Hydrophobic Interaction Mode Research Articles

Hemisynthesis of Pentacyclic Triterpenoids from Diospyros foxworthyi with In vitro and In silico Anti-malarial Evaluation

Abstract: A total of twelve pentacyclic triterpenoid derivatives based on betulin (1) and lupeol (2) scaffolds isolated from Diospyros foxworthyi were hemisynthesized by acylation or acetylation reactions with appropriate acid chloride or acetic anhydride. The structures of the hemisynthesised compounds were characterised by means of FT-IR, 1D- and 2D-NMR, as well as HRMS analysis. These compounds were assayed for in vitro anti-malarial studies by inhibition of β-hematin formation assay with chloroquine as a positive control. Compounds 1d and 2f showed the strongest potential as β-hematin formation inhibitors with IC50 values of 6.66 ± 1.36 and 11.89 ± 0.15 μM, respectively, compared with the positive control (chloroquine; IC50 = 37.50 ± 0.60 μM). In silico molecular docking simulations were performed using AutoDock Vina for compounds 1d and 2f to investigate the binding interactions and free energy of binding (FEB) with the hemozoin supercell crystal structure (CCDC number: XETXUP01). The findings revealed several hydrophobic interaction modes between the 1d, 2f and hemozoin, with calculated FEBs of -8.4 ± 0.2 and -8.9 ± 0.0 kcal mol-1, indicating strong and favourable interactions.

Tenacigenin B ester derivatives from Marsdenia tenacissima actively inhibited CYP3A4 and enhanced in vivo antitumor activity of paclitaxel

Ethnopharmacological relevanceMarsdenia tenacissima (Roxb.) Wight et Arn is a vine distributed in southwest area of China and used in folk medicine for treatment of tumors. Recent decades of studies on this plant reveal its synergistic effects with certain anticancer drugs in cancer therapy. In our previous study, an extract ETA which contains total aglycones made from M. tenacissima significantly enhanced antitumor activity of paclitaxel in tumor-bearing mice. However, the effective constituents in ETA and the underlying mechanisms remain unclear. Aim of the studyReveal the active components in ETA as well as the molecular mechanism in enhancing antitumor efficacy of paclitaxel. Material and methodsMain constituents in ETA were purified by chemical methods. Effects of the purified constituents on metabolic activity of CYP450 enzymes were evaluated in human liver microsomes. Ability of the constituents to enhance antitumor activity of paclitaxel were investigated in nude mice bearing HeLa tumors. Pharmacokinetic study was performed in SD rats. Molecular docking was carried out for investigation of drug-protein interactions. ResultsThree main C21 steroidal aglycones, 11α-O-tigloyl-12β-O-acetyl-tenacigenin B (MT1), 11α-O-2-methylbutanoyl-12β-O-tigloyl-tenacigenin B (MT2) and 11α-O-2-methylbutanoyl-12β-O-acetyl-tenacigenin B (MT3), together with tenacigenin B (MT4) was prepared from ETA. Among them, MT1, MT2 and MT3 strongly inhibit the metabolic activity of CYP3A4. MT2 also showed inhibitory effects on CYP2C8, CYP2B6 and CYP2C19. In HeLa tumor xenografts, MT1, MT2 and MT3 (30 mg/kg) did not affect tumor growth themselves, but significantly enhanced paclitaxel-induced growth inhibition. In addition, coadministration of MT2 with paclitaxel resulted in significant reduction of liver CYP2C8. In pharmacokinetic study, MT2 significantly increased the blood concentration of paclitaxel with increased AUC value by 2.2–5.3 folds. Molecular docking analysis suggested hydrophobic interaction modes of tenacigenin B derivatives with CYP3A4, and also the essential roles of the C-11 and C-12 ester groups for effective interaction with CYP3A4. ConclusionOur study proves that, 11α-O-tigloyl-12β-O-acetyl-tenacigenin B, 11α-O-2-methylbutanoyl-12β-O-tigloyl-tenacigenin B and 11α-O-2-methylbutanoyl-12β-O-acetyl-tenacigenin B, which are the main constituents of ETA, are active inhibitors of CYP3A4 with potential to increase therapeutic efficacy of anticancer drugs that are substrates of CYP3A4. Tenacigenin B derivatives with C-11 and C-12 ester group substitutions, or at least a large part of them, are active components in ETA and M. tenacissima to enhance in vivo antitumor efficacies of paclitaxel.

