Column Chromatography Matrices

• Column chromatography separates substances through liquid percolation in a packed column.
• Separation occurs via direct interaction of solute components with the stationary phase or solute adsorption.

Matrices in Column Chromatography

• The matrix supports the stationary phase.
• Matrix material should have high mechanical and chemical stability, allow good flow rate, have variable particle sizes and be chemically inert.
• Commonly used matrices: cellulose, dextran, agarose and polyacrylamide and Polystyrene and Silica.
• Cellulose is a hydrophilic polysaccharide of -1,4-linked D-glucose units.
• For matrix purposes cellulose is cross-linked with epichlorohydrin, controlling pore size.
• Dextran is a hydrophilic polysaccharide of -1,6-linked D-glucose units.
• For matrix purposes Dextran is cross-linked with epichlorohydrin.
• Dextran is less stable to acid hydrolysis but stable up to pH 12, and marketed as Sephadex and Sephacryl.
• Agarose is a polysaccharide of 3,6-anhydro-1-galactose and D-galactose units.
• Agarose is cross-linked with 2,3-dibromopropanol, forming gels stable in pH 3-14, and has good flow.
• Agarose materials must be suspended in water or buffer to avoid irreversible changes; include Sepharose and Bio-gel A.
• Polyacrylamide is a polymer of acrylamide monomer, cross-linked with N,N-methyl-bisacrylamide.
• Polyacrylamide is stable in pH 2-11, e.g., Bio-gel P.
• Polystyrene is a polymer of styrene, cross-linked with divinylbenzene.
• Polystyrene matrices have high stability over all pH ranges, used in ion exchange and exclusion chromatography.
• Silica is a polymeric material from orthosilicic acid, hydrophilic due to silanol groups.
• For matrix use, excess silanol groups are removed with trichloromethyl silane and are stable in pH 3-8.

Column Packing

• A column (2.5 x 100 cm) is held vertically with a glass wool plug at the end.
• Before packing, the gel swells in a solvent; heat-accelerated swelling requires cooling before packing.
• Cold-prepared slurry de-aerated under vacuum.
• Allow gel settling; remove half the supernatant and mix with solvent into a slurry/suspension.
• Constant stirring is needed while carefully pouring the slurry into the column using a glass rod.
• Add all slurry at once to ensure even packing.
• After the gel settles, run eluent/buffer for an extended time before sample application to achieve fine packing.

Sample application

• The application of sample to a prepared column should be done carefully.
• Mobile phase can be removed by suction or drained to the column bed.
• The sample should then be carefully applied with a pipette for it to slowly run into the column.
• A small volume of mobile phase should be slowly released into the column a few times for it to increase 2-5cm in the column.
• The column is then connected to a large reservoir with adjusted flow and steady height for the mobile phase.
• Application of a high-density sample (e.g., 1% sucrose) from 2 cm above, will allow it to sink to the bottom immediately.
• A capillary tube of syringe can be used to pass the sample to the column surface
• Capillary tubes of syringes are the preferred method of sample application

Separation Efficiency Factors

• Separation efficiency of column chromatography can be affected by drastic factors; dimension of column, particle size of column packing, quality of solvents, temperature of the column, flow rate, packing of the column.
• Column efficiency is improved by increasing the length/width ratio.
• Length to width ratio of 10:1 to 100:1 has been found most satisfactory for preparative separations.
• Long columns are used for closely related compounds.
• Large column diameters are used to accommodate large amounts of samples.
• Decreasing the particle size of the adsorbent improves separation.
• Particle sizes of 100 to 200 mesh range is recommended for both adsorption and partition.
• Solvents with low viscosity are generally used in high efficiency separations.
• The flow rate of solvent is inversely proportional to viscosity, so solvents with low viscosity should be selected.
• Adsorption decreases at higher temperatures, increasing the speed of elution.
• Column chromatography is done at room or low temperatures (5-10°C).
• Medium flow rates are preferred; slow flow rates cause zone spreading; high flow rates lead to non-equilibrium and tailing.
• Column packing is very important, with poorly packed columns leading to zone spreading or tailing.

