Eluent Solution Research Articles

Determination of adenosylhomocysteine in urine of immunodeficient children

A sensitive anion exchange separative procedure is described for the determination of adenosylhomocysteine in urine. Anionic components of the urine are first removed at a pH of 5 on a short anion exchange column, formate form. The column wash is then adjusted to pH 10 and applied to a longer anion exchange column, bicarbonate form. Elution is carried out with a sodium chloride gradient with the eluant solution containing 0.01 m sodium bicarbonate and 0.01 m sodium tetraborate. Adenosylhomocysteine forms a borate complex, giving an additional negative charge, and the separation of adenosylhomocysteine from adenine is enhanced. Adenine and adenosylhomocysteine in eluant fractions are detected after conversion to the fluorescent etheno derivatives. Using this assay procedure, we have analyzed urine specimens from seven children with severe combined immunodeficiency diseases and from three children with milder immunodeficiency disorders. Three of the children with severe combined immunodeficiency had excretion levels of adenosylhomocysteine (0.6–1.8 nmole/μmole creatinine) that were more than three standard deviations above the excretion levels noted for normal children (0.14 ± 0.07 nmole/μmole creatinine). The possible significance of these findings is discussed.

A simple and effective solvent system for elution of gonadotropins from concanavalin A affinity chromatography

Elution of gonadotropins and other glycoproteins from concanavalin A has been previously performed with competitive ligands such as α-methyl- d-mannopyranoside and α-methyl- d-glucopyranoside. In comparative studies we found that 1 m NH 4OH was a highly effective eluent solution and gave nearly complete recovery of gonadotropin as revealed by protein contents, immunological, and biological activities. Advantages of 1 m NH 4OH over the sugar derivatives are its low cost and volatile nature, and its ability to irreversibly inactivate concanavalin A. The use of this system permits further steps of purification to be performed directly or after lyophilization without dialysis.

Abhängigkeit der Calcium-Isotopenseparation bei der Ionenaustauschchromatographie von der HNO3-Elutionsmittelkonzentration / Calcium Isotope Separation by Ion Exchange Chromatography in Dependence on the HNO3 Eluant Concentration

Abstract Using a strongly acidic cation exchanger resin, the dependence of the calcium isotope separation on the HNO3 eluant concentration is investigated on column experiments. The elementary separation effect of the isotope ratios 44Ca/40Ca and 48Ca/40Ca is determined. Up to HNO3 concentrations of about 1.5 M the lighter calcium isotopes are enriched in the resin phase, whereas, using more concentrated HNO3 solutions, the heavier isotopes are enriched in the ion exchanger. A comparison of these results with those using hydrochloric acid and perchloric acid show, that there is a strong dependence of the isotope separation on the concentration and the counter ion of the eluant solution

An alumina column 82Rb generator

The use of an alumina column for the adsorption of radioactive Sr for the generator production of 75-s 82Rb was evaluated in both batch and column experiments using commercially available 85Sr and cyclotron-produced 82Sr. Comparisons of alumina, Bio-Rex 70 and Chelex 100 ion exchangers were made to determine Sr adsorption, 82Rb elution yield and Sr breakthrough. The adsorption of Sr is similar for alumina and Chelex 100 but different for Bio-Rex 70. Alumina and Chelex 100 have a higher breakthrough of Sr in the early elution volumes. After continued elution with 200–300 ml of solution the breakthrough of 82Sr decreases and remains stable at a lower breakthrough value through a large number of elutions. However, with Bio-Rex 70 the breakthrough of 82Sr remains the lowest of the three ion exchangers in the early elution volumes and remains low through a moderate number of elutions after which Sr breakthrough begins to increase steadily. The alumina-column 82Rb generator was found to be superior to Bio-Rex 70 or Chelex 100 for low breakthrough of 82Sr after many elutions of 82Rb with up to 3.1. of eluent solution.

Fluorescent peptide mapping with microgram quantities of protein

A microbore column chromatograph utilizing constant pressure eluent and reagent solution pumps is combined with fluorescent detection using o-phthalaldehyde. This yields a versatile, highly sensitive method of separating and detecting peptide fragments obtained from microgram quantities of protein.

