How does hydrophobic interaction chromatography (HIC) work?
Hydrophobic interaction chromatography (HIC) is one of the most widely used methods for separating and purifying proteins in their native state. HIC also proves to be quite useful in isolating protein complexes and in studying protein folding and unfolding. So, how does HIC work? What are the principles behind this technique?
How do you separate proteins by hydrophobic interaction chromatography?
Proteins with different degrees of surface hydrophobicity can be separated using hydrophobic interaction chromatography. The proteins are bound to the hydrophobic ligand on the HIC resin in a binding buffer with a high salt concentration. When the ionic strength of the buffer is reduced, the interaction is reversed.
What do you need to know about hydrophobic ionization chromatography?
To understand the mechanism behind this type of chromatography, here are some things you definitely need to know. How Does HIC Work? In a nutshell, HIC (also known as 'salting out') separates protein molecules using the properties of hydrophobicity.
What are the advantages of hydrophobic surface chromatography?
The technique takes advantage of the hydrophobic areas located on the surface of proteins. HIC is an excellent complement to size exclusion and ion exchange chromatography in difficult sepa- rations, particularly those in which the impurities are of similar isoelectric point or molecular weight.
How does hydrophobic chromatography work?
Hydrophobic interaction chromatography (HIC) separates proteins according to differences in their surface hydrophobicity. HIC utilizes a reversible interaction between the proteins and the hydrophobic ligand of a HIC resin.
How does the hydrophobic interaction work?
The hydrophobic effect describes the energetic preference of nonpolar molecular surfaces to interact with other nonpolar molecular surfaces and thereby to displace water molecules from the interacting surfaces. The hydrophobic effect is due to both enthalpic and entropic effects.
How do hydrophobic interactions fold proteins?
A Hydrophobic Effect. The major driving force in protein folding is the hydrophobic effect. This is the tendency for hydrophobic molecules to isolate themselves from contact with water. As a consequence during protein folding the hydrophobic side chains become buried in the interior of the protein.
What is the mobile phase in hydrophobic interaction chromatography?
Hydrophobic interaction chromatography (HIC) is a technique used to separate proteins such as antibodies by hydrophobic interactions between proteins and stationary phase. The mobile phase is typically an aqueous buffer with high concentration.
Why do hydrophobic molecules tend to stay together?
Hydrophobic molecules don't actually interact directly with each other. They aggregate together because polar molecules (such as water) from a highly ordered cage around the non-polar molecule.
How do hydrophobic substances interact with water?
Thermodynamics of Hydrophobic Interactions Water molecules that are distorted by the presence of the hydrophobe will make new hydrogen bonds and form an ice-like cage structure called a clathrate cage around the hydrophobe.
How do you break hydrophobic interactions?
Hydrophobic interactions can be broken by either reducing the structure of water (e.g. adding organic solvents) or by making the hydrophobic regions more hydrophilic (e.g. by coating them in detergent).
How does hydrophobicity stabilize protein structure?
The arrangement of hydrophobic and hydrophilic side chains in a protein determines its folded structure. Because of the hydrophobic effect, hydrophobic side chains (purple) are sequestered to the protein's interior when it folds. Hydrophilic side chains (green) are usually left exposed on the protein's surface.
Where are hydrophobic interactions most likely to occur?
Proteins will behave similarly to phospholipids in water; the polar groups will form favorable interactions on the surface with water, while the hydrophobic groups will be in the core and away from the water molecules. Usually, amino acids with non-polar residues will be found in the core of proteins.
What is the difference between reverse phase and hydrophobic interaction chromatography?
The main difference between reverse phase and hydrophobic interaction chromatography is that the reverse phase chromatography (RPC) uses a more hydrophobic medium, which leads to more stronger interactions whereas the hydrophobic interaction chromatography (HIC) uses a less hydrophobic medium when compared to the ...
Which of the following are the main stages of hydrophobic interaction chromatography?
The Steps in Hydrophobic Interaction Chromatography The column is washed to remove non-bound proteins. The salt concentration is gradually lowered to start eluting proteins. The manipulation of salt gradients allows differential elution of the proteins - Proteins having the lowest hydrophobicity are eluted first.
Which matrix is used in hydrophobic interaction chromatography?
