Analytical Chemistry (Chromatographic Analysis) - Past Paper PDF

1 Analytical Chemistry (Chromatographic Analysis) Third year of Special Chemistry 2 Contents 1- Chapter 1: Analytical Methodologies 2- Chapter 2: Introduction of chromatography 3- Chapter 3: Electrophoreses 4- Chapter 4...

1 Analytical Chemistry (Chromatographic Analysis) Third year of Special Chemistry 2 Contents 1- Chapter 1: Analytical Methodologies 2- Chapter 2: Introduction of chromatography 3- Chapter 3: Electrophoreses 4- Chapter 4: Gas Chromatography (GC) 5- Chapter 5: High Performance Liquid Chromatography (HPLC) 6- Chapter 6: Planer Chromatography 3 Chapter 1 ANALYTICAL METHODOLOGIES Qualitative Analysis: 4 The qualitative analysis provides presence/absence information only (i.e., microbial screening). Quantitative Analysis: The quantitative Analysis provides a measure of concentration (a number plus units) 1- Classical methods (wet chemistry): i- Gravimetric analysis (based on mass of analyte containing precipitate) ii- Volumetric analysis (based on the volume of titrant required to reach endpoint) Classical methods are generally accurate and precise, but may be time consuming and are usually limited to analysis of reasonably high concentrations of analyte (i.e., > 1 ppm) 2- Instrumental methods measure some form of a sensor/detector signal (usually a voltage or current) that is related either directly or indirectly to the analyte concentration via a calibration process. Instrumental methods are generally accurate, precise and readily automated, however require careful calibration procedures. i- Electrochemical devices convert chemical concentrations directly into a measured voltage potential or electric current. examples: dissolved oxygen (DO) meter ion selective electrode (ISE) 5 ii- Spectrophotometric instruments involve the interaction of the analyte with electromagnetic radiation (light) examples: UV/vis (colorimetric) atomic absorption spectrophotometry (AAS) atomic emission spectrophotometry (AES) atomic fluorescence spectrophotometry (AFS) iii- Chromatographic instruments serve to separate and quantify members of a closely related series of analytes examples: gas chromatography (GC) high performance liquid chromatography (HPLC) ion chromatography (IC) capillary electrophoresis (CE) 3- Tandem Instrumental methods combine instruments in sequence, often coupling chromatographic separation with sensitive and selective detectors, such as mass spectrometry (MS) example: GC-MS/MS Gravimetric Analysis A precipitating reagent is added to convert a soluble analyte species into an insoluble solid that contains the analyte in some known combination. The solid precipitate is collected, dried and the mass recorded. Mass of precipitate is directly related to the mass of the analyte present in a known volume of sample. Volumetric Analysis 6 A measured volume of a reagent of known concentration (the titrant) is added to a sample until a specified change occurs – the ‘end-point’. The volume and concentration of titrant are related the concentration of the analyte in sample titrated. Types of titrations i- Acid/Base ii- Oxidation/Reduction iii- Precipitation iv- Complex ion formation Electrochemical Devices An electrical signal (voltage or current) is dependent on the concentration of the analyte. Meters must be calibrated using solutions of known concentration (standard solutions) and measurements must be temperature compensated. Spectrophotometric (Molecular and Atomic) 1- Molecular spectrophotometry (UV/Vis): A chemical reagent is added to the sample, which selectively combines with the analyte resulting in some measurable color change. The intensity the colour (as measured by the absorbance of light) is related to the concentration of analyte by Beer’s Law. 7 2- Atomic Spectrophotometry: A sample is ‘atomized’ in a flame or high temperature plasma. The atoms of each element absorb or emit discrete quanta of electromagnetic energy (light) characteristic of their individual identities. The intensity of light absorbed or emitted is related to the concentration of the element. Chromatographic Techniques An analyte or mixtures of analytes are separated on the basis of selective partitioning between a mobile and stationary phase. Analytes are then quantified with an internal non- selective detector Specialty and Tandem Instruments i- Turbidity meter (nephelometer) ii- Total Organic Carbon (TOC) Analyzer iii- Mass Spectrometer Detector for GC, HPLC and IC Field Portable Equipment/Instruments i- Digital titrators, pH and DO meters ii- Portable spectrometers (Hach DR 2000) 8 iii- Field GC-MS instruments Chapter 2 Introduction of Chromatography 9 Chromatography (from Greek Chroma, color and graph in to write is the collective term for a set of laboratory techniques for the separation mixtures. It involves passing a mixture dissolved in a "mobile phase" through a stationary phase, which separates the analyte to be measured from other molecules in the mixture based on differential partitioning between the mobile and stationary phases. Subtle differences in a compound's partition coefficient result in differential retention on the stationary phase and thus changing the separation. Chromatography may be preparative or analytical. The purpose of preparative chromatography is to separate the components of a mixture for further use (and is thus a form of purification). Analytical chromatography is done normally with smaller amounts of material and is for measuring the relative proportions of analyte in a mixture. The two are not mutually exclusive. Chromatography is a physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary (stationary phase) while the other (the mobile phase) moves in a definite direction. 