Ovine luteinizing hormone. V. Significance of flow-through peaks observed during chromatofocusing as revealed by various methods of sample preparation and application.

In a previous study [Keel et al., Biol, Reprod., 36 (1987) 1102] the ovine luteinizing hormone (oLH) in pituitary extracts was chromatofocused on pH 10.5-7 gradients after equilibration in 25 mM triethylamine-HCl, pH 11.0, by gel permeation. Under these conditions, some immunoreactive oLH flowed through the columns unrestricted and this was interpreted to represent extremely basic isoforms. However, when selected flow-through peaks were re-chromatofocused, each was contaminated with other isoforms of oLH. In order to clarify this dilemma, various methods of sample preparation and application were systematically compared. Consistent with previous observations, variable amounts of the immunoreactive oLH in pituitary extracts equilibrated in triethylamine by gel permeation, dialysis, flow dialysis or ion-retardation chromatography eluted as flow-through peaks when chromatofocused. In contrast, when the ionic components in the pituitary homogenization buffer were removed by these methods as well as ultrafiltration and the proteins were applied to the resin in the elution buffer (1:45 Pharmalyte 8-10.5-HCl, pH 7.0), none of the immunoreactive oLH in pituitary extracts eluted as a flow-through peak. Thus, it appears that oLH eluting as a flow-through peak results from incomplete binding of the hormone to the chromatofocusing resin when applied in triethylamine.     

Separation of proteins of nuclear ribonucleoprotein particles by high-performance liquid chromatography

High-performance liquid chromatography (h.p.l.c.) is used to fractionate the proteins of the 30–40 S nuclear ribonucleoprotein particles. The major core proteins of the particles are eluted from a SynChropak AX-300 anion-exchange column before the more acidic higher-molecular-weight minor particle proteins. Each of the three major core proteins which can be separated from the other particle proteins by preparative polyacrylamide gel electrophoresis are eluted from the SynChropak AX-300 column as one peak. Isoelectric focusing separates each of these three apparently homogeneous peaks into a series of charge isomers ranging in isoelectric pH (pI) from 5.5 to 9.0. The core proteins of the ribonucleoprotein particles have a strong affinity to each other and form aggregates. The elution of each of the charge isomers of the three major proteins in one peak and their elution from an anion exchanger before the elution of the more acidic higher-molecular-weight minor proteins of the nuclear ribonucleoprotein particles is explained by the formation of these aggregates. The separation of the total proteins of the nuclear ribonucleoprotein particles by h.p.l.c. is similar to the separation which can be obtained by preparative electrophoresis but the l.c. technique is simpler, substantially quicker, and adaptable to large-scale preparation.     

Separable Forms of Radioiodinated Ovine Lutropin (oLH) Subunits,Fractionated by Anion Exchange High Performance Liquid Chromatography and Analyzed by Radioimmunoassay

Abstract

Radioiodinated oLH α and oLH ß subunits were fractionated with the aid of high performance liquid chromatography (HPLC) using a Waters Protein Pak DEAE 5PW anion exchange column. The content of these subfractions differed in their binding maxima to their respective subunit antisera. An increase of the pH from 6.5 to 7.5 and 8.5 affected the chromatographic profile of 8-week-old radioiodinated α-subunit. Overall, material from the various radioactive peaks exhibited binding to ß-subunit antiserum in the range of 32.0% - 81.0%, depending on the storage time of the tracer and the pH. Shifting strategies, we either applied the labelled subunits to a Pharmacia gel filtration column or subjected them to cellulose adsorption prior to HPLC. The radioiodinated α- and β-subunits subjected to HPLC after gel filtration were both eluted in only one peak with respective immunoreactivities of 46.6% and 73.2%.

When radioiodinated β-subunit was applied first to a cellulose column and then to HPLC, the chromatographic profile showed two radioactive peaks with retention times of 5 min (73.2% immunoreactivity) and 7.5 min (43.0% immunoreactivity), respectively.

It was concluded that an 8-week-old-tracer i s useful in such studies, owing to its highly stable immunoreactivity after repurification on an anion exchange HPLC.     

