柱前衍生化-超高效液相色谱法快速测定酱油中的18种氨基酸

建立了一种6-氨基喹啉基-N-羟基琥珀酰亚氨基甲酸酯(AQC)柱前衍生,超高效液相色谱(UPLC)对酱油中18种氨基酸进行快速分离检测的方法。采用BEH C18色谱柱分离,在260 nm波长下检测,以乙酸铵-乙酸-乙腈-水和乙腈-乙酸为流动相,将流动相梯度和流速梯度相结合,在12 min内实现了18种氨基酸衍生物的分离。方法的线性回归系数(r2)均大于0.999,检出限为0.032～0.12 mg/L,日间相对标准偏差(RSD)为0.72%～4.05%,在酱油中18种氨基酸的加标回收率为90.2%～103.7%。该方法前处理过程简单,分离时间短,是检测酱油中氨基酸的有效手段,可用于酱油的质量评定。     

Colour pigments of Trichoderma harzianum. Preliminary investigations with thin-layer chromatography-Fourier transform infrared spectroscopy and high-performance liquid chromatography with diode array and mass spectrometric detection

The colour pigments of Trichoderma harzianum fermentation broth were separated and the main fractions were tentatively identified by reversed-phase thin-layer chromatography-Fourier transform infrared spectroscopy (RP-TLC-FT-IR), RP-HPLC-diode array detection and RP-HPLC-MS. It was established that the multistep gradient elution developed for RP-TLC separation of pigments can be successfully used as a pilot method for the rational design of gradient elution in RP-HPLC for the separation of the same pigments. FT-IR and MS measurements were unable to identify the exact chemical structures of the main pigment fractions, the presence of OH, =CH and C=O (RP-TLC-FT-IR) and OH and NH, substructures (RP-HPLC-MS) was confirmed. It was assumed that the main pigment fractions are oxidation polymers originating from monomer molecules containing polar substructures and double bonds in the alkyl chain which are liable for oxidation during the aerobic fermentation process.     

Resolution of three forms of superoxide dismutase by immobilised metal affinity chromatography.

A new method for separation of three forms of superoxide dismutase (SOD) using immobilised metal affinity chromatography (IMAC) is reported. Fe-, Mn- and Cu/Zn-SODs were eluted sequentially from Cu(2+)-IMAC column with an increasing gradient of a counter ion (NH+4) run in combination with an increasing pH gradient (6.8-7.8). The combined gradient elution method resulted in separation of SODs with high resolution, the three proteins being eluted in electrophoretically homogeneous forms. Similar preparation could not be achieved by either increasing gradient of a counter ion or decreasing pH gradients used separately. The described methodology has been successfully applied for separation of three SODs from a protozoan parasite, indicating that this combined gradient elution system for IMAC offers new possibilities for the high-resolution separation of proteins exhibiting only minor differences in their amino acid composition and structure.     

High-performance liquid chromatography utilization of ionic liquids as mobile phase additives for separation and determination of the isomers of amino benzoic acids

For the isomers of amino benzoic acid, including o-, m-, p-amino benzoic acid, the beneficial effects of using the ionic liquid, 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIm][BF₄]), as mobile phase additives on retention behavior and separation were investigated. Chromatographic separation of the o-, m-, p-amino benzoic acid was performed on a reversed-phase C18 column by ultraviolet detection at 245 nm. The effects of several chromatographic parameters, concentrations and pH values of [BMIm][BF₄] solutions, methanol concentration and length of alkyl chain on different ionic liquids, on the separation and determination of the isomers were evaluated. The optimized chromatographic conditions were established using an aqueous 0.5 μmol/L [BMIm][BF₄] solution (pH 3.0)/methanol (40:60, v/v) as mobile phase without need of gradient elution, with separation of three amino benzoic acids achieved within four min. The calibration curve showed good linearity over the tested range of 2 mg/L to 120 mg/L for the three isomers with a correlation coefficients of 0.9999. The recoveries of the three amino benzoic acids of spiked components were between 99.8% and 100%. The method has been successfully applied to the determination of p-amino benzoic acid in the pharmaceutical, Bromine Mitag Procaine Injection.     

