Gradient separations isocratic fraction

Size-exclusion chromatography combined with RP-HPLC-MS was employed for the separation of pyranoanthocyanins from red wine. Wine samples (10 ml) were acidified with 3 M HC1 to pH 1 then sodium bisulphite was added at a concentration of 400 g/1. After 15 min reaction time the treated wine was loaded into a gel column (200 X 15 mm i.d.). Pigments were eluted with 95 per cent ethanol followed with 100 per cent methanol. The various fractions were acidified to pH 1, concentrated and redissolved in water. HPLC-DAD was carried out in an ODS column (150 X 4.6 mm i.d. particle size 5 /nn) at 35°C. Solvents were 0.1 per cent aqueous TFA (A) and ACN (B). The gradient started with 10 per cent B for 5 min to 15 per cent B for 15 min isocratic for 5 min to 18 per cent B for 5 min to 35 per cent B for 20 min. The flow rate was 0.5 ml/min and analytes were detected at 520 nm. MS conditions were sheath and auxiliary gas were a mixture of nitrogen and... 

The selectivity of different stationary phase materials can be applied using columns in sequence to provide high-speed isocratic separations instead of gradient elution. An example for amino acids analysis is shown later in Figure 4.15, where the same eluent was used for all of the separations and the fraction containing the sample components of interest was switched from one column to another. 

As a rule, the sequence in which the columns are placed in a column-switching system has a marked effect on the results of a 2D separation. The final choice is dictated by the specific separation objectives. When subsequent fraction cuts have to be performed on the effluent from the first dimension, the column with a higher peak capacity should be placed into the first-dimension system and the flow rate should be matched to the fraction transfer switching period. The fractions transferred to the second-dimension column should be completely eluted before the subsequent fraction is transferred from the first to the second dimension. To increase the peak capacity in the first dimension, gradient elution is preferred to isocratic conditions. 

In cases where mass transfer is rapid, as is the case with most small molecule separations, then isocratic elution can offer advantages such as automatic fraction reprocessing and solvent recycle. However, with larger synthetic objectives the rate of mass transfer is comparatively low so isocratic elution leads to band broadening and subsequently to recovery of the peptide at high dilution. Most preparative HPLC based peptide separations are carried out under gradient and overload conditions that allow for maximum throughput in terms of time and quantity. 

For metabolite isolation, 1.5 liters of pooled urine were applied to a XAD-2 resin column first. The ethyl acetate extract obtained containing 85 % of the radioactivity was applied upon evaporation to semipreparative HPLC on a Zorbax RX C18 column (9.4 x 250 mm, 5 pm) using gradient elution. Fractions obtained were further separated by isocratic elution on the semipreparative column. The metabolite fractions obtained were finally purified by preparative thin-layer chromatography. Liquid chromatography/mass spectrometry (LC/MS) and LC/MS/MS analysis was applied to the isolated metabolite fractions for structure elucidation. 

In [84] a variable size simplex is u.sed to optimize the mobile phase composition in RP chromatography. The fractions of methanol and water in a methanol/water/acetonitrile mobile phase were optimized. The feasible experimental area was determined by carrying out a gradient elution separation and then calculating the boundaries of the variable space in which the simplexes should move to find an optimized isocratic separation. 

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