Base line up and down

[praveenpaliwal]

Hi,

I have one basic question. If i am using Acetic acid or formic acid (0.1% ) in HPLC mobile phase and run a gradient form 90% Aqueous to 10% organic modifier at low wave length (i. e.210 or 220nm ) the base line goes down after 4-5 min.
But when I am using H3PO4 (0.1% in water) and run the same gradient as above and same wavelength the base linne goes up.

What is the cause of this?

Praveen

[bisnettrj2]

Do you have the same amount of acid in the aqueous and organic constituents of the mobile phase? e.g. - 0.1% in H2O and 0.1% in Acetonitrile/Methanol?

[praveenpaliwal]

Yes I have

It is 0.1% Acetic Acid in water

Praveen

[bisnettrj2]

Ok, so you have 0.1% Acetic in water - do you also have 0.1% Acetic in Methanol or 0.1% Acetic in Acetonitril as well?

[praveenpaliwal]

Yes I have

[skunked_once]

When trifluoroacetic acid (TFA) is mixed with water, the UV absorbance at low wavelength is slightly lower than the absorbance of an equal percentage of TFA in acetonitrile. Measure the absorbance of your acid/water mixture and your acid/organic mixture to see if this is the case. If so, you will have to adjust the percentage of acid in either the aqueous or organic mobile phase.

[praveenpaliwal]

I think u don't understand what I am saying.
When I am using Acetic aid / TFA / Formic Acid or ammonium salt as buffer in water along with ACN or methanol and run a gradient with lower wavelength (210 / 220nm) base line moves below the zero value as concentration of organic modifier is increases.
Now when i am taking H3PO4 / KH2PO4 as a buffer in water and run the same gradient with same wave length base line moves up side as concentration of organic modifier is increases .

Now tell me what is the reason?

Praveen

[skunked_once]

Now I understand your question but I think that the answer is the same. The absorbance of your mobile phase additives mixed in water is different than the absorbance of the additives when mixed in organic solvents. In some cases, the absorbance in the organic is lower than when mixed with water and in other cases the absorbance is the organic is higher than when mixed with water. Perhaps others can verify this explanation or shoot it down altogether

[praveenpaliwal]

so as I understand when I mixed H3PO4 in water and run a gradient its absorbance is high so baseline moves up.
On the other hand when I mixed Acetic or formic acid / TFA in water and run a gradient it's absorbance gets lower and base line moves down

am I right

[HW Mueller]

Aqu. H3PO4 has less absorbance than organic, if you add organic the baseline goes up.
Aqu. formic, etc. + TFA have a high absorbnce, if the baseline goes down when organic is added it simply means that the add in solution has less of an absorbing substance so that the mixture absorbs less.

[praveenpaliwal]

Dr. Muller plz clearify it I think I am very near to the actual solution.

[Aqu. H3PO4 has less absorbance than organic, if you add organic the baseline goes up.
Aqu. formic, etc. + TFA have a high absorbnce, if the baseline goes down when organic is added it simply means that the add in solution has less of an absorbing substance so that the mixture absorbs less.]

Regards

Praveen[/quote]

[HW Mueller]

If the first solution has low absorbance, the 2nd solution has high absorbance, the baseline goes up.
If the first solution has a higher absorbance than the 2nd then the baseline goes down.