Elution Techniques

• Elution is removing the sample from the solid matrix using a suitable solvent.
• Total volume of material (solid and liquid) in the column is the bed volume.
• The volume of the mobile phase is known as void volume.
• The amount of liquid to produce a peak of a particular solute in the effluent is the elution/effluent volume.
• Retention time is the time taken for each material to emerge from the column.
• Elution volume and retention time are related to the flow rate of the mobile phase through the column.
• Columns can be eluted by simple isocratic elution, stepwise elution, or gradient elution methods.
• An isocratic elution uses a single liquid mobile phase.
• In an isocratic elution, a single solvent flows continually through the column.
• Isocratic elution is mainly used in gel permeation chromatography.
• Stepwise/batch elution is employed for preparative purposes.
• Stepwise elution is when the column is eluted with one solvent until a predetermined volume is used.
• A second solvent is added to elute separation in the column via stepwise elution.
• Stepwise elution is used in ion exchange and affinity chromatography.
• Gradient elution is done to increase the resolving power of the mobile phase and a continuous change to its pH, ionic strength or polarity.
• Gradient elution is a method where a solution is used to elute the column with a gradually increased concentration of one of the components.
• Gradient elution is used in adsorption, affinity and ion exchange columns.

Chromatogram

• Placing a detector at the column's end in column chromatography creates a symmetrical peak signal when plotted as a function of time/volume.
• A plot like this is a chromatogram.
• Peak positions show sample components and peak areas quantify their amounts.
• Column chromatography involves ion exchange, molecular sieve, and adsorption or partition phenomenon.
• In adsorption column chromatography the substances are preferentially adsorbed by the adsorbent (stationary phase) packed in the column
• In partition column chromatography, the components of a mixture distribute then get separated.

Adsorption Chromatography

• Developed by D.T. Day (1900) and M.S. Tswett (1906) in plant pigment investigations.
• The procedure was not commonly used by chemists until 1930.
• The rate of adsorption varies with the adsorbent used on different materials.
• Separation relies on the principle of selective adsorption.
• The mixture to separate is dissolved in a solvent and passed through a tube with an adsorbent.
• Components with greater adsorbing power adsorb in the upper column.
• The next component is adsorbed in the lower portion of the column, which has lesser adsorbing power than the first component.
• The process is continued as materials are separated and adsorbed in the various parts of the column.
• Passing the original or a suitable solvent slowly through the column can improve the separation.
• As the various bands become defined, the banded adsorbent is termed a chromatogram.
• A zone is the occupied portion of a column by a particular substance.
• Adsorption chromatography separates mixture components by adsorption efficiency onto the solid stationary phase.
• The most strongly adsorbed component forms the topmost band while the least strongly adsorbed material forms the lowermost band on the adsorbent column.
• The separation depends on the surface area because adsorption is a surface phenomenon.
• Distribution coefficient (K) is obtained by the formula: Amount of solute per unit of mobile phase/Amount of solute per unit of stationary phase.
• The distribution coefficient mainly depends upon temperature and substance concentration.

Types of Adsorbents

• Commercial Adsorbents; Fuller's earth, powdered charcoal, polystyrene beads, hydroxyapatite, silica or silica gel, and alumina or aluminium oxide gel

Fuller's Earth

• Mixture of minerals from clay deposits like Kaolinite, Montmorillonite, and Halloysite.
• Decolorizes food oils and removes wine pigments.
• Fuller's Earth's low cost, it is sold as Florisil.

Powdered Charcoal

• A very powerful adsorbent with properties that are highly dependent on the origin processes.
• Bone, blood, and coconut can be a resource material.
• Reproducibility is a problem with charcoal.
• Applying finely divided charcoal and preparing a densely packed column is troublesome.

Polystyrene Beads

• Modified Polystyrene polymers or polystyrene-divinyl benzene copolymers create tiny beads without charged groups.
• Controlled cross-linking creates beads that have known porosity and swelling characteristics.
• They function by adsorption as they lack ionized groups.
• Used commercially under the trade name “XAD resins" for alkaloids, steroids, etc.

Hydroxyapatite

• A basic calcium phosphate with the formula 3Ca3(PO4)2·Ca(OH)2.
• Hydroxyapatite preparation is tedious, the quality varies from batch to batch.
• The gel must not be allowed to dry out an freeze.
• Drying or freezing alters hydration and gel structure, useful in protein separation.

Silica or Silica Gel

• Very popular, binding a wide range of substances with moderate strength.
• Preparing Silica gel is done by precipitating hydrated silica from sodium silicate by adding acid.
• Then it is exhaustively washed dried and sized precisely.
• Handle with caution as it is not as stable as alumina to changes from mechanic damage.
• Silica can be dried at relatively modest temperatures.
• Silica comes in the form of crushed and powdered glass, which has very strong adsorptive properties for some proteins.

