Salting-out Chromatography Research Articles

Lipophilicity study of different cephalosporins: Computational prediction of minimum inhibitory concentration using salting-out chromatography

The chromatographic and lipophilicity characters of seven cephalosporins of different four classes (cephradine, cefaclor, cefprozil, cefixime, cefotaxime, ceftazidime and cefepime) were examined by salting out thin-layer chromatography (SOTLC). SOTLC using ammonium sulfate salt was employed to predict the lipophilicity of the proposed drugs via their retention behavior. The calculated RM0 values showed liner relationship with the molar concentration of ammonium sulfate in mobile phase in the range of 0.5–2.5 mol/L. Additionally, quantitative structure retention relationship (QSRR) was generated to figure out the relationship between the calculated chromatographic parameters (RM0 and C0) and log P of the studied cephalosporins. Good correlations were found between the chromatographically obtained retention parameters (RM0 and C0) and some molecular descriptors of the examined drugs. Furthermore, an efficient QSAR model was carried out using the calculated chromatographic parameters (RM0 and C0) and log P of the studied cephalosporins to predict minimum inhibitory concentration (MIC) and blood brain barrier (BBB) penetration of the examined drugs. The study was extended to separate and quantify the selected antibiotics in their pure forms and pharmaceutical formulations. Normal phase thin layer chromatographic (NP-TLC) method using a usable developing system of acetone: methanol: water: ammonium hydroxide: glacial acetic acid (90: 10: 18: 3: 2, by volume) was successfully applied to resolve the studied cephalosporins. Linearity was achieved in the range of 0.2–3 µg/mL for most of the studied antibiotics. The developed SOTLC method can be considered as a good start alternative to reversed phase thin layer chromatography (RP-TLC) for prediction of the lipophilic properties of examined cephalosporins. Moreover, the proposed NP-TLC densitometric method can be easily applied for quality control analysis of the chosen drugs and other structurally related components.

Биосовместимость кальцийфосфатных материалов биогенного происхождения при имплантации в область дефектов костей собак

Purpose To study the biocompatibility of implantation calcium phosphate (CP) materials from bovine bone tissue and its dependence on their composition Materials and methods The authors studied the biocompatibility of three implantation calcium phosphate materials obtained by an original technology on experimental animals (dogs). The materials obtained from bovine bone tissue by its demineralization and sedimentation out of the CP salts solutions. Serum proteins (SP) of the molecular mass of 20-30 kDa that were isolated from blood serum of the dogs with active osteogenesis were added to CP composition in order to improve the characteristics of biocompatibility. The isolation was performed using the methods of salting-out, dialysis and gel permeation chromatography. CP materials and their composites were implanted into the defects of long bone metaphyses. The study was performed using the methods of infrared spectroscopy, X-ray electron probe microanalysis, and light and scanning electron microscopy. Chemical composition of CP materials, reparative osteogenesis character and its intensity were determined quantitatively and qualitatively. The diameter of the implant particles of the tissues that filled the cancellous defect area were evaluated histomorphometrically. Results It was found that the implanted materials differed in biodegradation, osteoinduction and osteoconduction properties. CP materials that were the closest to bone tissue by their composition had a higher degree of biocompatibility Conclusion The proposed CP materials can be used in clinical practice for filling posttraumatic defects and correction of the pathological conditions accompanied by osteoporosis or bone loss.

Separation Mechanisms of Co(III) Complexes with EDTA-Type Ligands during Salting-Out TLC on Impregnated and Non-Impregnated Silica Gel

A separation mechanism of eight Co(III) complexes with ethylenediaminetetraacetate-type ligands belonging to two series during salting-out thin-layer chromatography on silica gel is investigated. The sorbent is impregnated with five poly(ethylene glycol)s with different molecular masses, and ammonium sulphate solutions are used as mobile phase. Additionally, on non-impregnated sorbent, mobile phases containing one of eight salts with ions of different lyotropic properties are used: kosmotropic Mg(2+) accompanied with SO(4)(2-) (kosmotrope), Cl(-) (border), NO(3)(-) (chaotrope), ClO(4)(-)(chaotrope), Cl(-) of Li(+) (kosmotrope), Na(+) (border), K(+) (chaotrope) and NH(4)(+) (chaotrope). Salting-out and salting-in effects were observed depending on salt nature. The combined retention mechanism of specific H-bonding and nonspecific hydrophobic interactions is proposed.

