Chromatography, general identification

Oils are mixtures of mixed esters with different fatty acids distributed among the ester molecules. Generally, identification of specific esters is not attempted instead the oils are characterized by analysis of the fatty acid composition (8,9). The principal methods have been gas—Hquid and high performance Hquid chromatographic separation of the methyl esters of the fatty acids obtained by transesterification of the oils. Mass spectrometry and nmr are used to identify the individual esters. It has been reported that the free fatty acids obtained by hydrolysis can be separated with equal accuracy by high performance Hquid chromatography (10). A review of the identification and deterrnination of the various mixed triglycerides is available (11). 

Generally we allowed various model compounds to react with alkali for varying lengths of time and then separated the reaction products by paper chromatography. For identification, the products were eluted from the paper, and spectral analyses were made of the eluates. 

HPLC-UV diode-array detection (DAD) or HPLC-MS techniques take advantage of chromatography as a separation method and DAD or MS as identification and/or quantification methods. Both DAD and MS can rapidly provide on-line UV and MS information for each individual peak in a chromatogram. In most cases, the identification of the peaks can be made directly on-line by comparison with literature data or with standard compounds. However, when no standard compounds are available, the rapid preliminary identification process becomes significantly more complex. The general identification problem in bioprocess monitoring described below exists in all cases when a new biotechnological process is initiated and when the product is not available as a reference substance. 

There are many books and other sources that discuss the retention index from theoretical and practical aspects. One comprehensive book is "The Sadtler Standard Gas Chromatography Retention Index Library" [15]. This series of book (from Volume 1 to Volvune 4) provides detailed data on the retention indices for more than 2,000 compoimds under varying isothermal and temperature programming GC conditions for the purpose of the general identification of unknown compoimds. 

This is an analysis frequently conducted on oil lubricants. Generally, the additive is known and its concentration can be followed by direct comparison of the oil with additive and the base stock. For example, concentrations of a few ppm of dithiophosphates or phenols are obtained with an interferometer. However, additive oils today contain a large number of products their identification or their analysis by IR spectrometry most often requires preliminary separation, either by dialysis or by liquid phase chromatography. 

More general techniques covering a wider range employ gas chromatography (Durand et al., 1987). This enables identification and analysis of the nearly 200 gasoline components whose octane numbers are known. 

A potentially general method of identifying a probe is, first, to purify a protein of interest by chromatography (qv) or electrophoresis. Then a partial amino acid sequence of the protein is deterrnined chemically (see Amino acids). The amino acid sequence is used to predict likely short DNA sequences which direct the synthesis of the protein sequence. Because the genetic code uses redundant codons to direct the synthesis of some amino acids, the predicted probe is unlikely to be unique. The least redundant sequence of 25—30 nucleotides is synthesized chemically as a mixture. The mixed probe is used to screen the Hbrary and the identified clones further screened, either with another probe reverse-translated from the known amino acid sequence or by directly sequencing the clones. Whereas not all recombinant clones encode the protein of interest, reiterative screening allows identification of the correct DNA recombinant. 

The right chromatography column should separate the sample sufficiently to enable identification or quantitative measurement of the components within a reasonable period of time. The resolution factor (Rs) for two sample components is determined by the width of the two peaks and the distance between the peak maxima. In general, Rs values of 1.0 are required for good qualitative or quantitative work, whereas Rs values >1.5 indicate baseline resolution for two components (3). 

Identification and quantification is obtained by combined high-resolution gas chromatography/mass spectrometry (GC/MS) methods after special cleanup procedures of the matrix, as shown later for sediments (see Figure 8.2). The cleanup methods for other matrices are similar. Quantification is obtained by addition of 13-C labeled standards before the cleanup procedure. In general, only the toxic isomers are identified and quantified. 

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