I have noticed something this week when running a Toluene GC direct injection method. constant temperature at 100 deg C for 15 minutes. i am using nitrogen as carrier and detector make up gas, hydrogen and air also used in detector for this assay. I have been running 50 injection charts. for the Original chart the first 45 or so injections showed unexpected retention time t = 3 mins (but the RT doesnt change across all 45 injections) and massive peak areas ~1,300,000. RT should be 4 mins. This run was put on several hours after both empty cylinders of Nitrogen were replaced with full ones. Also to note there has been no Helium all week so those lines are not at full capacity/or empty. the last 5 injections of the same chart showed a sudden shift to the expected retention time t = 4 mins and peak areas in line with previous week’s charts ~ 130,000 (ten fold decrease). This sample set was re-ran after tightening column nuts and inspecting glass liner but no cracks in liner. the rerun showed for the first 33 injections the expected RT t = 4 mins and expected peak areas - then suddenly on the 34 th injection the RT goes back to exactly what it was for the first injections of the original chart t ~ 3 mins with the same massive peak areas ~1,300,000.

I set up a new instrument with new column using same instrument method yesterday. I set up original instrument but modified instrument method to be “constant flow” as i noted in software "constant pressure" was selected. . New syringes and inlet liners were installed on both instruments. Both systems observed the exact same retention time shift at the same time this Friday morning. It is almost as if a different carrier gas is getting pumped in when this occurs. . An air blank was running on one instrument the same time a standard injection was running on the other instrument. the air blank showed a notable rise in the baseline. the standard injection showed the sharp change in retention time and increased peak areas. the next several injections were unchanged with both systems showing 3 mins retention time and massive peak areas. At the same time on both instruments, the retention time jumps back to 4 minutes and more expected peak areas are observed. This coincided with the delivery from BOC and the full Helium cylinders being hooked up to the manifolds. So despite my assay/instrument not requiring helium, if helium is not hooked up out in the compound area, my retention time will shift to 3 minutes/peak areas will increase. In total this has happened 4 times in as many runs and this has not been observed any other week across familiarization runs/robustness/specificity/precision runs and appears to coincide with when the Helium line on the neighbouring manifold is fitted with new full cylinders/Helium cylinders empty. I am not sure what the issue is but I will hold off on setting up my last two validation runs until after all lines are filled with all gases out in the compound.

Any feedback would be appreciated.