Chromatography forum은 최근에 알게된 아주 좋은 사이트이다. 전 세계 대학원생 + 연구원 + 심지어는 HPLC 책 저자 들이 다양한 질문에 다양한 답변을 해주는 곳이다. 마치 국내의 Bric과 비슷한 성격의 사이트라 하겠다.
http://www.chromforum.org <- 요기.
<About Inorganic analysis>
1. I have done some work in the where I used ELSD for inorganic anions and cations. You need to keep the temparature of the drift tube low for anions like Cl- to detect them with good sensitivity otherwise other ions are easy to detect as non-volatile.
<GHOST peak appears...>
Hello. For the past 2 months I have been having trouble with the a reversed phase HPLC method (see conditions below). It is running on Agilent system. A ghost peak has been appearing with very varying retention time of between 2 and about 30 minutes (It is not caryover from previous injection- run time has been extended to up to 1 hour). Peak shape varies from good peak shape to 'a big blob'. Peak height varies from 10mAU to about 30. Peak area of about 1200mAUs to 2000.
1.I would focus on the acid sample, as it appears that the peak correlates with an acid injection?. I suspect something is being deposited on the column, and the acid injection is partially redissolving it, and the most likely source is your samples. I assume you can't use a DAD to look at the UV spectrum?.
I would try injecting several different volumes of acid blank, and perhaps differing reduced concentrations of acid, up to 100 ul. I would also try different equilibrium times between injections, extending up to an hour, after first having ensured you had a typical area range for the peak by repeated injections of acid blank. I would also try increasing column temperature to see if peak changes size.
If the peak size increases with equilibrium time. I would suspect degasser contamination, mobile phase contamination ( filtration, reservoir sinters etc. component ( esp water, or any filtration you may been performing ).
I would try flushing the system with an acidic mobile phase ( eg gradient with 0.1% TFA, with 100ul injections of 1:1 mobile phase ) and then revert back to your analysis to see if the peaks have gone. If they have then focus on your sample preparation.
Your second comment about the same batch numbers refers to normal use,? as you also said you had tried different suppliers - water would be my main suspect, after any filtration equipment in filters in the instrument, and the equilibrium time experiment should help sort that out.
I would also very carefully examine the sample preparation, and ensure that you are not adding the peak, such as by immersing the pH electrode into the solution.
I would be a little concerned about acid-soluble material depositing in a non acid mobile phase, if the peaks are from your sample. You need to find what has changed from historical procedures. Changes such as increased mixing, warming, or ultrasonication could dissolve material that previously was insoluble.
Good luck,
2. For, Ghost peaks appearing from nowhere.
"When performing sonication, keep an eye on the temperature of the sonication bath". Some of these drug compounds are very sensitive to heat and can break up into components that can UV absorbe".
Use only HPLC grade components, as impurities can lodge on the head of your column, and can elute at a later time.
3. Watch out for the suction frits in your mobile phase reservoirs. They are often a source of gunk, especially the ones placed in aqueous phases. Someone suggested to me to first dry the frits (if they're SS) in an oven, then sonicate with water - EtOH - THF - EtOH - water or similar, to clean them up.
<Peaks appeared in blank runs....>
Do you agree ? what could be the reason of thoose peaks ?
Rq: S/N for thoose peaks are about 10.
<RP-peak tailing>
I recommend using a TFA gradient, but doing a 10-75%B. If you're lucky - you'll notice a trend (which will help you for
further optimization).
If you look at Imtakt's RP protein data (using Intrada WP-RP) - it usually starts with 10-20% organic:
Interferon: http://www.silvertonesciences.com/files/TI263E.pdf
Hemoglobin: http://www.silvertonesciences.com/files/TI272E.pdf
Bovine Serum (injected directly into the column):
http://www.silvertonesciences.com/files/TI274E.pdf
<TFA related problems>
[1] Do I need put TFA in mobile phase B??
The benefit of faster re-equilibration by using TFA in mobile phase B is pretty small (i.e. due to the small hydrophobicity of TFA your column will be equilibrated at about the same time with or without TFA in mobile phase B). Under certain circomstances you can get better base line by using TFA in mobile phase B but if this is not your problem and you still achieve separation of your analytes, you do not need TFA in mobile phase B...
As a bonus, you will avoid ACN polymerization which takes place in acidic pH...
[2] at low UV nm, baseline negative.
Dear all,
I'm using a HPLC / DAD method for peptides analysis. This method works with H2O + 0.1% v/v TFA and ACN + 0.088% v/v TFA, with gradient elution from about 50% B to 65% B in 20 min.
With this condition at 215 nm I've got a visible negative drift.
Is it normal? ( Unfortunately YES)
Why in some articles is the % of TFA in ACN equal to 0.1% and sometimes 0.085% or 0.088%? Are these differences really due to different kind of samples?
Thanks a lot!
=>TFA is, among other things, a weak ion pairing reagent; the amount that sticks to the stationary phase varies with the %ACN. Just to make things more complicated, TFA and ACN form a charge-transfer complex, and the absorbance spectrum of TFA shifts with changes in ACN concentration. The relative amounts of TFA in the A and B solvents can, in principle, be "tweaked" to minimize the baseline drift, but it's hard to get rid of it entirely. How much drift (and in which direction) can vary with the exact wavelength (i seem to recall that the TFA spectra have an isosbestic point at 214) and on the bandpass of the detector.
There was a fairly thorough write-up in LC-GC Europe a few years ago. I think it's still accessible on their web site:
http://www.lcgceurope.com/lcgceurope/article/articleDetail.jsp?id=45019