Sample preparation column chromatography

Figure 1. A typical sequence of sample preparation manipulations for a procedure of Soxhlet extraction followed by preparative column chromatography for a solid sample - e.g., glycolipids out of wheat flour. Note that equivalently complex processes are encountered with "simple liquid-solid or liquid-liquid extractions followed by a series of back extractions - e.g., pesticides from fish tissue. (Reproduced with permission by Hewlett-Packard, HP Publication (43)5091-2102E, (1991), in preparation.)...

The standard methods that one would use in sample preparation for chromatography include filtration, sedimentation, centrifugation, liquid-liquid extraction (LEE) open-column chromatography, and concentration/evaporation. Filtration for sample preparation may be performed on numerous occasions in a sample preparation protocol, with the first filtration being used to separate large-particulate matter from solvent. 

Although most HPLC applications utilize one or more sample preparation procedures, it is best to keep sample preparation as simple as possible [2]. Some simple procedures that have found success for protein and peptide samples are precipitation and chromatographic methods. This chapter reviews the commonly used methods and devices for sample preparation in chromatography of biomolecules, particularly proteins. The proper choice and execution of the methods can greatly affect the success of the analysis, especially in terms of recoveries [1,3,4]. Planning the sequence of procedures can minimize time and maximize efficiency a flowchart for sample preparation strategies was presented in LC-GC [5]. Table 1 summarizes some of the typical requirements for the preparation of a sample for injection onto an HPLC column. 

A common myth about preparative column chromatography, virtually worshipped by less experienced chemists, is that the longa the column the better the separation. Very few exceptions aside, the opposite is the truth. Provided the column is well and uniformly packed, one has a beder chance to successfully resolve a complex mixture using the same amount of sUica gel in a reasonably fat, short column, onto which the sample can be applied as a thinner layer than would be the case using a longer, smaU-diameta column. Commercially available, machine-packed, disposable silica gel columns have... 

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. 

Preparation of soil—sediment of water samples for herbicide analysis generally has consisted of solvent extraction of the sample, followed by cleanup of the extract through Uquid—Uquid or column chromatography, and finally, concentration through evaporation (285). This complex but necessary series of procedures is time-consuming and is responsible for the high cost of herbicide analyses. The advent of soUd-phase extraction techniques in which the sample is simultaneously cleaned up and concentrated has condensed these steps and thus gready simplified sample preparation (286). 

Preparative chromatography involves the collection of individual solutes as they are eluted from the column for further use, but does not necessarily entail the separation of large samples. Special columns can be designed and fabricated for preparative use, but for small samples the analytical column can often be overloaded for preparative purposes. Columns can be either volume overloaded or mass overloaded. Volume overload causes the peak to broaden, but the retention time of the front of the peak... 

Although on-line sample preparation cannot be regarded as being traditional multidimensional chromatography, the principles of the latter have been employed in the development of many on-line sample preparation techniques, including supercritical fluid extraction (SFE)-GC, SPME, thermal desorption and other on-line extraction methods. As with multidimensional chromatography, the principle is to obtain a portion of the required selectivity by using an additional separation device prior to the main analytical column. 

The primary method for detecting methyl parathion and metabolites in biological tissues is gas chromatography (GC) coupled with electron capture (BCD), flame photometric (FPD), or flame ionization detection (FID). Sample preparation for methyl parathion analysis routinely involves extraction with an organic solvent (e g., acetone or benzene), centrifugation, concentration, and re suspension in a suitable solvent prior to GC analysis. For low concentrations of methyl parathion, further cleanup procedures, such as column chromatography on silica gel or Florisil are required. 

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