Purification liquid chromatography

Purification of poloxamers has been extensively investigated due to their use in medical applications, the intention often being to remove potentially toxic components. Supercritical fluid fractionation and liquid fractionation have been used successfully to remove low-molecular weight impurities and antioxidants from poloxamers. Gel filtration, high-performance liquid chromatography (HPLC), and ultrafiltration through membranes are among the other techniques examined [5]. 

Branchini, B. R., and Rollins, C. B. (1989). High-performance liquid chromatography-based purification of firefly luciferases. Photochem. Photobiol. 50 679-684. 

Figure 5.6 Positive-ion electrospray spectrum obtained from the major component in the LC-MS analysis of a purified recombinant 62 kDa protein using a Cig microbore 50 X 1 mm column and a flow rate of 50 p.lmin . The starting buffer (buffer A ) was 0.1% TEA in water, while the gradient buffer (buffer B ) consisted of 0.1% TEA in acetonitrile-water (9 1 vol/vol). The running conditions consisted of 0% B for 5 min, followed by a linear gradient of 100% B for 55 min. Reprinted from J. Chromatogr., B, 685, McAtee, C. P., Zhang, Y., Yarbough, P. O., Fuerst, T. R., Stone, K. L., Samander, S. and Williams, K. R., Purification and characterization of a recombinant hepatitis E protein vaccine candidate by liquid chromatography-mass spectrometry , 91-104, Copyright (1996), with permission from Elsevier Science.

In contrast to other organothallium(I) compounds, cyclopentadienyl-thallium(I) is a remarkably stable compound. Samples can be stored in sealed bottles for months without appreciable decomposition occurring it is unaffected by water and dilute alkali and it is only slowly oxidized by air at room temperature. Cyclopentadienyltballium(I) was first prepared by Meister in 1956 by addition of freshly distilled cyclopentadiene to a suspension of thallium(I) sulfate in dilute potassium hydroxide solution 101, 102). A number of variations of this procedure have been described (5, 25, 34, 56), and the compound has been made in other ways 35, 56,110, 164), but Meister s preparation, in which the yield of crude product is greater than 90%, remains the method of choice. Purification of crude cyclopenta-dienylthallium(I) is best accomplished by vacuum sublimation, and purity of samples can readily be assessed by gas-liquid chromatography on silicone oil at 170° C using hydrogen as carrier gas (7). 

Although saponification was found to be unnecessary for the separation and quantification of carotenoids from leafy vegetables by high performance liquid chromatography (HPLC) or open column chromatography (OCC), saponification is usually employed to clean the extract when subsequent purification steps are required such as for nuclear magnetic resonance (NMR) spectroscopy and production of standards from natural sources. 

Mitcham, E.J., Gross, K.C., and Wasserman, B.P. (1991) Synthesis of uridinediphospho-[U- C]-D-galacturonic acid by enzyme particulate fractions and purification via high performance liquid chromatography. Phytochem.Anal. 2 112-115. 

Specifically for triazines in water, multi-residue methods incorporating SPE and LC/MS/MS will soon be available that are capable of measuring numerous parent compounds and all their relevant degradates (including the hydroxytriazines) in one analysis. Continued increases in liquid chromatography/atmospheric pressure ionization tandem mass spectrometry (LC/API-MS/MS) sensitivity will lead to methods requiring no aqueous sample preparation at all, and portions of water samples will be injected directly into the LC column. The use of SPE and GC or LC coupled with MS and MS/MS systems will also be applied routinely to the analysis of more complex sample matrices such as soil and crop and animal tissues. However, the analyte(s) must first be removed from the sample matrix, and additional research is needed to develop more efficient extraction procedures. Increased selectivity during extraction also simplifies the sample purification requirements prior to injection. Certainly, miniaturization of all aspects of the analysis (sample extraction, purification, and instrumentation) will continue, and some of this may involve SEE, subcritical and microwave extraction, sonication, others or even combinations of these techniques for the initial isolation of the analyte(s) from the bulk of the sample matrix. 

Kroeff, E. P., Owens, R. A., Campbell, E. L., Johnson, R. D., and Marks, R. I., Production scale purification of synthetic human insulin by reversed-phase high-performance liquid chromatography, /. Chromatogr., 461, 45, 1989. 

Warren, W. and Vella, G., Analysis and purification of synthetic oligonucleotides by high-performance liquid chromatography, in Oligonucleotide Synthesis Protocols, Agrawal, S., Ed., Humana Press, Totowa, NJ, 1993, 235. 

Titani, K., Sasagawa, T., Resing, K., and Walsh, K. A., A simple and rapid purification of commercial trypsin and chymostrypsin by reverse-phase high-performance liquid chromatography, Anal. Biochem., 123, 408, 1982. 

Figure 5.11. Generic approaches to identify interacting proteins within complexes. The complex is isolated from cells by affinity purification using a tag sequence attached to a protein known to be in the complex. Alternatively, the complex can be immunprecipitated with an antibody to one of the proteins in the complex. The proteins are resolved by polyacrylamide gel electrophoresis, proteolyzed, and the mass of the resulting peptides is determined by mass spectrometry. Alternatively, the proteins can be proteolyzed and the resulting peptides resolved by liquid chromatography. The peptide masses are then determined by mass spectrometry and used for database searching to identify the component proteins.

Several methodologies have been published for the determination of selected emerging contaminants in sewage sludge. The most recently reported methods are summarized in Table 1, comprising extraction of the sludge sample, subsequent purification of the extract, and final analysis by either gas or liquid chromatography (GC or LC) coupled to mass spectrometry (MS) or tandem mass spectrometry (MS-MS). 

A sensitive method was described for the detection and estimation of residues of niclosamide in bananas involving extraction of niclosamide, purification of the extract by solvent partition and column chromatography, formation of the hepta-fluorobutyryl derivative of 2-chloro-4-nitroaniline in 99% yield, and determination of the derivative by gas liquid chromatography with electron capture detection. 

Procedure Flavonoids are then further purified with 2 ml of methanolic HC1 (2 N), followed by centrifugation (2 min, 15 600 g), hydrolyzation of 150 il of suspension in an autoclave (15 min, 120 C). A reverse osmosis-Millipore UF Plus water purification system is used in high performance liquid chromatography (HPLC) with an autosampler. After injections of 5 pg of samples, the mobile phases flow at a rate of 1 ml/minute with isocratic elution in a column at 30 C. 

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