Many notes taken from "Molecular Biomethods Handbook" 1998. Edited by Ralph Rapley and John M. Walker. Humana Press, Totowa, NJ.

Brief History

The name derives from separation of plant pigments.

Early on, Egyptians used this technology to separate dyes on papyrus.

In 1906, the Russian botanist Tswett created the name for chromatography.

In 1944, Martin and Synge developed the technique of partition chromatography and received a Nobel Prize.

In 1960, Gel Chromatography was introduced.

General Principles of Chromatography

• Separation is achieved by distribution of particles between a stationary phase and a mobile phase.
• A stationary phase may be a solid, liquid, or solid/liquid mixture (where liquid is held stationary by a solid). The solid support is called the matrix.
• The mobile phase is called the solvent and it is usually a liquid, but may also be a gas.
• The compounds to be separated are considered the solute.

Examples of different stationary and mobile phases

• Gel filtration is used for molecular sieving using a stationary porous bead through which a liquid mobile phase move inside and inside the porous beads.
• Adsorption chromatography is where an adsorption equilibrium is set up between a stationary solid and a mobile liquid phase.
• In paper chromatography or countercurrent distribution (thin layer chromatography), the partition equilibrium is set up between a stationary liquid (or semi-liquid) phase and a mobile liquid phase.
• In Gas chromatography the partition equilibrium is set up between a stationary liquid and a mobile gas phase.
• Ion Exchange chromatography is based on an ion exchange equilibrium where the stationary phase is the ion exchange resin and the mobile phase is a liquid electrolyte phase.
• Affinity Chromatography is an equilibrium between a macromolecule and a small molecule for which it has high biological specificity (and hence affinity).

Size-Exclusion Chromatography

It is also known as gel-filtration chromatography and separates proteins and other biological macromolecules on the basis of molecular size. This type of chromatography is compatible with physiological conditions.

The solid-phase matrix consists of porous beads (100-250µm) that are packed into a column with a mobile-liquid phase flowing through the column. The mobile phase has access to both the volume inside the pores and the volume external to the beads. The high porosity typically leads to a total liquid volume of >95% of the packed column.

During separation by this method, large molecules remain in the volume external to the beads because they are unable to enter the pores. The resulting shorter flow path means that they pass through the column relatively rapidly, emerging early. Proteins that are excluded from the pores completely elute in what is designated the void volume, Vo. This is often determined experimentally by the use of a high-molecular weight component, such a Blue Dextran or calf thymus DNA. Small molecules that can access the liquid within the pores of the beads are retained longer and therefore pass more slowly through the column. The elution volume for material included in the pores is designated the total volume, Vt. This represents the total liquid volume of the column and is often determined by small molecules, such as vitamin B12.

The elution volume for a given protein will lie between Vo and Vt, and is designated the elution volume, Ve. A partition coefficient can be determined for each protein as Kav:

In size exclusion, the macromolecules are not physically retained, unlike adsorption techniques; therefore, the protein will elute in a defined volume between Vo and Vt. If the protein elutes before the void volume (Ve<Vo) this suggests channeling through the column owing to improper packing or operation of the column. If the protein elutes after the total volume (Ve>Vt), then some interaction must have occurred between the matrix and the protein of interest.

Size exclusion tends to be used at the end of a purification scheme when impurities are low in number and the target protein has been purified and concentrated by earlier chromatography steps. An exception to this is membrane proteins, where gel filtration may be used first because concentration techniques are not readily used and the material will be progressively diluted during the purification scheme.

Several parameters are important in size-exclusion chromatography.

