HPLC Gradient Chromatograms

FIGURE 21 Two HPLC gradient chromatograms (tryptic maps of lysozyme) illustrating the dramatic effect of flow rate (F), gradient time (t ), and void volume (Vg) on analysis time. Figure reprinted with permission from Reference 22. 

Figure 4B. Chromatogram of RP-HPLC gradient elution separation of OPA—Ac—Cys derivatives of standard protein amino acid enantiomers.

FIGURE 15 RP-HPLC-UV chromatogram (using a gradient method) of an aqueous solution of drug substance A after stressing at 70°C for 14 days at pH 7. 

FIGURE I6 HPLC-UV chromatograms of LY297802 after 3 days and 7 days in a photostability chamber (cool white fluorescent lamps, I7,000 lux). Conditions Zorbax RX-C8 column, 25 cm X 4.6 mm id, 5 mm A = buffer/acetonitrile 95/5, B = buffer/acetonitrile, 25/75 buffer = 25 mM potassium phosphate with pH adjusted to 6.5 with NaOH,UVdetection using photodiode array with Waters Maxplot 200-400 nm gradient elution from 0% A to 100% B in 30 min, I mL/min flow rate. 

Table 2.4 HPLC gradient program used for analysis of anthocyanins in grape skins extract by C18 (250 x 4mm, 5 pm) column (chromatogram in Figure 2.14) (flow rate 0.5mL/min).

Because of lingering questions concerning the possible presence of unresolved peaks in the HPLC chromatogram and the extent of total autofluorescence in collagen explained by LWl, the HPLC gradient was modified to increase its shallowness such... 

FIGURE 4.22 HPLC chromatogram of amino acids employing precolumn derivatiza-tion with OPA. Chromatography was carried out on an Ultrasphere ODS column using a complex tetrahydrofuran methanol 0.05 M sodium acetate (pH 5.9) 1 19 80 to methanol 0.05 M sodium acetate (pH 5.9) 4 1 gradient at a flow rate of 1.7 mL/min. 

A typical HPLC separation using a 15-cm column of 15,000 theoretical plates produces peak capacity (Giddings, 1991) of about 80-100 under isocratic conditions and up to 150 under gradient conditions in 1 h(Eq. 7.3, n peak capacity, A number of theoretical plates of a column, and fR and t retention time of the last and the first peak of the chromatogram, respectively). An increase in the number of separated peaks per unit time can be achieved by increased separation speed made possible by monolithic silica columns (Deng et al., 2002 Volmer et al., 2002). This has also been shown for peptides and proteins (Minakuchi et al., 1998 Leinweber et al., 2003). 

FIGURE 16.8 HPLC chromatogram of cytochrome c and myoglobin digest, using a 250 cm x 4.6 mm ODS C18 Vydac column and a linear mobile-phase gradient, 5-50% B, in 50 min. Buffer A was 0.1% TFA in water and buffer B was 0.1 TFA in acetonitrile. UV detection was carried out at 214 nm, at room temperature (reprinted with permission from Electrophoresis). 

Fig. 16.6. (a) HPLC chromatograms for reaction mixture A. HPLC conditions C18 (20 x 100 mm Nova-Pak TM), gradient 5-95% acetonitrile/water, with 0.1% TFA (b) SFC chromatogram for reaction mixture A. SFC conditions Diol column (21.2 x 150 mm, Berger Instruments), gradient 5-60% Methanol with 0.5% dimethylethylamine in carbon dioxide [11],... 

Fig. 2.3.5. Normal-phase HPLC chromatograms of commercial standards of (b) f-octylphenol and (a) 4-nonylphenol (85%). Column 100 X 4.6 mm2 Hypersil 3 NH2 (3 pm), gradient elution with re-hexane-2-propanol-H20, detection fluorescence, excitation 230 nm...

A simple and rapid RP-HPLC method was developed for the determination of retinoid in galenicals. Commercial preparations were diluted, filered and used for separation. Measurements were carried out in an ODS column (150 X 4.6 mm i.d. particle size 3 /xm). Solvents A and B were methanol-10 mM ammonium acetate (75 25, v/v) and methanol-THF (84 16, v/v), respectively. The flow rate was 0.8ml/min. Gradient conditions were 0-25 min, 0 per cent B 35 min, 100 per cent B, isocratic for 10 min. Typical chromatograms are shown in Fig. 2.37. The repeatability of peak area ranged between 0.48 -3.2 per cent for UV-DAD and 0.57 - 3.1 per cent for fluorescence detection. The reproducibility varied between 0.26 - 4.6 per cent. It was found that the method is precise, selective, sensitive and linear, therefore, it can be employed for the routine quality control of this class of drags [85],... 

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