高效液相色谱分离羊毛角蛋白

本文报道用自制的化学键合短烷基链大孔硅胶反相固定相的C_4柱分离羊毛角蛋白样品,并讨论了流动相流速及梯度斜率对分离的影响,同时还结合使用了凝胶色谱和高效疏水色谱。实验表明:本法可作为分离羊毛角蛋白的有力手段。     

Separation and indirect detection of alkyl sulfonates and sulfates

Iron(II) 1,10-phenanthroline, Fe(phen)3(2+), salts are used as mobile phase additives for the liquid chromatographic separation of alkyl sulfonates and sulfates on the reversed-phase PRP-1. As alkyl chain length increases retention increases. For a given chain length an alkyl sulfate is more retained than the corresponding alkyl sulfonate. Major elution variables that affect retention are mobile phase solvent and counteranion concentration. Indirect photometric detection is used to detect alkyl sulfonates and sulfates at 510 nm where Fe(phen)3(2+) salts absorb. Conditions for isocratic and gradient elution of multicomponent mixtures are described. Detection limits depending on analyte approached 0.1 nmol for isocratic elution and 3 nmol for gradient elution.     

Determination of 20 underivatized proteinic amino acids by ion-pairing chromatography and pneumatically assisted electrospray mass spectrometry.

A qualitative determination of 20 underivatized proteinic amino acids by LC-MS is reported. The need for chromatographic separation before mass spectrometry determination is demonstrated based on the study of several amino acid pairs which have some similar characteristics. Two suitable LC-MS systems are proposed for amino acid analysis. A preliminary optimization of these systems has been investigated using evaporative light scattering detection as these two detection modes have the same chromatographic requirements. The amino acid separation was achieved on a Purospher RP-18e or a Supelcosil ABZ+Plus column with tridecafluoroheptanoic acid or pentadecafluorooctanoic acid as volatile ion-pairing reagent in an acetonitrile-water mobile phase. In order to elute the most retained amino acids, an elution gradient based on simultaneously increasing the concentration of acetonitrile and decreasing the concentration of the ion-pairing reagent was used. The detection limits of the present work (without specialized optimization) varied from 0.5 to 1 mg 1(-1).     

Polyacrylamide gradient electrophoresis for protein purification on the milligram scale.

A preparative-scale electrophoretic technique for protein fractionation and elution on a discontinuous gradient of acrylamide is described, which permits the separation and elution of a pure protein from a mixture containing 4-20 electrophoretically different proteins. The sharpness of the gradient electrophoretic resolution is demonstrated by the separation of proteins consisting of bovine serum albumin polymers and lactate dehydrogenase and enzymes such as acid phosphatase. The compositions of various discontinuous gradients of acrylamide and their application to enzyme purification are discussed. It was found that 60% of the enzyme activity loaded on the gel is recovered after gel fractionation and elution.     

Wheat digalactosyldiacylglycerol molecular species profiling using porous graphitic carbon stationary phase

The potential of porous graphitic carbon stationary phase (PGC) was assessed for the separation of molecular species of digalactosyldiacylglycerol (DGDG). Detection was by an evaporative light scattering detector (ELSD). A conventional optimization strategy allowed definition of a quaternary non-aqueous mobile phase and separation of 9 wheat DGDG molecular species with isocratic elution: methanol/toluene/tetrahydrofuran/chloroform 64.3/21.5/13.7/0.5 v/v with 0.1% of triethylamine and a stoichiometric amount of formic acid. The molecular species were identified by LC/MS. The chromatographic behavior of DGDG on PGC was then compared to previous studies. The addition of a carbon double bond on the alkyl chain decreased the retention. This contribution was less important when the number of unsaturations increased in the alkyl chain. The consequence of this retention behavior with PGC was an elution order of molecular species which did not agree with the partition number as observed with C18 grafted stationary phases.     

