A rapid, isocratic method for phospholipid separation by high-performance liquid chromatography

A rapid, isocratic method for separating the most prevalent phospholipids by high-performance liquid chromatography is described. Baseline resolution of phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, lysophosphatidylcholine, and sphingomyelin is achieved in less than 40 min on a silica column. Lipids are injected in 10 microliter of chloroform-diethyl ether 1:2 (v/v) and eluted with a solvent mixture of acetonitrile-methanol-sulfuric acid 100:3:0.05 (v/v/v) at a flow rate of 1 ml/min. Neutral lipids and cardiolipin elute with the solvent front. Chromatography of a radioactive cell lipid extract indicates a recovery of better than 97%. The procedure is sensitive enough to permit the analysis of the main phospholipids present in a monolayer culture containing about 100 micrograms of cell protein.     

Application of preparative high-speed counter-current chromatography/preparative high-performance liquid chromatography mode in rapid separation of saponins

Combined with preparative high-performance liquid chromatography, high-speed counter-current chromatography was employed for isolation and purification of saponins from Gypsophila paniculata L. n-Hexane-n-butanol-methanol-0.02% TFA (1:9:1:9, v/v) was employed as solvent system and 210 nm was chosen as the wavelength of ultraviolet detection for the first time. The research tried to compare HSCCC with prep-HPLC, and further integrated their advantages to improve separation efficiency. Five known triterpene saponins were identified by 13C NMR and ESI-MS and their purities were all above 96%. The results demonstrated that adopted method was a feasible, economical and efficient technique for rapid preparative isolation of saponins.     

Rapid method for trehalulose production and its purification by preparative high-performance liquid chromatography

Trehalulose was produced with a good yield by enzymatic conversion of sucrose and easily purified by preparative HPLC using a single Ca2+-based column. In addition, the structure of this sugar was confirmed by 13C and 1H n.m.r studies.     

Preparative ion-exchange high-performance liquid chromatography of bacterial ribosomal proteins

We have developed analytical and preparative ion-exchange HPLC methods for the separation of bacterial ribosomal proteins. Proteins separated by the TSK SP-5-PW column were identified with reverse-phase HPLC and gel electrophoresis. The 21 proteins of the small ribosomal subunit were resolved into 18 peaks, and the 32 large ribosomal subunit proteins produced 25 distinct peaks. All peaks containing more than one protein were resolved using reverse-phase HPLC. Peak volumes were typically a few milliliters. Separation times were 90 min for analytical and 5 h for preparative columns. Preparative-scale sample loads ranged from 100 to 400 mg. Overall recovery efficiency for 30S and 50S subunit proteins was approximately 100%. 30S ribosomal subunit proteins purified by this method were shown to be fully capable of participating in vitro reassembly to form intact, active ribosomal subunits.     

Analytical and preparative high-performance liquid chromatography separation of flavin and flavin analog coenzymes

Reversed-phase high-performance liquid chromatography has been used to separate a number of flavin and flavin analogs at the riboflavin, FMN, and FAD coenzyme level. Analytical methods were developed which enable the facile determination of a particular flavin or mixture of flavins present. These methods also allowed the separation of oxidized from reduced forms of oxygen-stable flavin analogs. Past investigations have utilized enzymatically synthesized FAD analogs with the problem of potential contamination by other levels of the coenzyme or ATP a cosubstrate in the flavokinase/FAD synthetase reaction. Preparative methods show that all the potential reaction products may be separated from one another thereby allowing the rapid purification of these redox coenzyme analogs. To demonstrate the utility of this method, radiolabeled FAD and 1-deazaFAD were prepared and purified.     

Ionic-exchange high-performance liquid chromatography of Escherichia coli ribosomal small-subunit proteins

Ion-exchange high-performance liquid chromatography was applied to the separation of proteins from the 30S ribosomal subunit. The proteins present in each peak have been identified by polyacrylamide gel electrophoresis analysis. The purification has been made using either unmodified proteins or proteins specifically labeled at their SH group. The results clearly show that the method can be used to purify and identify ribosomal proteins.     

