蛋白质在合成阳离子交换剂上的色谱特性研究

用国产材料按间接法合成了螯合型弱阳离子交换剂,详细研究了合成填料的色谱性能,并与商品柱的分离效能进行了比较;在宽温度范围内研究了蛋白质在弱阳离子交换系统中的色谱热力学,测定了蛋白质在色谱过程变性时的热力学参数 (△H0和△S0) 和补偿温度β,提出用标准熵变△S0判断蛋白质的构象变化和用△H0与△S0的补偿关系鉴定蛋白质各变体在色谱系统保留机理的同一性。考察了螯合型弱阳离子交换剂与金属离子的作用,研究了蛋白质在金属螯合色谱中的保留机理。     

Assessment by monte carlo simulation of thermodynamic correlation of retention times in dual-column temperature programmed comprehensive two-dimensional gas chromatography

The correlation of retention times in comprehensive two-dimensional gas chromatography caused by correlation of enthalpy and entropy changes between two stationary phases, methylsilicone and poly(ethylene glycol), was examined using commercial GC software and in-house Monte Carlo simulation. The enthalpy change, deltaH0, and entropy change, deltaS0, of 93 compounds were predicted from isothermal one-dimensional gas chromatograms predicted by the software. These values then were mimicked by Monte Carlo simulation, which removed the strong correlation of deltaH0 and modest correlation of deltaS0 between the two phases. Retention times in a comprehensive two-dimensional gas chromatogram (GC x GC) and in simulations of it were predicted for typical dual-capillary temperature-programmed conditions using the actual, correlated values of deltaH0 and deltaS0 and their uncorrelated Monte Carlo counterparts, respectively. The uncorrelated deltaH0 and deltaS0 values caused the retention-time range of the simulations' second dimension to expand substantially beyond that in the GC x GC. Other simulations were developed using a restricted range of uncorrelated deltaH0 and deltaS0 values to mimic more closely the retention-time range of the GC x GC's second dimension. The intervals between nearest neighbor retention-time coordinates were calculated in both the latter simulations and the GC x GC. The intervals were larger in the simulations than in the GC x GC because the former contained uncorrelated coordinates and the latter contained correlated ones, which clustered along or near the diagonal. The retention times in the first dimension of the GC x GC were Poisson distributed, as assessed by statistical-overlap theory. In contrast, the two-dimensional reduced retention-time coordinates in the GC x GC were not Poisson distributed, because retention times were correlated. However, the reduced retention-time coordinates in the simulations were Poisson distributed.     

Retention Behavior of Proteins on Glutamic Acid-Boned Silica Stationary Phase

A glutamic acid-bonded silica (Glu-silica) stationary phase with cation-exchange properties was synthesized using l-glutamic acid as ligand and silica gel as matrix. The effects of solution pH value, salt concentration and metal ion on the retention of proteins were examined. The standard protein mixture was separated with a prepared chromatographic column and an iminodiacetic acid column, and compared. The influence of the binding capacity of an immobilized metal ion on the complexation of metal chelate column was studied. The results indicate that the obtained column displays cation-exchange characteristic and better separation ability for proteins. As fixing metal ion on the Glu-silica column, retention of proteins on the column is a cooperative interaction of metal chelate and cation-exchange. The stationary phase shows the typical metal chelate properties with the increase of the sorption capacity of immobilized metal ion.     

Ion chromatographic investigation of the ion-exchange properties of microdisperse sintered nanodiamonds

The possibility of using sintered diamonds as a stationary phase in ion chromatography has been evaluated. Bare sintered synthetic nanodiamonds demonstrated the properties of a weak cation-exchanger. The observed ion-exchange selectivity is similar to carboxylic type cation-exchangers. The regularities of retention of alkali, alkaline-earth and transition metal ions on a column packed with sintered nanodiamonds in dilute nitric acid were studied and the occurrence of chelating properties was noted. For the first time chromatographic separations of model mixtures of cations on diamonds have been obtained.     