The name hydrophobic interaction chromatography was introduced by Hjerten in 1973. The HIC exploits stationary phase with weakly hydrophobic ligands such as short chain alkyl and phenyl immobilized on a hydrophilic matrix.
How do hydrophobic and hydrophilic molecules interact with each other?
The biological molecules known as phospholipids have a hydrophilic "head" region and a nonpolar, hydrophobic "tail." These forces cause the phospholipid molecules to aggregate together so that the polar heads are oriented towards the water and the hydrophobic tails are buried inside.
How are hydrophobic bonds formed?
Hydrophobic bonds in proteins arise as a consequence of the interaction of their hydrophobic (i.e., "water-disliking") amino acids with the polar solvent, water. The hydrophobic amino acids are gly, ala, val, leu, ile, met, pro, phe, trp (see amino acid structures for reference).
How does hydrophobic effect helps stabilize DNA?
The research team said it has shown that the secret to DNA's helical structure may be that the molecules have a hydrophobic interior, in an environment consisting mainly of water. The environment is therefore hydrophilic, while the DNA molecules' nitrogen bases are hydrophobic, pushing away the surrounding water.
Where are hydrophobic interactions most likely to occur?
Proteins will behave similarly to phospholipids in water; the polar groups will form favorable interactions on the surface with water, while the hydrophobic groups will be in the core and away from the water molecules. Usually, amino acids with non-polar residues will be found in the core of proteins.
What are the three theories of hydrophobic interaction chromatography?
There are three major theories that may help explain the mechanism behind HIC – (1) the salting out theory, (2) the thermodynamic theory and (3) the surface tension or Van der Waals forces theory.
What forces increase hydrophobic interaction?
This theory suggests that the hydrophobic interaction in HIC is dependent on van der Waals forces between proteins and immobilized ligands since these forces increase as the ordered structure of water increases in the presence of salting out salts.
What happens when a non-polar molecule comes into contact with a polar solvent?
As such, when a non-polar molecule comes into contact with a polar solvent such as water, there will be an increase in the degree of order of the solvent molecules surrounding the hydrophobic molecule. As long as enthalpy does not increase significantly, this will produce a decrease in entropy and provide an overall positive change in the Gibbs energy. As such, the dissolution of a non-polar molecule in a polar solvent will not occur spontaneously since it is not thermodynamically favorable.
What is HIC in chemistry?
Hydrophobic interaction chromatography (HIC) is one of the most widely used methods for separating and purifying proteins in their native state. HIC also proves to be quite useful in isolating protein complexes and in studying protein folding and unfolding. So, how does HIC work? What are the principles behind this technique? To understand the mechanism behind this type of chromatography, here are some things you definitely need to know.
What happens to the Gibbs energy of a hydrophobic solvent?
As a consequence, entropy increases while the Gibbs energy decreases.
When you put two or more non-polar molecules in a polar environment, the hydrophobic surfaces of the?
However, when you put two or more non-polar molecules in a polar environment, the hydrophobic surfaces of the macromolecules will be hidden from the polar surrounding and the hydrophobic molecules will aggregate spontaneously. The highly structured solvent molecules surrounding the exposed surface of the hydrophobic molecules will be displaced towards the bulk of the solvent consisting of less structured molecules. As a consequence, entropy increases while the Gibbs energy decreases. Under such cases, hydrophobic interaction becomes a thermodynamically favorable process.
Which amino acid forms hydrophobic areas?
According to the salting out theory, the hydrophobic amino acids form protected hydrophobic areas upon folding in aqueous solution while the hydrophilic amino acids allow proteins to bond with the hydrogen molecules in the water.
What are the factors that affect hydrophobic interaction chromatography?
This section provides an overview of hydrophobic interaction chromatography with general considerations about various factors such as the ligand, matrix, salt concentration, pH, and temperature.
What happens to the hydrophobic region as solvation decreases?
As solvation decreases, hydrophobic regions that become exposed are adsorbed by the media. The more hydrophobic the molecule, the less salt is needed to promote binding. Usually a decreasing salt gradient is used to elute samples from the column in order of increasing hydrophobicity.
How to adsorb protein to HIC?
