Chromatography Forum

I am using a Waters Alliance/quattro micro and trying to run a gradient from 2-40% ACN:water. My needle wash solvent is 80:20 MeOH:water with some NaOH and the purge solvent is ACN. The wash cycle is purge-wash-purge. I am not getting as good a separation of my first two analytes as an app note that I have, and I am getting a peak eluting in the void that has the masses of my analytes. Could the 100% ACN plug in my needle be messing up the start of my gradient? Samples are also reconstituted in ACN.

If I am doing a 10 uL injection, how much solvent is actually being injected? 10 uL shouldn't have a pronounced effect, should it? Column is 50 x 4.6 mm, flow is 2.0 mL/min.

The most likely cause of the problem is not so much the needle wash, but the fact that you dissolve the analyte in a strong solvent, i.e. 100% MeCN.

How much your sample is diluted depends on the precolumn extra-column bandspreading, which is something that is usually kept to a minimum.

I suggest to redissolve the sample with a substantial fraction of water.

Thanks for the reply. I tried prepping the sample in water, 50:50 ACN:water, and ACN, and also tried 100% ACN and then 100% water as the needle wash. Best results were obtained with sample in water and water as the needle wash. Needle wash did have an effect, as well as the sample prep. I am going to try 10-25% ACN to see if I can get away with it.

I mean purge solvent, not needle wash.