Chromatographic suitability relative retention

Chromatographic System The HPLC system is equipped with a 240-nm detector and a 4.6-mm 25-cm column that contains packing LI (octadecyl silane bound to porous silica particles). The flow rate is about 0.8 mL/min. System Suitability Chromatograph the Standard Preparation, and record the peak responses as directed in the Procedure section. The relative retention times are about 0.8 for benzoylformic acid, 1.0 for mandelic acid, 2.5 for benzoic acid, 2.8 for benzaldehyde, and 3.7 for acetophenone. The tailing factor for each peak is not more than 2.0. The resolution between the benzoylformic acid peak and the mandelic acid peak, and between the benzoic acid peak and the benzaldehyde peak, is not less than 3.0. The relative standard deviation for replicate injections is not more than 1% for the mandelic acid peak. 

Chromatographic System (See Chromatography, Appendix IIA.) Use a liquid chromatograph equipped with a refractive index detector that can be maintained at a constant temperature of 25°, a 25-cm x 4.6-mm (id) column packed with 10- im porous silica gel bonded with aminopropylsilane (Alltech 35643, or equivalent), and a guard column that contains the same packing. Maintain the column at a constant temperature of 25° 2°, and the flow rate at about 2.0 mL/min. Inject 20 pL of System Suitability Preparation into the chromatograph, and record the peak responses as directed under Procedure. The relative standard deviation for replicate injections is not more than 2.0%, and the alpha-Cyclodextrin and beta-Cyclodextrin peaks exhibit baseline separation, the relative retention times being about 0.8 and 1.0, respectively. 

Procedure Separately inject suitable portions (about 10 (xL) of the Assay Preparation and the Standard Preparation into the chromatograph, record the chromatograms, and measure the responses for the major peaks. The elution pattern includes the higher-molecular-weight hydrogenated polysaccharides, followed by three individual peaks representing mal-totriitol, maltitol, and sorbitol. The relative retention times are about 0.38 for maltotriitol, 0.48 for maltitol, and 1.0 for sorbitol. Separately calculate the percentages, on the anhydrous basis, of maltitol and sorbitol in the portion of sample taken by the formula... 

Apparatus (See Chromatography, Appendix IIA.) Use a suitable gas chromatograph equipped with a 250-cm x 2-mm (id) glass column, or equivalent, packed with 3% OV-1 stationary phase on 100- to 120-mesh Gas Chrom Q, or equivalent, and with a flame ionization detector. Maintain the column at 175°, the injection port at 210°, and the detector at 230°. Relative retention times (min) D-anhydroglucoses (levoglucosan), pyranose form (3.7), furanose form (not present in standard) (4.3) n-octadecane (5.1) a-D-glucose (8.7) D-sorbitol (11.3) (3-D-glucose (13.3). 

Procedure Use a gas chromatograph equipped with a flame-ionization detector, a cold-on-column injector, a suitable deactivated precolumn, and a 10-m x 0.32-mm (id) capillary column coated with an apolar stationary phase 0.12-p.L film thickness. Program the column to heat to 60°, hold for 1 min, heat to 300° at 20°/min, and hold for 3 min. Set the flame-ionization detector to 320°. Chromatograph five injections of the Standard Preparation. Measure the responses. The relative standard deviation for each peak should be below 2%. The peaks for brassicasterol and campesterol should be baseline resolved (Rs > 1.0) and show no tailing. Measure the response of the Internal Standard Solution and all the individual sterols eluting in the relative retention window of 0.98 to 1.13. 

System Suitability Test Chromatograph five injections of the System Suitability Preparation, and measure the peak responses as directed under Procedure (below). The relative standard deviation for the peak response does not exceed 2.0%, and the resolution between mms-cholecalciferol and pre-cholecalciferol is not less than 1.0 (see System Suitability under Chromatography, Appendix IIA). The chromatograms obtained in this test exhibit relative retention times of approximately 0.4, 0.5, and 1.0, for pre-cholecalciferol, trans-cholecalciferol, and cholecalciferol, respectively. 

It has been shown that gas-Hquid chromatographic methods are particularly suitable for a quantitative characterization of the polarity of solvents. In gas-liquid chromatography it is possible to determine the solvent power of the stationary liquid phase very accurately for a large number of substances [98, 99, 259, 260]. Many groups of substances exhibit a certain dependence of their relative retention parameters on the solvation characteristics of the stationary phase or of the separable components. In determining universal gas-chromatographic characteristics, the so-called retention index, I, introduced by Kovats [100], is frequently used. The elution maxima of individual members of the homologous series of n-alkanes (C H2 +2) form the fixed points of the system of retention indices. The retention index is defined by means of Eq. (7-41),... 

Determination of Relative Reaction Ratios of Olefins Comparison of relative reaction rates was done by gas chromatographic analysis of reaction mixtures with addition of a suitable internal standard such as biphenyl. A mixture of the olefin to be compared and norbornene (both olefins in excess) were readed with a defidency of ethyl mercaptoacetate at various temperatures. Relative retention times were compared with those of independently prepared and characterized mercaptoacetate-olefin adducts. Comparison of the relative amounts of each product in the reaction mixture gave the relative, competitive reaction rate. The independently synthesized adducts were also examined to determine that... 

Chromatograph this system suitabUity solution, the standard solutions, and dissolution test samples by separately injecting equal volumes (about 50 p.1) of the solutions into the chromatograph, record the chromatograms, and measure the areas for the major peaks. For the system suitability solution, the relative retention times are about 0.7 for isonicotinic acid, 1.0 for pyrazinamide, and 1.8 for isoniazid and the resolution, R, between isonicotinic acid... 

System Suitability Chromatograph a suitable number of injections of the Assay Preparation, as directed under Calibration, to ensure that the resolution factor, R, between the major peaks occurring at retention times of approximately 0.50 (8-tocopheryl propionate) and 0.63 ((3- plus y-tocopheryl propionates), relative to hexadecyl hexadecanoate at 1.00, is not less than 2.5 (see System Suitability in High-Performance Liquid Chromatography under Chromatography, Appendix IIA). 

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