10 Discovery of Chromatography Chromatography is a physicochemical method for separation of complex mixtures was discovered at the very beginning of the twentieth century by Russian–Italian botanist M.S. Tswett. “On the new form of adsorption phenomena and its application in biochemical analysis” presented on March 21, 1903 at the regular meeting of the biology section of the Warsaw Society of Natural Sciences, Tswett gave a very detailed description of the newly discovered phenomena of adsorption-based separation of complex mixtures, which he later called “chromatography” as a transliteration from Greek “color writing”. Serendipitously, the meaning of the Russian word “Tswett” actually means color. Although in all his publications Tswett mentioned that the origin of the name for his new method was based on the colorful picture of his first separation of plant pigments, he involuntarily incorporated his own name in the name of the method he invented. The chromatographic method was not appreciated among the scientists at the time of the discovery, as well as after almost 10 years when L. S. Palmer in the United States and C. Dhere in Europe independently published the description of a similar separation processes. Prior to the 1970's, few reliable chromatographic methods were commercially available to the laboratory scientist. During 1970's, most chemical separations were carried out using a variety of techniques including open-column chromatography, paper chromatography, and thin- layer chromatography. However, these chromatographic techniques were inadequate for quantification of compounds and resolution between similar compounds. During this time, 11 pressure liquid chromatography began to be used to decrease flow through time, thus reducing purification times. The history of chromatography begins during the mid-19th century. Chromatography, literally "color writing", was used and named in the first decade of the 20th century, primarily for the separation of plant pigments such as chlorophyll. New types of chromatography developed during the 1930s and 1940s made the technique useful for many types of separation process. Some related techniques were developed during the 19th century (and even before), but the first true chromatography is usually attributed to Russian botanist M. Tswett, who used columns of calcium carbonate for separating plant pigments during the first decade of the 20th century during his research of chlorophyll. Relation between Analytical Chemistry and Chromatography The types of analysis can be distinguished in two ways: 1- Qualitative Analysis: To refer identity of product, i.e., it yields useful clues from which the molecular or atomic species, the structural features, or the functional groups in the sample can be identified. 2- Quantitative Analysis: To refer the purity of the product, i.e., the results are in the form of numerical data corresponding to the concentration of analyte. 12 Chromatography terms Analyte: is the substance to be separated during chromatography. Analytical chromatography: is used to determine the existence and possibly also the concentration of analyte(s) in a sample. A bonded phase: is a stationary phase that is covalently bonded to the support particles or to the inside wall of the column tubing. A chromatogram is the visual output of the chromatograph. In the case of an optimal separation, different peaks or patterns on the chromatogram correspond to different components of the separated mixture. Plotted on the x-axis is the retention time and plotted on the y-axis a signal (for example obtained by a spectrophotometer, mass 13 spectrometer or a variety of other detectors) corresponding to the response created by the analytes exiting the system. In the case of an optimal system the signal is proportional to the concentration of the specific analyte separated A chromatograph: is equipment that enables a sophisticated separation e.g gas chromatographic or liquid chromatographic separation. Chromatography: is a physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary which is stationary phase while the other (the mobile phase) moves in a definite direction. The eluent: is the mobile phase leaving the column. An eluotropic series: is a list of solvents ranked according to their eluting power. The mobile phase: is the phase which moves in a definite direction. It may be a liquid (LC), a gas (GC), or a supercritical fluid (supercritical-fluid chromatography, SFC). The mobile phase consists of the sample being separated/analyzed and the solvent that moves the sample through the column. In the case of HPLC the mobile phase consists of a non-polar solvent(s) such as hexane in normal phase or polar solvents in reverse 14 phase chromatography and the sample being separated. The mobile phase moves through the chromatography column (the stationary phase) where the sample through the chromatography column (the stationary phase) where the sample interacts with the stationary phase and is separated Preparative chromatography: is used to purify sufficient quantities of a substance for further use, rather than analysis Retention time: is the characteristic time it takes for a particular analyte to pass through the system (from the column inlet to the detector) under set conditions. Sample: is the matter analyzed in chromatography. It may consist of a single component or it may be a mixture of components. When the sample is treated in the course of an analysis, the phase or the phases containing the analytes of interest is/are referred to as the sample whereas everything out of interest separated from the sample before or in the course of the analysis is referred to as waste. Solute refers to the sample components in partition chromatography. The solvent refers to any substance capable of solubilizing other substance, and especially the liquid mobile phase in LC. 15 Stationary phase is the substance which is fixed in place for the chromatography procedure. Examples include the silica layer in thin layer chromatography Types of Chromatography Chromatography can be classified by various ways: (I) On the basis of interaction of solute to the stationary phase (II) On the basis of chromatographic bed shape (III) Techniques by physical state of mobile phase Techniques by chromatographic bed shape: 1- Column chromatography 2- Planar chromatography i- Paper