doubled peaks at tops on hplc agilent 1200 with DAD

[krystynakk]

hello everybody,

when I do hplc analysis using agilent 1200 system (with DAD) I have doubled peaks at tops whereas when I do the same using the same method and so on sample on varian system; I have tried different peakwidth (responsetime), flow rate, injection volume but with no results; do somebody have any idea what else I can change to solve the problem?

even using directly pharmacopeia methods (standard C18 RP method)gave the same resuls
the samples are normal compunds (for instance parabens, benzyl alcohol), no diastereoisomers, enantiomers,

[zokitano]

Several factors can cause peak splitting:

- presence of void in the column (did you use the same column with the Agilent and Varian system?)

- if your sample is diluted in solvent which "is stronger" than the mobile phase peak splitting can be seen (what is the composition of the mobile phase and in what solvent is your sample dissolved?) - but also probably is not the problem that you're facing with

- column overload (but i presume that you used different injection volumes and still there were splitted peaks in your chromatogram)

- if the solvent (or mixture of solvents) in which your sample is dissolved, is immiscible with the mobile phase (in that way both HPLC systems ought to show peak splitting)

- failure of your injector rotor (this may be the problem)

Hope this helps

Regards

[danko]

Or inappropriate or corrupted fitting to the column inlet (on the Agilent system).

Best Regards

[krystynakk]

my bet is something happened with agilent system, because when i use the same column, the same solvents, the same sample etc on varian system there is no peaks doubling

anyway, the split of peak does not happen always, sometimes does and sometimes (other methods, other column etc) not using agilent system; therefore, if it would be defect of the agilent system it should happen always, shouldn't it?

bu the way, about the fittings, there are standart steel fittinggs coloured green for the whole apparatus, when I asked if I can change them for others ( I meant different diameter of those fittings or using peek fittings, they said that is not appropriate to do it for that system, and if I planned to change hem I need to change all of fittings not only that before and after column)

[chromatographer1]

I suspect it might be a solvent strength problem which is not manifested when there is enough dead volume between the injector and the column to allow mixing of the sample solvent and the mobile phase.

If the splitting of the peak goes away when you dilute your sample in a more polar solvent or add water you have proof of concept.

Hope this is helpful,

Rodney George
Senior Research and Development Scientist
Gas Separations Research
Supelco
595 North Harrison Road
Bellefonte, PA 16823

814-359-5737 voice
814-359-5459 fax
rodney.george@sial.com

[danko]

The fitting could be fine for one column design but inappropriate for another.

I need to change all of fittings not only that before and after column)

Only the inlet fitting is relevant in this situation.

Best Regards

[zokitano]

I suspect it might be a solvent strength problem which is not manifested when there is enough dead volume between the injector and the column to allow mixing of the sample solvent and the mobile phase.

If the splitting of the peak goes away when you dilute your sample in a more polar solvent or add water you have proof of concept.

I agree with chromatographer1

Please tell us in what solvent (or solvent mixture) is your sample dissolved; and what is the composition of the mobile phase for the method (showing the peak splitting)

Regards

[danko]

Zokitano and Chromatographer 1, please note that Krystynakk has experimented with different injection volumes. Injecting lower volumes would’ve indicated a potential strong solvent issue.

Best Regards

[chromatographer1]

True, unless the reduction in volume was still not enough to allow mixing before the solvent plug reached the column.

I still recommend that the sample be diluted with a weaker solvent.

Still, your point has value and is noted.

best wishes,

Rod

[krystynakk]

but once again how can you explain that I have no problems on varian system;

moreover, I tried yesterday other fittings (peek tubings with 'universal' fittings), which I usually use on varian system, the problem did not disappear

i have tried regural tricks (changing injection volume, different concentrations etc) but it did not help

I have tried different column f.i. zorbax (agilent) and other manufacturer as well; it did not help; thats why I believe that something wrong is with that agilent system;

furthermore, when I was working couple months ago in different company also using agilent systems, there was no such problems, I was using there columns of different manufacturers and peek tubings with appropriate fittings;

and what about detector or UV cell (its 10 mm lenght)?