Preparation of a weak anion exchange/hydrophobic interaction dual-function mixed-mode chromatography stationary phase for protein separation using click chemistry.

In this study, 3-diethylamino-1-propyne was covalently bonded to the azide-silica by a click reaction to obtain a novel dual-function mixed-mode chromatography stationary phase for protein separation with a ligand containing tertiary amine and two ethyl groups capable of electrostatic and hydrophobic interaction functionalities, which can display hydrophobic interaction chromatography character in a high-salt-concentration mobile phase and weak anion exchange character in a low-salt-concentration mobile phase employed for protein separation. As a result, it can be employed to separate proteins with weak anion exchange and hydrophobic interaction modes, respectively. The resolution and selectivity of the stationary phase were evaluated in both hydrophobic interaction and ion exchange modes with standard proteins, respectively, which can be comparable to that of conventional weak anion exchange and hydrophobic interaction chromatography columns. Therefore, the synthesized weak anion exchange/hydrophobic interaction dual-function mixed-mode chromatography column can be used to replace two corresponding conventional weak anion exchange and hydrophobic interaction chromatography columns to separate proteins. Based on this mixed-mode chromatography stationary phase, a new off-line two-dimensional liquid chromatography technology using only a single dual-function mixed-mode chromatography column was developed. Nine kinds of tested proteins can be separated completely using the developed method within 2.0 h.

Two-stage chromatographic separation of aggregates for monoclonal antibody therapeutics

Aggregates of monoclonal antibody (mAb) therapeutics, due to their perceived impact on immunogenicity, are recognized as a critical quality attribute by the regulatory authorities as well as the industry. Hence, removal of aggregates is a key objective of bioprocessing. At present, this is achieved by a combination of two or more orthogonal chromatographic steps with possible modalities of ion exchange, hydrophobic interaction and mixed mode. A two-stage chromatographic purification process consisting of ion-exchange and hydrophobic interaction modes is proposed in this paper for effective and efficient control of aggregates for a mAb therapeutic. The proposed scheme does not require any intermediate processing of the process stream. Further, baseline separation is achieved for monomer and aggregates resulting in robust performance. This was possible because the proposed operational scheme allowed for an addition of selectivities of the two chromatography modes vs. the traditional two column scheme where part of the separation of aggregates achieved by the first column is lost upon pooling. The proposed process scheme yielded improved separation of aggregates (0% vs. 1–2%) at >95% recovery and reduced overall process time (6h vs. 14h) for a typical application. Further, clearance of host cell proteins was also shown to have improved with the suggested process scheme. Successful implementation of the proposed scheme has been demonstrated for two different monoclonal antibody therapeutic products.

Characterization and multi-mode liquid chromatographic application of 4-propylaminomethyl benzoic acid bonded silica—A zwitterionic stationary phase

4-Propylaminomethyl benzoic acid bonded silica (4-PAMBA-silica) was synthesized by reacting aminopropyl modified silica with 4-carboxybenzaldehyde and reducing the resulting Schiff base with sodium cyanoborohydride in situ. The structure of this bonded phase was confirmed by 13C cross polarization magic angle spinning (13C CP MAS) NMR. Elemental analysis indicated a coupling efficiency of about 79%. Chromatographic characterization of a 4-PAMBA-silica stationary phase revealed that at a mobile phase pH of 3.0, basic compounds were unresolved and co-eluted near the void volume, while aromatic sulfonates were retained and were well-resolved. By contrast, at a mobile phase pH of 7.0, the aromatic sulfonates were unresolved and eluted at the void volume, while basic compounds were retained and were well-resolved. To further understand the chromatographic retention mechanism the retention factors for a series of cationic and anionic compounds were measured at pH 7.0 and 3.0 as a function of the charge and concentration of competing ions in the mobile phase. A plot of the logarithm of the retention factor versus the logarithm of the eluent ion concentration was linear with a negative slope that is equal to the ratio of effective charges of the solutes and the eluent ions. This indicates that an ion exchange mechanism contributes to the separation of both cations and anions at pH 7.0 and pH 3.0, respectively. The increase in retention of alkanoic acids with their number of carbons at a mobile phase pH of 7.0 and exclusion of alkanoic acids at a mobile phase pH of 3.0 suggests that an ion exclusion mode and hydrophobic interaction mode are also operational with 4-PAMBA-silica. The amino acids, l-arginine, l-phenylalanine, and l-tyrosine were retained and well-resolved with a mobile phase containing a high concentration of organic solvent. This behavior was further studied by measuring the retention factors of polar and charged compounds as a function of the organic solvent content in the aqueous mobile phase. An increase in retention with decreasing water content in the mobile phase was observed, consistent with a hydrophilic interaction liquid chromatographic (HILIC) mode of separation.

