Chromatography and Biological Stains: II. Column Chromatographic Separation of the Colored Materials in Commercial Sudan III

Commercial Sudan III was separated into various fractions by the usual column chromatographic technics using Celite-silicic acid as the adsorbent and commercial petroleum ether (B) as the solvent. By analysis, using both spectrophotometric and paper strip chromatographic methods, it was possible to identify fractions from the commercial sample, including several fractions which were indubitably very highly purified Sudan III. In addition several other fractions containing mixtures of colored components have been isolated.     

A Simple Assay Procedure for Mixtures of Hematoxylin and Hematein

A simple and rapid method for the simultaneous quantitative analysis of mixtures of hematoxylin and hematein uses the molar extinction coefficients of the pure substances calculated by Lalor and Martin (1959). Absorbance measurements of the samples dissolved in methanol are made at wavelengths of 292 nm and 445 nm, the wavelengths of maximum absorption of hematoxylin and hematein respectively. The hematoxylin absorbance at 292 nm is corrected for the presence of hematein.

Using this method it was found that of 12 commercial samples labelled “hematoxylin” all contained at least 90% of the compound. Hematein contents of these samples fell in the range 0.1% to 6.8%. In 9 commercial samples labelled “hematein” the hematein contents fell in the range 1.2% to 90.7%. The hematoxylin contents of these samples fell in the range of 1.0% to 82.7%.

This paper describes also a chromatographic method for the identification of hematein and its oxidation products.     

High-performance liquid chromatographic determination of creatinine in serum, and a correlation of the results with those of the Jaffé and enzymic methods

A high-performance liquid chromatographic method for the determination of creatinine in control sera is reported, based on its separation from deproteinated serum components on the ion-exchange material HEMA Bio 1000 SB and ultraviolet detection at 230 nm. Groups of eleven to fourteen samples of human serum and several control materials were simultaneously analysed by the Jaffé, enzymic ultraviolet and enzymic peroxidase aminophenazone methods. Another group (52–115 sera) was analysed for correlations with spectrophotometric methods. The precision of the chromatographic method ranges between 2.0 and 1.0% (relative standard deviation) for serum creatinine concentrations of 115.1 to 471 μmol/l, respectively. A very good accuracy was found in analyses of reference materials Kontrollogen-L and -LP. Some results of analyses of the other control sera were higher and the other lower than those obtained by the Jaffé and enzymic methods, because both interferences and enzyme inhibitors were encountered. Correlations between the chromatographic and spectrophotometric methods were good.     

Monitoring of ethanol during fermentation of a lignocellulose hydrolysate by on-line microdialysis sampling, column liquid chromatography, and an alcohol biosensor

During a 70-h fermentation of a lignocellulose hydrolysate, the ethanol produced was monitored on-line using a microdialysis probe as an in situ sampling device. The dialysate components were then separated in a column liquid chromatographic system and the ethanol was selectively detected by an amperometric alcohol biosensor. The result was compared with two off-line analysis methods: one chromatographic method with refractive index (RI) detection and one enzymatic method based on spectrophotometric detection. The two methods base on enzymes were shown to give lower values than the chromatographic method based on RI detection, which is discussed n terms of selectivity. The investigated on-line setup was found to be a flexible system for monitoring of fermentations, allowing a sampling frequency of at least 12 h(-1) and with a delay between sampling and detection of less than 5 min. (c) 1994 John Wiley & Sons, Inc.     

Development and validation of RP-HPLC and ultraviolet spectrophotometric methods of analysis for the quantitative estimation of antiretroviral drugs in pharmaceutical dosage forms

