Paper Chromatographic Techniques Research Articles

Chromatographic separation of the degradation products of pralidoxime iodide (pyridine-2-aldoxime methiodide).

Abstract A satisfactory method for the separation and identification of in vitro breakdown products of pralidoxime iodide (PAM; pyridine-2-aldoxime methiodide; 2-hydroxyiminomethyl-N-metnylpyridinium iodide) by paper chromatographic techniques is described

Vegetable purgatives containing anthracene derivatives. XI. Further work on the aloin-like substance of Rhamnus purshiana DC.

Abstract The aloin-like substance formerly referred to as Compound A1, has been resolved into four closely allied anthraquinone derivatives by paper chromatographic and countercurrent techniques. Two of these substances have been isolated in pure form and their general properties, melting point, optical rotation, RF values and ultra-violet light absorption curves are recorded. Treatment with ferric chloride yields aloe-emodin from both, and each on mild hydrolysis, produces barbaloin. The names Cascaroside A and Cascaroside B are proposed. Preliminary work on the other two components has shown that they are based on a compound similar to barbaloin but a derivative of chrysophanol instead of aloe-emodin; the name chrysaloin is suggested for this substance.

Vegetable purgatives containing anthracene derivatives. VIII. The paper chromatography of certain anthraquinones and their glycosides.

Abstract A simple paper chromatographic technique, using toluene alone as a running solvent, is described for identifying free anthraquinone compounds in crude drugs. A second system is described for anthraquinone glycosides: the running solvent is the lower aqueous phase of a water: acetone: benzene mixture. This system was found particularly successful for cascara bark Rhamnus purshiana D.C. as it readily revealed the presence of at least three anthraquinone glycosides.

Paper chromatographic separation of the phloroglucinol derivatives from Dryopteris species.

Abstract A paper chromatographic technique, using buffered filter papers, is described for separating from Dryopteris ferns the phloroglucinol derivatives and some of their decomposition products. The RF values on papers buffered from pH 4.0 to pH 9.1 are given in the form of diagrams with the aid of which the appropriate buffer can be chosen depending on the mixture to be separated

Optimization of Subtilin Production by Bacillus Subitilis

In Pharmaceutical industries, several peptide antibiotics of importance are produced by Bacillus subtilis. Many antibiotics of importance are produced by B.subtilis species. Antibiotics are the biochemical secreted by microorganism which in low concentration inhibit the growth or kill other microorganism i.e. the antibiotics are “antimicrobial agent of microbial agent”. The antibiotics producers are widely spread in nature, where they play very important role in regulating the microbial population of soil, water, sewage, and compost. During this study antibiotic production ability of microorganism was detected by conventional method of crowded plate technique. Further identification and isolation of the antibiotic was carried out by the technique of paper chromatography and Bioautography1 and subtilin was identified to inhibit the growth of S.aureus. For the maximum production of the antibiotic the suitable parameters are optimized experimentally i.e. time, temperature, pH, media (carbon and nitrogen source). © 2011 IGJPS. All rights reserved.

Historical Note: Yackzan of Vanderbilt, an unacknowledged investigator in the discovery of ergothioneine in biological tissues

In 1909, Tanret in France, extracted ergothioneine (ET) from the ergot of rye. Its empirical formula is C9 H15 O2 N3 ; regarded as a betaine, and recognized as the only naturally occurring thiolimidazole amino acid. Within its structure is the sulfhydryl group which is the main effector of antioxidation. Its biological importance is its role as an antioxidant; but, to have physiological significance, a transporter (ONCT1) is needed to enter the plasma cell. In 1950, it was known that ET was present in blood but unknown whether it was present in biological tissues. Kamal Yackzan, a graduate student in the laboratory of Dr. Oscar Touster at Vanderbilt University, investigated this subject by (a)altering concentrations and ratios of existing reagents of prevailing methods and (b) using paper chromatographic analytical techniques and found quantifiable concentrations were found in liver, kidney, cardiac and spleenic tissues. In 1951, these results were recorded in an obscure publication of the University (1). Three years later while using column chromatography, Melville confirmed Yackzan's tissue findings and that the liver and kidney had the highest concentrations with the heart and the spleen having the lowest values (JBC. 206: 221, 1954). In the decades that have followed,ET has received research and commercial interest as a strong antioxidant with therapeutic value for select health disorders and diseases.___________________________________________________1. Yackzan, K. Paper chromatographic analysis for ergothioneine in tissue. In: Bulletin of Vanderbilt University Abstracts of Theses. Nashville, 1951, p. 56.

