Chromatography Forum

hi
i m using Atlantis c18 column. when i inject concentrated sample of lactic acid (80%w/w) i get very broad peak. how can i eliminate this prob

It could be due to the viscosity of the sample, or it could be due to an overload of the mobile phase. To address the first possibility, dilute the sample 10:1. With respect to mobile phase, what is the buffer that you are using and what is the concentration of the buffer?

hi Uwe
thankx for reply. Concentrated lactic acid soltion is highly viscous. yea it might be due to the concentrated solution of lactic acid. but i have to use only concetrated solution for getting oligomer of lactic acid. i m using 20mM sodium dihydrogen phosphate solution (pH=5 as mobile phase, flow rate =o.5ml/min).

phosphate pKas in water are listed at 2.15, 7.2 and 12.3. Try producing some fresh mobile phase between pH 2.5 - 3. If you want to stay at pH 5, acetate is suitable. Seeing as you're not using a volatile buffer, don't feel limited to 20 mM if you still can't get the result you want...

I agree with JA. Part of the problem is that you do not have any buffer capacity. The maximum buffer capacity is achieved at the pK of the buffer. It also increases with the concentration. I recommend to go to pH 2.15 with a 20 mM phosphate buffer, and increase the buffer concentration, if the peak is still distorted.

Hello Rakesh,

as far as I know there is a equilibrium between lactic acid, the cyclic ester (lactid) and the linear ester. You can only shift the equilibrium to the acid side when you are working under basic conditions. That means that you have to choose a column that is stable under strong basic conditions. I don't know if the pH-stability of 12 of some commercial available silica based columns is enough, or if you have to use polymer based columns that withstand a pH of 14.

But as so often, pH seems to be the key for success.

Hope this helps.

Dirk

Now thanks to Dirk's message, I understand what you are after. I do not think that the equilibrium between the acid and the esters is happening in the time frame of chromatography and at the concentrations used in chromatography. Once you have injected your sample, you should not have a problem detecting your compounds. You do not need to go to high pH, low pH is fine. Just keep your sample in the sample vial under the existing conditions. I also expect that you get better retention and separation at acidic pH.

Hi Uwe

thankx for giving me some valuable suggestion. i have analyesd the samples with dilute and concentarted solution of lactic acid both at pH 4.87. With dilute solution of lactic acid there is no problem gettng peak. but in case of concentarted solution of lactic acid problem of broad peak is taking place. i think there is some relationship between linear polymer format high cooncentration and lactic acid.

If you are truly polymerizing the LA, it makes sense that you would get a broad peak, if you have changed from a RP mode to a SE mode. Small or single LA molecules will elute normally while the oligomerized portions will elute as a function of the sizes of the oligomers, for which you will get some sort of normal distribution...

The broad peak though is not comming from an oligomer formation, the braod peak is due to the fact that you do not have a buffer in your mobile phase. Phospahte at pH 4.9 is not a buffer. You are misleading yourself if you think that the broad peak is due to oligomer formation.

Hello rakesh,

I totally agree with Uwe regarding the buffer capacity and the buffer range. You definitively should use a phosphate buffer in the range between 2.2 and 2.7. Nevertheless in your highly concentrated sample you will detect a mixture of free acid and different esters (mainly non-cyclic). So even if you have solved the problem with the broad peak you probably won't get a single peak. This leads me to the question what is the purpose of this assay. Do you want to determine the purity of lactic acid ?

Dirk

rakesh kumar, where does this oligomer come from? I just checked an old organic chem text, the lactide (cyclic dimer, six membered ring) mentioned by Dirk is formed on heating, it seems to be the favorite product (it´s thermodynamically and kinetically advantageous to form the lactid). So I would think that a trick is necessary to get oligomers.
I go with Uwe on this: no dimer, lactide, nor oligomer under your chrom. conditions. Also, Uwe´s suggestion of overload of some kind seems the most logical, in fact you almost delivered proof of that.