Introduction to HIC In a classical paper published in 1948 and entitled:Adsorption Separation by Salting Out",Tiselius [1]laid down the foundation for a separation method which is now popularly known as hydrophobic interaction chromatography(HIC).He noted that,".proteins and other substances which are precipitated at high concentrations of neutral salts (saltingout)often are adsorbed quitestrongly already insalt oution of lower concentration than is required for their precipitation,and that some adsorbents which in salt-free solutions show no or only slight affinity for proteins,at moderately high salt concentrations become excellent adsorbents".Since then,great en made 、。 1 ping almost ideals tationary phases for chromat (such as cellulose,cross-linked dextran(SephadexTM),cross-linked agarose(Sepharose CL,Sepharose High Performance and Sepharose Fast Flow),and in developing biizing ligands of choice2to sch matc vents which,in the beginning to the synthesi of a variety of hydrophobic adsorbents for biopolymer separations based on this previously rarely exploited principle. The first attempt at synthesizing such adsorbents was made by Yon [4]followed by Er-el et al.[5],Hofstee [6] and Shaltiel Er-el [7].Charac these early adsorbents showed a mixed ionic-hydrophobic character [8].Despite this,Halperin et al.[9]claimed that protein binding to such adsorbents was predominantly of a hydrophobic character.Porath et al.[10]and Hjerten et al.[11]later synthesized charge-free hydrophobic adsorbents and demonstrat ted that the bind ng f prote n was enhanced by high concentrations of neutral salts,as previously observed by Tiselius [1],and that elution of the bound proteins was achieved simply by washing the column with salt-free buffer or by decreasing the polarity of the eluent [6,10,111. Amersham Pharmacia Biotech w s first in pr roducing commercial HIC adsorbent (Phenyl and Octyl Sepharose CL-4B[12])of the charge-free type and has continuously followed this up with new developments in agarose matrix design by introducing new stable HIC media based on SuperoseTM,Sepharose Fast Flow and Sepharose High Performance,meeting various demands on chromatographic productivity,selectivity and efficiency. 11
11 Introduction to HIC In a classical paper published in 1948 and entitled: ‘‘Adsorption Separation by Salting Out’’, Tiselius [1] laid down the foundation for a separation method which is now popularly known as hydrophobic interaction chromatography (HIC). He noted that, ‘‘.proteins and other substances which are precipitated at high concentrations of neutral salts (salting out), often are adsorbed quite strongly already in salt solutions of lower concentration than is required for their precipitation, and that some adsorbents which in salt-free solutions show no or only slight affinity for proteins, at moderately high salt concentrations become excellent adsorbents”. Since then, great strides have been made in developing almost ideal stationary phases for chromatography (such as cellulose, cross-linked dextran (Sephadex™), cross-linked agarose (Sepharose™ CL, Sepharose High Performance and Sepharose Fast Flow), and in developing coupling methods for immobilizing ligands of choice [2,3] to such matrices. It was a combination of these two events which, in the beginning of 1970's, led to the synthesis of a variety of hydrophobic adsorbents for biopolymer separations based on this previously rarely exploited principle. The first attempt at synthesizing such adsorbents was made by Yon [4] followed by Er-el et al. [5], Hofstee [6] and Shaltiel & Er-el [7]. Characteristically, these early adsorbents showed a mixed ionic-hydrophobic character [8]. Despite this, Halperin et al. [9] claimed that protein binding to such adsorbents was predominantly of a hydrophobic character. Porath et al. [10] and Hjertén et al. [11] later synthesized charge-free hydrophobic adsorbents and demonstrated that the binding of proteins was enhanced by high concentrations of neutral salts, as previously observed by Tiselius [1], and that elution of the bound proteins was achieved simply by washing the column with salt-free buffer or by decreasing the polarity of the eluent [6, 10, 11]. Amersham Pharmacia Biotech was first in producing commercial HIC adsorbents (Phenyl and Octyl Sepharose CL-4B [12]) of the charge-free type and has continuously followed this up with new developments in agarose matrix design by introducing new stable HIC media based on Superose™, Sepharose Fast Flow and Sepharose High Performance, meeting various demands on chromatographic productivity, selectivity and efficiency. 1
