Short Silica Gel Column Research Articles

D-arabino-hex-1-enitol from the inactive fraction of Acalypha wilkesiana var. lace-acalypha (Muell &Arg.)

Different herbal preparations of Acalypha.wilkesiana var. lace - acalypha (Muell & Arg.) are employed in traditional medicine for the treatment and management of disease conditions such as wounds, tumors, hypertension, inflammations, skin infections, gastroenteritis amongst many others. Ethyl 3, 4, 5-trihydroxybenzoate (ethyl gallate) and 1, 2, 3-benzenetriol (pyrogallol) have previously been isolated from the active fractions of this plant. However, this present study was done to isolate compound(s) from one of the inactive fractions. Hence, a short silica-gel column chromatography of the inactive fraction (5A) furnished a compound designated as 3 [ R f 0.15; [ n ] 20 D 1.0300 ]. The structure of 3 has been established to be D-arabino-hex-1-enitol-1, 5-anhydro-2-deoxy (1, 5-anhydro-2-deoxy-D-enopyranose-arabino-hex-1-enitol) by a combination of 1 H NMR, 13 C NMR, MS and IR spectral techniques. Compound 3 recorded no antibacterial activity against B. subtilis , S. aureus and Ps. aeriginosa . However, it demonstrated very weak antibacterial activities against E. coli and S. typhi. which were slightly better than the activity furnished by 5A. Furthermore, it was observed that 3 was inactive against C. albicans . Surprisingly, the crude extract and butanol fraction generally demonstrated comparably stronger antimicrobial activities than 3 implying that the purification of the crude extract did not improve the activity demonstrated by 3 .

Synthesis of Dideoxymycobactin Antigens Presented by CD1a Reveals T Cell Fine Specificity for Natural Lipopeptide Structures

Mycobacterium tuberculosis survival in cells requires mycobactin siderophores. Recently, the search for lipid antigens presented by the CD1a antigen-presenting protein led to the discovery of a mycobactin-like compound, dideoxymycobactin (DDM). Here we synthesize DDMs using solution phase and solid phase peptide synthesis chemistry. Comparison of synthetic standards to natural mycobacterial mycobactins by nuclear magnetic resonance and mass spectrometry allowed identification of an unexpected alpha-methyl serine unit in natural DDM. This finding further distinguishes these pre-siderophores as foreign compounds distinct from conventional peptides, and we provide evidence that this chemical variation influences the T cell response. One synthetic DDM recapitulated natural structures and potently stimulated T cells, making it suitable for patient studies of CD1a in infectious disease. DDM analogs differing in the stereochemistry of their butyrate or oxazoline moieties were not recognized by human T cells. Therefore, we conclude that T cells show precise specificity for both arms of the peptide, which are predicted to lie at the CD1a-T cell receptor interface.

A new method of histamine colorimetry using 2,3-naphthalenedicarboxaldehyde on a silica–gel column cartridge

In our work, we found, by chance, that 2,3-naphthalenedicarboxaldehyde (NDA) acts as a color development reagent to histamine (HA) in the absence of a reductant, e.g., cyanide anion, even in a weakly acidic environment. The NDA-induced color development reaction to HA shows the maximum absorption of 552 nm at pH 6 after a minimal interval of 10 min. By using this chemical property, a simple and convenient new method of visual colorimetry of HA on a short silica–gel column cartridge (called an “HA cartridge” in this study, which consisted of 50 mg of silica–gel packed into a 1.0 ml disposable syringe) was developed. The new visual method involves extraction of a 5 g sample with 5% trichloroacetic acid solution (TCA, 35 ml), followed by neutralizing with 1 M sodium hydroxide. After loading the TCA extract (500 μl) into the HA cartridge, the cartridge was then washed with a 0.1 M phosphate buffer, pH 6 (200 μl) and water (200 μl × 3 times). When a solution of 1 mM NDA in acetonitrile (200 μl) was passed through the HA cartridge, the color inside the cartridge was observed to changed at 3 min after the NDA loading from white to indigo-blue, for concentrations of HA ranging from 25 to 1000 mg kg −1. This sequence requires only 5 min to perform.

