Hello,
In the chromatogram you can see that the second peak (retention at 4 min), which is an acetate peak, has huge tailing which is known for the sample that is measured but I need to know how I can separate the component that causes the tailing.
My mobile phase is MilliQ/H2SO4 (1000;0.5) using an H+ Ion exchange column with UV detection. Method uses a flow rate of 0.8 ml/min with a column temperature of 40 degrees for the first 8 minutes.
I think you can dilute your samples (low concentration) and reduce the ingestion volume. Also, the solvent that is used to prepare also affects.
попробуйте сделать градиент или почистить колонку, плохое разделение компонентов может и с этим быть связано. так же можно увеличить время и изменить скорость потока.
The problem is probably because of the interaction of your column with the component.
Check if it's linear and you will see that it's ok.
If you have a column cooler compartment try with the lowest possible temperature and optimise.
I think we need a little more information. Presumably the H+ Ion exchange column is something like a sulfonic acid in the H+ form, which is not usually appropriate for acetate.
If the peak you're considering is the one at about 2.2 minutes, there is not enough retention for an adequate separation (perhaps the mechanism includes adsorption or ion exclusion), and it could be a system peak.
A general approach, not often used, to partly separated peak without modifying the chromatographic parameters is to switch at approriate times between identical columns, with the same mobile phase (the "heart-cutting" advantage as described by Deans in about 1968). I wrote about it here : DOI:
- 10.13140/RG.2.2.17720.85763
Typically, tailing is an indication that the API is not 'happy' with the mobile phase system and the retention (separation) it provides. It can be resolved by using a stronger solvent (IPA instead of Methanol) or a higher percentage of organic (60% instead of 50% Methanol) in order to 'boot' the molecule off the column. This will lower you retention time and sharpen the peak.
Baris S.
You indicate above that this is a 'pre-made method'. Such methods may be compendial (official; air, food, water, pharmaceutical, etc.), taken from the open literature or established locally. Whatever the situation here, we need to know the source of the method, and to have enough information to establish whether the samples being analysed fall within its intended validated scope.
Ideally, we should have accessible references to the analytical development reports, which should help with proposing modifications; unfortunately, these are not always readily available, particularly with official methods.
Christopher R. Lee
The pre-made method I was talking about was for the calculation of acetyl levels which was done through the internal standard & acetate peak. I do understand what you mean with the previously made comment however I am a starter intern who deals with confidentiality.
Ideally I want an experts suggestion on what parameters I could adjust or change with the HPLC method details given above to get a sharp peak so that the peak area is more accurate (and not tailed) for this ''pre-made method'' that calculates the acetyl levels.
The H+ method and column (it's a DVSP like a carbohydrate column but in the H mode) was developed for small fatty acids like acetic acid (vinegar and cheese analysis). That there is acetyl group complicates the separation. You can first try a stronger concentration of acid (0.0075 M) but an organic solvent will swell the DVSP backbone. Then look for a C18 column that can resolve carbohydrates and use an organic solvent.
I don't think so! The H+ column is a specialty column for small chain fatty acids (like formic, acetic, propanoic, butyric, pentanoic sometimes) and has many manufacturers. As I suggested before you can try a small amount of organic solvent (maybe 5% Methanol which is still polar). You might get lucky! Good luck!
نحتاح معزفة المواد الموجودة مع المادة الاصلية المراد فصلها حتى نتوصل الى امكانية تحسين الفصل بتغيير نسب وقطبية النذيب المستهدم للازاحة
Your best chance is to change column and separation mode. This is one of several methods developed in our lab https://sielc.com/hplc-method-for-separation-of-acetic-acid-and-formic-acid
It works differently so it can change the profile of your separation.
Yury Zelechonok
Hi, question. I see that a buffer gradient H3PO4 - 0.05-0.5%, 5 min is done with this method. My current mobile phase is H2SO4 in h2o, would I get different results if I had a mobile phase with only H2O and add H2SO4 for 0.05-0.5%, 5 min like the method describes?
Yes you can try sulfuric acid or perchloric acid instead of phosphate. Also you may not need gradient at all. However method that I recommended is used different column.
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