Chromatography Forum

Hi !! Everyone..
My question is that like this..
I'm analyzing the substances whose UV-Abosrbance value are very low in a HPLC analysis. In order to increase their peak responses I dissolved the substances about 200mg in 10ml of Methanol each.
That means the working concentration is 20mg/mL.
Is this too high or will it be acceptable in validation procedure ?
It will be really helpful if someone give me advice about the limt of Working Concentration.... etc..

Also, their tailing factor become lager as much as I dissolve them more.
Is there any way I can improve their tailing factor?

Hi !! Everyone..
My question is that like this..
I'm analyzing the substances whose UV-Abosrbance value are very low in a HPLC analysis. In order to increase their peak responses I dissolved the substances about 200mg in 10ml of Methanol each.
That means the working concentration is 20mg/mL.
Is this too high or will it be acceptable in validation procedure ?
It will be really helpful if someone give me advice about the limt of Working Concentration.... etc..

Also, their tailing factor become lager as much as I dissolve them more.
Is there any way I can improve their tailing factor?

Did you check injecting the lowest concentration of your analyte in microgram/ml. You can not inject in mg/ml. Prepare 1mg/ml solution of your analyte and make further dilutions down. Inject the lowest amount and observe the peak. If it appears/doesn't appears...follow accordingly. You will find a concentration range in which your analyte concentration will be directly proportional to the area obtained for the peaks. The plot of concentration (ug/ml) vs area will be linear in this concentration range. With regression coefficient of < 0.9990. Validation will be be the next step after you establish the linearity range. Check you the potency of your analyte. If its high quite possible you may not find peak at lower concentrations. Check out the some reference articles and take them as a guide to start your working concentration.

For tailing- keep your injectable concentration as low as possible. Don't overload your column. Have sufficient washing. And even if you find tailing refer LCGC articles over tailing or book by Synder over HPLC method development.

Good Luck!

Regards,

Amaryl.

20mg/mL is a lot higher than a typical column can handle. If your tailing is due to overloading, a semi prep column would be the fix, but you would probably not like that route. Are there other detection methods available to you? If not, you may have to consider an internal standard coupled with derivitization...

Re going to a prep column: that may not help very much, because the larger column will result in more dilution of the analytes.

Assuming a 10 microliter injection, the mass on column is 200 micrograms -- this is almost certainly an overload (confirmed by the fact that the tailing increases with the loading).

Vanjas, you have probably already checked this, but look at the absorbance spectra of your compounds and see if you have enough signal in the "end absorbance" region (down around 200 nm). Working down there has its problems, especially in terms of interferences, baseline noise, etc., but sometimes can result in surprising sensitivity. Failing that, I would concur with DR: you need to look at another detection technique.

vanjas-

Is this too high or will it be acceptable in validation procedure ?

The concern more is about technically feasible rather acceptable for validation. Concentration in milligram per ml is certainly not feasible technically.

Concentration in microgram per ml may be detected in most cases while keeping detection wavelength low (190-210 nm). Interfernce, baseline noise certainly may pose problem in this range. However, sample cleaning is case to case basis rather a generalized. So you should look up if you can clean your actual samples efficiently (depends upon your matrix). Most HPLC methods of Cyclosporin use lower wavelength (~205 nm) for detection due to lack of UV absorbance.

Else you may need to look for derivatization or another instrumental tool for same.

Amaryl-

Check you the potency of your analyte. If its high quite possible you may not find peak at lower concentrations.

I am unable to get idea behind above statement. Can someone clarify..??

Else you may need to look for derivatization or another instrumental tool for same.

Amaryl-

Check you the potency of your analyte. If its high quite possible you may not find peak at lower concentrations.

I am unable to get idea behind above statement. Can someone clarify..??[/quote]

Sorry i missed the line low UV absorption. Happens when your work and interest area differ. Try working with ACN that works best at lower wavelength.

Well Jiten it was just my visual observation. Based on the dose of your analyte you can have an idea of your linearity range.

If the dose is low, linearity will be much lower in ug/ml range.

Regards,

Amaryl.

Dear Vanjas:

If UV-absorbance is low then... go after a high absorbance first! All other actions will be unsuccesful unless you can see your peak!

Therefore, you must try another wavelnght (as Tom referred), another detection method (RI, Fluorescence, electrochemical, ELSD, whatever!) or technique (GC, GC-MS, etc.).

One thing I have found and is underneath all comments here is that you must do some homework before injecting something to your system.

Knowing more about your molecules would help finding the column, detection method and concentrations allowed.

The reason I keep try to stick to UV-method is just because of Validation.
I think other detection methods such as (RI, Fluorescence, electrochemical, ELSD) don't show reproducibility, accuracy, etc. and I need to concern about the method validation ICH guideline..

^^

Will it be reproducible in other method?

Dear Vanja:

Any detection method is reproducible if you keep all variables reproducible.

I use to say as a joke during training that "a variable that varies the same way all the time is a constant".

Therefore, you can use any detection method, taking care with the linearity and RSD along all concentrations of analytes you are looking at.

Dear Vanja:

Any detection method is reproducible if you keep all variables reproducible.

I use to say as a joke during training that "a variable that varies the same way all the time is a constant".

Therefore, you can use any detection method, taking care with the linearity and RSD along all concentrations of analytes you are looking at.

:: points to CAD detector thread :: Some detection methods do not follow Beard's law (those that are not a function of absorbance). Such detectors do not always provide linearity... Anything UV will be linear until you run it down near the upper noise threshold or up to where the cell width allows saturation of the PM. It's best to determine these points with a linear regression of several standard solutions.

Hi !! Everyone..

Also, their tailing factor become lager as much as I dissolve them more.
Is there any way I can improve their tailing factor?

Yes .Tailing factor will be affected by concentration.You have to either reduce the concentration of your solution or you have find most optimized method that can sustain your concentration.However such high concentrations for standard solutions as in this case pose problems during validation.20mg/ml is too heavy for the column to give you reliable results as it some times leads to peak spiltting.

As for as detection methods are concerned , if your method is validated with certain detector i think you may not get problems.However i observed one thing during our routine analysis.While we are working with flouroscence detector we observed that every time we do analysis on it we get different values for the standard but however the trend for the test is matching with other intervals.

Vanjas,
It is not clear from your post , whether you are doing Assay of said compound or Impurity determination using 20 mg/ml conc ? I have seen even USP using 2 mg/ml concentration with 100 microltr injection for a RI detection . If you are interested in quantification of some impurities in the drug , you can inject provided your analyte peak ( impurity ) is not affected. If you are doing Assay of the drug with external standard , you should consider the diluting as indicated in other replies to acceptable tailing.
try injecting more of a diluted sample to see the difference in tailing.

good luck

JM