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PubMed1984

Potentials of capillary gas chromatography in toxicology today.

The usefulness, the applicability and the limitations of capillary gas chromatography in various types of toxicological analysis were discussed. Technical parameters of capillary gas chromatography, such as type of injector, type of column with regard to material, size, and stationary phase were assessed in view of the requirements of toxicological analysis. The main advantage of capillary gas chromatography, i.e. high separation power, is unfortunately counterbalanced by low loadability and/or speed of analysis. In the present situation capillary gas chromatography is a method of choice in drug screening despite some disadvantages, but in drug-oriented analysis the conventional chromatography on packed; columns is probably more useful.

Chromatography, Gas↗
PubMed1979

Aldosterone 18-glucuronide excretion determined with and without chromatography in human hypertensives.

24 h-urine excretion of aldosterone-18-glucuronide was determined with and without preceding paper chromatography in patients with essential hypertension (n = 50). Radioimmunoassay was performed with a highly specific antibody against aldosterone. Urine specimens determined without chromatography gave significantly higher values than aliquots which were estimated at the same time with chromatography. A highly significant correlation was found between the two methods (r = 0.91, p less than 0.0001). Without chromatography, mean over estimation in samples with concentrations in the low and middle range was on average 32 and 24% respectively, whereas aldosterone concentrations in the upper range showed a 14% over estimation. The RIA procedure for aldosterone without chromatography as here described is suitable for hypertension screening programmes to exclude known forms of primary hyperaldosteronism in which elevated aldosterone excretion rates have to be differentiated from values lying within the normal range.

Aldosterone↗
PubMed1983

Ca2+-binding proteins: a comparative study of their behavior during high-performance liquid chromatography using gradient elution on reverse-phase supports.

Reverse-phase high-performance liquid chromatography has been shown to be applicable to the isolation of Ca2+-binding proteins, specifically parvalbumins, from tissue extracts or from preparations first purified by "conventional" chromatography. Through an investigation of the behavior of a series of Ca2+-binding proteins as a function of buffer composition, pH, and organic eluant it has been possible to define mild conditions allowing for chromatography of the proteins in their native states. The elution positions of parvalbumins were not observed to correlate with the "overall" protein hydrophobicity, calculated using hydrophobicity values for the individual amino acids, thus indicating that factors such as hydrophobic/hydrophilic surface areas are important in determining the degree of association with the support. The usefulness of reverse-phase chromatography as an analytical tool for determining protein homogeneity is illustrated. Samples which had been isolated via "conventional" chromatography methods, and thought to be homogeneous, were observed to contain multiple species of the same protein.

Animals↗
PubMed1984

Complete separation of the triplet components of neurofilament by DE-52 column chromatography depends upon urea concentration.

In the separation of the triplet components of neurofilament (P 200, P 160, and P 68) by DE-52 column chromatography in the presence of urea, it was revealed that the efficiency of separation depended upon urea concentration. When chromatography was performed in the presence of 8 M urea and a linearly increasing sodium phosphate concentration from 10 to 400 mM at pH 6.8, P 160 and P 68 were eluted in the same peak, although P 200 was eluted faster. P 160 and P 68 were partially separated with 6 M urea, and completely separated with 4 M urea. But, under these conditions, P 200 was eluted at the same position as contaminated glial fibrillary acidic protein (GFA). From these results, two methods were recommended for the complete separation of the triplet components of neurofilament and GFA by DE-52 column chromatography. In one method, chromatography was performed in the presence of 8 M urea at first, and then P 160 and P 68 were separated in the presence of 4 M urea. In the other method, chromatography was performed with a linearly decreasing urea concentration from 8 to 0 M.

Amino Acids↗
PubMed1991

Electrochemical detection of proteins in high-performance liquid chromatography using on-line, postcolumn photolysis.