A preparative high performance liquid chromatography method for separation of lecithin: comparison to thin-layer chromatography

We have developed a high performance liquid chromatography (HPLC) method to separate lecithin from other phospholipid classes and to obtain lecithin from biologic materials. The separation was performed on a preparative 10-micron Spherisorb column with an optimized solvent system consisting of the following components: acetonitrile, isopropanol, methanol, water, and trifluoroacetic acid. The advantages of this method are the use of an isocratic solvent system limited to about 30 min and the very good separation of the phosphatidyl-choline fraction from the sphingomyelin fraction. Furthermore, the HPLC method has a better recovery rate than the thin-layer chromatography method, and it can be run under automatic control.     

Reverse-phase high-performance liquid chromatography of Escherichia coli ribosomal small subunit proteins

Reverse-phase high-performance liquid chromatography has been explored as an approach for the separation of the proteins of the 30 S subunit of Escherichia coli ribosomes. The majority of these proteins are of similar molecular weight and isoelectric point, making separation by size exclusion or ion exchange difficult. With the use of an octadecasilyl silica column and a trifluoroacetic acid-acetonitrile solvent system, the 21 proteins of the 30 S subunit have been separated into 15 peaks. The yield of total protein recovered from the column was ≥85%. The proteins present in each peak have been identified by polyacrylamide gel electrophoretic analysis of the peaks as well as by comparison with the relative retention volumes of known purified 30 S proteins on the column. The results clearly show that this method is a powerful and rapid technique for the identification and purification of 30 S proteins. Analysis of [³H]puromycin-labeled 30 S subunit protein provides an illustrative example of its utility for affinity labeling studies.     

A novel preparative method for the isolation of avidin and riboflavin-binding glycoprotein from chicken egg-white by the use of high-performance liquid chromatography.

A method for the rapid preparative isolation of highly purified avidin using h.p.l.c. on a powerful cation-exchanger TSK SP-5PW column has been developed. The method is based on the following properties of avidin: solubility at a high (NH4)2SO4 concentration, stability and relatively low solubility in organic solvents, as well as the strongly cationic nature of the molecule. Riboflavin-binding glycoprotein may be isolated as by-product.     

A rapid high-performance liquid chromatography purification method of lodinated polypeptide hormones

A reverse-phase high-performance liquid chromatography procedure is presented for the separation of a variety of monoiodinated peptides from the corresponding unlabeled and diiodinated hormones. In the case of peptides lacking Met residues, susceptible to oxidation by the chloramine-T-labeling method, chloramine T treatment alone had no effect on elution position of unlabeled hormone. For those hormones containing Met, however, more than 90% of the peptide was transformed into the earlier-eluting oxidized form after 20 to 40 s of chloramine-T treatment. Upon iodination, monoiodinated derivatives were well separated from the corresponding chloramine-T-treated standards, the extent of separation being decreased with increased molecular weight. Application of this technique to the purification of monoiodinated ¹²⁵I-γ-melanocyte-stimulating hormone permitted a threefold increase in titer and sensitivity of a γ-melanocyte-stimulating hormone radioimmunoassay system.     

A rapid purification procedure for tetrodotoxin derivatives by high-performance liquid chromatography

A simple procedure for purification of tetrodotoxin (TTX) derivatives by high-performance liquid chromatography is described. Chemically oxidized TTX, C₁₁-nortetrodotoxin (nor-TTX), was purified and collected by reverse-phase chromatography. The separation of nor-TTX from unreacted TTX was excellent and recovery of nor-TTX was more than 90%. The isolated nor-TTX was further coupled with lysine, and the coupled product was purified again by high-performance liquid chromatography on a cation-exchange column. The separation of all compounds required less than 15 min. The uv monitoring at 230 nm allowed the detection of TTX derivatives at the 2- to 3-ng level.     

