High-pressure Ion-exchange Chromatography Research Articles

Opportunities and challenges of high-pressure ion exchange chromatography for nuclide separation and enrichment

With the rapid development of the nuclear industry, more-stringent requirements are proposed for high-level radioactive waste liquid treatment and the enrichment of isotope products. High-pressure ion exchange chromatography has been widely accepted for the fine separation of elements and nuclides due to its advantages, such as high efficiency, environmental friendliness, ease of operation, and feasibility for large-scale industrial applications. Here, we summarized the evolution of high-pressure ion exchange chromatography and the relevant research progress in ion exchange equilibrium and related separation technology. The prospects for application of high-pressure ion exchange chromatography to rare earth elements, actinide elements and isotope separation were discussed. High-pressure ion exchange chromatography represents a promising strategy for the extraction of rare earth elements and actinide elements from high-level radioactive waste liquid, as well as being an effective method for the automated production of high purity isotope products with great environmental benefits. The separation of radionuclides from radioactive liquid are essential for not only the recovery of valuable radionuclides, but also the purification treatment of radioactive waste water. High-pressure ion exchange chromatography has been widely accepted for the fine separation of elements and nuclides due to its advantages including high efficiency, environmental friendliness, ease of operation, and feasibility for large-scale industrial applications.

High-precision measurement of phenylalanine and glutamic acid δ15 N by coupling ion-exchange chromatography and purge-and-trap continuous-flow isotope ratio mass spectrometry.

Nitrogen isotopic compositions (δ15 N) of source and trophic amino acids (AAs) are crucial tracers of N sources and trophic enrichments in diverse fields, including archeology, astrobiochemistry, ecology, oceanography, and paleo-sciences. The current analytical technique using gas chromatography-combustion-isotope ratio mass spectrometry (GC/C/IRMS) requires derivatization, which is not compatible with some key AAs. Another approach using high-performance liquid chromatography-elemental analyzer-IRMS (HPLC/EA/IRMS) may experience coelution issues with other compounds in certain types of samples, and the highly sensitive nano-EA/IRMS instrumentations are not widely available. We present a method for high-precision δ15 N measurements of AAs (δ15 N-AA) optimized for canonical source AA-phenylalanine (Phe) and trophic AA-glutamic acid (Glu). This offline approach entails purification and separation via high-pressure ion-exchange chromatography (IC) with automated fraction collection, the sequential chemical conversion of AA to nitrite and then to nitrous oxide (N2 O), and the final determination of δ15 N of the produced N2 O via purge-and-trap continuous-flow isotope ratio mass spectrometry (PT/CF/IRMS). The cross-plots of δ15 N of Glu and Phe standards (four different natural-abundance levels) generated by this method and their accepted values have a linear regression slope of 1 and small intercepts demonstrating high accuracy. The precisions were 0.36‰-0.67‰ for Phe standards and 0.27‰-0.35‰ for Glu standards. Our method and the GC/C/IRMS approach produced equivalent δ15 N values for two lab standards (McCarthy Lab AA mixture and cyanobacteria) within error. We further tested our method on a wide range of natural sample matrices and obtained reasonable results. Our method provides a reliable alternative to the current methods for δ15 N-AA measurement as IC or HPLC-based techniques that can collect underivatized AAs are widely available. Our chemical approach that converts AA to N2 O can be easily implemented in laboratories currently analyzing δ15 N of N2 O using PT/CF/IRMS. This method will help promote the use of δ15 N-AA in important studies of N cycling and trophic ecology in a wide range of research areas.

Effect of Autohydrolysis Pretreatment Conditions on Sugarcane Bagasse Structures and Product Distribution Resulting from Pyrolysis

AbstractPyrolysis has been increasingly perceived as a promising technology to produce biofuel precursors (bio‐oil) from agricultural residuals; however, there is a significant quality gap between a bio‐oil and the fuels used for transportation. In this study, we autohydrolyzed pretreated sugarcane bagasse at three different conditions (180 °C–10 min, 180 °C–40 min, 200 °C–40 min), then we investigated the effect of this pretreatment on a subsequent pyrolysis stage. High‐pressure ion‐exchange chromatography (HPIC) and the 13C cross‐polarization/magic angle spinning (CP/MAS) solid‐state nuclear magnetic resonance (NMR) revealed that the autohydrolysis pretreatment significantly disrupted the hemicellulose fractions in the sugarcane bagasse and caused the breakage of lignin ether linkages in the sugarcane bagasse feedstocks. As the 31P NMR results indicated, the autohydrolysis pretreatment removed carboxylic acid groups up to 66.7 %, which could significantly address the corrosion problem of bio‐oils. Heteronuclear single quantum correlation (HSQC) analysis suggested that the autohydrolysis pretreatment effectively lowered the presence of the oxygenated aromatic compounds in the bio‐oils. Gel permeation chromatography (GPC) analysis of the bio‐oils indicated that the oils from severely pretreated sugarcane bagasse pyrolyzed at a low temperature (i.e., 400 °C) contained lower‐molecular‐weight components similar to those present gasoline products.