A high-performance liquid chromatographic and an UV spectrophotometric method were developed and validated for the quantitative determination of three antiretroviral drugs viz. Lamivudine, Stavudine and Nevirapine that constitute one of the first line regimens in antiretroviral therapy. The different analytical performance parameters such as linearity, precision, accuracy, specificity, limit of detection (LOD) and limit of quantification (LOQ) were determined according to International Conference on Harmonization ICH Q2B guidelines. Chromatography was carried out by isocratic technique on a reversed-phase C-18 SYMMETRY column with mobile phase based and optimized depending on the polarity of the molecules. The UV spectrophotometric determinations were performed at 270, 265 and 313 nm for Lamivudine, Stavudine and Nevirapine, respectively. The linearity of the calibration curves for each analyte in the desired concentration range is good (r(2)>0.999) by both the HPLC and UV methods. Both the methods were accurate and precise with recoveries in the range of 97 and 103% for all the three drugs and relative standard deviation (R.S.D.) <5%. Moreover, the accuracy and precision obtained with HPLC correlated well with the UV method which implied that UV spectroscopy can be a cheap, reliable and less time consuming alternative for chromatographic analysis. The proposed methods are highly sensitive, precise and accurate and hence were successfully applied for the reliable quantification of API content in the commercial formulations of Lamivudine, Stavudine and Nevirapine.     

Purification and analysis of the major components of chum salmon protamine contained in insulin formulations using high-performance liquid chromatography

A simple high-performance liquid chromatographic method has been developed for the rapid purification and analysis of protamine components contained in insulin formulations. Only a single step is needed to separate peptides whose compositions, sizes, and unusual isoelectric points (pI 13.8) are nearly identical. The method involves their isocratic separation on a reversed-phase column using a pH 2 phosphate buffer and a low acetonitrile content as an eluant. The purified chum salmon components were analyzed by amino acid analysis, solid-phase amino acid sequencing, carboxypeptidase B digests, insulin complexation analysis, and a mass spectrophotometric procedure which gives an accurate mass of the intact peptides. This HPLC purification technique may also be applicable to protamines and other highly basic peptides isolated from other sources.     

Influence of sex, slaughter weight and carcass weight on “non-carcass” and carcass quality in segureña lambs

The effects of sex, slaughter weight and carcass weight on carcass characteristics and meat quality traits were evaluated using 100 Segureña lambs. The management of all lambs was similar prior to slaughter at 19–25 kg. Slaughtered animals with a hot carcass weight below 20 kg were assigned to class B, and those greater than 22 kg to class C. Carcass weight had a significant influence on “non-carcass” components, dressing percentage, subjective carcass conformation, fat deposits, carcass fatness, bone and most carcass measurements. Sex had a significant effect on age at slaughter, “non-carcass” components, rib measurements, dressing percentage, fat deposits, and neck and shoulder percentage. As the weight increased, the carcass measurements also increased. Concurrently, while improving the conformation indices of the carcass, leg and dressing percentages, neither the commercial cuts of the animal nor tissue composition was significantly affected. Sex primarily affected the quantity of all types of fat deposits.     

A coupled optical assay for adenine phosphoribosyltransferase and its extension for the spectrophotometric and radioenzymatic determination of 5-phosphoribosyl-1-pyrophosphate in mixtures and in tissue extracts

A reliable assay was developed to characterize crude cell homogenates with regard to their adenine phosphoribosyltransferase activities. The 5-phosphoribosyl-1-pyrophosphate (PRPP)-dependent formation of AMP from adenine is followed spectrophotometrically at 265 nm by coupling it with the following two-stage enzymatic conversion: AMP + H2O----adenosine + Pi (5'-nucleotidase); adenosine + H2O----inosine + NH3 (adenosine deaminase). The same principle was applied to develop a spectrophotometric and a radioenzymatic assay for PRPP. The basis of the spectrophotometric assay is the absorbance change at 265 nm associated with the enzymatic conversion of PRPP into inosine, catalyzed by the sequential action of partially purified adenine phosphoribosyltransferase, commercial 5'-nucleotidase, and commercial adenosine deaminase, in the presence of excess adenine. In the radiochemical assay PRPP is quantitatively converted into [14C]inosine via the same combined reaction. Tissue extracts are incubated with excess [14C]adenine. The radioactivity of inosine, separated by a thin-layer chromatographic system, is a measure of PRPP present in tissue extracts. The radioenzymatic assay is at least as sensitive as other methods based on the use of adenine phosphoribosyltransferase. However, it overcomes the reversibility of the reaction and the need to use transferase preparations free of any phosphatase and adenosine deaminase activities.     