Cation-exchange high-performance liquid chromatography: separation of highly basic proteins using volatile acidic solvents

The chromatographic behavior of a number of globular proteins was studied on a Bio-Sil TSK CM-2-SW weak cation exchange HPLC column under acidic conditions. A linear gradient of 0-1 M NH4Ac in 1 M HOAc, inducing a convex pH gradient from 2.4-4.8, resulted in an excellent separation of highly basic proteins. For these proteins a linear relationship between isoelectric point and retention time was determined experimentally. The effect of pH and the ion composition of the eluting buffer system on this linear correlation was studied. Although the exact basis for protein separation on the CM-2-SW column at low pH is not clear yet, both the pH-dependent net positive charge per unit surface area and most likely the relative percentage of arginine in the total number of basic residues contribute to this separation. Because of the high resolving power and the high protein recovery obtained in a system using only acidic volatile buffer solutions, the cation exchanger is particularly suitable for the purification of nanogram amounts of acid-stable basic growth factors. The present sterile conditions (1 M HOAc/NH4Ac system, pH less than 4) and the easy removal of salt by lyophilization facilitate the detection of these proteins by biological assays.     

A high-performance liquid chromatography method for determining transition metal content in proteins

Transition metals are common components of cellular proteins and the detailed study of metalloproteins necessitates the identification and quantification of bound metal ions. Screening for metals is also an informative step in the initial characterization of the numerous unknown and unclassified proteins now coming through the proteomic pipeline. We have developed a high-performance liquid chromatography method for the quantitative determination of the most prevalent biological transition metals: manganese, iron, cobalt, nickel, copper, and zinc. The method is accurate and simple and can be adapted for automated high-throughput studies. The metal analysis involves acid hydrolysis to release the metal ions into solution, followed by ion separation on a mixed-bead ion-exchange column and absorbance detection after postcolumn derivatization with the metallochromic indicator 4-(2-pyridylazo)resorcinol. The potential interferences by common components of protein solutions were investigated. The metal content of a variety of metalloproteins was analyzed and the data were compared to data obtained from inductively coupled plasma-atomic emission spectroscopy. The sensitivity of the assay allows for the detection of 0.1-0.8 nmol, depending on the metal. The amount of protein required is governed by the size of the protein and the fraction of protein with metal bound. For routine analysis 50 microg was used but for many proteins 10 microg would be sufficient. The advantages, disadvantages, and possible applications of this method are discussed.     

A simple and rapid method of quantitative analysis of phosphoamino acids by high-performance liquid chromatography

A high-performance liquid chromatographic system for the separation of nonradiolabeled phosphoamino acids and orthophosphate by ion-pair reverse-phase chromatography has been developed. By the use of low-ionic-strength phthalate buffers at pH 6.3, the phosphoamino acids can be visualized by virtue of this uv-active eluant. The technique is sensitive to 200 pmol of phosphoamino acid and has been shown to be directly applicable to the analysis of isolated phosphoproteins.     

Improved procedure for the separation of phospholipids by high-performance liquid chromatography

We describe a rapid and efficient high-performance liquid chromatography procedure for the separation of phospholipids. The separation is accomplished on a microparticulate silica gel column using isocratic elution and UV detection at 203 nm. The solvent mixture is acetonitrile—methanol—85% phosphoric acid(130:5:1.5, v/v). Complete separation is achieved within 30 min of phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, phosphatidyldimethylethanolamine, lysophosphatidylethanolamine, phosphatidylcholine, lysophosphatidylcholine and sphingomyelin. The method is suitable for the analysis of phospholipids in tissue extracts.     