Biological Characterization of a Pituitary Monkey Lutropin Preparation after Chromatofocusing or after High Performance Anion Exchange Chromatography

Abstract

The Pharmacia Fast Protein Liquid Chromatography System equiped with a Mono P HR 5/10 chromatofocusing column was used to characterize the distribution of different bioactive forms of a cynomolgus (Macaca bascicularis) pituitary lutropin preparation. The results were compared with the profiles obtained after running the preparation on a Waters DEAE anion exchange column using eluants in the pH range from 6.0 to 8.0. The distribution of preserved biological activity were studied in respective eluted fractions from the different experiments of the applied preparation. Seventy-six percent of the bioactivity was recovered after chromatofocusing whereas preserved bioactivity after ion exchange chromatography ranged between 61–78% at pH 6.5 to 7.5. No bioactivity was restored after elution at pH 6.0 or 8.0.     

Separation and measurement of clofibroyl coenzyme A and clofibric acid in rat liver after clofibrate adminstration by reversed-phase high-performance liquid chromatography with photodiode array detection

A method to identify and quantitate clofibric acid and clofibroyl coenzyme A (CoA) products in rat liver was developed using reversed-phase high-performance liquid chromatography. The system was developed with baseline separation of clofibroyl-CoA from clofibric acid using isocratic elution, with a mobile phase consisting of 52% methanol and 28 mM potassium phosphate buffer (pH 4.2). With this high methanol concentration, the large amount of UV-absorbing materials present in the liver extracts were eluted earlier than the investigated compounds. Clofibroyl-CoA has a characteristic absorbance spectrum with distinct peaks at 260 and 230 nm, while clofibric acid showed only a distinct peak at 230 nm. Using an on-line photodiode array detector, the spectra could be recorded during analysis without interrupting the flow of the mobile phase. This spectral analysis identification possibilities and evaluation of the purity of the chromatographic peaks. In a perchloric extract of rat liver, the recovery of clofibric acid and clofibroyl-CoA added to the liver extract ranged from 70 to 80%. A linear relationship was observed between clofibric acid and clofibroyl-CoA concentration and the area of their peaks in the chromatogram. The detection limit of the method was lower than 5 pmol for both compounds when the absorbance was recorded at 230 nm. The method could be used without modification for the estimation of clofibroyl-CoA and clofibric acid in biological extracts.     

Analytical- and preparative-scale separation of molecular variants of alpha-fetoprotein by anion-exchange chromatography on Monobead resins.

A rapid and reliable purification procedure is described that is useful for both analytical detection and quantitative recovery of milligram amounts of individual molecular variants of mouse alpha-fetoprotein (AFP). The appropriate separation conditions were developed with an analytical-size Mono Q anion-exchange column linked to an automated Fast Protein Liquid Chromatography system. Effective separations of fetal-derived AFP variants was accomplished within 20 min under mild conditions with an L-histidine buffer. Employing the optimal separation conditions established on the Mono Q HR 5/5 column we upscaled the procedure by using a preparative Mono Q HR 16/10 column in order to obtain milligram quantities of each molecular variant of AFP. Seven distinct isomeric forms of AFP could be recovered on the preparative anion exchanger in a highly reproducible manner. Each of the seven protein peaks eluted from the Mono Q column were confirmed to be distinct isoforms of AFP by isoelectric focusing and Western blotting developed with monospecific anti-AFP antisera. This method in its scaled up version offers the benefit of providing milligram quantities of immunochemically pure AFP isomers for structure and function studies.     

Effect of buffer concentration on gradient chromatofocusing performance separating protiens on a high-performance DEAE column

Gradient chromatofocusing is a recently developed chromatographic technique that overcomes the limitations of conventional chromatofocusing. This technique employs a HPLC gradient system and simple low-molecular-mass buffer components to generate linear or other function pH gradients on ion-exchange columns. Results of the present work show a superior separation of beta-lactoglobulin A and B in gradient chromatofocusing compared to salt gradient chromatography using the same DEAE column, with an optimized resolution of 2.3 obtained with gradient chromatofocusing compared to 1.1 obtained with NaCl gradients at constant pH. A significant advantage of the gradient chromatofocusing technique over the conventional chromatofocusing technique is its ability to employ a relatively wide range of buffer concentrations in the mobile phase, the effect of which is studied in the present work. Five proteins (conalbumin, ovalbumin, bovine serum albumin, beta-lactoglobulin A and B) are chromatographed on a DEAE-polymethacrylate HPLC anion-exchange column using the same approximately linear pH gradient profile but different mobile phase buffer concentrations. Results show a significant effect of buffer concentration on peak width, separation factor and resolution. For example, resolution increases from 1.5 to 2.3 in the separation of beta-lactoglobulin A and B when the concentration of each of the components in the 100% elution buffer is increased from 6.25 to 25.0 mM (with the same outlet pH gradient). This separation trend is also seen in the chromatography of ovalbumin from a commercial source, noting a progressive increase in resolution of two peaks in the sample (resolution increased from 0.7 to 2.4) when the concentration of each of the components in the 100% elution buffer is increased from 6.25 to 37.5 mM (same outlet pH gradient). The gains in the resolution are attributed to an increase in the separation factor, since the peak widths are generally noted to also increase with increased buffer concentration. These results point to a significant interplay between buffer concentration and pH, which is not effectively exploited in either conventional chromatofocusing or in conventional ion-exchange chromatographic procedures employing salt gradient elution at constant pH. Gradient chromatofocusing has the ability of optimizing both parameters, thus providing it with unique capabilities in protein separations.     

