Fronting

[syx]

Dear Chromatographers,

How to overcome fronting peak? We have problem with PVP25 as excipient in our formula. It has huge-long front side that disturb the integration due to floating baseline. The other peaks are in normal shape.
We use a RP-18 column with a mixture of pH 2.5 phosphate buffer - methanol (contains 0.5% TEA) as mobile phase in gradient run.
Oven temperature 25 C, flow 1 ml/min.
What should we do to repair the shape of PVP25?

I have a simple idea to resolve the problem: eliminate PVP25 from the formula, but the formulator disagree.

Best regards,
SYX

[Supercritical]

Syx...yes reformulating is the best idea!

Fronting in this case is most certainly due to overloading. You need to reduce the amount (pvp25) you are injecting somehow. Some column manufacturers claim to be able to load more onto a column so you could investigate that some. Could also try a sample cleanup (SPE?) pre-injection. Choice of detector could be helpful to not see this component, but the effect of its eluetion may still be noticable.

[DR]

Can you change the wavelength? If your analytes of interest absorb at higher wavelengths than PVP does, you mey be able to get around it by running at a higher wavelength (but if PVP is really loading the column, RTs between samples & standards may vary some - solution is to add PVP to standards if RT differences are unacceptable).

[ravi]

have you tried different temps or sample diluents? What is the dimensions of your column?

[syx]

We have 2 active substances in the dosage form, 2 mg and 0.25 mg/5 ml. For sample solution I dilute 5 ml of syrup to 20 ml with initial mobile phase.
I use 2 wavelengths for detection: 223 nm (1 - 10 min) and 240 (10 - 15 min). On 223 nm, the baseline is floating after t0 due to PVP25.
the peak of PVP is eluted at the same time of 2nd substance. I cannot change the wavelength, they have small absortion in the concentration I used.
I have tried to reduce gradient slope and ... PVP peak is wider!
I have tried temp 25 - 40 C, 25 is the best for separation.
We use 150-4.6 mm column.
Supercritical, do you have experience with SPE to a sample with polymer? It may be a good option.

[Uwe Neue]

1. What is the molecular weight of PVP 25?
2. Can you precipitate it while keeping your analytes solubilized?

[syx]

PVP25 has approximate mw 30000.

Solubility
PVP25:
freely soluble in water, in alcohol and in methanol, slightly soluble in acetone.

Substance 1:
very soluble in water, freely soluble in alcohol, in methanol and in methylene chloride, soluble in chloroform, slightly soluble in benzene, and in ether.

Substance 2:
practically insoluble in water, sparingly soluble in ethanol, in acetone, in dioxane, and in methanol, very slightly soluble in methylene chloride, in chloroform, and in ether.

I do not know how to precipitate PVP25. But I have found identification method for PVP in USP that using precipitation method:
A: To 10 ml of a solution (1 in 50) add 20 ml of 1N HCl and 5 ml K-dichromate: an orange-yellow precipitate is formed.
B: Dissolve 75 mg of cobalt nitrate and 300 mg of ammonium thiocyanate in 2 ml of water. To this solution add 5 ml of a solution of Povidone (1 in 50), and render the resulting solution acid by the addition of 3N HCl: a pale blue precipitate is formed.

I am not sure that those reagents are safe for the active substances and the column.

Picture 1: Chromatogram of PVP25


Picture 2: Chromatogram of sample solution, red arrows are active substances: respectively, substance 1 and 2.

[Uwe Neue]

Based on the information that you gave, I would try acetone as a precipitant. I have to admit though that I would be uncomfortable knowing that an undesired ingredient coelutes with one of my actives.

[syx]

Yes, my target now is separating substance 2 and PVP25. I have tried to reduce slope of gradient after 7.5 min, but the result: wider peak of PVP.
I will try different proportion of acetone in diluent to get optimum condition.
I will report here. Thank you.

[maris]

As I understand PVP 25 it's a Polyvinylpyrrolidone 25 -i.e. a polymer consists of molecules with molecular weight distribution in range with the average molecular weight value of 25000. So, it not surprising at all, that you observe the "fronted" peak - you have here not a single molecular moiety, but poor separated mixture eluted as a broad peak. For such a complex mixture of different molecular weight polymers like PVP 25 the obtained picture isn't bad at all!

[syx]

You're right, maris. Do you have an experience to tame this peak?

[maris]

I have no idea about the nature of your active substances, what can be done is to try to move the analyts peaks by changing the pH (PVP-25 should be insensitive), addition of ion paring reagent etc.

[syx]

..., I would try acetone as a precipitant.

PVP25 is still soluble in acetone...
In Merck Index I have found that it is insoluble in ether. I think it will be difficult because ether was not immiscible with mobile phase.

Note:
The syrup contains 250 mg PVP25 per 5 ml.

PVP... formulators love it, I hate it!

[adam]

Seems like nobody mentioned one of the most obvious solutions.

Try to increase the resolution of the method: weaker mobile phase, longer column (maybee even smaller particles if pressure will allow).

[syx]

I have tried to change the organic component to acetonitrile in the percentage that has same strength (40% to 70% acetonitril in 15 minutes). PVP is eluted in the front, and disturbs the peak of substance 1.
I blend the composition of the organic solvents to get 20% ACN/25% MeOH to 35%ACN/30% MeOH in 15 minutes. Substance 1 has k' = 1.29.
I changed wavelength to 250 nm to reduce baseline effect of PVP.