chromatography ii- Thin layer chromatography 16 iii- Displacement chromatography Techniques by physical state of mobile phase: 1. Gas chromatography 2. Liquid chromatography 3. Affinity chromatography 4. Supercritical fluid chromatography Techniques by separation mechanism: 1. Ion exchange chromatography 2. Size exclusion chromatography16 Special techniques: 17 1. Reversed-phase chromatography 2. Two-dimensional chromatography 3. Simulated moving-bed chromatography 4. Pyrolysis gas chromatography 5. Fast protein liquid chromatography 6. Countercurrent chromatography 7. Chiral Chromatography Chromatographic bed shape 1. Column Chromatography Column chromatography is a separation technique in which the stationary bed is within a tube. The particles of the solid stationary phase or the support coated with a liquid stationary phase may fill the whole inside volume of the tube (packed column) or be conce ntrated on or along the inside tube wall leaving an open, unrestricted path for the mobile phase in the middle part of the tube (open tubular column). Differences in rates of movement through the medium are calculated to different retention times of the sa mple. In expanded bed adsorption, a fluidized bed is used, rather than a solid phase made by a 18 packed bed. This allows omission of initial clearing steps such as centrifugatio n and filtration, for culture broths or slurries of broken cells. 19 2. Planer Chromatography Planar chromatography is a separation technique in which the stationary phase is present as or on a plane. The plane can be a paper, serving as such or impregnated by a substance as the stationary bed (paper chromatography) or a layer of solid particles spread on a support such as a glass plate (thin layer chromatography). Different compounds in the sample mixture travel different distances according to how strongly they interact with the stationary phase as compared to the mobile phase. The specific Retention factor (Rf) of each chemical can be used to aid in the identification of an unknown substance. 2.1. Paper Chromatography Paper chromatography is a technique that involves placing a small dot or line of sample solution onto a strip of chromatography paper. The paper is placed in a jar containing a shallow layer of solvent and sealed. As the solvent rises through the paper, it meets the sample mixture which starts to travel up the paper with the solvent. This paper is made of cellulose, a polar 20 substance, and the compounds within the mixture travel farther if they are non-polar. More polar substances bond with the cellulose paper more quickly, and therefore do not travel as far. 2.2. Thin layer Chromatography Thin layer chromatography (TLC) is a widely employed laboratory technique and is similar to paper chromatography. However, instead of using a stationary phase of paper, it involves a stationary phase of a thin layer of adsorbent like silica gel, alumina, or 20 cellulose on a flat, inert substrate. Compared to paper, it has the advantage of faster runs, better separations, and the choice between different adsorbents. For even better resolution and to allow for quantification, high-performance TLC can be used. 2.3 Displacement Chromatography The basic principle of displacement chromatography is, “A molecule with a high affinity for the chromatography matrix (the displacer) will compete effectively for binding sites, and thus displace all molecules with lesser affinities”. There are distinct differences between displacement and elution chromatography. In elution mode, substances typically emerge from a column in narrow, Gaussian peaks. Wide 21 separation of peaks, preferably to baseline, is desired in order to achieve maximum purification. The speed at which any component of a mixture travels down the column in elution mode depends on many factors. But for two substances to travel at different speeds, and thereby be resolved, there must be substantial differences in some interaction between the biomolecules and the chromatography matrix. Operating parameters are adjusted to maximize the effect of this difference. In many cases, baseline separation of the peaks can be achieved only with gradient elution and low column loadings. Thus, two drawbacks to elution mode chromatography, especially at the preparative scale, are operational complexity, due to gradient solvent pumping, and low throughput, due to low column loadings. Displacement chromatography has advantages over elution chromatography in that components are resolved into consecutive zones of pure substances rather than “peaks”. Because the process takes advantage of the nonlinearity of the isotherms, a larger column feed can be separated on a given column with the purified components recovered at significantly higher concentrations. Physical state of mobile phase 1. Gas Chromatography Gas chromatography (GC), also sometimes known as Gas-Liquid chromatography, (GLC), is a separation technique in which the mobile phase is a gas. Gas chromatography is 22 always carried out in a column, which is typically "packed" or "capillary". Gas chromatography (GC) is based on a partition equilibrium of analyte between a solid stationary phase (often a liquid silicone-based material) and a mobile gas (most often Helium). The stationary phase is adhered to the inside of a small-diameter glass tube (a capillary column) or a solid matrix inside a larger metal tube (a packed column). It is widely used in analytical chemistry; though the high temperatures used in GC make it unsuitable for high molecular weight biopolymers or proteins (heat will denature them), frequently encountered in biochemistry, it is well suited for use in the petrochemical, environmental monitoring, and industrial chemical fields. It is also used extensively in chemistry. 