Chromatography of microbial cells using continuous supermacroporous affinity and ion-exchange columns

Continuous supermacroporous chromatographic columns with anion-exchange ligands [2-(dimethylamino)ethyl group] and immobilized metal affinity (IMA) ligands (Cu2+-loaded iminodiacetic acid) have been developed allowing binding of Escherichia coli cells and the elution of bound cells with high recoveries. These poly(acrylamide)-based continuous supermacroporous columns have been produced by radical co-polymerization of monomers in aqueous solution frozen inside a column (cryo-polymerization). After thawing, the column contains a continuous matrix (so-called cryogel) with interconnected pores of 10-100 microm in size. The large pore size of the matrix makes it possible for E. coli cells to pass unhindered through a plain column containing no ligands. E. coli cells bound to an ion-exchange column at low ionic strength were eluted with 70-80% recovery at NaCl concentrations of 0.35-0.40 M, while cells bound to an IMA-column were eluted with around 80% recovery using either 10 mM imidazole or 20 mM EDTA solutions, respectively. The cells maintain their viability after the binding/elution procedure. These preliminary results indicate that microbial cells can be handled in a chromatographic mode using supermacroporous continuous columns. These columns are easy to manufacture from cheap and readily available starting materials, which make the columns suitable for single-time use.     

Continuous separation of maslinic and oleanolic acids from olive pulp by high‐speed countercurrent chromatography with elution‐extrusion mode

In this work, a continuous high‐speed countercurrent chromatography method has been developed on the basis of elution‐extrusion mode and this method was successfully applied to the separation of maslinic and oleanolic acid from the extract of olive pulp. In the process of ‘elution’, the sample solution was continuously loaded into the column and the maslinic acid was steadily eluted out in this step while highly retained oleanlic acid always stayed in the column. In the process of ‘extrusion’, the oleanlic acid was pushed out of the column with the stationary phase. In this way, we achieved a large sample loading. A total of 120 mL sample solution (about 89.55% of the column volume) which contains 600 mg olive pulp extract was pumped in the apparatus by a constant‐flow pump and the maslinic and oleanolic acids were largely separated within 120 min. Both of these two compounds presented high yields and high purities (271.6 mg for maslinic acid with 86.7% and 83.9 mg oleanolic acids with 83.4%).     

Theoretical background of short chromatographic layers. Optimization of gradient elution in short columns

Although linear salt gradient elution ion-exchange chromatography (IEC) of proteins is commonly carried out with relatively short columns, it is still not clear how the column length affects the separation performance and the economics of the process. The separation performance can be adjusted by changing a combination of the column length, the gradient slope and the flow velocity. The same resolution can be obtained with a given column length with different combinations of the gradient slope and the flow velocity. This results in different separation time and elution volume at the same resolution. Based on our previous model, a method for determining the separation time and the elution volume relationship for the same resolution (iso-resolution curve) was developed. The effect of the column length and the mass transfer rate on the iso-resolution curve was examined. A long column and/or high mass transfer rate results in lesser elution volume. The resolution data with porous bead packed columns and monolithic columns were in good agreement with the calculated iso-resolution curves. Although the elution volume can be reduced with increasing column length, the pressure drop limits govern the optimum conditions.     

High performance liquid chromatographic determination of bound sulfide and sulfite and thiosulfate at their low levels in human serum by pre-column fluorescence derivatization with monobromobimane.

A sensitive and specific high performance liquid chromatographic method for the determination of sulfide, sulfite, and thiosulfate was established. Inorganic sulfur anions were converted into fluorescent derivatives with monobromobimane. The derivatives were separated on a coupled column chromatography with a reversed-phase octadecyl silica column connected with a weakly basic anion exchanger column by isocratic elution with acetic acid solution (pH 3)-acetonitrile (13:3, v/v) containing 25 mM NaClO4. The method was applied to the determination of bound sulfide and sulfite and thiosulfate in normal human serum. Thiosulfate could be determined directly by use of an ultrafiltered sample. For the determination of bound sulfide and sulfite, the pretreatment step with continuous flow gas dialysis was effective for the sample after releasing sulfide and sulfite by reduction with dithiothreitol. The limits of quantification by the present method were 0.05 microM for thiosulfate, 0.5 microM for bound sulfide, and 0.2 microM for bound sulfite.     

