Chromatography Forum

Hello,

I'm new in HPLC. We had to buy one 3 months ago in order to analyse some new products.
I'm a little affraid because for our standards, retention time and area are bigger than it was at the beginning.
For each level of standard, area has doobled and there is a retention time modification : 4.85 => 5.
We made new standards each month.
We haven't change method, colum or solvant...

What can be the reason of that modifications?

Thank you.

CJour,

More detail concerning your troubles would be helpful. Is the detection UV? Do you perform post-run column cleanup? What is the general nature of your sample(s), weak acid, weak base, neutral compound? What is your eluent, does it include a buffer, how do you prepare your eluent? Are you certain prep of standard and/or injection volume hasn't changed...easiest explanation is often that an error of this magnitude isn't due to the instrument.

Please see what you think...but in general, more information rather than less helps people to help you troubleshoot.

We use ELSD detector.

Before each run, we clean out column 10mn with 100% acetronitrile.
Our standards are DDAB and Triamin to analyse products which contain DDAC, CAT or Triamin.
Dilution slovant is 60/40 water/acetronitril with 0,05% TFA. We use a gradiant of Acetronitrile and water during for analysis.
Injection volume is 10µL and haven't been changed. Drift, sample and column temperatures stay unchanged.


RT and area evolutions were quite progressive.

Hi CJour,

Okay then. Is DDAB didecyldimethylammonium bromide? I've not the foggiest idea concerning triamin--is this a single compound?

Kindly assume I know nothing of your analytes...most important is not their exact identity, but are they polar, ionic, neutral, acidic/basic...what is/are there chemical nature(s)? I can help best if I have an idea of what you're separating and how...abbreviations do not help me out.

You clean out the HPLC column with acetonitrile, very well...is this a reversed-phase column? Seems that it is based on the choice of eluent. What are the dimensions of the column, length and internal diameter? Is the stationary phase completely porous, or a superficially-porous material? Your samples are dissolved in 60/40 water/acetonitrile...what is the gradient flow profile of your separation...does it start at 60/40 water/acetonitrile and increase in percent organic, for example? And there are no buffers in there with the water?

Sample and column temp have remained unaltered, good...how do these temperatures compare with the ambient temperature of the lab?

Peak area may change due to a variety of things...could be as simple as an error in preparation. Could be settings on the ELSD. Could be changes in flow rate of eluent. Need more detail here.

Retention time may change due to a variety of things...if the gradient program and eluent flow rate have remained unchanged, could be leaks in the pumping system. Could be aging of the stationary phase. You clean out the column with 100% acetonitrile...how long do you equilibrate the system with the initial gradient eluent composition before starting a run of samples?

Guess what I'm asking for is additional details so I may better help you.

Hi mattmullaney and thank you for your attention

You are right for DDAB.
Triamin is N-(3-aminopropyl)-N-dodecylpropane-1,3-diamine.

Equipmemnt information : Waters Alliance e2695 - Column : Symmetry C18, 75x4.6mm; 3.5um - Detector ELSD

For gradiant :
Time Water Acetonitril
0 80 20
5 50 50
13 10 90
18 80 20

We equilibrate during about 30 mn in order to obtain a 0 signal on detector.

Temperature in Lab is hot (25°C due to clim problem) but the evolution of RT and area have begun before hot temperature.

My thanks,

So your analytes of interest are both polar...perhaps you should consider buffering your mobile phase, that may help greatly to improve the repeatability of the analytes' retention times. Buffered mobile phases may even help with the consistency of retention times of neutral species depending upon the sample matrix. Perhaps a simple pH 2.5 phosphate buffer, 25 mM? Reasonable starting point. Another variable to consider with keeping consistent retention times is temperature...if the lab is warm...when the samples are running, can the lab cool down? This may increase analyte retention times...Consider setting a Column Temperature.

Your column will have a volume of about 0.848 mL or so with the stationary phase inside. Please Consider your Flow Rate here and the retention times of your analytes. When do they elute during your gradient program? All between 5 and 13 minutes? In general, it is a good idea to inject a sample solvent composition no greater in percent organic than the starting composition of your gradient program...yes, I understand that one may get away with doing this, but generally if a minimum of 5-6 "column-volumes" of mobile phase are able to pass through the column prior to the elution of your first peak of interest.

I'll have to think a bit more about the increase in peak areas...please, see what you think, so far.

Thank for your patience.

TFA is not enough to buffer (~2pH)?

Moreover, phosphate buffer is a problem with ELSD detector, isn't it?

Column temperature is fixed at 30°C and remains stable like sample temperature (20°C).
But we haven't stuff to warm the little lab where is our HPLC because it is a separated room...
Today HPLC room is at 27°C and thus solvant temperature are high... Can it create huge variations?


RT for Triamin is about 5mn and 11mn for DDAB.

If the pump is giving a lower flow than expected, the RT and peak area will increase.

That is ar least valid for UV, I assume that it is the same for ESLD?

Hi CJour, Mattias,

For solute-dependent detectors (UV, fluorescence, electrochemical, and ELS, among others), Mattias is absolutely correct. If the flow is being affected in any way to lower levels, peak response/area would increase. Twice the peak area is a lot, though...the nebulization of the analytes in the sample also have a distinct role in ELSD...if those setting have been modified or changed somehow, that could make a differencel.

CJour is also correct, only volatile buffers such as trifluoroacetic acid, ammonium formate, ammonium acetate, acetic acid, ammonium carbonate and ammonium hydroxide could be used with ELSD, or perhaps heptafluorobutyric acid...modifiers that would work with MS as well.

Have to run, will keep thinking about the problem...