Ion-exchange chromatography in lunar organic analysis

Although ion exchange chromatography has been used in separating amino acids from mineral salts, quantitative recovery has not been possible for the basic amino acids or for subnanomole concentrations of amino acids.As an analytical tool for amino acid analysis, ion-exchange chromatography has made it possible to resolve a relatively complex mixture of amino acids in less than an hour with detection limits of less than 10^–12 moles of amino acids. Reasonable specificity for amino acids is achieved by multiple wavelength detection of the reaction product found with ninhydrin. Unequivocal specificity must be obtained in conjunction with other methods such as mass spectrometry.In the analysis of subnanomole levels of amino acids, it is necessary to carry both reagent blanks and low-level amino acid standards through the entire sample preparation step since both contamination and selective losses occur and must be monitored.     

Ion-exchange high-performance liquid chromatography of oligodeoxyribonucleotides using formamide

The superiority of buffer systems containing formamide for the ion-exchange high-performance liquid chromatographic separation of oligodeoxyribonucleotide mixtures generated in solid-phase syntheses is illustrated. The resolutions achieved are compared to those achieved with the same mixtures in other eluting solvents. The use of formamide systems is recommended for oligodeoxyribonucleotide purification in general and is particularly valuable where the oligonucleotide of interest is highly self-complementary and/or rich in deoxyguanosine residues.     

Ion-exchange and affinity chromatography costs in alpha-galactosidase purification

The purification of alpha-galactosidase from soybean seeds is a five to six-step procedure consisting of cryoprecipitation, acid precipitation and ammonium sulfate fractionation followed by two or three chromatography steps. The procedures, while not optimized, were carried out in a manner that resulted in 414-515-fold purification, as reported previously. The costs of two purification sequences were compared. In the best case, the preparative-scale costs of stationary phase, reagents, and hardware were $790 per million enzyme units, excluding labor. Stationary phase costs predominated over extraction, chromatography reagent, and eluent costs when the stationary phase is replaced after 10-40 cycles of use. However, if stationary phase life exceeds 50-200 cycles, stationary phase costs become similar in magnitude to eluent and reagent costs. Labor costs, which are process-specific and difficult to estimate, exceed all other costs by a factor of 10-50 at a small scale of operation and constitute a major cost, regardless of scale. This case study provides equations and a frame-work for carrying out a first comparison of costs for multistep purification sequences. Column life, throughput, and scale of operation were found to determine not only the magnitude, but also the relative contributions, of the different components that make up purification costs. This analysis shows that there are major opportunities for reducing purification costs through the development of less expensive stationary phases and the implementation of intelligent process control and automation for process scale chromatography.     

Ion-exchange thin-layer chromatography and paper ionophoresis of dinucleoside monophosphates

The thin-layer chromatography of dinucleoside monophosphates on plates coated with DEAE-cellulose powder and on plates coated with cellulose impregnated with polyethyleneimine, together with their ionophoretic mobilities on DEAE-cellulose paper, is described. It is shown that the 2′→5′ dinucleoside monophosphates can be separated from the corresponding 3′→5′ isomers by ion-exchange thin-layer chromatography and paper ionophoresis. The method is very sensitive and can replace the commonly used enzymic hydrolysis for analyzing the nature of the phosphodiester linkage of a given dinucleoside monophosphate.     

Ion-exchange high-performance liquid chromatography analysis of oligodeoxyribonucleotide phosphorothioates.

Oligodeoxynucleotide-containing phosphorothioate backbones have been used to regulate viral as well as cellular gene expression. The studies carried out in tissue culture have shown promising results on the use of oligonucleotide phosphorothioates as antiviral agents and, at present, study is underway to develop these oligonucleotide analogues as chemotherapeutic agents. To analyze and purify oligonucleotide analogues, high-performance liquid chromatography using weak anion exchange column has been described. The separation of oligonucleotide phosphorothioate is found to be length dependent.     

Ion-exchange chromatography of biologically important phosphate esters and other compounds

Borate complexed sugars, sugar phosphates, and nucleotides present in tissue extracts were separated and quantitated in 4 hr. An anion-exchange resin column and a programmed borate/acetate buffer gradient were used. Sugar residues were determined by a very sensitive orcinol/H₂SO₄ reaction. Samples required little preparation, and recovery of standard compounds added to tissue extracts was quantitative.     

