Carbon content of the column

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Dear experts,
I have few conceptual doughts in chromatography

1. Will carbon content of the column material influences the retention time of the molecule, if so how?

2. What is the role of SDS as ion-pairing reagent in improving the retention of highly ionized molecules (mobile phase also contains pH 3.5 buffer, TBA+ and acetonitrile)?

3. Amoung the amide C16 column and RP C18, which is preferred for a highly ionized molecule?

Expecting answers............

Regards
Venugopal K
1. Will carbon content of the column material influences the retention time of the molecule, if so how?


If two columns have the same specific surface and porosity, and if reversed-phase is the dominant retention mechanism, then retention time will be longer on the column with the higher carbon loading. Note that the first condition is rarely met, and so carbon load, by itself, provides little useful information.


2. What is the role of SDS as ion-pairing reagent in improving the retention of highly ionized molecules (mobile phase also contains pH 3.5 buffer, TBA+ and acetonitrile)?


The use of both anionic (SDS) and cationic (TBA) ion-pair reagents simultaneously is unusual. The effect on retention in such a system can be anything, depending on the charge and pKa of the analyte.


3
. Amoung the amide C16 column and RP C18, which is preferred for a highly ionized molecule?


That depends on the pH, the pKa of the ionized molecule, on what you are trying to separate it from, and on whether or not you use ion-pair reagents.

Expecting answers............


It would have been more diplomatic to add some explanation as to why these questions are important to you. :) The experts who answer questions here on the Forum do it voluntarily and without obligation.
-- Tom Jupille
LC Resources / Separation Science Associates
tjupille@lcresources.com
+ 1 (925) 297-5374

Dear Tom Jupille,

These questions are relavent to the topic "Separation of zwitter ionic drug molecule". In which i faced problem in separation of solvent front and drug. When i used TBA+ and SDS in combination my retention time is increased by 1 min. Which is achievement for all of us. Based on the suggestions of the experts in forum i worked and succeded. I am looking forward still improve the retention time. Before going to work on it i want to clear some doughts. Which i asked in this forum. Thank you for your valuble suggestions.

When TBA+ alone is used the retention time reduced as compared to simple pH 3.5 phosphate buffer. When i used TBA+ and SDS in combination my retention time is increased by 1 min as compared to simple pH 3.5 phosphate buffer. Even this combination is reported in JC B in 2002 and 2003 articles. If you want i will rpovide those articles for you.

Why i am asking regarding amide and RP C18 columns is to purchase a new column and use ion-paring reagents. i want to take forum help in this.

Best regards
Venugopal K

kvgpal,

Have you tried only SDS without TBA? In general the use of both ion-pairing reagent is not recommended. In the pH range that you work and knowing the pka's of your product, you probably get retention only from the SDS and not the TBA...

Venugopal,

Here is the problem why you can't get complete and good advise from this board (or at least not from me).

1. I do not understand what the mobile phase composition is i.e the % acetonitrile that you are using. You say that you want to get more retention... unless I know your solvent composition, I can not answer the question. My colleagues gave you some advice, but it appears that everybody assumes that you are working close to 100% water, which may not be the case at all, and it you still use ACN, why don't you go to a higher water content to increase retention.

2. If I understand you correctly, you are working with a potential zwitterion as sample at a pH where you MAY have a dominant positive charge, and you added cationic and anionic ion-pair reagents to your mobile phase, which increased the retention by 1 minute or so. What does this mean? What is your retention factor (at least approximately)? If it is under 1, then you got a long way to go to get to a reasonable spot. If you still use acetonitrile in the mobile phase, you may want to reduce it or remove it. You may want to go to a lower pH to get a more clear ion-pair retention mechanism, and you may want to consider an ion-pairing reagent with a lower pK (such as HFBA), if this is where the problem is.

3. At this moment, I would not consider purchasing a new column, and I can not give you good advise what to purchase. I have learned that you got a lot of tailing using a 125 mm RP column, which I translate (=guess) to be a Lichrosorb RP something. If this is indeed what you are using, I would first recommend to buy a modern C18 based on a high-purity silica, maybe a phase with an embedded polar group, but there are issues with these as well. On the other hand, you now have a good peak shape on the column that you have been using, so why change?

Bottom line: a better description of the probelm will result in better advice.
So: what is your mobile phase now, and what is your retention factor?

Can all of you who now all of this info please explain to me in a more simpler manner what it is that you are saying? I would greatly appreciate it if you would also give me places to go to for information on all of the aboved talk. Any thing at this point would be greatly appreciated.
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