Semi-preparative Column Research Articles

Anion-exchange high-performance liquid chromatography of human serum apolipoproteins

The rapid separation of seven urea-soluble apolipoprotein species from delipidated human serum very low density lipoproteins (VLDL) and high density lipoproteins (HDL) has been achieved by high-performance liquid chromatography on an anion-exchange column of Syn-Chropak AX 300. Effluent chromatographic peaks were detected by absorbance at 280 nm in a flow-through cell. Peaks corresponding to apolipoproteins AI 1, AI 2, AII, CI, CII, CIII 1, and CIII 2 were identified by amino acid analysis, gel electrophoresis, and isoelectric focusing. Maximum efficient loading of semipreparative columns (250 × 9.0 mm) was established to be ca. 20 mg HDL apolipoprotein. Minimum detectable protein was shown to be ca. 1 μg on an analytical-scale column (300 × 4.5 mm). Chromatographic resolution is comparable to that of conventional DEAE-cellulose column chromatography. The ratio of apoAI 1 to apoAI 2 was considerably greater in high-performance liquid chromatography, suggesting that the variants seen in conventional chromatography and isoelectric focusing are in part artifactual.

Rapid analysis of labeled globin chains without acetone precipitation or dialysis by high-pressure liquid chromatography and ion-exchange chromatography

Human red blood cells were incubated with tritiated leucine. The cells were washed several times, lysed, and aliquots of the lysates were acidified with trifluoroacetic acid and applied on reversed-phase columns to separate the α-, β-, G γ-, and A γ- globin chains. The ratios of the labeled non-α- to α-globin chains separated by this method were practically identical to those obtained using the standard method of globin chain precipitation with acidified acetone. Hemoglobins present in very small amounts or containing chains which are not readily separated in the reversed-phase system can be purified first using a semi-preparative column of carboxymethyl-Sepharose. The hemoglobins of cell lysates are eluted using linear gradients of NaCl in bis-Tris buffer, and the fractions containing the desired hemoglobin can be applied directly on reversed-phase columns without sample concentration or acetone precipitation. Both chromatographic systems utilize solutions which are very stable at room temperature, and the columns can be easily regenerated and used many times. This results in a considerable reduction of the time and the cost of analysis of labeled globin chains. This system can be easily applied to the common hemoglobins A, F, C, and S.

Separation of steroidal estrogens and their major unconjugated metabolites by high performance liquid chromatography

A high performance liquid Chromatographic method is described for the rapid, non-destructive separation of a number of physiologically important steroidal estrogens, including the labile catechol estrogens. This procedure uses a “Diol” column and gradient elution to separate in a single run, estrogens ranging from 2-methoxy estrone. one of the least polar C 18 steroids, to estriol, one of the most polar. Simpler, isocratic conditions, are provided for the separation of estrogens of similar polarity. A semipreparative column of similar composition was used for the purification of samples containing 25 to 50 mg of individual steroids.

Comparative Studies on Tree Pollen Allergens

The aqueous extract of alder (<i>Alnus incana</i>) pollen was found to consist of a protein mixture amounting to 3% of the dry pollen weight. Separation by gel permeation chromatography on an AcA-54 column resulted in 6 fractions designated chronologically to their relative elution volumes (AI 1 to AI 6). The immunological reactivity of these fractions was demonstrated in both IgE and IgG test systems. Being both immunochemically and quantitatively dominant fraction AI 4 was selected for further purification by a semipreparative isoelectric focusing column. A well-resolved and apparently homogeneous component with a pI value of 4.78 was eluted from the IEF column. The homogeneity of this fraction was demonstrated by one sharp band in thin layer IEF, one single band in HVE, one distinct sharp peak in HPLC and a symmetrical precipitate in CIE. The molecular weight was estimated to 22,500 daltons. Fraction pI 4.78 was shown to be a major allergen of alder pollen, it elicited a 1.17 histamine wheal reaction in skin prick test at a concentration of 1 mg·ml<sup>––</sup><sup>1</sup> and showed a significantly high RAST inhibition in all of the reaginic sera used. Fraction pI 4.78 was found to be an adequate model allergen of alder pollen well suited for further immunochemical characterization.