Identification of major alkaloids and steroidal saponins in rat serum by HPLC‐diode array detection‐MS/MS following oral administration of Huangbai‐Zhimu herb‐pair Extract

Huangbai-Zhimu herb-pair (HBZMHP) is a widely used Chinese traditional medicine formula in treating various diseases; however, its active components have remained unknown. In this paper, serum chemistry and combined high-performance liquid chromatography (HPLC), diode-array detection and mass-spectrometry (MS) techniques were used to study the constituents of HBZMHP extract absorbed into rat serum after oral administration. A total of nine characteristic HPLC peaks in the TIC chromatograms were identified as magnoflorine (1), menisperine (2), palmatine (3), berberine (4), timosaponin N or timosaponin E1 (5), timosaponin D (6), timosaponin BIII, anemarsaponin C or xilingsaponin B (7) timosaponin BII (8) and timosaponin AIII (9). All of the identified peaks were constituents of HBZMHP extract. The results narrow the range of active compounds to be found in HBZMHP extract, and pave the way for the follow-up action mechanism research.

Extraction Paper Chromatography Technique for the Radionuclidic Purity Evaluation of 90Y for Clinical Use

Yttrium-90 used for therapy should be of very high radionuclidic (RN) purity (>99.998%) as the most probable contaminant, strontium-90, is a bone seeker with a maximum permissible body burden of 74 kBq (2 microCi) only. None of the current known methods of RN purity estimations is adequate to reliably measure the 90Sr RN impurity at such low levels. Our aim was to develop a reliable technique to accurately determine the amount of 90Sr in 90Y used for therapy. This new technique combines chelate-based extraction with paper chromatography using paper impregnated with 2-ethylhexyl, 2-ethylhexylphosphonic acid (KSM-17), which is a 90Y-specific chelator. A PC strip impregnated with KSM-17 at the point of spotting is used for chromatography. Upon development with normal saline, 90Sr moves to the solvent front leaving 90Y completely chelated and retained at the point of spotting. The activity at the solvent front (90Sr) is quantified by liquid scintillation counting, and the data are compared with the total applied activity to provide the RN purity of the test solution. The method has a sensitivity of > or =74 kBq (2 microCi) of 90Sr per 37 GBq (1 Ci) of 90Y. This novel, innovative, and simple technique offers a reliable solution to the unanswered problem of estimation of 90Sr content in 90Y used for cancer therapy.

On-line identification of the constituents of Buyang Huanwu decoction in pig serum using combined HPLC–DAD–MS techniques

Buyang Huanwu decoction (BYHWD) is a widely used Chinese traditional compound medicine that has proved effective in treating cerebrovascular illnesses; however, its active substances have remained unknown. In this paper, serum chemistry and combined high-performance liquid chromatography (HPLC), photodiode-array detection and mass-spectrometry techniques were used to study the constituents of BYHWD from pig serum after oral administration. A total of 45 characteristic HPLC peaks were detected from serum containing drug. The chemical structures of nine of the peaks were tentatively elucidated as 7,3'-dihydroxy-4'-methoxyisoflavone-7-O-glucuronide (P1), 7-hydroxy-4'-methoxyisoflavone-7-O-glucuronide (P2), 7,2',4'-trihydroxy-3'-methoxyisoflavane-7-O-sulphate (P3), 3-hydroxy-9,10-dimethoxypterocarpan-3-O-glucuronide (P4), 7,2'-dihydroxy-3',4'-dimethoxyisoflavane-7-O-glucuronide (P5), 3-hydroxy-9,10-dimethoxypterocarpane-3-O-sulphate (P6), 4(1H)-quinolinone (P7 or P8), 4-hydroxyquinoline (P8 or P7) and oleic acid (P9). All of the identified peaks, with the exception of P9, were metabolites of the constituents of BYHWD in vivo.

FUNCTIONAL POLYSACCHARIDES FROM LILIES

The present study is concerned with physical and chemical characterization of polysaccharides in fresh lilies. Plant polysaccharides have been found to have many clinical benefits, including lowering blood sugars. Consumption of fresh lilies as food in China and other part of the world is rapidly increasing. Understanding the physical and chemical properties of lily polysaccharides is the first step toward further clinical studies. Fresh lilies were extracted using a hot water extraction method. Several techniques were used to remove residual proteins. DEAE cellulose columns were used to purify the isolates. Paper and gas chromatography techniques were used to determine the compositions and molecular weights of the polysaccharides. Two fractions of lily polysaccharides, namely LP1 and LP2, were obtained. It was found that LP1 contained mainly glucose and mannose (glucose:mannose = 1:2.46) with an average molecular weight of 79,400, and LP2 contained glucose, galactose, mannose, arabinose, and galacturonic acid (1:0.73:2.6:1.8:0.84) with an average molecular weight of 18,150.

