Figure 26.3: A linear gradient of acetonitrile. 0 – 10 minutes is 0% B that allows binding of the analytes to the column; from 10 – 40 minutes, the acetonitrile concentration linearly increases at 3.33% per minute causing elution of the analytes; during 40 – 50 minutes, the column is washed with 100% B causing elution of the molecules that might have not eluted during the linear gradient; during 50 – 55 minutes, the solvent composition returns to 0% B; and during 55 – 65 minutes, the column is equilibrated with 100% A making it ready for next injection.
- 21. Once the column is equilibrated with solvent A i.e. once you see a very stable baseline, set the absorbance to ZERO (for PDA detector, it becomes zero for all the wavelengths).
22. In the appropriate software window, include the sample information, the file name for the chromatogram, and the directory where the chromatogram is to be stored.
23. Turn the injector knob to the ‘Load' mode and inject 100 μl of the solvent used for sample dissolution using a clean injection syringe.
24. Turn the injector knob to ‘Inject' mode and take out the injection syringe. Turning the injector knob starts the HPLC run. It is, however, possible to start the run at desired time following sample injection.
Figure 26.4: Injector in ‘Inject' mode (panel A), an injection loop (panel B), an injection syringe (panel C), and a C18 column (panel D) |