A rapid screening procedure for 2-aminonaphthalene in natural, synthetic, and refined crudes

The mutagen, 2-aminonaphthalene, which is the dominant aromatic amine species in several fossil fuels, may be isolated readily by using a sequential high-performance liquid chromatographic procedure. An initial isolation is achieved on a semi-preparative scale aminosilane column and the final separation is completed with a nonpolar stationary phase column on an analytical scale with a pH-adjusted solvent. The use of fluorescence detection permits discrimination (at least 100-fold) against the nonmutagenic isomer, 1-aminonaphthalene. The overall recovery of 2-aminonaphthalene, as determined by scintillation counting of a radioactive 1-aminonaphthalene tracer, typically exceeds 50% and can reach 92% depending on both the sample matrix and the solubility of the isolate in acetonitrile. The minimum detectable quantity is 0.06 μg g -1 of crude sample.

Semi-Preparative High-Performance Liquid Chromatographic Separation of Potato Glycoalkaloids

Abstract A semi-preparative high-performance liquid chromatographic method has been developed to separate crude mixtures of the potato glycoalkaloids α-chaconine, α-solanine, commersonine and demissine. Milligram quantities of each substance can be obtained within an 8 hour period. A Zorbax semi-preparative NH2 column and a solvent system of tetrahydrofuran-water-acetonitrile (55:20:25) were employed for the separation. The flow rate was 1.0 ml/min. Glycoalkaloid separations were monitored using both refractive index and ultraviolet detection (215 nm). Further analyses of these glycoalkaloids were done using analytical high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC) to check compound purity and identity.

Immunonephelometric quantification of serum vitamin D-binding globulin

Rapid quantification of serum vitamin D-binding globulin (VDBG) is described. An immunonephelometric method was developed and compared with the conventional immunodiffusion method for VDBG. A correlation coefficient of 0.896 (P less than 0.001) was found with a regression line y = 0.965x + 0.011 (n = 23). The within-assay and day-to-day coefficient of variation of the immunonephelometric method was 6 and 11% at 0.150 g/l (low VDBG concentration), 5 and 7% at 0.230 g/l (normal), and 4 and 8% at 0.570 g/l (high). Levels of VDBG and total 25-hydroxyvitamin D (25(OH)D, 25(OH)D2 + 25(OH)D3) were analysed from 33 serum samples obtained in April-May 1980 from 14-month-old children undergoing vitamin D substitution. 25(OH)D was extracted from 0.1-0.5 ml serum followed by semipreparative silica column chromatography and final quantification by a competitive protein-binding method. The VDBG mean value was 0.308 +/- 0.068 g/l (SD) and 25(OH)D had a mean value of 121 +/- 70 nmol/l (SD). There was no significant correlation between the levels of VDBG and 25(OH)D. The results of this study in general agreed well with other findings.

Liquid chromatographic determination of benzo[a]pyrene in natural, synthetic, and refined crudes

Benzo(a)pyrene (BaP) is isolated and quantitated by using a sequential high-pressure liquid chromatographic (HPLC) procedure. The sample is first injected onto a semipreparative HPLC column containing a bonded polar aminocyano (PAC) packing material, from which a BaP-enriched fraction is obtained. This isolate is then reinjected onto a Zorbax ODS reversed-phase analytical-scale column, and fluorescence detection is used to quantitate BaP. This procedure is applicable to samples with BaP concentrations ranging from 0.02 to 500 ..mu..g/g. The precision is nominally +- 6% (relative standard deviation), and the accuracy compares favorably with that displayed by more tedious methods. Recoveries, as determined by counting a radioactive BaP tracer, usually exceed 95%. Two samples may be processed per person-day.

Synthesis and biological properties of enzyme-resistant analogues of substance P.