Sample molecules containing hydrophobic and hydrophilic regions are applied to an HIC column in a high-salt buffer. The salt in the buffer reduces the solvation of sample solutes. As solvation decreases, hydrophobic regions that become exposed are adsorbed by the media. The more hydrophobic the molecule, the less salt is needed to promote binding. Usually a decreasing salt gradient is used to elute samples from the column in order of increasing hydrophobicity. Sample elution may also be assisted by the addition of mild organic modifiers or detergents to the elution buffer.
What is HIC in chemistry?
HIC is a useful separation technique for purifying proteins while maintaining biological activity due to the use of conditions and matrices that operate under less denaturing conditions.
What pH is used for HIC?
pH. HIC mobile phases are typically in the neutral pH range from 5–7 and buffered with sodium or potassium phosphate. In general, the strength of the interaction between proteins and the media decreases with increasing pH as a result of increased charge of the protein due to the titration of acidic groups.
What happens when a buffer is hydrophobic?
The more hydrophobic the molecule, the less salt is needed to promote binding.
What are the effects of sodium, potassium, and ammonium sulphates on HIC?
Though sodium, potassium or ammonium sulphates produce relatively higher precipitation effects, these salts effectively promote ligand-protein interactions in HIC. Most bound proteins are eluted by washing with water or dilute buffer at near neutral pH. Effect of anions and cations on protein precipitation. pH.
What is hydrophobic interaction chromatography?
Hydrophobic interaction chromatography (HIC) is a valuable tool used in protein purification applications. HIC is used in the purification of proteins over a broad range of scales—in both analytical and preparatory scale applications. HIC is used to remove various impurities that may be present in the solution, including undesirable product-related impurities. In particular, HIC is often employed to remove product aggregate species, which possess different hydrophobic properties than the target monomer species and can often be effectively removed using HIC. In this chapter, we provide a description of the basic theory of HIC and how it is used to purify proteins in aqueous-based solutions. Following the theoretical background, the latest in HIC adsorbent technology is described, including a list of commonly used and commercially available adsorbents. The basic procedures for using HIC adsorbents are described next, in order to provide the reader with useful starting points to apply HIC in protein purification applications.
What is HIC in chromatography?
HIC is most commonly employed when aggregated protein species need to be separated from a more desirable monomeric form. HIC often possesses superior selectivity for removal of aggregate species, compared to other forms of chromatography, including ion exchange and affinity (see Using ion exchange chromatography to purify a recombinantly expressed protein, Purification of His-tagged proteins, Affinity purification of a recombinant protein expressed as a fusion with the maltose-binding protein (MBP) tag, Purification of GST-tagged proteins, Protein Affinity Purification using Intein/Chitin Binding Protein Tags, Immunoaffinity purification of proteins and Strep-tagged protein purification ). HIC may also provide superior selectivity for the removal of undesirable misfolded or variant forms of a protein.
What is the difference between RPC and HIC?
Separation of proteins by hydrophobic interaction chromatography (HIC) is similar to RPC and utilizes hydrophobic interactions between the proteins in a mixture and the hydrophobic ligands (usually butyl, octyl, or phenyl groups) attached to an immobile matrix. HIC is different to RPC, however, in its use of aqueous mobile phases of high ionic strength and neutral pH that do not denature proteins. The ligands in HIC are also less hydrophobic and less densely bound to the solid matrix than in RPC matrices.
How are HIC and RPLC related?
In theory, hydrophobic interaction chromatography (HIC) and RPLC are closely related, as in both techniques separation is based on hydrophobic interactions between the surface of an analyte and the stationary phase. However, in application the techniques are very different. The solid phase used in RPLC is characteristically more hydrophobic than that used in HIC. Therefore, RPLC results in stronger interactions between solute and solid phase compared to HIC. For elution from RPLC, organic solvents must be used. In comparison, the weaker hydrophobic interactions present using HIC can be disrupted by decreasing the concentration of salt in the mobile phase. HIC offers an alternative system to exploit hydrophobic properties of molecules in a more polar and less denaturing environment [12]. The HIC stationary phase consists of a nonionic group (octyl-, butyl-, hexyl-, phenyl-, propyl-) fused to an inert matrix, such as cross-linked agarose or sepharose. The mobile phase consists of a phosphate buffer, pH 7 and a salt such as potassium chloride, ammonium sulfate, or ammonium tartrate. A mobile phase containing a stronger salt, such as ammonium sulfate versus potassium chloride, causes a greater degree of protein denaturation, resulting in a greater degree of hydrophobic binding [12].