Purification and partial characterization of a shed 66 kDa melanoma-associated antigen identified by autologous antibody

We have previously reported the isolation of a 66 kDa melanoma-associated antigen, identified by autologous antibody, in serum and unfractionated spent tissue culture media by Western blot analysis. The antigen, detected by autologous serum S150, was found to be broadly represented on melanoma, glioma, renal cell carcinoma, neuroblastoma and head and neck carcinoma cell lines. S150 did not react with bladder or colon carcinoma, fetal fibroblasts, pooled platelets, lymphocytes and red blood cells, autologous cultured lymphocytes or fetal calf serum. To further characterize the antigen, spent tissue culture media, obtained from autologous melanoma cell line, Y-Mel 84:420, was separated by an isoelectric focusing column. Unabsorbed control serum S150 was noted to have a maximum titer of 1:2040 against autologous melanoma cells as measured by protein A hemadsorption. Following isoelectric focusing the greatest decrease in autologous antibody titer (30-fold) occurred with fractions having a pI between 2 and 3. Further resolution of the antigen was accomplished with high-pressure ion-exchange chromatography. One of these fractions showed a significantly higher concentration of antigen and was distinctly resolved from bulk serum albumin. Subsequent Western blot analysis, with autologous antibody, of the isolated antigen-containing fraction, confirmed the presence of a single 66 kDa band. Exposure of the antigen, purified by high-pressure ion-exchange chromatography, to neuraminidase ablated recognition by autologous antibody and suggests that sialic acid is present on the protein and may be part of the antigenic epitope. Binding of antigen, obtained following DEAE anion exchange chromatography, was noted to lectins derived from Triticum vulgaris, Dolichos biflorus and Lycopersicon esculentum. Preparative purification of the antigen was accomplished by anion exchange followed by lectin affinity chromatography with a Dolichos biflorus column. Antigen obtained following lectin affinity chromatography subjected to SDS-PAGE and silver stain revealed a single band at 66 kDa. We conclude that a melanoma-associated antigen detected by autologous antibody in spent tissue culture media is an unusually acidic glycoprotein (pI 2-3).

Clostridium botulinum type D neurotoxin: Purification and detection

K. S. De Jongh C. L. Schwartzkoff and M. E. H. Howden. Clostridium botulinum type D neurotoxin: purification and detection. Toxicon 27, 221–228, 1989.—A method is reported for the purification of type D botulinum toxin using a combination of low and high pressure ion exchange chromatography. The procedure produced homogeneous toxin in its free form in 3 days, with a specific toxicity in mice of 5.4 × 10 7 ld 50/mg protein. Polyclonal antibodies against the pure toxin were raised in rabbits and detected the toxin in both ELISA and western blotting. The antibodies also detected type C1 botulinum toxin using these techniques, confirming the presence of cross-reacting antigenic determinants in these two proteins.

Purification and partial sequence analysis of murine B cell growth factor II (interleukin 5).

Murine B cell growth factor II (BCGF-II/interleukin 5) was purified from the conditioned media of the helper T cell line D10 . G4 . 1. The purification scheme consisted of sequential batch adsorption onto trimethylsilyl-controlled pore glass beads, high pressure ion exchange chromatography, and reverse phase high pressure liquid chromatography. The purified BCGF-II had a relative molecular weight of 45,000 when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. Identical analysis of BCGF-II under reducing conditions yielded a m.w. of 22,500, suggesting that native BCGF-II exists as a homodimer. The NH2-terminal amino acid sequence of the purified lymphokine was determined by automated Edman degradation. A single amino acid sequence of 24 residues was obtained that, upon comparison, was contained within the cDNA pSP6K-mTRF23 recently described as encoding murine BCGF-II/T cell-replacing factor. The NH2-terminal methionine in mature BCGF-II is found at position 21 of the amino acid sequence predicted from the cDNA pSP6K-mTRF23. This finding supports the contention of Kinashi et al. (Kinashi, T., N. Harada, E. Severinson, T. Tanabe, P. Sideras, M. Konishi, C. Azuma, A. Tominaga, S. Bergstedt-Lindqvist, M. Takahashi, F. Matsuda, Y. Yaoita, K. Takatsu, and T. Honjo. 1986. Nature 324:70) that amino acids 1-20 serve as the signal sequence for the BCGF-II gene. The ability of BCGF-II to stimulate the proliferation of the B cell lymphoma BCL1 was used to assess the potency of the lymphokine. BCGF-II at 13.5 pM induced 50% of the maximal proliferative response in the BCL1 cells; concentrations as low as 2 pM were still effective in stimulating the growth of the cells. Assuming that the amount of BCGF-II necessary to mount a 50% response in the BCL1 assay is defined as one unit of activity, then the purified BCGF-II has a specific activity of 16.5 U/ng of protein.

