High-performance Liquid Chromatography (HPLC)
High-performance liquid chromatography (HPLC) is a separation process used to isolate the individual components of various organic mixtures. The technique can be used to determine the qualitative nature and quantitative concentration of various species within a sample at 0.1 mM level.
(1) Turn on the software, and click on the “Method”, choose the method of “startup_CO2 reduction”. Then click the icon of “download”.
(2) Click the icon of “Status”, which is close to “Method”. Then turn on the VWD and RID detector for the HPLC, just click the icon of “on” and wait for the detector icon to turn green, showing “Idle”. Note: plan to use at least 60 minutes before running samples.
(3) Afterwards, click the “Method” icon, and select the method of “CO2 reduction”. Then click the “Status” icon, and wait for the column pressure rises up beyond150 bars.
(4) You can key in the sample information either in "Single Sample Analysis" or "Sequence". If you have many samples fall under the same project, "Sequence" is more convenient.
(5) Then the running system can be started up after clicking “Run”. The whole characterization process of each sample will take ~1 h.
(6) Go back to the “Status” and click the button “Submit Shut Down”. This program will flush the system before shutting down the pump and detectors.
Remarks:
In our lab, the mobile phase solution is 1.0 mM H2SO4, and the stationary phase is de-ionized water. The chemical species for calibration in DI water are formic acid, acetic acid, ethanol, and propanol (0.1, 1, 10, 100 mM for line fitting calibration). The retention time of chemical species in other stationary phases (KHCO3, KOH, H2PO3) may shift.
Before testing our sample, DI water samples should be run two times to ensure the system (e.g., temperature & pressure) is fully stabilized. The background should be flattened out, instead of keeping raising at different retention times.
In general, you should transition between normal and reverse phase solvent systems by choosing an intermediate solvent that is miscible with both systems, purging the system, and gradually introducing new eluent(s). Note that you should label and separate solvents that are used for eluent and washing to prevent cross-contamination.