INSTITUTE OF MEDICAL BIOCHEMISTRY FIRST FACULTY OF MEDICINE, CHARLES UNIVERSITY IN PRAGUE. Chromatography. in biochemistry

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1 INSTITUTE OF MEDICAL BIOCHEMISTRY FIRST FACULTY OF MEDICINE, CHARLES UNIVERSITY IN PRAGUE Chromatography in biochemistry Prof. RNDr. Věra Pacáková, CSc. Faculty of Natural Sciences, Charles University in Prague 2008/2009

2 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. 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 (e.g., 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). 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

3 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. 1.1): c1 K D = (1.1) c 2 where K D is the distribution constant and c 1, c 2 are the concentrations of a component in both phases. Total time that the separated components spend in the chromatographic system (retention time t R ), can be divided into a time spent in the stationary phase (retention time t S also called reduced retention time t R ) and time spent in the mobile phase (dead retention time t M ). A recording of the separation, a chromatogram, is shown in Fig. 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, c s and c m, and to the volumes of both phases, V S and V M : ts c S VS = (1.2) t c V M m m Ratio of times spent in both phases is called retention factor k: ts VS = k = K D (1.3) t V M M Ratio of concentrations in both stationary and mobile phases remain constant and is called distribution constant K D. 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 F m, the retention volume V R, i.e., the volume of the mobile phase necessary for the component elution, is obtained V = t F (1.4) R R m From the chromatogram in Fig. 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

4 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 p i = 100 (1.5) A j j where p i is the relative amount (in %) of component i, j A 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. 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: A V = c (1.6) i s c i As Vi s where c i is the concentration of component i, V i is the sample volume, to which a known volume of standard, V s, is added, A i and A s are peak areas of component i, and of the added standard s. c s 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

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 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. The density of liquid is 10 3 times higher and the viscosity 10 2 times higher than those of gases. Diffusion is 10 5 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 10 4 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 µm 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, i.e., 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). Choice of the stationary and mobile phase depends on the chemical character of analytes. If the stationary phase is an adsorbent (usually silica gel) and the separation mechanism is adsorption, then the method is called adsorption chromatography. Due to polar character of silica gel, a non-polar hydrocarbon (e.g., hexane) is selected as a mobile phase and polar solvents (e.g., water, alcohols, in concentration lower than 1 %) are added to increase its 5

6 polarity. The adsorption chromatography is suitable, e.g., for separation of isomers (for example phenols). The most commonly used is a chromatography on non-polar chemically bound phases (reversed-phase chromatography, RPC). Mechanism of separation is complicated and not quite understood. It is a combination of dissolution and adsorption. The phases consist of an inert support with chemically bound functional groups, for example non-polar butyl, octyl, octadecyl (the most common), but more polar groups such as phenyl, cyanopropyl, amino propyl or chiral selectors for separation of enantiomers can be applied. Aqueous solutions of methanol, acetonitrile and of other solvents are used as mobile phases in the separation of non-polar analytes. Gradient of organic solvents is applied during gradient elution. In case of ionizable substances we suppress their dissociation by ph adjustment (using buffers, trifluoroacetic acid etc.) It is recommended to add ion-pairing agents (organic acids or bases) that form neutral ion pairs with ionic analytes and to carry the analysis on non-polar stationary phases (ion-pair chromatography). Ionic compounds can also be separated by ion exchange chromatography (IEC). Ionexchanger with ionizable functional groups, -SO 3 - (strong cation exchanger), -COO - (weak cation exchanger), -N + (CH 3 ) 4 (strong anion exchanger) and -NH 3 + (week anion exchanger) are use as stationary phases. Ionic analytes are exchanged with H + or OH - ions of ion exchanger. Buffers are used as mobile phases; eventually gradients of increased ph or salt concentration are applied. Ion exchange chromatography can be applied only to ionic compounds, i.e., strong acids and bases and their salts. However if the dissociation of weak acids and bases is increased by a ph change the ion exchange chromatography can also be applied to weak electrolytes. Amino acids are very often analyzed by IEC. The analyses are usually carried out in acidic medium with gradient elution, where amino acids are present in the form of cations. Size-exclusion chromatography (gel, gel permeation, SEC) is a method of choice for analysis of high-molecular mass compounds. Separation is based on size-exclusion effect. Separation depends on both the relationship of the size and shape of analytes, and size and shape of pores of the stationary phase. The smaller the molecules, the more they are retained in the stationary phase. Mobile phase is inert and does not participate in the separation. Solute retention is directly proportional to negative value of logarithm of its relative molecular mass M r : M r k 1 k2 log M r = (1.7) where k 1 and k 2 are constants. Size exclusion chromatography can be used not only to separation but also for determination of molecular mass according to Eq. (1.7). Determination of M r requires a calibration with standards of known molecular mass. The method can be applied to analysis of polypeptides, proteins, synthetic polymers and other high-molecular mass compounds, etc. Affinity chromatography is based on bio-specific interaction of biologically active compounds with complementary ligands, which are used as stationary phases after their binding to suitable support. Interactions of analytes with affinity ligands are very strong; a change of ph or elution strength of the mobile phase (usually buffer solutions) has to be 6