Purification and characterization of murine protoporphyrinogen oxidase.

The penultimate enzyme of the heme biosynthetic pathway, protoporphyrinogen oxidase (EC 1.3.3.4), has been purified to apparent homogeneity from mouse liver mitochondria. The purification involves solubilization from mitochondrial membranes with sodium cholate followed by ammonium sulfate fractionation and gel filtration on a Sepharose CL-6B column. The eluate is adjusted to 0.67 M (NH4)2SO4 and loaded onto a phenyl-Sepharose column. After salt washes, the enzyme is eluted with 0.5% sodium cholate and 0.5% Brij 35. The final step is high-pressure ion-exchange chromatography on a DEAE-5PW column. The purified protein has a molecular weight of approximately 65,000 by gel filtration chromatography on Sepharose CL-6B in the presence of 0.5% sodium cholate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows a single band corresponding to a molecular weight of 65,000. The absorption spectrum of the purified enzyme shows no evidence of a chromophoric cofactor. Purified protoporphyrinogen oxidase has a Km for protoporphyrinogen IX of 5.6 microM with a Vmax of 2300 nmol mg-1 h-1. It utilizes meso- and hematoporphyrinogen at about 10% the level of protoporphyrinogen. The pH optimum is broad with a maximum at 7.1. There is no stimulation or inhibition by any tested divalent cations, and sulfhydryl reagents have no inhibitory effect on the purified enzyme.

Age-related variations in the distribution of crystallins within the bovine lens

The native water-soluble proteins of equator, anterior cortex, posterior cortex and nucleus from bovine lenses in the age range 0·3–33·7 years were analyzed by high-pressure gel-permeation chromatography and high-pressure ion-exchange chromatography. Unlike the equator and cortices, the nucleus shows a gradual decrease in α-crystallin proportion with age which is not compensated for by an increase in HM-crystallin. The β H 6-crystallin species, almost the only β H-component in the youngest lens, is largely replaced by at least four fractions with higher and lower molecular weights in the older lenses. In the nucleus a β L-component (39 000 MW) increasingly seems to replace the major β L-crystallin ( β L 2, 50 000 MW). Moreover, a switch in the synthesis of monomeric crystallins is demonstrated. This study clearly reveals an age-related increase in the size heterogeneity of the native soluble crystallins with age.

Effect of activated charcoal administration on acetylcysteine serum levels in humans

The effect of orally administered activated charcoal on the absorption of acetylcysteine in three men was studied. Each of the three volunteers was given 140 mg/kg acetylcysteine as a single oral dose. Blood samples were taken at 0, 0.5, 1, 1.5, 2, 4, 6, and 8 hours after acetylcysteine ingestion. Following a one-week washout period, each subject was given orally 50 g of Activated Charcoal, USP, 15 minutes before a 140-mg/kg acetylcysteine oral dose. Blood samples were taken. Acetylcysteine content in the plasma was determined with a high-pressure ion-exchange chromatography system using a gold-mercury electrochemical cell. No statistically significant differences were found between total areas under the curves, peak concentrations, or time of peak concentrations of acetylcysteine given with or without charcoal. Although acetylcysteine absorption was delayed in two patients, the differences were not significant. These data suggest that acetylcysteine absorption is not impaired by activated charcoal administration. This conflicts with previously published in vitro data; therefore, it is recommended that activated charcoal should not be administered concomitantly with acetylcysteine as a usual procedure until more data are available.

Determination of 5-fluorouracil in human plasma by high-pressure ion-exchange chromatography.

