Peak Area decrease
Posted: Tue Jan 20, 2009 4:23 pm
Hi all!
I am running a RP method using a Varian Reverse Phase PLPS 150x4.6mm 5um with mobile phase of 20% ACN + 0.1% TFA (A) and 90%2-propanol + 0.1% TFA (B)at a flow rate of 0.5mL/min of 100% A to 100%B in 60minutes with a reequilibration step of 25 minutes with 100%A after the gradient. I am having two significant problems that maybe related to eachother but I am not sure.
1. I am seeing what I think is carryover. I believe it to be carryover because I run at blank buffer injection at the beginning and a blank at the end. The blank at the beginning is clean the blank at the end always shows a fairly significant peak usually with a peak are of about 1000000. I have tried to dilute in the mobile phase and I have run a small test of blank, 3 samples, and then three blanks. The first blank shows a peak and the next two do not. I have also tried changing the needle wash to different concentrations currently it is at 20%Methanol but I have tried different conc of 20% two propanol as well.
2. My second problem started about a month ago, my calibration curve I run a 0.2mg/mL ref std at 25, 50, 75, 100, 150uL. Since december I have seen a significant decrease in peak area and therefore my slope. It is causing my unknown concentrations to show up high. We have also run a qELISA to confirm that my the concentrations I am getting are incorrect. I have run a injector volume test and it is coming out to be the exact amount that I injected
I appreciate any help! Please let me know if you need any more information.
I am running a RP method using a Varian Reverse Phase PLPS 150x4.6mm 5um with mobile phase of 20% ACN + 0.1% TFA (A) and 90%2-propanol + 0.1% TFA (B)at a flow rate of 0.5mL/min of 100% A to 100%B in 60minutes with a reequilibration step of 25 minutes with 100%A after the gradient. I am having two significant problems that maybe related to eachother but I am not sure.
1. I am seeing what I think is carryover. I believe it to be carryover because I run at blank buffer injection at the beginning and a blank at the end. The blank at the beginning is clean the blank at the end always shows a fairly significant peak usually with a peak are of about 1000000. I have tried to dilute in the mobile phase and I have run a small test of blank, 3 samples, and then three blanks. The first blank shows a peak and the next two do not. I have also tried changing the needle wash to different concentrations currently it is at 20%Methanol but I have tried different conc of 20% two propanol as well.
2. My second problem started about a month ago, my calibration curve I run a 0.2mg/mL ref std at 25, 50, 75, 100, 150uL. Since december I have seen a significant decrease in peak area and therefore my slope. It is causing my unknown concentrations to show up high. We have also run a qELISA to confirm that my the concentrations I am getting are incorrect. I have run a injector volume test and it is coming out to be the exact amount that I injected
I appreciate any help! Please let me know if you need any more information.