Development of High Pressure Liquid Chromatography (HPLC) for Fractionation and Fingerprinting of Petroporphyrin Mixtures

Abstract

A method is described which permits the analysis of fossil porphyrin mixtures by HPLC. This method involves the fractionation of the demetallated petroporphyrin mixtures on silica columns followed by rechromatography of the trapped fractions on ODS columns. This coupling of the two modes of HPLC provides a fast and effective method for the separation of isomeric porphyrins and for the petroporphyrin fingerprinting of geological (oil/shale) samples. Representative examples of analysis are discussed in terms of the potential applicability of the technique in the areas of structure elucidation and geochemical correlations.     

Preparative HPLC for the Facile Isolation of Drug Glucuronide Conjugates from Crude Extracts of Urine

Abstract

Reverse-phase preparative HPLC has been used to advantage for the isolation of crystalline quantities of drug glucuronide conjugates from crude urinary extracts. Following chronic administration of large doses of diazepam, levorphanol and hydroxyethylflurazepam to dogs, urine was collected and the water soluble drug conjugates adsorbed on a column of Amberlite XAD-2 resin. In each instance elution with methanol and solvent evaporation yielded a crude oil (approx. 3 g) which was chromatographed in one portion on either PrepPAK 500 C₁₈ (Waters) or Magnum 40 ODS-3 (Whatman) columns using aqueous methanol solvent systems. A greater than 90% purification was achieved in this single initial chromatographic step. Employing a combination of subsequent semi-preparative HPLC steps on either C₁₈ or silica gel columns, milligram quantities of the glucuronides of oxazepam, levorphanol and hydroxyethylflurazepam were isolated. The isolation procedures provide a general approach for obtaining milligram quantities of intact drug conjugates which may otherwise be difficult to obtain by chemical synthesis. Such conjugates can be used as authentic standards in the quantitation of certain drug metabolites in biological media during pharmacokinetic/biopharmaceutic studies.     

Rapid Sara Seprations by High Performance Liqid Chromatography

Abstract

This paper presents both rapid analytical and preparative high performance liquid chromatographic (HPLC) techniques for separating liquid-fuel type materials into saturates, aromatics, resins, and asphaltenes (SARA). The preparative method, an adaptation of a technique developed by Jewell, et. al. (1, 4), significantly decreases analysis time. The analytical technique utilizes HPLC to achieve the same separations in less time.     

Hydrocarbon Groups Type Analysis of Petroleum Products by HPLC on Specific Stationary Phases

Abstract

The hydrocarbon group types analysis of a large number of petroleum products by HPLC equipped with columns of suitable selectivity is described. An effective approach to the factors influencing the specificity of the columns was developped and stationary phases were synthetised in fonction of the products to be separated. All new phases were characterized by elemental, ²⁹Si and ¹³C NMR analyses. The potentialities of theses phases were illustrated by analysis of selected samples either of fundamental or of industrial interest.     

Preparative Liquid Chromatographic Method for the Characterization of Minor Constituents of Lubricating Base Oils

Abstract

In an effort to isolate, identify, and measure the properties of the active ingredients in a lubricating base oil, a high performance liquid chromatography (HPLC) separation scheme has been developed. The preparative mode of production is necessary to yield sufficient amounts of minor constituents for property measurements in terms of friction, wear, and oxidation characteristics.

In friction and wear control, the polarity of the molecular species is more important than the function groups in the species. Therefore the design of the separation scheme is based on the relative polarity of various functional groupings. Because the effort is directed towards identifying key components rather than analysis of the major compositions, mass recovery requirement is critical.     

Preparative Hydrophobic Interaction Chromatography of Proteins Using Ether Based Chemically Bonded Phases

Abstract

This paper examines the use of 15–20 micron wide-pore silica-based ether bonded phases for the preparative hydrophobic interaction chromatography of proteins. In particular, silyl ethers are immobilized on large particle silica in an analogous manner to previously developed ether bonded 5 um analytical supports. The preparative supports are reproducibly prepared and exhibit constant chromatographic retention for at least five months of continual use. Preparative columns can be operated for protein chromatography with peak shapes and capacity as predicted by the Snyder gradient elution model. Moreover, similar retention times are obtained relative to those on the 5 um analytical columns, enabling the direct transition and scale-up of separation. Gradient optimization is seen to directly parallel that performed on 5 um bonded ether analytical columns. Acceptable chromatographic resolution was obtained with sample capacity of >15 mg protein/ml column volume using a repetitive injection technique. A column clean-up strategy is examined for rapid and safe removal of contaminants. An illustrative example of use of the bonded ether preparative columns is made by application to soybean trypsin inhibitor purification. Initial results are presented on a column-switching method for the analytical monitoring of preparative separation.     