Six analogues of substance P were synthesized with the aim of developing a metabolically stable peptide that would retain the biological activity of substance P. A recently isolated and characterized substance-P-degrading enzyme from human brain with a high specificity for substance P described in the preceding paper in this journal was used as a model for the enzymatic inactivation of substance P. The synthetic analogues were designed to protect the peptide bonds on the carboxyl side of residues 6, 7 and 8 of substance P, which represent the sites of cleavage by substance-P-degrading enzyme. To test for increased enzymatic resistance, the analogues were incubated with the enzyme, the digests were separated on a high-performance liquid chromatography reverse-phase column and the peptide fragments were collected and identified by amino acid analysis. Of the analogues described, an heptapeptide analogue of residues 5-11, less than Glu-Gln-Phe-MePhe-MeGly-Leu-MetNH2, showed almost complete resistance both towards substance-P-degrading enzyme and to degradation on exposure to rat hypothalamic slices. This analogue was about a third as potent as substance P in competing for binding to receptor sites for this peptide in rat brain membranes and a tenth as potent in eliciting contractions of the guinea pig ileum. The peptides were synthesized using the solid-phase technique with polydimethylacrylamide as a solid support and the coupling was achieved with pre-formed symmetrical anhydrides in dimethylacetamide. Fluorenylmethyloxycarbonyl was used as an alpha-amino protecting group in conjunction with t-butyloxycarbonyl as an epsilon-amino protecting group. Ammoniolytic cleavage from the resin was followed by stepwise elution from an SP-Sephadex column, deprotection with trifluoroacetic acid and chromatography on a Bio-Rex 70 ion-exchanger. The peptides were finally purified on a semi-preparative reverse-phase column.

High-performance liquid chromatographic separation and isolation of quinidine and quinine metabolites in rat urine

A procedure for the separation and isolation of the urinary metabolites of quinidine and quinine by reversed-phase high-performance liquid chromatography is described. Nine metabolites of quinidine and eight metabolites of quinine were detected in the urine of male Sprague-Dawley rats after a single dose of quinidine or quinine (50 mg kg −1). Following extraction from urine, the metabolites were separated on either an analytical or a semi-preparative reversed-phase column by gradient elution. After isolation and derivatization, the metabolites were analyzed by gas chromatography and gas chromatography—mass spectrometry.

The use of high performance liquid chromatography for the analysis and monitoring of iron carbonyl complexes in a reaction mixture

The suitability of high performance liquid chromatography (HPLC) as an analytical and preparative tool for the analysis and isolation of the products resulting from a reaction of diironnonacarbonyl [Fe 2(CO) 9] with di-t-butyl-sulfurdiimine [DBSD] was investigated. Reserved phase chromatography with octyl-modified silica as stationary phase and methanol/water mixtures as mobile phase showed a high selectivity for the separation of the products, and by use of gradient elution the separation of 17 products was completed within 30 min, which meant that the progress of the reaction could be monitored, A semi-preparative column (dimensions 250 X 10 mm) was useful in isolating some of the products; with this column 7 mg of reaction products could be injected without loss in efficiency.

High-performance liquid chromatographic separation of carbamazepine metabolites excreted in rat urine.

A procedure for the separation and isolation of the urinary metabolites of carbamazepine by reversed-phase high-performance liquid chromatography is described. After extraction from urine, the metabolites were separated on either an analytical or semi-preparative C18 mu Bondapak column by gradient elution with methanol-water-acetic acid. Following derivatization the metabolites isolated by the use of the semi-preparative column were analyzed by gas chromatography and gas chromatography-mass spectrometry.

Concentration and analysis of trace impurities in styrene monomer

Trace levels of impurities in styrene monomer are collected at the exit end of a semi-preparative gas chromatographic column by passing the effluent gas into a tube containing the porous polymer Tenax. The major component, styrene, is not permitted to enter the adsorbent. The concentrated impurities are then thermally desorbed and analyzed by capillary gas chromatography and mass spectrometry. Of the more than 100 peaks present, 60 have been identified.

Optimisation of liquid chromatographic performance on columns packed with microparticulate silicas

A modular packing system for packing analytical and semi-preparative scale columns has been constructed. Columns were packed with microparticulate silicas using different slurry media. Optimum results were obtained with carbon tetrachloride as the slurry medium using a packing pressure of 20.7 MPa (3000 p.s.i.g.) and a slurry concentration in the range 1–10% w/v. Various methods of sample introduction were investigated. Highest efficiencies were obtained using syringe injection, but comparable performance can be achieved with valve injection if a split-stream technique is used. Plate heights of between 2.5 and 3.5 particle diameters were attained with 11-, 6- and 3.7-μm particle size silicas in 0.10-m × 4.5-mm-I.D. columns. The use of wider-bore, 9.5-mm-I.D., columns gave plate heights of 2 particle diameters and up to 30 effective plates per second at a linear velocity of 1.5 mm/sec.