Direct detection of proteins in high-performance liquid chromatography electrochemistry (LCEC) is difficult. By using on-line, postcolumn photolysis, proteins now can be detected by LCEC at microgram per milliliter levels. The compatibilities of size exclusion chromatography (SEC), reversed-phase chromatography (RPC), ion-exchange chromatography (IEC), and hydrophobic interaction chromatography (HIC) with photolysis-electrochemical detection is described for proteins together with the analytical figures of merit. Inherent from the advantages of electrochemical detection, the method is sensitive and selective.

Chromatography, High Pressure Liquid↗
PubMed1983

DEAE-Affi-Gel Blue chromatography of human serum: use for purification of native transferrin.

Human serum was subjected to chromatography on DEAE-Affi-Gel Blue which combines ion-exchange and pseudo-ligand-affinity chromatography in a 0.02 M phosphate buffer, pH 7.0. All serum proteins were bound with the exception of transferrin, IgG (immunoglobulin G) and trace amounts of IgA. After a second step of Sephadex G-100 gel chromatography, or affinity chromatography against goat anti-human IgG F(ab')2 coupled to AH-Sepharose 4B, IgG and IgA were removed. The transferrin obtained was homogeneous and of high yield (greater than 80%), and was unaltered as judged by analyses of molecular weight, isoelectric point, iron-binding capacity, antigenicity, and ability to bind to high-affinity specific cellular receptors. Thus, DEAE-Affi-Gel Blue chromatography may be used as the basis for a simple, rapid, two-step method for the purification of large amounts of native transferrin from serum.

Chromatography, Affinity↗
PubMed1982

Identification of an arene oxide metabolite of 2,2',5-5'-tetrachlorobiphenyl by gas chromatography-mass spectroscopy.

Tritiated 2,2'5,5'-tetrachlorobiphenyl (3H-TCB) was incubated with phenobarbital(PB)-induced rat liver microsomes in the presence of an epoxide hydrase inhibitor and brominated analog (BrAO) of the expected metabolic intermediate, 2,2',5,5'-tetrachlorobiphenyl-3,4-oxide (TCBAO). A putative arene oxide intermediate (3H-AO), which was radiolabeled, was separated from 3H-TCB and BrAO by column chromatography, high pressure liquid chromatography (HPLC) and thin-layer chromatography (TLC), and was analyzed for TCBAO by methods that were independent of radiometric techniques. The retention times (Rt's) of TCBAO and 3H-AO on two gas chromatography (GC) columns were the same, both before and after acid catalyzed rearrangement. 3H-AO was further characterized by rearrangement to a mixture of 3- and 4-hydroxy-TCB that was identified by gas chromatography-mass spectroscopy (GC-MS). The rate of TCB metabolism and the production of 3H-AO by liver microsomes from a PB-induced, adult male rhesus monkey was less than that observed with rat microsomes. The 3H-AO from the monkey was also characterized as TCBAO by rearrangement to the characteristic TCB phenols that were analyzed by GC-MS using selective ion monitoring. This study is the first in which an arene oxide of a polychlorinated biphenyl (PCB) was actually isolated as a mammalian metabolite and subjected to direct chemical analysis.

Animals↗
PubMed1975

Lipidex chromatography in the radioimmunoassay of serum and urinary cortisol.

A highly specific method for the determination of cortisol in human serum and urine is described. The sample is first extracted with diethyl ether/ethyl acetate (1 : 1, by vol.), then chromatographed on a highly lipophilic derivative of Sephadex (hydroxyalkoxypropyl Sephadex, Lipidex) in light petroleum/chloroform (1 : 1, by vol.), and finally cortisol is measured by radioimmunoassay using a cortisol-21-BSA antiserum. Bound and unbound radioactivities are separated using dextran-coated charcoal technique. The 8 a.m. values (mean +/- S.D.) of cortisol among 11 young females and 16 young males were 152 +/- 32 ng/ml (range 111-235) and 185 +/- 21 ng/ml (103-232), respectively. The respective values at 4 p.m. were 84 +/- 29 ng/ml (26-117) and 84 +/- 36 ng/ml (32-172). The importance of Lipidex chromatography was demonstrated with assays of serum samples from children with congenital adrenal hyperplasia; without chromatography cortisol values were 6 times those with chromatography. Specific cortisol assays from pregnancy serum also necessitated Lipidex chromatography. Among 11 young men and 9 young women the mean daily urinary cortisol excretion was 56 +/- 26 mug (32-109) and 60 +/- 24 mug (39-109), respectively. Specific urinary cortisol determination could not be achieved without Lipidex chromatography.