A method has been developed for the measurement of 5-fluorouracil by isocratic high-pressure ion-exchange liquid chromatography. Chlorpheniramine was added to the standards and samples before extraction with 16% isopropanol in ethyl acetate. The extracts were then chromatographed using a mobile phase of 0.014 mol/l ammonium dihydrogen phosphate buffer, pH 3.5, at a flow rate of 1 ml/min. Ultraviolet detection at 266 nm was used, and quantitation was by measurement of the peak height ratios of 5-fluorouracil to chlorpheniramine.

Presence of four toxins in red tide infested clams and cultured [formula omitted] cells

Four toxins have been isolated by Sephadex G-15 and high pressure ion exchange chromatography from the soft shell clams, Mya arenaria , which were collected during 1972 and 1974 red tide outbreaks on the New England coast. One of the toxins was saxitoxin and the rest seem to be new toxins. The same toxins were isolated from the extract of cultured Gonyaulax tamarensis .

Separation of some chloramphenicol intermediates by high-pressure ion-exchange chromatography

High-pressure cation-exchange chromatography was used to separate three of the by-product compounds formed in the chloramphenicol production process. Baseline separation can be achieved within 8.5 min employing a 1-m column packed with Zipax SCX pellicular cation exchanger and 0.05 M sodium sulphate solution as eluent at pH 1.1 in a Varian Aerograph LC 4020 liquid chromatograph.

High-pressure ion exchange chromatography.

High-pressure ion exchange chromatography.

3] Separation, purification and analysis of cyclic nucleotides by high pressure ion exchange chromatography

High-pressure anion exchange chromatography has been used to measure cyclic AMP (cAMP) produced by adenylate cyclase and to measure cAMP in tissues and in patient urine. In addition, it has been shown useful in the measurement of cyclic GMP (eGMP) and cAMP analogs. The anion exchange resin used in this method is that originally described by Horvath. Anion exchange resin is polymerized upon small glass beads and has been termed “pellicular” resin. The glass bead matrix eliminates changes in column volume when the ionic strength is altered and markedly reduces the bleed of ultraviolet absorbing materials from the resin. The ability to obtain good results at the picomole level is dependent upon optimal performance of the mechanical and electronic systems.

High-Pressure Ion Exchange Chromatography as Applied to the Separation of Complex Biochemical Mixtures

Abstract Interest in liquid column chromatography, including ion exchange chromatography, as a separation method has increased markedly in the past few years. Numerous new automated analytical techniques, as well as applications for preparative or production-scale separations, have been developed. This has been particularly true in the areas pertaining to separation and analysis of biochemical mixtures such as physiologic fluids. The recent trend in ion exchange chromatography has been toward achieving two goals: high-resolution analysis in the case of complex mixtures, and high-speed separation when simpler mixtures are involved. In either case, the use of small-diameter ion exchange resin particles coupled with high flow rates (and, in some cases, long columns) requires operation at relatively high column inlet pressures, since the pressure drop through the ion exchange column is dependent on factors such as resin particle size, flow rate, and column length.

Identification of doxapram metabolites using high pressure ion exchange chromatography and mass spectroscopy.

Abstract1. High pressure ion exchange chromatography has been used for separation of metabolites of doxapram from whole dog urine. Thin layer chromatography was used for additional separation and clean-up.2. The structures of the metabolites thus isolated were determined by mass spectroscopy.3. Seven metabolites were separated and identified.

Computerized Automation in Acquisition and Processing of Patterns from High-Pressure Ion-Exchange Chromatography

Abstract Fully automated generation, recording, storing, and processing of complex chromatograms demand sophisticated laboratory instrumentation and computer implementation. In the high-resolution liquid-chromatographic system for body fluids analyses we use in our clinical laboratory, the chromatograph is interfaced to a 2114 Hewlett-Packard minicomputer that accomplishes, in an on-line mode, analog-to-digital conversion, reductive averaging of sampled data, transformation from transmittance to absorbance units, and storage of the digitized spectrum on a peripheral disk. The information on disk is eventually transferred to a library of spectral files resident on a direct-access disk associated with an IBM 360/ Model 65 computer. These data files serve as the input source for program COCOA, written in Fortran IV for the IBM 360 computer, which provides the following processing: (a) iterative polynomial smoothing; (b) sectionally linear baseline tracking; (c) peak and envelope detection and delineation; (d) least-squares gaussian resolution of up to five component peaks in an envelope; and (e) location, size, and shape quantification of peaks. The capability and merits of program COCOA as an off-line procedure on a maxicomputer for the analysis of chromatograms are discussed.