2. Liquid Chromatography Liquid chromatography (LC) is a separation technique in which the mobile phase is a liquid. Liquid chromatography can be carried out either in a column or a plane. Present day liquid chromatography that generally utilizes very small packing particles and a relatively high pressure is referred as high performance liquid chromatography (HPLC). In the HPLC technique, the sample is forced through a column that is packed with irregularly or spherically shaped particles or a porous monolithic layer (stationary phase) by a liquid (mobile phase) at high pressure. HPLC is historically divided into two different sub- classes based on the polarity of the mobile and stationary phases. Technique in which the stationary phase is more polar than the mobile phase (e.g. toluene as the mobile phase, silica as 23 the stationary phase) is called normal phase liquid chromatography (NPLC) and the opposite (e.g. water-methanol mixture as the mobile phase and C18 = octadecylsilyl as the stationary phase) is called reversed phase liquid chromatography (RPLC). Ironically the "normal phase" has fewer applications and RPLC is therefore used considerably more 3. Affinity chromatography Affinity chromatography is based on selective non-covalent interaction between an analyte and specific molecules. It is very specific, but not very robust. It is often used in biochemistry in the purification of proteins bound to tags. These fusion proteins are labeled with compounds such as His-tags, biotin or antigens, which bind to the stationary phase specifically. After purification, some of these tags are usually removed and the pure protein is obtained. Affinity chromatography often utilizes a biomolecule's affinity for a metal (Zn, Cu, Fe, etc.). Columns are often manually prepared. Traditional affinity columns are used as a preparative step to flush out unwanted biomolecules. However, HPLC techniques exist that do utilize affinity chromatography properties. Immobilized Metal Affinity Chromatography (IMAC) is useful to separate aforementioned molecules based on the relative affinity for the metal (Dionex IMAC). Often these columns can be loaded with different metals to create a column with a targeted affinity. 24 4. Supercritical fluid chromatography Supercritical fluid chromatography is a separation technique in which the mobile phase is a fluid above and relatively close to its critical temperature and pressure. Based on the mode of separation 1- Normal phase chromatography: It was one of the first kinds of HPLC that chemists developed. In this type stationary phase used is polar in nature and the mobile phase used is non-polar and nonaqueous in nature. If the affinity between the stationary phase and the analyte increases the selection time (RT) of the analyte also increases and vice versa. The interaction strength depends not only on the functional groups in the analyte molecule but also on steric factors. The effect of steric on interaction strength allows this method to resolve (separate) structural isomers. 2. Reverse phase chromatography In reverse phase technique, a non-polar stationary phase is used and the mobile phase is polar in nature. Hence polar components get eluted first and nonpolar compounds are retained for a longer time. Since most of the drugs and pharmaceuticals are polar in nature, they are not 25 retained for a longer time and eluted faster, columns used in the mode of chromatogram are ODS (Octadecyl silane) or C18, C8, C4, etc. 3. Partition chromatography Partition chromatography was the first kind of chromatography that chemists developed. The partition coefficient principle has been applied in paper chromatography, thin layer chromatography, gas phase and liquid-liquid applications. Partition chromatography uses a retained solvent, on the surface or within the grains or fibers of an "inert" solid supporting matrix as with paper chromatography; or takes advantage of some additional columbic and/or hydrogen donor interaction with the solid support. Molecules equilibrate (partition) between a liquid stationary phase and the eluent separate analytes based on the polar differences is known as Hydrophilic interaction chromatography (HILIC). Partition HPLC has been used historically on unbonded silica or alumina supports. Each works effectively for separating analytes by relative polar differences. However, HILIC has the advantage of separating acidic, basic and neutral solutes a single chromatogram. 26 Based on principle of separation: 1. Adsorption chromatography: When a mixture of compounds (adsorbate) dissolved in the mobile phase (eluent) moves through a column of stationary phase (adsorbent) they travel according to their relative affinities. The compound which has more affinity towards stationary phase travels slower, if less affinity towards stationary phase travels faster. 2. Ion exchange chromatography: It is the process by which a mixture of similar charged ions can be separated using ion exchange resin. There is a reversible exchange of ions between the ions present in the column. And those present in the ion exchange resin. For cations, cation exchange resin and for anions, an anion exchange resin is used. 3. Size exclusion chromatography It is the process by which mixture of compounds with molecular sizes are separated by using gels. The gel used acts as molecular sieve. It can be separated by steric and diffusion effects of pores in the gels. The compound can separate according to the molecular sizes and the stationary phase is a porous matrix. Eg: separation of proteins and polysaccharides. 