Chromatographic preparation

Procedure Inject 3 xL of each of the Standard Solutions into the gas chromatograph. Prepare a calibration curve by plotting the concentration, Cs, in milligrams per milliliter, of each Standard Solution versus its peak response, and draw the best straight line. Similarly inject 3 (xL of the Sample Preparation. Record the peak response for dimethyl sulfoxide for the Sample Preparation, and determine its concentration from the standard curve. Calculate the concentration of dimethyl sulfoxide, in milligrams per kilogram, in the sample taken by the formula... 

A mixture of 6-chloro-9-(2,3,5-tri-0-acetyI-)3-D-ribofuranosyl)purin-2-amine (0.293 g, 0.685 mmol), anhyd, distilled pentyl nitrite (1.53 mL, 11.4 mmol), and dry CCL (30 niL) was heated at reflux temperature under Nj for 24 h. The solvent was then removed and the resulting residue taken Up in CH,Cl2 (3 mL) and chromatographed (preparative layer silica gel plates, developed twice with i-PrOH/CH CL, 1 50). Elution of the only significant band Rf = 0.29) with MeOH/CH CIj gives, after crystallization (EtOH), colorless needles yield 0.2 g (66%) rap 139 -t4t°C. 

Fig. 6.2 Chromatographic preparative separation ofthe enantiomers of thalidomide on a chiral polyacrylamide phase (from [18]).

Ault, J. A., Schofield, M. C., Johnson, L. D., and Waltz, R. H. (1979). Automated gel pennealion chromatographic preparation of vegetables, fmits, and crops, for organophophate residue determination utilizing flame photometric detection. J. Agric. Food Chein. 21, 825-828. 

Ware GM, Price G, Carter L, Eitenmiller RR (2000) Liquid chromatographic preparative method for isolating ergot alkaloids, using a particle-loaded membrane extracting disk. J AOAC Int 83 1395-1399... 

Warneke C, de Gouw JA, Kuster WC, et al. Validation of atmospheric VOC measurements by proton transfer reaction- mass spectrometry using a gas chromatographic preparation method. Environ Sci Teehnol. 2003 37 2494-501. 

Berglof JH, Eriksson S, Curling JM. Chromatographic preparation and in vitro properties of albumin from human plasma. J Appl Biochem 1983 5(4—5) 282—92. 

O. Tenberken, H. Thiermann, F. Worek and G. Reiter, Chromatographic preparation and kinetic analysis of interactions between tabun enantiomers and aeetylcholinesterase, Toxicol Lett, 2010,195,142-146. 

Chromatographic Separation of a Mixture of o- and p-Nitroaniline. Prepare a glass tube A (Fig. 24) in which the wider portion has a diameter of 3 cm. and a length of ca. 30 cm. the narrow portion at the base has a diameter of 5-7 mm. Wash the tube thoroughly (if necessary, with chromic acid, followed by distilled water and ethanol) and then dry. Insert a small plug of cotton-wool P as shown just within the narrow neck of the tube it is essential that this plug does not project into the wider portion of the tube. Clamp the tube in a vertical position. 

Cholestenone. Place a mixture of 1 0 g. of purified cholesterol and 0-2 g. of cupric oxide in a test-tube clamped securely at the top, add a fragment of Dry Ice in order to displace the air by carbon dioxide, and insert a plug of cotton wool in the mouth of the tube. Heat in a metal bath at 300-315° for 15 minutes and allow to cool rotate the test-tube occasionally in order to spread the melt on the sides. Warm with a few ml. of benzene and pour the black suspension directly into the top of a previously prepared chromatographic column (1) rinse the test-tube with a little more benzene and pour the rinsings into the column. With the aid of shght suction (> 3-4 cm. of mercury), draw the solution into the alumina column stir the top 0 -5 cm. or so with a stout copper wire to... 

Precision The precision of a gas chromatographic analysis includes contributions from sampling, sample preparation, and the instrument. The relative standard deviation due to the gas chromatographic portion of the analysis is typically 1-5%, although it can be significantly higher. The principal limitations to precision are detector noise and the reproducibility of injection volumes. In quantitative work, the use of an internal standard compensates for any variability in injection volumes. 