CHEMOMETRIC CHARACTERIZATION OF MODEL COMPOUNDS RETENTION IN SALTING-OUT THIN LAYER CHROMATOGRAPHY ON CELLULOSE

The current paper presents a second part of chemometric characterization of retention behavior of thirty-five simple model compounds in salting-out chromatography on cellulose. Twelve inorganic salts, dissolved in high concentrations (0.5–4 mol/l) in water, were used as mobile phases. The resulting retention dataset was subjected to Principal Component Analysis (PCA). First principal component (PC) (explaining majority of variance) is connected with compound contribution, independent of the salt used. The differences between the salts are modeled by the second PC. Third PC explains mainly differences in retention slopes, whereas the fourth PC models weak minor differences. The remaining PCs contain only noise.

Purification and Characterization of α-Glucosidase I from Japanese Honeybee (Apis cerana japonica) and Molecular Cloning of Its cDNA

alpha-Glucosidase (JHGase I) was purified from a Japanese subspecies of eastern honeybee (Apis cerana japonica) as an electrophoretically homogeneous protein. Enzyme activity of the crude extract was mainly separated into two fractions (component I and II) by salting-out chromatography. JHGase I was isolated from component I by further purification procedure using CM-Toyopearl 650M and Sephacryl S-100. JHGase I was a monomeric glycoprotein (containing 15% carbohydrate), of which the molecular weight was 82,000. Enzyme displayed the highest activity at pH 5.0, and was stable up to 40 degrees C and in a pH-range of 4.5-10.5. JHGase I showed unusual kinetic features: the negative cooperative behavior on the intrinsic reaction on cleavage of sucrose, maltose, and p-nitrophenyl alpha-glucoside, and the positive cooperative behavior on turanose. We isolated cDNA (1,930 bp) of JHGase I, of which the deduced amino-acid sequence (577 residues) confirmed that JHGase I was a member of alpha-amylase family enzymes. Western honeybees (Apis mellifera) had three alpha-glucosidase isoenzymes (WHGase I, II, and III), in which JHGase I was considered to correspond to WHGase I.

Purification and substrate specificity of honeybee, Apis mellifera L., alpha-glucosidase III.

Alpha-glucosidase III, which was different in substrate specificity from honeybee alpha-glucosidases I and II, was purified as an electrophoretically homogeneous protein from honeybees, by salting-out chromatography, DEAE-cellulose, DEAE-Sepharose CL-6B, Bio-Gel P-150, and CM-Toyopearl 650M column chromatographies. The enzyme preparation was confirmed to be a monomeric protein and a glycoprotein containing about 7.4% of carbohydrate. The molecular weight was estimated to approximately 68,000, and the optimum pH was 5.5. The substrate specificity of alpha-glucosidase III was kinetically investigated. The enzyme did not show unusual kinetics, such as the allosteric behaviors observed in alpha-glucosidases I and II, which are monomeric proteins. The enzyme was characterized by the ability to rapidly hydrolyze sucrose, phenyl alpha-glucoside, maltose, and maltotriose, and by extremely high Km for substrates, compared with those of alpha-glucosidases I and II. Especially, maltotriose was hydrolyzed over 3 times as rapidly as maltose. However, maltooligosaccharides of four or more in the degree of polymerization were slowly degraded. The relative rates of the k0 values for maltose, sucrose, p-nitrophenyl alpha-glucoside and maltotriose were estimated to be 100, 527, 281 and 364, and the Km values for these substrates, 11, 30, 13, and 10 mM, respectively. The subsite affinities (Ai's) in the active site were tentatively evaluated from the rate parameters for maltooligosaccharides. In this enzyme, it was peculiar that the Ai value at subsite 3 was larger than that of subsite 1.

Thin-Layer Chromatography Experiments That Illustrate General Problems in Chromatography

A number of chromatography experiments using cellulose thin layers and aqueous eluents are proposed to illustrate:1. How chromatography can be used for "pattern recognition" of the inks of felt-tip pens.2. The principle of displacement chromatography of the constituents of the antiseptic Mercurochrome.3. Salting-out chromatography for the separation of halides using a nearly saturated ammonium sulfate solution as eluent.4. The impossibility to use chromatography alone as a method of identification: Methyl Orange travels differently in presence and absence of cyclodextrins or starches in the sample to be analysed.

Salting-out thin layer chromatography of transition metal complexes: A comparative study of the effect of increased number of CH2 groups in chelate rings

The regularities previously established in salting-out thin-layer chromatography on silica gel of Co(III) complexes, in which the side chains of a specific chelate ring were successively increased in length, were also found to be valid for two series of Co(III) complexes of the EDTA type, in which different chelate rings were enlarged by one CH2 group. It was found that in the case of complexes of the EDTA type the salting-out efficiencies were practically the same for all members of a series, while the values of the separation factors were paractically independent of the ammonium sulphate concentration in the solvent system used. The results obtained were consistent with the mechanism of nonspecific hydrophobic interactions. The method used can be applied to the separation of the members of the series of complexes that we investigated.

