Impregnated Silica Gel Plates Research Articles

Retardation of some drugs in thin-layer chromatographic systems with impregnated silica gel plates with hen's egg white and bovine serum albumin

The influence of impregnation the chromatographic plate adsorbent layer, silica, with hen's egg white albumin (OVA) or bovine serum albumin (BSA) on the retention of some popular medicines (paracetamol, aminophenazone, theophylline, caffeine, acetanilide, ciprofloxacin, tramadol, acetylsalicylic acid, acebutolol) is investigated. The effect of composition and buffer pH of the mobile phase on solute separation selectivity is also studied. The chromatographic systems with and without above mentioned albumins and their influence on investigated drug retention are compared. In general, it has been turned out that retention of tested medicines in systems with the sorbent impregnated with albumin significantly increase relative to those with non-impregnated.

Thin-layer chromatographic method for separation of wheatgrass pigments on sucrose impregnated silica gel plates

One of the most important aspects of the selected analytical method is avoiding the degradation of the target compounds during the analysis of the samples. Chromatographic techniques, particularly thin-layer chromatography (TLC), are widespread in this field. However, difficulties arise in applying chromatographic methods, such as separation efficiency, and stability of target compounds. The application of silica gel as stationary phase might be an appropriate choice [2], but at the same time, the degradation of the pigment compounds is fast, so the analysis has to be done relatively quickly [3]. In order to avoid degradation, a column filled with sucrose was used as adsorbent for isolation of chlorophylls [4, 5] and home-made thin-layer plates for analysis [6–8]. However, the resolution was rather limited. The aim of this investigation was to find a rapid and simple analytical method for the separation of the pigments originated from commercial wheat grass extracts without any degradation product generated during the analysis of the samples. The sucrose immersed silica gel system fulfilled all of these requirements. 2 Experimental

Development of efficient SPE–TLC method and evaluation of biological interactions of contraceptives with progesterone receptors

TLC–SPE methodologies were developed to ascertain biological interactions of norethindrone acetate and dydrogesterone contraceptives with plasma progesterone receptor proteins. TLC solvent system for plain and Cu(II) impregnated silica gel plates was n-hexane-n-butanol (90:10, v/v), which took 20min to run up to 10.0cm. The best separation was on Cu(II) impregnated plates due to maximum difference in Rf values and compact spots. The optimized SPE conditions were pH 2.0 and 3.0 of phosphate buffer (50mM) for norethindrone acetate and dydrogesterone, respectively. The flow rate of plasma and eluting solvent (methanol) through C18 cartridge was 0.10mL/min. The interactions of these contraceptives with progesterone receptor proteins were analysed by TLC–SPE results, which were supported by modelling using PyMOL and Autodoc4 softwares. The dydrogesterone was found to be bound strongly than norethindrone acetate. Attempts have been made to discuss the drugs’ interactions at chemo-supramolecular level.

Thin-layer chromatography of glucose and sorbitol on Cu(II)-impregnated silica gel plates

A thin-layer chromatographic (TLC) separation of glucose and sorbitol on Cu(II)-impregnated silica gel plates with n-propanol: water (4:1) v/v as developer and potassium permanganate as detecting reagent has been worked out. The new impregnant is completely insoluble in water and thus enables the use of an aqueous developer. The Rf-values are 55 and 10 for glucose and sorbitol, respectively.

The Salvage of Deoxycytidine into dCDP-Diacylglycerol by Macrophages and Lymphocytes

Extracellular deoxycytidine (CdR) was previously shown to be salvaged into water soluble [1] and also into lipidic [2] precursors of phospholipids in stimulated lymphocytes and in lymphoma cells [3]. In this paper we have described that non-dividing murine macrophages salvaged not only 5- 3H-CdR but also tritiated thymidine( 3H-TdR) mainly into the pools as nucleotides. Chlorprosazine shifted the CdR salvage into a lipidic compound of the cells which was identified as 3H-dCDP-diacylglycerol (dCDP-DAG). After 5- 3H-CdR labeling the lipid/DNA ratio was eleven times higher in macrophages than in tonsillar lymphocytes. Thin layer chromatography (TLC) on borate impregnated silica gel plates gave clear separation of CDP-DAG from dCDP-DAG supporting that the extracellular precursor for it is exclusively deoxycytidine and not ribocytidine. No interconversion between deoxy- and ribocytidine could be observed neither in lymphocytes nor in macrophages.