Adrenal Glands↗
PubMed1986

Study of anti-DNA antibodies prepared by DNA cellulose or Cibacron blue chromatography.

We prepared anti-DNA antibodies from sera of lupus patients by either DNA cellulose or by Cibacron blue chromatography. Eluates from both columns were studied with respect to recovery of IgG, recovery, purification and specificity of anti-DNA activity. An attempt was made to raise rabbit anti-idiotypic antibodies against both eluates. DNA cellulose chromatography--if DNA leakage was prevented--yielded 58% recovery and 58-fold purification of the anti-DNA activity present in the original purified IgG sample. 1% of loaded IgG was recovered. Cibacron blue chromatography yielded 32% recovery and 1.1-fold purification of the anti-DNA activity. 29% of loaded IgG was recovered. Eluates of Cibacron blue were not pure as shown by their high binding activity against an unrelated antigen, tetanus toxoid. Eluates from DNA cellulose were pure and did not show anti-tetanus toxoid activity. Rabbit anti-idiotypic antibodies could be raised only against eluates of DNA cellulose suggesting that the eluates of Cibacron blue did not contain enough idiotypes to induce anti-idiotypic antibodies. The characterization of the rabbit anti-idiotypic antibodies showed that it contained two populations, one against site-specific idiotypes and the other against framework idiotypes. Anti-DNA antibodies prepared by Cibacron blue had idiotypes similar to those prepared by DNA cellulose. The present study demonstrates that DNA cellulose chromatography--if leakage of DNA is prevented--can yield excellent recovery and purification of anti-DNA activity. Anti-DNA antibodies prepared by DNA cellulose were enriched and could induce anti-idiotypic antibodies in rabbits. Tube chromatography on Cibacron blue yielded poor recovery and minimal enrichment of anti-DNA activity.

Animals↗
PubMed1985

Isolation of high-density lipoproteins by immunoaffinity column chromatography from hog plasma.

High density lipoprotein (HDL) was isolated from hog plasma by a simple immunoaffinity column chromatography procedure using immobilized anti-apolipoprotein AI. The composition of HDL isolated by immunoaffinity chromatography was nearly identical to that of a control sample that was isolated by an alternate method utilizing ultracentrifugation and gel chromatography. The HDL isolated by immunoaffinity chromatography had a larger number of polypeptide components that the control as indicated by acrylamide gel electrophoresis in the presence of urea. When the HDL isolated by immunoaffinity chromatography was applied to a heparin-agarose column the amount of protein retained was approximately twice that of the control. These findings indicate that the ultracentrifugation procedure probably induced the loss of apolipoprotein E containing components from the HDL complex.

Animals↗
PubMed1987

Column liquid chromatography of integral membrane proteins.

Biological membranes have as a major function the compartmentation of biological processes in cells and organelles. They consist of a bilayer of phospholipid molecules in which proteins are embedded. These integral membrane proteins, which cross the bilayer once or several times, generally have a higher than average hydrophobicity and tend to aggregate. Detergents are needed to remove integral membrane proteins from the lipid bilayer and they have to be present during further chromatographic purification. Predominantly, four modes of HPLC have been used alone or in combination for the purification of integral membrane proteins. These are based on differences of proteins in size (size-exclusion chromatography, SEC), electrostatic interaction (ion-exchange chromatography, IEC), bioaffinity (bioaffinity chromatography, BAC) and hydrophobic interaction (reversed-phase chromatography, RPC, and hydrophobic-interaction chromatography, HIC). SEC, IEC, BAC and HIC are used under relatively mild conditions, and buffer systems generally contain a non-ionic detergent. RPC generally has a denaturing effect on the protein and should preferably be used for the purification of integral membrane proteins smaller than 50 kD.