A simple paper chromatographic technique for detection of different types of histones in the unirradiated and gamma irradiated states in vitro

Paper chromatographic technique has been employed to detect low levels (20-25 μg/ml) of H 1 , H 3 and H 4 histones under physiological pH range in the native, heat denatured and gamma irradiated states (10-50 Gy). The R t values obtained for these histones under different experimental conditions have rendered insight about the configurational alterations.

In yeast the export of small glycopeptides from the endoplasmic reticulum into the cytosol is not affected by the structure of their oligosaccharide chains.

A "quality control" system associated with the endoplasmic reticulum (ER) that discriminates between misfolded proteins and correctly folded proteins is present in a variety of eukaryotic cells, including yeast. Recently, it has been shown that misfolded proteins that are N -glycosylated in the lumen of the ER are transported out of the ER, de-N-glycosylated by a soluble peptide: N -glycanase (PNGase) and degraded by action of the proteasome. It also has been shown that small N -glycosylatable peptides follow a fate similar to that of misfolded proteins, i.e., glycosylation in the lumen of the ER, transport out of the ER, and de- N -glycosylation in the cytosol. These processes of retrograde glycopeptide transport and de- N -glycosylation have been observed in mammalian cells, as well as in yeast cells. However, little is known about the mechanism involved in the movement of glycopeptides from the ER to the cytosol. Here we report a simple method for assaying N -glycosylation/de- N -glycosylation by simple paper chromatographic and electrophoretic techniques using an N -glycosylatable(3)H-labeled tripeptide as a substrate. With this method, we confirmed the cytosolic localization of the de- N -glycosylated peptide, which supports the idea that de- N -glycosylation occurs after the export of the glycopeptide from the lumen of the ER to the cytosol. Further, we found that the variations in the structure of the oligosaccharide chain on the glycopeptide did not cause differences in the export of the glycopeptide. This finding suggests that the mechanism for the export of small glycopeptides may differ from that of misfolded (glyco)proteins.

A paper chromatographic technique for quantitative evaluation of hydrolysed and ionic components in 201Tl radiopharmaceuticals

A reported paper chromatographic technique has been extended to delineate the hydrolysed components in 201Tl radiopharmaceuticals for the determination of radiochemical purity. Detailed radiochemical purity analysis of 201TlCl has been demonstrated using mini paper chromatographic systems. The technique is also useful in evaluation of the radiochemical purity of 67Ga and 111In radiopharmaceuticals.

A miniaturized rapid paper chromatographic procedure for quality control of technetium-99m sestamibi

A miniaturized rapid paper chromatographic technique (MRPC) for quality control of a technetium-99m sestamibi preparation was developed and compared with the manufacturer's and Hung et al. techniques. The MRPC system involves the use of 6.0x0.5cm Whatman 3MM paper strip developed in ethyl acetate. The procedure was completed within 3min while that of the manufacturer and Hung techniques took 30-35 and 4min respectively. The Rf range of 99mTc-sestamibi using MRPC was 0.55-0.75 while that of the other two techniques was 0.9-1.0. The results indicate that MRPC can be used to separate 99mTc-sestamibi from any 99mTc contaminant that migrates with the solvent front. The MRPC is a fast and effective chromatographic technique for routine quality control testing of 99mTc-sestamibi preparation.

Chelating resin-impregnated paper chromatography, applications to trace element collection of ferrous and ferric ions, and determination by differential pulse anodic stripping voltammetry

An ion-exchange approach to the preparation of chelating resin is demonstrated whereby a typical sulfonated chelating agent, 7-iodo-8-hydroxy quinoline-5-sulfonic acid, is immobilized as counterions on a piperazinium polyelectrolyte matrix. The resulting chelate forming resin has been used to effect the selective separation of ferrous as well as ferric ion from a known mixture containing other trace elements without any complication of the leaching of either chelating ligand or resin from the stationary support. The chelating resin-impregnated paper chromatographic technique followed with differential pulse anodic stripping analysis is described for the preconcentration, separation, and recovery of divalent and trivalent ions of iron from the various heavy metals in aqueous phases. The combination of chelation and paper chromatography involves a differential migration procedure which provides a technique for the separation of analyte ions quantitatively without any interference from the complex matrices.