Regioselective oxidative coupling of 4-hydroxystilbenes: synthesis of resveratrol and epsilon-viniferin (E)-dehydrodimers.

Treatment of 5-[2-(4-hydroxyphenyl)vinyl]benzene-1,3-diol (resveratrol) with an equimolar amount of silver(I) acetate in dry MeOH at 50 degrees C for 1 h followed by chromatographic purification with a short silica gel column allowed the isolation of its (E)-dehydrodimer, 5-[5-[2-(3,5-dihydroxyphenyl)vinyl]-2-(4-hydroxyphenyl)-2,3-dihydrobenzofuran-3-yl]benzene-1,3-diol, as a racemic mixture in high yield. The present method was applicable to the oxidative dimerization of 4-hydroxystilbenes such as trans-styrylphenol and 5-[6-hydroxy-2-(4-hydroxyphenyl)-4-[2-(4-hydroxyphenyl)vinyl]-2,3-dihydrobenzofuran-3-yl]benzene-1,3-diol (epsilon-viniferin) leading to the corresponding 2-(4-hydroxyphenyl)-2,3-dihydrobenzofurans possessing various types of biological activities.

Phase-transfer-catalyzed alkylation of guanidines by alkyl halides under biphasic conditions: a convenient protocol for the synthesis of highly functionalized guanidines.

An operationally straightforward and efficient method for the alkylation of carbamate-protected guanidines with various alkyl halides and mesylates is described. This protocol proceeds via deprotonation of the acidic N-carbamate hydrogen of the guanidine under biphasic conditions using a catalytic amount of a tetrabutylammonium salt as a phase-transfer catalyst. In this manner, highly functionalized guanidines can be obtained. The reaction is tolerant of a wide range of functional groups on both the alkyl halide and guanidine component. In addition, the reaction is sufficiently mild such that simple aqueous workup and filtration through a short silica gel column yields the substituted guanidines in high purity. In conjunction with the EDCI-mediated guanylation of disubstituted thioureas with amines, phase-transfer catalyzed alkylation of guanidines via a one-pot, three-component synthesis of substituted guanidines was achieved.

A new analytical procedure to differentiate virgin or non‐refined from refined vegetable fats and oils

The effects of individual steps of industrial refining on the alteration of triacylglycerides (TAG) are reported. The level of dimer triglycerides, normally not present in crude oils, increased after each refining step, especially after steam-washing and desodorisation. A good correlation between the applied temperatures and dimer triglycerides content was found. The forming of dimer triglycerides starts at 90 °C and increases corresponding to the extension of thermal treatment like normal heating or desodorisation. The data for various types of vegetable oils demonstrate that there is no clear-cut different tendency to form dimers. Heated oils with different contents of linoleic acid produced nearly the same amount of dimers. Other criteria, like the determination of trans fatty acids, steradienes, or the UV-absorption, were found not to be appropriate to detect a thermal treatment at temperatures below 150—170 °C. The formation of steradienes mainly depends on the total sterol contents, the percentage of added bleaching earth, and its acidity and moisture. Over 160 commercial vegetable oil samples were analysed to obtain a data range on the content of dimerised triglycerides. Mostly, vegetable oils Iabelled as non-refined (which may be steam-washed) did not exceed dimer contents of 0.1%. Virgin vegetable oils did not contain dimers (< 0.05%). The content of dimer triglycerides in vegetable oils was determined by a new method via clean-up on a short silica gel column, followed by size-exclusion HPLC with refractive index detection.