[zokitano]

but once again how can you explain that I have no problems on varian system;

moreover, I tried yesterday other fittings (peek tubings with 'universal' fittings), which I usually use on varian system, the problem did not disappear

As previously said by Chromatographer1, having not enough dead (or system dwell) volume in Agilent 1200 system cannot provide appropriate mixing of the sample injected and the mobile phase (but peak splitting will result only if your sample solvent is stronger than the initial composition of the mobile phase)

I can recall that Agilent 1200 has system dwell volume of about 0.5mL, but if you have the Rapid Resolution configuration the system dwell volume may be less than that (< 0.5mL)
I don't know what is the system dwell volume of your Varian HPLC system.

furthermore, when I was working couple months ago in different company also using agilent systems, there was no such problems, I was using there columns of different manufacturers and peek tubings with appropriate fittings;

Yes, but tell us did you use the same method/same assay then as now?
Did you use Agilent 1200 Series HPLC system in your previous company or other older Agilent models?

i have tried regural tricks (changing injection volume, different concentrations etc) but it did not help

Ok, then probably you're not facing with this "dilution" problem of your sample, but still can you provide more information about the solvents used for preparation of your sample and your mobile phase composition?

Regards

[Hollow]

maybe there is a void volume on the "innner parts" of the Agilent system.
Probably checking or re-connecting the entire flow path helps.

We get (probably) something simliar on a Waters UPLC when we tested one about a year ago. After returning the system after the test period they checked the problem and answered "void volume at injector"

see Thread, Page1, down
http://www.sepsci.com/chromforum/viewto ... highlight=

[mbicking]

Many good suggestions and thoughts. It is encouraging for the world of chromatography that we have so many people trying to help a colleague with so many comments. Even if they don't solve this problem, other readers can learn a great deal from following this discussion.

But no real answers yet. The fact that the problem is not consistent or reproducible makes it very difficult to solve.

Some additional questions:
1. Are all the peaks split, or just some? Are the early eluting peaks split more than later eluting ones?
2. I want to confirm that you are using stainless steel fittings with stainless steel nuts. If you are using PEEK tubing or fingertight fittings, perhaps the tubing is not positioned all the way into the column end, leaving a gap. This problem would change every time you removed the fitting.
3. Do you have another test mixture that you could inject? It would be interesting to see if the same problem is observed with compounds that are known to show good chromatographic performance.

[krystynakk]

now it happens, when I use methanol: water around 1:1 (v/v), but it happenned before when I was using acetonitrile:1%acetic acid (gradient or not) as well

I used different samples of different products for the analyses

small peaks have never splitted (lets say blelow 500 mAU), but higher have splitted (sometimes their heights are 1000 mAU, and sometimes 3000 mAU); so that does not happen all the time;

I use steel fittings with steel nuts, but I do not like them (I must put a lot of effort to make no leak connecting column )

when I was working at other company they used the same systems as I have now (the same for me it means agilent 1200 and the same chemstation, the same type of peek tubings etc.; at my present work I have choosen myself the agilent 1200 system because I have wanted to work on the same apparatus as I was worked before)

I do not know if it helps, but when I turn on agilent, only the diodes of column thermostat and DAD, for a while (1-2s) are red, then they change to usual yellowish colour

[zokitano]

now it happens, when I use methanol: water around 1:1 (v/v), but it happenned before when I was using acetonitrile:1%acetic acid (gradient or not) as well

I used different samples of different products for the analyses

small peaks have never splitted (lets say blelow 500 mAU), but higher have splitted (sometimes their heights are 1000 mAU, and sometimes 3000 mAU); so that does not happen all the time;

Ok then, I see that your peaks are pretty high (1000-3000mAU) and I think that you're dealing with column overloading for those compounds (in some extent). Are these peaks asymmetrical (showing front tailing)?
Column overload can cause peak splitting as stated above.

I do not know if it helps, but when I turn on agilent, only the diodes of column thermostat and DAD, for a while (1-2s) are red, then they change to usual yellowish colour

This normally happens during the initialization of the modules.

Best regards