Flow injection analysis system for the supervision of industrial chromatographic downstream processing in biotechnology

Sugar beet molasses is a natural resource for various products used in daily life, ranging from sucrose to amino acids for pharmaceutical industry. The separation of molasses into these high value components is performed on a large scale by ion exchange/exclusion chromatography. A biosensor system was set up for the “in time” analysis of serine and sucrose during molasses desugarisation. -Serine was analysed with the multi-enzyme system -serine dehydratase/lactic dehydrogenase and photometric detection of the NADH consumed. Sucrose was determined with invertase/mutarotase/glucose oxidase and the oxygen consumed was monitored amperometrically. An analysis could be performed within 2–5 min by directly injecting samples from the chromatographic process into the flow injection analysis system. The determination range for the sucrose analysis was 0–2.5 gl⁻¹ and for the analysis of -serine 0–0.5 gl⁻¹. The standard deviation for the measurement of -serine was 1.7%.     

Analysis of enzymatic steroid conversions by high pressure liquid chromatography.

Enzyme catalyzed introduction of the 1–2 double bond into a steroid can be monitored through spectrophotometric changes accompanying electron acceptor reduction or through paper or thin-layer chromatographic analysis of the reaction product. The spectrophotometric method is not applicable to cases in which the oxidized form of the electron acceptor is continually regenerated. In studying such cases, we have found high pressure liquid chromatography (HPLC) to be a method of direct analysis more convenient than paper chromatography or tlc. Use of a water based eluant and a reverse phase column for the HPLC analysis allows direct injection of a sample of the aqueous reaction solution after acidification, and no extraction with an organic solvent is necessary.     

Lipid composition of mitochondria from bovine heart, liver, and kidney

Highly purified preparations of mitochondria from bovine heart, liver, and kidney were isolated and characterized by electron microscopy, oxidative phosphorylation ability, cytochrome c reductase activity, and cytochrome content. Components of lipid extracts of the preparations were determined by thin-layer chromatography, diethylaminoethyl-cellulose column chromatography, and spectrophotometric procedures. The major phospholipids were identified by their chromatographic behavior, IR spectrometry, and paper chromatography of their hydrolysis products. The lipid content of the mitochondria paralleled that of the components of the electron transfer chain, heart mitochondria being richest and liver mitochondria poorest in lipid. Heart mitochondria contain equal concentrations of coenzyme Q and cholesterol (1%); the highest cholesterol content (4.7%) was found in mitochondria from kidney. The phospholipids of mitochondria from the three organs were qualitatively and quantitatively very similar. The major polar lipid components (cardiolipin, choline glycerophosphatides, and ethanolamine glycerophosphatides) were present in a molar ratio of 1:4:4. It is suggested that mitochondria from different sources contain characteristic lipids, mainly phospholipids, of which cardiolipin is particularly diagnostic of the source of the mitochondria.     

Separation and characterization of cardiolipin molecular species by reverse-phase ion pair high-performance liquid chromatography-mass spectrometry

An improved high-performance liquid chromatography-mass spectrometry method for the separation and characterization of cardiolipin molecular species is presented. Reverse-phase ion pair chromatography with acidified triethylamine resulted in increased chromatographic retention and resolution when compared with chromatography without acidified triethylamine. Using a hybrid triple quadrupole linear ion trap mass spectrometer to generate MS/MS spectra revealed three regions within each spectrum that could be used to deduce the structure of the cardiolipin molecular species: the diacylglycerol phosphate region, the monoacylglycerol phosphate region, and the fatty acid region. Cardiolipin standards of known composition were analyzed and exhibited expected chromatographic and mass spectral results. Two minor components in commercial bovine heart cardiolipin, (with the same molecular weight but different chromatographic retention times), were shown to differ by fatty acid composition: (C18:2)₂(C18:1)₂ versus (C18:2)₃(C18:0)₁. These compounds were then analyzed by HPLC-MS³ to examine specific diac ylglycerol phosphate generated fatty acid fragmentation. Also, two commercial sources of bovine heart cardiolipin were shown to have minor differences in cardiolipin species content. Cardiolipin isolated from rat liver, mouse heart, and dog heart mitochondria were then characterized and the relative distributions of the major cardiolipin species were determined.     

