Gradient elution flow rate effect

Figure 3 Flow rate effects in gradient elution. Separation of peptide mixture (lysozyme digest) by reversed-phase gradient elution. Gradient time 30 min 8-40% acetonitrile-water (0.1% TFA) gradient Cg column, (a) 0.5mL/min and (b) 1.0 mL/min. (From Ref. 6.)...

Gradient elution is a procedure whereby the conditions under which the sample is eluted are progressively varied throughout the separation so as to speed up the process. This can be achieved by altering the composition of the mobile phase or increasing the temperature or flow rate. The effect is to elute components more rapidly in the latter stages and sharpen their elution profiles. Stepwise elution is a similar procedure in which elution conditions are changed at predetermined times rather than continuously. 

Fig. 5. Effect of the flow rate on the separation efficiency. Separation of a protein mixture at six different flow rates (40,80,120,160,200 and 240 ml/min) normalized to the elution volume. Conditions Column 80 ml CIM DEAE Tube Monolithic Column Mobile phase buffer A 20 mM Tris-HCl buffer, pH 7.4 buffer B 20 mM Tris-HCl buffer + 1 M NaCl, pH 7.4 Gradient 0-100% buffer B in 200 ml Sample 2 mg/ml of myoglobin (peak 1), 6 mg/ml of conalbumin (peak 2) and 8 mg/ml of soybean trypsin inhibitor (peak 3) dissolved in buffer A Injection volume 1 ml Detection UV at 280 nm. (Reprinted with permission from Podgornik A, Barut M, Strancar A, Josic D, Koloini T (2000) Anal Chem 72 5693)...

Fig. 14 a, b. Effect of gradient steepness on the very fast separation of polystyrene standards in a molded monolithic poly(styrene-co-divinylbenzene) column (Reprinted with permission from [121]. Copyright 1996 Elsevier). Conditions column, 50 mm x8 mm i.d., mobile phase, linear gradient from 100% methanol to 100% tetrahydrofuran within a 1 min b 12 s, flow rate, 20 ml/min, peaks represent polystyrene standards with molecular weights of 9200,34,000 and 980,000 (order of elution), 3 mg/ml of each standard in tetrahydrofuran, injection volume 20 pi, UV detection, 254 nm... 

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. 

FIGURE 5.4 Effect of the gradient dwell volume, V7>. the elution volume, Vj, in reversed-phase chromatography. Solute neburon, retention equation (Equation 5.7) with parameters a=A, m = 4. Linear gradients 2.125% methanol/min (a) from 57.5% to 100% methanol in water in 20min ( i = 50) (b) from 75% to 100% methanol in water in 11.75 min (k = 10). Vg uncorrected calculated from Equation 5.8, Vg + Vg, Vg, added to Vg uncorrected, Vg corrected calculated from Equation 5.21. (A) A conventional analytical C18 column, hold-up volume y ,= ImL flowrate l.OmL/min. (B) A microbore analytical C18 column, hold-up volume y = 0.1mL flow rate 0.1 mL/min. 

FIGURE 5.7 Effects of binary and ternary gradient elution with methanol and acetonitrile on separation selectivity in RP HPLC. Column LiChrosorb RP-C18, 5 pm, 300x4.0mm i.d. flow rate ImL/min UV detection, 254nm. Sample 4-cyanophenol (1), 2-methoxyphenol (2), 4-fluorophenol (3), 3-fluorophenol (4), 3-methylphenol (5), 4-chlorophenol (6), 4-iodophenol (7), 2-phenylphenol (8), and 3-ferf-butylphenol (9). 

Fig. 15. Memory effect. Gradient elution of 500 pg phosphorylase kinase on a RP 18 column (250 x 4.6 mm dP = 5 pm). Mobile phase A 0.1% TFA in water B 0.08% TFA in acetonitrile gradient program 0 % B (0-8 min), 46 % (9 min) 68 % (24 min), 75% (33 min) flow rate 1 ml/min. The lower chromatogram was obtained upon sample injection the five blank gradients were performed immediately after the initial separation. Molar mass of the subunits a — 132,000 daltons P — 113,000 y - 43,000 5 - 16,680. (From Ref. 66> with permission)...

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