Reversed-phase thin-layer chromatography in the parametrization of lipophilicity of some series of arylaliphatic acids

In the series of arylacetic acid and β-aryl-n-butyric acids, chromatography was carried out on a thin layer of silica gel impregnated with silicone oil, with 50% acetone as mobile phase. A separation mechanism in this system was evaluated using relationships between R_M values and the concentration of the lipophilic solvent in the silica-gel layer. It was found, that both partition and adsorption mechanisms participate, and that the adsorption effect increases with decreasing lipophilicity of the acids. The dichotomy of the mechanism manifests itself in the non-linear course of the relationships between R_M values and π parameters, or fragmental constants f derived from the partition system n-octanol—water. Such relationships can be expressed by a single quadratic dependence between lipophilic parameters and R_M values, or by two separate linear expressions with different slopes for different regions of substituent lipophilicities. The linear dependence between R_M and π at the lower range of lipophilicity is most probably made possible by significant linear dependence between π parameters and molecular surface areas of the substituents.     

Reversed-phase partition thin-layer chromatography of rat liver lecithins to yield eight simple phosphatidyl cholines

The four fractions obtained by argentation thin-layer chromatography of intact rat liver lecithins can be further subdivided by reversed-phase partition thin-layer chromatography on hydrophobic kieselguhr. The resultant eight fractions contain virtually only one saturated and one unsaturated acid each.     

Reversed-phase chromatography of urinary metabolites of paracetamol using ion suppression and ion pairing

High-performance liquid chromatography (HPLC) has proven particularly useful for the study of paracetamol metabolism. Two alternative methods were developed using reversed-phase C₁₈ columns. A rapid ion suppression technique was used for the analysis of free paracetamol, paracetamol mercapturic acid and cysteine conjugate in urine samples obtained from isolated perfused rat kidney preparations, which has conveniently demonstrated the oxidative metabolic capacity of the kidney towards paracetamol. A somewhat longer, but higher resolution, ion-pair HPLC procedure was developed for the analysis of paracetamol metabolites in urine samples from experimental animals. The ion-pairing solvent was composed of tetrabutylammonium hydroxide, Tris and EDTA buffered to pH 7.2 with phosphoric acid. Gradient programming was further used to enhance resolution. Using this system two new metabolites, the sulphate and glucuronide conjugates of 3-thiomethyl-paracetamol were detected and routinely determined along with other known paracetamol metabolites, viz. free paracetamol, paracetamol sulphate, glucuronide, mercapturic acid, and cysteine conjugates, 3-methoxyparacetamol glucuronide and sulphate, p-aminophenol and its O-glucuronide and O-sulphate conjugates. Phenolic O-substituted glucuronide and sulphate conjugates of N-hydroxyparacetamol were also separated.     

Reversed-phase high-performance liquid chromatography procedure for the simultaneous determination of S-adenosyl-l-methionine and S-adenosyl-l-homocysteine in mouse liver and the effect of methionine on their concentrations

An improved reversed-phase high-performance liquid chromatography (HPLC) procedure with ultraviolet detection is described for the simultaneous determination of S-adenosyl-l-methionine (SAM) and S-adenosyl-l-homocysteine (SAH) in mouse tissue. The method provides rapid resolution of both compounds in a 25-μl perchloric acid extract of the tissue. The limits of detection in 25-μl injection volumes were 22 and 20 pmol for SAM and SAH, respectively. The limits of quantitation in 25-μl injection volumes were 55 and 50 pmol for SAM and SAH, respectively, with recovery consistently >98%. The assay was validated over linear ranges of 55–11 000 pmol for SAM and 50–10 000 pmol for SAH. The intra-day precision and accuracy were ≤6.4% relative standard deviation (RSD) and 99.9–100.0% for SAH and ≤6.7% RSD and 100.0–100.1% for SAM. The inter-day precision and accuracy were ≤5.9% RSD and 99.9–100.6% for SAH and ≤7.0% RSD and 99.5–100.1% for SAM. Compared to earlier procedures, the HPLC method demonstrated significantly better separation, detection limit and linear range for SAM and SAH determination. The assay demonstrated applicability to monitoring in mice the time-course of the effect of methionine on SAM and SAH levels in the liver. Administering methionine to mice increased by 10-fold the liver concentration of SAM and SAH within 2 h, which then rapidly decreased to the control levels by 8 h. This indicated that methionine was promptly converted to SAM and then rapidly catabolized into SAH. Thus, the metabolism of methionine to SAM should be considered in the supplementation of methionine to maintain SAM levels in the body.     

