. L.R. Snyder, J. L Glajch,  J. J. Kirkland, “Practical HPLC Method Development”, Wiley-Interscience: New York, 1988.

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