27 Chapter 3 Electrophoresis Electrophoresis 28 The term electrophoresis means any experimental technique that is based on movement of charged particles (ions, molecules, macromolecules) in electric field in liquid medium. Any electrically charged particle dissolved in aqueous solution, when placed to a constant electric field, will start to migrate towards the electrode bearing the opposite charge; the speed of the particle movement will be directly proportional to the applied voltage and particle charge, but inversely proportional to the particle size. Any molecules that differ in size and/or charge can be separated from each other in this way. The electrophoretic analysis can in principle be applied to any particles that are charged under given experimental condition, such as small cations or anions, organic acids, amino acids, peptides, saccharides, lipids, proteins, nucleotides, nucleic acids, even the whole subcellular particles or the whole cells. In practice, however, the by far commonest subjects of electrophoretic separation are proteins and nucleic acids. Electrophoresis of macromolecules is normally carried out by applying a thin layer of a sample to a solution stabilized by a porous matrix. Under the influence of an applied voltage, different species of molecules in the sample move through the matrix at different velocities Discovery of electrophoresis 29 The movement of particles under spatially uniform electric field in a fluid is called electrophoresis. In 1807, Ferdinand Frederic Reuss observed clay particles dispersed in water to migrate on applying constant electric field for the first time. It is caused by a charged interface present between the particle surface and the surrounding fluid. The rate of migration of particle depends on the strength of the field, on the net charge size and shape of the molecules and also on the ionic strength, viscosity and temperature of medium in which the molecules are moving. As an analytical tool, electrophoresis is simple, rapid and highly sensitive. It is used analytically to study the properties of a single charged species and as a separation technique. It provides the basis for a number of analytical techniques used for separating molecules by size, charge, or binding affinity, example- for the separation of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or protein molecules using an electric field applied to a gel matrix. Gel matrix used mainly is polyacrylamide and agarose. DNA Gel electrophoresis is usually performed for analytical purposes, often after amplification of DNA via PCR, but may be used as a preparative technique prior to use of other methods such as mass spectrometry, RFLP, PCR, cloning, DNA sequencing, or Southern blotting for further characterization. 30 Techniques of electrophoresis Gel electrophoresis Gel electrophoresis can provide information about the molecular weights and charges of proteins, the subunit structures of proteins, and the purity of a particular protein preparation. It is relatively simple to use and it is highly reproducible. The most common use of gel electrophoresis is the qualitative analysis of complex mixtures of proteins. Microanalytical methods and sensitive, linear image analysis systems make gel electrophoresis popular for quantitative and preparative purposes as well. The technique provides the highest resolution of all methods available for separating proteins. Polypeptides differing in molecular weight by as little as a few hundreds of daltons and proteins differing by less than 0.1 pH unit in their isoelectric points are routinely resolved in gels. Agarose gel electrophoresis In particular, agarose gel electrophoresis is generally used to separate DNA (single- stranded, double-stranded, and supercoiled) and RNA. Since DNA is negatively charged, it migrates in an electric field toward the positively charged cathode. The agarose matrix retards DNA migration roughly proportionally to DNA length when the DNA being separated is small. Longer oligonucleotides have a harder time traveling through the matrix, while shorter oligonucleotides (and small molecules such as ATP) breeze right through it. 31 Types of agarose There are a few different types of agarose available. For analytical purposes, such as running digested plasmids to see whether a ligation was successful, you can usually use agarose from USB. However, if you want to recover your DNA and/or perform some in-gel reactions, you should use the low melting agarose (the Nu Sieve GTG, etc). These specific agarose protocols are usually provided with the reagent and are available online. Agarose can be used for isoelectric focusing and separation of large proteins or protein complexes. Agarose is a highly purified polysaccharide derived from agar. For protein IEF applications, the critical qualities arelow EEO and good clarity at the working concentration. When used in a thin horizontal format for IEF, agarose gels must be supported on a plastic backing and cooled during electrophoresis. Agarose is normally purchased as a dry powder. It dissolves when the suspended powder is heated to near boiling and it remains liquid until the temperature drops to about 40 ºC, when it gels or “sets.” There are specific types of agarose that have melting and gelling temperatures considerably lower than those of standard agarose. These properties improve the recovery of material from a gel after separation for subsequent enzymatic treatments of the separated material. The pore size and sieving characteristics of a gel are determined by adjusting the concentration of agarose in the gel. The higher the concentration, the smaller the pore size. Working concentrations are normally in the range of 0.4–4% w/v. 32 Agarose gels are relatively fragile and should be handled carefully. The gels are hydrocolloids, held together by hydrogen and hydrophobic bonds, and tend to be somewhat brittle (Fig 1.5a). An agarose gel should always be handled with some form of support for the entire gel, such as a tray or wide spatula, because the gel will break if it bends too far. Agarose and polyacrylamide gels are cross-linked, sponge like structures. Although they are up to 99.5% water, the size of the pores of these gels is similar to the sizes of many proteins and nucleic acids. As molecules are forced through the gel by the applied voltage, larger molecules are retarded by the gel more than are smaller molecules. For any particular gel, molecules smaller than a matrix-determined size are not retarded at all; they move almost as if in free solution. At the other extreme, molecules larger than a matrix-determined size cannot enter the gel at all. Gels can be tailored to sieve molecules of a wide range of sizes by appropriate choice of matrix concentration. The average pore size of a gel is