The commercial availability of well-characterized HIC adsorbents opened new s[12,13] membrane-bound proteins [14],nuclear proteins [15],receptors [16],cells [17],and recombinant proteins[18,19]in research and industrial laboratories.These adsorbents were also used for the reversible immobilization of enzymes [20]and liposomes [21]. The principle for protein adsorption to HIC media is complementary to ior exchange chromatography and gel filtration.HIC is even sensitive enough to be influenced by non-polar groups normally buried within the tertiary structure of proteins but exposed if the polypeptide chain is incorrectly folded or damaged(e.g.by proteases).This sensitivity can be useful for separating the pure native protein from other forms. Altogether this makes HIC a versatile liquid chromatography technique,being a logical part of any rational purification strategy,often in combination with ion exchange chromatography and gel filtration.HIC has also found use as an analytical tool to detect protein conformational changes. HIC requires a minimum of sample pre-treatment and can thus be used effectively in combination with traditional protein precipitation techniques. Protein binding to HIC adsorbents is promoted by moderately high concentration of anti salts,which also have a stabilizing influence on protei Elution isachieved by alinear or stepwise ecrease in the coneation of salt in the adsorption buffer.Recoveries are often very satisfactory. humber of mechanisms have been proposed for HIC over the years and factors that affect the binding of proteins ents have been investigated.These aspects will be briefly outlined in this handbook.Greater emphasis has been given to practical considerations on how to make optimal use of Amersham Pharmacia Biotech range of HIC products. 12
12 The commercial availability of well-characterized HIC adsorbents opened new possibilities for purifying a variety of biomolecules such as serum proteins [12, 13], membrane-bound proteins [14], nuclear proteins [15], receptors [16], cells [17], and recombinant proteins [18, 19] in research and industrial laboratories. These adsorbents were also used for the reversible immobilization of enzymes [20] and liposomes [21]. The principle for protein adsorption to HIC media is complementary to ion exchange chromatography and gel filtration. HIC is even sensitive enough to be influenced by non-polar groups normally buried within the tertiary structure of proteins but exposed if the polypeptide chain is incorrectly folded or damaged (e.g. by proteases). This sensitivity can be useful for separating the pure native protein from other forms. Altogether this makes HIC a versatile liquid chromatography technique, being a logical part of any rational purification strategy, often in combination with ion exchange chromatography and gel filtration. HIC has also found use as an analytical tool to detect protein conformational changes. HIC requires a minimum of sample pre-treatment and can thus be used effectively in combination with traditional protein precipitation techniques. Protein binding to HIC adsorbents is promoted by moderately high concentrations of anti-chaotropic salts, which also have a stabilizing influence on protein structure. Elution is achieved by a linear or stepwise decrease in the concentration of salt in the adsorption buffer. Recoveries are often very satisfactory. A number of mechanisms have been proposed for HIC over the years and factors that affect the binding of proteins to such adsorbents have been investigated. These aspects will be briefly outlined in this handbook. Greater emphasis has been given to practical considerations on how to make optimal use of Amersham Pharmacia Biotech range of HIC products
Principles of HIC Theory The discussions that follow in this chapter will be limited to the non-charged type of HIC adsorbents. The many theories that have b en proposed for HICare essentially based upon those derived for interactions between hydrophobicsoues and water (2)but none of them has enjoyed universal acceptance.Whatis common to all is the central role played by the structure-forming salts and the effects they exert on the individual components solute,solvent and adsorbent)of the omatographic system tobri ring about the binding of solute to adsorbent.In view of this,Porath(24)proposed"salt-promoted adsorption"as a general concept for HIC and other types of solute-adsorbent interactions occuring in the presence of moderately high concentrations of neutral salts. Hofstee(6)and later Shaltiel (7)proposed"hydrophobic chromatography"with the implicit assumption that the mode of interaction between proteins and the immobilized hydrophobic ligands is similar to the self association of small aliphatic organic molec cules in water.Porath et al (1)suggested a salting-out effect in hydrophobic adsorption,thus extending the earlier observations of Tiselius(1).They also suggested that".the driving force is the entropy gain arising from structure changes in the water surrounding the interacting hydrophobic groups".This concept was later extended and formalized by Hjerten(25)who based