Development of sensitive radioiodinated anti-sense oligonucleotide probes by conjugation techniques

Radioactive and nonradioactive oligonucleotide (ON) probes have been used in a variety of studies of in vitro hybridization for locations of specific genes and determination of the level of mRNA transcription in activated and proliferative cells. Nuclease-resistant phosphorothioate derivatives of oligonucleotides are also finding potential therapeutic applications in intact cells and animal models. In this study, we have developed new techniques of radioiodination by the conjugation of oligonucleotides with p-methoxyphenyl isothiocyanate (PMPITC) and optimized the conditions of the radioiodination, e.g. concentration of reactants, oxidants, pH, time, and temperature. We have used a 25-mer actin mRNA probe as a model substrate for radioiodination. The ON probe was modified by conjugation with the addition of aminohexyl (AH) group at the 5'-terminal. Both the techniques of preiodination of PMPITC followed by conjugation and radioiodination of PMPITC-conjugated AHON were evaluated; the latter was found to be more convenient. The optimum pH of radioiodination of PMPITC with 125I was 6.5; on the other hand, the optimum pH for PMPITC-conjugated AHON was 8.5. The conditions of conjugation were optimized with 125I-labeled PMPITC. The techniques of paper chromatography and HPLC with a Spherogel column were developed for the purification and characterization of these oligonucleotide probes. Without PMPITC conjugation, the yield of direct radioiodination of the same oligonucleotide was very low (4-6%); the yield in our optimized technique is 50-60%. Radioiodination of conjugated oligonucleotides is a simple and efficient route to radiolabeled probes for hybridization studies.

Evaluation in vitro and in vivo of two labelling techniques of different 99mTc-dextrans for lymphoscintigraphy

Five dextrans with different molecular weights and charges were labelled with 99mTc. The labelling methods presented by Henze et al. (1982a) and Ercan et al. (1985) were compared. The labelling efficiency was tested with gel column chromatography scanning (GCS), gel chromatography (GC) combined with the Anthrone test, paper chromatography (PC) and thin layer chromatography (TLC). The GCS technique always indicated a lower labelling efficiency than the PC and TLC techniques, which was due to a more optimal separation of the radioactive components. Gel chromatography in combination with the Anthrone test made it easy to identify the different radiochemical components in contrast to the other methods. Dextran solutions were injected subcutaneously bilaterally at the xiphoid processes in rabbits. The injection sites were massaged for 30 s. Uptake in the parasternal lymph nodes was registrated with a scintillation camera. The animals were killed and dissected at the end of the study. This investigation shows that the labelling method of Ercan et al. gives the highest labelling efficiency. Furthermore, the final pH (4.5) for the dextran solution makes it more useful for injection. For quality control of 99mTc-labelled dextran we recommend the Anthrone test as a complement to GC because it is a quick and simple method of determining the dextran content.

Untersuchungen zur Dynamik der Polyphenol-und Phytoalexinsynthese fäuleinfizierter Kartoffelknollen

Host pathogen interactions between potato tubers and soft rot bacteria Erwinia carotovora ssp. atroseptica respectively dry rot fungi Fusarium spp. were investigated by a special descending paper chromatographic technique for the eluation of soluble compounds from the infected tissue region. The continuously formed sugars, phenolics and phytoalexins determined by thin layer or gas chromatography after diffusing into the eluate. As fresh harvested as stored potato turners produced phenolics and phytoalexins after infection. The uninfected controls produced only low levels of these compounds.

Pathways of inorganic carbon fixation in the endosymbiont‐bearing lucinid clam Lucinoma aequizonata. Part 2. Analysis of the individual contributions of host and symbiont cells to inorganic carbon assimilation

AbstractThe incorporation of 14C from H14CO−3 into acid‐soluble metabolites was examined in the endosymbiont‐containing gills and purified symbionts of the lucinid clam Lucinoma aequizonata. HPLC, paper chromatography, and enzymatic techniques were used to identify and quantify compounds into which label was incorporated in continuous and pulse‐labeling experiments. Both symbiont and host cells fix carbon in considerable quantities; however, each incorporates carbon into a distinctly different subset of compounds. In intact gills the initial carbon fixation product is malate. Label subsequently accumulates in succinate, glucose phosphate, glutamate, alanine, and glycogen. In the symbiont cells carbon is incorporated into aspartate, 3‐phosphoglycerate, malate, and citrate. Purified symbionts incorporate carbon into the same compounds in the same proportions as do symbionts that are exposed to labeled H14CO−3 while still within the host tissues. Under the conditions tested, purified symbionts released no significant quantities of labeled compounds into the incubation medium.

Separation Studies of As(III), Sb(III) and Bi(III) by Reversed-Phase Paper Chromatographic Technique

Abstract Reversed-phase paper chromatographic separations of As(III), Sb(III) and Bi(III) have been camed out on Whatman No 1 filter paper lmpregnated with tnphenylphosphine oxide as stationary phase and using organic complexmg agents such as sodium acetate, sodium succinate and sodium malonate solutions as active mobile phases. Results for the separation of binary and ternary mixtures are reported and the method has been successfully applied to the separation and detection of these elements present in real samples and at ppm level concentration.