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Effective Protein Separation Using Chromatography

The separation of intact proteins in proteomics remains a challenge with scientists inventing more reliable methods that not only are easier to conduct but that are effective in separating the protein molecules. Among the methods that are used in protein separation, the most common ones include the reversed phase chromatography (RPC) and the hydrophobic interaction chromatography (HIC).

The hydrophobic interaction chromatography process involves the separation of biomolecules, peptides, and proteins through a mechanism that works on the basis of the degree of the hydrophobicity of the molecules (Valeja, 2014). The technique involved in HIC includes non-denaturing which means that the inherent form of the protein is usually maintained after the separation. This process is important in the research work since it has been used for further research in the biological characteristics of the divisional proteins (Yang, 2015).

The procedure involved in the HIC separation includes interacting the stationary phase and that of the hydrophobic exhibited on the surface of the protein. The overall effect of this stationary phase involves the retention mechanisms which are affected by the adsorption-desorption equilibrium. All processes in HIC happen in the presence of the salts. In this case, the presence of the high concentration of salts, the proteins is forced to bind in the stationary phase. By decreasing the salt concentration and increasing hydrophobicity, the proteins are eluted which makes the separation possible (Yang, 2015). The conjugated protein and the proteins which are separated by the hydrophobic interaction chromatography is the best example. There are some of the proteins like the antibody drug conjugates which require systematic methods in the separation mechanisms. This means that they require optimal conditions for their separation to reduce these non-denaturing processes to the associated proteins. One important point to note is that the whole procedure requires a low ionic strength buffer which should be weak as per the required standard to allow the elution of the protein from the column without necessarily using the organic modifier as the elution buffer. There are some salts that affect the hydrophobic interactions and stationary phase. These interactions include the adsorption of the protein to the HIC media. De-adsorption of the proteins can be attained by lowering the salt concentration gradually. The contained samples will elute by the systematic order of hydrophobicity (Yang, 2015).

Reversed-phase chromatography is one of the oldest ways of protein separation. This process involves the use of liquids in the column which acts as the adsorbent. The adsorbent material or liquid usually used in this chromatography is usually hydrophilic. In this RPC method, the hydrophilic ligand is used which acts by attaching itself in the adsorbent and the hydrophobic patches. This process involves the covalent bond attachment between the two ligands and patches. The whole process takes place in the presence of the aqueous mobile phase. The attachment leads to the strong interactions of the hydrophobic processes which rely on the use of additives and the organic solvents to desorb the proteins. Desorption processes have been associated with denaturing the associated proteins, the main cause being the strong covalent bond created. Scientists initially used the RPC technique to separate relatively small portions of the organic molecular components in the hydrocarbon phase which exhibited to be more or less dissolved.

A very good example is the purification of the polypeptides which showed massive improvements when the process was applied. Scientists gained interest in reverse phase chromatography because it had high resolving power. There was much effect in the results that were identified when another category of proteins was used. Organic solvents proved to work well with the process because of the size; organic solvents are relatively small compared to the proteins. When the peptides and proteins were separated using the process, the separation proved difficult because they were large in comparison to the initial organic solvents. One profiting characteristic of the organic solvent is that they are subject to partitioning which is different from the proteins. This research work entails the comparison of the analytical methods, both the hydrophobic interaction chromatography and the reverse phase chromatography, for successful separation of protein in top-down proteomics.


Reverse phase chromatography

The separation of the proteins using the reverse phase chromatography (RPC) method is divided into stages. The stages are subdivided into five stages. The first stage is equilibrating the chromatographic processes in the reverse phase medium. To equilibrate the process involves ensuring that the components are in the correct proportionality suitable in the initial mobile phase. These conditions are the pH, correct ionic strength, and the polarity. The conditions are sometimes known as the hydrophobicity of the mobile phase. Organic modifiers such as the acetonitrile are used mostly in the experiment to control the polarity of the mobile phase. In some cases, trifluoroacetic acid may be used as the ion pairing agent. In this case, keen emphasis should be put on the polarity because it is one of the considerations when initiating the process and the mobile phase A is supposed to be low enough to the required standard. The low polarity is supposed to dissolve the partially hydrophobic solute and it should also be set high enough to create the binding processes of the solutes to the matrix involving the reverse phase chromatography.

