Aliquote 20µl of the sample into a spin filter tube and dissolve it in 60µl of the solvent, Buffer A, which is used at the beginning of the run (like dissolves like). The spin filter removes any large particulates (debris, precipitate, etc.) that may not dissolve in the solvent, as the column only allows small (less than 2µm) particulates and filters out the large particulates. This could create blockage in the column filter when there are too many large particulates to filter out, creating higher pressure in the system. Smaller particulates in the system increases the column resolution of the sample and displays a more distinctive peak.
Spin the tube at 10,000 rpm at 4 degrees Celsius for one minute in a microfuge. Sample preparation is important to maximize the elution yield and system efficiency.
Start the system by turning on the computer and the HPLC system. The computer is directly connected to the HPLC system to receive data from the detector. After the computer receives the signal from the system, the HPLC program will be opened.
Wash the system with Buffer A at 5ml/min for 20 minutes. This is to prevent buffer crystallization around the valves and to wash out any particulates that remained in the system. During this process, check and tighten for leaks and drips to prevent pressure loss or bubble formation. Check the liquid levels of Buffer A and B.
The injection site has a switch that can be manually flipped between "Load" and "Inject." For the "Load" position, inject the sample into the injection site with a syringe. Bubbles may be formed at the barrel of the syringe, which will cause errant spikes to occur in the chromatogram. Tap the barrel and expel the bubbles. Other methods include to rinse the syringe with a stronger solvent before loading or to reload the sample in the syringe by depressing and repressing the plunger carefully to prevent more bubble formation in the sample.
Type the sample information into the program prior to sample run to ensure the results are associated with the correct sample.
Flip the switch to "Inject" for the program to run and the sample will begin running through the system. Injection must be done quickly to prevent pressure buildup in the system. The sample will be run through a 5-loop system before going to the column. This is to ensure that the sample is dissolved in Buffer A prior to entering the column.
Each sample run takes approximately 28 minutes, in accordance to the solvent type and pressure gradient that is programmed for the run.
Collect the sample once a spike is seen in the program. This will be the sample that is purified from the compounds that are stuck in the column.
During the second half of the sample run, the program will introduce a second buffer (Buffer B) into the system to elute the high abundant proteins bound to the column. Near the end of the run, Buffer A is reintroduced to the system. This regenerates the column for the next sample run.