Chromatography Forum

I am trying to setup my HPLC for lactode determination. I run the calibration standard consisting of Melezitose and Lactose but I am surprised to see the chromatogram. I am getting two peaks but at the same time they are going towards negtave side too. I think it is problem of detector? Can any one any help me where is the problem.

More information is needed before we can be of help

What are your chromatography conditions?

what type detector are you using?

Can you post an example of the chromatogram?

We're good, not telepathic, just good

Thanks for reply. Here ia all about my LC system/conditions
Water 1525 Binary pump.
Varian Prostar 9042 RI detector
Biorad HPX-87P anion exchange column with guard cartridge.
Mobile phase is water
Flow rate is 0.6 ml
Column temperature is 60 Centigrade as I cant get higher than this from my comlum heater.
I have chromatogram, but how I can post it here. Can you tell me how I can post.

To post chromatograms...

viewtopic.php?f=1&t=2617

Dear All, thanks for your help and reply. I have uploaded the image of chromatogram. Please guide me why I am getting positive and negative peak at same time for my calibration standard. I have already stated all the chromatographic conditions. Here is the link of chromatogram.
http://tinypic.com/view.php?pic=2i9oy83&s=7
Thanks for your time and help

Here's the OP's chromatogram:

Start by injecting a blank...

. . . and then inject a much smaller sample (like 1/10); my guess would be that you're overloading the detector. Those peaks look like first derivatives.

It looks like there are three preaks here. First the negative peak starts, a positive peak sits right in it. Just before the negative peak comes back to the baseline the second positive peak emerges. (I have seen this many times, especially at tm, with UV detection). This has been sort of suggested already: Inject all the sample components one at a time + blank, also vary amount/conc. injected.

Hi,
I haven' used the varian system. But while working on GPC with shimadzu, such peaks come adjacent to Main peak whenever I keep R flow ON. That is reference detector saturation ON.

Thanks dear all. I am injecting 20 microliter of my calibration standard. But any how I will inject in more less quantity as you advised and then will let you know. But it is really surprising for me that at same time i am getting both positive and negative peaks of almost same same respobse on both sides. Should I change the detector? Thanks

Those peaks are almost perfect differentials. Is there a "differential" mode setting on the detector? Is there a chance that you have the plumbing arranged so that sample is going through both the reference and sample side of the cell? Either of those could generate that kind of waveform.

Thanks tom,
No there is no differentail mode set. This thing is also in my mind, taht may be there si some problem in the solenoid valve and the sample is flowing both from sample cell and reference cell and I am getting negative peaks of asme height. As trhis is used detector which we bought it online so I think there sis ome problem and sample is passing through both sample cell and reference cell.

It certainly looks to me as if the sample is passing through both the sample and reference cells. Definitely check that solenoid.