Ion-exchange displacement chromatography of serum proteins, using carboxymethyldextrans as displacers

A system of displacers comprising carboxymethyldextrans with progressively higher content of carboxyl groups forms a displacement train in which absorbed proteins find positions according to their affinities for the adsorbent, an anion exchanger. Because little or no salt need be used, effluent fractions can be evaluated directly by gel electrophoresis. Application to the fractionation of serum proteins is demonstrated.     

Immunoaffinity chromatography of enzymes.

Immunoaffinity chromatography of enzymes represents an attractive purification technique suitable for one-step and large-scale purification of enzymes to homogeneity. Monoclonal and polyclonal antibodies can be used equally well. The broad use of the technique is restricted by the harsh elution conditions which are often required. The efforts to overcome these limitations and to optimize the method are reviewed, viz. proenzyme purification, purification of enzymes as part of multienzyme complexes carried out by a mild dissociation step, specific elution by substrates and effectors, enzyme stabilization, electrophoretical desorption and negative elution by adsorbing impurities from the crude extract, and hypotonic elution. The current practice is discussed considering antibody and enzyme selection, optimization of elution conditions, and washing steps using different media. Representative examples are given for various approaches.     

Ion-exchange chromatography of a dinucleotide preparation from controlled alkaline hydrolysis of ribonucleic acids

With the aim of preparing the 16 possible ribodinucleotides derived from the four principal ribonucleotides, we made a kinetic study to determine the optimum conditions for a partial alkaline hydrolysis of RNA. Satisfactory results were obtained when the hydrolysis of RNA (1g.) in 0.2m-sodium hydroxide (100ml.) at 37 degrees was ceased approx. 10min. after the start of the reaction. The relative yields of the monomer, dimer and trimer fractions were approx. 41:30:15 in E(260) units, indicating that the reaction conditions were reasonably close to those required from kinetic considerations. The partial alkaline hydrolysate was chromatographed on a column of DEAE-Sephadex A-25 in the presence of 7m-urea. The dinucleotide fraction thus obtained was subjected to a subsequent chromatography on Dowex 1 (X2) under acidic conditions to separate the mixture according to base composition and base sequence. The results were satisfactory, and most of the 32 dinucleotides [i.e. 16 XpYp(3') plus 16 XpYp(2')] were fractionated by this single chromatographic procedure. The present method should be useful for further study of oligonucleotides as a time-saving method for the simultaneous preparation of a variety of dinucleotides. Further, examination of the present chromatographic pattern has provided several empirical criteria useful for the identification of oligonucleotides other than dimers appearing in the elution profile of Dowex 1 (X2) column chromatography under similar conditions.     

Ion-exchange chromatography of sulfur amino acids on a single-column amino acid analyzer.

Examination of the extent of production of the ninhydrin-colored derivative, Ruhemann's purple, under automated conditions of a single-column amino acid analyzer by several classes of sulfur-containing amino acids revealed a wide variation in the color factors relative to leucine. These ranged from 0.02 for the methyl ester of cysteine to 2.19 for D-homocystine. Color yields obtained by the manual ninhydrin reaction are generally lower than the corresponding values obtained on the amino acid analyzer. The elution positions ranged from 5.12 min for cysteic acid to 84.9 min for l-cystine dimethyl ester. The observed behavior of these compounds in the ninhydrin reaction is rationalized in terms of structural and electronic factors which they exhibit in reacting with ninhydrin to form the visible dye. Such an analysis should make it possible to predict ninhydrin color factors, and possibly also elution times, of structurally related compounds.     

Ligand-exchange chromatography of proteins and enzymes

Current state of the ligand-exchange chromatography (metal chelate affinity chromatography) of proteins and enzymes is reviewed. This technique is based on the ability of proteins to bind metal ions immobilized on chelate gels. The influence of pH, composition of buffer, type of stationary ligand and nature of metal ions on the chromatographic behaviour of proteins is discussed.     

Ion-exchange chromatography on cholesterol oxidase from Actinomyces lavendulae on a Biocarb-type carboxyl cationite

Reversible sorption on the Biocarb-D carboxyl cationite of cholesterol oxidase extracted from the mycelium of Actinomyces lavendulae was examined. Sorption at pH 5.0 and desorption at pH 5.5 maintained 80% of the enzyme activity and increased seven-fold the specific activity as calculated per protein. By gel chromatography on Sephadex G-150 it was shown that the process was accompanied by a significant decrease in the number of inactive proteins.