Chromatography complex samples

In chromatography, complex samples are introduced onto a column comprised of a solid support that is coated with a stationary phase (having a special chemical formulation) through which a mobile phase (a gas in GC and a liquid in LC) of variable composition passes. The premise is that the components of the injected mixture are adsorbed initially onto the stationary phase followed by a sequential release of analytes into the mobile phase as its properties are altered by temperature (in GC) or solvent composition (in LC). 

Time, Cost, and Equipment Analysis time can vary from several minutes for samples containing only a few constituents to more than an hour for more complex samples. Preliminary sample preparation may substantially increase the analysis time. Instrumentation for gas chromatography ranges in price from inexpensive (a few thousand dollars) to expensive (more than 50,000). The more expensive models are equipped for capillary columns and include a variety of injection options and more sophisticated detectors, such as a mass spectrometer. Packed columns typically cost 50- 200, and the cost of a capillary column is typically 200- 1000. 

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. 

Figure 10.3 Gas cliromatograms of a cold-pressed lemon oil obtained (a) with an SE-52 column in the stand-by position and (b) with the same column showing the five heart-cuts (c) shows the GC-GC chiral chromatogram of the ti ansfeired components. The asterisks in (b) indicate electric spikes coming from the valve switcliing. The conditions were as follows SE-52 pre-column, 30 m, 0.32 mm i.d., 0.40 - 0.45 p.m film tliickness cairier gas He, 90 KPa (stand-by position) and 170 KPa (cut position) oven temperature, 45 °C (6 min)-240 °C at 2 °C/min diethyl-tert-butyl-/3-cyclodextrin column, 25 m X 0.25 mm i.d., 0.25 p.m film thickness cairier gas He, 110 KPa (stand-by position) and 5 KPa (cut position) oven temperature, 45 °C (6 min), rising to 90 °C (10 min) at 2 °C/min, and then to 230 °C at 2 °C/min. Reprinted from Journal of High Resolution Chromatography, 22, L. Mondello et al, Multidimensional capillary GC-GC for the analysis of real complex samples. Part IV. Enantiomeric distribution of monoterpene hydrocarbons and monoterpene alcohols of lemon oils , pp. 350-356, 1999, with permission from Wiley-VCH.

The open-column technique is commonly applied in the case of crude oils (being the least complex geochemical organic mixtures). MPLC, high-pressure liquid chromatography (HPLC), and PTLC are more often applied to more complex samples, especially those dominated by more polar compounds, such as hydrothermal bitumens or samples showing terrestrial organic matter input, such as extracts or pyroly-sates of coals of various ranks. 

Specifically for triazines in water, multi-residue methods incorporating SPE and LC/MS/MS will soon be available that are capable of measuring numerous parent compounds and all their relevant degradates (including the hydroxytriazines) in one analysis. Continued increases in liquid chromatography/atmospheric pressure ionization tandem mass spectrometry (LC/API-MS/MS) sensitivity will lead to methods requiring no aqueous sample preparation at all, and portions of water samples will be injected directly into the LC column. The use of SPE and GC or LC coupled with MS and MS/MS systems will also be applied routinely to the analysis of more complex sample matrices such as soil and crop and animal tissues. However, the analyte(s) must first be removed from the sample matrix, and additional research is needed to develop more efficient extraction procedures. Increased selectivity during extraction also simplifies the sample purification requirements prior to injection. Certainly, miniaturization of all aspects of the analysis (sample extraction, purification, and instrumentation) will continue, and some of this may involve SEE, subcritical and microwave extraction, sonication, others or even combinations of these techniques for the initial isolation of the analyte(s) from the bulk of the sample matrix. 

A variety of formats and options for different types of applications are possible in CE, such as micellar electrokinetic chromatography (MEKC), isotachophoresis (ITP), and capillary gel electrophoresis (CGE). The main applications for CE concern biochemical applications, but CE can also be useful in pesticide methods. The main problem with CE for residue analysis of small molecules has been the low sensitivity of detection in the narrow capillary used in the separation. With the development of extended detection pathlengths and special optics, absorbance detection can give reasonably low detection limits in clean samples. However, complex samples can be very difficult to analyze using capillary electrophoresis/ultraviolet detection (CE/UV). CE with laser-induced fluorescence detection can provide an extraordinarily low LOQ, but the analytes must be fluorescent with excitation peaks at common laser wavelengths for this approach to work. Derivatization of the analytes with appropriate fluorescent labels may be possible, as is done in biochemical applications, but pesticide analysis has not been such an important application to utilize such an approach. 

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. 

Preparative chromatography is a powerful tool to isolate working quantities of compounds from complex samples. Preparative GC, which is normally carried out on packed columns, is useful for components with a b.p. < 450 °C and levels > 100 ppm (impossible for < lOppm questionable for 10-100ppm). Total amount needed is 2 p,L. 

Larsson, L. Saraf, A. Use of gas chromatography-ion trap mass spectrometry for the detection and characterization of microorganisms in complex samples. Mol. Biotechnol. 1997, 7, 279-287. 

The example shown in Fig. 3.4g uses off-line column switching to combine exclusion and reverse phase chromatography for the separation of pesticides from a complex sample matrix. 

The concept of peak capacity is rather universal in instrumental analytical chemistry. For example, one can resolve components in time as in column chromatography or space, similar to the planar separation systems however, the concept transcends chromatography. Mass spectrometry, for example, a powerful detection method, which is often the detector of choice for complex samples after separation by chromatography, is a separation system itself. Mass spectrometry can separate samples in time when the mass filter is scanned, for example, when the mass-to-charge ratio is scanned in a quadrupole detector. The sample can also be separated in time with a time-of-flight (TOF) mass detector so that the arrival time is related to the mass-to-charge ratio. 

In chromatography, the separation efficiency of any single separation method is limited by the efficiency and selectivity of the separation mode, that is, the plate count of the column and the phase of the selected system. Adding more columns will not overcome the need to identify more components in a complex sample, due to the limitation of peak capacities. The peak capacity in an isocratic separation can be described, following Grushka (1970), as given in Equation (17.1) ... 

The principal analytical methods for complex samples are those that separate the mixture by differential migration and then detect the separated components. The separation methods are chromatography, electrophoresis, and field flow fractionation the detection methods—which need not be selective but must be sensitive—include absorption, laser-induced fluorescence, electrochemistry, and mass... 

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