• Pumps: An important factor in size exclusion is a reproducible and accurate flow rate. The most commonly used pumps are peristaltic pumps.
• Column: Resolution of molecular separation is dependent on column length. Columns tend toward being long and thin, typically 70-100 cm long. In some instances, the length of the column required to obtain a satisfactory separation exceed that which can be packed into a commercially available column (>1 m). In these cases, columns can be packed in series. The tubing connecting the columns should be as narrow and as short as possible to avoid zone spreading.
• Detectors: Protein elution is most often monitored by absorbance in the ultraviolet range, either at 280 nm, which is suitable for proteins with aromatic amino acids, or at 206 nm, which detects the peptide bond. Fluorescence detection either by direct detection of fluorescent tryptophan and tyrosine residues or after chemical derivatization have been used, as have refractive index, radiochemical, electrochemical, and molecular size (by laser-light scattering).
• Fraction collectors: For the detector to reflect as near as possible in real time the fraction collector, the volume between the detector and the fraction collector should be minimal.
• Buffers: Size-exclusion matrices tend to be compatible with most aqueous-buffer systems even in the presence of surfactants, reducing agents, or denaturing agents. All buffers used in size exclusion should ideally be filtered through a 0.2 µm filter and degassed by low vacuum or sparging with an inert gas, such as helium. The majority of protein separations performed using size exclusion are carried out in the presence of aqueous-phase buffers. Size exclusion of proteins in organic phases is not normally undertaken but is sometimes used for membrane-protein separations.
• Selection of matrix: In size exclusion chromatography, the pore diameter controlling the separation is selected for the relative size of proteins to be separated. The beads used for size exclusion have a closely controlled pore size, with a high chemical and physical stability. They are hydrophilic and inert to minimize chemical interactions between the solutes (proteins) and the matrix itself. Many matrices retain a residual charge owing to, for example, sulfate groups in agarose or carboxyl residues in dextran. The ionic strength of the buffer should be kept at 0.15-2.0M to avoid electrostatic or Van Der Waals interactions that can lead to non-ideal size exclusion.

Practical Procedures

• Flow rates: Conventional low-pressure size exclusion matrices tend to operate at linear flow rates of 1-15 cm/h. Too high a flow rate leads to incomplete partitioning and band spreading. Conversely, very low flow rates may lead to diffusion and band spreading.
• Separation of proteins: It is not only the molecular weight that is important in size exclusion, but also the hydrodynamic volume or the Stokes radius of the molecule. Globular proteins appear to have a lower molecular size than proteins with a similar molecular weight, which are in a a-helical form. Gel-filtration columns are often loaded at relatively high concentrations of protein, such as 2-20 mg/ml.
• Column cleaning and storage: They can be cleaned in situ or as loose gel in a sintered-glass funnel. Common general-cleaning agents include nonionic detergents, such as Triton X-100, for lipids and 0.2-0.5M NaOH for proteins. In extreme circumstances, contaminating protein can be removed by use of enzymatic digestion (pepsin for proteins and nucleases for RNA and DNA). The gel should be stored in a buffer with antimicrobial activity, such as 20% (v/v) ethanol or 0.02-0.05% (w/v) sodium azide.

Ion Exchange Chromatography

Ion-Exchange Chromatography

Proteins contain charged groups on their surfaces that enhance their interaction with solvent and hence their solubility. At physiological pH, some of these charged groups are cationic (positively charged, e.g., lysine), whereas others are anionic (negatively charged, e.g., aspartate). Because proteins differ from each other in their amino-acid sequence, the net charge possessed by a protein at physiological pH is determined ultimately by the balance between these charges (i.e. negatively charged proteins possess more negatively charged groups than positively charged groups). This also underlies the different isoelectric points (pIs) of proteins. Ion-exchange chromatography separates proteins first on the basis of their charge type (cationic or anionic) and, second, on the basis of relative charge strength (e.g. strongly anionic from weakly anionic).

The basis of ion-exchange chromatography is that charged ions can freely exchange with ions of the same type. In this context, the mass of the ion is irrelevant. Therefore, it is possible for a bulky anion like a negatively charged protein to exchange with chloride ions. This process can later be reversed by washing with chloride ions in the form of a NaCl of KCl solution. Such washing removes weakly bound proteins first, followed by more strongly bound proteins with greater net negative charge.

Like most column chromatography techniques, ion exchange chromatography requires a stationary phase, which is usually composed of insoluble, hydrated polymers, such as cellulose, dextran, and agarose.

The ion-exchange groups are immobilized on this stationary phase. Some of the anion exchangers are quaternary ammonium, quaternary aminoethyl and diethylaminoethyl (DEAE). Some of the cation exchangers are sulfopropyl, methyl sulfonate and carboxymethyl.

Practical requirements

• Buffers: Buffers used will depend on the characteristics of the protein of interest. For proteins not previously purified by DEAE-cellulose chromatography, buffer selection may be helped if the pI of the protein of interest is known. In general, proteins will bind to anion exchangers at pH values above their pIs, while binding to cation exchangers, such as CM-cellulose, will occur at pH values below their pIs. Phosphate, Tris, and other common buffers are used in ion-exchange chromatography in concentrations of 10-50mM.
• Chromatography: In general, approximately 70% of rat-liver cytosolic proteins will bind to DEAE-cellulose at pH 7.5. If the protein of interest has an alkaline pI, then passage through such a column at this pH might be a useful first step in the purification. Only highly acidic proteins will bind at pH 10.0. The column is developed by applying a suitable salt gradient, for example 0-100 mM NaCl in 10mM Tris buffer, pH 8.0. Fractions are collected in a fraction collector and assayed for the protein of interest, protein concentration, and conductivity. Although shallower (e.g. 0-100, 300-400 mM) gradients generally give better resolution, steeper gradients (e.g., 0-1 M) can be used in initial experiments to identify elution positions of proteins of interest.