The second step is the implementation of the solute separation. This involves the sample salutes which are dissolved in the mobile phase. The main consideration in this stage is the equilibration of the chromatographic bed. This process ensures that the sample is passed through the column at a standard flow rate which permits the optimal binding of the sample. The sample application causes the separation processes to occur in the column. The chromatography bed which is formed in the process is washed by the mobile phase A. The purpose of washing in this instance is to remove any unbound solute to the column. Under the same process, mobile phase A also removes the unwanted solutes or components from the chromatographic bed. In the end, the bound solutes which need to be desorbed are removed. Desorption requires the adjusting of the mobile phase polarity which necessitates the elution of the solutes from the column. The whole process is simplified where the mobile phase’s polarity is reduced. It also associates the increase of the organic modifier in the mobile phase to detach the bound solutes. In this, the concentration of the organic modifier should be looked at and maintained at the constant concentration. This concentration is important in the final mobile phase, also known as the mobile phase B.

During the whole process it is required that the pH of the mobile phase A and B should be kept at a constant regulation. This particular constant is supposed to be the same pH from the start of the process to the end. The increase in the hydrophobicity in the mobile phase will lead to the decrease in the polarity of the mobile phase and it is reached by raising the 100% linear gradient in phase A. What differentiates phase A with phase B is that, Phase A has less organic modifier. Hence, the increase in the linear gradient causes a shift from mobile phase A to mobile phase B which contains the maximum concentration of organic modifier. A unique feature in this stage is that the bound solutes desorbs into their relative inherent state from the reverse phase medium.

The fourth stage involves the removal of un-desorbed solutes from the column. This removal is necessitated by the process of changing the composition of the mobile phase B which is characterized by the transformation of mobile phase B to nearly 100% organic modifier. In case the researcher or the acclaimed individual may want to reuse the column, in that case, he may decide to remove the un-desorbed solutes so as to restart the process again. The change in phase composition ensures the total removal of all bound substances in the column. The fifth stage involves the re-equilibration processes of the used chromatographic mediums. The re- equilibration involves changing back the mobile phase B into the initial mobile phase A (Valeja, 2015).

The achievement in the reverse phase chromatography technique is affected by gradient manipulation, which means that changing the gradient from 100% initial mobile phase to the 100% latest mobile phase. There is also the consideration of the organic modifier’s amount regulation in both the mobile phase A and the mobile phase B. This amount varies a lot in the whole process and contains five percent or lesser than that in the mobile phase A, as compared to the mobile phase B which has 95% or more.

Hydrophobic interaction chromatography

The hydrophobic interaction chromatography entails the usage of the ligand media whose characteristics is that the media contain alkyl and aryl groups which are embedded in an inert matrix. The matrix also contains the spherical particles and is porous. The porous nature of the HIC provides the maximum internal surface area as the ligand plays an important role in the hydrophobicity of the medium. The initial stage involves packing of the medium into the packed bed. In order to fill the pores in the matrix, the buffer is used which also equilibrates the packed bed. This packing is necessitated to ensure that results are of high performance. The process may entail usage of small prepacked columns for example RESOURCE HIC test kit or Hitrap HIC selection kit which ensures better separation if used in the gradient elution. The interaction processes are necessitated by the contact between the protein and the medium. This interaction requires relatively high salt concentration of about one to two molar of the ammonium sulphate or 3-molar sodium chloride. The buffer which contains the type of the salt and its concentration is highly selected to ensure that the target protein is bound to the matrix. This means that the way this mechanism works is related to hydrophobicity, where the less hydrophobic proteins are easily passed through the matrix in the column. It is also assumed that the impurities will not be bound by the matrix and that they too must pass through the matrix leaving behind the more hydrophobic proteins. Any separation by the HIC requires the optimal binding capacity of the matrix since the higher the binding capacity, the better the end product.

Selectivity is also one of the conditions to be considered, the salts to be used should be regarded to obtain the selected proteone. The selected sample should resemble the buffer to be used and this includes the relevant conditions. A very good example is that of performing buffer exchange as the buffer ion should resemble the pH, and in this course, the pH should be manipulated to obtain the desired results. The next phase to watch is the stability window of the selected proteins. Some proteins work best under different salt concentrations. A highly concentrated salt composition can precipitate the required protein and this may make the separation impossible. To determine this range of the window stability, the sample is passed through the testing tube at various concentrations to obtain the supernatant. This is to be closely checked to ensure that the required product reaches the standard.

The sample is passed through the column to ensure that it is bound and then the washing process follows. This separation is achieved by ensuring that non-bound proteins are washed away from the matrix together with the impurities. Then the process of evolution follows where proteins are eluted by reduction of the salt concentration from the matrix. The concentrated and purified forms of the proteins are variedly eluted under different concentrations of the salt gradient. The proteins with the lowest degree of hydrophobicity are the first ones to be eluted from the column and those proteins with the highest degree will be eluted last from the column. Some sample proteins may act impossible to unbind and the wash step in the buffer with the salt-free composition is used. This removes all the remaining proteins of the sample in the column, the success of the experiment is actualized when all the protein components are eluted from the column. To start the next phase in the elution of the protein components, the column is passed through re-equilibration. Some elusion substances might be used if the hydrophobic interaction may be perceived to be harsh. The first step may involve washing the samples using the 0.5 to 1.0 sodium hydroxide followed by 70% ethanol and lastly 30 isopropanol. At the end of every experiment, the column must be washed with water or even a salt-free buffer. The re-equilibrating of the column should use a high salt concentration starting buffer.