Behavior of Some Benzene Derivatives on Progressively Dried Fe(lll)-Impregnated Silica Gel Plates

Abstract The influence of progressive drying of Fe(III)-hydroxy oxide support on the behaviour of hydroxy and carboxy benzene derivatives in thin layer chromatography was investigated. The results show that different complexes are formed depending on the kind of substances and/or condition applied.

The Behaviour of Some Benzene Derivatives on Thin Layers of Aluminum Oxide - Comparation with Plain and Fe(III)Impregnated Silica Gel

Abstract The behaviour of some carboxy and hydroxy benzene derivatives related to humic material was examined by thin layer chromatography on aluminium oxide as the support and water as developer. Their Rf-values were compared with those on plain and Fe(III)-impregnated silica gel plates.

Polyamines stimulate the phosphorylation of phosphatidylinositol in rat mast cell granules

Rat mast cell granules were obtained by homogenization of highly purified rat mast cells and isolated in a Percoll gradient. Diphosphoinositide (DPI) synthesis in rat mast cell granules was assayed by measuring the incorporation of 32P from [gamma 32P] ATP into DPI in the absence of exogenous phosphatidylinositol (PI). Lipids were isolated with methanol/chloroform/HCl and were separated by thin-layer chromatography on oxalic acid impregnated silica gel plates. DPI areas were identified by staining with iodine, scraped and measured for 32P radioactivity. The addition of polyamines, spermine and spermidine, to the granules caused an increase of DPI synthesis, which can be catalyzed by PI kinase. This effect of polyamines in the DPI synthesis was in a dose-dependent manner and maximal effects were observed at 1 mM spermine and 10 mM spermidine, respectively.

Inhibitory effect of adenine nucleotides and anti-allergic drugs on phosphorylation of phosphatidylinositol in rat mast cell granules.

Rat mast cell granules were obtained by sonication of highly purified rat mast cells and isolated in a Percoll gradient. Phosphorylation of endogenous phosphatidylinositol in rat mast cell granules, which is catalyzed by phosphatidylinositol kinase in the granules, was assayed by measuring the incorporation of 32P from [gamma 32P]ATP into phosphatidylinositol 4-phosphate. Lipids were isolated with methanol/chloroform/HCl and were separated by thin-layer chromatography on oxalic acid impregnated silica gel plates. Phosphatidylinositol 4-phosphate areas were identified by staining with iodine, scraped and measured for 32P radioactivity. The phosphorylation reaction was inhibited by 50-500 microM adenosine, ADP and 500 microM AMP in a concentration-dependent manner. Among several anti-allergic drugs investigated. 100-1000 microM theophylline and 10-100 microM azelastine inhibited the phosphorylation reaction, but disodium cromoglycate and ketotifen had little effect.

The Behavior of Some Carboxy and Hydroxy Benzene Derivatives on Thin Layers of Plain and Fe(III)-Impregnated Silica-Gel

Abstract The behavior of some carboxy and hydroxy benzene derivatives related to lignin and humic materials was examined by thin layer chromatography on plain and Fe(III)-impregnated silica-gel plates, in three solvent systems. The mobility differences on impregnated and plain plates, expressed as Ri values, were calculated and can be used as one of the parameters for identification of the compounds tested.

Changes in polyphosphoinositide metabolism during mediator release from stimulated rat mast cells

The metabolism of the polyphosphoinositides, diphosphoinositide (DPI) and triphosphoinositide (TPI), was studied during mediator release from rat mast cells. Serosal mast cells were purified by density gradient centrifugation and prelabeled with 32PO 4. Incorporation of 32PO 4 into DPI and TPI was determined by thin-layer chromatography on oxalic acid impregnated silica gel plates. 32PO 4 incorporation into DPI and TPI was increased by Concanavalin A (ConA) or compound 48/80. The concentration of ConA causing a half-maximal increase in DPI labeling was less than that required for a comparable change in histamine release. The increases in DPI labeling and histamine release in response to ConA were enhanced by phosphatidylserine. The addition of α-methylmannoside to mast cells after challenge with ConA rapidly halted DPI and TPI labeling. The results of these studies indicate that changes in the metabolism of polyphosphoinositides may be an intrinsic part of the biochemical mechanisms that control mediator release from mast cells.