Ion Exchange Chromatography

Principle:

• Ion Exchange chromatography relies on the reversible exchange of ions in solution with ions electrostatically bound to an insoluble support media. There can be two types of functional groups covalently attached to the support beads. These are called anion exchangers (resin with positive functional groups) or cation exchangers (resin with negative functional groups).
• Separation on ion exchange chromatography columns is based on charge density. Start with exchange resin with bound buffer counter ions (e.g. Cl^-). The sample proteins are then added to the column and the charge opposite to the functional group are bound to exchanger, replacing buffer counter ions; any neutral or like (positive) charged molecules pass through without binding. The bound molecules are sequentially eluted, often using increasing amounts of counter ions (e.g. a gradient from 0.0- 0.4 M KCl). Proteins with fewer negative charges eluted first (and replaced by gradient ions), followed by proteins with greater quantity of negative charges.

Selecting the adequate ion exchanger

• Selection of ion exchanger depends on the charge of the protein of interest. In the case of proteins, they have both negative and positive charges, thus their net charge which depends upon pH (can make protein more negative by raising pH or more positive by lowering pH) dictates their binding to the ion exchanger. When selecting the appropriate pH, the primary consideration is the stability of the protein. If the protein is stable at pHs above the isoelectric point, then use anionic (positive) exchanger. If protein more stable below isoelectric point, chromatograph on cation (negative) exchanger. If range of stability extends 1 pH unit above and below isoelectric point, either an anion or cation exchanger can be used. If you do not know the isoelectric point and thus the range of stability, mix known concentrations of protein with small amount of cationinc and anionic resins at different pHs. Determine amount bound by measuring concentration in supernatant (protein assay). If you have a mixture of proteins, you may have to perform some assay to determine where your protein of interest is (bound or in supernatant)..
• Types of ion exchange resins. The supporting matrix determines the flow properties, ion accessibility, chemical and mechanical stability. Three groups generally used are (1) polystyrene, polyacrylic, or polyphenolic resins, (2) cellulose resin, (3) dextran (Sephadex) or Agarose (Serpharose). Functional groups can be strongly, intermediately or weakly basic (anion (+)) or acidic (cation (-)) exchangers.
• Classified as weak or strong depending upon the range of pH values over which it remains charged (strong ion exchangers are charged over a wider pH range than weak exchangers). Weak exchangers lose their charge below 6 (for cationic) or above 9 (for anionic) exchangers.
• Buffers used will depend on the characteristics of the protein of interest. For proteins not previously purified by DEAE-cellulose chromatography, buffer selection may be helped if the pI of the protein of interest is known. In general, proteins will bind to anion exchangers at pH values above their pIs, while binding to cation exchangers, such as CM-cellulose, will occur at pH values below their pIs. Phosphate, Tris, and other common buffers are used in ion-exchange chromatography in concentrations of 10-50mM.
• Strongly basic anionic exchangers are quaternary aminoethyl (QAE) QAE-Sephadex and quarternary ammonium (Bio-Rad AG 1, Dowex 1. A weakly basic anion exchanger is diethyaminoethyl (DEAE), DEAE-Cellulose, DEAE-Sepharose.
• Strongly acidic cationic exchanger are those having sulfonic groups such as Bio-Rad AG 50, Dowex 50. Weakly acidic cation exchangers contain carboxyl groups such as carboxymethyl (CM-cellulose).
• The choice of using a strong vs weak cation or anion exchanger depends on the pH of the molecule. A weakly acidic molecule which requires very low or very high pH for ionization will require the use of a strong ion exchanger because it functions at extremes of pH. There are also stability considerations. If the substance is stable to extremes of pH (eg. amino acids), strongly acidic or basic ion exchangers can be used. If substance is labile (eg. proteins) a weaker exchanger of low charge density is advisable because the molecules can be eluted by more gentle conditions of pH and ionic strength. For separation of molecules with high charge from those with small charge it is best to use weak exchange resins. If the charge differences between molecules is very small-weak exchanger produces more resolution.
• Volume and shape of column. The amount of sample to be loaded on the column recommended is 10-20% of the column capacity. Use a short wide (fat) column . Long narrow columns often offer less resolution, because the eluted ionic molecules may have to travel long distances down column and may diffuse.
• Shape and size of gradient. A gradient of increasing ionic strength can be used. Less often, a pH gradient is used. A continuous or step gradient can be used. A continuous gradient will elute symmetrical peaks and produce better resolution. A step gradient may be useful if one has a few components with large differences in elution (charge). Decreasing the slope of the gradient (increasing the volume of the gradient) will lead to better resolution.
• In general, approximately 70% of rat-liver cytosolic proteins will bind to DEAE-cellulose at pH 7.5. If the protein of interest has an alkaline pI, then passage through such a column at this pH might be a useful first step in the purification. Only highly acidic proteins will bind at pH 10.0. The column is developed by applying a suitable salt gradient, for example 0-100 mM NaCl in 10mM Tris buffer, pH 8.0. Fractions are collected in a fraction collector and assayed for the protein of interest, protein concentration, and conductivity. Although shallower (e.g. 0-100, 300-400 mM) gradients generally give better resolution, steeper gradients (e.g., 0-1 M) can be used in initial experiments to identify elution positions of proteins of interest.