Effect of pH and salts on the binding of free amino acids to the corn protein zein studied by thin-layer chromatography

Summary. The interaction of free amino acids with the corn protein zein was studied by thin-layer chromatography carried out on cellulose layers covered with zein and the effect of pH and salts on the strength of interaction was elucidated. Only the binding of Arg, His, Lys, Orn and Trp to zein was verified, other amino acids were not retained. Retention of Arg, His, Lys and Orn decreased linearly with increasing concentration of salts the mobile phase indicating the hydrophilic character of amino acid–zein interaction. Both alkaline and acidic pH influenced the strength of binding. Principal component analysis indicated the different character of the influence of pH and salts on the interaction. The results suggest that these amino acid residues may account for the binding of other peptides and proteins to zein.     

Electron-microscope study of the effect of chlorhexidine on Pseudomonas aeruginosa.

Electron micrographs of cytological damage to log phase Pseudomonas aeruginosa caused by low consentrations of chlorhexidine indicate an action primarily on the cytoplasmic membrane at concentration of 2.0--3.0 micrograms/ml chlorhexidine, and on the cytoplasmic membrane plus layers external to it at concentrations greater than 3.0 micrograms/ml. Evidence of two types of resistance to chlorhexidine is presented.     

Reversed-phase liquid chromatography with ultraviolet detection for simultaneous quantitation of indinavir and propranolol from ex-vivo rat intestinal permeability studies

A simple, rapid, sensitive and specific reversed-phase high performance liquid chromatographic (RP-HPLC) method involving ultraviolet detection (lambda = 210 nm) was developed for analysis of indinavir along with propranolol in samples obtained from ex vivo intestinal permeability studies. Chromatography was carried out on C-18 column with mobile phase comprising of phosphate buffer-acetonitrile (68:32, v/v) pumped at flow rate of 1 ml/min. The proposed method has a short run time of 12 min and involves a simple sample preparation for the purpose of reducing permeability model artifacts and to concentrate the samples. Fluorescein was used as internal standard. The proposed method has been validated with regard to specificity, detection limit, recovery, accuracy and precision. For both the drugs, method was found to be selective, linear (R(2) approximately 0.999), accurate (recovery = 100-105%) and precise (<3% R.S.D.) in the range of 2-20 microg/ml. The limit-of-detection and limit-of-quantification of the method were 40 ng/ml and 100 ng/ml for indinavir, and 30 and 80 ng/ml for propranolol, respectively. Indinavir, a widely prescribed HIV protease inhibitor, suffer from bioavailability problems where involvement of P-glycoprotein mediated drug efflux may play a significant role. The proposed method was successfully applied for intestinal permeability of indinavir to estimate the contribution of P-glycoprotein in limiting its oral bioavailability. The advantage of the developed method lies in the simultaneous determination of propranolol, a passive integrity marker, routinely employed in permeability studies and its selectivity in presence of various P-gp modulators and permeability markers.     

The effect of pH and ionic strength on the electrophoretic separation of acidic glycosaminoglycans

Using the electrophoretical methods applied to this study it is possible to determinate the dissociation constants (pK) of acid glycosaminoglycans containing a carboxylic group. The pK-values of the six acid glycosaminoglycans separated from animal connective tissues determined in this work were: hyaluronic acid (HA), pK = 3.0; chondroitin sulfate A (CS-A), pK = 2.8; chondroitin sulfate C (CS-C), pK = 3.3; dermatan sulfate (CS-B), pK = 3.3; heparatin sulfate (HeS), pK = 3.1 and heparin (HeP), pK = 2.4 and were measured at a constant ionic strength of I = 0.164 (NaCl) and at 10 ± 2°C.Variation of ionic strength showed that physiological conditions seem to be most suitable for the electrophoretic separation of the glycosaminoglycans studied. A decrease of ionic strength causes increasing mobility but less accurate spots. In the case of increasing ionic strength the results are vice versa.The second spot for HA very often appeared when pH values higher than 2 were used for electrophoresis. The spot had the same form as the original, high intensity, but an undecided migration in the pH range near the pK value of HA (3.0).     

