Derivatisation post-column

Unlike pre-column derivatisation, the post-column derivatisation reaction need not reach completion, although the extent of reaction must be reproducible (Gfeller et al., 1977). It is this requirement, coupled with the desire to limit band spreading, which has led to reactor design being a crucial factor (Schwedt, 1979). Three reactor 

Examples of pre-column derivatisation agents (reproduced from Bristow (1976), with permission) 

Dansyl chloride Amino acids, phenols amines, peptides Fluorimetry (ex,340 em,510) 

Phenacyl bromide Fatty acids, phospholipids prostaglandins UV 

Fluorescamine Amino acids, peptides, primary amines Fluorimetry (ex,395 em,490) 

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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. 

Post-column on-line derivatisation is carried out in a special reactor situated between the column and detector. A feature of this technique is that the derivatisation reaction need not go to completion provided it can be made reproducible. The reaction, however, needs to be fairly rapid at moderate temperatures and there should be no detector response to any excess reagent present. Clearly an advantage of post-column derivatisation is that ideally the separation and detection processes can be optimised separately. A problem which may arise, however, is that the most suitable eluant for the chromatographic separation rarely provides an ideal reaction medium for derivatisation this is particularly true for electrochemical detectors which operate correctly only within a limited range of pH, ionic strength and aqueous solvent composition. 

After passing through the column, the separated solutes are sensed by an in-line detector. The output of the detector is an electrical signal, the variation of which is displayed on a potentiometric recorder, a computing integrator or a vdu screen. Most of the popular detectors in hplc are selective devices, which means that they may not respond to all of the solutes that are present in a mixture. At present there is no universal detector for hplc that can compare with the sensitivity and performance of the flame ionisation detector used in gas chromatography. Some solutes are not easy to detect in hplc, and have to be converted into a detectable form after they emerge from the column. This approach is called post-column derivatisation. 

The following pages give tables of substances that are electrochemically detectable after pre-column derivatisation (PRE-CD) or post-column derivatisation (POST-CD), (enzymatic-) reaction ((ENZ.-)POST-CR) or irradiation (HPLC-hv-EC) with corresponding data on reagents, enzymes, etc. The detection potentials are versus AgCl unless otherwise stated. 

Post-Column Derivatisation There are certain stubborn and fairly difficult components that are not easily detectable in HPLC. Therefore, such component(s) have to be appropriately converted into their corresponding detectable form once they emerge from the column. 

Simoes-Pires, C. A., Queiroz, E. F., Henriques, A. T., and Hostettmann, K., Isolation and on-hne identification of antioxidant compounds from three Baccharis species by HPLC-UV-MS/MS with post-column derivatisation. Phytochemical Analysis 16(5), 307-314, 2005. 

Some investigators have produced organic chelate complexes from the metals after they have been separated in the inorganic form on the column, but before entry into the detector, ie post-column derivatisation, while other investigators form the organic complexes before the sample is applied to the separation column, ie pre-column derivatisation. 

Apart from the above reasons derivatisation is also required in the analyses of many of the newer pesticides which have one or more functional groups which need protection in order to facilitate GC work. Also the increased use of high pressure liquid chromatography (HPLC) necessitates pre- or post-column derivatisation since the choice of HPLC detectors is limited compared with GC. 

Often it is required to detect compounds with no or only very weak chromophores such as sugars and amino acids. Refractive index detectors and mass sensitive detectors can be used but they are relatively insensitive in the context of biological sample concentrations. Indirect detection using a UV or fluorescent eluent can also be employed. However, the most common approach is the use of derivatisation. Derivatisation of some chemically reactive moiety on the analyte can be performed in two modes. In post-column derivatisation the sample is separated first and then reacted with a flowing stream of derivatising reagent being pumped into... 

This is by no means the only approach, however. The Moore and Stein protocol employs post-column derivatisation with ninhydrin to give a blue colour whose intensity can be measured spectrophotometrically, to provide quantitative detection of each of the separated components. 