Other properties of ion exchange resin

• The capacity of exchanger (molecules bound per unit weight of exchanger) are affected by the porosity -- polystyrene, Sephadex, Sepharose have functional groups on outside, and inside the beads (porous), whereas cellulose has functinal groups only on surface. The degree of cross-linking influences the capacity--high degree of x-linking---> smaller pore size---> higher capacity (more surface area)--good for separation of small molecules (eg. amino acids, peptides, nucleotides). If using porous matrix with larger molecules, chose matrix with larger pores (e.g. agarose-Sepharose)
• The mesh size (diameter of resin beads). The smaller beads---> greater capacity---> slower flow rate (because beads packed closer together)--> may get spreading of bands (less resolution). Larger beads--may get better resolution by less capacity

Applications of ion exchangers

• Polystyrene and polyphenolic ion exchange resins (trade names Dowex, Bio Rad AG, Bio Rex, Chelex) are more often used to separate small such as amino acids (basis of amino acid analyzer), small peptides, nucleotides, N-bases, cyclic nucleotides, organic acids (e.g. intermediates of respiration). Large molecules do not penetrate beads very well.
• The cellulose ion exchangers are commonly used for proteins, including enzymes, polysaccharides and nucleic acids. DEAE-cellulose, CM-cellulose and phosphocellulose are the most commonly used.
• Polydextran and agarose, e.g. DEAE-Sephadex, CM-Sephadex are used to separate proteins, hormones, tRNAs, polysaccharides.
• Use of ion exchanger in conjunction with gel filtration. In protein purification techniques both ion exchange and gel filtration chromatography can be used. Ion exchange can be used on rather crude homogenates and can be adopted for large capacity separations. Afterwards, gel filtration chromatography can be used for finer separation and molecular weight determination.

Affinity Chromatography

Principle:

• The separation is based on the biological function of the molecule to be separated or purified rather than by size or charge characteristics of the molecule. The ligand, a molecule complementary to the one you want to isolate is immobilized on an insoluble support (the "matrix").
• A mixture containing the desired molecule is run through the column and the desired molecule binds
• specifically to the ligand, and the undesirable molecules in the mixture, the ones which do not
• bind, are washed from the column. Then the molecule adsorbed to the ligand is desorbed (eluted)
• by changing the conditions of binding