Procedure for the quantification of rider peaks.

Rider peaks are small peaks which are not well resolved from a large and asymmetrical neighbour but sit on its trailing side. The usual case is a large, tailed peak which is eluted just in front of the small peak, although the opposite situation can also occur (a small peak in front of a large peak with fronting). The common integration techniques. i.e. separating the peaks by vertical drop or by a tangent and determining area or height, give erroneous results. We propose a method for their quantification with low error. It is necessary to set up a "two-dimensional" calibration by varying both concentrations, i.e. of the large peak and of the rider. This leads to a series of linear equations which describe the rider size, as found by the integrator, as a function of the size of the large peak. The y-axis intercepts i of these equations show a linear relationship with the concentration x of the rider analyte, whereas the slopes s follow a quadratic relationship. These equations can be used to solve the equation y = s(x) x z + i(x) for x (y and z are the integrated peak size of the rider and the large peak, respectively). The procedure was tested with computer-generated peak pairs as well as with HPLC separations of 2,3-dimethylaniline (large tailing peak) and 2,3-dimethylphenol (symmetrical rider peak).     

曲线拟合定量法在高效液相色谱手性分离中的应用

 首次将曲线拟合定量法用于解决高效液相色谱手性分离中重叠峰的定量问题。通过比较百树菊酯 8个立体异构体的实验谱图和拟合谱图 ,证明曲线拟合定量法与传统的中切法相比 ,可获得更令人满意的结果。     

Characterization of each isoform of a F(ab')2 by capillary electrophoresis.

Free solution capillary electrophoresis was investigated for the characterization of an M(r) 100 000 purified F(ab')2. Optimization of the experimental conditions allowed the identification of five separated peaks, suggesting the presence of isoforms which differed by only 0.2 pH unit. This heterogeneity was still detectable with 80 amol of protein. After a preparative separation by chromatofocusing, identification of each form was performed for the first time by capillary electrophoresis. A quantitative and qualitative correlation with isoelectric focusing showed that free solution capillary electrophoresis represents a sensitive method for revealing subtle differences in charge, even for large proteins.     

Pseudohypericin and hypericin in St. John's wort extracts. Breakdown of pseudohypericin

Photodiode array detector-coupled HPLC analysis of extracts of St. John's wort (Hypericum perforatum) reveals two main compounds: pseudohypericin (PH) and hypericin (H) (peaks A and B respectively). When the pH of the extracts is raised, peak A irreversibly disappears whereas peak B does not change. This instability at high pH means that alkaline conditions should be avoided during extraction and raises a question concerning the bioavailability of pseudohypericin from St. John's wort extract-containing medicinal products designed for oral administration.     

Liquid Chromatographic-Electro-Chemical Technique for Determination of Ethylenethiourea Residues

Abstract

A high performance liquid chromatographic-electrochemical (HPLC-EC) technique was developed to selectively determine ethylenethiourea (ETU) at residue levels without derivatization. ETU was eluted from a C-8 column with water, a phosphoric acid electrolyte solution was added to the column eluate, and then ETU was detected with an electrochemical detector containing a Au/Hg working electrode. The HPLC-EC system produced a sharp chromatographic peak for ETU that was detected by the Au/Hg electrode at an applied potential of +0.36 V. With detector sensitivity adjusted so that 10 ng ETU produced a 50% full scale deflection peak (1% baseline noise), the detector's response was linear from 2 to 400 ng ETU. No peaks were observed in potato and spinach controls, and only small apparent ETU peaks of 7 and 3 ppb, respectively, were found in apple and grape controls. Detector response was equivalent to 90% of actual ETU added (0.1 ppm) to purified spinach extracts. Crop coextractives from apples, grapes and potatoes did not affect detector response to ETU at the 0.1 ppm fortification level.     