When using the hydrophobic interaction chromatography method, the surface hydrophobic properties of the protein molecules that are targeted are utilized. The hydrophobic groups that are in the matrix react with those of the proteins in the sample. The separation of the mixture of proteins is achieved due to the variation in the nature of the surface hydrophobic properties of the proteins. The reversed phase chromatography is different from the HIC in that it uses the ion pairing technique to allow for the reverse phase chromatography of the solutes that are charged. This method has a high resolution.

Advantages and disadvantages of methods

Hydrophobic interaction chromatography as a method is advantageous since the method can be easily performed and the results are reproducible. The steps involved in the hydrophobic interaction chromatography can also be easily automated. The atomization of most of the process makes the process of separating the proteins very fast. The binding of proteins in a hydrophobic interaction chromatography is not usually affected by a high concentration of salt in the buffer. Therefore, this attribute makes the method very useful and applicable in purification schemes. HIC is preferred in the case whereby the biological activity of the proteins is of concern and importance. In ion exchange chromatography and size exclusion, HIC can be used alongside in order to achieve separations that are difficult, for example, in cases where the involved impurities are of the same molecular weight or of the same isoelectric points. The HIC method is also preferred in instances where the proteins that are closely related have recognition sites which are very similar and indistinguishable by use of affinity ligands.

HIC is disadvantageous in the sense that at times, the process has a low mass protein recovery hence may not be very advantageous when doing the mass spectrometry. Additionally, the method could once in a while face the unfolding of proteins which is irreversible. The recovery of some biological active proteins may be affected due to the use of the hydrophobic ligands in the HIC process which are strong. However, a solution to the effect of strong ligands is the use of weak hydrophobic ligands, for example, alkyl to avoid the denaturation of proteins to higher degrees. These weak ligands are also used to ensure the recovery of proteins is higher than the recovery percentage that is made possible by the strong ligands. However, a balance has to be struck so that the hydrophobicity level is sufficient enough to achieve efficient bonding.

The reversed phase chromatography (RPC) also has several advantages as a method for separation and purification in proteomics. First, the method excellently can effectively separate the hydrophobic-natured protein molecules with a good rate of recovery as well as resolution. Charged solutes can also be separated using the reverse phase in the mobile phase of the process due to the presence of the ion pairing modifiers. An example of the charged solutes includes hydrophilic peptides. RPC gives the researcher a wider range of separation conditions with which to work with. The method provides the researcher with either the option of binding the contaminants as the desired solutes are permitted to pass freely through the column or the option of binding the contaminants and allowing the desired molecules to run freely through the column by manipulating the conditions in the initial mobile phase. Dilute samples can, therefore, be applied to the initial mobile phase column. The method is generally not suitable when analyzing small polar analytes. Among the two methods, the most preferred of them was hydrophobic interaction since the biological activity of the proteins can be preserved and the resolution can be adjusted (Xiu, 2014).

Data analysis for the hydrophobic interaction chromatography in top-down proteomics:

Modern top-down proteomics has led to the usage of the compatible techniques in the chromatography including the mass spectrometry. This technique is used to separate the proteins which are unstable in nature in the mobile phases. Hydrophobic interaction chromatography is associated with better separation mechanisms with high resolutions. It provides the non-denaturing condition of the proteins but does not require the high concentrations of the nonvolatile salts. One of the preferred hydrophilic in the separation of the protein composition include the MS- compatible concentration, this concentration is known to have ammonium acetate salts.

There has been a recommendation of Online HIC-MS due to the analysis of qualitative and quantitative data. In the online HIC-MS process complex lysate cell and standard protein is used. The protein mass spectra show minimal charge distribution state in line with the inherent MS.

Quantitative results

With sample loading for the two chromatography processes being the same, the data was analyzed using the RPC/MS OR THE HIC-RPC/MS software. Given that the two techniques are used in the separation of the protein, the experiment carried out revealed the low numbers in each processes, though the HIC was found more effective than RPC. The HIC technique has high resolution mechanism but the salt is incompatible to the mass spectrometer, leading to the recording of lower numbers of the separated proteins. Some of the protein separates were also not identified by the RPC technique.


Despite the effectiveness of the HIC when compared to the RPC method from existing research, using a two dimensional approach that utilizes both the HIC and the RPC can be adopted to give an even more effective method of separating proteins.