Chromatography Forum

I use WATERS HPLC for dye analysis. The dye is two unknown molecules.

My system:
Xbridge C18
A : Ammonium Carbonate pH 9.8
B : ACN

230nm

I tried the method for a few times. The ratio between the two dye molecules is changong from time to time.

What do thing may the problem be???

Thank you,
Noam

The ratio between the two dye molecules is changong from time to time.

What exactly do you mean? You observed change in the ratio of your peaks' areas or change in ratio of their retention times?

we will need more information in order to understand what is going on

what is your procedure? what exactly is the experiment that you are conducting?
are those results of controlled samples, standards, unknown samples etc...
what is exactelly the nature of the change as zokatino asks? what ratio?
how do you prepare your samples?
is this a gradient or isocratic run?

things like that

Hi guys,

Thanks for your help.

1.The area ratio is the variable that causes me troubles. My intension is to determine this ratio.

2. It is a gradient.
3. sample preparation : 1ml of the ink/50ml (water)

Thanks
N

Dear Noam,

What type of method for quantification are you using for your analyses? Normalization or internal standard or something else...
Do these changes in the peak ratio vary in same direction during time? Or are they random changes? What kind of injection mode are you using? Manually or by autoinjector/autosampler?
Is it possible that your sample solution changes the concentration ratio among compounds of interest due to degradation, evaporation of the solvent etc..?

Give us some information to get the picture

Regards

at first glance i would look into the sample preparation eventhou it looks simple.
what is your entire ink made off (if you know)?
maybe there is a solubility issue. how do you mix your 1 in 50 ml of sample? for how long? have you checked samples mixed at various mixing time or different mixing methods?