Animals↗
PubMed1991

Scanning thin-layer chromatography-liquid secondary ion mass spectrometry and its application for investigation of drug metabolites.

Thin-layer chromatography-mass spectrometry including both direct and scanning thin-layer chromatography-liquid secondary ion mass spectrometry, has been used to obtain good separation and good mass spectra of non-volatile and thermally unstable mixtures. The methods are very simple, useful and easy to perform, and they can overcome the problems associated with indirect and direct coupling of liquid chromatography and mass spectrometry. Scanning thin-layer chromatography-liquid secondary ion mass spectrometry can be carried out like gas chromatography-mass spectrometry, and gives good quality chromatograms and mass spectra. These methods were used to analyse drug metabolites and mixtures of other organic compounds.

Aminoglycosides↗
PubMed1993

Affinity chromatography with nucleic acid polymers.

Column chromatography utilizing polynucleotides immobilized on solid support is reviewed. This form of affinity chromatography is used for the isolation of polynucleotides and polynucleotide binding proteins, and to a lesser extent for analysis. Several specific applications within these categories have been widely used in the biomedical sciences. Poly(A) mRNA is routinely isolated using oligo(dT) or oligo(dU) supports. Many DNA binding proteins, including transcription factors, restriction endonucleases, and proteins involved in DNA repair, replication, recombination, and transposition have been purified using DNA affinity chromatography. Recently, DNA supports suitable for use in high-performance liquid chromatography have been described and utilized. The current usage of DNA affinity chromatography is reviewed and potential future uses for this technology are speculated upon.

Animals↗
PubMed2004

Purification of pharmaceutical-grade plasmid DNA by anion-exchange chromatography in an RNase-free process.

Anion-exchange is the most popular chromatography technique in plasmid DNA purification. However, poor resolution of plasmid DNA from RNA often results in the addition of bovine-derived ribonuclease (RNase) A to degrade RNA impurities which raises regulatory concerns for the production of pharmaceutical-grade plasmid DNA. Low capacity for plasmid of most commercial media is another issue affecting the suitability of anion-exchange chromatography for large-scale processing. This study reports the use of anion-exchange chromatography to remove RNA in an RNase-free plasmid purification process. Resolution was achieved through careful selection of adsorbent and operating conditions as well as RNA reduction steps before chromatography. Dynamic capacity for plasmid was significantly increased (to 3.0mg/ml) so that it is now possible to envisage the large-scale manufacturing of therapeutic-grade plasmid DNA in the absence of added RNase using anion-exchange chromatography as a polishing step.

Anion Exchange Resins↗
PubMed2006

Exploiting heparin-binding properties of MoMLV-based retroviral vectors for affinity chromatography.

Chromatography is deemed the most promising technology for large-scale purification of viral vectors. The authors have previously shown that heparin affinity chromatography could be successfully employed for the purification of VSV-G pseudotyped Moloney murine leukemia virus (MoMLV)-derived vectors giving excellent results in terms of recovery of active particles, reproducibility and selectivity. In this study, the authors examined whether the ability of retrovirus particles to specifically bind to heparin ligands is restricted to VSV-G pseudotypes produced by 293-based packaging cells. It is shown that VSV-G deficient retrovirus particles are captured by a heparin chromatography column as efficiently as VSV-G containing particles. Most strikingly, RD114 pseudotyped retrovirus particles derived from a HT1080-based cell line were found to bind heparin with the same affinity as 293-derived VSV-G pseudotypes. RD114 pseudotyped retrovirus particles were successfully isolated using heparin affinity chromatography obtaining good recoveries of functional particles (43%). These results indicate that heparin affinity chromatography can be extended to the purification of retroviral vectors produced by different packaging cell lines independently of the Env-protein used for pseudotyping.

Cell Line↗
PubMed2005

Effects of liquid chromatography mobile phases and buffer salts on phosphorus inductively coupled plasma atomic emission and mass spectrometries utilizing ultrasonic nebulization and membrane desolvation.