A new analytical procedure to differentiate virgin or non-refined from refined vegetable fats and oils

The effects of individual steps of industrial refining on the alteration of triacylglycerides (TAG) are reported. The level of dimer triglycerides, normally not present in crude oils, increased after each refining step, especially after steam-washing and desodorisation. A good correlation between the applied temperatures and dimer triglycerides content was found. The forming of dimer triglycerides starts at 90 °C and increases corresponding to the extension of thermal treatment like normal heating or desodorisation. The data for various types of vegetable oils demonstrate that there is no clear-cut different tendency to form dimers. Heated oils with different contents of linoleic acid produced nearly the same amount of dimers. Other criteria, like the determination of trans fatty acids, steradienes, or the UV-absorption, were found not to be appropriate to detect a thermal treatment at temperatures below 150—170 °C. The formation of steradienes mainly depends on the total sterol contents, the percentage of added bleaching earth, and its acidity and moisture. Over 160 commercial vegetable oil samples were analysed to obtain a data range on the content of dimerised triglycerides. Mostly, vegetable oils Iabelled as non-refined (which may be steam-washed) did not exceed dimer contents of 0.1%. Virgin vegetable oils did not contain dimers (< 0.05%). The content of dimer triglycerides in vegetable oils was determined by a new method via clean-up on a short silica gel column, followed by size-exclusion HPLC with refractive index detection.

The use of furans in natural product syntheses

Abstract

Analysis of stereoisomeric C 27-bile acids by high performance liquid chromatography with fluorescence detection

A method for differentially measuring the 24-hydroxylated stereoisomeric intermediates (3α,7α,12α,24-tetrahydroxy- and 3α,7α,24-trihydroxy-5β-cholestan-26-oic acids) and related C 27-bile acids in β-oxidation of bile acid biosynthesis has been developed by high performance liquid chromatography with fluorescence detection. The method involved the derivatization of the above intermediable C 27-bile acids into fluorescent esters with 3-(4-bromomethylphenyl)-7-diethylaminocoumarin, a newly synthesized labeling reagent for carboxylic acids. The fluorescent derivatives were subjected to a short silica gel column to eliminate interfering products prior to analysis by high performance liquid chromatography. The separation of the 16 kinds of bile acids containing stereoisomers was carried out using a reversed-phase Inertsil C8-column by gradient elution and detected with a fluorometer (Ex. 400 nm, Em. 475 nm). The linearity of calibration curve for each bile acid was from 0.5 to 250 pmol ( r = 0.999) and the detection limits were about 15 fmol at a signal-to-noise ratio of 3. The method was applied to the determination of intermediates in β-oxidation of bile acid biosynthesis using rat liver homogenate. The results showed that two stereoisomers of 24-hydroxylated C 27-bile acids were predominantly produced, indicating the formation of the isomers by the cis-hydration with water.

Alteration of steryl ester content and positional distribution of fatty acids in triacylglycerols by chemical and enzymatic interesterification of plant oils

AbstractSteryl ester content of refined and interesterified corn, soybean, and rapeseed oils has been measured via clean‐up on a short silica gel column, followed by high performance liquid chromatography with evaporative light‐scattering mass detector. Chemical interesterification, catalyzed by sodium methoxide, led to random positional distribution of fatty acids in triacylglycerols and some increase in the steryl ester content of all three oils. Enzymatic interesterification, catalyzed by the immobilized lipase from Rhizomucor miehei (Lipozyme), resulted in a distinct reduction in steryl ester content, but essentially no alteration in positional distribution of fatty acids in triacylglycerols occurred. Formation of steryl esters during chemical and enzymatic interesterification was also examined by radioactive tracer technique with [4‐14C]β‐sitosterol added as marker to refined rapeseed oil and measurement of the radioactive steryl esters formed. Chemical interesterification of rapeseed oil resulted in moderate formation (10% of total radioactivity) of radioactive β‐sitosteryl esters. Enzymatic interesterification of the oil, catalyzed by Lipozyme, led to little formation of radioactive β‐sitosteryl esters, whereas with the lipase from Candida cylindracea high proportions (&gt;90% of total radioactivity) of 14C‐labeled β‐sitosteryl esters were formed.

