Size Exclusion Chromatography

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Size Exclusion Chromatography

INTRODUCTION

Size exclusion chromatography was first developed by Lathe and Ruthven in 1955. In 1959, Porath and Flodin described the separation of the macromolecules on cross-linked poly dextrane gels. J.C. Moore of Dow Company proposed the separation of the synthetic polymers on cross-linked polystyrene gels in organic mobile phases. This method is the chromatographic method in which the molecules are separated based on the size. SEC is also called as gel permeation chromatography (GEC) and gel filtration chromatography. The separation of the biomolecules in aqueous or organic mobile phase is called as the gel filtration chromatography. The separation of the organic polymers in non-aqueous mobile phase is known as GEC.

PRINCIPLE

The main principle involved in the SEC is diffusion through the pores present in the stationary phase with solute and solvent molecules and then removed with the continuous flow of mobile phase.

Principle separation of SEC

Principle separation of SEC

The main principle is the separation mainly based on the sizes and shapes of the molecular sieves with porous material that is gels are commonly employed. High molecular weight compounds are eluted first and the low molecular weight compounds are eluted later.

Separation technique

Separation technique

THEORY

The sample is introduced into the column packed with gel or porous particle packing material (stationary phase) and carried out by the solvents (mobile phase). In case of GEC, non-polar organic mobile phase is used whereas in gel filtration chromatography aqueous mobile phase is used. The theory is nothing but the particles of the different sizes and shapes present in the sample elute through the stationary phase with different rates by mobile phase.

The partition ratio between the stationary phase and mobile phase is defined by the distribution coefficient (KD)

(KD) = (Vc – Vo) /Vi

where Ve is the elution volume that is volume of the solvent between the injection and elution; Vo is the void volume that is the porous particles occupied volume; Vi is the inner volume that is the volume of the solvent held in the packing material.

Distribution coefficient is independent of the pH, ionic strength and concentration of the solvent. Low molecular weight samples diffuse freely into the gel have the distribution coefficient values between 0.8 and 1.0. Greater than 1 of distribution coefficient value indicates adsorption of the substance into the stationary phase which indicates the less elution rate. The value of 0 indicates that there is no absorption.

The total volume is given by the following equation:

Vt = Vg + Vi + Vo

where Vt is the total volume; Vg is the volume of the polymer gel; Vi is the volume of solvent held in the pores; Vo is the free volume which is present outside of the particles.

SEC is based on the amount of time that the molecules spend in the stationary phase.

INSTRUMENTATION

The instrumentation of the SEC contains the following components. They are as follows:

• Mobile phase reservoir
• Pump
• Injector
• Column
• Detector
• Recorder

Flow chart of the sixe exclusion chromatography instrument

• Mobile phase reservoir: Glass or stainless steel reservoirs are commonly employed as reservoirs. The only precaution should be taken is that initially degassing has to be done by the degasser for the removal of dissolved gases.
• In GPC, the commonly employed mobile phases are of non-polar organic mobile phases.
• Example: Tetra hydro furan, toluene, dimethylformamide, trichloro benzene, hexa fluoro propanol, etc.
• To improve the GPC separations, additives are added to improve the efficiency of separation.
• Example: An antioxidant is added to the trichloro benzene to increase the stability in high temperatures.
• In GFC, the commonly employed mobile phases are aqueous mobile phases. In general, buffer solutions are used.
• Example: Sodium acetate buffer, sodium phosphate buffer, Sodium borate buffer, etc.
• Based on the separation of the compounds, the following mobile phases are used:
1. For the separation of the synthetic elastomers: toluene is used as mobile phase.
2. For the separation of the poly vinyl carbons: epoxy resins-tetra hydro furan is used as the mobile phase.
3. For the separation of the poly olefins: trichloro benzene is used as the mobile phase.
4. For the separation of the proteins and polysaccharides: water and buffer solutions are used as the mobile phase.
• Pumps: This is mainly used for the continuous flow of mobile phase through the injector with constant pressure of 6,000 psi. The flow rate is ranging from 1 to 10 ml per minute. There are two types of pumps commonly employed in the SEC. They are as follows:
1. Syringe pumps: works as a normal syringe and delivers the mobile phase by the pulse less flow.
2. Reciprocating pumps: in this, single piston pumps are cheap but these are not suited for the SEC. The dual piston pumps are easy to maintain and frequently used in the SEC.
• Injector: This is manly used for the injection of the solvent through the column. The sample is dissolved in the solvent. Volumes of the mobile must be small that is 1–500 μl.
• Column: Smooth bore steel or heavy walled glass tubing is used as column. Column temperature is maintained by the thermostats (105 °C) and is cooled by fitting it in the cooling jacket containing water. The column is packed with the porous silica or organic gels such as styrene and divinyl benzene. Based on the internal diameter and length of the column, they are divided as the following:
1. Analytical columns: 8 mm in diameter and 300 mm in length.
2. Microcolumns: 4.6 mm in diameter and 250 mm in length.
3. Preparative columns: 20 mm in diameter and 300 or 600 mm in length.
• The following are the materials used in the column packing:
1. Agarose
2. Sephadex
3. Dextrin
4. Polyacrylamide
• The cross-linked polymers are used as stationary phases for the packing of the columns. There are mainly two types of stationary phases used in the SEC. They are as follows:
1. Soft gels such as poly acrylamide gels or dextran gels are used. These gels are mainly used for the separation of the proteins. These are prepared by imbibing the gel into the liquid for the complete swelling.
2. Rigid gels such as polystyrene gels which are mainly used for the separation of the non-polar polymers.
• The following are the precautions for SEC column handling:
1. Same mobile phase should be run through the column because new solvent requires recalibration of the column.
2. Columns never be operated in backward direction.
3. Care should be taken for connecting the columns.
4. Flow rate is minimised to enhance the column efficiency.
• Detectors: The detectors are mainly used to detect the sample constituents and concentration of the sample. The detectors should posses the following ideal characters:
1. Adequate sensitivity
2. Stability and reproducibility
3. Short time response
4. Low zone broadening
5. High reliability
6. Non-destructive
• The SEC detectors are of the following types:
1. Concentration sensitive detectors
2. Example: Refractive index detectors
3.                 UV detectors
4.                 Infrared detectors
5. Molecular weight sensitive detectors
6. Example: Low-angle light scattering detectors.
7.                 Multi-angle light scattering detectors.
• The following are the commonly employed detectors in the SEC:
• Refractive index detectors: Measures displacement of beam with respect to the photo sensitive surface of the detector. The main advantage its high reliability. The main disadvantages are expensive and temperature sensitive.
• UV detector: Measures the absorbed radiation within the range of 190–380 nm. The advantages are high sensitivity and linearity over wide ranges. The main disadvantage is only applicable to the samples absorbs the UV radiation.
• Fluorescent detectors: Measures the fluorescent compounds. The main advantages are high sensitivity and the high selectivity. The main disadvantage is only compound which shows the fluorescence are measured. Less applicability to the samples.
• Conductivity detector: This measures the conductivity of the column effluent.
• Electrochemical detector: This is based on the reduction or oxidation of the compound.
• Evaporative detectors: The compound is detected after evaporation of the total solvent present in the eluent.
• Example: Flame ionisation detector.
• Recorders: Recorders are commonly employed for the interpretation and recording the data obtained from the detector.

ELUTION PROCEDURE

The porous gels are packed into the column and it is filled with the liquid buffer in the case of gel filtration chromatography and filled with the organic solvents in the case GEC. The buffer or organic solvent added to the column calibrates the column. Then the sample dissolved in the mobile phase is applied from the top to the bottom of the column and allow the mixture of the sample solution from top to the bottom of the column. The larger molecules elute first and smaller molecules elute later. The following increases the separation efficiency:

• Increased column length increases the separation efficiency. This can be achieved by the connecting more numbers of column.
• Reduced flow rates enhance the separation efficiency.
• Alternate pumping by using number of pumps enhances separation efficiency.

COLUMN PARAMETERS

• Number of theoretical plates (N):
• N = 5.54(Ve/W1/2) × 1000/L
• where Ve is the peak elution volume; W1/2 is the peak width of half-peak height; L is porous bed height.
• Symmetry factor (As):
• As = b/a
• where a is the first peak width at 10% height; b is the second peak width at 10% height.