What is HIC in chemistry?
The discovery of hydrophobic interaction chromatography (HIC) resulted from an attempt to make affinity columns.38 This fortunate accident uncovered a unique mode of protein chromatography. On the surface HIC resembles reversed-phase chromatography 27,39,40 in that the protein binds to the column through hydrophobic interactions in an aqueous solvent. Both resin types consist of a stationary phase with a hydrophobic surface. Thereafter the two techniques diverge. 23 HIC resins are typically constructed from polysaccharide or polymeric material. Reversed-phase resins are typically bonded silicas. HIC resins have a lower density of substitution and they tend to be less hydrophobic than reversed-phase media.
Why is HIC important?
The HIC can directly deal with a sample containing high salt concentration, so it is promising for the processing of samples obtained from salting-out precipitation or IEC elution. Because hydrophobic interaction strength can be readily adjusted by altering salt concentration in mobile phase, HIC is an important method in the bioseparations of therapeutic proteins, DNA vaccines, and hydrophobically tagged proteins.
Why are ligands important for HIC?
Moreover, because hydrophobic interaction is proportional to ligand hydrophobicity and coupling density on the surface, ligand density should be varied according to the ligand hydrophobicity.
What is the purpose of hydrophobic interaction chromatography?
Hydrophobic interaction chromatography (HIC) has less harsh chromatographic conditions than RPC and can be used to preserve the biological activity of enzymes.
What is hydrophobic interaction membrane chromatography?
Hydrophobic interaction membrane chromatography was used for the separations of model mixtures of proteins on membranes modified with C4 and C8 groups [29]. The separations were similar to those obtained using a packed column. The advantage of adsorptive membranes was that the pressure used was lower by as much as two orders of magnitude than that required to reach the same flow rate in a packed column, although a high binding capacity was still achieved. Separations obtained with a continuous linear gradient were used to design a stepwise gradient profile, which decreased both time and mobile phase in the separations of proteins [94].
How is hydrophobic selectivity influenced?
Instead of the addition of salt, hydrophobic selectivity was influenced through the use of a more hydrophobic ligands, and modulating the pH of the mobile phase that alters the surface charge and hydrophobicity of the protein [80 ].
What is the interaction between hydrophobic proteins and a HIC medium?
The interaction between hydrophobic proteins and a HIC medium is significantly influenced by the presence of certain salts in the running buffer. The most frequently used salt for the preparation of the mobile phase is (NH 4) 2 SO 4. [35]. The high salt concentration enhances the interaction between the hydrophobic components of the sample and the chromatography medium, and the lower salt concentration weakens the interaction. These interactions are a result of the presence of side chains of hydrophobic amino acids. They do not form hydrogen bonds with water and are not surrounded by water molecules. As a result of this phenomenon, the hydrophobic interaction depends on the behavior of the water molecules rather than on direct attraction between the hydrophobic molecules with the support.
What is HIC based on?
HIC is based on the interaction between hydrophobic ligands (aliphatic or aromatic) and hydrophobic residues on a protein surface. Protein adsorption onto the stationary phase increases with increasing salt concentration and elution is achieved by decreasing salt concentration [ 73 ].
Why is reverse phase chromatography important?
Due to the excellent resolving power, convenience, versatility, stability, and reproducibility , reversed-phase liquid chromatography (RPLC) is one of most important techniques for protein separations and the method of choice for peptide separations. RPLC has been applied on the nano, micro, and analytical scales and can be scaled up for preparative purification at the industrial scale [41,42]. In contrast to reversed phase chromatography, mechanisms of the ion-exchange and hydrophobic-interaction chromatography for peptide and protein separation is based on differences in surface hydrophobicity or surface charge [41]. Because of its compatibility with mass spectrometry (MS), RPLC is an indispensable tool in proteomic research. The increase in resolution offered by LC separation greatly enhances MS detection of sample components, and high-resolution separation reduces ion suppression in MS [42].