Characterization of alkyl polyglycosides by both reversed-phase and normal-phase modes of high-performance liquid chromatography

Alkyl polyglycosides today represent the most important sugar surfactant. Nonionic sugar surfactants produced via different synthetic routes are mixtures of alkyl homologues, oligomers, anomers and isomers. Alkyl homologues and oligomers of alkyl mono- and diglucosides were separated by reversed-phase high-performance liquid chromatography (HPLC) with methanol-water as the mobile phase using a gradient elution. The gradient was optimized in respect to a simultaneous separation of alkyl glycosides according to their alkyl chain length and alkyl polyoxyethylene glucosides with regard to their length of the polyoxyethylene spacer. The separation of alkyl glycosides into alpha- and beta-anomers was carried out by normal-phase HPLC with isooctane-ethyl acetate (60:40, v/v)-2-propanol in the gradient mode. Light scattering detection was used. Matrix-assisted laser desorption ionization time-of-flight mass spectra of alkyl glucosides and dodecyl glucosides with oxyethylene spacer groups are presented.     

Ion-exchange liquid chromatographic analysis of bisphosphonates by on-line post-column photochemical reaction and spectrophotometric detection

A simple ion-exchange high-performance liquid chromatographic method was developed and employed for the analysis of bisphosphonate compounds in dosage formulations using on-line post-column photochemical reactions. The method used molybdate as the post-column reagent to react with the photolyzed bisphosphonate to form phosphomolybdate for enhanced spectrophotometric detection. A bisphosphonate compound, 2-thioethane-1,1-bisphophonic acid, was selected to evaluate the separation using both isocratic and gradient elution methods, along with the effects of other experimental parameters including mobile phase composition, flow-rate and post-column reagent concentration. The gradient elution method showed improved resolution and detection sensitivity compared to the isocratic elution method. The optimized gradient method was simple, reproducible, and specific to bisphosphonate compounds. It was successfully employed for the stability study of the bisphosphonate compound in pharmaceutical dosage formulations.     

Chromatographic studies of human lung elastin digestion products obtained by leucocyte elastase: comparison between newborn and adult soluble fragments

Human insoluble elastin was prepared from newborn lungs and digested by leucocyte elastase. The soluble fragments were compared to those obtained from adult lung elastin in a previous work (Smyrlaki et al., 1986). Gel filtration on a Bio-Gel P-100 column of newborn elastin allowed the separation of fraction F1N (Mr's 30,000-10,000) which was eluted later than the excluded fraction F1A (Mr's 80,000-30,000) previously isolated from adult elastin. The difference in the sizes of the large peptide fragments originating from both elastins was also shown on SDS-PAGE. Reversed phase HPLC was performed on a C18 column using a multi-step gradient elution procedure. Different patterns were observed for the high (F1N) and the low (F2N) molecular size fragments of newborn elastin. The same peak distribution was obtained with adult elastin. Comparison of the amino acid compositions of the most retained peaks (3, 4 and 5), derived from fractions F1N and F1A, showed analogies for the contents of the major nonpolar amino acids and crosslinks. Thus, this procedure allowed the separation of typical fragments of elastin which might be released in vivo by leucocyte elastase during pulmonary diseases.     

阴离子交换-积分脉冲安培检测法分离测定氨基酸注射液中的氨基酸和葡萄糖

 用阴离子交换 积分脉冲安培检测法测定了氨基酸注射液中 1 7种氨基酸和葡萄糖。研究了氨基酸和葡萄糖在阴离子交换中的保留行为。采用了优化的水、NaOH和NaAc三元梯度淋洗条件。在优化的梯度淋洗条件和积分脉冲安培检测条件下 ,氨基酸和葡萄糖的检出限为 0 3pmol～ 1 0 3pmol,线性范围约为 2个数量级。样品加标回收率为 88 3 %～ 1 0 4 6 %。方法简单、灵敏、准确。     