determined by the percentage of solids in the gel and, for polyacrylamide, the amount of cross-linker and total amount of polyacrylamide used Polyacrylamide, which makes a small-pore gel, is used to separate most proteins, ranging in molecular weight from 200,000, and polynucleotides from 5 × 106 base pairs 33 long. Whichever matrix is selected, it is important that it be electrically neutral. Charged matrices may interact chromatographically with molecules and retard migration. The presence of fixed charged groups on the matrix will also cause the flow of water toward one or the other electrode, usually the cathode. This phenomenon, called electro endosmosis (often abbreviated EEO in supplier literature), usually decreases the resolution of separation. Polyacrylamide gels electrophoresis Polyacrylamide gels are physically tougher than agarose gels. The gel forms when a mixed solution of acrylamide and cross-linker monomers copolymerize into long chains that are covalently cross-linked. The gel structure is held together by the cross-linker (Fig 1.5b). The most common cross-linker is N,N'-methylene bisacrylamide (“bis” for short). Other cross- linkers that can be cleaved after polymerization are available (e.g.N,N'-bis-[acryloyl]-cystamine can be cleaved by disulphide reducing agents); they aid in recovering separated species from the gel by allowing the polymerized acrylamide to be solubilized. Because polymerization of acrylamide is a free-radical catalyzed reaction, preparation of polyacrylamide gels is somewhat more complex than that of agarose gels. Some of the technical issues are discussed in the following sections. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is the most commonly practiced gel electrophoresis technique used for proteins. The method provides an 34 easy way to estimate the number of polypeptides in a sample and thus assess the complexity of the sample or the purity of a preparation. SDS-PAGE is particularly useful for monitoring the fractions obtained during chromatographic or other purification procedures. It also allows samples from different sources to be compared for protein content. One of the more important features of SDS-PAGE is that it is a simple, reliable method with which to estimate the molecular weights of proteins. SDS-PAGE requires that proteins be denatured to their constituent polypeptide chains, so that is limited in the information it can provide. In those situations where it «is desirable to maintain biological activity or antigenicity, non-denaturing electrophoresis systems must be employed. However, the gel patterns from non-denaturing gels are more difficult to interpret than are those from SDS-PAGE. Non-denaturing systems also give information about the charge isomers of proteins, but this information is best obtained by isoelectric focusing (IEF; see the entry on IEF in the AES website). An IEF run will often show heterogeneity due to structural modifications that is not apparent in other types of electrophoresis. Proteins thought to be a single species by SDS-PAGE analysis are sometimes found by IEF to consist of multiple species. A true determination of the purity of a protein preparation is obtained with two- dimensional polyacrylamide gel electrophoresis (2-D PAGE) that combines IEF with SDS- PAGE. Since 2-D PAGE is capable of resolving over 2,000 proteins in a single gel it is important as the primary tool of proteomics research where multiple proteins must be separated for parallel analysis (see the Application Focus on 2-D PAGE on this website). Proteins can be definitively identified by immunoblotting, which combines antibody specificity with the high 35 resolution of gel electrophoresis. Finally, gel electrophoresis lends itself to protein purification for which purpose35various devices have been developed. Polymerizing the gel The free-radical vinyl polymerization of acrylamide gel can be initiated either by chemical peroxide or by a photochemical method. The most common method uses ammonium persulphate as the initiat or peroxide and the quaternary amine, N,N,N',N'-tetramethylethylene diamine (TEMED) as the catalyst. For photochemical polymerization, riboflavin and long-wave UV light are the initiator, and TEMED is the catalyst. Shining long-wavelength ultraviolet light on the gel mixture, usually from a fluorescent light, starts the photochemical reaction. Photochemical polymerization is used when the ionic strength in the gel must be very low, because only a minute amount of riboflavin is required. It is also used if the protein studied is sensitive to ammonium persulphate or the by-products of peroxide-initiated polymerization. Polymerization of acrylamide generates heat. Rapid polymerization can generate too much heat, causing convection inconsistencies in the gel structure and occasionally breaking glass plates. It is a particular problem for high-concentration gels (>20%T). To prevent excessive heating, the concentration of initiator-catalyst reagents should be adjusted so that complete polymerization requires 20–60 min. 36 Chapter 4 37 Gas Chromatography Gas chromatography Is a common type of chromatography used in analytical chemistry for separating and analyzing compounds that can be vaporized without decomposition. Typical uses of GC include testing the purity of a particular substance, or separating the different components of a mixture (the relative amounts of such components can also be determined). In some situations, GC may help in identifying a compound. In preparative chromatography, GC can be used to prepare pure compounds from a mixture. Gas chromatography is in principle similar to column chromatography (as well as other forms of chromatography such as HPLC, TLC), but has several notable differences. First, the process of separating the compounds in a mixture is carried out between a liquid stationary phase and a gas mobile phase, whereas in column chromatography the stationary phase is a solid and the mobile phase is a liquid. (Hence the full name of the procedure is "Gas–liquid 38 