What are the four proteins that are soluble in HIC?
To and Lenhoff [37] investigated the contribution of protein solubility to retention in HIC using isocratic elution with four commercially available agarose media and four model proteins (ribonuclease A, lysozyme, myoglobin, and ovalbumin). Various retention trends (type of adsorbents, type and concentration of salt, pH) were tested as a function of the properties of each protein and the importance of protein-surface interaction or conformational change for protein binding was found.
What is hydrophobic interaction chromatography?from sciencedirect.com
Hydrophobic interaction chromatography (HIC) separates proteins according to differences in their surface hydrophobicity. HIC utilizes a reversible interaction between the proteins and the hydrophobic ligand of a HIC resin.
What is HIC in chromatography?from sciencedirect.com
HIC is most commonly employed when aggregated protein species need to be separated from a more desirable monomeric form. HIC often possesses superior selectivity for removal of aggregate species, compared to other forms of chromatography, including ion exchange and affinity (see Using ion exchange chromatography to purify a recombinantly expressed protein, Purification of His-tagged proteins, Affinity purification of a recombinant protein expressed as a fusion with the maltose-binding protein (MBP) tag, Purification of GST-tagged proteins, Protein Affinity Purification using Intein/Chitin Binding Protein Tags, Immunoaffinity purification of proteins and Strep-tagged protein purification ). HIC may also provide superior selectivity for the removal of undesirable misfolded or variant forms of a protein.
What is the difference between RPC and HIC?from sciencedirect.com
Separation of proteins by hydrophobic interaction chromatography (HIC) is similar to RPC and utilizes hydrophobic interactions between the proteins in a mixture and the hydrophobic ligands (usually butyl, octyl, or phenyl groups) attached to an immobile matrix. HIC is different to RPC, however, in its use of aqueous mobile phases of high ionic strength and neutral pH that do not denature proteins. The ligands in HIC are also less hydrophobic and less densely bound to the solid matrix than in RPC matrices.
How are HIC and RPLC related?from sciencedirect.com
In theory, hydrophobic interaction chromatography (HIC) and RPLC are closely related, as in both techniques separation is based on hydrophobic interactions between the surface of an analyte and the stationary phase. However, in application the techniques are very different. The solid phase used in RPLC is characteristically more hydrophobic than that used in HIC. Therefore, RPLC results in stronger interactions between solute and solid phase compared to HIC. For elution from RPLC, organic solvents must be used. In comparison, the weaker hydrophobic interactions present using HIC can be disrupted by decreasing the concentration of salt in the mobile phase. HIC offers an alternative system to exploit hydrophobic properties of molecules in a more polar and less denaturing environment [12]. The HIC stationary phase consists of a nonionic group (octyl-, butyl-, hexyl-, phenyl-, propyl-) fused to an inert matrix, such as cross-linked agarose or sepharose. The mobile phase consists of a phosphate buffer, pH 7 and a salt such as potassium chloride, ammonium sulfate, or ammonium tartrate. A mobile phase containing a stronger salt, such as ammonium sulfate versus potassium chloride, causes a greater degree of protein denaturation, resulting in a greater degree of hydrophobic binding [12].
Why is HIC important?from sciencedirect.com
The HIC can directly deal with a sample containing high salt concentration, so it is promising for the processing of samples obtained from salting-out precipitation or IEC elution. Because hydrophobic interaction strength can be readily adjusted by altering salt concentration in mobile phase, HIC is an important method in the bioseparations of therapeutic proteins, DNA vaccines, and hydrophobically tagged proteins.
What happens when the ionic strength of the buffer is reduced?from cytivalifesciences.com
When the ionic strength of the buffer is reduced, the interaction is reversed. Therefore, the protein with the lowest degree of hydrophobicity is eluted first.
Why are ligands important for HIC?from sciencedirect.com
Moreover, because hydrophobic interaction is proportional to ligand hydrophobicity and coupling density on the surface, ligand density should be varied according to the ligand hydrophobicity.
What is the correlation between temperature and hydrophobic interactions?
Temperature – There is a direct and positive correlation between temperate and the affinity of hydrophobic interactions. High temperature also influences the structure and solubility of proteins. Temperature is seldom used to modulate elution of molecules in HIC. pH – The mobile phases used in HIC mostly have a neutral pH range of 5 – 7.
