Chromatography Forum

Posted: **Tue Jul 27, 2010 1:21 am**

Dear All,
I am just looking for your kind help. I am setting up a used waters HPLC 1525 system. When I am checking my system I am unable to get a stable baseline on acquisition bar. I am using normal phase column with hexane as mobile phase. I know I have to equilibrate the column for 20 column volumes or almost 1 hour. But even then baseline starts from 3 digits after decimal pints (AU) i.e. 0.001 AU and start going on up to 5 or 6 digits after decimal (AU) i.e. 0.00005 or 0.000003 AU and I am not getting it stable. It appears like a noisy baseline. I degassed my mobile phase, checked the flow cell and even run the system without the column. There is now air bubble in flow cell but when I by pass the flow cell from detector way by taking the flow cell out, I get stable baseline. Please tell me where is problem? Should I call waters service person. I will be really thankful.

Posted: **Tue Jul 27, 2010 2:07 am**

1. what is the temperature in your lab? (hexane has a very low boiling temperature)
2. what is your column type?

Posted: **Tue Jul 27, 2010 2:15 am**

Thanks for reply. My lab temperature is between 25 to 27 centigrade and I am using silica column 5 micron 4.6 x 250 mm. Even I by passed the column.

Posted: **Tue Jul 27, 2010 8:25 am**

Sorry, I donÂ´t understand what is happening. I am entering here as that 0.000003 AU cought my eye. Did I miss some very recent advances in UV detector development?

Posted: **Tue Jul 27, 2010 3:22 pm**

I think HW hit the nail on the head. Check the noise specification for that detector; detector specs are typically given for "no-flow" operation; in real-life (i.e., with solvent flowing), expect maybe 5x more noise.

Posted: **Tue Jul 27, 2010 5:49 pm**

Hi Tom and HW,
Thansk for reply. It means you guys think that my UV detector is very sensitive that is why resolution (AU) on Y axis reaches to 5 digits after decimal. If this is the case then can you tell me how I can decrease the sensitivity to keep the AU upto 3 digits after decimal. Plz guide me .

Posted: **Wed Jul 28, 2010 2:14 pm**

I didnÂ´t think anything except that I did not understand what you are describing (what happened) and how you get such a low absorption (or absorption difference?).

Posted: **Wed Jul 28, 2010 2:35 pm**

I dont think this has something to do with HPLC, its just the software that displays the chromatogramm. I don't know your software. I guess right-clicking on the chromatogramm will offer you some options. switch off autoscale.

Alex

Posted: **Wed Jul 28, 2010 4:02 pm**

maybe he wants to quantify some peaks in the 0.5mAU range. than this can be of problem...

otherwise it think you should just make an injection of your standard in the concentration you're expecting your samples and see if you get a nice peak with a good signal to noise ratio.

As Alex said, what you probably see is a very high zoom-in to your (stable) baseline, and you will always see some fluctuations in this range
(I don't know how much is common in normal phase applications, but I'm mostly fine with about 0.1 mAU noise under HPLC-RP conditions)

In contrast, a baseline which is just a straight line without any noise would make me worry a lot more...

Chromatography Forum

Base line problem in waters HPLC

Base line problem in waters HPLC