Retention mechanism of fatty alcohol ethoxylates in reversed-phase liquid chromatography

Fatty alcohol ethoxylates (FAEs) are widely used nonionic surfactants that have distributions in both alkyl and poly(ethylene oxide) (PEO) chain length. Generally, two-dimensional liquid chromatography technique is required for the complete characterization of both distributions. By selecting a proper stationary and mobile phase condition, however, we can obtain fully resolved chromatograms of a FAE sample (Brij 30) with respect to both alkyl and PEO chain length by using a single reversed-phase C18 column and aqueous acetonitrile mobile phase. FAEs show a peculiar reversed-phase liquid chromatography (RPLC) retention behavior with an aqueous-organic mobile phase, the retention mechanism of which has not been fully elucidated. For a fixed alkyl chain length, FAEs with higher-molecular-mass PEO block elutes first and the van't Hoff plot of the retention factor shows a curvature. The unique retention behavior can be understood from the opposite thermodynamic characteristics associated with RPLC retention of PEO block and alkyl chain: the sorption process of PEO to the non-polar stationary phase shows deltaH(o) > 0 and deltaS(o) > 0 while the alkyl chain shows deltaH(o) < 0 and deltaS(o) < 0 in contrast. The relative magnitude of the two contributions can change the elution order of the FAE. Therefore the often found, inverted elution order of FAEs (the early elution of FAEs with longer PEO block) is due to the positive enthalpic interaction of PEO blocks, which is a characteristic of the hydrophobic interaction. And the curvature of the van't Hoff plots was analyzed assuming the temperature dependent thermodynamic variables.     

2-芳基丙酸类手性药物色谱拆分的热力学研究

以0.5%和1.0%(体积分数)正丙醇-50 mmol/L磷酸盐缓冲液(pH 6.41)为流动相,温度74~313 K,在Chiral-AGP柱手性固定相上,考察了萘普生和布洛芬对映体在手性柱上的保留和分离行为。在实验范围内,温度升高对分离不利,随着温度的升高,对映体的保留时间、分离度和选择性因子都减少;萘普生对映体的分离度均比布洛芬大;流动相含1.0％正丙醇时,萘普生对映体和布洛芬对映体达到完全分离应控制的最高温度分别为298和288 K。用ln k对1/T作图得到的Van’t Hoff曲线都具有良好的     

Retention mechanism of poly(ethylene oxide) in reversed-phase and normal-phase liquid chromatography

The retention behavior of low- and high-molecular-mass poly(ethylene oxide) (PEO) in reversed-phase (RP) and normal-phase (NP) liquid chromatography was investigated. In RPLC using a C18 bonded silica stationary phase and an acetonitrile-water mixture mobile phase, the sorption process of PEO to the stationary phase showed deltaH(o) > 0 and deltaS(o) > 0. Therefore, PEO retention in RPLC separation is an energetically unfavorable, entropy-driven process, which results in an increase of PEO retention as the temperature increases. In addition, at the enthalpy-entropy compensation point the elution volume of PEO was very different from the column void volume. These observations are quite different from the RPLC retention behavior of many organic polymers. The peculiar retention behavior of PEO in RPLC separation can be understood in terms of the hydrophobic interaction of this class of typical amphiphilic compounds with the non-polar stationary phase, on the one hand, and with the aqueous mobile phase, on the other. The entropy gain due to the release of the solvated water molecules from the PEO chain and the stationary phase is believed to be responsible for the entropy-driven separation process. On the other hand, in NPLC using an amino-bonded silica stationary phase and an acetonitrile-water mixture mobile phase, PEO showed normal enthalpy-driven retention behavior: deltaH(o) < 0 and deltaS(o) < 0, with the retention decreasing with increasing temperature and PEO eluting near the column void volume at the enthalpy-entropy compensation point. Therefore, high-resolution temperature gradient NPLC separation of high-molecular-mass PEO samples can be achieved with relative ease. The molecular mass distribution of high-molecular-mass PEO was found to be much narrower than that measured by size-exclusion chromatography.     