A High Performance Preparative Procedure for the Isolation of Degradation Products from D-Xylose

Abstract

An improved method for the preparative isolation of 3,8 dihydroxy-2-methylchromone, formed by degradation of D-xylose at 100°C is presented. From the ethyl acetate extractable part of the reaction mixture the chromone was isolated by preparative HPLC in less than one hour using a highly cross-linked dextran gel.     

Preparative Isolation of Destruxins from Metarhizium Anisopliae by High Performance Liquid Chromatography

Abstract

A method has been developed for preparative isolation of destruxins, a group of major insecticidal cyclodepsipeptides from culture broth of Metarhizium anisopliae. Prepurification of the crude extract by flash chromatography on silica gel followed by HPLC on reverse phase C18 Column using linear gradients of acetonitrile-water permitted the isolation of ten destruxins.     

Use of the Peak Shaving-Recycle Technique for Separation of Labdadiene and Labdatriene Isomers by HPLC

Abstract

Separation on a preparative scale of labdadiene and labdatriene isomers by extensive use of the peak shaving-recycle technique is described. The procedure can be used to effect rapid and high yield separations of closely related isomers with α ～1 using a commercial instrument (Prep LC/system 500) and commercially available columns.     

Determination of hydrocarbons in water by evanescent wave absorption spectroscopy in the near-infrared region

An home-made EFA (evanescent field absorbance)-sensor has been tested for the determination of hydrocarbons in water. The investigations have been performed both with crude oil emulsions and petrol solutions. Cuvette and evanescent wave spectra of crude oil and petrol in the near-infrared region are presented and discussed. The concentration of aromatic compounds in crude oil can be determined semiquantitatively by the standard addition method. The sorption behaviour of the hydrocarbons in the cladding of the fiberoptic sensor has been investigated and a correlation between the sensor signal and the concentration of the aqueous hydrocarbon emulsion/solution could be shown. The desorption of the enriched molecules after the measurements is also presented. The petrol molecules evaporate in ambient air so that the sensor is easily regenerated. In case of oil measurements the hydrocarbon molecules cannot be removed by rinsing the sensor with clear water or by evaporating them in ambient air. It has to be regenerated by washing it with a high volatile solvent instead.     

Reversed-Phase,Ion-Pair Separation of L-Methionine and L-Methionine Dipeptides Complexed with Palladium (II)

Abstract

A high performance liquid chromatographic (HPLC) method is described for separation of L-methionine and L- methionine dipeptides complexed with palladium (II). Under isocratic conditions, at room temperatures, with the appropriate selection of counter-ion (cetyltrimethylammonium or trioctylmethylammonium), it was possible by ion-pairing reversed phase chromatography to resolve the palladium (II) complexes studied. Stainless steel and polythylene columns were used. The chroma-tograms from both the two different materials made columns indicate about the same ratio of capacity factor of the palladium (II) complexes.     

A review of: “HPLC of Biological Compounds-A Laboratory Guide,by William S. Hancock & James T. Sparrow, (Volume 26 of the Chromatographic Science Series,Jack Cazes,Editor), Marcel Dekker,Inc., New York,NY, 1984, 361 pp., £39.75 (US)”.

Abstract

The analytical and preparative separation of the saturated, monoene, diene and triene constituents of cardanol and cardol have been achieved by reverse phase HPLC on a C₁₈ column using gradient elution.     

Analysis of Cell-Wall-Associated Enzyme Complexes by High Performance Liquid Chromatography

Abstract

The feasibility, efficiency and advantages of the HPLC method for the analysis of enzyme mixtures, associated with cell walls, is demonstrated with two different types of macroporous columns.     

Chemical Studies on Tobacco Smoke LXVII. Quantitative Determination of Alkaloids in Tobacco by Liquid Chromatography

Abstract

An HPLC method was developed for the quantitative determination of individual alkaloids of the basic fraction of tobacco extracts. Two reverse phase RP-18 columns were used, eluting with a gradient of aqueous triethylamine phosphate buffer (pH 7.56) and acetonitrile.