chromatography", referring to the mobile and stationary phases, respectively.) Second, the column through which the gas phase passes is located in an oven where the temperature of the gas can be controlled, whereas column chromatography (typically) has no such temperature control. Finally, the concentration of a compound in the gas phase is solely a function of the vapor pressure of the gas. 2. ORIGINS OF GAS CHROMATOGRAPHY The development of GC as an analytical technique was pioneered by Martin and Synge 1941; they suggested the use of gas-liquid partition chromatograms for analytical purposes. When dealing with liquid-liquid partition chromatography, they predicted that the mobile phase need not be a liquid but may be a vapor. Very refined separations of volatile substances on a column in which a permanent gas is made to flow over a gel impregnated with a non-volatile solvent would be much faster and thus, the columns much more efficient and separation times much shorter. 39 3. WHY CHOOSE GAS CHROMATOGRAPHY The two main chromatographic techniques used in modern analytical chemistry are Gas Chromatography (GC) and High Performance Liquid Chromatography (HPLC). HPLC uses a liquid mobile phase to transport the sample components (analyte) through the column, which is packed with a solid stationary phase material. In contrast, gas chromatography uses a gaseous mobile phase to transport sample components through either packed columns or hollow capillary columns containing a polymeric liquid stationary phase. In most cases, GC columns have smaller internal diameter and are longer than HPLC columns. GC has developed into a sophisticated technique since the pioneering work of Martin and James in 1951, and is capable of separating very complex mixtures of volatile analytes. GAS INLETS: Gas is fed from cylinders through supply piping to the instrument. It is usual to filter gases to 40 ensure high gas purity and the gas supply may be regulated at the bench to ensure an appropriate supply pressure. Gas filters: required for a GC instrument with Flame Ionization (FID) detector. Required gases might Carrier: (H2, He, N2) Make-up gas: (H2, He, N2) Detector Fuel Gas: (H2/Air, Ar, Ar/CH4, N2) depending on the detector type The purity of the carrier gas is also frequently determined by the detector, though the level of sensitivity needed can also play significant role. 41 GC instrument 1- PNEUMATIC CONTROLS: The gas supply is regulated to the correct pressure (or flow) and then fed to the required part of the instrument. Control is usually required to regulate the gas coming into the instrument and then to supply the various parts of the instrument. A GC fitted with a Split/Splitless inlet, capillary GC column and Flame Ionization detector may have the following different gas specifications: 42 Carrier gas supply pressure, column inlet pressure (column carrier gas flow), inlet split flow, inlet septum purge flow, detector air flow, detector hydrogen flow, detector make-up gas flow. Modern GC instruments have Electronic Pneumatic pressure controllers – older instruments may have manual pressure control via regulators. 2- COLUMN: In GC, retention of analyte molecules occurs due to stronger interactions with the stationary phase than the mobile phase. This is unique in GC and, therefore, interactions between the stationary phase and analyte are of great importance. The interaction types can be divided into three broad categories:  Dispersive  Dipole  Hydrogen bonding The sample is separated into its constituent components in the column. Columns vary in length and internal diameter depending on the application type and can be either packed or capillary. Packed columns (typical dimension 1.5 m x 4 mm) are packed with a solid support coated with immobilized liquid stationary phase material (GLC). Capillary columns (typical dimension 30m×0.32mm×0.1mm film thickness) are long hollow silica tubes with the inside wall of the column coated with immobilized liquid stationary phase material of various film thickness. Many different stationary phase chemistries are available to suit a host of applications. Columns may also contain solid stationary phase particles (GSC) for particular application types. 43 3- COLUMN OVEN: Temperature in GC is controlled via a heated oven. The oven heats rapidly to give excellent thermal control. The oven is cooled using a fan and vent arrangement usually at the rear of the oven. A hanger or cage is usually included to support the GC column and to prevent it touching the oven walls as this can damage the column. The injector and detector connections are also contained in the GC oven. For Isothermal operation, the GC is held at a steady temperature during the analysis. In temperature programmed GC (pTGC) the oven temperature is increased according to the temperature program during the analysis. GC temperature 44 4- DETECTOR: The detector responds to a physicochemical property of the analyte, amplifies this response and generates an electronic signal for the data system to produce a chromatogram. Many different detector types exist and the choice is based mainly on application, analyte chemistry and required sensitivity – also on whether quantitative or qualitative data is required. Detector choices include:  Flame Ionization (FID)  Electron Capture (ECD)  Flame Photometric (FPD)  Nitrogen Phosphorous (NPD)  Thermal Conductivity (TCD  Mass Spectrometer (MS) 5- Data System: The data system receives the analogue signal from the detector and digitizes it to form the record of the chromatographic separation known as the ‘Chromatogram’ (Figure 4). The data system can also be used to perform various quantitative and qualitative 45 The following information gives an indication of the type of sample (analyte) analyzed by either GC and HPLC and relative strengths and limitations of each technique. GC: SAMPLES ANALYZED BY GC MUST BE VOLATILE (HAVE A SIGNIFICANT VAPOR PRESSURE BELOW 250 °C) o Derivatization to increase volatility is possible but can be cumbersome and introduces possible quantitative