Chromatography Forum

greetings,

My baseline is descending through the run from positive to negative number.

mobile phase is
mobile A: 95% pH2.8 KH2PO4 + 5% acetonitrile
mobile B: 55% pH2.8 KH2PO4 + 45% acetonitrile

gradient:
0 min 92% A
15 min 50% A

thanks a lot!

By the way, my retention time changed from 4.1 to 3.9 min after I prepared another mobile phase A. Is it because of PH or Acetonitrile components?

I had similar observation. You may refer to the following link for more info.

http://www.sepsci.com/chromforum/viewto ... sc&start=0

best

duantech

Hi Zimanili,

Your baseline is exactly as expected. The descending baseline is caused by the decrease of phosphate over the gradient. It’s a refractive index thing.

By the way, my retention time changed from 4.1 to 3.9 min after I prepared another mobile phase A. Is it because of PH or Acetonitrile components?

It could be any number of the following 3 cases:

1. More acetonitrile in the freshly prepared eluent A.
2. Higher phosphate concentration
3. higher pH

By the way retention time change from 4.1 to 3.9 is actually not that bad, but if it is a problem for you, then look at the precision of the weighing and pH adjustment.

Best Regards

thanks a lot!

danko

typically for reversed phase columns, the gradients would be for longer time, the organic phase increases and phosphate buffer phase decreases. Do we expect such decreases for baselines?

By the way, it is not DAD detector and I can not set reference wavelength. Duantech

thanks

Ho, Zimanli,

Sorry for not having asked for the detector info.

If the detector is not a DAD or MWD, it could be a different case. You may look into it a bit more. According to Agilent's Detector manual, such baseline drifting is common for dispersive detectors. Also, I did not observe this with a VWD detector. With VWD, the baseline went up when gradient was started.

What's the wavelength?

best

duantech

the organic phase increases and phosphate buffer phase decreases. Do we expect such decreases for baselines?

Hi Zimanli,

Yes, the describe baseline is indeed expected in the case of phosphate (or another non UV active salt) decrees over the gradient.
However, if your mobile phase contained TFA or formic acid, for instance, then you would experience an ascending baseline (at f. ex. 214 nm) when the organic component is increased.
This is due to genuine UV absorbance as opposed to your current experience.

Best Regards

my detector is VWD. it is set to be at 210nm.
Say if I inject blank with isocratic and the baseline is flat, then I can confirm it is my phosphate, right?

Thank you everyone

my detector is VWD. it is set to be at 210nm.
Say if I inject blank with isocratic and the baseline is flat, then I can confirm it is my phosphate, right?

As Danko said previously, the background absorption of your mobile phase comes from the acetonitrile present in it. Increasing the content of acetonitrile in the mobile phase causes increase of the background absorption and vice versa. Phosphates don't absorb on this wavelength.

Yes, if you run isocratic than you should observe flat baseline if everything is ok.

Regards

OK, let’s clarify the things a bit, so that no misunderstandings arise.

1. Descending baseline means lower absorbance.
2. In the actual case none of the mobile phase components absorb energy at 210 nm.

So, what happens is: The potassium phosphate in the eluent A is more concentrated compared to the eluent B. This means that the light beam going through the flow cell gets curved somehow away from it’s intended track, which is “translatedâ€

I believe that the VWD standard cells have conical flowpaths, and thus will correct slightly for the RI effects by avoiding some wall losses, as the exit is larger than the inlet. If the flow path is shorter, the RI effects will also be reduced.

I'm not sure whether DAD cells have the same design. I also believe the micro cells for the VWD are not conical, and the DAD cells may be of that design.

Please keep having fun,

Bruce Hamilton

Maybe zimanli just has a dirty column which is slowly coming clean?

Maybe it helps...

Try to prepare both mobile phases separtatelly with the same buffer concentration.
(weight the same amount of salt, dissolve in water, titrate with phosphoric acid, add acetonitrile)

Maybe zimanli just has a dirty column which is slowly coming clean?

Then I would expect an ascending baseline under the gradient.
Anyway it’s easy to investigate the idea. Substituting the column with a connector and running the gradient should do the trick.

Best Regards

Danko, that was stated on the premise that what goes up has to come down (sometime, in spite of a gradient).