Automatic Analysis for Sugars by Liquid Chromatography

A quantitative determination by a simple detection system for carbohydrates with elution chromatography was developed. This method depends upon the detection of ultraviolet absorbing chromophere, with absorption maximum in the 260-270 mμ region, produced by heating the carbohydrates in the eluent solution without reagent. In the product from fructose, the existence of furfural and formic acid was confirmed . The heating condition and eluant buffer (conc., pH) affected the intensity of ultraviolet absorption produced. Under the condition of heating below 190°C and shorter than 20 min, the greater efficiency was obtained at higher temperature and longer period . Higher concentration and neutral pH of borate buffer were more effective . The molar absorption coefficient at 260 mp of mono- and oligosaccharides heated in a solution of 0.8 M borate buffer pH 7.0 for 9.5 min at 190°C was 500-3000 . The coefficient of variation of this method was less than 3.5%. A heating period was obtained by adjusting the diameter and length of teflon tube connected with the chromatographic column. In order to decrease the extra column effect, it was effective to decrease the diameter of tube (0.5 mm) and heating period, and increase the heating temperature . Several examples for mono- and oligosaccharides and oligodextrines were shown.

Molecular aggregation in poly(vinyl chloride)

PVC synthesized in a bulk polymerization at −25°C was fractionated by preparative GPC. The eluent THF solutions consist largely of dissociated molecules. Following precipitation into methanol, recovered PVC fractions contain aggregated molecules and are partially insoluble in THF. The soluble portions produce distorted chromatograms characteristic of molecular aggregation. The aggregate fraction increases with molecular weight. Heating recovered PVC fractions at 90°C in THF was adequate to dissociate the aggregates. Following recovery, fractions of PVC do not show crystalline x-ray diffraction patterns. The PVC fractions were crystallizable by subsequent annealing at 150°C to approximately 15% crystallinity, largely independent of molecular weight.

A new sensitive ultraviolet detection system for carbohydrates eluted during column chromatography

A detection system for carbohydrates eluted from chromatographic columns has been developed that is simple and sensitive. It relies upon the production of an ultraviolet-absorbing chromophore by reaction of the carbohydrate in the eluent solution dynamically with sulfuric acid.

Ion exchange chromatography of proteins. Artifact separations resulting from inadequate pH control.

Abstract The ion exchange chromatography of wheat proteins on carboxymethyl cellulose is described. As the concentration of buffer in the eluant is reduced, a higher concentration of sodium ion is required to elute a give protein component; and concurrently the pH of the eluant is observed to deviate from the original pH of the eluant. It is suggested that these effects are related and that they arise from the ion exchange process itself as a result of the non equivalence of eluant ions and charged protein groups. The general conclusion that eluant solutions for protein ion exchange chromatography should be effectively buffered to reduce the possibility of artifact separations also appears to apply to chromatography with DEAE cellulose and the other adsorbents.

Mixing device for generating simple chromatographic gradients

A linear gradient device consisting of two concentric reservoirs of equal cross-sectional area, in which the initial and limiting eluent solutions are maintained in hydrostatic equilibrium, may be constructed from several combinations of commercial acrylic resin cylinders. By the use of nesting cylinders, the areas of the reservoirs may be reduced independently, allowing an assortment of nonlinear gradients to be produced by simple adjustment of the area ratio of the two reservoirs.

The Separation of Tellurite, Selenite and Sulfite Ions by Anion-Exchange Resins

Abstract 1. The distribution coefficients of tellurite, selenite and sulfite in neutral nitrate solution and alkaline hydroxide solution were measured and the results suggest the presence of an acid salt ion HXO3− in neutral solution. 2. By the addition of ammonia to the eluant solution, the distribution coefficient of these salts in nitrate solution was increased, but in hydroxide solution it was decreased. 3. By the addition of ethanol to the eluant solution containing nitrate and ammonia, the distribution coefficient of sulfite increased, but that of tellurite and selenite remained constant even up to 75%. On the contrary, in the addition of ethanol to the hydroxide solution, the distribution coefficient of sulfite decreased, while that of tellurite and selenite increased at first and then decreased. 4. By using these results a complete separation of tellurite, selenite and sulfite was accomplished by the anion exchange chromatographic method using an alkaline eluant.

The Separation of Sulfate, Sulfite, Thiosulfate and Sulfide Ions with Anion-Exchange Resins

Abstract 1. The distribution coefficients of sulfate, thiosulfate, sulfite and sulfide in the nitrate solution were measured. 2. By the addition of ethanol or acetone to the neutral eluant solution, the adsorb-ability of sulfate, sulfite, thiosulfate and sulfide was increased, while that of sulfite in alkaline acetone solution showed a marked decrease. 3. By the use of these results a mutual separation of sulfate, thiosulfate, sulfite and sulfide was effected by the anion-exchange chromatography.