Phorbol Myristate Acetate Stimulates Formation of Diphosphoinositide in Rat Mast Cell Granules

Rat mast cell granules were obtained by homogenization of highly purified rat mast cells and isolated in a Percoll gradient. Diphosphoinositide (DPI) synthesis in rat mast cell granules was assayed by measuring the incorporation of 32P from [gamma 32P]ATP into DPI in the absence of exogenous phosphatidylinositol (PI). Lipids were isolated with methanol/chloroform/HCl and were separated by thin-layer chromatography on oxalic acid impregnated silica gel plates. DPI areas were identified by staining with iodine, scraped and measured for 32P radioactivity. The addition of phorbol myristate acetate (PMA) to the granules caused an increase of 32P incorporation from [gamma 32P]ATP in the DPI fraction, which can be catalyzed by PI kinase. This effect of PMA in the DPI synthesis was dose dependent and maximal effects were observed at 10 ng/ml.

ラットｍａｓｔ ｃｅｌｌか粒ｐｈｏｓｐｈａｔｉｄｙｌｉｎｏｓｉｔｏｌ ｋｉｎａｓｅに及ぼすａｄｅｎｉｎｅ ｎｕｃｌｅｏｔｉｄｅと抗アレルギー薬の影響

Rat mast cell granules were obtained by homogenation of highly purified rat mast cells and isolated in a Percoll gradient. PI kinase in rat mast cell granules were assayed by measuring the incorporation of 32P from [γ32P] ATP into diphosphoinositide (DPI) in the absence of exogenous PI. Lipids were isolated with methanol chloroform/HCl and were separated by thin-layer chromatography on oxalic acid impregnated silica gel plates. DPI areas were identified by staining with iodine, scraped and measured for 32P radioactivity. The enzyme was inhibited by 50-500μM adenosine, 5-500μM ADP and AMP. Among several anti-allergic drugs investigated, 10-1000μM theophylline and 1-100μM azelastine inhibited the enzyme, but 1-100μM disodium cromoglycate and ketotifen had little effect.

TLC Resolution of Amino Acids in a New Solvent and Effect of Alkaline Earth Metals

Abstract A new solvent system for the resolution of 15 component mixture of amino acids and the effect of alkaline earth metals on the resolution is reported, the TLC was carried out using plain and impregnated silicagel plates and, solutions of amino acids in tris buffer and pretreated with metal hydroxides.

Rapid resolution of phenylthiohydantoin amino acids by thin-layer chromatography on silica gel plates impregnated with transition metal ions

The resolution of a 15-component mixture of phenylthiohydantoin (PTH) amino acids using metal ion impregnated silica gel plates is reported. The spots are located by exposing the chromatograms to an iodine chamber. The method provides a rapid, simple, and less expensive chromatographic system, provides resolution for certain difficult combinations, and leaves the PTH amino acids unaltered chemically.

ラットｍａｓｔ ｃｅｌｌリン酸化反応に関する研究（補遺／その１） ｍａｓｔ ｃｅｌｌか粒ｔｒｉｐｈｏｓｐｈｏｉｎｏｓｉｔｉｄｅ

Granules were isolated from sonicated rat mast cells on Percoll gradient and incubated with [γ32P] ATP in the presence of Mg2+ and exogenous diphosphoinositide. After stopping the reaction, lipid extraction of the intact membrane granules was performed with acidic medium and phospholipids were separated by thin layer chromatography on 1% (w/v) oxalic acid and potassium oxalate impregnated silica gel plates. Considerable amounts of radioactivity were found to be incorporated into TPI on both plates. Extensive washing of the granules did not significantly affect the 32P incorporation into TPI. The radioactivity in TPI was associated with the granule themselves and it is apparent that TPI is produced in rat mast cell granule.