Alumina or aluminum oxide gel

• Alumina is a very powerful adsorbent that is widely used.
• It is prepared as a precipitate of aluminium hydroxide by addition of sodium hydroxide to some soluble aluminium salts.
• The gelatinous precipitate is then washed, dried and sized.
• Can be made acidic, basic or neutral.
• Anhydrous state (Al2O3) is achieved by heating it strongly
• Alumina is mechanically strong.
• It has the advantage of not crumbling to still finer particles when manipulated for packing into columns.

Preparation of Column

• Dry Packing Method is used when pouring in powdered substance into the tube in small lots and the tube is then tapped.
• Wet Method is the second method and it closes the bottom of the tube and is then filled with the solvent.
• In wet method powdered material is added in small quantities and settles against the solvent's viscosity.
• Interlocking of air bubbles must be avoided in both methods.

Separation Procedure

• The components can be separated as distinct bands along the adsorbent column.
• Extrusion method stops the running solvent when separation of bands has occurred, this is how elution is carried out.
• The column is drained, extracted and kept horizontally on a table with knife cuts separating the bands.
• The scrapings are then extracted by a suitable technique using solvent.
• Liquid chromatography involves continuous elution for subsequent component washing.

Applications of Adsorption Chromatography

• Adsorption chromatography is used for the separation of the following:
• Polycyclic aromatic compounds, phenols, amines, etc.
• Urinary 17-ketosteroids and their glucuronides.
• Plasma cortisol.
• Aliphatic hydrocarbons from aromatic hydrocarbons.
• Geometrical isomers.
• Technical products in a highly purified state.

Affinity Chromatography

• Useful for the isolation of proteins, polysaccharides, nucleic acids and other classes of naturally occurring compounds
• Affinity chromatography exploits functional specificities of biological systems
• Affinity chromatography can achieve separations that are otherwise difficult using other techniques rapidly.
• A complex mixture of substances can be purified simply using an affinity column.
• Separations use specific affinity of a substance and a specifically binding molecule ligand.
• Covalently coupling a binding molecule (macromolecule or small molecule) to an insoluble matrix synthesizes the column.
• The column can specifically adsorb the substance to be isolated.
• Elution of the desired substance is done by changing the conditions to avoid binding.
• The ligand can be directly attached to the matrix.

Matrix Properties

• Contains groups suited for ligand binding.
• Exhibits good flow properties.
• Stable during macromolecule binding and elution.
• Low interaction with other macromolecules.
• Matrix materials: cross-linked dextrans, agarose, polyacrylamide gel, and polystyrene.
• Cross-linked dextrans are used for Commercial Sephacryl S.
• Agarose is used for Commercial Sepharose or Bio-Gel A.
• Polyacrylamide gel is for Commercial Bio-Gel P.
• Polystyrene is used for Commercial Bio-Beads.

Experimental Procedure

• The column packing has a ligand-treated matrix.
• The buffer must contain cofactors needed for ligand-macromolecule interaction.
• The sample is applied and fractionated.
• The macromolecule binds and gets immobilized.
• Non-specifically bound contaminants are removed with buffer.
• The purified compound is recovered from the either specifically or non-specifically.
• Non-specific elution is achieved by a change in either pH or ionic strength.
• Acetic acid or ammonium hydroxide can be used in elution to change the state of ionization of groups.
• Collected fractions are readjusted to have reduced chances in protein denaturation.
• An ionic strength change causes macromolecule elution, with 1M NaCl used towards that purpose.
• Protein is purified from low molecular species through exclusion chromatography.

Applications of Affinity Chromatography

• Used to purify a wide range of enzymes and other proteins, including receptor proteins and immunoglobulins
• Lectin-sepharose isolates glycoproteins, separates lymphocytes, and fractionates T-cells.
• 5'-AMP- and 2',5'-ADP-sepharose purifies NAD+, NADP+-, and ATP-dependent dehydrogenases and kinases.
• Protein A-agarose is used to purify human immunoglobulin G (IgG) from serum or cell extracts.
• Cibacron-blue agarose can bind with affinity nucleotides as its structurally similar.
• Cibacron-blue agarose is used for kinases, dehydrogenases, albumin, DNA polymerase and coagulation factors isolation.
• Poly(A)-agarose binds nuclear mRNA and fractionates ribonucleoproteins and viral mRNA.
• Lysine agarose binds ribosomal RNA and the protein, plasminogen.
• Heparin-agarose is used to purify bone collagenase, hepatitis B surface antigen, uterine estradiol receptor, murine myeloma DNA polymerase, ribosomes, adrenal tyrosine hydroxylase and several androgen receptors.

