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Chromatography - Univerzita Karlova

INSTITUTE OF MEDICAL BIOCHEMISTRY. FIRST FACULTY OF MEDICINE, CHARLES UNIVERSITY IN PRAGUE. Chromatography in biochemistry Prof. RNDr. V ra Pac kov , CSc. Faculty of Natural Sciences, Charles University in Prague 2008/2009. Separation methods Separation is the distribution of a mixture into individual components, usually with the aim to isolate them in a chemically pure form. In many cases the components are not isolated but their resolution is recorded and qualitatively and quantitatively evaluated. Separation methods play a key role in a number of branches. The requirements put on separation methods are steadily growing. Chromatography belongs to the most important form of separation methods. Chromatography is a separation method based on the different migration of solutes through a system of two diverse phases, one of which is mobile and the other stationary. Chromatographic methods can be classified according to: A.

5 Liquid Chromatography Liquid chromatography (LC) is a separation method, in which the mobile phase is a liquid. Stationary phase can be an adsorbent; a liquid coated or chemically bound on a support, an ion exchanger, a size-exclusion phase, etc. Usually the stationary phase is placed in a

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Transcription of Chromatography - Univerzita Karlova

1 INSTITUTE OF MEDICAL BIOCHEMISTRY. FIRST FACULTY OF MEDICINE, CHARLES UNIVERSITY IN PRAGUE. Chromatography in biochemistry Prof. RNDr. V ra Pac kov , CSc. Faculty of Natural Sciences, Charles University in Prague 2008/2009. Separation methods Separation is the distribution of a mixture into individual components, usually with the aim to isolate them in a chemically pure form. In many cases the components are not isolated but their resolution is recorded and qualitatively and quantitatively evaluated. Separation methods play a key role in a number of branches. The requirements put on separation methods are steadily growing. Chromatography belongs to the most important form of separation methods. Chromatography is a separation method based on the different migration of solutes through a system of two diverse phases, one of which is mobile and the other stationary. Chromatographic methods can be classified according to: A.

2 Mobile phase arrangement liquid Chromatography (LC) - mobile phase is a liquid Gas Chromatography (GC) - mobile phase is a gas. B. Stationary phase arrangement Column Chromatography stationary phase is placed in a column Planar techniques: Paper Chromatography (PC) stationary phase is a special paper, either as such or modified with other compounds. Thin-layer Chromatography (TLC) stationary phase is spread on a solid flat support ( , glass plate or aluminum foil). C. The process, which prevails in separation (usually several physical and chemical processes take place in separation but one of them prevails). Partition Chromatography separation is based on different solubility of sample components in a stationary phase (a liquid ) and in a mobile phase (a liquid or a gas). Adsorption Chromatography separation is based on different abilities of components to adsorb on the surface of stationary phase (a solid).

3 Ion-exchange Chromatography separation is based on exchange of the ionic sample with the ionic group of the stationary phase and is governed by electrostatic interaction. Size exclusion Chromatography (gel Chromatography ) components are separated according to the size and shape of their molecules as well as the size and shape of the pores of the stationary phase (size-exclusion effect). The large molecules elute at the beginning, and the small molecules at the end. Affinity Chromatography separation is based on molecular recognition. Only those components, which are complementary to stationary phase, are adsorbed by their affinity. Affinity interactions are very strong. Theoretical introduction Let us suppose that two components A and B, present in a homogeneous mixture, should be separated. In order to separate component A from component B, we have to create a new phase in such a way that component A will be retained in one phase and component B will be 2.

4 Moved to the other phase. The common basis of the separation method is interfacial equilibrium. Separation can be carried out in one step (one equilibrium between two phases is attained) or in multi-step (continuous) mode, where the equilibrium is repeated many times. Chromatographic methods are continuous methods. In chromatographic methods one phase is stationary while the other is mobile. Distribution between two phases 1, 2 is governed by Nernst s distribution law (Eq. ): c KD = 1 ( ). c2. where KD is the distribution constant and c1, c2 are the concentrations of a component in both phases. Total time that the separated components spend in the chromatographic system (retention time tR), can be divided into a time spent in the stationary phase (retention time tS also called reduced retention time t R ) and time spent in the mobile phase (dead retention time tM). A recording of the separation, a chromatogram, is shown in Fig.

