Pre-columns

GC using chiral columns coated with derivatized cyclodextrin is the analytical technique most frequently employed for the determination of the enantiomeric ratio of volatile compounds. Food products, as well as flavours and fragrances, are usually very complex matrices, so direct GC analysis of the enantiomeric ratio of certain components is usually difficult. Often, the components of interest are present in trace amounts and problems of peak overlap may occur. The literature reports many examples of the use of multidimensional gas chromatography with a combination of a non-chiral pre-column and a chiral analytical column for this type of analysis. 

Figure 10.1 Analysis of racemic 2,5-dimethyl-4-hydroxy-3[2H]-furanone (1) obtained from a strawbeny tea, flavoured with the synthetic racemate of 1 (natural component), using an MDGC procedure (a) dichloromethane extract of the flavoured strawbeny tea, analysed on a Carbowax 20M pre-column (60 m, 0.32 mm i.d., 0.25 p.m film thickness earner gas H2, 1.95 bar 170 °C isothermal) (b) chirospecific analysis of (1) from the sti awbeny tea exti act, ti ansfened foi stereoanalysis by using a pemiethylated /3-cyclodextrin column (47 m X 0.23 mm i.d. canier gas H2, 1.70 bar 110 °C isothemial). Reprinted from Journal of High Resolution Chromatography, 13, A. Mosandl et al., Stereoisomeric flavor compounds. XLIV enantioselective analysis of some important flavor molecules , pp. 660-662, 1990, with permission from Wiley-VCH.

Figure 10.2 MDGC-MS differentiation between the enantiomers of theaspiranes in an aglycone fraction from puiple passion fruit DB5 pre-column (25 m X 0.25 mm i.d., 0.25 p.m film thickness canier gas He, 0.66 ml/min oven temperature, 60-300 °C at 10 °C/min with a final hold of 25 min) permethylated /3-cyclodextrin column (25 m X 0.25 mm i.d., 0.25 p.m film thickness canier gas He, 1.96 ml/min 80 °C isothermal for 20 min and then programmed to 220 °C at 2 °C/min). Reprinted from Journal of High Resolution Chromatography, 16, G. Full et al., MDGC- MS a powerful tool for enantioselective flavor analysis , pp. 642-644, 1993, with permission from Wiley-VCH.

EC, electrochemical detection Flu, fluorescence detection MS, mass specu-omeu-ic detection pre-Flu, fluorescence detection after pre-column derivatization post-Flu, fluorescence detection after post-column derivatization UV, UV absorbance detection. 

In principle, on-line SPE-LC can be automated quite easily as well, for instance, by using Such programmable on-line SPE instrumentation as the Prospekt (Spark Holland) or the OSP-2 (Merck) which have the capability to switch to a fresh disposable pre-column for every sample. Several relevant applications in the biomedical field have been described in which these devices have been used. Eor example, a fully automated system comprising an autosampler, a Prospekt and an LC with a UV... 

LC-MS with on-line SPE using a RAM pre-column with an internal ODS phase was described by van der Hoeven et al. (95) for the analysis of cortisol and prednisolone in plasma, and arachidonic acid in urine. The samples were injected directly and the only off-line pretreatment required was centrifugation. By using the on-line SPE-LC-MS system, cortisol and related compounds could be totally recovered and quantified in 100 p.1 plasma within 5 min with a typical detection of 2 ng/ml (Figure 11.6(b)). The RAM-type of sorbents, in which the outer surface of the particles is covered with aj-acid glycoprotein, also appear to be useful for direct SPE of... 

B. Johansson, Simplified quantitative determination of plasma phenytoin on-line pre-column high-perfor mance liquid immunoaffinity cliromatogr aphy with sample pre-purification , J. Chromatogr. 381 107-113 (1986). 

A. Farjam, G. J. de Jong, R. W. Frei, U. A. Th Brinkman, W., Haasnoot, A. R. M. Hamers, R. Schilt and F. A. Huf, Immunoaffinity pre-column for selective on-line sample pre-tr eatment in liigh-perfor mance liquid cliromatogr aphy determination of 19-nortestosterone , J. Chromatogr. 452 419-433 (1988). 

Figure 12.13 Illustration of isothermal dual capillary column clnomatography used for separation of UV photolysis products of methyl isopropyl ether, (a) Heait-cut and hack-flushing at preseparation clnomatogram 1, PPG pre-column (20 m X 0.25 mm i.d.) 55 °C, 0.2 har N2 3p.L. Clnomatogram 2, Marlophen main column (100 m X 0.25 mm i.d.) 1.5 har N2 sample, heait-cut from chromatogram 1. (h) Obtained under the same conditions as (a), hut without capping of the heait-cut. Reprinted with permission from Ref. (19).

The packed column section contains a stripper pre-column (column 1), which separates the Cg+ fraction by back-flushing all compounds above -pentane in one peak. HjS, CO2, C2, O2, N2 and Cj are trapped in columns 3 and 4, while C3-C5 hydrocarbons elute from column 2 to the TCD. The remaining components are... 

Multidimensional LC has also been used to determine ursodeoxycholic acid and its conjugates in serum (14). These compounds are used in the treatment of cholesterol gallstones, hepatitis and bilary cirrhosis. These authors employed a traditional (10 X 4 mm) pre-column and a micro-bore (35 X 2 mm) analytical column that were interfaced by using a six-port switching valve. 

Recently, multidimensional GC has been employed in enantioselective analysis by placing a chiral stationary phase such as a cyclodextrin in the second column. Typically, switching valves are used to heart-cut the appropriate portion of the separation from a non-chiral column into a chiral column. Heil et al. used a dual column system consisting of a non-chiral pre-column (30 m X 0.25 mm X 0.38 p.m, PS-268) and a chiral (30 m X 0.32 mm X 0.64 p.m, heptakis(2,3-di-(9-methyl-6-(9-tert-butyldimethylsilyl)-(3-cyclodextrin) (TBDM-CD) analytical column to separate derivatized urinary organic acids that are indicative of metabolic diseases such as short bowel syndrome, phenylketonuria, tyrosinaemia, and others. They used a FID following the pre-column and an ion trap mass-selective detector following the... 

Multiple cryogenic traps between the pre-column and the analytical column were developed in 1993 by Ragunathan et al. (23). These authors found that a multiple trap configuration provided the necessary resolution and automated sampling for simultaneous GC/IR and GC/MS determination of very complex mixtures. A... 

Figure 15.8 Multidimensional GC-MS separation of urinary acids after derivatization with methyl chloroformate (a) pre-column cliromatogram after splitless injection (h) Main-column selected ion monitoring cliromatogram (mass 84) of pyroglutamic acid methyl ester. Adapted from Journal of Chromatography, B 714, M. Heil et ai, Enantioselective multidimensional gas chromatography-mass spectrometry in the analysis of urinary organic acids , pp. 119-126, copyright 1998, with permission from Elsevier Science.

Derivatization techniques are divided into pre-column and post-column techniques. Post-column derivatization is especially useful to enhance the detection of compounds, whilst pre-column derivatization is the method of choice for enan-tioseparations via derivatization. 

Pre-column derivatization offers some general advantages ... 

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. 

Pre-column off-line derivatisation requires no modification to the instrument and, compared with the post-column techniques, imposes fewer limitations on the reaction conditions. Disadvantages are that the presence of excess reagent and by-products may interfere with the separation, whilst the group introduced into the molecules may change the chromatographic properties of the sample. 

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