Column minimum elution time

The optimum value for (k A), that will provide the maximum resolution and consequently, the minimum elution time, (for an unoptimized column) has been deduced by Grushka and Cook, (14) and Katz et al (15) to be about 2.5. As a result, the composition of the mobile phase is usually adjusted to achieve this value for (k1) if it is at all possible. 

The scale of preparative HPLC is normally larger than that of conventional HPLC. Therefore, a practical starting point is to develop an analytical separation that optimizes the isolation conditions. Optimization of the analytical method implies seeking conditions that combine maximum resolution of the peak of interest and minimum elution time, under the restriction of a limited pressure drop. The optimized conditions determine the column, mobile phase, flow rate, and sample loading capacity for the particular column. The conditions may be either normal phase or reverse phase. The mobile phase should be chosen carefully to avoid salt complexation with the compound to be isolated. Volatile acid salts such as trifluoroacetic acid, formic acid, and acetic acid are acceptable mobile phase additives, and the ammonium counterion is preferred for pH adjustment to any of these acids. 

The scale of preparative chromatography is larger than that of conventional analytical chromatography. Therefore, a practical starting point is to develop an analytical separation that optimizes the isolation conditions. Optimization of the analytical method implies seeking conditions which combine maximum resolution of the peak of interest and minimum elution time, under the restriction of a limited pressure drop. " The optimized conditions determine the column, mobile... 

In the optimisation process, resolution and elution time are the two most important dependent variables. The goal is to conduct a separation of the components of interest in the minimum time without neglecting the time it will take for the instrument to come back to its initial state and be ready for the next analysis. Chromatography corresponds to a slow type of analysis. If the resolution is excellent, optimisation can still be conducted in order to save time in the analysis. This can be done using a shorter column, knowing that resolution varies with the square root of the length of the column (cf. N in equation (1.26)). 

Each sample to be analyzed was dissolved in tris(ethylenediamine)cadmium dihydroxide (1 mL) by stirring overnight, and then water (1 mL) was added. A 1-mL aliquot (concentration < 1.0%) was applied to the column, and elution proceeded with a pressure head of 100 cm and flow rate of 10 mL/h. A Turner 111 fluorometer (excitation filter 2A plus 47B and emission filter 8 plus 65A) fitted with a flow-through door allowed for automatic continuous monitoring of carbohydrates as they were eluted. Relative fluorescence was automatically recorded on a linear strip recorder. Fractions of 3 mL were collected on a FC-80K Gilson microfractionator. Typically, each sample was analyzed several times, usually at different concentrations, to ensure the reproducibility and accuracy of the data. A calibration run using the labeled dextrans was performed a minimum of one time per week. 

The 2 -hydroxychalcones or flavanones (1.0 mmol, dissolved in a minimum amount of ethyl acetate) were added to silica gel supported InBr3 (2.0 g, 15-20 mol%) and the solvent was removed. The dry mixture was heated with stirring at 130-140 °C in an inert container for different periods of time. The reaction mixture was directly applied onto a silica gel column and eluted with a mixture of ethyl acetate-hexane (1 1). 

Both systems described above have limitations. In the parallel mode either double pumps or dual valves (as shown in Figs. 4.7, 2.6 d) 9] have to be used to control the sequential elution of the columns. This is not necessary in the alternating mode. However, the. sample consumption is usually larger for the latter because the time for. sample changeover is determined by the sample loading time, and not by the normally shoner elution time. On the other hand, unless separate pumps are used for loading and elution, both systems lack the possibility of independent control over the sample changeover time. Therefore, if minimum sample consumption is required, a more complicated three-pump system will be necessary. ... 

The DEQUEST 2060 C pentaphosphonate component separated to 95% radiochemical purity in a single pass with a minimum of yield sacrifice. Based on later work with the other phosphonates, we now feel that 99% radiochemical purity is probably obtainable on a single pass under a modified elution strategy. But we elected to composite several 95% pure fractions and put them through the columns a second time to reach the higher purity. 

During the A time, a minimum of 80 % of the dodecane must elute from the polar column. The A time is too short or the A flow is too low if the dodecane elution is less than 80 %. The A time is too long or the A flow is too high if benzene or trans-decalin, or both, elute during the A... 

The combined acetone extracts were extracted six times with one-fourth volume of ethylene dichloride and the ethylene dichloride extract was evaporated under vacuum to leave the steroid residue. This steroid residue was taken up in a minimum of methylene chloride and applied to the top of a column packed with 30 grams of silica which had been previously triturated with 21 ml of ethylene glycol. Then various developing mixtures, saturated with ethylene glycol, were passed over the column. Cuts were made as each steroid was eluted as determined by the lowering of the absorption of light at 240 mp on the automatic chromatographic fraction cutter. 

Purification of the conjugates may be done by reverse phase HPLC separation. Dry the reaction solution under a nitrogen stream and reconstitute in a minimum volume of acetonitrile/water (1 1, v/v). Apply the sample to a 5 pm Cig-silica HPLC column (250 X 4.6 mm, Nucleosil). Elute with a gradient of water to acetonitrile at a flow rate of 1 ml/minute over a time course of 30 minutes. Free BNAH and BNAH-glycan derivatives can be monitored by absorbance at 275 nm. The conjugate peak also will be positive for carbohydrate by reaction with orcinol, which can be detected by spray after spotting a small eluted sample on a TLC plate. 

The crude residue is applied to the column head using a minimum of dichloromethane. The submitters use flash-grade (230-400 mesh) silica gel purchased from E. Merck and a column 10 cm in diameter. TLC values for 2 are Rf = 0.18 (hexane acetone = 3 1) and for 3 (see Note 10) are Rf = 0.08 (hexane acetone = 3 1), employing Whatman K6F silica gel TLC plates 60 A. In a typical purification, collecting 200-mL fractions, 2 would elute in fractions 9-18 and 3 in fractions 20-30. The checkers found that immediate purification of the crude residue was necessary. Yields decreased dramatically with time between isolation and purification. Furthermore, the activity (related to the degree of dehydration) of the silica gel greatly affected yields. Only half the amount of silica mentioned above was used by the checkers to get the reported yields. When the full amount was used, the yield decreased to 40-50%. 

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