Atomic spectrometry, specifically inductively coupled plasma atomic emission spectrometry (ICP-AES) and mass spectrometry (ICP-MS) show promise for heteroatom-based detection of pharmaceutical compounds. The combination of ultrasonic nebulization (USN) with membrane desolvation (MD) greatly enhances detection limits with these approaches. Because pharmaceutical analyses often incorporate liquid chromatography, the study herein was performed to examine the effects of solvent composition on the analytical behaviors of these approaches. The target analyte was phosphorus, introduced as phosphomycin. AES response was examined at the 253.7 nm atom line and mass 31 ions were monitored for the MS experiments. With pure aqueous solutions, detection limits of 5 ppb (0.5 ng in 0.1 mL injection volumes) were obtained with ICP-MS. The ICP-AES detection limit was 150 ppb. Solvent compositions were varied from 0 to 80% organic (acetonitrile and methanol) with nine buffers at concentrations typically used in liquid chromatography. In general, solvents and buffers had statistically significant, albeit small, effects on ICP-AES sensitivities. A few exceptions occurred in cases where typical liquid chromatography buffer concentrations produced higher mass loadings on the plasma. Indications are that isocratic separations can be reliably performed. Within reasonable accuracy tolerances, it appears that gradient chromatography can be performed without the need for signal response normalization. Organic solvent and buffer effects were more significant with ICP-MS. Sensitivities varied significantly with different buffers and organic solvent content. In these cases, gradient chromatography will require careful analytical calibration as solvent and buffer content is varied. However, for most buffer and solvent combinations, signal and detection limits are only moderately affected. Isocratic separations and detection are feasible.

Acetonitriles↗
PubMed2000

Perfusion chromatography: an emergent technique for the analysis of food proteins.

Perfusion chromatography is a technique arised to overcome the problem associated with mass transfer in the separation of large molecules such as proteins by high-performance liquid chromatography (HPLC). Perfusion media are constituted by two set of pores: throughpores (6000-8000 A) and diffusive pores (800-1500 A) which enable better access of macromolecules to the inner of the particle by the combination of convective and diffusive flow. As a consequence, times required for a chromatographic separation are reduced. Perfusion media are available in different chromatographic modes: reversed-phase, ion-exchange, hydrophobic interaction, and affinity. From the theoretical models developed to explain the dynamic of retention of solutes in perfusive supports, it was derived that efficiency of a separation was independent of the flow-rate and only depended slightly on the particle diameter. Furthermore, loading capacity was also independent of the superficial velocity. All these advantages have promoted the use of this chromatographic technique for the separation of biomolecules both in analytical and preparative chromatography. Characteristics of perfusion chromatography make this technique very interesting for the analysis of food proteins. Perfusion chromatography enables the assessment of protein composition of a foodstuff at sufficient speed and low cost to be suitable in routine analysis.

Chromatography, Liquid↗
PubMed2001

Performance comparison of suspended bed and batch contactor chromatography.

In some applications, the purification and recovery of biomolecules is performed via a cascade of batch adsorption and desorption stages using agitated contactors and related filtration devices. Suspended bed chromatography is a recent process-scale innovation that is applicable to these separations. This hybrid technique exploits the benefits of combining batch adsorption in an agitated contactor with elution in an enclosed column system. To some extent, the process is similar to batch contactor chromatography but can be fully contained and significantly quicker. The process has two steps; first the fluid containing the sample is mixed with the adsorbent in a stirred tank. Second, the slurry suspension is transferred directly into a specialized column, such as an IsoPak column. The media with the adsorbed product is formed as a packed bed, whilst the suspension liquid is passed out of the column. The product is then eluted from the packed bed utilizing standard column-chromatography techniques. The performance of the suspended bed and the agitated contactor operations are demonstrated both by full-scale experimental results and process simulations. The purification of ovalbumin from a hen-egg white feedstock by anion-exchange chromatography was used as a case study in order to prove the concept. With the availability of both pump-packed systems and shear-resistant media, suspended bed chromatography is a better alternative for a range of applications than the traditional batch separations using agitated contactors.

Adsorption↗