咪唑类离子液体与酪氨酸相互作用及机理的密度泛函理论研究

Ionic liquids (ILs) are thermally and chemically stable and have adjustable structures, which gives them the potential to be used as green, efficient biomolecular solvents. Given the critical role of ILs in dissolving biomolecules, the mechanism of interaction between them deserves further study. Herein, density functional theory (DFT) calculations, using the SMD implicit water solvent model, were employed to study the interaction and mechanism between a hydrophobic zwitterionic amino acid (Tyr) and a series of imidazolium ILs with different alkyl chain lengths and methylation sites. The contributions of hydrogen bonding (H-bonding), electrostatic effects, induction, and dispersion to the intermolecular interactions were determined by combining the symmetry-adapted perturbation theory (SAPT), the atoms in molecules (AIM) theory, and reduced density gradient (RDG) analysis. The results indicate that the H-bonding between the IL cation and Tyr is stronger than that between the IL anion and Tyr; however, the binding between either ion and Tyr is dominated by electrostatic effects. By contrast, the difference between the induction and dispersion forces is small when methylation occurs on the C2 site of the imidazolium cation; whereas, it is significantly large when methylation takes place on the N3 site. This is rationalized by the interaction patterns that vary based on the methylation site. H-bonding and π⁺-π stacking interactions between the imidazole and benzene rings are dominant during C2-methylation, while H-bonding and C_Alkyl-H…π interactions between the alkyl chain and benzene ring are dominant during N3-methylation. Increasing the side alkyl chain length has different effects on the interaction energy to cations with different methylation sites. During N3-methylation, when the side alkyl chain length increases from 4 to 12, there are significant van der Waals interactions between the Tyr benzene and the side alkyl chain. However, these van der Waals interactions are inapparent when methylation takes place on the C2 site. Finally, the synergetic effect of the H-bonding and the interaction between the benzene and the side alkyl chain for C2-methylation is greater than the H-bonding and the interaction between the imidazole and benzene rings for N3-methylation, when the side alkyl chain length n > 9. Therefore, the interaction strength and mechanism in these imidazolium-Tyr complexes can be regulated by changing the methylation site and the side alkyl chain length of the cation. Further study of ion-pair and Tyr reveals that the change tendency of the interaction energy of IL-Tyr systems is consistent with that of cation-Tyr cases, and the ion pair further stabilizes the binding with Tyr. These results illustrate the interaction mechanism of IL-Tyr systems and provide a novel strategy for the design and screening of functional ILs for amino acid extraction and separation in the future.     

Fractionation and separation of human salivary proteins by pH-gradient ion exchange and reversed phase chromatography coupled to mass spectrometry

In the present work, a 2-D capillary liquid chromatography method for fractionation and separation of human salivary proteins is demonstrated. Fractionation of proteins according to their pI values was performed in the 1-D employing a strong anion exchange (SAX) column subjected to a wide-range descending pH gradient. Polystyrene-divinylbenzene (PS-DVB) RP columns were used for focusing and subsequent separation of the proteins in the 2-D. The SAX column was presaturated with a high pH buffer (A) consisting of 10 mM amine buffering species, pH 9.0, and elution was performed with a low pH elution buffer (B) having the same buffer composition and concentration as buffer A, but pH 3.5. Isoelectric point fractions eluting from the 1-D column were trapped on PS-DVB trap columns prior to back-flushed elution onto the PS-DVB analytical column for separation of the proteins. The 1-D fraction eluting at pH 9.0-8.7 was chosen for further analysis. After separation on the RP analytical column, nine RP protein fractions were collected and tryptic digested for subsequent analyses by MALDI TOF MS and column switching capillary LC coupled to ESI TOF MS and ESI QTOF MS. Eight proteins and two peptides were identified in the pH 9.0-8.7 fraction using peptide mass fingerprinting and uninterpreted MS/MS data.     

High-performance chromatofocusing using linear and concave pH gradients formed with simple buffer mixtures. II. Separation of proteins

The separation of proteins using high-performance chromatofocusing with linear or concave pH gradients formed using simple mixtures of buffering species in the elution buffer is investigated experimentally. The separation achieved is comparable to that using polyampholyte elution buffers with these types of systems. More specifically, protein band widths at one half of the band height in the range between 0.1 and 0.025 pH units were observed, and good resolution was achieved of protein variants differing by a single amino acid residue in separation times of 30 min or less. An especially useful elution buffer is investigated that contains only four buffering species and that produces a linear pH gradient in the range between pH 9.5 and 6.0 when used together with a particular high-performance column packing made specifically for chromatofocusing. This elution buffer and column packing combination is evaluated by using it for the chromatofocusing of equine myoglobin and human hemoglobin variants. Additional applications are described in which a polyethyleneimine derivatized silica column packing and a pH gradient that is concave in shape are used for the separation of proteins in an E. coli cell lysate.     

Purine and pyrimidine compounds in murine peritoneal macrophages cultured in vitro.