Formation and migration of zones in overloaded preparative liquid-solid chromatography

Summary An equilibrium sandwich chamber for continuous thinlayer chromatography was used to study overloaded systems of the heptane + methylene chloride-silica type. Mixtures of two or three dyes were used as the model samples. Wide starting zones were formed using a glass distributor (frontal chromatography stage), then the movement of the zones was recorded during continuous elution. The effect of sample concentration and volume on the maximum separation yield was investigated. Band compression effects are illustrated for samples dissolved in solvents having a low eluent strength. Satisfactory analogy was found to separations in preparative column chromatography. Good separation yield was obtained for frontal + elution TLC: depending on the differences in the R_F values of the components, 6–16 mg samples were completely separated on 0.5×100×75 mm layers containing ca. 1 g of silica.Presented at the Eighth International Symposium on Liquid Chromatography, Baden-Baden, 3–7 May 1983.     

Application of capillary reversed-phase high-performance liquid chromatography to high-sensitivity protein sequence analysis.

A continuous gradient elution method for capillary column (less than 0.32 mm I.D.) liquid chromatography was developed. Gradient eluent from a microbore liquid chromatograph was split ahead of the injector so that an accurate percentage (2-3%) of the mobile phase delivered by the pump flowed through the capillary column. The outlet of the column was connected to a length of 0.075 mm I.D. fused-silica capillary tubing which, in turn, was connected to a 6-mm optical path length longitudinal capillary flow cell. Fused-silica capillary columns of 0.32 mm I.D. were slurry-packed efficiently with 7-microns spherical, 300 A pore size, C8 bonded-phase particles, and evaluated in terms of their ability to resolve mixtures of proteins, peptides or phenylthiohydantoin (PTH)-amino acid derivatives. The gradient elution profiles agreed with those obtained using microbore (less than 2.1 mm I.D.) and larger bore columns. The minimum detectable amounts for proteins and PTH-amino acids on 0.32 mm I.D. capillary columns were 50 pg and 25 fmol, respectively. At a flow-rate of 3.6 microliters/min, proteins and peptides were recovered from the capillary columns in volumes of about 2-8 microliters. The use of a multiple-wavelength, forward-optics detector for identifying tryptophan- and tyrosine-containing peptides is discussed.     

Capillary electrochromatography of proteins with polymer-based strong-cation-exchanger microspheres

Monodisperse poly(glycidyl methacrylate-divinylbenzene) microspheres were functionalized with propyl sulfonic acid moieties to obtain beads negatively charged in a wide pH range. They were packed into fused-silica capillary of 50 micro, I.D. in order to separate proteins by capillary electrochromatography (CEC). Baseline separation of four basic proteins as well as three cytochrome c variants with an average column efficiency of 60,000 theoretical plates was obtained under isocratic elution conditions. The high efficiency is attributed to the uniformity of the column packing and the hydrophilic surface coverage of the polymer beads derived from the functionalization process. The effect of pH and salt concentration on protein separations was investigated and the results showed that the CEC separation mechanism is the combination of chromatographic retention and electrophoretic migration. Moreover, the column packed with the strongly acidic poly(glycidyl methacrylate-divinylbenzene) beads was also suitable for protein separations by micro-HPLC with a salt gradient. The comparison between the two kinds of elution modes shows that the column described here exhibited higher peak efficiency with isocratic elution in CEC than with gradient elution in micro-HPLC.     

填充柱超临界流体色谱系统中的溶剂效应

 考察了填充柱超临界流体色谱法 (SFC)中的样品溶剂及连续进样等因素对化合物保留行为变化的影响规律。以超临界 CO2 或含低体积分数甲醇的 CO2 为流动相时 ,氨基柱上组分的保留时间随着样品溶剂的极性增大而增大 ,而溶剂对 C1 8柱上组分的保留时间影响不大 ;在 C1 8柱上 ,溶剂对连续进样的后续效应不强 ;而在氨基柱上 ,甲醇溶液的后续效应比丙酮、氯仿溶液的后续效应强。当甲醇的体积分数大于 1 .0 %时 ,溶剂的效应明显减弱。这种变化规律对填充柱 SFC的合理进样并获得重现性良好的色谱数据具有实际意义。     

Development of dual gradient column in liquid chromatography

The dual gradient column, in which both the chemical property of the stationary phase and the flow velocity in the mobile phase are heterogeneous longitudinally along the column, is developed to obtain the mobile phase gradient-like elution in an isocratic condition. Here, the step-wise dual gradient columns were prepared by connecting an inlet column (I.D. 50 microm, packed with ODS) serially to an outlet column (I.D. 100-200 microm, packed with the mixture of ODS and C1 [9:1]). The retention behavior of alkylbenzenes was able to be controlled in the dual gradient column depending on the variation in the flow velocity. Moreover, the change in retention behavior induced by the flow velocity variation for the dual gradient columns was quite different from that by the variation in organic modifier content of the mobile phase in isocratic elution for a single gradient column and can induce the similar effect with an ordinary gradient elution in a mobile phase composition.     