Coupling the ligand to the matrix

• The matrix is an insoluble support that should contain a sufficient amounts of a suitable group to which the ligand can be covalently linked. The matrix should be stable under conditions of attachment, should be stable during binding of the macromolecule and during elution of macromolecule. That is it should tolerate extremes of pH, ionic strength and high concentration of denaturants often used to elute the macromolecules. The matrix should have low non-specific adsorption and should have good flow properties. In some cases high porosity is desirable to increase binding of the macromolecule.
• The matrices most commonly used are agarose (Sepharose 4B, Bio Get A) and polyacrylamide; less often used are cellutlose and polystyrene.
• The most generally used coupler for the ligand to matrix is the cyanogen bromide-activated agarose (trade name CNBr-activiated Sepharose 4B). The cyanogen bromide reacts with amino groups of the ligand. Other media couple to ligands containing carboxyl, amino, thiol, or hydroxyl groups.
• Some matrices have spacer arms--sequences extending from the matrix--ligands bind to reactive groups at end of spacer arm. Advantage of media with spacer arms: where ligand is small and molecule to be bound is large, there may be steric hindrance from the media--the ligand is partially buried within the media support and may not be able to bind the molecule effectively. With the spacer arm, the ligand is extended away from the media so the desired molecule can bind to it.
• The ligand should have affinity for the molecule to be separated in the range of 10^-4 to 10^-8 M in solution. Interactions with dissociation (binding) constants greater than 10^-4 M may be too weak to bind the molecule to the ligand. If the dissociation constant is lower than 10^-8 M (tight binding which takes small number of molecules for interaction), then it may be difficult to elute the molecule of interest.
• Coupling procedure--exact procedure depends upon the properties of the matrix and interactions of ligand and binding molecule. In general: swell and wash gel, interact ligand with matrix (covalently bound), block groups not bound to ligand and wash gel to remove ligand not covatentty bound.

Affinity chromatography procedures

• Overall procedure: pack gel (matrix with ligand attached) in suitable column. Apply sample--volume of sample not critical if tight binding occurs. Weakly bound substances should be applied in small volume (about 5% of bed volume) to give the desired molecule(s) a chance to bind without being washed through the column. Wash column with about 10 column volumes of the starting buffer to remove non-bound substances.
• Molecules are bound to the ligand by various types of interactions including hydrogen bonding, electrostatic, and hydrophobic interactions. Therefore, for elution, one needs to use reagents that weaken these interactions.
• There are two types of elution methods: (1) Non-selective elution is often used with highly specific adsorbants. Change in pH--usually a decrease in pH--alters the degree of ionization of the charged groups. Change in ionic strength (higher or lower salt concentration)--may use gradient elution. (2) Selective elution--often used with group specific adsorbants and when binding affinities are rather low it is best to use a molecule which is complementary to the one to be eluted. The complementary molecule will either compete with the desired molecule for binding to the ligand, or will have a high affinity for the desired molecule, bind, then "pull" off the desired molecule.
• You may use a batch procedure as a form of affinity chromatography. Mix gel with molecules in a microcentrifuge tube. Invert the tube over and over to allow desired molecule(s) to bind. Centrifuge to pellet gel. Remove supernatant containing nonbound molecules. Wash several times by adding buffer, interacting buffer with gel, then recentrifuging and removing supernatant containing more non-bound molecules; then elute with change in ionic strength, pH, or selectively, with complementary molecule.

Applications

• Isolation of poly A containing messenger RNAs using oligo dT cellulose.
• Lectins (carbohydrate binding proteins isolated from plant seeds) can recognize glycoproteins and glycolipids. A column can be made with the lectin Concanavalin A (Con A). Con A Sepharose beads bind to specific types of glycoproteins. This type of molecules can be eluted with sugars recognized by the particular lectin.

High Performance (Pressure) Liquid Chromatography (HPLC)

