HPLC gradient separation

The results are qualitatively evaluated by making overlay plots. An example of an HPLC gradient separation for a typical assay and impurities analysis is shown in Figure 13a. The selectivity is evaluated on five different lots of the selected stationary phase. As can be observed, apart from two minor selectivity issues (indicated by the arrows in Figure 13a), the reproducibility on different lots of stationary phases is acceptable. 

Fig. 2. HPLC ion exchange gradient separation of samples (25 ug of uronic acid) of smaller (G7) citrus pectin oligomers (a), larger (G12) citrus pectin oligomers (b), and the B. fruit extract (c). Detection was by PAD. For each sample the peak co-eluting with the standard galacturonate oc-tamer was designated peak 8, and the remaining peaks were numbered consecutively.

Fig. 3. QAE-Sephadex gradient separation of the B fruit extract. An 18 mg (uronic acid equivalents) sample of extract was dissolved in 20 ml of 125 mM imidazole-HCl buffer (pH 7.0) and applied to the column. The column was then eluted with 50 ml 125 mM buffer followed by a 125 mM to 1.5 M buffer gradient (500ml), and, finally, 50 ml of 1.5 M buffer. Fractions of 5 ml were collected and assayed for uronic acids. Groups of fractions (26-41, 45-50, 53-75 and 84-100) were pooled, concentrated by ultrafiltration and analyzed by HPLC.

The two major approaches for HPLC purification are fast gradient separation and parallel purification. Yan et al.178 utilized the former (Figure 1.51). The purification lab received a 96-well plate containing synthesized products at 0.1 to 0.2 mmol/well. A Hydra 96-probe liquid handler prepared QC plates for all samples that were analyzed with a MUX-LCT eight-channel parallel LCMS instrument at a throughput of 2000 samples/day. Only samples with purities above 10% were purified on a... 

The case stndy of the SP development of an HPLC assay method for an OTC prodnct derived from natural materials illustrates the importance of optimizing both the extraction and filtration conditions to fully recover all the labile active components in the formulation. The HPLC gradient assay method, which separates all active ingredients from other... 

FIG U RE 1.13 Gradient separation of polypeptides on silica rod column and particle-packed columns. Mobile phase velocity 4mm/s, gradient 5%-60% ACN in the presence of TFA, gradient time 5min, columns (a) silica rod column, (b) Capcellpak SG (5 pm), (c) LiChrospher WP 300 RP-18e (5 pm), (d) nonporous NPS-ODS-1 HPLC column (1.5pm) (e) polymer-based TSKgel Octadecyl-NPR (2.5pm). (Reprinted from Minakuchi, H. et al., J. Chromatogr. A, 828, 83, 1998. Copyright 1998, with permission from Elsevier.)... 

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

Table F1.3.1 HPLC Gradient for Anthocyanin Separation on Silica Cis Columns...

Table F1.3.3 HPLC Gradient for Anthocyanidin Separation on C-is Silica Columns...

A reversed-phase HPLC column (typically Cl 8 or C30) is required for HPLC separations. Because the flow rate into the continuous-flow FAB-MS or LSIMS source must be <10 pl/min, either a capillary column must be used or else the flow must be split postcolumn. For narrow-bore HPLC columns operated at 200 pl/min, the split ratio would be 30 1. Isocratic or gradient separations may be used. A syringe pump is usually necessary for capillary columns, but standard HPLC pumps are sufficient for applications using narrow-bore columns. 

Which type of instrument should be used Is the method for one specific model from a specific vendor or should it be used by all models from all vendors This is especially important for HPLC gradient methods, because different instruments may have different delay volumes, ranging from 0.5 up to 8 ml. This can have a tremendous impact on the separation and elution order of the compounds. 

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