Both preparative and analytical GPC were employed to analyze a standard (NBS 706) polystyrene sample. Fractions were collected from the preparative column, the solvent was evaporated away, and the weight of each polymer fraction was obtained. The molecular weights of each fraction were obtained usmg an analytical gel permeation chromatograph calibrated in terms of both and M. The following data were obtained ... 

Monobasic acids are determined by gas chromatographic analysis of the free acids dibasic acids usually are derivatized by one of several methods prior to chromatographing (176,177). Methyl esters are prepared by treatment of the sample with BF.—methanol, H2SO4—methanol, or tetramethylammonium hydroxide. Gas chromatographic analysis of silylation products also has been used extensively. Liquid chromatographic analysis of free acids or of derivatives also has been used (178). More sophisticated hplc methods have been developed recentiy to meet the needs for trace analyses ia the environment, ia biological fluids, and other sources (179,180). Mass spectral identification of both dibasic and monobasic acids usually is done on gas chromatographicaHy resolved derivatives. 

Potency of hGH preparations is quantitatively deterrnined, in terms of mass per vial, by one or more chromatographic procedures (50). Biopotency is calculated from the mass-based potency using a conversion factor, typically 3 lU/mg. Traditionally a bioactivity assay using hypophysectomized rats has been used to determine potency however, the imprecision of this assay has resulted in its use only as a semiquantitative indicator of bioactivity (1), sometimes referred to as a bioidentity test. 

Analytical Techniques. Sorbic acid and potassium sorbate are assayed titrimetricaHy (51). The quantitative analysis of sorbic acid in food or beverages, which may require solvent extraction or steam distillation (52,53), employs various techniques. The two classical methods are both spectrophotometric (54—56). In the ultraviolet method, the prepared sample is acidified and the sorbic acid is measured at 250 260 nm. In the colorimetric method, the sorbic acid in the prepared sample is oxidized and then reacts with thiobarbituric acid the complex is measured at - 530 nm. Chromatographic techniques are also used for the analysis of sorbic acid. High pressure Hquid chromatography with ultraviolet detection is used to separate and quantify sorbic acid from other ultraviolet-absorbing species (57—59). Sorbic acid in food extracts is deterrnined by gas chromatography with flame ionization detection (60—62). 

Silyl Ethers. The preparation of per- O-trimethyl silyl ethers of sucrose is generally achieved by reaction with chi orotrimethyl sil ane and/or hexamethyldisila2ane in pyridine (25,26). However, this reaction is not selective and in general per-trimethyl silyl ethers are only used as derivatives for gas chromatographic studies. 

Manufacture. HFS containing 42% fmctose is produced commercially by column isomerization of clarified and refined dextrose hydrolyzate using an immobilized glucose isomerase. Enriched symp containing 90% fmctose is prepared by chromatographic separation and blended with 42% HES... 

Gas Chromatography Analysis. From a sensitivity standpoint, a comparable technique is a gas chromatographic (gc) technique using flame ioni2ation detection. This method has been used to quantify the trimethylsilyl ester derivative of biotin in agricultural premixes and pharmaceutical injectable preparations at detection limits of approximately 0.3 pg (94,95). 

Preferably, high pressure Hquid chromatography (hplc) is used to separate the active pre- and cis-isomers of vitamin D from other isomers and allows their analysis by comparison with the chromatograph of a sample of pure reference i j -vitainin D, which is equiUbrated to a mixture of pre- and cis-isomers (82,84,85). This method is more sensitive and provides information on isomer distribution as well as the active pre- and cis-isomer content of a vitamin D sample. It is appHcable to most forms of vitamin D, including the more dilute formulations, ie, multivitamin preparations containing at least 1 lU/g (AOAC Methods 979.24 980.26 981.17 982.29 985.27) (82). The practical problem of isolation of the vitamin material from interfering and extraneous components is the limiting factor in the assay of low level formulations. 

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