his theory on the well known thermodynamic relationship:AG=AH-TAS.He proposed that the displace ment of the ordered water molecules surrounding the hydrophobic ligands and the proteins leads to an increase in entropy(AS)resulting in a negative value for the change in free energy(AG)of the system.This implies that the hydrophobic ligand-protein interaction is thermodynamically favourable,as is illustrated in Fig.1. An alternative theory is based on the parallelism between the effect of neutral salts in salting out(precipitation)and HIC(26,27).According to Melander and Horvath (27),hydrophobic interaction is accounted for by increase in the surface tension of ater arising from the structure forming salts dissolved in it.In fact,a combination of these two mechanisms seems to be an obvious extension and has been exploited long 13
13 Principles of HIC Theory The discussions that follow in this chapter will be limited to the non-charged type of HIC adsorbents. The many theories that have been proposed for HIC are essentially based upon those derived for interactions between hydrophobic solutes and water (22,23), but none of them has enjoyed universal acceptance. What is common to all is the central role played by the structure-forming salts and the effects they exert on the individual components (i.e., solute, solvent and adsorbent) of the chromatographic system to bring about the binding of solute to adsorbent. In view of this, Porath (24) proposed ‘‘salt-promoted adsorption’’ as a general concept for HIC and other types of solute-adsorbent interactions occuring in the presence of moderately high concentrations of neutral salts. Hofstee (6) and later Shaltiel (7) proposed ‘‘hydrophobic chromatography’’ with the implicit assumption that the mode of interaction between proteins and the immobilized hydrophobic ligands is similar to the self association of small aliphatic organic molecules in water. Porath et al. (10) suggested a salting-out effect in hydrophobic adsorption, thus extending the earlier observations of Tiselius (1). They also suggested that ‘‘. . .the driving force is the entropy gain arising from structure changes in the water surrounding the interacting hydrophobic groups’’. This concept was later extended and formalized by Hjertén (25) who based his theory on the well known thermodynamic relationship: DG = DH - TDS. He proposed that the displacement of the ordered water molecules surrounding the hydrophobic ligands and the proteins leads to an increase in entropy (DS) resulting in a negative value for the change in free energy (DG) of the system. This implies that the hydrophobic ligand-protein interaction is thermodynamically favourable, as is illustrated in Fig. 1. An alternative theory is based on the parallelism between the effect of neutral salts in salting out (precipitation) and HIC (26,27). According to Melander and Horvath (27), hydrophobic interaction is accounted for by increase in the surface tension of water arising from the structure – forming salts dissolved in it. In fact, a combination of these two mechanisms seems to be an obvious extension and has been exploited long 2
Fig.1. Close to the surface of the +s= LndH).the water molecules H are more highly ordered than w hydrophobic ligand and solute P=Polymer matri olec =Lig polymer less W-Wat 0 es in the buk olute molecule ng of before HICadsorbents were synthesized(28).Finally,Srinivasan and Ruckenstein(29) have proposed that HICis due to van der Waals attraction forces between proteins and immobilized ligands.The basis for this theory is that the van der Waals attraction forces between protein and ligand increase as the ordered structure of water increases in the presence of salting out salts. HIC vs RPC In theory,HIC and reverse-phase chromatography (RPC)are closely related LC techniques.Both are based upon interactions between solvent-accessible non-polar groups s (bvd. rophobic patches)on the surface of biomolecules and the hydrophobic ligands(alkyl or aryl groups)covalently attached to the gel matrix.In practice however,they are different.Adsorbents for RPC are more highly substituted with hydrophobic ligands than HIC adsorbents.The degree of substitution of HIC adsorb ents is usually in the range of 10-50um ml ge elof CCalkyl or simple aryl ligands,compared with several hundred umoles/ml gel of C-( .alkyl ligands usually used for RPCadsorbents.Consequently,protein binding to RPCadsorbents is usually eryrrong,which require the use of no-polar solvents for their elurion.KPC ha e applications in analytical and preparativ separations of mainly peptides and low molecular weight proteins that are stable in aqueous-organic solvents. In summary,HIC is an alternative way of exploiting the hydrophobic properties of proteins,working in a more polar and less denaturing environment Compared with RPC,the polarity of the complete system of HIC is increased by decreased ligand density on the stationary phase and by addingsalt to the mobile phase. 4
