Six Steps To Purify Hplc

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HPLC systems are frequently used by pharma companies. These need to be regularly maintained and purified to ensure their smooth functioning. In case you are facing problems with purifying HPLC, mentioned below are the steps that will help you purify your HPLC systems. You can also ask an HPLC replacement parts service provider to offer you the necessary guidance. Well, here are the steps-

Take Aliquote 20µl of the sample in a spin filter tube. Next, dissolve it in 60µl of the solvent, Buffer A, that is used at the beginning of the run. Now, the spin filter will remove large particulates (for instance debris, precipitate, etc.) which may not dissolve in the solvent. This is because the column allows less than 2µm particulates. It will flawlessly filter out the large particulates. This can also create blockage in the column filter when there are too many large particulates that need to be filter out, creating higher pressure in the system. Smaller particulates in the system increase the column resolution of the sample and display a more distinctive peak. You need to spin the tube at around 10,000 rpm at 4 ...
... degrees Celsius for a minute in a microfuge. You should always remember that the sample preparation is important to maximize the elution yield and system efficiency.
Next, you need to start the system by turning on the computer including the HPLC system. You will find that the computer is directly connected to the HPLC system to receive data from the detector. When the computer receives the signal from the system, it is only then the HPLC program will be opened.
Now you need to wash the system with Buffer A at 5ml/min for 20 minutes. This is done to prevent buffer crystallization around the valves and also to wash out any particulates which have been remained in the system. Make sure that you check and tighten for leaks and drips to prevent pressure loss or bubble formation. You also need to check the liquid levels of Buffer A and B.
The next step involves the sample run, herein the injection site has a switch which can be manually flipped between "Load" and "Inject." For the "Load" position, you need to inject the sample into the injection site using a syringe. One may find bubbles being formed right at the barrel of the syringe, which will cause errant spikes to occur in the chromatogram. Now you need to tap the barrel and expel the bubbles. Further, you need to type the sample information into the program before the sample run. Why so? Well, this is done to make sure that the results are in connection with the desired sample.
Now, you need to go for the switch “Inject” so that the program can run and the sample, too, starts functioning via the system. Here it is always important to remember that injection must be done rapidly, so that pressure does not get build up. The sample needs to run via a five loop system. This is mainly done to ensure that the sample is dissolved in Buffer A prior to entering the column. Remember that each of the sample will take 28 mins approx to run. This is in accordance to the solvent type and pressure gradient which is programmed for the run.
Lastly, you need to collect the sample once a spike is seen during the program. Now, this is the sample which is purified from compounds that were stuck in the column. In the next half of the sample run, program has a second buffer (Buffer B) into the system for eluding the high abundant proteins bound to the column. You will notice that while the run ends Buffer A is reintroduced to the system. This will regenerate the column for the next sample run.

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