Currently a period of rapid chromatography heyday is undergoing, its scope is extremely wide and includes sectors such as biotechnology, medicine, forensics, organic synthesis, environmental monitoring, obtaining ultrapure substances, analysis of space objects and more. A special place in the list of chromatographic methods takes liquid chromatography - the most versatile method of analysis based on non-destructive separation of substances. The latter allows applying liquid chromatography not only as an analytical, but also as a unique technological method for the isolation and purification of substances, when other methods are ineffective. One should also not forget that the very discovery and subsequent development of chromatographic methods started with liquid chromatography. There is a separate branch of liquid chromatography dubbed biomedical HPLC attached to biology and medicine. Objects of its research are the analysis of endogenous substances and drugs in biological fluids (blood, urine, saliva). Most of the researches in clinical biochemistry and clinical pharmacokinetics are successfully implemented using biomedical HPLC.
Amino acid analysis in biological fluids, primarily homocysteine, appears to be very important clinical problem. Hyperhomocysteinemia increases the risk of early development of atherosclerosis, thrombosis, coronary and cerebral blood vessels, and is a predictor of death (Barker, n.d.). Existing analytical techniques are complex and costly, both in time and in finance. So, providing a simple and reliable technology is required. For example, thiols, including homocysteine, cysteine and glutathione, are transparent to UV rays, and are not captured by the chromatographic apparatus. This, incidentally, is tr...
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Kim, Taewoo, Trey L Arnold, Kyle A Leland, Aimee M Morey, Department of Chemistry, USAF Academy, CO 80840
...its role as a coenzyme for L-methylmalonyl-CoA mutase and methionine synthase (e). Serum vitamin B12 less than 145 pg/mL is considered deficient, and is commonly caused by pernicious anemia and atrophic gastritis (k) (h). B12 is measured with a competitive-binding immunoenzymatic assay that measures concentration based on an inverse relationship to absorbance (i). Methylmalonic acid and homocysteine are associated metabolites that increase in concentration with B12 deficiency and act as disease markers (b). The Schilling test is a less common method that involves administration of oral radiolabeled B12, saturation of B12 binding proteins, and subsequent detection of radiolabeled B12 in urine to assess absorption rate (g). Current laboratory methods are rapid and specific, providing clinicians with accurate information in the diagnosis of vitamin B12 deficiency.
In preparing for the quantitative test for the Bio-Rad protein assay, a spectrophotometer was switched on. Ten test tubes were used and that the known and unknown protein samples were tested duplicate. Tubes one and two were the 0.2 mg/ml protein, three and four 0.3 mg/ml protein, five and six 0.6 mg/ml protein, seven and eight 0.9 mg/ml protein, and lastly nine and ten
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Analysis of a total of eighteen samples (six samples at each of the three test levels) which were prepared by adding known amounts of standardized chlorine solution to 0.1% sulfamic acid collecting solution.
Introduction: Within this experiment we wish to facilitate a greater understanding of the concepts of experimental design and quantifying techniques. Specifically, this lab will allow us to gain an enhanced understanding of the isolation of a protein using differential solubility, which allows us to separate and purify various proteins using high concentrations of a specific salt so that they may be studied in great detail. Last week we separated our desired protein using ammonium sulfate. Since we have already extracted the desired protein, we will begin quantifying the amount using the Bradford Protein Assay. Because it is a dye-binding assay, we will use the spectrophotometer to measure the absorbance of various dilutions of a protein: this will comprise our standard curve. We will then compare the absorbance of our extracted protein from l...
Chromatography is the laboratory technique for separating mixtures into their components for analyzing, identifying, purifying or quantifying the mixtures or components. The mixture dissolved in the mobile phase (gas or liquid) is passed through the stationary phase (liquid or solid), which separates the analyte from the other molecules in the mixture. The differences in the migration rate of the compounds in these two phases effects the separation.
The term chromatography refers to different methods of molecular separation between a mobile phase and a stationary phase based on various physio-chemical properties. There are many types of chromatography that are used as analytical tools in environmental science, forensics, metallurgy, biology, etc. Some common examples are thin layer chromatography (TLC), gas chromatography (GC), high performance liquid chromatography (HPLC) and ion chromatography. Ion chromatography (IC) was introduced as an analytical technique by Small, Stevens, and Bauman in 1975. According to IUPAC in IC “separation is based on differences in the ion exchange affinities of the individual analytes. If inorganic ions are separated and can be detected by conductivity detectors or by indirect UV detection then this is also called ion chromatography” (Eith 17).
...s the change in the temperature of both of these batches, 6°C for the pure, and 13°C for the crude. In this final sub-section of the Characterization of Aspirin, the values of absorbance were recorded. Initially, 0.0566 grams and 0.0590 grams of pure and crude Aspirin respectively were obtained and each individually placed into beakers (400 milliliter) and had 250.0 milliliters of distilled water added to them. From each beaker, a tiny amount of the just dissolved solutions was transferred to a cuvette, one cuvette for each type of aspirin. Each cuvette was placed into the ultraviolent spectroscopy mechanism which was connected to a computer and absorbance spectrum values were obtained at 298 nm (Figure 5) (0.1987 pure aspirin, and 0.9549 crude aspirin).
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23. S. Alwarappan, S. Boyapalle, A. Kumar, C.-Z. Li and S. Mohapatra, J. Phys. Chem. C, 2012, 116, 6556–6559
HPLC (High Performance Liquid Chromatography) is an analytical technique which separates a complex mixture of components into its specific individual components. It is a powerful tool in analysis, as it combines high speed with extreme sensitivity compared to traditional methods of chromatography because of the use of a pump which creates a high pressure and forces the mobile phase to move with the analyte in high speed. It is been used as a principle technology in various automated analyzers used for diagnostic purpose.
Zein, N. N., & Edwards, K. (2009). The Cleveland Clinic guide to liver disorders. New York: Kaplan Pub.
Due to the nature of amino acids, a titration curve can be employed to identify
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