Chromatography Forum

Hi to all of you experts and non! As far as i am a little bit new to the HPLC systems i would like to help me with the injections! I am trying using a Dionex RP-HPLC system (Ultimate-3000) with manual injection port and UV-DAD detector.The question is how quick i have to turn the injection valve from the LOAD position to the INJECT position and back to the LOAD position?The loop is 20μL and the flow is 1mL/min.After flashing the loop i inject the sample and do i have to leave the syringe inside the port before turning to the INJECTION port? A detailed description of the whole procedure would be very helpful.Another query is about the levels of standards.What is the recomended levels for a calibration curve? 5ppm-50ppm is good or can i go to a more expanded standard i.e 150ppm? I am going to determine DEHP in plastics/Sludge...
Thank you very much!

The question is how quick i have to turn the injection valve from the LOAD position to the INJECT position and back to the LOAD position?The loop is 20μL and the flow is 1mL/min.After flashing the loop i inject the sample and do i have to leave the syringe inside the port before turning to the INJECTION port? A detailed description of the whole procedure would be very helpful.Another query is about the levels of standards.What is the recomended levels for a calibration curve? 5ppm-50ppm is good or can i go to a more expanded standard i.e 150ppm? I am going to determine DEHP in plastics/Sludge...
Thank you very much!

I haven't done manual HPLC injections in at least 15 years.The easiest way is to choose a loop size and have that be the full injection, like 20ul in your case, the theory being that one cannot then overfill the loop, and easier than partial loop injection. Yes, load the loop with excess to get your 20ul fill, then leave the syringe in and turn to the inject position, and leave the syringe there.

As to your question on phthalate concentrations: do a linearity study and see how far you get decent linearity on your system.

Thank you very much for the reply.After turning to the injection position should i return the valve to the load position immediately because i think if i am not very quick the pressure will arise too much and stop the procedure for safety reasons!

You need to leave the valve in the "inject" position for long enough to flush the loop; I usually go with 3x the loop volume. So, for a 100 microliter loop and a flow rate of 1 mL/min, that would be 300 microliters = 0.3 minutes = 20 seconds or so.

The pressure should be about the same with the valve in either position. If it's substantially higher in the "inject" position, then you have a plumbing problem (kink in the loop? partial blockage? bad fitting?).

Thank you very much Tom! That's the answer I was looking for! I check Rheodyne site and says about 10 loop volumes...but I think 3 loops will be OK. The pressure is the same to either inject or load but I understand that I have to turn the valve back to the "load" position for the second injection after the first flushing very quick in order not to block the flow rate and rise up the pressure and stops. That's the reason ,probably, for getting 5ppm standard with very small height! I do not take all the sample from the loop....