Epoxy-based monoliths for capillary liquid chromatography of small and large molecules

A versatile epoxy-based monolith was synthesised by polycondensation polymerisation of glycidyl ether 100 with ethylenediamine using a porogenic system consisting of polyethylene glycol, M(w) = 1000, and 1-decanol. Polymerisation was performed at 80 °C for 22 h. A simple acid hydrolysis of residual epoxides resulted in a mixed diol-amino chemistry. The modified column was used successfully for hydrophilic interaction liquid chromatography (HILIC) of small molecule probes such as nucleic acid bases and nucleosides, benzoic acid derivatives, as well as for peptides released from a tryptic digest of cytochrome c. The mixed-mode chemistry allowed both hydrophilic partitioning and ion-exchange (IEX) interactions to contribute to the separation, providing flexibility in selectivity control. Residual epoxide groups were also exploited for incorporating a mixed IEX chemistry. Alternatively, the surface chemistry of the monolith pore surface rendered hydrophobic via grafting of a co-polymerised hydrophobic hydrogel. The inherent hydrophilicity of the monolith scaffold also enabled high performance separation of proteins under IEX and hydrophobic interaction modes and in the absence of non-specific interactions.

Separation of proteins from human plasma by sample displacement chromatography in hydrophobic interaction mode

Sample displacement chromatography (SDC) in reversed-phase and ion-exchange modes was introduced approximately 20 years ago. This method was first used for the preparative purification of peptides and proteins. Recently, SDC in ion-exchange mode was also successfully used for enrichment of low-abundance proteins from human plasma. In this paper, the use of SDC for the separation of plasma proteins in hydrophobic interaction mode is demonstrated. By use of two or more columns coupled in series during sample application, and subsequent elution of detached columns in parallel, additional separation of bound proteins was achieved. Further low-abundance, physiologically active proteins could be highly enriched and detected by ESI-MS/MS.

Assembly of Graphene Oxide–Enzyme Conjugates through Hydrophobic Interaction

Biochemical and biomedical applications of graphene oxide (GO) critically rely on the interaction of biomolecules with it. It has been previously reported that the biological activity of the GO-enzyme conjugate decreases due to electrostatic interaction between the enzymes and GO. Herein, the immobilization of horseradish peroxidase (HRP) and oxalate oxidase (OxOx) on chemically reduced graphene oxide (CRGO) are reported. The enzymes can be adsorbed onto CRGO directly with a tenfold higher enzyme loading than that on GO, and maximum enzyme loadings reach 1.3 and 12 mg mg(-1) for HRP and OxOx, respectively. Significantly, the more CRGO is reduced, the higher the enzyme loading. The CRGO-HRP conjugates also exhibit higher enzyme activity and stability than GO-HRP. Excellent properties of the CRGO-enzyme conjugates are attributed to hydrophobic interaction between the enzymes and the CRGO. The hydrophobic interaction mode of the CRGO-enzyme conjugates can be applied to other hydrophobic proteins, and thus could dramatically improve the performance of immobilized proteins. The results indicate that CRGO is a potential substrate for efficient enzyme immobilization, and is an ideal candidate as a macromolecule carrier and biosensor.

Preparation of poly(N-isopropylacrylamide)-grafted well-controlled 3D skeletal monolith based on E-51 epoxy resin for protein separation

A novel type of poly(N-isopropylacrylamide) grafted E-51 epoxy-based monoliths in a 100 mm × 4.6 mm I.D. stainless steel column with well-controlled three-dimensional skeletal structures has been prepared and proposed for the separation of proteins. The grafted PNIPAAm chain via surface-initiated atom transfer radical polymerization was successfully performed. The proposed method provided a new route to modify the E-51 epoxy-based monoliths for widening their applications. Meanwhile, the temperature and the salt concentration responses of the grafted monolithic columns were investigated. Under the salt gradient, six proteins were well separated in hydrophobic interaction mode. Moreover, for further confirming the application of the prepared monolith was meaningful for proteome analysis in actual system, the separation of human serum sample was performed.

