Basic chromatographic parameters

The most essential component in any liquid chromatograph is the column and the chromatographic packing contained therein. This is where the sample is separated into its individual components. The solute molecules are in an equilibrium between the column packing (stationary phase) and the eluent (mobile phase), and it is this equilibrium which governs the separation. In some types of chromatography this equilibrium involves an interaction between the solute and the column packing, but this is not the case in true SEC, as will be seen later. 

If a slug of a solution of a pure component is injected on to any liquid chromatographic column, it should pass through the column and emerge with an unaltered profile at the end. However, this does not happen in practice. Diffusion occurs, and the sample is diluted, with a consequent broadening of the sample zone. This phenomenon gives rise to the concepts of efficiency and resolution which will now be discussed. 

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The chief analytical purpose of resorting to chemical reactions is to simplify the solution of specific analytical problems and to extend the area of application of gas chromatography. In analytical reaction GC the column efficiency and the characteristics of the detector used generally remain invariable. However, as a result of chemical conversions of the sample components, derivatives are formed with different separation characteristics and detection limits, which generally leads to changes in the basic chromatographic parameters with respect to the initial compounds. 

Basic Considerations Linking Retention, Resolution, and Peak Efficiency with the Other Chromatographic Parameters... 

Thus the evaluation of the basic process parameters reduces to the determination of the amount of the element contained in the initial and resultant components of the raw material and the end product. Such values can be established in different ways, some of which were discussed by Pankov and Khripin [175], To obtain a value proportional to the amount of carbon in the key component, the zones of compounds separated in a chromatographic column are converted into carbon dioxide or methane. 

Chromatographic parameters for basic amino acid separations... 

This optimization entails a large reduction in the number of measured chromatographic parameters per stationary phase 252 (7 compounds x 3 injections X 3 columns x 2 mobile phases x 2 chromatographic parameters) instead of 1890 (14 compounds x 3 injections x 5 columns x 3 mobile phases x 3 chromatographic parameters). Furthermore, in the analysis of basic compounds, only the mobile phase at pH 7.0 is effectively necessary to achieve a good evaluation of silanol masking capacity of chromatographic supports. A second mobile phase (at pH 3.0) may be required only when an assessment of the hydrophobic properties of stationary phases is needed. 

The optimization of chromatographic separations can generally be seen as a compromise between speed, i.e., to produce the largest possible amount of data or substance per unit time, and resolution, i.e., to produce the highest possible quality of data or purity of substance. Obviously the goal for optimization differs according to the purpose of the separation and also between scale of operation. Therefore, different parameters are critical for different situations. Still, some basic rules for optimization may be applied. 

Seleetion of basic parameters of chromatographic process (stationary phase, proper pure solvents according to Snyder s classification, and vapor phase)... 

Valko et al. [37] developed a fast-gradient RP-HPLC method for the determination of a chromatographic hydrophobicity index (CHI). An octadecylsilane (ODS) column and 50 mM aqueous ammonium acetate (pH 7.4) mobile phase with acetonitrile as an organic modifier (0-100%) were used. The system calibration and quality control were performed periodically by measuring retention for 10 standards unionized at pH 7.4. The CHI could then be used as an independent measure of hydrophobicity. In addition, its correlation with linear free-energy parameters explained some molecular descriptors, including H-bond basicity/ acidity and dipolarity/polarizability. It is noted [27] that there are significant differences between CHI values and octanol-water log D values. 

Figure 1.3—Chromatographic peaks, a) Retention time b) Distribution of the peak c) Significance of the three basic parameters and features of a Gaussian distribution d) Example of a real chromatogram that shows the elution of components leading to peaks that resemble Gaussian distributions.

As mentioned above, the basic principle of NLC is the same as for conventional techniques. The separation is identified and characterized by measuring retention times, capacity, separation, and resolution factors. Therefore, it is necessary to explain the chromatographic terms and symbols by which the chromatographic speciation can be understood and explained. Some of the important terms and equations of the chromatographic separations are discussed below. The chromatographic separations are characterized by retention (k), separation (a), and resolution factors (Rs). The values of these parameters can be calculated by the following standard equations [92]. 

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