Liquid chromatography, pre-column

Derivatization may be performed before the sample is injected into the liquid chromatograph (pre-column), as in the derivatization reactions used with gas chromatography. In liquid chromatography the derivatization may, as an alternative, be performed post-column, i.e., the reagent is added to the effluent from the column. The advantages and disadvantages of these two derivatization strategies are discussed in section 4.3.3. 

In liquid chromatography, in contrast to gas chromatography [see Section 9.2(2)], derivatives are almost invariably prepared to enhance the response of a particular detector to the substance of analytical interest. For example, with compounds lacking an ultraviolet chromophore in the 254 nm region but having a reactive functional group, derivatisation provides a means of introducing into the molecule a chromophore suitable for its detection. Derivative preparation can be carried out either prior to the separation (pre-column derivatisation) or afterwards (post-column derivatisation). The most commonly used techniques are pre-column off-line and post-column on-line derivatisation. 

Figure 2, Block diagram of a liquid chromatograph. A, solvent reservoir B, filter C, pump D, pulse dampener (optional) E, pre-column (used only in liquid-liquid chromatography) F, pressure gauge G, infector H, column I, detector J, fraction collector K, recorder or oth readout device.

High-performance liquid chromatography (HPLC) with a micellar mobile phase or with a selective pre-column or reaction detection system has also been used to determine alkylenebis(dithiocarbamaes). ° Zineb and mancozeb residues in feed were determined by ion-pair HPLC with ultraviolet (UV) detection at 272 nm. These compounds were converted to water-soluble sodium salts with ethylenediaminetetra-acetic acid (EDTA) and sodium hydroxide. The extracts were ion-pair methylated with tetrabuthylammonium hydrogensulfate (ion-pair reagent) in a chloroform-hexane solvent mixture at pH 6.5-8.S. The use of an electrochemical detector has also been reported. ... 

Katayama, M., Masuda, Y., and Taniguchi, H., Determination of alcohols by high-performance liquid chromatography after pre-column derivatization with 2-(4-carboxyphenyl)-5,6-dimethylbenzimidazole,/. Chromatogr., 585,219, 1991. 

The most popular current techniques for amino acid analysis rely on liquid chromatography and there are two basic analytical methods. The first is based on ion-exchange chromatography with post-column derivatization. The second uses pre-column derivatization followed by reversed-phase HPLC. Derivatization is necessary because amino acids, with very few exceptions, do not absorb in the UV-visible region, nor do they possess natural fluorescence. 

The process of chemically modifying the test molecules before the separation procedure is known as preparing derivatives or pre-column derivatization. In liquid chromatography this is done to permit the test molecules to be more easily detected after separation and to increase the sensitivity of the detection system and less frequently to alter the separation process. 

The molecular absorption intensity of polar compounds is usually small, but highly sensitive detection can be obtained after pre- or post-column derivatiza-tions. The use of ultraviolet absorption or fluorescence-active counter-ions makes it possible to achieve highly sensitive detection of polar compounds and enhance the capability of ion-pair liquid chromatography. For example, N,N-dimethylprotriptyline has been used as a counter-ion for carboxylic acids12 and picric acid for quaternary amines13 in normal-phase ion-pair partition liquid chromatography. Phenethylammonium, cetylpyridinium, l-phenethyl-2-pyco-linium, and naphthalene-2-sulfonic acid have been used for sulfonic acid and alkyl amines detection.14,15 Ion-pair post-column extraction was applied on-line for fluorescence detection.16... 

Song Y, Quan Z, Evans JL, Byrd EA, Liu YM. 2004. Enhancing capillary liquid chromatography/tandem mass spectrometry of biogenic amines by pre-column derivatization with 7-fLuoro-4-nitrobenzoxadiazole. Rapid Commun Mass Spectrom 18 989. 

King LE, Vieth R. Extraction and measurement of pamidronate from bone samples using automated pre-column derivatization, high-performance liquid chromatography and fluorescence detection. J Chromatogr B 1996 678 325-330. 

Ishii, D., Hibi, K., Asai, K., Nagaya, M., Mochizuki, K., and Mochida, Y., Studies of micro high-performance liquid-chromatography. 4. Application of micro pre-column method to analysis of corticosteroids in serum. Journal of Chromatography 156(1), 173-180,1978. 

Determination of serum cortisone by reversed-phase liquid chromatography using pre-column sulfuric acid-ethanol fluorescence derivatization and column switching. (159)... 

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