Separation of aldicarb,aldicarb sulfoxide,and aldicarb sulfone on unmodified silica with reverse-phase eluents

Summary Aldicarb, aldicarb sulfoxide, and aldicarb sulfone were chromatographed on an octyl-silica bonded-phase column and on an unmodified silica column using acetonitrile/water mobile phases. The elution order of the analytes from the silica column was different from that using the octyl-silica bonded phase and allowed confirmation of residues of aldicarb sulfoxide in citrus nectar. Isocratic elution of the unmodified silica column allowed rapid sample throughput.     

Single nucleotide polymorphism detection method by temperature-gradient affinity chromatography using a single-stranded oligo-DNA coupled column

We developed an affinity chromatographic method for simple single nucleotide polymorphism (SNP) detection by use of a single-stranded DNA-coupled column and temperature gradient elution, utilizing the difference in thermal stability between hybridized double-stranded DNAs with and without mismatched base-pairs in the course of temperature gradient elution. We studied experimentally and theoretically the elution behavior of DNAs with and without SNPs in this chromatography and proposed a numerical calculation method based on a thermodynamic dissociation model. The effects of the column volume, flow rate of eluent and heating rate of the column on elution profiles were clarified. For designing DNA ligands, mismatched base-pair positions favorable for detection of SNPs were also explored by use of hybridized DNAs coding a part of the human TP53 gene.     

离子色谱-抑制电导法分别测定海水中阴离子和阳离子

采用离子交换-抑制电导法测定海水中阴、阳离子。采用抑制电导可以降低淋洗液的背景电导,又可以增加被测离子的电导值,改善信噪比。采用的电化学自身再生抑制器,由连续电解水产生抑制淋洗液所需要的H^＋或者OH^-,加上电场引力,能用于高客量分离柱所用的淋洗液浓度和梯度淋洗。在试验条件下,利用阴离子和阳离子分离柱,配合抑制电导检测,可以同时分离和测定海水中7种阴离子和6种阳离子。且都可以得到很好的线性和较低的检出限。     

Column-switching high-performance liquid chromatographic detection of pholcodine and its metabolites in urine with fluorescence and electrochemical detection.

A sensitive and selective method for the detection of pholcodine and its metabolite morphine in urine using high-performance liquid chromatography is described. It involves on-line clean-up of urine on a trace enrichment column packed with a polymeric strong cation-exchange material. Pholcodine and its metabolites were separated on two analytical columns with different selectivities. Pholcodine was detected by a fluorescence detector and morphine was detected electrochemically. One system, based on reversed-phase chromatography, applied a polystyrene-divinylbenzene column and gradient elution. The other system was based on normal-phase chromatography with a silica column and isocratic elution. Morphine was confirmed to be a metabolite of pholcodine by reversed-phase chromatography and electrochemical detection. Two unidentified metabolites of pholcodine were separated from pholcodine by normal-phase chromatography and detected by fluorescence detection.     

Cyclodextrin biospecific-like displacement in dye-affinity chromatography

Interactions between Cibacron Blue F3GA (CB F3GA), as a model of triazine dye, and 2-hydroxypropyl-beta-cyclodextrin (HP-beta-CD), as a model of cyclodextrin, were investigated by monitoring the spectral shift that accompanies the binding phenomena. Matrix analysis of the difference spectral titration of CB F3GA with HP-beta-CD revealed only two absorbing species, indicating a host-guest ratio of 1:1. The dissociation constant for this HP-beta-CD-CB F3GA complex, Kd, was found to be 0.43 mM. The data for HP-beta-CD forming inclusion complexes with CB F3GA were used to develop the concept of competitive elution by inclusion complexes in dye-affinity chromatography. When this concept was applied to the elution of L-lactate dehydrogenase from a CB F3GA affinity matrix, it was shown to be an effective elution strategy. It provided a 15-fold purification factor with 89% recovery and sharp elution profile (0.8 column volumes for 80% recovery), which is as good as that obtained by specific elution with NADH (16-fold, 78% recovery and 1.8 column volumes). In addition, the new elution strategy showed a better purification factor and sharper elution profile than traditional non-specific elution with KCl (4.5-fold, and 1.4 column volumes). Hence, competitive elution by inclusion complexes may be a promising strategy for eluting proteins with high recoveries and purification factors in dye-affinity chromatography.     