7 used. This method is mainly used for isolation and purification of enzymes and their inhibitors, antibodies, nucleic acids and other biologically active compounds. Instrumentation Scheme of liquid chromatograph is presented in Fig.2. Liquid mobile phase is placed in a reservoir. If a gradient elution is going to be used, another solvent is added and a programming device creates a required gradient. Mobile phase is pumped using pulseless pumps to the injector and then to the chromatographic column. Components separated in a column are detected in a detector. The detector signal is measured and evaluated by computer, which can feed back the chromatographic system. Fig. 2: Scheme of liquid chromatograph Pumps ensure constant mobile phase flow. Due to a high hydrodynamic resistance of the columns the pumps have to work at pressures as large as tens of MPa, and their running has to be pulseless. Reciprocated pumps are mostly used. Sampling multi-way valves with inner or outer loops are applied for injection. They enable the injection of liquid samples ranging from tens of nanolitres to hundreds of microlitres. Analytical columns are stainless steel (most often) or glass tubes of inner diameter between 1 to 10 mm and length of 5 to 50 cm. Capillary columns with diameters less than 1 mm are used as well. Columns are homogeneously packed with stationary phase. Various detectors are available in LC. The most common is the spectrophotometric detector. It can work in both UV and visible regions of the spectrum; eventually at any moment of analysis the whole spectrum can be recorded (diode array detector, DAD). Volume of the detection cell has to be as small as possible to prevent dispersion of separated solute zones. The UV detector is practically universal, very sensitive and its response is linear over a broad concentration range. The mobile phase has to be transparent in a given spectral region. Fluorescence detector is selective and more sensitive than UV detector. It can be applied to substances with native fluorescence or to substances that can be converted to fluorescent derivatives. Application of mass spectrometric detector (MS) is growing. It enables direct identification of substances on the basis of their mass spectra and very sensitive quantitation. 7

8 Application Liquid chromatography is used in all cases when GC fails or when its application is complicated, e.g., for non-volatile compounds. Their GC analyses would require high working temperature or their treatment to volatile derivatives. A broad range of compounds can be analyzed by LC, e.g., low molecular anions and cations (ion chromatography), polar and non-volatile compounds such as amino acids, nucleic acids, drugs and their metabolites, polyaromatic hydrocarbons and high-molecular mass substances such as proteins, synthetic polymers etc. Flat-bed Chromatography Paper chromatography (PC) and thin-layer chromatography (TLC) belong to this group. PC and TLC are separation methods with limited efficiency (ca. 300 theoretical plates). However, they are widely used for isolation of natural substances and for determination of their purity, in organic synthesis, molecular biology, microbiology, toxicology, and in other research areas, because they are simple and cheap, and require minimal instrumentation. Paper chromatography is at present replaced by TLC. A modern variant of TLC, highperformance thin-layer chromatography (HPTLC) employs efficient stationary phases with uniform small particles (similar to HPLC) and corresponding instrumentation for automatic injection, development, and detection. Precision and accuracy of quantitative evaluation in HPTLC is comparable to either GC or HPLC. PC and TLC have the same arrangements of the stationary phase: it is placed on flat support, contrary to column techniques. In paper chromatography the stationary phase is formed by a special type of paper, in thin-layer chromatography the solid stationary phase is coated in a thin layer on suitable plate. Mobile phases are similar as in LC, i.e., solvent mixtures, sometimes with the addition of acids, bases, or buffers. Analogously to other chromatographic techniques, PC and TLC are based on distribution of analytes between two phases stationary and mobile. These phases are in contact with the third phase formed by mobile phase vapors. Basic quantity that characterize the position of separated zones, is the retardation factor R F, defined as a ratio of distances reached by the sample component d i, and the front of the mobile phase, d m (Fig. 3): di R F = (1.8) d m d i distance from the start reached by analyte, d m the distance reached by mobile phase front. The R F values are within limits <0, 1>. If R F = 0, the analyte does not migrate, if R F = 1, the substance migrates with solvent front. It is difficult to obtain reproducible values of R F, especially if stationary phase, mobile phase and its vapor are not in equilibrium. That is why the standard substances have to be used in identification based on R F values. Driving forces of mobile phase movement are capillary forces. They are bigger in narrow interparticle channels and the mobile phase flow is higher there. On contrary, the wider pores are filled more slowly and the mobile phase layer in them is larger. Mobile phase flow in PC 8

