HPLC methods Development for basic compounds

[tom jupille]

Uwe, my "back of the envelope" estimate for peak capacity here is somewhere around 50 (assuming a target resolution of 2 and 10,000 plates). The problem is getting those 15 peaks separated -- which means a focus on selectivity.

Having said that, I agree with your suggestion about starting at a lower %B, given the relatively early elution of the first peak, then using gradient steepness, mobile phase chemistry, and column chemistry to adjust the spacing of the peaks. Only difference is I'd add temperature to the list.

[SIELC_Tech]

dcobice

can you rovide us with the names of basic compounds your are analysing - just want to know what are you dealing with in terms of the difference in basicity and hydrophobicity.

[Uwe Neue]

Tom.

My calculation is based on baseline resolution, but it makes no difference in the overall judgement. For a resolution of 2, my estimate translates to a peak capacity around 90. There is no way that he can get to the same spot with a shorter run time with a 3 micron column and the available pressure, unless he changes the selectivity drastically. To go to acetonitrile will on one hand reduce the pressure and narrow the peaks, at the same time, it will scramble up selectivity enough so one can start from a fresh spot (and a more promising spot to boot).

[tom jupille]

Uwe, other things being equal, I'd trust your spreadsheet more than my estimate :

P ≈ 2 ΔΦ N^½ [k* /(1 + k*)] (for resolution of 1)

Assuming a 10,000-plate column, and a k* of 4 (to make the math easier) that's:

2 x (0.5) x (100) x (4/5) ≈ 80

I think I specified a resolution of 2 in my post, which cut that number in half (okay, so I rounded up from 40 to 50).

We're at least in the same ballpark. Either way, there's enough room to separate 15 peaks, the problem is to avoid having the first peak elute early and then to get the right selectivity to separate these 15, so I think your suggestion is dead-on.

[dcobice]

Thank you very much for your reply!!! but Its imposible to use acetinitrile in the mobile phase because Acetonitrile and TFA react with any compound of the impurities profile and a artefact peak appears near the active peak.I tried to use Acetonitrile because of things taht you told me but for these reason ITS imposible.These artefact peaks increase the heigt during time..... the area was about 15000.

Thanks again!!! and Have a good year!!!!!

[dcobice]

I try to do these, but Its a bit problem when talk about selective of the 15 peaks!!!

[SIELC_Tech]

Any reverse phase column provides you only with one type of interaction (hydrophobic). Ion-exchange column will provide you with ion-exchange interaction. When you have a mixed mode column you can combine these interactions and use it for your benefits. You can either run isocratic conditions keeping organic content or ion strength of the mobile phase the same, do gradient by changing organic content (increasing concentration of ACN or MeOH), do gradient of ion-strength (increasing concentration of buffer) or do double gradient (changing both concentration of organic component and concentration of the buffer of the mobile phase). This approach allows you to create two dimensional variation of the mobile phase and give you more flexibility. We were able to separate over 50 compounds using double gradient on Primesep columns (amines, amino acids, organic acids). Selectivity of the column can also be modified by changing the nature of the additive (ammonium formate, ammonium acetate, phosphate buffers, TFA, sulfuric acid, etc.). Please check the following

http://allsep.com/Technology_RetentionO ... pounds.php

http://allsep.com/Applications_By_Compound.php (Library of Applications)

Here is the method development guide for mixed mode columns:

http://www.hplcmethoddevelopment.com/

The beauty of this approach is that you can scale it up to preparative separation and use a volatile mobile phase to isolate you compounds

http://allsep.com/Technology_Preparativ ... graphy.php

I am associated with a column mmanufacturer so my advice might be bias so if you like you can explore other people suggestions ( HILIC, ion-pairing or any other approach) - it is the matter of choice.

Happy New Year to Everybody on this Board

[Uwe Neue]

I do not know what you see, but acetonitrile is not reactive. This may have something to do with the quality of the acetonitrile that you have.

Also in one of your previous replies you complained about the slope in the baseline. What wavelength are tou using to monitor the chromatogram, and why did you select this wavelength?

[dcobice]

I do not know what you see, but acetonitrile is not reactive. This may have something to do with the quality of the acetonitrile that you have.