Denatured Thermodynamics of Proteins in Weak Cation－exchange Chromatography

The thermostability of some proteins in weak cation-exchange chromatography was investigated at 20—80 ℃. The results show that there is a fixed thermal denaturation transition temperature for each protein. The appearance of the thermal transition temperature indicates that the conformations of the proteins are de-stroyed seriously. The thermal behavior of the proteins in weak cation-exchange and hydrophobic interaction chromatographies were compared in a wide temperature range. It was found that the proteins have a higher thermostability in a weak cation-exchange chromatography system. The thermodynamic parameters (△H^0,△S^0) of those proteins were determined by means of Van′t t Hoff re|ationship(lnk′-1/T). According to stan-dard entropy change(△S^0) , the conformational change of the proteins was judged in the chromatographic pro-cess. The linear relationships between △H^0 and △S^0 can be used to evaluate “compensation temperature“ (β) at the protein denaturation and identify the identity of the protein retention mechanism in weak cation-ex-change chromatography.     

Effects of temperature on retention of chiral compounds on a ristocetin A chiral stationary phase

The isocratic retention of enantiomers of chiral analytes, i.e. tryptophan, 1,2,3,4-tetrahydroisoquinoline and gamma-butyrolac tone analogs, was studied on a ristocetin A chiral stationary phase at different temperatures and with different mobile phase compositions, using the reversed-phase, polar-organic and normal-phase modes. By variation of the both mobile phase composition and the temperature, baseline separations could be achieved for these enantiomers. The retention factors and selectivity factors for the enantiomers of all investigated compounds decreased with increasing temperature. The natural logarithms of the retention factors (ln k) of the investigated compounds depended linearly on the inverse of temperature (1/T). van't Hoff plots afforded thermodynamic parameters, such as the apparent change in enthalpy (deltaH(o)), the apparent change in entropy (deltaS(o)) and the apparent change in Gibbs free energy (deltaG(o) ) for the transfer of analyte from the mobile to the stationary phase. The thermodynamic parameters (deltaH(o), deltaS(o) and deltaG(o)) were calculated in order to promote an understanding of the thermodynamic driving forces for retention in this chromatographic system.     

硅胶基质弱阳离子交换剂的合成及蛋清中溶菌酶的分离

用间接法合成了硅胶基质弱阳离子交换剂（ＸＩＤＡＣＥ－ＷＣＸ）,详细研究了合成填料的色谱性能,并与商品柱（Ｓｈｉｍ－Ｐａｃｋ ＷＣＸ－１和Ｐｏｌｙ ＣＡＴＡ）进行了比较。结果表明：合成柱比商品柱具有较高的分辨能力。通过考察合成填料的疏水性、蛋白质的保留值与等电点（ｐＩ）的关系、流动相的ｐＨ值和盐浓度对蛋白质保留行为的影响,论证了合成填料的离子交换特征。利用合成的色谱填料从蛋清中分离出纯度较高的溶菌酶     

Gas chromatographic determination of the interconversion energy barrier for dialkyl 2,3-pentadienedioate enantiomers

