Post-chromatographic Derivatization

Department of Chemical Pathology, Bernhard Baron Memorial Research Laboratories, Queen Charlotte s and Chelsea Hospital, London 

Edited by K. Blau and J. M. Halket 1993 John Wiley Sons Ltd 

The choice of detection reagents depends more than anything on the chemical properties of the selected compounds of interest, and chemical modifications can be made not only by the use of reagents but also by 

To detect the separated components To provide specificity To discriminate in favour of selected compounds of interest To amplify the sensitivity of detection To achieve quantitation 

These principles have been applied very widely, as will be described, and examples will be given of how specific problems might be solved. This overview is meant to be illustrative rather exhaustive it may suggest possible solutions to problems that readers may have and point to potential fresh approaches. 

---

Ritter, W. Post-Chromatographic Derivatizations in Quantitative (HP)TLC , Proc. 2nd Int Synq>. lustrum. High ifomu Thm-Lay Chmmalogr. (HPTLC) p. 100-113, Interlaken, 1982. 

Tertiary aliphatic amines are rather difficult to detect, but they can be detected at 254 run after quatemization with p-NBBr [66]. Pilocarpine and isopilocarpine were derivatized in a sealed ampoule for 24 h at 40 °C, then separated by ion-pair HPLC. Tertiary amines can also be determined by post-chromatographic derivatization. Kudoh et al. described a method [67] involving derivatization with 1% dtric acid in acetic anhydride at 120 C, separation on a chemically bonded silica gel Nucleosil 5N(CH3)2, column, and detection at 550 nm. 

A post-chromatographic derivatization method has been developed for penicillins, and was applied to the analysis of amoxicillin [119]. The compound was extracted from plasma and urine and separated by RP-HPLC with phos-plate buffer (pH 8.0) methanol (92 8, v/v) as the mobile phase. The derivatization was accomplished with an aqueous solution of imidazole (33%) and mercuric chloride (0.11%), pH 7.2, containing Brij 35 (0.12%). The derivative was detected at 310 nm. 

In liquid chromatography, including HPLC, Schlabach and Weinberger [15] classify analytes as co-operative if they lend themselves to methods of detection such as UV or visible absorption, fluorescence, changes in refractive index, electrochemical detection or other instrumental procedures. Analytes are classed as nonco-operative if such detection methods are not applicable or lack the necessary sensitivity, and in general it is such analytes that are the prime candidates for post-chromatographic derivatization. The scope of post-chromatographic procedures is summarized in Table 2. 

Table 2 Post-chromatographic derivatization in liquid chromatography...

Liquid chromatographic systems are particularly suited to post-chromatographic derivatization, because the vast majority of chemical reactions are done in solution or in the liquid phase. Another reason in that liquid chromatographic separations can very often be achieved without any need for derivatization before the chromatographic step, whereas derivatization may be needed for detection or quantitation (or both) afterwards. 

Table 3 Requirements for post-chromatographic derivatization in liquid chromatographic systems...

Figure 2. Simple basic post-chromatographic derivatization manifold. The detector may be a photometer, fluorimeter, electrochemical detector or any other appropriate type of instrument.

A significant advance in post-chromatographic derivatization to fluorescent products came with the introduction of fluorescamine by Udenfriend s group [150], and this was applied to a post-column system by Stein ei al [151], leading to very much greater sensitivity of... 

This brief account, taken together with applications mentioned in earlier sections of the chapter, is by no means exhaustive, but reflects the extent and scope of post-chromatographic derivatizations. However, in spite of its kaleidoscopic character some principles do stand... 

As will be abundantly clear from the rest of this handbook, derivatization for chromatography is still most usually done before the chromatographic separation step, because the choice of derivative includes as one of its aims conversion to compounds with improved chromatographic properties. However, this chapter describes many situations where post-chromatographic derivatization has been found necessary or desirable, not only as an alternative to pre-chromatographic derivatization, but also as a complementary additional procedure. 

Cimpean G (2001) Pre- and post-chromatographic derivat-ization. In Nyiredy Sz (ed.) Planar Chromatography, pp. 410-445. Budapest Springer. 

The preparation of derivatives for TLC has been reviewed by Gasparic (1961) and Edwards (1980). Derivatization has been used in the TLC of amino acids where certain amino acids are easily converted into the so-called DANS forms (Jimethyl amino naphthalene suUbnyl) (Pataki and Niederweiser, 1967). These authors have studied a variety of amino acid derivatives on silica gel. The use of both pre- and post-chromatographic derivatization in TLC applications in food analysis has been discussed (Shalaby, 1996). For a recent update on the use of derivatization in TLC, see Cserhati and Forgacs (1996). 

Factors relative to the quality of a chromatogram, such as preparation of the sorbent layer, development procedure (isocratic or gradient elution), and post-chromatographic derivatization. 

---