Enantioselectivity of Azalanstat and Its Ketal Tosylate Intermediate in Chiral High Performance Liquid Chromatography Separations

Abstract Separation of the stereoisomers of azalanstat (RS-21607-197), a substituted imidazolyl-1,3-dioxolane which acts as a potent inhibitor of lanosterol 14α-demethylase in cholesterol biosynthesis, and its ketal tosylate intermediate was achieved by chiral high performance liquid chromatography. Resolution of all four stereoisomers of azalanstat was accomplished by normal phase separation of the diastereomers on a short silica gel column coupled on-line to a Chiralpak AS amylose carbamate phase chiral column for resolution of the enantiomers. A reversal in elution order of the cis-(2S,4S) and cis-(2R,4R) enantiomers of the ketal tosylate intermediate of azalanstat was exhibited by an Ultron ES-OVM ovomucoid protein bonded-phase column with a change in organic modifier from ethanol to acetonitrile. This unusual enantioselectivity in the chiral reverse phase separation of the ketal tosylate was attributed to a putative change in binding domains or recognition sites on the ovomucoid protein as a function of the organic modifier of the mobile phase. Resolution of all four stereoisomers of the ketal tosylate was achieved by use of a Chiralpak AD amylose carbamate phase column in the normal-phase mode.

Minor constituents of vegetable oils during industrial processing

AbstractWe report the effects of individual steps of industrial refining, carried out in Brazil, on the alteration of selected minor constituents of oils, such as corn, soybean, and rapeseed oils. Total sterols, determined by capillary gas chromatography (GC), decreased by 18–36% in the fully refined oils, compared with the crude oils. The total steradienes, dehydration products of sterols, were determinedvia a simple clean‐up on a short silica gel column, followed by high‐performance liquid chromatography (HPLC) with ultraviolet detection. The level of steradienes, normally not present in crude oils, increased after each refining step, especially after deodorization. Thus, the content of steradienes increased after deodorization by about 15‐ to 20‐fold in corn and soybean oils, and by about 2‐fold in rapeseed oil. The total steryl esters were also determinedvia clean‐up on a short silica gel column, followed by HPLC with evaporative light scattering mass detection. A minor decrease in the level of steryl esters was observed after complete refining. The individual tocopherols and tocotrienols were determined by HPLC with a fluorescence detector. The level of total tocopherols and tocotrienols decreased by about 2‐fold after complete refining of corn oil and by about 1.5‐fold in soybean and rapeseed oils. In all three cases, maximum reduction of tocopherols was observed after the deodorization step. The level of polymeric glycerides, determinedvia clean‐up on a short silica gel column followed by size‐exclusion HPLC, increased to some extent (0.4–1%) during refining. The level oftrans fatty acids, determined by capillary GC, also increased to a substantial extent (1–4%) after refining.

Fluorimetric determination of diarrhetic shellfish toxins in scallops and mussels by high-performance liquid chromatography

The fluorimetric determination of okadaic acid (OA) and dinophysistoxin-1 (DTX-1), the principal toxins of diarrhetic poisoning, is reported. The digestive glands of mussels or scallops were homogenized with 2-propanol. OA and DTX-1 were extracted from the homogenate, with hexane-ethyl acetate and labelled with 2,3-(anthracenedicarboximido)ethyl trifluoromethanesulfonate in dry acetonitrile. After cleaning up by passage through a short silica gel column, the fluorescent derivatives were determined by HPLC. The derivatives were at first separated on a Develosil Ph-5 column, and only the target fraction obtained was introduced into a Develosil ODS K-5 column by a valve-switching device. Both toxins were determined in the range 2.5–500 pg, and the detection limits were 0.8 pg (OA) and 1.3 pg (DTX-1) with a signal-to-noise ratio of 3.

Formulation, in vitro dissolution, and ocular bioavailability of high- and low-melting phenylephrine oxazolidines.

