Resolution problem

[S.M.PANDYA]

Dear Experts

I am facing problem in system performance test for Resolution .

Column : Octadecylsilanized Silica gel . ( we have used YMC ODS )

For this test Propyl Parahydroxybenzoate is to be used with product .

Mobile Phase : 4.4 ml of HClO4 +1.5 g of NaOH in 950 ml water . Adjust pH to 2.0 with NaOH solution & make volume 1000 ml . To 700 ml of this solution add 300 ml of acetonitrile .

Limit : Not Less Than 12 . ( We are getting 9.88 ) .

We are getting RSD of peak area 0.90 % which is within limit . ( Limit is Not more Than 4 % )

Retention time should be about 6 min. & we are getting 6.08 min.

Could you please advise why we are facing this problem ?

Also we need advise for solution of this problem .

[philippem]

Dear Mr Pandya,

In order to give you some advise, we need more information I guess.
What is your other component, at what time do both components elute ?

You are speaking about system performance test for resolution ( separation between two peaks) Your criterium is 12 ! Baseline separation, based on the formulae to calculate the resolution, is already achieved at a value of 1.5 ( assuming both peaks have the same height and peakwidth)

Stating your limit is not less than 12 for resolution is some what over killed ?

regards

philippe

[dhru]

Dear Mr. Pandya

some time if your column is not good than also you can not get resolution.
can you try on other newer column with same make.
if this method is offical and you got resolution in past than i think you just change the column and take new one

[S.M.PANDYA]

Dear Mr.philippe

Thanks for yr prompt reply .

My product & Propyl Parahydroxybenzoate elutes at 6.18 min. & 11.748 min. respectively .

Peak height is 26916 ( Area is 435010 ) for product ( 6.18 min. )

Peak height is 11972 ( Area is 377068 ) for Propyl Parahydroxybenzoate( 11.75 min. )

[S.M.PANDYA]

To : Mr.Dhru

Yeh you are correct . It is already under analysis on another make column .

This method is recently published & hence checking for the first time for our product .

[dhru]

Dear Mr. Pandya

i hope you get the resolution

[philippem]

Dear Mr Pandya,

If this products are so well separated (by the way what is the peak width of the compenents, should be around 0.45 min I guess) why should you determine the resolution on those peaks .
Could it be that other products are eluting in between ?
I think one should look for the critical separation in your sample and put a specification on that separation.

Philippe

[Consumer Products Guy]

Sounds like you're making this way too tough, even though you don't state what your product is. Is your goal to assay the propyl paraben or both that AND your material simultaneously? When we assay for propyl paraben here we simply use acetic acid in the aqueous along with the ACN; your system requires filtration of the mobile phase (or you should) and either flushing or constant pumping to avoid precipitation. How locked-in are you to your current mobile phase?

[tom jupille]

First of all, I will reiterate the various comments that a requirement for Rs of 12 seems a bit excessive.

Nonetheless, if it's a validated method and that's the requirement, then you have to live with it (or revalidate the new method).

Resolution is affected by two things: retention times and peak widths. The retention time for propylparaben meets the specs. Is there a specification for retention time of the product peak? If so, how does that compare with your actual retention time of approx 12 minutes?

If your product retention time matches the specification, then the problem is that your peaks are too wide. If you have run the method successfully on your instrument using the specified (standard) column, then you should check the particle size of the new column. It should be the same or smaller than that of the standard column. If you have never run the standard column on your system, then your problem could be anything: particle size, a defective column, temperature variations, excessive injection volume, excessive dead volume in connecting tubing , etc., etc.

If your product retention time does not match the specification (product elutes earlier than the specification), then you will have to adjust the mobile phase chemistry (probably by tweaking the pH). Depending on how much you tweak (and what your SOPs call for), this may be regarded as a modification to the method (rather than an "adjustment") and may thus require revalidation. C18 columns are not equivalent, regardless of what the USP says! If this were my problem, I would run the method on the originally specified column.

[S.M.PANDYA]

To : tom jupille

Thanks for your reply . Following is my reply to yr message .

I agree with u for requirement for Rs of 12 seems a bit excessive.

Peak width of Product as per USP : 5 % 0.68 & 10 % 0.54
Peak width of Propyl paraben as per USP : 5 % 1.26 & 10 % 1.018

Product Retention time is specified i.e. about 6 min. & I get it at 6.18 min. Hence it is matching .

I am testing my product as per Japanese Pharmacopoeia & in this monograph it column's phase is mentioned & not the brand name .
Column : Octadecylsilanized Silica gel 5 microns. ( we have used YMC ODS 5 microns )

Please advise .

[Mark Tracy]

I suspect the original authors (or their bosses) were a bit confused. They probably meant to specify relative retention (t2/t1) or selectivity (t2-t0)/(t1-t0), but it came out resolution. Either that or their SOP for new methods required a resolution spec even when it was irrelevant. Sorry you are stuck with it.

Make sure that all the extra-column effects are minimized: connecting tubing, flow cell, injector loop, etc. Also check your detector settings and reduce the filter response constant as much as possible without excessive noise.

Since the column is underspecified, you could use a 3µ or 4µ particle size and make the peaks narrower without violating the method.

[Uwe Neue]

If it is a USP method: what are the column dimensions specified?

[S.M.PANDYA]

Dear experts

I am also interested to write my problem to Japanese Pharmacopoeia also . I wrote them on their email ID mentioned in JP but till date no reply from JP .

If anybody knows the other email ID than jp@nihs.go.jp , please advise me here or any other way to get information / advise for problem related to Japanese Pharmacopoeia , please also advise .