Gas Liquid Chromatography (GLC)

• Also known as Gas Chromatography (GC)
• Gas Liquid Chromatography (GLC) is a type of partition chromatography in which the mobile phase is a carrier gas, usually an inert gas such as helium or nitrogen.
• A gas chromatograph uses a thin capillary fiber known as the column, through which different chemicals which pass at different rates depending on various chemical and physical properties.
• As chemicals exit the column, they are detected and identified electronically.
• The Function to separate and concentrate different components in order to maximize detection signal.
• A stationary phase of a high-boiling liquid material is supported on an inert granular solid.
• The column is maintained in an oven at a temperature to separate compounds in an evaporating state.
• The basis for separation depends on the partition coefficients of volatilized compounds between the liquid and gas phases.
• Compounds are carried through the column by the carrier; a recorder then marks the peak as each separable compound passes.

Instrumental Components of GLC

• Carrier gas: nitrogen, helium, argon, and carbon dioxide.
• Sample injection port
• Columns: Packed and capillary (open tubular).
• Stationary chemicals: high-boiling organic compounds
• Stationairy phase examples: polyethylene glycols, methyl phenyl silicon gum and esters of adipic, succinic and phthalic acids.

Advantages of GLC

• The great length of the column in Gas Liquid Chromatography allows for easier lower molecular weight compound separation.
• Sensitivity enables a concentration of 10-12 gram to be detectable for many substances.
• Separation is frequently achieved in less than a minute which dramatically increases the speed.
• Using a non-destructive detector, GLC can be used for preparative purposes.

Uses of GLC

• GLC is widely used for both qualitative and quantitative analyses.
• GLC provides a high speed of resolution, reproducibility and high sensitivity.
• GLC separates partition compounds between liquid/gas phases.
• Thousands of volatile organic compounds can be separated.
• Non-volatile substances can be can also be separated if converted into volatile ones via oxidation, acylation, and alkylation.
• Biological samples of Alcohols, esters, fatty acids and amines can get separated by GLC
• High sensitive reading concentrations of individual elements of Carbon and hydrogen.
• Identification of functional groups
• For example, to identify alkoxy groups, use iodination to form an alkyl iodide
• The position of the double bond can then be determined by cleaving the bond by oxidation.

High Performance Liquid Chromatography (HPLC)

• High-Performance Liquid Chromatography is a chromatographic technique used to separate, identify, and quantify components in a liquid sample.
• HPLC uses high pressure to pass liquid through a stationary phase-packed column.
• HPLC Provides high precision and sensitivity in separating complex mixtures.
• Mobile phase is a liquid solvent in a solvent reservoir.
• The mobile phase is moved through the system by a pump at high pressure.
• As separation occurs, the compounds pack into a stationary phase (silica/polymer beads) to form a column.
• As the components leave this column, they are detected by a detector (e.g., UV, fluorescence, and conductivity).
• Data processors process the signal and generates a chromatogram.
• During operation the sample is injected into the mobile phases.
• The mobile phase moves the sample through the column with the stationary phases.
• The components of the sample interact differently, causing them to separate.
• As the separated components leave the column, they are detected and analyzed by the detector.

HPLC Types

• Normal Phase HPLC has Polar stationary phase and Non-polar mobile phase.
• Normal Phase HPLC is mainly used for polar compounds.
• Reversed Phase HPLC has Non-polar stationary phase with a polar mobile phase.
• Reversed Phase HPLC is more commonly used for non-polar compounds.

Applications of HPLC

• Pharmaceutical: High analytical for active ingredients, impurities, and drug formation.
• Environmental: Helpful for detection in pollutants across water, soil, and air samples.
• Food and Beverage Industry: Monitors quality and analyzes food additives, preservatives, and contaminants.
• Forensic Science: Important to analyse toxins, drugs in biological samples.
• Biotechnology: Used in separation for nucleic acids, protein and peptides.

Advantages of HPLC

• High resolution and sensitivity.
• Capable of separating complex mixtures.
• Quick and reliable.
• Wide range of applications in different fileds.

Limitations of HPLC

• Expensive for both equipment and maintenance.
• Requires skilled operation and maintenance.
• Limited volatile components analysis.

HPLC Chromatogram Example

• The description should be an example chromatogram.
• X-axis: Retention time.
• Y-axis: Detector response (peak height or area).
• An explanation of each peak represents a different compound eluting from the column at different times.