14 Fig. 1. Close to the surface of the hydrophobic ligand and solute (L and H), the water molecules are more highly ordered than in the bulk water and appear to ‘‘shield off’’ the hydrophobic ligand and solute molecules. Added salt interacts strongly with the water molecules leaving less water available for the ‘‘shielding off’’ effect, which is the driving force for L and H to interact with each other. before HIC adsorbents were synthesized (28). Finally, Srinivasan and Ruckenstein (29) have proposed that HIC is due to van der Waals attraction forces between proteins and immobilized ligands. The basis for this theory is that the van der Waals attraction forces between protein and ligand increase as the ordered structure of water increases in the presence of salting out salts. HIC vs RPC In theory, HIC and reverse-phase chromatography (RPC) are closely related LC techniques. Both are based upon interactions between solvent-accessible non-polar groups (hydrophobic patches) on the surface of biomolecules and the hydrophobic ligands (alkyl or aryl groups) covalently attached to the gel matrix. In practice, however, they are different. Adsorbents for RPC are more highly substituted with hydrophobic ligands than HIC adsorbents. The degree of substitution of HIC adsorbents is usually in the range of 10–50 mmoles/ ml gel of C2 –C8 alkyl or simple aryl ligands, compared with several hundred mmoles/ml gel of C4 –C18 alkyl ligands usually used for RPC adsorbents. Consequently, protein binding to RPC adsorbents is usually very strong, which requires the use of non-polar solvents for their elution. RPC has found extensive applications in analytical and preparative separations of mainly peptides and low molecular weight proteins that are stable in aqueous-organic solvents. In summary, HIC is an alternative way of exploiting the hydrophobic properties of proteins, working in a more polar and less denaturing environment. Compared with RPC, the polarity of the complete system of HIC is increased by decreased ligand density on the stationary phase and by adding salt to the mobile phase. L + H S L H S + W P=Polymer matrix S=Solute molecule L=Ligand attached to polymer matrix H=Hydrophobic patch on surface of solute molecule W=Water molecules in the bulk solution P
Factors affecting HIC The main parameters to consider when selecting HIC media and optimizing separation processes on HIC media are: Ligand type and degree of substitution ·Type of base matrix Type and concentration of salt ·pH ·Temperature ·Additives Type of ligand The type of immobilized ligand (alkyl or aryl)determines primarily the protein adsorption selectivity of the HIC adsorbent(6,7,30).In general,straight chain alkyl ocarbon)ligands show pure”hydropho ic cha cter while aryl ligands show a mixed mode behaviour where both aromatic and hydrophobic interactions are possible(30).It is also established that,at a constant degree of substitution,the protein binding capacities of HIC adsorbents increase with increased alkyl chain length (Fig.2A)(30,31).The charged type HICadsorbents(6,7)show an additional mode of interaction,which will not be discussed here.The choice between alkyl or aryl ligands is empirical and must be established by screening experiments for each individual separation problem. chain length and in HIC.In Fig.2A a 10 20 30 n-Alkyl chain length Degree of substitution (umol ligand/ml gel) 15
15 Factors affecting HIC The main parameters to consider when selecting HIC media and optimizing separation processes on HIC media are: • Ligand type and degree of substitution • Type of base matrix • Type and concentration of salt • pH • Temperature • Additives Type of ligand The type of immobilized ligand (alkyl or aryl) determines primarily the protein adsorption selectivity of the HIC adsorbent (6,7,30). In general, straight chain alkyl (hydrocarbon) ligands show ‘‘pure’’ hydrophobic character while aryl ligands show a mixed mode behaviour where both aromatic and hydrophobic interactions are possible (30). It is also established that, at a constant degree of substitution, the protein binding capacities of HIC adsorbents increase with increased alkyl chain length (Fig. 2A) (30,31). The charged type HIC adsorbents (6,7) show an additional mode of interaction, which will not be discussed here. The choice between alkyl or aryl ligands is empirical and must be established by screening experiments for each individual separation problem. Fig. 2. The effect of alkyl chain length and degree of substitution on binding capacity in HIC. In Fig. 2A it is assumed that the degree of substitution is the same for each alkyl chain length shown. A B C C C 10 20 30 n-Alkyl chain length Degree of substitution (µmol ligand/ml gel) Binding capacity (mg protein/ml gel) 4 68