Simplified method for simultaneous determination of diazepam and its metabolites in urine by thin-layer chromatography and direct densitometry

A direct densitometric method for determination of diazepam and its metabolites in urine was developed. The proposed procedure involves acid hydrolysis of urine specimens, thereby converting diazepam and its metabolites into benzophenones [2-methylamino-5-chlorobenzophenone (MACB) and 2-amino-5-chlorobenzophenone (ACB)]. It is followed by extraction with chloroform—isopropanol (3:1, v/v). The two benzophenones were separated on thin-layer chromatography plates using hexane—diethyl ether—acetic acid (80:10:10) as a mobile phase. Quantitation of the MACB and ACB spots was achieved by direct ultraviolet densitometry. The limit of detection was 0.5 μg per ml of urine for both benzophenones. The proposed method is simple, rapid, reproducible and has been found to be effective for direct determination of diazepam and its metabolites in urine.     

Charge transfer chromatography used to study the interaction between synthetic phospholipids and nonylphenyl-nonylglycolate

The interaction between 8 synthetic phospholipids and the nonionic tenzide nonylphenyl-nonylglycolate was studied by charge transfer chromatography. The method has been improved by carrying out the determination at different organic phase concentrations of eluent and by extrapolating the interactive strength back to pure water. It was suggested that two types of interaction of commensurable strength may exist between the tenzide and the phospholipids; hydrophil-hydrophil interactions between the polar head groups of phospholipids and the hydrophilic ethylene-oxide chain of tenzide; hydrophob-hydrophob interactions between the fatty acid chains of phospholipids and the alkyl chain of tenzide. This last effect strongly depends on the length of the fatty acid chain, but it is independent of the presence of double bonds in the lipophilic region of phospholipid.     

Reversed-phase boronate chromatography for the separation of O-methylribose nucleosides and aminoacyl-tRNAs

Boronate forms an anionic complex with the cis-2′,3′ hydroxyls of unsubstituted ribonucleosides and the 3′-terminal adenosine of unacylated tRNAs, but not with ribosesubstituted nucleosides such as 2′-O-methylnucleosides and aminoacyl-tRNAs. We have synthesized phenyl boronates with hydrophobic side chains of about 1-nm-long and coated inert 10-μm solid beads of polychlorotrifluoroethylene with this material. This matrix complexes easily with compounds containing free cis-hydroxyls, but not with their O-alkyl or O-acyl derivatives. This permits the separation of mammalian and bacterial amino-acyl-tRNAs from uncharged tRNAs and O-methyl nucleosides from ribose-unsubstituted nucleosides in one chromatographic step, as the substituted members of each group do not undergo boronate complex formation and are thus not as much retarded in passing through the column. Complex formation between ribofuranoses and the boronate matrix appears to be enhanced by the hydrophobic “tail” of the boronate compound, by the high ionic environment of the solvent, and by the hydrophobic nature of the inert support. This method of one-step purification of tRNAs on reversed-phase boronate columns has been tested for several tRNAs specific for amino acids of different hydrophobicity and ionic character. The results indicate that each tRNA tested can be purified with appreciable purity (70–95%) and high yield (80%). However, recovery of the queuine base containing aminoacyl-tRNAs is only about 6% of the applied material. Several other boronate matrices have also been synthesized using cellulose, agarose. Sepharose, or porous glass beads as the inert support with different lengths of the spacer arm. Cellulose with a 1-nm-long spacer arm is satisfactory not only for the separation of aminoacyl-tRNAs and O-methylribose nucleosides, but also for the separation as a group of tRNAs containing the base of Q, queuine. However, other inert supports are unsatisfactory because of a non-specific binding of the tRNAs.     

Influence of salt and pH on the hydrophobicity parameters of antisense nucleosides studied by reversed-phase thin-layer chromatography and multivariate mathematical-statistical methods.

