Separation of Complex Sample Mixtures

Special solutions are required for the separation of very complex sample mixtures, which are the daily challenge in contemporary bioanalytics. The main focus of this chapter is an introduction to the theory and technical solutions in multidimensional chromatography. Furthermore, a selected real example is used to demonstrate the significant increase in separation power that may be achieved by a clever coupling of different separation mechanisms. In order to encourage the reader to realize unconventional approaches, the key issues are discussed and it is shown that reproducible results can be achieved. 

It is standard that more than a thousand proteins are present in a biological sample such as a cell lysate. Besides the state-of-the-art separation technique, 2D-gel electrophoresis, liquid-phase separation techniques are beginning to play a major role in proteomics. Their advantages are reproducibility, automation, speed, and their suitability for direct coupling with mass spectrometry. 

HPLC Made to Measure A Practical Handbook for Optimization. Edited by Stavros Kromidas Copyright 2006 WILEY-VCH Verlag GmbH Co. KGaA, Weinheim ISBN 3-527-31377-X 

The limitations of single-stage separation systems have been recognized for many years. In order to describe separations of a multicomponent mixture, the concept of peak capacity was introduced, which is defined as the maximum number of components, n, that can be placed side by side in the available separation space. 

In this formula, r is the number of standard deviations taken as equaling the peak width (typically 4), and k is the retention factor of the last peak in a series. Modem, high-resolution chromatographic systems yield peak capacities which are calculated to be in the range of 100-300. The peak capacity is a theoretical value that will never be attained since components in a complex mixture are usually not uniformly distributed, appear randomly, and are overlapping each other. Assuming that the number of components in a mixture can be estimated, when the number of components equals the peak capacity of the system used, the maximum number of visible peaks will be equivalent to 37% of the system s peak capacity. 

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These long lengths make possible very efficient separations of complex sample mixtures. Figure 6.1 is a typical chromatogram of a standard text mixture on a 30-m fused silica OT. Notice the sharp symmetrical peaks obtained for polar, acidic, and basic compounds. 

Possibility of measuring mass spectra of complex sample mixtures directly (without extensive separation or sample cleanup)... 

Various comprehensive HPLC systems have been developed and proven to be effective both for the separation of complex sample components and in the resolution of a number of practical problems. In fact, the very different selectivities of the various LC modes enable the analysis of complex mixtures with minimal sample preparation. However, comprehensive HPLC techniques are complicated by the operational aspects of transferring effectively from one operation step to another, by data acquisition and interpretation issues. Therefore, careful method optimization and several related practical aspects should be considered. 

Transfer of proteins onto nitrocellulose or PVDF membranes is usually performed after separation of complex protein mixtures by polyacrylamide gel electrophoresis (PAGE). Proteins can be separated on the basis of their molecular weight or isoelectric point, under reducing or nonreducing conditions, and it is therefore for the investigator to establish the most appropriate separation conditions for particular samples. Full details of PAGE can be found in refs. 4 and 5, and the reader is encouraged to use these as sources of further details... 

Mixed beds of stationary phases in a single column have not found much application in GSC. However, for the separation of complex gas mixtures, more than a single column is often used in a configuration that involves switching valves, so that only part of the sample is subjected to a second column (see also section 6.1). 

For the interpretive optimization of the primary (program) parameters in the programmed analysis of complex sample mixtures it may well be sufficient to optimize for the major sample components. This may be done if it is assumed that the primary parameters do not have a considerable effect on the selectivity, so that if the major sample components are well spread out over the chromatogram, the minor components in between these peaks will follow suit automatically, and if it is assumed that the minor peaks are randomly distributed over the chromatogram. The major chromatographic peaks can be separated to any desired degree if optimization criteria are selected which allow a transfer of the result to another column. 

Multidimensional chromatography separations are currently one of the most promising and powerful methods for the fractionation and characterization of complex sample mixtures in different property coordinates. This technique combines extraordinary resolution and peak capacity with flexibility, and it overcomes the limitations of any given single chromatographic method. This is the ideal basis for the identiflcation and quantification of major compounds and by-products, which might adversely affect product properties if not detected in time. 

The GC-IRMS technique is based on the separation of complex organic mixtures on a capillary GC column. A splitter at the end of the GC column sends >95% of the sample to a combustion or pyrolysis tube. The remainder is sent to an optional flame ionization detector (FID), ion trap MS, or vented to the atmosphere. 

Separations of complex FAME mixtures, with chain lengths up to 24 carbons and up to six double bonds, including aU co3 and co6 acids, can be achieved on capillary columns of polyethylene glycol or Carbowax type columns (usually of dimensions 25-30 m long, 0.25 mm i.d., and a film thickness of 0.2 xm) as recommended in the AOCS official method Ce lb-89 (American Oil Chemists Society, 2005). A GC profile of FAME from a fish oil/sunflower oil mixture on a column of this type is shown in Figure 2.3. These columns are preferred because of the stability, and the predictable order in which all compounds elute. An analysis on this type of column would normally be the first step for a sample of unknown composition. Typically in our laboratOTy, the following conditions are used an... 

Chromatography is the best technique for the separation of complex mixtures. Frequently, samples to be analysed are very complex, so the analyst has to choose more and more sophisticated techniques. Multidimensional separations, off-line and recently on-line, have been used for the analysis of such complex samples. 

In the analysis of complex PAH mixtures obtained from environmental samples, reversed-phase LC-FL typically provides reliable results for only 8-12 major PAHs (Wise et al. 1993a). To increase the number of PAHs determined by LC-FL, a multidimensional LC procedure is used to isolate and enrich specific isomeric PAHs that could not be measured easily in the total PAH fraction because of interferences, low concentrations, and/or low fluorescence sensitivity or selectivity. This multi-dimensional procedure, which has been described previously (Wise et al. 1977 May and Wise 1984 Wise et al. 1993a, 1993b), consists of a normal-phase LC separation of the PAHs based on the number of aromatic carbon atoms in the PAH, thereby providing fractions containing only isomeric PAHs and their alkyl-substituted isomers. These isomeric fractions are then analyzed by reversed-phase LC-FL to separate and quantify the various isomers. 

An important application of carbon-skeleton gas chromatography is the simplification of the analysis of complex samples such as polychlorinated biphenyls, polybrominated biphenyls and polychloroalkanes [709-711], These complex mixtures of halogenated isomers produce multiple peaks when separated by gas chromatography, making quantitation difficult. The isomers have identical carbon skeletons, resulting in a very simple chromatogram after hydrodechlorination. 

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