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Chromatography

Characteristics of elution tR – retention time Retention time is the time for a sample to travel through a chromatographic column t0 – hold-up time Hold-up time is the time necessary for the mobile phase to travel through a chromatographic column k’ – retention factor Retention time (tR) can be used for identification purposes but this characteristic depends on the flow rate. The retention factor (k’) is the parameter that does not depend on the flow rate. Therefore it is more preferable for qualitative analysis

Figures of merit for separation Selectivity (a) Resolution (Rs) Selectivity (a) shows potential of a given stationary phase to the separation of a pair of compounds. It can give a false concept of a real resolution as the latter depend on the widths of peaks. The resolution (Rs) value takes into account the dispersion of peaks, since is more useful for the selection of media for chromatographic separation. Selectivity does not depend on the widths of peaks whereas Resolution does.

Theoretical plate concept and the broadening of chromatographic peaks There are several reasons causing the broadening of chromatographic peaks. Those are (1) slow kinetics of mass transfer, (2) diffusion in the pores of the adsorbent particles, and (3) axial dispersion in the interparticle volume. The lower the rate of mass transfer processes, the broader the chromatographic peak. N = 400 N = 100 A parameter characterizing the broadening of chromatographic peaks is the number of theoretical plates N. It relates to the apparent dispersion Da of a peak as N = uL/2Da (u is the linear flow velocity, L the length of a column). The larger N, the narrower the peak N = 50 Retention time depends only on the affinity of an analyte to the stationary phase. However the width of a chromatographic peak is a function of kinetics of mass transfer processes in a chromatographic column. This dependence is reflected in a concept of the theoretical plate. According to the plate theory, a chromatographic column is divided by a number of small sections (plates). The length of a plate is enough for adsorption equilibrium to be established under dynamic conditions, that is while a flow of an analyte passes through the column.

N Resolution and the broadening of chromatographic peak Dependence on N N N = 300 Rs = 0.6 N = 1000 Rs = 1.0 N = 5000 Rs = 3.2

Resolution and the Loading Loading is the amount of a sample applied to chromatographic separation Loading = 1

Resolution and the Loading

Resolution and the Loading

Types of chromatography with respect to loading Maximal Loading Separation Column diameter ~ mg – mg ~ 100 mg ~ g Analytical Semi-preparative Preparative 2 - 5 mm 10 – 20 mm 20 – 50 mm Amount of an adsorbent needed for the separation of a certain amount of a sample can be evaluated according to an approximate rule 1:20. We need 20 g of an adsorbent per 1 g of a sample.

Comparison Analytical vs. Preparative chromatography Analytical requirements Precision Accuracy Sensitivity Reproducibility Robustness Diluted samples, expensive solvents and adsorbents Preparative requirements Recovery Product purity Productivity Costs Concentrated samples, cheap solvents and adsorbents

Chromatography Planar Column TLC (Thin layer chromatography) Paper

Thin layer chromatography Phase systems for TLC Adsorbent Eluent Typical analytes Polar adsorbents Silica (SiO2)x Alumina (Al2O3) Non-polar adsorbents Polymers ODS-grafted silica non-aqueous solvents aqueous solvents non- or medium-polar organics polar and ionogenic organics

Silica gel Structure of silica Silica gel is a granular porous form of silica Due to the surface hydroxyls it has a strong affinity towards polar organics that are able to hydrogen bonding and strong dipole-dipole interactions. Such compounds will longer retain on the silica whereas non-polar components will be eluted first

Silica gel Nontreated silica Acid washed analysis of acidic components Base washed analysis of basic components

ODS modified Silica gel ODS – octadecylsilane hydrophobic surface Hydrophobic (non-polar) compounds are stronger retained whereas hydrophilic compounds are eluted first

TLC Plate Support 200 mm for analytical chromatography 1000 mm for preparative chromatography The adsorbent layer is prepared from an adsorbent, a binder, and, optionally, a fluorescent indicator Adsorbent layer Support (Metallic or glass)