Chromatography column variables

Many of the possible column combinations that are useful in 2DLC are listed in Chapter 5. Besides the actual types of column stationary phases, for example, anion-exchange chromatography (AEC), size exclusion chromatography (SEC), and RPLC, many other column variables must be determined for the successful operation of a 2DLC instrument. The attributes that comprise the basic 2DLC experiment are listed in Table 6.1. 

The choice of column should be made after careful consideration of mode of chromatography, column-to-column variability, and a number of other considerations [3-5]. A short discussion on columns and column packings is given below. The column packings may be classified according to the following features [2] ... 

Treatment of wood with multi-component systems is likely to result in separation of the components when large wood samples are treated. This has been likened to the action of a chromatography column (Schneider, 1995). This is a significant problem that is often only encountered during scale-up of laboratory-based studies, where satisfactory results were previously obtained on small wood samples. Similarly, treatment of large wood samples can often lead to considerable variability in results due to inhomogeneous distribution, which again may not be evident with small samples treated under laboratory conditions. 

The apparatus consists of a chromatography column and a flow controller (Fig. 2.141). This figure is reproduced from the original literature report, to illustrate the description of the technique below, but the equipment is commercially available (e.g. Aldrich Chemical Co., May and Baker Ltd). The flow controller is a simple variable bleed device for precise regulation of the elution rate and is constructed from a glass/Teflon needle valve. Eluate fractions are collected in... 

In contrast to GC, liquid chromatography hyphenated with mass spectrometry (LC-MS) does not require a derivatization step before sample analysis. Separation of metabolite from sample matrix is achieved using chromatography columns with various stationary phases of different physicochemical characteristics. LC-MS is more often used than GC-MS because it is more suitable for unstable compounds, compounds difficult to derivatize, and nonvolatile compounds [6, 7]. Therefore, a wider range of metabolites with various physicochemical properties can be determined using LC-MS. Moreover, the sample pretreatment procedure is much simpler, which can have a great impact on minimization of analytical variability. 

The most commonly used method to measure smells is with an organoleptic panel, i.e. a group of, typically, 4 to 8 people who sniff professionally the headspace of an odorant compound. In some cases, the headspace is first passed down a gas chromatography column and then the panel smells the separate compounds as they are slowly eluted Irom the end of the column - this is known as an olfactometer. However, organoleptic panels are expensive to train, take a considerable amount of time and effort to detect the compounds, and are subject to considerable variability -perhaps a factor of 3 or more Ifom panel to panel. Consequently, there has been considerable effort to employ other headspace techniques that are well known in the field of analytical chemistiy, such as ... 

The formation of aptamers rests upon a technique termed SELEX (systematic evolution of ligands by exponential enrichment). It involves the synthesis of a DNA molecule with constant sections at both ends and a randomly variable segment of nucleotides in the middle. A variable segment as short as 10 nucleotides has a combinatorial library of 4 ° or about 10 different sequences a variable sequence of 30 nucleotides generates a combinatorial library of 4 or about 10 different sequences. The DNA mixture so synthesized is then enzymatically transcribed to a combinatorial RNA library. This mixture is applied to a chromatography column in which the substance of interest (e.g., amino acid, protein, drug) is bound. RNA molecules in the combinatorial library that do not bind with the target compound are simply washed out of the column. 

According to Eqs. (1) and (2), the retention time in GC depends on several variables (a) the chemical nature of the phase system and its temperature, as reflected by the distribution coefficient (b) the ratio of the phase volumes in the column Fs/ Fm and (c) the value of fo. In the practice of chromatography, these variables are used to maximize the component separation and the speed of analysis. 

The high degree of sensitivity, selectivity, and efficiency of gas chromatography allows the elucidation of a complete profile of the volatile components of distilled spirits. The wide selection of chromatographic columns and techniques, such as gc-ms, gc-ftir, and gc-ms-ftir, has allowed the chemist to routinely identify and quantify individual constituents on a parts-per-biUion level. The two most critical variables in the analysis of volatile components of distilled spirits by gas chromatography are the selection of a suitable chromatographic column and of the most appropriate detector. 

Flow markers are often chosen to be chemically pure small molecules that can fully permeate the GPC packing and elute as a sharp peak at the total permeation volume (Vp) of the column. Examples of a few common flow markers reported in the literature for nonaqueous GPC include xylene, dioctyl phthalate, ethylbenzene, and sulfur. The flow marker must in no way perturb the chromatography of the analyte, either by coeluting with the analyte peak of interest or by influencing the retention of the analyte. In all cases it is essential that the flow marker experience no adsorption on the stationary phase of the column. The variability that occurs in a flow marker when it experiences differences in how it adsorbs to a column is more than sufficient to obscure the flow rate deviations that one is trying to monitor and correct for. 

---