Derivatisation the detectabdity of certain compounds can be improved by chemical derivatisation prior to the HPLC analysis, or after the separation but prior to the detection (post-column derivatisation) the latter can be easily automated and is shared commercially by several manufacturers. 

On the other hand, the more sophisticated variable wavelength UV-Vis detector wiU allow the detection of carbohydrates in the low wavelength (190-225 nm) region. Carbohydrates such as fructose, glucose, sucrose, etc. which are transparent at 254 or 280 nm, required either RI detection or post column derivatisation before the advent of the variable wavelength detector. 

A. Caballo-Lopez, M.D. Luque de Castro, Continuous ultrasound-assisted extraction coupled to on line filtration—solid-phase extraction—column liquid chromatography—post column derivatisation—fluorescence detection for the determination of N-methylcarbamates in soil and food, J. Chromatogr. A 998 (2003) 51. 

By reduction of the scope of the investigation to the non-polar (in general more toxic) organotins and accepting a relative high detection limit, a simple robust method can be used. If the concentration is high enough, the extraction can be followed by a relatively simple measurement. Compano etal. (1995) described an extraction with methanol for triphenyltin and tributyltin followed by HPLC-separation, post-column derivatisation and fluorimetric detection. 

Determination continuous ultrasound-assisted extraction coupled to on line filtration-SPE-column hquid chromatography—post column derivatisation—fluorescence detection LOD 12 ng/g and LOQ 40 ng/g carbamates at 1 /xg/g spiked level recoveries similar to those provided by the EPA 8318 method repeatability 3.1% reproducibility 7.5%... 

Pre- or post-column derivatisation involving chemical reaction of the analyte. 

An example of post-column derivatisation is the reaction of aminoacids with ninhydrin (Figure 6.31) again a chromophoric unit is present in the sample derivatives. 

Post-column derivatisation techniques, though imposing restrictions on the reagents and solvents that can be used has the advantages that it may be readily incorporated as part of a fully automated system and thus will... 

The internal standardisation technique actually increases the analytical error due to the measurement of two peak areas and should be reserved for samples undergoing pretreatment of pre- or post-column derivatisation to account for variable sample recovery or conversion. Quantitative analysis when applied to gradient elution systems affords reduced accuracy and precision due to the practical disadvantages of constancy of flow, reproducibility of gradient formation and solvent mixing-demixing. 

Orthopthalaldehyde (OPA). To achieve post-column derivatisation with OPA a reaction coil (4 mm x 0.3 mm I.D.) is usually inserted in-line after the point of mixing with the fluorogenic reagent. Since the dead volume of the coil contributes to band broadening effects, the solution is not as good as with the pre-column system. Slower flow rates are required to resolve certain residues which thereby increases the analysis time. However, the main advantages of OPA derivatisation (which is also used in conventional analysers) are the reproducibility and the fact that, unlike ninhydrin, a heating device is not required for the reaction. 

Several different types of detectors have been used for the quantitation of the biogenic amines however, currently the most popular techniques are electrochemical detection and fluorescent detection, usually coupled with pre- or post-column derivatisation techniques. The specific details of these detection techniques are discussed elsewhere (Chapter 3) and in the present section only the basic principles as applied to the biogenic amines will be reviewed together with a comparison between the two techniques in terms of their respective advantages and disadvantages. 

Normal phase chromatography using a Lichrosorb-NH2 stationary phase in combination with a post-column derivatisation procedure has been used for the resolution and detection of thiamine and its derivatives. Using a mobile phase of acetonitrile-90 mM potassium phosphate, pH 8.4 (60 40), good separations of thiochrome, thiochrome monophosphate, pyrophosphate and triphosphate were obtained (Ishii et al., 1979). The sensitivity of detection using mixtures of the thiochrome derivatives was approximately 1 pmol. 

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