Reverse Phase Chromatography (RPC)

Reverse Phase Chromatography is a special, yet powerful column chromatography that involves the use of hydrophobic adsorbents. Normal phase chromatography normally uses hydrophilic stationary phase. This technique uses the hydrophobic adsorbent, which are the aliphatic chains that are attached to the silica beads.

Brief Introduction

The gel bead is surrounded by a layer of lipophilic aliphatic chains of carbon, which are the ligands. The sample is applied to an aqueous solvent. The peptides that are adsorbed to the gel bead are eluted out from the column by decreasing the organic solvent concentration of the column, thus creating an increasing organic solvent gradient. The elution of peptide from the column is in the trend of increasing hydrophobicity. The peptide with the lowest hydrophobicity would get eluted out first.

Advantage

1. High resolution
   
2. Speed
   
3. High capacity, which is the best method to concentrate samples
   

 

Disadvantage

1. Solvents
   

   The organic solvents are toxic, which have the possibility of denaturing the labile proteins.
   

2. Silica-based
   

   Hence, it is unstable under alkaline condition.

Gel Filtration (Size-Exclusion Chromatography)

Gel filtration of protein is the separation of protein based on size and shape of the protein. The protein sample will pass through a column packed with gel beads. Each bead contains pores that have size that is comparable to the size of the protein molecules that we want to separate. Each bead has its own working range, 10kD to 600 kD. If the protein molecule us too large, it will get eluted from the column as a void volume. Only the smaller protein can e fractionated.

In gel filtration, the gel matrix should be physically and chemically stable. It is also paramount that the gel matrix should not have adsorptive properties, which means inert. In order to prevent the adsorptive property of the gel matrix, eluent of certain ionic strength should be used. However, the pH and composition of the eluent is not important as the separation technique is based on size and shape. In short, upon application of the eluent, the structure and activity of the protein should be preserved. In the separation of folded protein, chaotropic agent such as urea can be used for fractionation of the proteins.

Factor affecting the outcome of gel filtration:

1. Sample volume
   
2. Column length
   

It should be noted that the ration of sample volume to column length would affect the resolution of the gel filtration. Larger ratio would result in decreased resolution. Hence, in order to obtain high resolution, it is wise to either apply smaller sample volume or increase the column length. Sample concentration would not affect the resolution of the chromatography. However, it is inevitable that gel filtration process would result in dilution of the sample. Hence, the sample should be as concentrated as possible. However, it should be noted that the concentration of the sample should be within the limit of the viscosity, as well as solubility of the column.

Ion-Exchange Chromatography (IEC)

The principle behind Ion-Exchange Chromatography is that we are creating a competition between the target ions and other ions of the same charge to bind to the adsorbent matrix of the column, which has opposite charge. Hence, we are exploiting the fast that other than at isoelectric point, by changing the pH, we are able to obtain protein in ionic form, where it is positively charged or otherwise. The ionic protein would have to compete with the other ions present in the matrix to bind to the adsorbent beads. The separation of the proteins depends on the reversible adsorption of charged solute molecules to immobilized ion-exchange groups of opposite charge.

Steps in IEC:

1. Equilibration
2. Sample application and washing
3. Elution
4. Washing and re-equilibration

Equilibration
The process of equilibration is to ensure that all the adsorbent beads are in equilibration to the counter ions. Usually, for anion exchanger, Cl^- ion is used while for cation exchanger, Na⁺ is used.
Strong exchanger means that the exchanger remains charged over a large pH range while weak exchanger means the exchanger remains charged over a small pH range.
Sample Application and Washing
Before the sample is inserted into the column, we should dissolve the protein sample into the starting buffer and adjust the pH. As IEC has the ability to concentrate the protein sample, the sample applied into the column can be of any volume, and also maximize the buffering capacity of the buffer.
Upon application of sample, the proteins that carry appropriate charge would displace the counter ions and bind reversibly. The protein that binds the strongest to the column is usually found closest to the top of the column. The column would then be washed with the starting buffer to wash out the protein samples or component that do not adsorb and they are recovered in a breakthrough peak. Sometimes, in the process of adsorption, protein bands can be observed.

Figure 4.14 above shows that the α decreases with increasing salt concentration. As the salt concentration is increasing, the protein band is becoming sharper. When salt is added, desorption occurs and proteins start to move down the column. However, the proteins are not moving as fast as the salt passing by. Hence, each part of the protein band experiences increase in salt concentration. At the same time, the edge of the band is sharpen due to the protein concentration effect.