The enantiomers of dialkyl 2,3-pentadienedioate undergo interconversion during gas chromatographic separation on chiral stationary phases. In this paper the on-column apparent interconversion kinetic and thermodynamic activation data were determined for dimethyl, diethyl, propylbutyl and dibutyl 2,3-pentadienedioate enantiomers by gas chromatographic separation of the racemic mixtures on a capillary column containing a polydimethylsiloxane stationary phase coupled to 2,3-di-O-methyl-6-O-tertbutyldimethylsilyl-beta-cyclodextrin. A deconvolution method was used to determine the individual enantiomer peak areas and retention times that are needed to calculate the interconversion rate constants and the energy barriers. The apparent rate constants and interconversion energy barriers decrease slightly with an increase in the alkyl chain length of the dialkyl 2,3-pentadienedioate esters. The optimum conformation of the dialkyl 2,3-pentadienedioate molecules, their separation selectivity factors and apparent interconversion enthalpy and entropy data changes with the alkyl chain length. The dependence of the apparent interconversion energy barrier (deltaG(app)(a-->b), deltaG(app)(b-->a)) on temperature was used to determine the apparent activation enthalpy (deltaH(app)(a-->b), deltaH(app)(b-->a)) and apparent entropy (deltaS(app)(a-->b), deltaS(app)(a-->b)) (where a denotes the first and b second eluted enantiomer). The comparison of the activation enthalpy and entropy (deltaS(app)(a-->b), deltaS(app)(a-->b)) indicated that the interconversion of dialkyl 2,3-pentadienedioate enantiomers on the HP-5+Chiraldex B-DM column series is an entropy driven process at 160 degrees C. Data obtained for dimethyl 2,3-pentadienedioate enantiomers on the HP-5+Chiraldex B-DM column series at 120 degrees C (deltaG(app)(a-->b) = 123.3 and deltaG(app)(b-->a) = 124.4 kJ mol(-1)) corresponds (at the 95% confidence interval) with the value of deltaG(#) = 128+/-1 kJ mol(-1) found at this temperature by gas chromatography using a two-dimensional stop flow technique on an empty capillary column [V. Schurig, F. Keller, S. Reich, M. Fluck, Tetrahedron: Asymmetry 8 (1997) 3475].     

流动相组成、浓度和pH对蛋白质在金属螯合柱上的保留特性的影响

 系统研究了流动相中盐的性质和浓度、溶液 pH以及竞争配体对蛋白质在金属螯合色谱中保留值的影响。导出了描述蛋白质在金属螯合色谱中保留特征的数学表达式 ,提出用洗脱强度指数ε表征盐溶液的洗脱能力。根据不同色谱条件下蛋白质的保留特性 ,发现蛋白质在金属螯合色谱中的保留是配位、静电和疏水的协同作用。对与蛋白质强结合的金属螯合柱 ,以配位作用为主 ,静电作用为辅 ;对弱结合的金属柱 ,以静电作用为主 ,配位作用为辅。在流动相中加入高浓度非成络盐 ,可增强蛋白质和固定相间的疏水作用。     

Effects of Immobilized Metal Ion on Retention Behaviors of Proteins in Metal Chelate Affinity Chromatography

The chromatographic behaviors of proteins on iminodiacetic acid (IDA) column with and without immobilized metal ion were examined in detail. Comparing the effects of pI, solution pH, and salt concentration on retention of proteins in cation‐exchange chromatography (CEC) and metal chelate affinity chromatography (MCAC), the retention mechanism of proteins was investigated in MCAC. By aid of observing the retention characteristics of proteins on naked IDA and metal chelate columns in high concentration salt‐out salt solution, the hydrophobic interaction in MCAC and the influence of metal ion on it were proved. In terms of the comparison of the thermodynamics of proteins in CEC and MCAC, the thermostability, the conformational change entropy Δ(ΔS⁰) and enthalpy Δ(ΔH⁰), compensation temperature β, the driving force and caloritic effect of proteins in MCAC were discussed. The identity of retention mechanism at protein thermal denaturation in CEC and MCAC was demonstrated by using the compensation relationship between ΔH⁰ and ΔS⁰.     