www.chem.vt.edu/chem-ed/sep/lc/hplc.html

• Basic principle of HPLC: In theory, the resolution increases with increasing length of column and of theoretical plates/unit length. If the size of the matrix particles decreases, this increases surface area increasing the number of theoretical plates. However, flow rate drops as particle size decreases, and a slow flow rate leads to more time for diffusion which leads to loss of resolution. Therefore, one can reduce the diffusional spreading by reducing the transit time of mobile phase by using high pressure. This pressure forces liquid through the bed faster. Fine particles and high pressure maintain high flow rate. The fine rigid particles used are about 10µm in diameter. Stainless steel columns 20-50 cm length 1-4 mm diameter pressure up to 8000 psi. Columns of 10-20 mm diameter are used for preparative higher capacity columns, and are typically 50-200 psi. In general, two factors to consider when selecting a column size are the efficiency and sample-loading capacity. In the case of proteins, column length contributes little to efficiency. In addition, longer columns may have an adverse effect on protein recovery. The choice of column diameter should be based on required sample capacity. Columns of 1mm-2.1mm internal diameter are best suited to submicrogram levels of material, thus minimizing sample loss and also increasing sensitivity of detection. For analytical applications in the microgram to low milligram range, columns of 4.6mm internal diameter (analytical) are best.
• HPLC results in high resolution, that is, sharp peaks. It also yields rapid separation (minutes to 1 hour). Can be used with very small amounts of material (sample size 0.01 — 0.1 ml). HPLC can be analytical or preparative. HPLC can be used for all types of chromatography discussed so far (size exclusion chromatography, ion exchange chromatography, affinity chromatography). In addition, two other types of separation developed for HPLC are reversed phase chromatography and paired-ion chromatography.
• Reverse phase HPLC: In a normal phase there is a polar solid support and an organic relatively non-polar mobile phase solvent. Highly polar molecules are strongly attracted to the stationary phase so they stick strongly and are hard to elute (have long retention times and peak trailing). Reversed phase uses non-polar stationary phase and polar mobile phase. Reverse phase column chromatography uses matrix bound non-polar hydrocarbon chains. The most common are n-alkyl-bonded C4, C8 and C18. In general, C4 and C8 phases are preferable for more hydrophobic samples and the C18 phase for hydrophilic samples. If the proteins of interest are too strongly retained, a C4 column can be used. The polar molecules now have higher affinity for mobile phase and are eluted quickly. Reverse phase HPLC (RP-HPLC) is good for polar biomolecules such as drugs and their metabolites, peptides, vitamins, polyphenols, steroids. This technique is used clinically to separate molecules in serum, urine, etc.
• Paired ion chromatography: Some highly polar compounds such as amino acids, organic acids and catecholamines do not adhere at all to stationary phase (non-polar). Therefore, the molecules are paired with their counterions giving a less polar species which will initially stick to non polar stationary phase but can still be eluted, apparently by a type of ion-exchange mechanism. The ionization is suppressed by chromatography at higher (or lower) pHs (depending upon charge).

Matrix for HPLC

• Column packing materials need to be of a small uniform size and rigid (to withstand pressure flow). Theoretically, column performance should increase with decreasing particle size. Columns with particle sizes of 5-10µm show little difference in performance. Columns with particle sizes of 2-3µm are now available and can give much greater resolution. However, any gain in column efficiency can be outweighed quickly by increased column back-pressure, susceptibility to plugging, and shorter column lifetime. Three types of matrix exist.
• Microporous supports. Micropores exist within particles 5-10µm in diameter. The standard pore size for separation of small molecules is 80 Angtrons but for the separation of proteins a pore size of 300 Angstrons has become accepted as the norm. For proteins >100Kda, pore sizes of >1000 Angstrons may be even better (used in size exclusion chromatography).
• Pelicular support (superficially porous). Porous particles coated onto inert solid core such as a glass bead about 40 µm in diameter. Stationary phase supports often used are silica, alumina, polystyrene (used in size exclusion chromatography).
• Bonded phase support. Stationary phase chemically bonded to inert support (ion exchange, affinity, reverse phase).

Sample injection.

• The sample is injected through syringe either directly onto top of column or onto a small plug of inert material above the column matrix either under pressure or the pressure can be stopped and the sample loaded. The sample can also be injected through a loop injector.

Thin layer Chromatography

• The paper is the stationary phase. A non-aqueous solvent (mobile) phase is allowed to travel along the paper. When the solvent reaches the solute, according to distribution coefficient, a distribution between the 2 phases occurs. Essentially the more soluble a solute is in the mobile phase, the further the molecules will travel along the paper. You have probably used this method to separate amino acids and plat pigments.
• Two dimentional (2-D) paper chromatography can be used in "finger printing" where the analysis of tryptic peptide digests are carried out in 2-D.
• Thin layer chromatography (TLC) is similar to paper chromatography except that the stationary phase is a layer of sorbent spread over a glass, foil or plastic surface. The mobile phase is primarily by partitioning although adsorption of the molecules to the stationary phase is also involved. The adsorbents are very fine particles of silica, cellulose, etc. The advantages of TLC over paper chromatography are the following: (1) that you can separate very small amounts of material; (2) TLC has greater solving power (better separation is due to very fine particles that create a large surface area and due to the high ratio of sorbent to solute; (3) wide choice of solvents; (4) easy detection of spots; (5) Easy isolation of substance separated (i.e. by scraping).
• 2-D TLC is similar to paper chromatography in that two solvents are used for separation.

Gas Chromatography
Look at the following website: http://www.shu.ac.uk/schools/sci/chem/tutorials/chrom/gaschrm.htm

Quatination of Proteins:

Beer's law

http://www.shu.ac.uk/schools/sci/chem/tutorials/molspec/beers1.htm

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