Viral clearance studies on new and used chromatography resins: Critical review of a large dataset

Viral clearance studies for naïve and maximally cycled chromatographic resins used for cGMP recombinant protein production are reviewed for three products, comprising 10 different chromatographic steps, including affinity, ion exchange, immobilized metal ion affinity, and hydrophobic interaction modes. Thirty-two separate studies were conducted (over 90 runs in total). No consistent reductions in model virus clearance were observed with used resins. The results address the reproducibility of virus clearance studies conducted by different scientists over several years at multiple contract labs. The log reduction values (LRVs) are typically within 0.5 LRVs for new and used resin, but varied as much as 2 LRVs for resins showing no functional deterioration. This relatively large difference is not believed to reflect resin changes, but highlights the challenges encountered in modeling column clearance. Production column performance and cleaning efficacy are demonstrated for these steps by trending mock runs, impurity removal and product recovery. No deterioration in cGMP column performance is seen over the established resin lifetimes, confirming that the resin regeneration and sanitization procedures restore the resins to a suitable initial state without damage. It is proposed that for some chromatography steps, the combination of lab-scale cycling studies confirming consistent performance throughout the resin lifetime and monitoring of cGMP manufacturing preclude the need for virus clearance studies on maximally cycled resin.

Hydrophobic interaction ligand selection and scale-up of an expanded bed separation of an intracellular enzyme from Saccharomyces cerevisiae

A prototype Streamline-Phenyl matrix was evaluated in a hydrophobic interaction mode for the direct recovery of alcohol dehydrogenase (ADH) from yeast cell homogenate. At 5% breakthrough of ADH, a yield of 100% was obtained for a dynamic expanded bed capacity of 240 U(ADH)/ml matrix with a purification factor of 9.2. This compared with a dynamic capacity of 3013 U(ADH)/ml matrix for the packed bed equivalent and a purification factor of 18. In both systems the purification factor was found to increase simultaneously with a decrease in yield as the load of homogenate or breakthrough of ADH was increased. The expanded bed mode of operation conferred considerable robustness with respect to process fouling. No loss in yield was seen over five cycles of repeat loading with an unclarified homogenate. By contrast the packed bed media showed a decrease in yield from 86 to 56% over the same period. Successful scale up of the expanded bed protocol for a 20% breakthrough was demonstrated over a fourfold increase in column diameter. The application of hydrophobic interaction chromatography mediated expanded bed adsorption and its scale-up is discussed in the context of large-scale operations.

A hydrophobic interaction site for lysozyme binding to polyethylene glycol and model contact lens polymers

Proton nuclear magnetic resonance ( 1H-NMR) spectroscopy is used to identify a preferred binding site for uncharged hydrophilic polymers on the surface of hen egg-white lysozyme. Chemical shift titrations show that exchangeable proton signals from amino acids Arg-61, Trp-62, Trp-63, Arg-73, Lys-96 and Asp-101 are selectively perturbed upon binding of poly(ethylene oxide), poly(ethylene glycol) and poly(ethylene-co-propylene oxide). The greatest binding-induced chemical shift changes are observed for Trp-62, Arg-61 and Arg-73 at the edge of the active site cleft of the protein, consistent with a predominantly hydrophobic interaction mode involving the polymer ethylene moieties. The more hydrophilic species poly(dihydroxypropyl methacrylate) causes similar but substantially smaller chemical shift effects than the other polymers, confirming the nature of the interaction. A dissociation constant of 76±5 m m is determined for the poly(ethylene glycol)–lysozyme complex. The relatively low affinity of the protein–polymer interactions compared to oligosaccharide substrate binding suggests that lysozyme activity is minimally affected by these materials.

Application of Compact Porous Disks for Fast Separations of Biopolymers and In-Process Control in Biotechnology

Production and downstream processing in biotechnology requires fast and accurate control of each step in the process. Improved techniques which can be validated are required in order to meet these demands. For these purposes, chromatographic units containing compact porous disks for fast separation of biopolymers were developed and investigated with regard to their performance and speed. The problems that have, in the past, arisen from the use of wide and flat separation units, such as membranes and disks, have chiefly been those of sample distribution and large void volumes before and behind the unit. Improvements in the construction of the cartridge have led to better performance of the compact porous disks and faster separation. Using these disks, three calibration standard proteins could be separated within less than 1 min by an anion-exchange, cation-exchange, and hydrophobic interaction mode. Such units can be used for in-process control in production and downstream processing of biopolymers, as was shown in experiments involving the purification of α(1)-antitrypsin and clotting factor IX and the immobilization of enzyme glucose oxidase on an epoxy-activated compact porous disk.