The method was optimized and compared favorably with existing quantitative techniques in regard to time factors, sensitivity and accuracy.     

High Performance Liquid Chromatongraphy of ABBOTT-53385 in Dog,Rat, and Human Plasma

Abstract

A simple, sensitive and reproducible high-performance liquid chromatographic (HPLC) method was developed to monitor plasma ABBOTT-53385 (I) levels in dogs, rats and humans. The samples were first supplemented with the internal standard, then extracted on Bond Elut® extraction columns. They were analyzed by reverse-phase HPLC with UV detection at 240 nm. The calibration curve was rectilinear over the range of 0.05–2.0 μg/ml and the interday variance was less than 4%. The limit of detection for this method was about 0.03 μg/ml.     

A Simple Liquid Chromatographic Method for Analysis and Isolation of the Unsaturated Components of Anacardic Acid

Abstract

Good analytical and preparative separation of the saturated, monoene, diene and triene components of anacardic acid have been achieved by reverse phase HPLC on a C₁₈ column by isocratic elution with methanol-aqueous acetic acid.     

Separation of Methylated Purines by High Pressure Liquid Chromatography

Abstract

This paper reports a method for the separation and measurement of methylated purines of interest to carcinogenesis studies by high-pressure liquid chromatography (HPLC) following their column chromatographic isolation from collected urine samples. HPLC was evaluated on three different cation-exchange columns, with optimum conditions obtained on a Partisil 10-SCX column employing isocratic elution with 0.25M (NH₄)H₂PO₄ at pH 4.0. This column was also found to be useful for the separation of mono-methylguanine isomers. Application is shown to the analysis of rat urine following animal treatment with methyl methanesulfonate.     

Rapid isolation of omega‐3 long‐chain polyunsaturated fatty acids using monolithic high performance liquid chromatography columns

Eicosapentaenoic and docosahexaenoic acids are important bio‐active fatty acids in fish oils. Monolithic HPLC columns both in the polymeric cation exchange (silver‐ion) and RP formats were compared with corresponding packed columns for the isolation of these acids from tuna oil ethyl esters. Monolithic columns in both formats enabled rapid (typically 5–10 min) separations compared with packed columns (30 min). Polymeric monolithic silver‐ion disc column rapidly furnished mixtures of eicosapentaenoic and docosahexaenoic esters (90% purity) within 5–10 min, but was unable to resolve individual esters. A preparative version of the same column (80 mL bed volume) enabled isolation (>88% purity) of 100 mg quantities of eicosapentaenoic and docosahexaenoic esters from esterified tuna oil within 6 min. Baseline separation of eicosapentaenoic and docosahexaenoic esters was achieved on all RP columns. The results show that there is potential to use polymeric monolithic cation exchange columns for scaled‐up preparation of eicosapentaenoic and docosahexaenoic ester concentrates from fish oils.     

Determination of Folic Acid in Commercial Diets by Anion-Exchange Solid-Phase Extraction and Subsequent Reversed-Phase HPLC

Abstract

Folic acid at the μ/g level is determined in total nutritional diets by concentration on disposable commercial anion exchange columns followed by elution with a concentrated salt solution and subsequent reversed-phase HPLC with absorbance detection at 365 nm. The method is specific for folic acid with respect to some known degradants and electrochemically generated oxidation products.     

Applications of Fast High Performance Liquid Chromatography in Pharmaceutical Analysis

Abstract

Fast HPLC offers a definite time advantage for analyses such as content uniformity which requires, in general, a routine analysis of as many as 30 samples and for the analysis of dosage forms of developmental drugs. The columns for Fast HPLC can be used with a minimal amount of modification to conventional HPLC hardware. The resolution obtainable with a Fast HPLC column is poorer than that of a conventional column. (All references to conventional columns in the article imply, unless otherwise indicated, those columns 15-30 cm in length and 3.9-4.6 mm i.d.) Attempts to modify a mobile phase to improve the resolution of a closely eluting peak resulted in retention times not too different from typical conventional columns. Although microbore columns offered improved resolution and a savings in both solvent and sample consumption, the utility of these columns is limited by the need for specialized hardware. Since the sample volume is not a limiting factor in a typical pharmaceutical analysis and overall cost savings from lesser solvent consumption are not significant in a majority of cases as solvent can be recycled, it is concluded that the Fast HPLC columns can play a more important role than the currently available microbore columns in laboratories engaged in pharmaceutical analysis, particularly at the product development stage.