errors o Most GC analytes are under 500 Da Molecular Weight for volatility purposes o Highly polar analytes may be less volatile than suspected when dissolved in a polar solvent or in the presence of other polar species due to intermolecular forces such as hydrogen bonding. o 4. Gas Chromatography Separation Mechanism 46 In Gas Chromatography (GC) the mobile phase is a gas and the stationary phase is either a solid - Gas solid chromatography (GSC) or an immobilized polymeric liquid - Gas Liquid Chromatography (GLC). Of the two types of GC, GLC is by far the most common as will be seen. (Figure 5) shows a typical separation process in GC. Each sample component ‘partitions’ between the gaseous mobile phase and liquid stationary phase (often coated onto the inner wall of a long thin capillary tube). The rate and degree of partitioning depends upon the chemical affinity of the analyte for the stationary phase and the analyte vapor pressure – which is governed by the column temperature. From it can be seen that component A has a lower affinity for the stationary phase and therefore is moved through the column more quickly than component B, which spends more of its time in the stationary phase – in this way separation is achieved. 47 IN GC, ANALYTE SEPARATION IS ACHIEVED BY OPTIMIZING THE DIFFERENCES IN STATIONARY PHASE AFFINITY AND THE RELATIVE VAPOR PRESSURES OF THE ANALYTES. IN PRACTICE THESE PARAMETERS ARE MANIPULATED BY CHANGING THE CHEMICAL NATURE OF THE STATIONARY PHASE AND THE COLUMN TEMPERATURE THE DISTRIBUTION COEFFICIENT (PARTITION COEFFICIENT) (KC) The ‘distribution coefficient’ measures the tendency of an analyte to be attracted to the stationary phase (Equation 1). Large Kc values lead to longer retention analyte times. The value of Kc can be controlled by the chemical nature of the stationary phase and the column temperature. [CS] [CM] WHERE: CS = CONCENTRATION OF ANALYTE IN THE STATIONARY PHASE AND CM = CONCENTRATION OF ANALYTE IN THE MOBILE PHASE 5. THE GAS CHROMATOGRAPH Instrumentation for Gas chromatography has continually evolved since the inception of the technique in 1951 and the introduction of the first commercial systems in 1954. Most modern commercial GC systems operate in the following way : 48 o An inert carrier gas, such as helium, is supplied from gas cylinders to the GC where the pressure is regulated using manual or electronic (pneumatic) pressure controls o The regulated carrier gas is supplied to the inlet and subsequently flows through the column and into the detector o The sample is injected into the (usually) heated injection port where it is volatilized and carried into the column by the carrier gas o The sample is separated inside the column - usually a long silica based column with small internal diameter. The sample separates by differential partition of the analytes between the mobile and stationary phases, based on relative vapor pressure and solubility in the immobilized liquid stationary phase o On elution from the column, the carrier gas and analytes pass into a detector, which responds to some physicochemical property of the analyte and generates an electronic signal measuring the amount of analyte present o The data system then produces an integrated chromatogram o Gas chromatography uses ovens that are temperature programmable. The temperature of the GC oven typically ranges from 5 °C to 400 °C but can go as low as -25 °C with cryogenic cooling 49 6. THE CHROMATOGRAM AS THE COMPONENTS ELUTE FROM THE COLUMN THEY PASS INTO A DETECTOR – WHERE SOME PHYSICOCHEMICAL PROPERTY OF THE ANALYTE PRODUCES A RESPONSE FROM THE DETECTOR. THIS RESPONSE IS AMPLIFIED AND PLOTTED AGAINST TIME – GIVING RISE TO A ‘CHROMATOGRAM’) Components (such as the injection solvent) that are not retained within the column elute at the ‘dead time’ or ‘hold up time’ t0. There are various ways of measuring this parameter using unretained compounds such as methane or hexane. THOSE COMPOUNDS (ANALYTES AND SAMPLE COMPONENTS) THAT ARE RETAINED ELUTE AS APPROXIMATELY ‘GAUSSIAN’ SHAPED PEAKS LATER IN THE CHROMATOGRAM. RETENTION TIMES PROVIDE THE QUALITATIVE ASPECT OF THE CHROMATOGRAM AND THE RETENTION TIME OF A COMPOUND WILL ALWAYS BE THE SAME UNDER IDENTICAL CHROMATOGRAPHIC CONDITIONS. THE CHROMATOGRAPHIC PEAK HEIGHT OR PEAK AREA IS RELATED TO THE QUANTITY OF ANALYTE. FOR DETERMINATION OF THE ACTUAL AMOUNT OF THE COMPOUND, THE AREA OR HEIGHT IS COMPARED AGAINST STANDARDS OF KNOWN CONCENTRATION 50 7. GC ADVANTAGES AND DISADVANTAGES Gas chromatography has several important advantages which are listed opposite. GC techniques produce fast analyses because of the highly efficient nature of the separations achieved – this will be studied further in the Band Broadening Section. It can be argued that modern GC produces the fastest separations of all chromatographic techniques. A column has been produced to separate 970 components within a reasonable analysis time - proving that very complex separations may be carried out using GC. By using a combination of oven temperature and stationary phase chemistry (polarity) very difficult separations may also be carried out – including separations of chiral and other positional isomers. GC is excellent for quantitative analysis with a range of sensitive and linear detectors to choose from. GC is however limited to the analysis of volatile samples. Some highly polar analytes can be derivatized to impart a degree of volatility, but this process can be difficult and may incur quantitative errors. A practical upper temperature limit for conventional GC columns is around 350-380 °C. Analyte boiling points rarely exceed 400 °C in GC analysis and the upper Molecular Weight is usually around 500 Da. ADVANTAGES o Fast analysis. 51 o High efficiency – leading to high resolution. o Sensitive detectors (ppb). o Non-destructive – enabling coupling to Mass Spectrometers (MS) - an instrument that measures the masses of individual molecules that have been converted into ions, i.e. molecules that have been electrically charged. o High quantitative accuracy (