Preparation of a monolithic column for weak cation exchange chromatography and its application in the separation of biopolymers

A procedure for the preparation of a monolithic column for weak cation exchange chromatography was presented. The structure of the monolithic column was evaluated by mercury intrusion. The hydrodynamic and chromatographic properties of the monolithic column--such as back pressures at different flow rates, effects of pH on protein retention, dynamic loading capacity, recovery, and stability--were determined under conditions typical for ion-exchange chromatography. The prepared monolithic column might be used in a relatively broad pH range from 4.0 to 12.0 and exhibited an excellent separation to five proteins at the flow rates of both 1.0 and 8.0 mL/min, respectively. In addition, the prepared column was first used in the purification and simultaneous renaturation of recombinant human interferon gamma (rhIFN-gamma) in the extract solution with 7.0 mol/L guanidine hydrochloride. The purity and specific bioactivity of the purified rhIFN-gamma in only one chromatographic step were obtained to be 93% and 7.8 x 10(7) IU/mg, respectively.     

金属螯合亲和色谱中的疏水作用

通过考察盐溶盐和盐析盐浓度对蛋白质在IDA裸柱和金属螯合柱上保留行为的影响,详细研究了金属螯合色谱中的疏水作用,疏水作用的发生、形成的条件以及不同条件下对蛋白质保留值的贡献。实验结果表明,在高浓度和低浓度的盐溶盐以及低浓度盐析盐中,蛋白质在金属螯合柱上的保留主要受静电和配位作用控制,而疏水作用对蛋白质的保留影响很小。对弱亲和性的金属螯合柱以静电作用为主,其大小可用参数Q表征;对强亲和性的IDA-Cu（Ⅱ）柱以配位作用为主。仅在高浓度的盐析盐中,金属螯合柱才呈现较强的疏水作用,支配蛋白质保留。实验证明,金属螯合色谱中疏水作用主要来自固定相间隔臂中的疏水碳链和盐析盐对蛋白质的增疏作用,利用这种疏水作用有可能改善金属螯合色谱分离的选择性。     

GC separation of 2-substituted ethyl propionate enantiomers on permethylated and 2,6-dimethyl-3-pentyl beta- and gamma-cyclodextrin stationary phases

In this work, the capillary gas chromatographic enantiomer separation of eight congeneric compounds with general formulae CH3-HCX-COOC2H5 (where X = Cl, Br, I, CN, OH, OC2H5, OC6H5 and NHCOCF3) on four different permethyl- and 2,6-di-O-methyl-3-O-pentyl- beta- and gamma-CD stationary phases has been studied. The separation of enantiomers was evaluated in terms of the interactions of the X substituent of studied derivatives, as well as the nature of the 3-O-alkyl group in the 2,6-di-O-methyl-3-O-alkyl-CDs and the CDs cavity size. The differences in thermodynamic data [deltaH and -deltaS] obtained for studied compounds and the selectivity of modified beta- and gamma-cyclodextrin phases in gas chromatographic separation were evaluated. deltaH values were compared with a deltaH value of an achiral standard (ethyl propionate, where X = H) in order to obtain the contribution of a particular substituent to the overall interaction energy. It was shown that the variation in the enantiomeric separation with temperature and the retention order of these compounds on a given cyclodextrin capillary column depends on the nature of the substituents bonded to stereogenic carbon atom. It was found that the temperature dependencies of selectivity factors, In a on 1/T, were both linear as well as non-linear, inter alia depending on the number of glucopyranose units of the CD derivatives. The enantiospecific thermodynamic data [delta21(deltaH)] and [-delta21(deltaS)] which characterize the chiral recognition in the separation system were used to gain more insight into the mechanistic aspects of the enantioseparations on permethylated and 2,6-di-O-methyl-3-O-pentyl-beta- and gamma-cyclodextrins.     

