Hydrophobic Interaction Chromatography

Advance Separation

Assignment (1)

SAJJAD K. A.

Faculty of Engineering & built Environment

Advance Separation ( KKKK6524 ) Assignment (1)

Title (Gel filtration + Hydrophobic Interaction Chromatography)

Student : SAJJAD KHUDHUR ABBAS Matric Number :

P81540

Supervisor : Prof. Madya NOORHISHAM BIN TAN KOFLI 1|Page

Advance Separation

Assignment (1)

SAJJAD K. A.

1- Gel filtration Introduction Gel filtration (also referred to as size exclusion chromatography, SEC) separates molecules according to differences in size as they pass through a gel filtration medium packed in a column. Unlike ion exchange or affinity chromatography, molecules do not bind to the chromatography medium so buffer composition does not directly affect resolution (the degree of separation between peaks). Consequently, a significant advantage of gel filtration is that conditions can be varied to suit the type of sample or the requirements for further purification, analysis or storage without altering the separation. Gel filtration is well suited for biomolecules that may be sensitive to changes in pH, concentration of metal ions or cofactors and harsh environmental conditions. Separations can be performed in the presence of essential ions or cofactors, detergents, urea, guanidine hydrochloride, at high or low ionic strength, at 37°C or in the cold room according to the requirements of the experiment. Purified proteins can be collected in any chosen buffer. A key step towards successful separation is selecting the correct medium.

Purification by gel filtration To perform a separation gel filtration medium is packed into a column to form a packed bed. The medium is a porous matrix of spherical particles with chemical and physical stability and inertness (lack of reactivity and adsorptive properties). The packed bed is equilibrated with buffer which fills the pores of the matrix and the space between the particles. The liquid inside the pores, or stationary phase, is in equilibrium with the liquid outside the particles, or mobile phase. Samples are eluted isocratically so there is no need to use different buffers during the separation. However, a wash step using the running buffer is usually included at the end of a separation to remove molecules that may have been retained on the column and to prepare the column for a new run. Gel filtration can be used directly after ion exchange, chromatofocusing, hydrophobic interaction, or affinity, since the buffer composition will not generally affect the final separation. For further details on using gel filtration in a purification strategy. Figure below illustrates the separation process of gel filtration .

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Advance Separation

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SAJJAD K. A.

In the figure up , Process of gel filtration (A) Schematic picture of a bead with an electron microscopic enlargement. (B) Schematic drawing of sample molecules diffusing into bead pores. (C) Graphical description of separation I. Sample is applied on the column, II. The smallest molecule (yellow) is more delayed than the largest molecule (red). III. The largest molecule is eluted first from the column. Band broadening causes significant dilution of the protein zones during chromatography.

Gel filtration in a purification strategy The three phase purification strategy of Capture, Intermediate Purification and Polishing (CIPP) is used in both the pharmaceutical industry and in the research laboratory to ensure faster method development, a shorter time to pure product and good economy. This chapter gives a brief overview of this approach, which is recommended for any multistep protein purification. The Protein Purification Handbook from GE Healthcare is recommended as a guide to planning efficient and effective protein purification strategies. As shown in Figure 8.1, an important first step for any purification is correct sample preparation and this is covered in more detail in Appendix 3. Gel filtration is often used for desalting and buffer exchange during sample preparation using Sephadex G-25, and samples volumes up to 30% of the total column volume can be applied. In high-resolution mode, gel filtration is ideal for the final polishing steps in a purification when sample volumes have been reduced (sample volume significantly influences speed and resolution in gel filtration). Samples are eluted isocratically (single buffer, no gradient) and buffer conditions can be varied to suit the sample type or the requirements for subsequent purification, analysis or storage, since buffer composition does not directly affect resolution.

Sample preparation and CIPP purification strategy. The purification strategy according to CIPP Imagine the purification has three phases: Capture, Intermediate Purification and Polishing. Each phase may include one or more purification steps.Assign a specific objective to each step within the purification process.The problem associated with a particular purification step will 3|Page

Advance Separation

Assignment (1)

SAJJAD K. A.

depend greatly upon the properties of the starting material. Thus, the objective of a purification step will vary according to its position in the process. In the capture phase the objectives are to isolate, concentrate and stabilize the target product. The product should be concentrated and transferred to an environment that will conserve potency/activity. During the intermediate purification phase the objective is to remove most of the bulk impurities, such as other proteins and nucleic acids, endotoxins and viruses.

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Hydrophobic Interaction Chromatography

Hydrophobic interaction chromatography (HIC) is an alternative to reversed phase chromatography for exploiting the hydrophobic properties of proteins. The addition of a salt to the mobile phase buffer and sample solution promotes protein-medium interactions. The proteins are adsorbed to the medium in a mobile phase containing a high concentration of salt. Most of the bound proteins are effectively desorbed by simply washing with water or a dilute, near neutral buffer. Because HIC employs a more polar, less denaturing environment than RPLC, it is becoming popular for protein purification, often in combination with ion exchange or gel filtration chromatography. The commercial availability of well characterized HIC matrices offers new possibilities for purifying a variety of biomolecules, such as serum, membrane, recombinant, and nuclear proteins, and receptors. The technique is sufficiently sensitive to be influenced by nonpolar groups normally buried within the tertiary structure of the protein, but which are exposed if the peptide chain is incorrectly folded or damaged. This sensitivity is useful for separating the native protein from other forms. The protein adsorption selectivity of an HIC medium is primarily determined by the type of immobilized ligand (protein-binding molecule) on the support. In general, alkyl ligands exhibit only hydrophobic character, while aryl ligands exhibit mixed mode behavior – both aromatic and hydrophobic interactions are possible. Very hydrophobic proteins are generally applied to the least hydrophobic media; hydrophilic proteins are purified on the most hydrophobic media. The appropriate HIC medium can reduce salt consumption, and thus lower cost. The lowest possible salt concentration should be used to bind the protein to the ligand. This often depends on the salt chosen. For example, compared to ammonium sulfate or sodium sulfate, an up to four times higher concentration of sodium chloride may be needed to obtain the same binding. The salt concentration should be below that which will precipitate the proteins in the crude feed stock. A 1 M solution of ammonium sulfate is most commonly used. A decreasing salt gradient can be used to increase protein resolution.

Application of HIC HIC is typically used in combination with other purification methods  

First step in purification from biological medium After salt (ammonium sulfate) precipitation 4|Page

Advance Separation

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Assignment (1)

SAJJAD K. A.

After ion-exchange chromatography

Example. Process for large-scale purification of mouse IgG1

References: GE Healthcare, Gel filtration Principles and Methods, Handbooks from GE Healthcare Hydrophobic Interaction Chromatography PRINCIPLES AND METHODS, Amersham Pharmacia biotech

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