ラットｍａｓｔ ｃｅｌｌリン酸化反応に関する研究 第１報 か粒膜ｄｉｐｈｏｓｐｈｏｉｎｏｓｉｔｉｄｅ

Granules were isolated from sonicated rat mast cells on a Percoll gradient and incubated with [γ 32P] ATP in the presence of Mg2+. After stopping the reaction, lipid extraction of the intact membrane granules was performed with acidic medium and phospholipids were separated by thin layer chromatography on 1% (w/v) oxalic acid and potassium oxalate impregnated silica gel plates. Considerable amounts of radioactivity were found to be incorporated into DPI. The radioactivity in DPI was associated with the granule themselves and not detected in the granule supernates. Extensive washing of the granules did not significantly affect DPI formation and areas of the Percoll gradient containing cytosol fraction had no demonstrable DPI forming activity and did not increase the response when added back to the granules. When suspensions of intact and broken membrane granules which contained approximately equivalent amounts of granule membrane proteins were compared with respect to DPI formation, broken membrane granule preparations were found not to exceed in DPI production than intact membrane granules, suggesting that DPI formation is occuring on the cytoplasmic side of granule membranes.

ラットｍａｓｔ ｃｅｌｌリン酸化反応に関する研究 （第８報） 活性化ｍａｓｔ ｃｅｌｌよりのｐｏｌｙｐｈｏｓｐｈｏｉｎｏｓｉｔｉｄｅ遊離とその局在

Serosal mast cells were purified by bovine serum albumin density gradient centrifugation and prelabeled with 32PO4. Incorporation of 32PO4 into PPI, diphosphoinostide (DPI) and triphosphoinositide (TPI), was determined by thin-layer chromatography on oxalic acid impregnated silica gel plates. 32PO4 incorporation into DPI in mast cell supernates as well as pellets was increased by concanavalin A. As a control for possible artifactual formation of DPI in the supernates, supernates from labeled stimulated or unstimulated cells were incubated with intact or broken membrane granules from unlabeled cells isolated from sonicated mast cells on a Percoll gradient. No obvious 32PO4 incorporation into PPI in the granules was seen. The question of how much of the labeled PPI is present in the granule fraction was considered. Studies in unstimulated mast cells labeled with 32PO4, including controls for artifactual incorporation of label during granule isolation, suggest that at least 40% of the total cellular radiolabel in PPI is in the granules. The results of these studies indicate that changes in PPI metabolism may be a intrinsic part of the biochemical mechanisms that control mediator release from mast cells.

The Use of Sodium Borate Impregnated Silica Gel Plates for the Separation of 3-0-Methyl Catecholamines from Their Corresponding Catecholamines

The use of sodium borate impregnated silica gel plates for the chromatographic separation of the catecholamines noradrenaline, adrenaline, and isoprenaline from their respective 3-0-methylated derivatives, normetanephrine, metanephrine, and methoxy-isoprenaline, is described. The parent catecholamines remain at the origin of the plates while the 3-0-methylated derivatives concentrate in discrete bands at the upper edge of the borate impregnated area (the "borate front").

Nicht methylenunterbrochene cis,cis-Octadecadiensäuren Minor Constituents of Human Milk, VI Non Methylene Interrupted cis,cis-Octadecadienoic Acids

The non methylene interrupted cis,cis-octadecadienoic acids from human milk fat were pre­pared by thin layer chromatography on AgNO3 impregnated silicagel plates. The position of double bonds was determined by hydroxylation of the double bonds with OsO4 followed by silylation. The corresponding O-trimethylsilylether derivatives of the fatty acid methyl esters were analyzed by gaschromatography mass spectrometry. 14 isomers of the following series could be identified: C18:2 ⊿9. (12.13.14.15.16.17) ; C18:2 ⊿ (6.7.8.9.).12; C18:2 ⊿ (10.11.12.).15 (⊿18.15) ; C18:2 ⊿5.9-, ⊿6.10; ⊿10.14; (⊿12.16). Possible biosynthetic pathways are discussed.