5 1. Detector response is plotted on axis y, while retention time is on axis x. Components are eluted in the form of elution curves or peaks. Ratio of retention times spent in both phases is proportional to the ratio of component concentrations in the stationary and mobile phases, cs and cm, and to the volumes of both phases, VS and VM: tS c V. = S S ( ). t M c m Vm Ratio of times spent in both phases is called retention factor k: tS V. = k = KD S ( ). tM VM. Ratio of concentrations in both stationary and mobile phases remain constant and is called distribution constant KD. Substances, which differ in their distribution constants, will move with different velocities and will separate. If the retention time is multiplied by mobile phase flow rate Fm, the retention volume VR, , the volume of the mobile phase necessary for the component elution, is obtained V R = t R Fm ( ). From the chromatogram in Fig.

6 1, we get the information about compound quality (retention time), quantity (from peak area or height) and separation efficiency. Qualitative analysis in Chromatography usually relies on the agreement of retention data of separated compounds (reduced retention time t R and, retention factor k etc.) with standard compounds. When using mass spectrometric detector the separated compounds can be identified on the basis of their mass spectra. 3. Fig. 1: Chromatogram Quantitative analysis is carried out by evaluation of the peak area or height. Various techniques are available. Method of internal normalization is based on the evaluation of all peak areas or height and calculation of the relative amounts of individual components in the mixture: Ai pi = 100 ( ). Aj j where pi is the relative amount (in %) of component i, A. j j is total area of all peaks. Known amounts of analyzed sample and standard are analyzed under identical experimental conditions and corresponding peak areas compared using the method of absolute calibration.

7 Method of internal standard is based on the addition of known amount of standard substance to the sample (standard must not be present in the original sample). Concentration of component i is established by the evaluation of peak areas of both sample and standard components: Ai Vs ci = cs ( ). As Vi where ci is the concentration of component i, Vi is the sample volume, to which a known volume of standard, Vs, is added, Ai and As are peak areas of component i, and of the added standard s. cs is the concentration of the standard s. This method is widely used, especially when experimental conditions are not properly controlled and when a sample pretreatment is required. 4. liquid Chromatography liquid Chromatography (LC) is a separation method, in which the mobile phase is a liquid . Stationary phase can be an adsorbent; a liquid coated or chemically bound on a support, an ion exchanger, a size-exclusion phase, etc.

8 Usually the stationary phase is placed in a column, but can be spread on a flat bed as well. In the latter case, it is either a paper paper Chromatography (PC) or it is placed on the surface of flat plates thin-layer Chromatography (TLC). A great number of compounds can be separated using liquid Chromatography (approximately 85 % of all known compounds polar, non-polar, nonvolatile, low- and high-molecular mass compounds), in contrary to gas Chromatography which is limited to volatile compounds. While the mobile phase in GC is an inert gas and does not participate in the interactions with the analytes, in LC the intermolecular interaction are much stronger than in gases and the mobile phase then competes with the stationary phase for analytes. The choice of the mobile phase in LC influences the distribution coefficients and thus the whole separation process. Other differences between GC and LC come from different properties of both gases and liquids.

9 The density of liquid is 103 times higher and the viscosity 102 times higher than those of gases. Diffusion is 105 times slower in liquids. It comes from the properties of the liquids that lower flow rates have to be used in LC and the analyses are slower in comparison with GC. Regarding the low compressibility of liquids it is possible to work at high pressures without a loss of separation efficiency. The retention data in GC have to be corrected for the compressibility of gases. Surface tension in liquids is 104 times higher than in gases and enables the use of the ascending techniques in TLC and PC. Column Chromatography In classical column Chromatography the mobile phase moves without external pressure, by gravitation forces only. Analyses can last several hours and the separation efficiency is low. In order to reach efficient separation we have to use stationary phases with small particles (10.)

10 M and less) of uniform size, which are homogenously packed in a column. However these phases create a large resistance to mobile phase flow and therefore high pressures have to be applied (tens of MPa). The method that uses such column packaging in combination with high pressures is called high performance liquid Chromatography , HPLC. Separation in LC can be influenced by the choice of both mobile and stationary phases. It is simpler and cheaper to change the composition of the mobile phase, , to change the solvent type, a pH and an ionic strength of the mobile phase, etc., than to change the chromatographic column. First of all the mobile phase has to dissolve the analytes. An old rule says that similar is dissolved in similar. The mobile phase should not interfere with the detection. We can work either at constant composition of the mobile phase (isocratic elution) or change the composition during analysis (gradient elution).


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