Preparation of poly(vinyltetrazole) chain-grafted poly(glycidymethacrylate-co-ethylenedimethacrylate) beads by surface-initiated atom transfer radical polymerization for the use in weak cation exchange and hydrophilic interaction chromatography

A novel stationary phase for weak cation exchange (WCX) and hydrophilic interaction chromatography (HILIC) was prepared with surface-initiated atom transfer radical polymerization (SI-ATRP). Vinyltetrazole was grafted onto the surface of the beads in water medium with the polyglycidylmethacrylate beads (P_GMA/EDMA) previously modified with 2-bromoisobutryl bromide as the macromolecule initiators and CuCl as catalyst. The poly(vinyltetrazole)-grafted beads obtained with different atom transfer radical polymerization (ATRP) formulations were tried as chromatographic packings in ion-exchange chromatography. The results showed that the prepared columns could separate the tested proteins with high efficiency and high capacity, and the retention time of protein had a positive relationship with increasing the chain lengths of the grafted poly(vinyltetrazole) (PVT). The prepared column was also found to be able to separate nucleosides by hydrophilic interaction chromatographic mode.     

Displacement chromatography of biomolecules

Displacement chromatography was used for the preparative-scale separation of peptides, antibiotics, and proteins. The feed components were both purified and concentrated during the separation processes. The components of a peptide mixture were separated on a reverse-phase analytical column using 2-(2-butoxyethoxy) ethanol as the displacer. The use of organic modifiers in the carrier along with an elevated column temperature of 45 degrees C enabled the efficient separation of relatively hydrophobic peptides by displacement chromatography. In addition, the throughput of the process was significantly increased by carrying out the separation at an elevated flow-rate with no adverse effect on product purity. The antibiotic cephalosporin C was isolated from impurities in a fermentation broth using 2-(2-butoxyethoxy)ethanol as the displacer along with a step change in column temperature. The proteins cytochrome c and lysozyme were purified on a weak cation-exchanger column using cationic polymers as the displacers. While polymers of 60 and 20 kilodaltons were both found to be good displacers for these proteins, only the lower molecular weight polymer was readily removed from the column by standard regeneration techniques.     

连续棒状弱阳离子交换柱的合成及其对蛋白质保留行为

以甲基丙烯酸缩水甘油酯为单体,乙二醇二甲基丙烯酸酯为交联剂在空管柱内就地聚合制备了一种聚合物连续棒状色谱基质,并通过“在线”化学改性将其修饰为弱阳离子交换柱。考察了该色谱柱的孔结构特征、表面亲水性能、对标准蛋白的分离效果和ｐＨ值对保留行为的影响以及色谱柱的重现性。结果表明,该色谱柱对蛋白的分离性能及重现性良好,柱寿命不短于半年。对溶菌酶的活性回收率达９６．２％。流动相流速为８．０ｍＬ／ｍｉｎ条件下     

New alpha-amino phenylalanine tetrazole ligand for immobilized metal affinity chromatography of proteins

A new chelating compound has been developed for use in the immobilized metal affinity chromatographic (IMAC) separation of proteins. The bidentate ligand, alpha-amino phenylalanine tetrazole, 4, was synthesized via a five-step synthesis from N-fluorenylmethoxycarbonyl phenylalanine and then immobilized onto silica through the epoxide coupling procedure. The binding behavior of the resulting IMAC sorbent, following chelation with Zn2+ to a density of 183 micromol Zn2+ ions/g silica, was characterized by the retention of proteins in the pH range of 5.0-8.0, and by the adsorption behavior of lysozyme with frontal chromatography at pH 6.0 and 8.0. The prepared column showed the separation ability to four test proteins and the retention time of these proteins increased with an increase in pH. From the derived isotherms, the adsorption capacity, qm, for the binding of lysozyme to immobilized Zn2+-alpha-amino phenylalanine tetrazole-silica was found to be 1.21 micromol/g at pH 6.0 and 1.20 micromol/g sorbent at pH 8.0, respectively, whilst the dissociation constants KD at these pH values were 5.22x10(-6) and 3.49x10(-6) M, respectively, indicating that the lysozyme was retained more stable under alkaline conditions, although the binding capacity in terms of micromole protein per gram sorbent remained essentially unchanged.