Pigment Diversity in Freshwater Phytoplankton. I. A Comparison of Spectrophotometric and Paper Chromatographic Methods

Spectrophotometric and paper chromatographic analyses of the pigments in the phytoplankton were made from early spring till the end of summer in two small Dutch freshwater lakes. It was found that pigment diversity cannot be adequately estimated by MARGALEF'S pigment ratio nor by polychromatic spectrophotometric methods. The pigments detected with the paper chromatographic method were: chlorophyll-a, chlorophyll-b, chlorophyll-c, phaeophytin-a (traces), phaeophorbide-a, Mg-containing chlorophyll-derivatives, carotene, lutein, violaxanthin, neoxanthin (traces), fucoxanthin, diadinoxanthin, diatoxanthin (traces), peridinin and keto-carotenoids (traces). It is suggested to distinguish between a richness-component and an evenness-component of pigment diversity.     

Monitoring of the glucose concentration during microbial fermentation using a novel mass-producible biosensor suitable for on-line use

A flow-injection analysis system was combined with a mass producible, disposable biosensor and was used to monitor glucose concentrations during several microbial fermentations. The biosensor was manufactured using thick film, screen printing technology. Unlike previous devices of this type, the biosensor was designed to make multiple measurements over extended periods rather than to operate as a “one-shot” sensor. One yeast, one lactic acid, and three E. coli bioreactor cultures were tested using either defined or complex media. Results from the sensor were compared with a standard spectrophotometric test kit. In samples containing glucose concentrations within the range of the biosensor and the test kit, good correlations were obtained between the two methods. In addition to glucose, microbial growth and pH were recorded.     

Human plasma lipofuscin melanins formed from tryptophan metabolites

Incubation of human plasma with the tryptophan metabolites 3-hydroxy-DL-kynurenine (3HK) or 3-hydroxyanthranilic acid (3HAA) yields soluble brown pigments with the fluorescence spectrophotometric and paper chromatographic properties of plasma lipofuscin (PL). An alternative to the recognized tyrosine pathway of melanogenesis is therefore demonstrated in human plasma.     

Quantification of carnitine and specific acylcarnitines by high-performance liquid chromatography: application to normal human urine and urine from patients with methylmalonic aciduria, isovaleric acidemia or medium-chain acyl-CoA dehydrogenase deficiency

This paper describes the development of a high-performance liquid chromatographic method for the quantitation of free carnitine, total carnitine, acetylcarnitine, propionylcarnitine, isovalerylcarnitine, hexanoylcarnitine and octanoylcarnitine in human urine. Carnitine and acylcarnitines were isolated from 10 or 25 μl of urine using 0.5-ml columns of silica gel, derivatized with 4'-bromophenacyl trifluoromethanesulfonate and separated by high-performance liquid chromatography. Using 4-(N,N-dimethyl-N-ethylammonio)-3-hydroxybutanoate (“e-carnitine”) as the internal standard, standard curves (10–300 nmol/ml) were generated. Carnitine and acylcarnitines were quantified (when they were present) in normal human urine and the urine of patients diagnosed with one of three different disorders of organic acid metabolism: methylmalonic aciduria, isovaleric acidemia, and medium-chain acyl-CoA dehydrogenase deficiency.     

A kinetic epoxidation assay for chloroperoxidase

Chloroperoxidase exhibits a wide variety of enantioselective epoxidation reactions. Until now, the epoxidation activities have been mainly evaluated using elaborate gas chromatographic methods. This paper reports a rapid and convenient spectrophotometric assay for CPO. The disappearance of indene by catalytic epoxidation is monitored at 250 nm and this is used as an index of enzyme activity. This method will prove to be highly useful in large-scale screening of mutants.     

A simple preparation of an enzyme reactor producing nicotinamidemononucleotide

Summary An enzyme reactor which produces nicotinamidemononucleotide is easily prepared by adsorption of NAD pyrophosphatase to phosphocellulose. The separation and purification of the mononucleotide is achieved in a single chromatographic step. The spectrophotometric data of purified NMN and its cyanide adduct were redetermined.