Fast, comprehensive online two-dimensional high performance liquid chromatography through the use of high temperature ultra-fast gradient elution reversed-phase liquid chromatography

A new approach to high speed, comprehensive online dual gradient elution 2DLC (LCxLC) based on the use of ultra-fast, high temperature gradient elution reversed phase chromatography is described. Entirely conventional gradient elution instrumentation and columns are assembled in a system which develops a total peak capacity of about 900 in 25 min; this is equivalent to roughly one peak/2 s. Each second dimension gradient is done in a cycle time of 21 s and the peak retention times measured for a set of twenty six indole-3-acetic acid (IAA) derivatives are reproducible to 0.2 s. Each peak eluting from the first dimension column is sampled at least twice across its width, as the corresponding peaks on the second dimension column appear in two or three consecutive second dimension chromatograms, clearly indicating that there is little loss in the resolution gained in the first dimension separation. Application to the separation of the low molecular weight components of wild-type and mutant maize seedlings indicates the presence of about 100 peaks on a timescale of 25 min. Compelling illustrations of the analytical potential of fast, high temperature 2DLC are evident in the clear presence of nine distinct peaks in a single second dimension chromatogram from a single quite narrow first dimension peak, and the great power of 2DLC to solve the "analytic dynamic range" problem inherent in the measurement of small peaks that are neighbors to a gigantic peak.     

Preparation of samples for high-performance liquid chromatography of inositol phosphates.

A simple method is described for the removal of extraneous material from tissue extracts prior to anion-exchange high-performance liquid chromatography of inositol phosphates. Samples are prepared by extraction with trichloroacetic acid or perchloric acid followed by removal of the excess acid. The extracts are then passed through small Dowex-50 cation-exchange columns and eluted with water. Dowex-50 pretreatment removes most of the ultraviolet absorbing material and cations from the samples but does not alter the content of inositol phosphates. This treatment results in improved reliability of chromatography, especially with respect to weakly retained molecules such as adenosine 5'-phosphate and the isomers of inositol monophosphate. In addition, sample pretreatment improves the useful lifetime of the analytical anion-exchange columns.     

Effect of subambient temperature on RP-HPLC of β-carotene isomers

Summary (E)-β-carotene and three mono-(Z)-isomers (9(Z)-, 13(Z)-, 15(Z)-) were eluted isocratically on a VYDAC 201TP54 column, using column temperatures between +30°C and −7°C. In this temperature range, their elution order changed dramatically with decreasing temperature. Nearly baseline separation of the four isomers within one hour was achieved at −7°C. For laboratory routine, temperatures of 9±2°C can be recommended. Thus, a good resolution of β-carotene- and lycopene-isomers (e.g. in tomato products) within 30 minutes is possible.     

Comparison by gel filtration chromatography and reversed-phase high-performance liquid chromatography of the immunoreactive growth hormone composition of a human pituitary extract

The immunoreactive growth hormone composition of a pituitary extract has been compared by conventional gel filtration chromatography (pH 8), and reversed-phase high-performance liquid chromatography (pH 2) on a wide-pore (300 A) short-chain column. By gel filtration chromatography, four peaks of immunoreactivity were obtained, labelled "monomer", "dimer", "aggregate" and "void". However, by high-performance liquid chromatography all of these fractions were themselves shown to be multicomponent mixtures. The "monomer" peak contained at least two forms (M1 and M2). The "dimer" fraction contained three peaks, two of which co-eluted with M1 and M2, and a third component, D. Similarly, the aggregate fraction contained M1, M2, D and a fourth component, A. The "void", in contrast, contained mostly M1 and M2 with very little D. One interpretation of these results is that M1 (the 22K molecular weight monomeric form) and M2 (a chemically modified form of M1) are present in all molecular weight fractions in loosely bound aggregates which break up under acidic conditions. D and A are probably oligomeric forms of growth hormone (possibly a dimer and higher molecular weight species, respectively).     

Application of HPLC and Dual Wavelength Detection to the Analysis of Phenolic Compounds in Apples

Abstract

High-performance liquid chromatography, coupled with a programmable UV/visible detector, was applied to the identification and quantification of the phenolic compounds of apple tissue. The identity and purity of unknown components eluted from the HPLC column were established by comparing retention times and absorbance ratios with those of authentic compounds. In Golden Delicious apples, components with retention times agreeing with those of authentic catechin, epicatechin, chlorogenic acid, protocatechuic acid, p-hydroxybenzoic acid, p-coumaric acid, ferulic acid, and sinapic acid were found. However, comparison of absorbance ratios with those of authentic compounds suggested that many of the components that eluted in this system as single, symmetrical peaks were mixtures rather than individual compounds. This method helped to eliminate inaccurate identifications or faulty assumptions about homogeneity of HPLC “peaks”, errors that can occur when traditional HPLC methods that rely solely on detection at a single wavelength are employed. The method should have general applicability to plant extracts.     

