Derivatization in chromatography

What is the purpose of derivatization in chromatography Give an example. 

The two major examples of derivatization in chromatography are the formation of volatiles for GC and the creation of UV detectivity or fluorescence in LC. Each will be discussed briefly. 

Most recently derivatization on SPE has been tried with good success. A review study by Zhou and co-workers (1992) outlines recent examples of silica based, solid-phase reagents (SPR) for derivatizations in chromatography. Solid-phase reagents can be made on many types of matrices, including silica, alumina, and organic polymers. Silica is most suitable because of the large surface area available for modification. 

Zhoul, F. X., Thome, J. M., and Krull, I. S. 1992. Silica based, solid phase reagents for derivatizations in chromatography. Trends Anal. Chem., 11 80-85. 

A book [45], special issues of chromatographic journals [453, 454] and reviews [459-470] have been devoted to the formation of derivatives of compounds. In chromatography there are several reasons why chemical derivatization has become useful. Firstly, derivatives are often formed to impart a measurable characteristic to a given compound. Secondly, derivatization is widely used to improve the chromatographic behaviour of compounds. A third use of derivatization in chromatography is for confirmation of identity — this is how Lawrence [455] assessed the importance of reaction pre-chromatographic methods. 

Johnson ME, Carpenter TS. The use of solid-phase supports for derivatization in chromatography and spectroscopy. Appl Spectrosc Rev 2005 40 391-412. 

Despite their importance, gas chromatography and liquid chromatography cannot be used to separate and analyze all types of samples. Gas chromatography, particularly when using capillary columns, provides for rapid separations with excellent resolution. Its application, however, is limited to volatile analytes or those analytes that can be made volatile by a suitable derivatization. Liquid chromatography can be used to separate a wider array of solutes however, the most commonly used detectors (UV, fluorescence, and electrochemical) do not respond as universally as the flame ionization detector commonly used in gas chromatography. 

Pfeffer, W. D. Yeung, E. S. Anal. Chem. 1986, 58, 2103. Lawrence, J. F. Frei, R. W. Chemical Derivatization in Liquid Chromatography, Elsevier Scientific New York, 1976. 

The most general method for the simultaneous analysis of oxyanions by gas chromatography is the formation of trimethylsilyl derivatives. Trimethylsilyl derivatives of silicate, carbonate, oxalate, borate, phosphite, phosphate, orthophosphate, arsenite, arsenate, sulfate and vanadate, usually as their ammonium salts, are readily prepared by reaction with BSTFA-TMCS (99 1). Fluoride can be derivatized in aqueous solution with triethylchlorosilane and the triethylfluorosilane formed extracted into an immiscible organic solvent for analysis by gas chromatography [685). 

N. A. Parris, Instrumental Liquid Chromatography, a Practical Manual on High-Performance Liquid Chromatographic Methods (Journal of Chromatography Library, Vol. 27), Elsevier, Amsterdam, 2nd revised ed., 1984 J. Drozd, Chemical Derivatization in Gas Chromatography (Journal of Chromatography Library, Vol. 19), Elsevier, Amsterdam, 1981 J. F. Lawrence and... 

R. W. Frei, Chemical Derivatization in Liquid Chromatography (Journal of Chromatography Library, Vol. 7), 1976, 1st reprint 1983, Elsevier, Amsterdam, 1983. 

Hawk, G.L. and Little, J.N., Derivatization in Gas and Liquid Chromatography in Zymark Sample Preparation Program (SPF CT 1 103), Zymark Corporation, Hopkinton, MA, 1982. 

Drozd, J. (1981). Chemical Derivatization in Gas Chromatography. Amsterdam, Elsevier. 

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. 

Derivatives of amino acids (Table 9.10) are required because amino acids are not themselves sufficiently volatile for gas-liquid chromatography and difficulties may be encountered in the choice and method of derivatization. In the past no single column was normally capable of resolving the derivatives of such a diverse group of compounds but the introduction of fused silica capillary columns has resulted in considerably improved resolution. 

Another difficulty in the gas chromatographic separation of amino acids is the choice of detector and it may be necessary to split the gas stream and use two different detectors. The flame ionization detector, which is commonly used, is non-specific and will detect any non-amino acid components of the sample unless purification has been performed prior to derivatization. In addition the relative molar response of the flame ionization detector varies for each amino acid, necessitating the production of separate standard curves. As a consequence, although gas chromatography offers theoretical advantages, its practical application is mainly reserved for special circumstances when a nitrogen detector may be useful to increase the specificity. 

Unlike the previous techniques, sensitivity is not an issue for AAA. There are few interfering substances because the method involves hydrolysis, derivatization, and chromatography with detection at a unique wavelength. Most excipients will not affect the hydrolysis step, but one has to be careful to ensure that the amino acids used to quantitate the protein are not destroyed. In addition, it must be determined if the excipients interfere with the derivatization chemistry or the chromatography. A BSA standard in the same buffer formulation is routinely run in parallel to the target protein to ensure the accuracy of the method. 

Abe I, Fujimoto N, Nishiyama T, Terada K, Nakahara T. 1996. Rapid analysis of amino acid enantiomers by chiral-phase capillary gas chromatography. J Chromatogr A 722 221. Ahuja S. 1976. Derivatization in gas chromatography. J Pharm Sci 65 163. 

Wong JTF, Baker GB, Coutts RT. 1988. Rapid and simple procedure for the determination of urinary phenylacetic acid using derivatization in aqueous medium followed by electron-capture gas chromatography. J Chromatogr B Biomed Appl 428 140. 

Mitamura K, Shimada K. 2001. Derivatization in liquid chromatography/mass spectrometric analysis of neurosteroids. Se Pu 19 508. 

The reaction of amines and amino acids with orthophthaldehyde has been widely used in postcolumn and precolumn derivatization in analyses of foods (99-104) and in analyses of peptides from biological samples. Figure 2 (87) presents a chromatogram for OPA derivatives of tryptic peptides from two proteins. The sensitivity of the method was on the order of picomoles. The authors have themselves performed postcolumn OPA derivatization of low-molecular-weight peptides from blue cheeses separated by reversed-phase chromatography (86). 

In order to increase the sensitivity and selectivity of detection, a number of UV-absorbing or fluorescent derivatives have been prepared. Several reasons can be given for the use of deriva-tization in chromatography to enhance solute volatility (GC), to enhance separation, and to enhance detectability. The selection of a derivative can often become much more difficult than the actual derivatization itself. In all areas of chromatography these effects have been succesfully applied to difficult or otherwise impossible separation. 

Some of the essential advantages of derivatization in liquid chromatography are summarized below ... 

The concept of derivatization in liquid chromatography is relatively new. The introductory chapter is therefore intended to familiarize the novice in this field with the basic technique of using chemical reactions and labeling procedures to enhance the sensitivity, specificity and separation properties of liquid chromatography. 

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