Salting-out thin-layer chromatography of transition metal complexes. IV: Effect of sorbent on the behaviour of mixed aminocarboxylato cobalt (III) complexes

From literature data for 14 mixed aminocarboxylato cobalt(III) complexes, the effect of the sorbent on RM values, salting-out efficiency (m) and separation factors (α) of adjacent members of homologous series was investigated in salting-out, thin-layer chromatography on silica gel, polyacrylonitrile and cellulose sorbents, using ammonium sulphate solutions as developers. For complexes belonging to the same homologous series, linear dependence was found between RM,m and log α values, respectively, on two different sorbents. Polyacrylonitrile was shown to be the most suitable sorbent for the separation of the complexes since it gave the highest values for RM,m and α.

Linear relation between RM values obtained in salting-out planar chromatography and mol % ammonium sulphate in aqueous solvent systems

Existence of a linear relation between RM and mol % ammonium sulphate in an aqueous solvent system, established earlier for mixed aminocarboxylato cobalt(III) complexes in salting-out, thin-layer chromatography on silica gel, polyacrylonitrile and cellulose, was verified by linear regression analysis of the corresponding data reported in the literature for 108 organic compounds separated on cellulose. The same regularity was also found for seven inorganic anions.

Salting-out thin-layer chromatography of transition metal complexes. III: Investigation of the behaviour of mixed aminocarboxylato cobalt(III) complexes on cellulose

Salting-out thin-layer chromatography of fourteen mixed aminocarboxylato cobalt(III) complexes (three homologous series) on cellulose has been investigated with eight aqueous ammonium sulphate solutions. A pronounced salting-out effect, with reversed-phase order, was observed with bis(aminocarboxylato) complexes, whereas with mono(aminocarboxylato) complexes such a behaviour was noticeable only at higher salt concentrations. In both cases a positive linear dependence between the RM values of the complexes and mol % of the salt in the solvent system, and between the RM values and the number of carbon atoms in the hydrocarbon part of the complexes belonging to the same homologous series, were found. These phenomena were explained by the mechanism of non-specific hydrophobic interactions.

Purification of an Iron-Containing Superoxide Dismutase from A Citrus Plant,Citrus Limonum R

A cyanide-insensitive superoxide dismutase was purified to apparent homogeneity from lemon leaves (Citrus limonum R). The enzyme was isolated from leaf extracts by ammonium sulfate salting-out, and ion-exchange, gel filtration and hydroxylapatite column chromatography. The purified Fe-SOD had a specific activity of about 1,500 U/mg and represents approximately 1.6% of the total soluble protein in lemon leaf extracts. A molecular weight of 47,500 was determined for the enzyme. Analytical gel electro-focusing of the purified preparation revealed the presence of two isozymes with pI values of 5.13 and 4.98. Metal analysis showed the presence of 1 g-atom of iron and 0.5 g-atom of manganese per mol of enzyme. The visible and UV absorption spectra of the Citrus enzyme were similar to those reported for other iron-containing SODs from different origins. The significance of the presence of Fe-SOD in higher plants is briefly discussed.

Adsorption chromatography on cellulose : I. Salting-out chromatography of organic compounds

The adsorption on cellulose of a wide range of organic compounds from aqueous ammonium sulphate solutions was studied. The adsorption increased with increasing concentration of ammonium sulphate for all the compounds examined, and plots of their R M values against the logarithm of the activity of ammonium sulphate gave essentially parallel curves in the range from 1 M to saturated ammonium sulphate. It is concluded that the main mechanism of adsorption is a non- specific hydrophobic interaction.

Purification and characterization of pyruvate: ferredoxin oxidoreductase from the anaerobic protozoon Trichomonas vaginalis

The pyruvate: ferredoxin oxidoreductase from the anaerobic protozoon Trichomonas vaginalis is an extrinsic protein bound to the hydrogenosomal membrane. It has been solubilized and purified to homogeneity, principally by salting-out chromatography on Sepharose 4B. Low recoveries of active enzyme were caused by inactivation by O2 and the irreversible loss of thiamin pyrophosphate. It is a dimeric enzyme of overall Mr 240,000 and subunit Mr 120,000. The enzyme contains, per mol of dimer, 7.3 +/- 0.3 mol of iron and 5.9 +/- 0.9 mol of acid-labile sulphur, suggesting the presence of two [4Fe-4S] centres, and 0.47 mol of thiamin pyrophosphate. The absorption spectrum of the enzyme is characteristic of a non-haem iron protein. The pyruvate: ferredoxin oxidoreductase from T. vaginalis is therefore broadly similar to the 2-oxo acid: ferredoxin (flavodoxin) oxidoreductases purified from bacterial sources, except that it is membrane-bound.