Why is HIC more popular than other chromatography techniques?
HIC separates and purifies protein molecules on the basis of their hydrophobicity – it is more popular than other chromatography techniques for the separation of proteins as it employs a less denaturing environment compared to them. Therefore the biological activity of the proteins is kept intact.
How to desorbed bound proteins?
Bound protein molecules can be effectively desorbed by washing with a dilute buffer or water.
What is HIC used for?
HIC can also be effectively used to remove impurities or product aggregate species in aqueous solutions as it exploits the difference in hydrophobic properties of the aggregates and the target molecules. It is often used in combination with techniques such as ion exchange or gel filtration chromatography. YouTube. Subscribe.
What is the role of salt in HIC?
The salt promotes interaction between the hydrophilic and hydrophobic regions of the protein and the medium by reducing the solvation of sample molecules and exposing their hydrophobic regions. The amount of salt needed to promote binding is inversely ...
Why is the column washed?
The column is washed to remove non-bound proteins.
Does pH affect protein binding?
Whilst pH can have an effect on the degree of protein binding, it is not considered significant enough to use pH gradients for the elution of solute molecules.
What is hydrophobic interaction chromatography?from americanpharmaceuticalreview.com
Hydrophobic interaction chromatography (HIC) separates proteins according to differences in their surface hydrophobicity. HIC utilizes a reversible interaction between the proteins and the hydrophobic ligand of a HIC resin.
Which ligands are most commonly used for hydrophobic ligands?from americanpharmaceuticalreview.com
The most commonly used hydrophobic ligands are phenyl and alkyl groups of varying chain lengths and differing hydrophobicity. For samples with low hydrophobicity, ligands with high hydrophobicity are required to ensure adequate retention.
Why is HIC important?from americanpharmaceuticalreview.com
HIC has been widely used in the purification and characterization of biomolecules after its development. With the introduction of ADCs in the past decade, HIC analyses have witnessed a resurgence, especially for ADC drug distribution analysis. ADCs in particular are ideally suited to analysis via HIC as they possess exposed pockets of hydrophobic moieties which serve as a mechanism of separation for HIC. Drug distribution on the ADCs is a critical parameter as DAR0 species are by definition subpotent and can even block binding sites on the tumor cells from other higher DAR species leading to reduced efficacy. Conversely, over drugged ADCs often exhibit increased aggregation and have been associated with increased toxicity. Thus HIC performs an essential analysis for ADCs, which is difficult to replicate with other analytical techniques, especially in a QC environment.
How does HIC work?from americanpharmaceuticalreview.com
Following equilibration of the column with a high-salt start buffer , sample molecules are injected to the column. Exposed hydrophobic regions of the sample molecules cause the molecules to partition onto the stationary phase. As the salt concentration is reduced in the elution step, sample molecules with weaker hydrophobic interactions with the stationary phase partition into the mobile phase and elute from the column. Overall, this results in the elution of sample molecules with the lowest exposed hydrophobicity first, followed by those with more exposed hydrophobicity. After all of the sample molecules are eluted, a “saltfree” wash step is introduced to remove any hydrophobic compounds which did not elute during the elution step. Based on the mechanism of elution and sequence of steps, the following parameters are typically varied in development of a HIC separation: column, salt identity and amount, temperature, and pH 6,7.
What happens when the ionic strength of the buffer is reduced?from cytivalifesciences.com
When the ionic strength of the buffer is reduced, the interaction is reversed. Therefore, the protein with the lowest degree of hydrophobicity is eluted first.
What is the drug component of an ADC?from americanpharmaceuticalreview.com
The drug component of an ADC is typically a hydrophobic small molecule conjugated onto an exposed region of the mAb; therefore ADC species with more conjugated drug molecules have more regions of hydrophobicity, making HIC analyses uniquely suited to monitor ADC drug distribution.
How to reduce salt concentration in solvent?from nestgrp.com
Decrease the salt concentration of the solvent. Use small amounts of sucrose, glycol, or propanol in solvent A & B (<5%) Use of Octyl Glucoside, CHAPS, and CHAPSO work well (below their CMC) although use of hydrophobic detergents is not recommended.