High performance ion exchange chromatography of human plasma lecithin:cholesterol acyltransferase

Highly purified monomeric human plasma lecithin:cholesterol acyltransferase (LCAT), completely free of apolipoprotein D, has been chromatographed on a MonoQ HR 5/5 anion exchanger. LCAT eluted as symmetrical peaks after 12.8 min and 14.8 min at pH 5.0 and pH 6.0, respectively, using a linear NaCl gradient. The corresponding concentrations of NaCl effecting desorption of LCAT from the anion exchanger were 125 mM and 175 mM. At both pH values human serum albumin eluted earlier and was well separated from the enzyme. Rechromatography of LCAT in the eluates from these experiments at acid pH, on high performance gel filtration, demonstrated absence of aggregation. The nonspecific adsorption during anion exchange chromatography at pH 5.0 and pH 6.0 was negligible, as demonstrated by a linear relationship between injected amounts of LCAT and recorded peak areas for a 2-20 micrograms protein range. Zone immunoelectrophoresis assay indicated unaltered immunoreactivity of the eluted LCAT.     

Proteomic analysis of rat plasma by two-dimensional liquid chromatography and matrix-assisted laser desorption ionization time-of-flight mass spectrometry

The proteomic analysis of plasma and serum samples represents a formidable challenge due to the presence of a few highly abundant proteins such as albumin and immunoglobulins. Detection of low abundance protein biomarkers therefore requires either the specific depletion of high abundance proteins using immunoaffinity columns and/or optimized protein fractionation methods based on charge, size or hydrophobicity. Here we describe a two-dimensional (2D) liquid chromatography separation method for the fractionation of rat plasma. In the first dimension proteins were separated by chromatofocusing according to their isoelectric point (pI). In the second dimension, proteins were further fractionated by non-porous, reversed-phase chromatography according to their hydrophobicity. The data from both separations was displayed as a 2D protein expression map of pI versus retention time (relative hydrophobicity). Both separations were carried out on the ProteomeLab PF 2D system (Beckman Coulter), an instrument platform that provides a high degree of automation and real-time monitoring of the separation process. The reproducibility of the first-dimension separation was evaluated in terms of pH gradient formation. The second-dimension separation was evaluated in terms of peak retention times on the reversed-phase column. We found in four consecutive chromatofocusing separations that the pH gradient differed by less than 0.2 pH units at any time during the elution step. Second dimension retention times of peaks from identical pI fractions differed by less than 7 s in six consecutive separations. Each 2D separation generated a total of 540 fractions which were analyzed by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). We detected approximately 275 peptides and proteins with molecular masses ranging from 3 to 225 kDa. Most fractions were found to contain multiple low and high molecular weight proteins. Differential display of 2D protein expression maps from retinol-sufficient and -deficient rat plasma samples identified a fraction with several proteins that appeared to be down-regulated in the vitamin A-deficient animal. Quantitative proteomic analysis of complex samples such as plasma is still a difficult task. We discuss the potential of this approach for biomarker discovery and address the experimental challenges that remain.     

Peak distortion in the column liquid chromatographic determination of omeprazole dissolved in borax buffer.

Injection of a sample containing omeprazole dissolved in borax buffer (pH 9.2) into a reversed-phase liquid chromatographic system consisting of a mixture of acetonitrile and phosphate buffer (pH 7.6) as the mobile phase and a C18 surface-modified silica as the solid phase resulted under special conditions in split peaks of omeprazole. The degree of peak split and the retention time of omeprazole varied with the concentration of borax in the sample solution and the ionic strength of the mobile phase buffer as well as with the column used. Borax is eluted from the column in a broad zone starting from the void volume of the column. The retention is probably due to the presence of polyborate ions. The size of the zone varies with the concentration of borax in the sample injected. In the borax zone the pH is increased compared with the pH of the mobile phase, and when omeprazole (a weak acid) is co-eluting in the borax zone its retention is affected. In the front part and in the back part of the borax zone, pH gradients are formed, and these gradients can induce the peak splitting. When the dissolving medium is changed to a phosphate buffer or an ammonium buffer at pH 9 no peak distortion of omeprazole is observed.