The lipophilicity and specific hydrophobic surface area of 12 8-substituted 2'-deoxyadenosine and 17 5-substituted-2'-deoxyuridine derivatives were determined by reversed-phase thin-layer chromatography in ion-free eluents and in eluents containing sodium chloride, sodium acetate and acetic acid. The strength and selectivity of the effect of eluent additives were separated by use of spectral mapping technique followed by two-dimensional nonlinear mapping. The relationship between the structural characteristics and hydrophobicity parameters was elucidated by stepwise regression analysis. Eluent additives exert a considerable influence on both hydrophobicity parameters. The effect of sodium chloride and acetic acid was higher than that of sodium acetate. The strength and selectivity of the sensitivity of nucleosides towards eluent additives significantly depended on the character of the ring structure and on the length of the apolar alkyl chain. The influence of the degree of unsaturation and the branching of the alkyl substituent was negligible.     

Electron microscope study of the effect of benzalkonium, chlorhexidine and polymyxin on Pseudomonas cepacia

Electron micrographs of Pseudomonas cepacia cell grown in nutrient broth show an external membrane which is distinctly wavy, when compared with similar preparations of Pseudomonas aeruginosa, and which is not affected by growing in the presence of broth containing benzalkonium (10 microgram/ml), chlorhexidine (10 microgram/ml) or polymyxin (25 units/ml). Both benzalkonium (10 microgram/ml) and chlorhexidine (10 microgram/ml) damage the cytoplasmic membrane of P. cepacia cell grown in the presence of the chemicals. Contrasts are shown between the effect of polymyxin (chlorhexidine and benzalkonium) on the outer membrane of P. cepacia and P. aeruginosa.     

Liquid chromatography method for simultaneous analysis of amino acids and biogenic amines in biological fluids with simultaneous gradient of pH and acetonitrile

A liquid chromatography method for simultaneous analysis of amino acids, polyamines, catecholeamines and metanephrines in human body fluids after derivatization with 9-fluorenylmethyloxycarbonyl chloride was developed. The chromatographic behavior of analytes at different pH of mobile phase was studied. Successful baseline resolution of all analyzed compounds was achieved using simultaneous gradient of pH and organic modifier in reverse phase mode of HPLC within 36 min. The repeatability of the proposed procedure in respect of retention time and peak area, expressed as RSD, ranges from 0.06 to 1.64% and 0.4 to 7.6%, respectively. The method linearity in the range of 1-200 microM for amino acids and in the range of 0.1-20 microM for polyamines, catecholeamines and metanephrines was found to be with correlation coefficients higher than 0.994. The limit of quantification (LOQ) was assessed to be in the range of 2.6-10 pmol for amino acids and 2-4 pmol for polyamines, catecholeamines and metanephrines.     

The electrogenic ion pump revealed by the external pH effect on the membrane potential of Nitella. Influences of external ions and electric current on the pH effect

The influences of external ions and electric current on thepH effect of the membrane potential of Nitella are described.Results are explained in terms of a pH-dependent electrogenicion pump. The reduction of membrane resistance with a pH rise is attributedto the pump current being potential-dependent. (Received July 9, 1974; )     

High-performance liquid affinity chromatography and in situ fluorescent labeling on thin-layer chromatography of glycosphingolipids

A method combining high-performance liquid affinity chromatography and in situ fluorescent labeling on thin-layer chromatography is introduced for determination of glycosphingolipids. Glycolipids in crude extract from rat liver were separated quantitatively from neutral lipids and phospholipids with a phenylboronic acid-derivatized silica gel column. Glycolipids were eluted quantitatively with approximately 98% of crude extract recovered. This column is useful for selective cleanup of glycosphingolipids in crude extract from tissue. Simultaneously, a fluorometric determination of glycosphingolipids with 7-amino-4-methylcoumarin after NaIO4 oxidation on a TLC plate was introduced and its condition was optimized. Glycolipids in amounts ranging from 1 to 100 pmol are easily detectable and give linear responses over the respective ranges. The method is fast and useful for the determination of glycolipids from small amounts of biological samples and requires a minimum amount of about 1 mg of biological specimen for determination of glycolipids.