SEC peak

FACTORS AFFECTING SEC SEPARATIONS

• Column packing material particle size
• Column length
• Column diameter
• Packing of the column
• Mobile phase selection
• Run time
• Temperature
• Pressure

ADVANTAGES OF SEC

• Short and well-defined separation.
• Narrow bands with high sensitivity.
• High selectivity.
• High resolution.
• Relatively easy separation.

DISADVANTAGES OF SEC

• Scale of chromatogram is short.
• Inapplicability to the samples of similar sizes
• Standards are required for the analysis.
• High cost.
• Interferences like dust, solvent concentration are not detected.

APPLICATIONS

• Used in the determination of glycosides.
• Example: Digitalis glycosides determination.
• Used in the determination of the structure of the proteins.
• Example: Tryptophan determint alanine determination.
• Used in the fractionation of the proteins and water soluble polymers.
• Example: Tryptophan determint alanine determination.
• Used in the determination of molecular weight of newly synthesised drugs.
• Example: Tryptophan determint alanine determination.
• Used in the determination of sample molecules shape.
• Example: Tryptophan determint alanine determination.
• Used in the characterisation of starch.
• Example: Tryptophan determint alanine determination.
• Used in the purification of the proteins.
• Example: Tryptophan determint alanine determination.
• Used in the desalting analysis.
• Example: Tryptophan determint alanine determination.
• Used in the separation of the low molecular weight compounds from the complexes.
• Example: Dextran is separated from the corn syrup oil.

GEL PERMEATION CHROMATOGRAPHY

This method is mainly used to separate the components based on their size. The gel permeation chromatograph is proposed by J.C. Moore in 1964. This method is mainly used for the separation of the polymers. In this method, organic solvent is used as the mobile phase and the polymer beads which form gels when imbibed in the solvent are used as the stationary phase.

Principle

Gel permeation chromatography (GPC) separation is mainly based on the size or molecular weight of the components present in the sample solution. In this method, the separation is mainly based on the physical or chemical interaction of the solute molecules with the stationary phase which is a porous bed of the packing material packed in the column. The large molecules are eluted first and the small molecules are eluted later. The main difference of GPC when compared to the gel filteration chromatography is the use of the non-aqueous organic solvent as the mobile phase.

Diagram of the GPC separation of the molecules

Diagram of the GPC separation of the molecules

If the analytes are not retained completely or eluted with the free volume of the presence of the outside particles (Vo), then the analytes are completely retained by holding the solvent in the pores (Vi). The total volume is given by the following equation:

Vt = Vg + Vi + Vo

where Vg is the volume of the polymer gel.

Theory

The main requirement for the GEC is that the analyte should not interact with the surface of the stationary phases. The main theory involved in the GEC is that the particles of the sample of different sizes elute through the stationary phases at different rates based on their molecular sizes and molecular weights. Thus the particles with the same size are eluted together. This is mainly based on the distribution of the solute particles between the stationary phase and the mobile phase.

Instrumentation

The instrumentation of the SEC contains the following components. They are as follows:

• Mobile phase reservoir
• Pump
• Injector
• Column
• Detector
• Recorder

Flow chart of the sixe exclusion chromatography instrument

• Mobile phase reservoir: Glass or stainless steel reservoirs are commonly employed as reservoirs. The only precaution should be taken is that initially degassing has to be done by the degasser for the removal of dissolved gases.
• In GPC, the commonly employed mobile phases are of non-polar organic mobile phases.
• Example: Tetra hydro furan, toluene, dimethylformamide, trichloro benzene, hexa fluoro propanol, etc.
• To improve the GPC separations, additives are added to improve the efficiency of separation.
• Example: An antioxidant is added to the trichloro benzene to increase the stability in high temperatures.
• In GFC, the commonly employed mobile phases are aqueous mobile phases. In general, buffer solutions are used.
• Example: Sodium acetate buffer, sodium phosphate buffer, Sodium borate buffer, etc.
• Based on the separation of the compounds, the following mobile phases are used:
1. For the separation of the synthetic elastomers: toluene is used as mobile phase.
2. For the separation of the poly vinyl carbons: epoxy resins-tetra hydro furan is used as the mobile phase.
3. For the separation of the poly olefins: trichloro benzene is used as the mobile phase.
4. For the separation of the proteins and polysaccharides: water and buffer solutions are used as the mobile phase.
• Pumps: This is mainly used for the continuous flow of mobile phase through the injector with constant pressure of 6,000 psi. The flow rate is ranging from 1 to 10 ml per minute. There are two types of pumps commonly employed in the SEC. They are as follows:
1. Syringe pumps: works as a normal syringe and delivers the mobile phase by the pulse less flow.
2. Reciprocating pumps: in this, single piston pumps are cheap but these are not suited for the SEC. The dual piston pumps are easy to maintain and frequently used in the SEC.
• Injector: This is manly used for the injection of the solvent through the column. The sample is dissolved in the solvent. Volumes of the mobile must be small that is 1-500 μl.
• Column: Smooth bore steel or heavy walled glass tubing is used as column. Column temperature is maintained by the thermostats (105 °C) and is cooled by fitting it in the cooling jacket containing water. The column is packed with the porous silica or organic gels such as styrene and divinyl benzene. Based on the internal diameter and length of the column, they are divided as the following:
1. Analytical columns: 8 mm in diameter and 300 mm in length.
2. Microcolumns: 4.6 mm in diameter and 250 mm in length.
3. Preparative columns: 20 mm in diameter and 300 or 600 mm in length
• The following are the materials used in the column packing:
1. Agarose
2. Sephadex
3. Dextrin
4. Polyacrylamide
• The cross-linked polymers are used as stationary phases for the packing of the columns. There are mainly two types of stationary phases used in the SEC. They are as follows:
1. Soft gels such as poly acrylamide gels or dextran gels are used. These gels are mainly used for the separation of the proteins. These are prepared by imbibing the gel into the liquid for the complete swelling.
2. Rigid gels such as polystyrene gels which are mainly used for the separation of the non-polar polymers.
• The following are the precautions for SEC column handling:
1. Same mobile phase should be run through the column because new solvent requires recalibration of the column.
2. Columns never be operated in backward direction.
3. Care should be taken for connecting the columns.
4. Flow rate is minimised to enhance the column efficiency.
5. Detectors
• The detectors are mainly used to detect the sample constituents and concentration of the sample. The detectors should posses the following ideal characters:
1. Adequate sensitivity
2. Stability and reproducibility
3. Short time response
4. Low zone broadening
5. High reliability
6. Non-destructive
• The SEC detectors are of the following types:
1. Concentration sensitive detectors
2. Example: Refractive index detectors
3.                 UV detectors
4.                 Infrared detectors
5. Molecular weight sensitive detectors
6. Example: Low-angle light scattering detectors
7.                 Multi-angle light scattering detectors

Mobile phase: In mobile phase, non-aqueous organic solvents are used. The following are the ideal requirements of the solvent:

1. Solvent should be dry.
2. Solvent should be degassed to remove the air.
3. Sample should be used the same solvent for the sample preparation.

Stationary phases: Gels are used as stationary phases in the GPC. The following are the ideal requirements of the stationary phases:

1. It should be of uniform size and shape.
2. It should be inert.
3. It should be stable.
4. It should be free from inorganic particles.

FACTORS AFFECTING GPC SEPARATION

• Increasing the column length will enhance the resolution, and increasing the column diameter increases the capacity of the column.
• Proper column packing is important to maximise resolution. An overpacked column can collapse the pores in the beads, resulting in a loss of resolution.
• An under packed column can reduce the relative surface area of the stationary phase accessible to smaller species, resulting in those species spending less time trapped in pores.
• Unlike affinity chromatography techniques, a solvent head at the top of the column can drastically diminish resolution as the sample diffuses prior to loading, broadening the downstream elution.

ADVANTAGES OF GPC

• Well-defined separation time.
• Applicable to large molecules separation.
• Simple to handle.
• High selectivity.
• High sensitivity.
• Less time consuming.

DISADVANTAGES

• Limited number of peaks.
• Less resolution.
• Interferences are more.
• Broad peaks.
• Prefiltration of the sample is required.

APPLICATIONS

• Used in the determination of the molecular weight of the polymers.
• Used in the determination of the blood substitutes.
• Used in the analysis of the binding agents in the tablet formulations.
• Used in the study of the different drug delivery systems.
• Used in the estimation of the thickening agents.
• Used in the estimation of the coagulants such as heparin.
• Used in the determination of the quaternary structure of the purified proteins.
• Used in polymers synthesis.