Importance of pH in IEC
Actually, the pH of the micro-environment of the ion-exchanger differs slightly from the pH of the starting buffer added to the column. Hence, the effect of pH in Ion-Exchange Chromatography cannot be underestimated due to Donnan's effect. Due to Donnan's effect, the proton in the adsorbent matrix can be repelled or attracted. Hence, for anionic ion-exchanger, normally the pH is 1 unit higher while for cationic ion-exchanger, the pH is 1 unit lower. The lower of ionic strength of the buffer results in greater the difference in pH due to Donnan's effect. Hence, it would have great impact on the stability of the protein, especially enzyme, which is a function of pH. Enzyme protein usually prefers mild alkaline solution than mild acidic environment. For anionic ion-exchanger, denatured due to high pH is normally rare. Hence, in order to prevent the denaturation due to low pH in cationic-exchanger, it is wise to dilute the sample first before application into the column.

Elution
Desorption of the proteins can be done by:

1. Changing the ionic strength
2. Changing the pH
3. Inclusion of new ionic species

Choosing between isocratic elution, stepwise elution and gradient elution?
Isocratic elution
Isocratic elution is done using solvent that has constant composition throughout the elution process. Such elution method can only be used on proteins with known chemical properties and the proteins are run in repeated trials.
Stepwise elution
Stepwise is the serial application of isocratic elution. Such a method might not work efficiently in protein separation because sometimes the ionic strength of the buffer used at any changes in the concentration might be too high, or too low, resulting in false separation. If the buffer used is too weak, it might result in sample being separated into broad peak while if the buffer used is too strong, it will result in the several different proteins elute at the same time.
Gradient elution
Gradient elution is by increasing the concentration of buffer added to the column gradually. At low salt concentration, protein that are weakly attached to the adsorbent would be desorbed and eluted out, while at higher salt concentration, the second protein would get eluted, and so on. The problem of having broad elution peak in isocratic elution is actually minimize in gradient elution as the elution power of the salt solution increases as the gradient increases.
Linear gradient is highly recommended. However, the difference lies in the fact that whether it is a short steep linear gradient or a long shallow gradient. There is no perfect choice of gradient. For short steep linear gradient, the peak produced would be sharp and faster separation but with the compromise of having short retention distance between the peaks. For long shallow linear gradient, maximum separation of the peak is achieved but the separation will be long and the peak broadening might result.
The gradient elution can be created by mixing the initial buffer and final buffer together initially. Gradually, the proportion of final buffer increased while the proportion of initial buffer decreases along the process of elution.
Washing and re-equilibration
The column should be washed as it might contain denature proteins as well as other components such as lipid. The presence of such components might affect the efficiency of the column in the future re-use, present as contaminant in the future run, or reduce adsorption.


Advantages of Using IEC
IEC is the most popular chromatography method used to separate protein, due to:

1. High resolution
   
2. High protein-binding capacity
   
3. Versatility as there are various kinds of IEC available and the pH and buffer condition vary.
   
4. Straight-forward as the separation is based on charge
   
5. Ease of peformance

Hydrophobic Interaction Chromatography (HIC)

Hydrophobic interaction chromatography is exploiting the special chemical property of protein that there are extensive hydrophobic patches on the protein surface, in addition to expected hydrophilic regions. The hydrophobic patches are the part on the protein surface that would bind to the hydrophobic ligands on the adsorbent, which are in favour of hydrophobic interaction in the presence of high salt concentration. Hence, the type and concentration of salt play an important role in the efficiency of the HIC. The influence of the salt on the hydrophobic interaction follows the Hofmeister series. Such non-ionic interaction is enhanced by the salting-out principle. In salting-out, the main cause of aggregation is the strengthening of hydrophobic interaction between proteins. The presence of high concentration of salt causes the formation of water cavity. The formation of water cavity with a salt that gives a high surface tension needs a bigger input of energy than the salt that gives low surface tension. It is the salt that gives the highest surface tension that provides greatest hydrophobic interaction.

 

Similar to other chromatography, HIC also follow the following steps:

1. Equilibration
   
2. Sample application
   
3. Washing
   
4. Elution
   

Sample Application

The sample has to be in a salt in order to enhance hydrophobic interaction. Most commonly used salt are ammonium sulphate or sodium sulphate, but sometimes chloride salts are used.

Elution

The process of elution can be done by:

1. Changing the concentration of salt
   
2. Changing the pH of the buffer
   
3. Changing the polarity of the solvent
   

Elution process can be carried out by decreasing the concentration of salt, either a downward gradient or by stepwise lowering. As resolution is not high, gradients may not achieve any better result than stepwise process. Changing the pH of the buffer for the process of elution might not work as it is impossible to predict how the strength of the hydrophobic interaction between a protein and HIC adsorbent will effect upon change in pH. Besides that, solvent that can lower the polarity of the column such as ethanol and ethylene glycol can be used for elution of strongly-bound protein. Such solvent can be added after the salt has been removed from the column.