9 and TLC is not constant and cannot be controlled and optimized. This is the reason of poor reproducibility of R F values and low separation efficiency.. d m d i Fig. 3: Chromatogram in PC or TLC Experimental procedure in PC and TLC: Sample is applied in the form of spots or bends on the start near the edge of the paper or plate using microcapillaries. Then the edge of the paper or plate is immersed into a mobile phase, which is placed in a chamber saturated with mobile phase. The mobile phase is permitted to elevate by the pores of the paper in PC or the stationary phase in TLC. This procedure of development is called ascending, and it is the most common one. In PC the descending development is used as well. Circular development can also be applied (see Fig. 4). Fig. 4: Various development techniques in PC and TLC 9

10 Usually a simple development is carried out but if no separation occurs the multi development can be used. Plate developed in one direction is turned over 90 o, dried and the development is repeated again but with different solvents (two-dimensional development). The mobile phase moves the analytes, which are retained by stationary phase for different times. The analysis is interrupted before the mobile phase reaches the end of the paper or plate; the paper or plate is removed from the chamber, dried, and the place where the samples are applied (start), the position of spots (zones) and solvent front are marked. In some cases when solutes migrate very slowly the overflow technique is applied. Then the chromatogram is evaluated, i.e., separated components are visualized (detected), identified, and quantitated. If the analytes are naturally colored, they are visible on chromatogram. If not, the spots have to be visualized. The plate or paper are sprayed with, or immersed into various agents (e.g., concentrated sulfuric acid, iodine, permanganate, ninhydrin), and examined in UV light, employing the natural fluorescence or quenching of fluorescence. A fluorescence indicator is an ingredient of the stationary phase on the plate and the components, which absorb at the same wavelength (quench the fluorescence), are expressed as black spots on fluorescent layer. Indicators are usually excited at 254 nm (less often at 366 nm). Quantitative evaluation is either direct or indirect. In indirect semi-quantitative method the spot is cut from a paper or scratched from a plate and then extracted with a suitable solvent. Concentration of a component in extract is then determined by e.g., spectrophotometrically or by any other method. Direct methods involve measurement of spot area (logarithm of spot area is proportional to concentration), radiochemical methods (if compounds are radioactively labeled) and a densitometry. Scanning photodensitometers are mostly used and provide the most precise results. They transfer the intensity of spot color to chromatogram with peaks and their area is proportional to the amount of analytes in spots. The whole spectrum can be recorded using densitometers. If densitometer is applied the affirmation that TLC is only a semiquantitative method is no longer valid. While the stationary phase in PC is formed by water sorbed on a paper, the same materials as in column chromatography are used in TLC: adsorbents (strongly polar silica gel is the most widely used stationary phase in TLC), ion exchangers and chemically modified phases spread in a thin-layer on a plate. Choice of the stationary phase depends on the character of separated substances (if they are hydrophilic or hydrophobic). As far as the substances are dissolved in water and are of ionic character it is important whether they are acidic or basic. Adsorbents like silica gel, aluminum oxide (alumina), polyamide, or acetylated cellulose are applied for lipophilic (hydrophilic) substances. Cellulose, ion exchangers, kieselguhr, polyamides, and chemically bound reversed phases are used for analyses of hydrophilic compounds. Adhesion of a stationary phase to the TLC plate is enhanced by additives (e.g., starch). A mixture of solvents is used as mobile phases in PC and TLC. Their composition depends on the structure of analytes and in TLC also on the stationary phase used. Their selection is governed by a rule similar dissolves in similar. There is a great number of recommended mobile phases for given analytes and stationary phases. A mixture of butanol-acetic acid-water in ratio 4:1:5 is used in PC in analyses of polar compounds. Butyl acetate, diisopropyl ether or chloroform with the addition of small amounts of polar solvents and water are suitable for medium polar analytes. Non-polar stationary phases, e.g., a paper impregnated with paraffin oil in combination with mobile phases consisting of aqueous 10