Extracts of murine peritoneal macrophages were analysed by ion-pair reversed-phase high-performance liquid chromatography during incubation at 37 degrees C in vitro. Four-step gradient elution was applied to an ODS column (250 x 4.6 mm I.D.) at a flow-rate of 1.3 ml/min, allowing the separation of hypoxanthine, inosine, guanosine, adenosine, IMP, CDP, AMP, GDP, UDP, ADP, CTP, GTP, UTP and ATP within 50 min. Samples of 0.4 . 10(6)-0.5 . 10(6) cells were washed twice with RPMI 1640 medium and extracted with perchloric acid. Nucleotide concentrations of murine peritoneal macrophages did not change during incubation for 4 days in vitro.     

Comparison of the chiral resolution on cis-trans isomeric chiral stationary phases derived from (S)-1-(1-naphthyl)ethylamine

A pair of cis-trans isomeric chiral stationary phases (CSPs) derived from (S)-1-(1-naphtyl)ethylamine was prepared. The chromatographic behaviours on both CSPs with regard to the resolution of enantiomeric amino acids, amino alcohols, amines, and carboxylic acid were studied. According to separation factors, the trans-CSP showed better chiral recognition ability for the separation of most analytes chosen in this study. Three homologous series of the alkyl esters of racemic amino acids were resolved on both CSPs using n-hexane-2-propanol and n-hexane-dichloromethane as mobile phases. The trans-CSP also showed better enantioselectivity for the resolution of homologues. A reverse of elution order was observed for the resolution of the homologous series of phenylglycine alkyl esters on both CSPs. It was found that the relationship between the separation factor and the alkyl chain length of the ester homologous series depended upon the components of mobile phase. A higher magnitude of difference between the two CSPs in enantioselectivity for the resolution of a given homologue was obtained when n-hexane-dichloromethane was used as a mobile phase. A chiral recognition process, in which steric repulsion, face-to-face π-π interaction, face-to-edge π-π interaction and hydrogen bonding interaction were involved, was also suggested to describe the separation of enantiomeric homologues on both CSPs. This study clearly indicates that the chiral resolution is influenced by the geometry of the double bond in a CSP.     

Development of a rapid and convenient method to separate eight ginsenosides from Panax ginseng by high-speed counter-current chromatography coupled with evaporative light scattering detection

Ginsenosides exhibit diverse biological activities and are major well-known components isolated from the radix of Panax ginseng C.A. Meyer. In the present work, a rapid and facile method for the separation and purification of eight ginsenosides from P. ginseng by high-speed counter-current chromatography coupled with evaporative light scattering detector (HSCCC-ELSD) was successfully developed. The crude samples for HSCCC separation were first purified from ginseng extract using a macroporous resin; the extract was loaded onto a Diaion-HP20 column and fractionated by methanol and water gradient elution. The ginsenosides-protopanaxadiol (PPD) and protopanaxatriol (PPT) fractions were subsequently eluted with 65 and 80% methanol and water gradient elution, respectively. Furthermore, these two fractions were separated by HSCCC-ELSD. The two-phase solvent system used for separation was composed of chloroform/methanol/water/isopropanol at a volume ratio of 4:3:2:1. Each fraction obtained was collected and dried, yielding the following eight ginsenosides: Rg(1), Re, Rf, Rh(1), Rb(1), Rc Rb(2) and Rd. The purity of these ginsenosides was greater than 97% as assessed by HPLC-ELSD, and their structures were characterized by electrospray-ionization mass spectrometry (ESI-MS) and nuclear magnetic resonance spectroscopy. This is the first report regarding the separation of the ginsenosides Rh(1), Rb(2) and Rc from P. ginseng by HSCCC.     

各种氨基酸衍生物在Chirasil—Val固定相上的气相色谱分离

本文叙述氨基酸及其对映体的全氟酰基烷基脂(O-异丙基、-异丁基、-正丁基;N(O,S)-三氟乙酰基,TFA、-七氟丁酰基,HFB)在Chirasil-Val上的洗脱特性。随烷基取代增大,保留时间增加;用HFB代替TFA,对映体的分离因子降低。此外,DL-氨基酸与另丁醇衍生,生成的对映及非对映衍生物在Chirasil-Val上可拆分成四个色谱峰。