Enantiomeric separation of imidazolinone herbicides using chiral high-performance liquid chromatography

Chiral high-performance liquid chromatography (HPLC) is one of the most powerful tools to prepare enantiopure standards of chiral compounds. In this study, the enantiomeric separation of imidazolinone herbicides, i.e., imazethapyr, imazapyr, and imazaquin, was investigated using chiral HPLC. The enantioselectivity of Chiralpak AS, Chiralpak AD, Chiralcel OD, and Chiralcel OJ columns for the three analytes was compared under similar chromatographic conditions. Chiralcel OJ column showed the best chiral resolving capacity among the test columns. The resolved enantiomers were distinguished by their signs of circular dichroism detected at 275 nm and their structures confirmed with LC-mass spectrometric analysis. Factors affecting the chiral separation of imidazolinones on Chiralcel OJ column were characterized. Ethanol acted as a better polar modifier than the other alcohols including 2-propanol, 1-butanol, and 1-pentanol. Although the acidic modifier in the mobile phase did not influence chiral recognition, it was necessary for reducing the retention time of enantiomers and suppressing their peak tailing. Thermodynamic evaluation suggests that enantiomeric separation of imidazolinones on Chiralcel OJ column is an enthalpy-driven process from 10 to 40 degrees C. This study also shows that small amounts of pure enantiomers of imidazolinones may be obtained by using the analytical chiral HPLC approach.     

Simple and rapid method for determining nicotinamide adenine dinucleotide synthetase activity by high-performance liquid chromatography

A method is described for the determination of nicotinamide adenine dinucleotide synthetase (NADS) activity in human blood. Using high-performance liquid chromatography (HPLC), the formed NAD is separated from the substrates and the other blood components in less than 13 min. The activity of NADS determined by HPLC is closely correlated with that determined by the conventional spectrophotometric method, which requires two steps of enzyme reaction. The present method is simple and reliable and facilitates the routine analysis of NADS activity.     

A rapid preparative method for isolation of neutral and acidic glycosphingolipids by radial thin-layer chromatography

An efficient method to separate neutral and acidic glycosphingolipids (GSLs) from their mixtures within a short period (45-60 min) and with low consumption of solvents (chloroform-methanol-water, 60/35/8 (v/v/v); 250-500 ml) has been developed. This method utilizes a centrifugal thin-layer chromatograph (Chromatotron) and the GSL mixtures (30-400 mg) are applied to glass plates coated with a 1-mm layer of silica gel 60 PF-254. The method (radial thin-layer chromatography) is rapid and simple and the recovery of glycosphingolipids is high (70-80%).     

Enantiomer separation of triazole fungicides by high-performance liquid chromatography

Enantiomer separation is one of the most important prerequisites for the investigation of environmental enantioselective behaviors for chiral pesticides. In the present study, the enantiomer separation of 7 triazole fungicides, i.e., hexaconazole (1), triadimefon (2), tebuconazole (3), diniconazole (4), flutriafol (5), propiconazole (6), and difenoconazole (7), were evaluated using normal phase high-performance liquid chromatography. Chrialcel OD column and Chrialcel OJ column were used. The influence of column temperature was studied for the optimization of the resolution as well as the type and percentage of organic modifier in the mobile phase. The retention factors for the enantiomers of all investigated compounds decreased as the temperature increased. The natural logarithms of the selectivity factors (lnalpha) of hexaconazole (1), tebuconazole (3), flutriafol (5), propiconazole (6) and difenoconazole (7) depended linearly on the inverse of temperature (1/T) while the corresponding values for triadimefon (2) and diniconazole (4) kept unchanged in the studied temperature range 10-35 degrees C. Van't Hoff plots afforded thermodynamic parameters, such as the apparent change in enthalpy DeltaH degrees , the apparent change in entropy DeltaS degrees and the apparent change in DeltaDeltaH degrees and DeltaDeltaS degrees . The thermodynamic parameters (DeltaH degrees , DeltaS degrees , DeltaDeltaH degrees and DeltaDeltaS degrees ) were calculated in order to provide an understanding of the thermosynamic driving forces for enantioseparation. The established method shows perspective to be used for preparing micro-scale amount of pure enantiomers of the chiral triazoles studied.