Chromatography of B prostaglandins on beta-cyclodextrin silica: application to analysis of major E prostaglandins in human seminal fluid

A new chiral stationary phase (CSP) was developed for the direct optical resolution of enantiomeric amino acid derivatives. The CSP was readily prepared by a three-step reaction carried out in a pre-packed aminopropylsilyl silica gel column. In the first step, a solution of disuccinimido carbonate (DSC) was delivered through the pre-packed column to give a succinimido carbamyl aminopropylsilyl-bonded, activated-carbamate type silica gel (ACsil) column. Through the column was then delivered a solution of pentaethylenehexamine to afford a polyamine-bonded column. Finally, a solution of optically active succinimido (S)- or (R)-naphthylethyl carbamate was delivered through the polyamine column, to give a naphthylethylurea multiple-bonded CSP. p-Bromophenylcarbamyl derivatives of enantiomeric protein amino acids were resolved on these CSPs by elution with an aqueous mobile phase. Simultaneous analysis of these amino acid enantiomers by means of gradient elution was also accomplished.     

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.     

Fast cleanup method for the analysis of Sudan I-IV and para red in various foods and paprika color (oleoresin) by high-performance liquid chromatography/diode array detection: focus on removal of fat and oil as fatty acid methyl esters prepared by transesterification of acylglycerols

A fast and effective cleanup method was developed for the analysis of Sudan I, II, III, IV, and Para Red (Sudan dyes) in various foods and paprika color (oleoresin) by high-performance liquid chromatography (LC) with a diode array detector (DAD). Removal of fat or oil in fatty sample was a critical point for reducing the volume of the final sample solution in order to obtain a sufficient level of the analytes. Separation of fat or oil from the dyes with a silica gel solid-phase extraction (SPE) column seemed unfeasible, because elution profiles of oil, fat, and the dyes were similar. Finally, fat and oil were separated from the dyes by elution from the SPE column with n-hexane, not as intact compounds but as fatty acid methyl esters prepared by direct transesterification of acylglycerols in fat and oil, leaving the dyes on the column. The dyes were eluted with n-hexane-diethyl ether (9 + 1). Gradient elution with water and tetrahydrofuran was used for separation on a C18 column by LC. Measurement of spectral of 0.5 microg/g of Sudan dyes in foods and 1 microg/g in paprika color (oleoresin) with the DAD was achieved.     

Isolation of lanthanides from spent nuclear fuel by means of high performance ion chromatography (HPIC) prior to mass spectrometric analysis

Pure and complete fractions of neodymium, samarium, europium, gadolinium and dysprosium were isolated by means of high performance ion chromatography, using a cation exchange column and gradient elution with alpha-hydroxyisobutyric acid solutions (α-HIBA). Intermediate precision and robustness of the isolation method was investigated, identifying eluent pH as the most important parameter. Investigation of the elution behaviour of the most important fission and activation products and actinides indicated that preventing the accumulation of cesium on the cation exchange column required further isocratic elution with a higher concentrated α-HIBA solution after elution of the lanthanides. A sample matrix free of carbon was achieved by means of acid digestion, followed by UV photo-oxidation, resulting in samples suitable for mass spectrometric analysis.

     

Chemical compositional separation of styrene-methyl methacrylate copolymers using high-performance liquid chromatography with liquefied carbon dioxide as eluent

Chromatographic separation of styrene-methyl methacrylate (MMA) copolymers depending on the chemical composition was studied using liquefied carbon dioxide as an adsorption promoting solvent, and tetrahydrofuran, chloroform containing ethanol as a desorption promoting solvent in the mobile phase and the column packed non-bonded silica gel by a solvent gradient method. With the increase of MMA content, the elution was retarded indicating that the typical normal-phase type of adsorption occurred. The effects of type of desorption solvents, molecular mass of sample, and column temperature on the elution were investigated.