11 solution of alcohols saturated with stationary phases are used in PC for analyses of non-polar substances. Due to different separation mechanisms in TLC various mobile phases are applied and for their selection the same rules apply as in HPLC. Gas Chromatography Gas Chromatography (GC) is a separation method, in which the mobile phase is an inert gas. Stationary phase is placed in a column. It can be either an adsorbent or a liquid chemically bound to support (most often to inner walls of capillary column). Separation is therefore based either on adsorption or on partition equilibrium. Gas chromatography is an important chromatographic method due to its advantages: high sensitivity, high separation efficiency, speed and simplicity of analysis. The instrumentation is available for an acceptable price. The GC method can be applied to gaseous and volatile compounds, eventually to substances, which can be converted in defined way to volatile compounds (by derivatization, pyrolytic degradation and by other method). Instrumentation Block scheme of gas chromatograph is presented in Fig. 5. Mobile phase (carrier gas) is led from the carrier gas source (pressurized bomb with nitrogen, helium or hydrogen) through cleaning and drying devices and flow regulator to injector. Samples are injected by injection syringe through septum. After injection of sample into carrier gas stream the sample is led into the column where the separation takes place. Separated sample components are further led to a detector; the detector signal is then amplified and evaluated, usually by a computer. Injectors, columns, and detectors are independently thermostatted. Analysis can be carried out at constant temperature (isothermally) or the temperature can be changed according to a program set beforehand (temperature programming). Capillary columns from fused silica with an inner diameter of 0.1 to 0.5 with chemically modified stationary phase with film thickness up to 1.0 µm and length of 30 m are exclusively used in GC. Chemically bounded poly (dimethyl)siloxanes with various amounts of further functional groups, usually phenyl or cyanopropyl, are used as stationary phases in GC. Poly (dimethyl)siloxane phase is nonpolar; presence of phenyl groups (in the range 5-50 %) increases the polarity of stationary phase. Separation efficiency reaches thousands of plates per 1 m of the column. Nonpolar substances are best separated on nonpolar stationary phase while the polar ones on polar phase. On nonpolar phases the retention is governed by boiling points of analytes (the lower the boiling point, i.e., the higher the volatility, the shorter retention). In case of polar substances, in addition to nonpolar, polar interactions take place as well. Various detectors are available in GC. The most commonly used is the sensitive flame ionization detector (FID). Substances eluted from the column are burnt in a hydrogen-oxygen flame where they are ionized. Current formed is amplified and measured. Flame ionization detector gives response to almost all organic substances. Universal detector (for both organic and inorganic substances) is the thermal conductivity detector (TCD), which measures the differences in the thermal conductivity of analytes and the carrier gas. The sensitivity of this detector is low. Electron-capture detector (ECD) is selective for substances containing electronegative atoms, mainly chlorine, bromine, fluorine, and it is very sensitive. It is often employed in the analyses of polychlorinated environmental contaminants. Using the electron-capture detector, the identification of analytes is possible only on the bases of retention data agreements with standards. Similarly to HPLC, the mass spectrometric detector has an important place. It is a universal and very sensitive detector. It provides not only quantitative content of the component in analyzed sample, but also information about their structure. Its usage is 11

12 prerequisite in analyses of complicated mixtures of unknown composition, e.g., in analysis of unknown drugs or their metabolite. Fig. 5: Scheme of gas chromatograph Application Gas chromatography is mainly used in analyses of gases, volatile organic compounds, but also for less volatile compounds. Commercial stationary phases are stable at high temperatures (up to C and more), so that it is possible to analyze for example polychlorinated biphenyls, pesticides, steroids etc. Polar compounds, such as amino acids and fatty acids, can be analyzed by GC after their derivatization. Various derivatives are used, e.g., alkyl and silyl ethers and esters, methyl derivatives etc. High-molecular mass compounds, e.g., synthetic polymers, can be thermally decomposed under defined conditions to low-molecular mass fragments (monomers, oligomers, etc.), which are characteristic for a given polymer. 12