Salting-out chromatography on unsubstituted Sepharose CL-6B as a convenient method for purifying proteins from dilute crude extracts : Application to horseradish peroxidase

Salting-out chromatography on unsubstituted Sepharose CL-6B as a convenient method for purifying proteins from dilute crude extracts : Application to horseradish peroxidase

Striking differences of physical properties for two different phenylalanyl-tRNA synthetases inCyanophora paradoxa

Extracts of Cyanophora paradoxa contain 2 activities for phenylalanyl-tRNA synthetase (PRS), when assayed with yeast and eubacterial tRNA as substrate. These enzymes can be separated by salting-out chromatography. Subsequent gel filtration revealed a striking difference in Mr between the 2 enzymes. Whereas the Mr of the enzyme aminoacylating yeast tRNA is 260 000, typical for PRS from lower eukaryotic organisms, the second enzyme has a unique Mr of 80 000. It aminoacylates eubacterial tRNA, but shows no immunological relationship to the corresponding enzyme from Escherichia coli.

Striking differences of physical properties for two different phenylalanyl-tRNA synthetases in Cyanophora paradoxa

Extracts of Cyanophora paradoxa contain 2 activities for phenylalanyl-tRNA synthetase (PRS), when assayed with yeast and eubacterial tRNA as substrate. These enzymes can be separated by salting-out chromatography. Subsequent gel filtration revealed a striking difference in Mr between the 2 enzymes. Whereas the Mr of the enzyme aminoacylating yeast tRNA is 260 000, typical for PRS from lower eukaryotic organisms, the second enzyme has a unique Mr of 80 000. It aminoacylates eubacterial tRNA, but shows no immunological relationship to the corresponding enzyme from Escherichia coli.

Purification and properties of phenylalanyl-tRNA synthetase from a higher plant (Phaseolus vulgaris).

Phenylalanyl-tRNA synthetase from beans (Phaseolus vulgaris) was purified 2 800-fold to homogeneity with a 16% overall yield by salting-out chromatography, salting-out affinity chromatography, gel filtration and chromatography on DEAE-cellulose and hydroxylapatite. This combination minimizes potentially harmful effects of proteinases and products of the secondary metabolism of a green plant during the early steps. The molecular mass is 260 000 Da with a subunit structure of alpha 2 beta 2 (alpha = 59 000, beta = 70 000 Da). Enzymatic activity was optimal with 20mM Mg2+ and 10mM KCl at pH 6.5 and pH 8.5, depending on the buffer substance. Kinetic measurements at low temperature and steady-state kinetics indicate that the esterification of tRNA or a step preceding it, but not the activation, are rate-determining at pH 7.65. The cognate tRNAPhe is exclusively aminoacylated at the 2'-OH group. tRNAs from Escherichia coli and bean chloroplasts are not aminoacylated. No immunological relationship of the plant enzyme to other phenylalanyl-tRNA synthetases was revealed by immuno-diffusion and immunotitration with polyclonal antibodies raised against the enzymes from E. coli, yeast and hen liver. ATP analogs revealed a unique pattern of substrate properties with indication of conservation of ATP binding in the form of an ATP-Mg2+ complex in the anti-conformation with a coordination of the cation to the nitrogen in position 7 of the purine moiety.

Purification by salting-out chromatography and properties of phenylalanyl-tRNA synthetase from turkey liver.

Purification by salting-out chromatography and properties of phenylalanyl-tRNA synthetase from turkey liver.

Automated purification of human erythrocytic 6-phosphogluconate dehydrogenase.

Human 6-phosphogluconate dehydrogenase (6PGD) was purified from hemolyzate by group affinity chromatography on 2',5'-ADP-Sepharose, followed by buffer exchange chromatography on Sephadex G-25 and finally salting-out chromatography on Sepharose 6B. An apparatus was assembled from commercially available elements, in which the purification procedure can proceed and be completed unattended and under fully automatic control. The weekly production of the set-up is at present 10 mg of pure 6PGD. The choice of the purification of procedure and the advantages of automation are discussed.