Derivatization in HPLC

The second benefit in the earlier example illustrates the primary application of derivatization in HPLC. Inspection of Table 13.2 suggests that if we want to derivatize for improved selectivity and sensitivity, introduction of fluorescence should be our first choice. Table 13.3 lists a number of fluorescence derivatization agents and their target functionalities. Generally, we wish to derivatize only one group at a time. 

9-Fluorenylmethyl chloroformate (FMOC) Primary and secondary amines 260 305 

Source Katz, E. etal., eds., Handbook of HPLC, Marcel Dekker, Inc., New York, 1998. 

Derivatization can be employed to advantage with BCD detection as weU. Both precolumn or postcolumn reactions may be anployed to produce electroactive compounds analogons to the formation of fluorescent derivatives. The UV-induced reaction chamber for ECDs described in Section 15.4 and the dual electrode detector oxidation-reduction series described at the end of Section 13.1.4.5 may be regarded as nonreagent instrumental forms of the principle of postcolumn derivatization. 

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Reaction detectors are a convenient means of performing online postcolumn derivatization in HPLC. The derivative reaction is performed after the separation of the sample by the column and prior to detection in a continuous reactor. The mobile phase flow is not interrupted during the analysis and reaction, although it may be augmented by the addition of a secondary solvent to aid the reaction or to conform to the requirements of the detector. Reaction detectors are finding increasing application for the analysis of trace components in complex matrices where both high detection sensitivity and selectivity are needed. Many suitable reaction techniques have been published for this purpose [641-650]. 

The main purpose of derivatization in HPLC is to improve detection specifically when determining traces of solutes in complex matrices, for example ... 

Uihlein, M. Schwab, E. A novel reactor for photochemical post-column derivatization in HPLC. Chromatographia 1982, 15, 140. 

Post-Column, On-Line, Continuous Photolytlc Derivatizations in HPLC-Electrochemical Detection (EC) and FIA-EC ... 

In 1994, only 15% of EPA method validations (tolerance method validation and environmental chemistry method validations) that involved GC were carried out using GC/MS. In 2002, this number is reversed in that 85% of the GC methods that were validated by both programs used GC/MS. Many of the compounds investigated in these method trials were polar compounds, and hence these compounds required derivatization in order to be amenable to GC. One common methylating agent is (trimethylsilyl)diazomethane, which is used, for example, to methylate the sulfonamide flumetsulam. As opposed to HPLC/MS, where derivatization is often not necessary, the GC/MS procedure involves an extra step to methylate this compound, under dry conditions, prior to determination by GC/MS. 

Advances have been achieved in recent years, such as the use of CL reagents as labels to derivatize and sensitively determine analytes containing amine, carboxyl, hydroxy, thiol, and other functional groups and their application in HPLC and CE [35, 36], the synthesis and application of new acridinium esters [37], the development of enhanced CL detection of horseradish peroxidase (HRP) labels [38], the use of immobilization techniques for developing CL-based sensors [39-42], some developments of luminol-based CL in relation to its application to time-resolved or solid-surface analysis [43], and the analytical application of electrogenerated CL (ECL) [44-47], among others. 

A study of fifty-five aliphatic, aromatic and heteroclyclic amines showed that twenty-eight of them could be detected in a FIA system at concentrations in the range of 1.0 x 10-10 to 4.0 x ] 0 6 M (SNR 3, 20 XL injection), without derivatization, by HPLC-CLD, taking profit of the chemiluminescence produced in the presence of aryl oxalate and sulforhodamine 101 (41). The method was applied to the determination of histamine (6) in fish146. See reaction 24 in Section IV.G. 

Fluorescence is one of the most sensitive detection methods in HPLC analyses. However, as ginsenosides do not contain a suitable fluorescence chromophore they have to be derivatized before detection. Shangguan... 

Molins-Legua C, Campinc-Falco P, Sevillano-Cabeza A, Ped-r6n-Pons M. 1999. Urine polyamines determination using dansyl chloride derivatization in solid-phase extraction cartridges and HPLC. Analyst 124 477-482. 

Chiral separation of drng molecules and of their precursors, in the case of synthesis of enantiomerically pure drugs, is one of the important application areas of HPLC in pharmaceutical analysis. Besides HPLC, capillary electrophoresis (CE) is another technique of choice for chiral separations. Chapter 18 provides an overview of the different modes (e.g., direct and indirect ones) of obtaining a chiral separation in HPLC and CE. The direct approaches, i.e., those where the compound of interest is not derivatized prior to separation, are discussed in more detail since they are cnrrently the most frequently used techniques. These approaches require the use of the so-called chiral selectors to enable enantioselective recognition and enantiomeric separation. Many different molecnles have been nsed as chiral selectors, both in HPLC and CE. They can be classified into three different groups, based on their... 

The secondary structure, the mesopores, is similar to the internal structure of standard HPLC particles. This secondary structure provides the surface for retention. The standard pore size is in the order of 13 nm, resulting in a specific surface area of about 300 mVg. Due to the lower ratio of retentive structure to interstitial space, the retentivity of monoliths and the preparative loadability tends to be significantly lower than the retentivity and loadability of packed beds of 10-nm particles. Since the monolithic columns described here are made from silica, they can be derivatized in the same way and with the same technology as silica-based particles. Also, the useful pH range is the same as for silica-based particles. 

Determination of Carbonyl Compounds in Drinking Water by DNPH Derivatization and HPLC... 

P-Blockers (BB) are widely used, in particular in the treatment of angina, hypertension, arrhythmia, and cardiac failure. Their determination in plasma may offer useful information in cases of intoxication. In addition, BB may be abused by athletes, particularly in disciplines where psychomotor coordination is important. Adequate GC repartition of these analytes require derivatization, therefore HPLC appears as a valid alternative for the efficient analysis of BB. 

To evaluate the enantiomeric purity of (S,S)/(R,R)-statine, racemic mixtures and en-antiomerically enriched samples are analyzed by HPLC after derivatization with 2,3,4,6-tetra-0-acetyl- 3-D-glucopyranosyl isothiocyanate (GITC, 51 Scheme 21 and Table 7).[49 The GITC method also allows the determination of the enantiomeric composition of ethyl esters of statine analogues.[44 491 Another assay to determine the optical purity of statine has been developed, and uses l-G1u-NCA to produce dipeptides with different retention times in HPLC.[18]... 

Supercritical fluid chromatography (sfc) combines the advantages of gc and hplc in that it allows the use of gc-type detectors when supercritical fluids are used instead of the solvents normally used in hplc. Carbon dioxide, n-petane, and ammonia are common supercritical fluids, For example, carbon dioxide employed at 7.38 MPa (72 9 atm) and 31.3°C has a density of 448 g/mL. Derivatization of primary and secondary amines using 9-fluorenylmethy] chlorofomiate to form a nonpolar, uv-absorbing derivative lias been reported. 

The derivatization of fatty acids, and of acidic substances in general, has been a problem in analytical-organic chemistry for many years. The classically used derivatives have been amides and esters. The main problem in using methyl esters of fatty acid in HPLC is that one cannot then use the UV detector, because of inadequate absorption. Thus a search for an alternate method of forming UV-sensitive derivatives has been initiated in several laboratories. 

Fig. 2 Configuration used for postcolumn derivatization detection in HPLC. E = eluent P = HPLC pump IV = injection valve CC = chromatographic column R = pump reagent RC = reaction coil D = detector.

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