The in vitro dissolution and the relative ocular bioavailability of high- and low-melting phenylephrine oxazolidines (HMP and LMP) from a nonaqueous suspension (silicone fluid) were compared. Stability-indicating HPLC assays were developed for evaluation of the prototype formulations, in which a normal-phase HPLC method was necessary for analysis of PO, while a reverse-phase HPLC method was required for analysis of the primary degradation product, phenylephrine (PE), following its separation from the formulation using a short silica gel column. PO was formulated as an ophthalmic suspension in silicone fluid (20 cs) because of its property of undergoing rapid hydrolysis in aqueous media. An experimental test system for measuring the dissolution characteristics of a water-immiscible multiparticulate suspension was designed to obtain the dissolution profiles of suspensions of HMP and LMP. The dissolution rates, which were nearly identical for LMP and HMP, were obtained assuming a quasi-infinite reservoir. A reverse-phase HPLC assay with fluorescence detection was used for measuring the concentrations of PE in aqueous humor and corneal samples. Statistical analysis of the bioavailability data showed that suspensions containing HMP and LMP were equal in extent of absorption following a single topical application to the rabbit eye. The results correlated well with the in vitro dissolution rates of the suspensions of HMP and LMP.

Determination of Ammonium and Other Major Cations in River and Rain Water by Ion Chromatography Using Silica Gel as an Ion Exchanger

The separation of NH4+ from Na+, K+, Mg2+ and Ca2+ on silica gel columns was investigated using natural water samples and lithium oxalate as a retention modifier. The pore size of silica gels was found to be the most important factor affecting the retention of the cations. When a 0.0025 mol dm−3 lithium oxalate solution of pH 7 was used as the mobile phase these cations could be completely separated on a short silica-gel column (Develosil 30-3, 35 mm×4.0 mm i.d.) within about 20min. NH4+ contents in river (0.01–0.27 ppm) and rain (1–1.8 ppm) water samples were determined successfully.

Determination of total hydrocarbons in sea water at the microgram level with a flow calorimeter

One litre of water is extracted with 10 ml of 1,2,2-trichlorotrifluoroethane and the extract is then concentrated to 100 μl. A 50-μl volume is injected into a high-performance liquid chromatographic system with a flow calorimeter that measures the absorbance of hydrocarbonds on porous glass beads. A short silica gel column in the system removes non-hydrocarbon material. A chromatogram is obtained within 3 min and only one peak has to be evaluated. The minimum detectable amount is 4.0±3.4 μg/l. These values can be improved by extracting larger volumes. The equipment used is fairly inexpensive and can readily be taken to sea.

The use of silica gel for the separation of traces of uranium

A simple chromatographic procedure for the separation of trace amounts of uranium has been developed. From the solution containing EDTA and tartaric acid at pH 5, uranyl ions are selectively retained on a short silica gel column and are later eluted with 3 M acetic acid. The uranium content is measured spectrophotometrically with arsenazo III directly in the eluate without further pH adjustment. The effects of aluminium and thorium are discussed in detail. The method is applied to the determination of microgram amounts of uranium in rocks and underground waters.

Mercuration of Diazomethane

As a possible route to bis-oxymethylmercury acetate, Hg(CH2CO2CH3)2, we have examined the reaction of diazomethane with mercuric acetate. Although a reaction has been stated to occur1 no details of the products were reported. The reaction in ether was slow and heterogeneous. There was no evidence of methylenation of the mercury–oxygen bond, as apparently occurs with arylmercury benzoates2, nor of reduction to mercurous acetate. Instead, when diazomethane (0.03 M) was slurried in ether with dry mercuric acetate (0.015 M), methyl acetate was formed (50 per cent after one day, 90 per cent after five days), together with an insoluble and dangerously explosive pale yellow solid (90 per cent), and small amounts of a soluble yellow oil. This oil, obtained by evaporation of the filtrate and separation from polymethylene on a short silica-gel column, is apparently bisdiazomethylmercury, Hg(CHN2), analogous to the long known bis(carboxyethyldiazomethyl)mercury3, Hg(CN2CO2Et)2, although the yield (5 per cent) is much lower. It showed a strong localized absorption at 2,105 cm−1, indicating a diazoalkane grouping, and evolved nitrogen with acids. Bromine decolorized it, giving mercuric bromide, nitrogen and some bromoform.