Chromatographic system performance

The concept of separation number (SN) in planar chromatography is a practical approach to the task of quantification of chromatographic system performance. According to this concept, such performance can simply be evaluated by calculating how many components of the separated mixture can be comfortably accommodated (i.e., without any overlap of adjacent components) along the direction of migration of the eluent. A convenient relationship proposed in Ref. 17 enables easy calculation of the numerical value of SN ... 

Having established that a finite volume of sample causes peak dispersion and that it is highly desirable to limit that dispersion to a level that does not impair the performance of the column, the maximum sample volume that can be tolerated can be evaluated by employing the principle of the summation of variances. Let a volume (Vi) be injected onto a column. This sample volume (Vi) will be dispersed on the front of the column in the form of a rectangular distribution. The eluted peak will have an overall variance that consists of that produced by the column and other parts of the mobile phase conduit system plus that due to the dispersion from the finite sample volume. For convenience, the dispersion contributed by parts of the mobile phase system, other than the column (except for that from the finite sample volume), will be considered negligible. In most well-designed chromatographic systems, this will be true, particularly for well-packed GC and LC columns. However, for open tubular columns in GC, and possibly microbore columns in LC, where peak volumes can be extremely small, this may not necessarily be true, and other extra-column dispersion sources may need to be taken into account. It is now possible to apply the principle of the summation of variances to the effect of sample volume. 

To reiterate the definition of chromatographic resolution a separation is achieved in a chromatographic system by moving the peaks apart and by constraining the peak dispersion so that the individual peaks can be eluted discretely. Thus, even if the column succeeds in meeting this criterion, the separation can still be destroyed if the peaks are dispersed in parts of the apparatus other than the column. It follows that extra-column dispersion must be controlled and minimized to ensure that the full performance of the column is realized. 

The analytical specifications must prescribe the ultimate performance of the total chromatographic system, in appropriate numerical values, to demonstrate the performance that has been achieved. 

A. Dossena, G. Galaverna, R. Coiradini and R. Marchelli, Two-dimensional liigh performance liquid chromatographic system foi the determination of enantiomeric excess in complex amino acid mixtures. Single amino acids analysis , ]. Chromatogr. 653 229-234 (1993). 

In carrying out a chromatographic separation, an analyst is concerned with whether the components of a mixture can be separated sufficiently for the analytes of interest, and this is not always all of them, to be identified and/or for the amounts present to be determined. Our ability to carry out these tasks successfully will depend upon the performance of the chromatographic system as a whole. 

The performance may be described in terms of a number of theoretical parameters, although the performance required for a particular analysis will depend upon the separation that is required. This, in turn, depends upon the similarity in the behaviour in the chromatographic system of the analyte(s) of interest to each other and to other compounds present in the mixture. 

How then does the performance of the chromatographic system affect the quality of the analytical information that may be obtained ... 

Mixed lateral interactions can occur in the case of the following pairs of molecules A. .. B, AB. .. A, AB. .. B, and ABj. .. ABj. Their appearance is even more importunate than that of the self-associative lateral interactions. With the self-asso-ciative lateral interactions alone, tailing of chromatographic bands lowers separative performance of a given chromatographic system, whereas with the mixed lateral... 

The main difference between the chromatographic process carried out in the linear and the nonlinear range of the adsorption isotherm is the fact that in the latter case, due to the skewed shapes of the concentration profiles of the analytes involved, separation performance of a chromatographic system considerably drops, i.e., the number of theoretical plates (N) of a chromatographic system indisputably lowers. In these circumstances, all quantitative models, along with semiquantitative and nonquantitative rules, successfully applied to optimization of the linear adsorption TLC show a considerably worse applicability. 

Procedures used vary from trial-and-error methods to more sophisticated approaches including the window diagram, the simplex method, the PRISMA method, chemometric method, or computer-assisted methods. Many of these procedures were originally developed for HPLC and were apphed to TLC with appropriate changes in methodology. In the majority of the procedures, a set of solvents is selected as components of the mobile phase and one of the mentioned procedures is then used to optimize their relative proportions. Chemometric methods make possible to choose the minimum number of chromatographic systems needed to perform the best separation. 

Zarzycki and coworkers [77] studied the influence of temperature on the separation of cholesterol and bile acids using reversed-phase stationary phases. The best chromatographic conditions for the separation of mnlticomponent samples of steroids were chosen. Experiments were performed on wettable plates with RP-18W and at the temperatnres of 5, 10, 20, 30, 40, 50, and 60°C. The studies showed (Figure 9.9) that the degree of separation in the high-temperature region can be increased by an improvement of the efficiency of the chromatographic system. However, a relatively weak retention-temperatnre response for the studied steroids was observed. 

The optimization of preparative and even micropreparative chromatography depends on the choice of an appropriate chromatographic system (adsorbent and eluent), sample application and development mode to ensure high purity, and yield of desirable compounds isolated from the layer. For the so-called difficult separations, it is necessary to perform rechromatography by using a system with a different selectivity. But it should be taken into account that achievement of satisfactory results frequently depends on a compromise between yield and the purity of the mixture component that is being isolated. 

These chromatographic performance tests are carried out in order to ensure that all impurities to be controlled are well separated from the substance to be examined (HPLC, GC andTLC). Forthis reason, preferably such reference substances are chosen which elute dose to the main compoxmd (HPLC, GC) or which have a similar Rf-value (TLC) but can stiU be separated. These may be structurally related compoimds which shall be separated with a minimum requirement for the resolution using the chromatographic system described, e.g. such as in the monograph for desmopressin (Monograph 07121999), Figure 5.3. 

High-performance liquid chromatographic system equipped with an automated column switching system... 

Standards and blanks are the usual controls used in analytical HPLC. Standards are usually interspersed with samples to demonstrate system performance over the course of a batch run. The successful run of standards before beginning analysis demonstrates that the system is suitable to use. In this way, no samples are run until the system is working well. Typically, standards are used to calculate column plate heights, capacity factors, and relative response factors. If day-to-day variability has been established by validation, the chromatographic system can be demonstrated to be within established control limits. One characteristic of good science is that samples... 

McKern, N. M., Edskes, H. K., and Shukla, D. D., Purificatiion of hydrophilic and hydrophobic peptide fragments on a single reversed phase high performance liquid chromatographic system, Biomed. Chromatogr., 7, 15, 1993. 

Takahashi, N., Takahashi, Y., Ishioka, N., Blumberg, B., and Putnam, F. W., Application of an automated tandem high-performance liquid chromatographic system to peptide mapping of genetic variants of human serum albumin, J. Chromatogr., 359, 181, 1986. 

Detectors are composed of a sensor and associated electronics. Design and performance of any detector depends heavily on the column and chromatographic system with which it is associated. Because of the complexity of many mixtures analysed and the limitation in regard to resolution, despite the use of high-resolution capillary columns and multicolumn systems, specific detectors are frequently necessary to gain selectivity and simplify the separation system. Many detectors have been developed with sensitivities toward specific elements or certain functional groups in molecules. Those detectors that exhibit the highest sensitivity are often very specific in response, e.g. the electron capture detector in GC or the fluorescence detector in LC. Because... 

Moment analysis is one of the simplest types of analysis and is useful for measuring the performance of the chromatography. Moments can be used to measure the same things that are measured in ID chromatographic systems these include the first, second, and third moments, which are more accurate than the related peak maximum, peak width, and peak asymmetry. In 2D, however, these values each have a component in each dimension and this can be easily determined in software-based measurement systems. 

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