Applied slalom chromatography improved DNA separation by the use of columns developed for reversed-phase chromatography

Improved resolution in slalom chromatography, a novel size-fractionation method discovered recently for relatively large DNA molecules (>5 kbp), was obtained by using columns generally employed for reversed-phase chromatography: i.e., two types of Capcell-Pak (methyl or phenyl-derivatized 5-μm microbeads), and five types of Hypersil-3 packings (trimethylsilyl, dimethyloctyl, cyanopropyl, octadecyl or phenyl-derivatized 3-μm microbeads). The resolution of 5–15-kbp DNA was significantly improved by employing these columns, though the separation characteristics differed. When Capcell-Pak columns were used with a normal low-salt eluting solvent (10 m M sodium phosphate, pH 6.8, 1 m M EDTA), chromatograms were obtained for λ/ HindIII fragments (a mixture of 0.1, 0.5, 2.0, 2.3, 4.4, 6.6, 9.4 and 23.1-kbp fragments) similar to those obtained previously with Asahipak GS-310 5-μm size-exclusion packings. However, when up to 0.2 M NaCl was added to the solvent, the DNA was increasingly retarded, particularly the 4.4, 6.6 and 9.4-kbp fragments, resulting in improved resolution in the low to middle molecular-mass range. The effect of salt was more significant with Capcell-Pak Phe than C1, although various features characteristic of slalom chromatography were preserved with both columns; i.e., dependency on DNA size, flow-rate, and temperature. This suggests that a mixed mode of separation, that is, slalom mode and hydrophobic-interaction mode, was operating. Although all of the Hypersil-3 packings showed significant adsorption of λ/ HindIII fragments under low-salt conditions, the fragments could be eluted with satisfactory yield and resolution by adding acetonitrile (>5%) to the solvent. Notably, these Hypersil-3 packings allowed resolution of a 4.4-kbp λ/ HindIII fragment from the flow-through fraction for the first time, possibly due to their small particle size. Thus, various packing materials developed for high-performance liquid chromatography proved to be applicable for slalom chromatography, though the eluting conditions still need to be refined. The results support the concept that slalom chromatography is based on a hydrodynamic phenomenon.

New porous organic microspheres for high-performance liquid chromatography

A process for producing spherical porous organic microspheres, based on urea-formaldehyde (UF) polymer, has been developed. These micropheres exhibit exceptional mechanical strength and resiliency and have minimal tendency toward shrinking or swelling in aqueous, organic, or hydroorganic media. The geometric parameters of the microspheres are conveniently adjusted by process variables, which allow precise control of pore and particle dimensions. The surface of the UF microsphere contains organic functional groups suitable for chemical modification so that ligands of choice may be covalently attached for operation in the anion-exchange or hydrophobic interaction modes.

Multimodal liquid chromatography columns for the separation of proteins in either the anion-exchange or hydrophobic-interaction mode

Several high-performance stationary phases suitable for protein chromatography were synthesized. Columns packed with these materials could be operated independently in either the anion-exchange or hydrophobic-interaction mode. Two approaches were used to prepare these materials. In the first method, a polyamine was adsorbed on the surface of macroporous silica and then crosslinked with a multifunctional oxirane. The hydrophobicity of the crosslinking agent and the extent of interconnection were used to modulate the electrostatic and solvophobic interactions. The second approach also utilized a crosslinked polyamine stationary phase; however, the forces of interaction were attenuated through controlled acylation of surface amines with a small anhydride molecule. The resolving ability of these columns, functioning in either mode, was comparable to commercial high-performance liquid chromatographic columns, designed to operate by a single retention mechanism. Column selectivity for proteins was completely different in each mode. Protein fractions collected from a multimodal column, operated in the anion-exchange mode, could be further purified by rechromatographing them on the same column in the hydrophobic-interaction mode. Utility of the multimodal column was demonstrated with the fractionation of several cytochromes and ferredoxins from the cyanobacterium Microcystis aeruginosa.