Also in one of your previous replies you complained about the slope in the baseline. What wavelength are tou using to monitor the chromatogram, and why did you select this wavelength?

hi, Thanks for you reply... the peak what I told you was appeared near the active peak, and we confirm these, monitoring the stalitity of the solution and the peak increase during time.Also the data was confirmed with mass spectra LC/MS/MS.The fragmentation profile was identical to an acetylation of an important Synthesis impurities.The quality of the acetonitrile was HPLC ( fisher chemicals).
The wavelength was 220 because of the absortion coeficient of the most important impurities.Sure that acetonitrile is better than metanol to change the selectivity, but I had these issue with AC-N.

Happy new year!!!!

thanks!!

[dcobice]

Any reverse phase column provides you only with one type of interaction (hydrophobic). Ion-exchange column will provide you with ion-exchange interaction. When you have a mixed mode column you can combine these interactions and use it for your benefits. You can either run isocratic conditions keeping organic content or ion strength of the mobile phase the same, do gradient by changing organic content (increasing concentration of ACN or MeOH), do gradient of ion-strength (increasing concentration of buffer) or do double gradient (changing both concentration of organic component and concentration of the buffer of the mobile phase). This approach allows you to create two dimensional variation of the mobile phase and give you more flexibility. We were able to separate over 50 compounds using double gradient on Primesep columns (amines, amino acids, organic acids). Selectivity of the column can also be modified by changing the nature of the additive (ammonium formate, ammonium acetate, phosphate buffers, TFA, sulfuric acid, etc.). Please check the following

http://allsep.com/Technology_RetentionO ... pounds.php

http://allsep.com/Applications_By_Compound.php (Library of Applications)

Here is the method development guide for mixed mode columns:

http://www.hplcmethoddevelopment.com/

The beauty of this approach is that you can scale it up to preparative separation and use a volatile mobile phase to isolate you compounds

http://allsep.com/Technology_Preparativ ... graphy.php

I am associated with a column mmanufacturer so my advice might be bias so if you like you can explore other people suggestions ( HILIC, ion-pairing or any other approach) - it is the matter of choice.

Happy New Year to Everybody on this Board

hi, i think that is very interesting to do 2D-LC, I will do sure , any experiments....

Thanks a lot for reply!!!

HAPPY New years!!!

If I will do any with these kind of 2D-LC platform, sure I will contact you.

[smiley]

to SIELC, Tom, and Uwe,
dcobice has 15 peaks in his sample, so do you guys have any good sample test mix that can mimic the situation to test either the slope calcultion and peak capacity?
the most i can find from column vendor is about 4-5 peaks, it may work fine to show a point but not enough to solve the practical situations like this guys' here.
and to SIELC, what do you think the difference b/w primesep column and HILIC mode column? and do you guys ever try your column in other mode of chromatography? or just one fixed mode?
thanks.

[Uwe Neue]

Smiley: I do not know what you are after, but the theory of gradient separations has been cracked a long time ago. These calculations are thus fairly solid. We have recently looked at peptide gradients published in J. Chromatogr. 1061 (2004), 183-194. A specific view of rapid, broad gradients has been published in Advances in Chromatography, Vol. 41, 93-136 (2001). There are a few c-grams in there with hundreds of peaks. Chromatography works the same way whether you have only a few peaks or you have a few hundred peaks

[Uwe Neue]

Smiley,

In the meantime, I looked up the two references which in my opinion are the gospel of reversed-phase gradient chromatography: Snyder, Dolan, Gant, J. Chromatogr 165 (1979), 3 and 31. Thye are good reading for anybody who wants to understand how reversed-phase gradients are working.

[pipettemonkey]

Smiley,

In the meantime, I looked up the two references which in my opinion are the gospel of reversed-phase gradient chromatography: Snyder, Dolan, Gant, J. Chromatogr 165 (1979), 3 and 31. Thye are good reading for anybody who wants to understand how reversed-phase gradients are working.

Do you, by chance, have electronic versions of these earlier publications?


I am particularly interested in learning the nuts and bolts involved in
numerical applications in reversed-phase HPLC.

andyfromelite@hotmail.com

[Uwe Neue]

No, I do not have these old publications in an electronic format. I have a publication on my own on method development in eletronic format and I am willing to pass it on to those that are interested.