Affinity Chromatography

Principle of Chromatography:

1. Equilibration
   
2. Sample application
   
3. Binding and washing
   
4. Desorption and elution
   

Generally, during the process of equilibration, the specific ligand would be attached to the matrix of the column. After that, the sample would be inserted into the column. Only target protein would binds to the ligand, which is bound onto the column matrix, while the non-target protein would be washed out. Finally, during the process of desorption, the target protein, which is attached to the ligand initially, would get eluted out simply by changing the eluent.

 

Choosing the Correct Ligand

These are the properties of the ligands that are highly favourable:

1. Form reversible complex with protein sample.
   
2. High complex constant for stable complex
   
3. Easy to dissociate the complex
   
4. Easy mobilization
   

The ligand chosen must be able to form reversible complex with the protein sample as you would want to obtain the purified protein back during the process of desorption at the end of the purification. However, the complex formed must be stable enough to give sufficient retardation (retention) to the column. In addition to the requirement that the complex formed between the ligand and the protein sample must be reversible, it should be easy to dissociate the complex by simply changing the medium or buffer of the column, without creating any change or denaturing the protein or ligand.

The types of ligand:

1. Monospecific low molecular weight ligand
   
2. Monospecific macromolecular weight ligand
   
3. Group specific low molecular weight ligand
   
4. Group specific macromolecular weight ligand
   

Monospecific low molecular weight ligand

Monospecific means that ligand binds to single or very small amount of protein in cell extract or body fluid.

Group specific low molecular weight ligand

The ligand used here are most likely enzymes and its analogues such as biomimetic dye, boronic acid derivatives, vitamins, amino acids, etc.

Monospecific macromolecular ligand

This kind of ligand is about groups of specific protein-protein interactions. Due to the high specificity of antibodies, antigen and antibodies is normally good candidate for Monospecific macromolecular ligand. The mobilized proteins are also known as immunoadsorbent. These immunoadsorbent can be used to purify cells, soluble proteins, peptides, solubilised membrane protein and also viruses. It is good to use antigen as ligand due to its uniform binding to the target protein. Besides that, the use of monoclonal antibodies allow a constant supply of a highly uniform antibody, which give rise to high reproducibility from batch to batch of immunoadsorbent. This is extremely advantageous as such uniform antigen binding would produce sharp desorption peak. However, the disadvantages of such ligand are mainly due to its high cost of production, as well as high risk of fouling. This is because the antibody will only recognize and bind to corresponding antigenic determinant of actual protein. Thus, the antibody would fail to observe if the protein has been modified or degraded. Furthermore, if the sample is crude extract, the sample might be denatured chemically via proteolysis.

 

Choosing the correct matrix

The following properties are important when choosing a matrix:

1. Macroporous
   
2. Hydrophilic and neutral
   
3. Presence of functional group for derivation
   
4. Chemically stable
   
5. Physically stable
   
6. Readily available
   

The best candidate would be spontaneously gel-forming galactan agarose, as it possesses most of the characteristic mentioned above for an ideal affinity chromatographic matrix. Although agarose gel might be less chemically and physically stable, it still can be used in affinity chromatography due to its chemical cross-linking of the physically cross-linked junction zones in the agarose gel structure.

Spacer arm

Spacer arm is a linker between the matrix and the ligand. If the length of the arm is too short, it might be ineffective. However, if the length of the arm is too long, it might result in non-specific binding due to hydrophobic interaction. The spacer arm can be added via the following two methods:

1. Immobilize a spacer arm with a terminal primary amine; increase the length by reaction with succinic anhydride.
   
2. Immobilize a spacer arm with a terminal carboxyl group, increase the length by reaction with 1,7-diamino-4-azaheptane with the aid of condensation reaction.
   

Ligand Immobilization

Action of the matrix can be done by introducing an electrophilic group into matrix. The ligand is either coupled directly to the activated matrix or otherwise. The deactivation of the matrix can be done by a large excess of suitable low molecular weight substance. When affinity adsorbents are prepared, a stable bond should be formed between matrix and the ligand to prevent leakage of ligand.

Evaluation of the Affinity Adsorbent

The ligand density can be used to evaluate the affinity adsorbent by a few methods. One of the methods is indirect method, which estimates the uncoupled ligand. The amount of immobilized ligand can be calculated by the difference between the amount of ligand originally added and the amount of ligand recovered in the liquid phase and pooled washing after finished coupling.

 

Desorption Process

The process of desorption is about changing the binding equilibrium for the absorbed substance from the stationary to the mobile phase. Affinity chromatography is all about the interaction between the sample proteins and the ligand, which is a combination of electrostatic, hydrophobic and hydrogen bonds interaction. Hence, any agent that can weaken such an interaction would be effective non-specific chemical used for desorption. However, during the process of desorption, it is always a compromise between the harshness and risk of denaturing the protein.