Denaturation and the effects of temperature on hydrophobic-interaction and reversed-phase high-performance liquid chromatography of proteins : Bio-gel tsk-phenyl-5-pw column

Cytochrome c, myoglobin and lysozyme, as well as two synthetic peptides, TM-22 and TM-36, were used to examine denaturation of protein structure on a hydrophobic-interaction column, the Bio-Gel TSK-Phenyl -5-PW high-performance liquid chromatography column. The first three proteins were chosen because they have a monomeric structure while both synthetic peptides, which have the sequence Ac-(Lys-Leu-Glu-Ala-Leu-Glu-Gly) inn-Lys-amide where n  3 for TM-22 and n  5 for TM-36, are dimeric under solvent conditions used for hydrophobic-interaction chromatography. Only TM-36 is dimeric under reversed-phase conditions. Thus, denaturation of both tertiary and quaternary structure can be examined. The column was operated in both reversed-phase and hydrophobic-interaction modes. This, in combination with temperature variation between approximately 0-50°C, provided conditions where denaturing effects of the support could be examined. In reversed-phase mode, cytochrome c, myoglobin, and TM-22 were eluted in a denatured form throughout the temperature range. In contrast, lysozyme and TM-36 eluted primarily in their native conformation at low temperatures, but experienced partial or total denaturation at higher temperatures. Significantly, all of the polypeptides studied were denatured at room temperature by a conventional reversed-phase column, the Altex Ultrapore RPSC C-3 indicating that the Bio-Gel TSK-Phenyl-5-PW column is less denaturing. In the hydrophobic-interaction mode, the dimeric structure of TM-22 was totally disrupted at all temperatures, while TM-36, myoglobin, and cytochrome c underwent various degrees of partial denaturation as the temperature increased. Comparison of the temperatures at which the various polypeptides underwent denaturation on the column with their normal melting temperatures (where half the molecules are unfolded) demonstrated that the hydrophobic column itself, rather than the temperature, was primarily responsible for denaturation. Hence, even relatively “gentle” hydrophobic columns can promote denaturation of protein structure. Since the tertiary and quaternary structures of most proteins are stabilized by hydrophobic interactions, the possibility of denaturation must always be taken into consideration when a hydrophobic column is used.

Purification of monoterpenyl glycosides by gel-permeation and hydrophobic-interaction chromatography on polyacrylamide (Bio-Gel P-2)

Hydrophobic interaction of the aglycone of monoterpenyl glycosides with the polyacrylamide matrix of Bio-Gel P-2 greatly retards the elution of these substances when chromatographed in dilute aqueous sodium chloride. This hydrophobic interaction is eliminated by inclusion of 15% acetonitrile in the eluant, thereby permitting conventional gel-permeation chromatography. Combination of these techniques by sequential chromatography on the same Bio-Gel column, in the hydrophobic interaction mode followed by the gel-permeation mode, provides a simple, yet mild and highly selective procedure for the purification of monoterpenyl glycosides from crude plant extracts. Examination of the chromatographic properties of β- d-glucopyranosides and β- d-galactopyranosides of a number of acyclic, monocyclic, and bicyclic monoterpenols indicates that the extent of hydrophobic interaction is of diagnostic value in determining the nature of the aglycone.

Wide-pore silica-based ether-bonded phases for separation of proteins by high-performance hydrophobic-interaction and size exclusion chromatography

This paper examines the use of wide-pore silica-based hydrophilic ether-bonded phases for the chromatographic separation of proteins under mild elution conditions. In particular, ether phases of the following structure  Si-(CH 2) 3-O-(CH 2-CH 2-O) n-R, where n  1, 2, 3 and R  methyl, ethyl or n-butyl, have been prepared. These phases can be employed either in high-performance hydrophobic-interaction or size-exclusion chromatography, depending on mobile phase conditions. In the hydrophobic-interaction mode, a gradient of decreasing salt concentration, e.g., from 3 M ammonium sulfate (pH 6.0, 25°C), yields sharp peaks with high mass recovery of active proteins. In this mode, retention can be controlled by salt type and concentration, as well as by column temperature. In the size-exclusion mode, use of medium ionic strength, e.g., 0.5 M ammonium acetate (pH 6.0) yields linear calibration of log (MW[ n]) vs. retention volume. Even at 0.05 M salt concentration, no stationary phase charge effects on protein elution are observed. These bonded-phase columns exhibit good column-to-column reproducibility and constant retention for at least five months of continual use. Examples of the high-performance separation of proteins in both modes are illustrated.