Unretained compound

[vikmurush]

I am trying to get retention of this compound. I appreciate any of your suggestions.

I tried C18 Zorbax Eclipse with MP acetonitrile 25% in water. It elutes about 1 min on 25 cm column. By increasing the aqueous portion in the mobilephase i did not see any improvement in the retention. Diluent water, column temp 30c and analysis LC using UV detector at 195nm. I need to have retention in order to separate placebo peaks from this compound. This is one of the degradant.

Thanks for your help

[danko]

Try HILIC

Best Regards

[mohan_2008]

1. Please let me know the pKa of the compound if you have info.
2. How much shift in RT do you require for this compound inorder to be away from the rest of placebo peaks.

Based on this info - I can send you an optimized method.

No need for Hilic to start with. Based on your requirement, we can tweak the RP method, to obtain just enough retention.

[vikmurush]

Thank you for your replies. I need at least 3min retention so that i can separate all the placebo peaks from this compound. I do not know pka at this time. I may need to look in to some ion pair reagents. I am thankful for any of the ideas you can suggest.

[mohan_2008]

Hi Vikmurush,

Since you require only a 3 minute RT, I would say try RP with the below modifications before you choose an ion pair.

Using an ion pair will definitely move up your RT as much as 6-10 minutes (based on my experience with the methods) approx and should solve the problem.

However try this:

1. Use this low pH combination mobile phase - 80% Methanol + 20% Water in pH 2.5 adjusted phosphate buffer (20 mM).
(I removed ACN from the equation since MeOH has a lower eluting strength)

2. Use a Zorbax SB Phenyl column 4.6 X 250 mm column (Pi-Pi interactions) with a 3.5 uM particle size.

3. Use ambient Column temperature or 23 C (thermostatted).

4. Use a lower flow rate of 0.5 mL/min.

I think by tweaking your method as above - you can hit the RT mark of 3 minutes.

Let me know if this works.

[Kostas Petritis]

Hmmm,

I do not think that anything that Mohan_2008 proposes will work... 80% Methanol eluting strength is too much and you already mentioned that increasing the aqueous portion you do not get any retention. I also do not see much potential for pi-pi interactions so a Phenyl column won't work and finally temperature and lower flow rates by themselves won't be any good.

My take would be either ion-pairing chromatography (assuming that you can use a low pH mobile phase and that your molecule can be ionized positively (which looks like it can) or use HILIC as danko suggested. You could also try to use a polar embendeed reversed phase column.

[Vlad Orlovsky]

Kostas,

I am not sure that this molecule is ionizable, it is phosphonamide which suppose to be neutral, unless ring is opening and forms zwitter-ion, IP will not work My guess is you will have retention on silica column at 85-95% ACN and buffer pH above 5 (to make silica more polar).

If it is basic in nature (which I don't believe) you can use Primesep 100 column similar to etanolamines:
http://www.sielc.com/compound_106.html

You will need ELSD to monitor this product

[vikmurush]

thank you for all of your replies. As i mentioned before this is one of the degradant. In API i can analyze as i mentioned using C18 Zorbax. For the product formulation i need to separate this product with placebo. For now the product method i am using is same conditions with 20mM phosphate buffer (pH 6.0) 75% with 25% acetonitrile. Since my wavelength set for 195nm i can not use methanol and it supresses the signal very largely. As per the product conditions i am getting about 9 min RT for the active peak and 6 min for the another degradant. This degradant retention can be changed by changing MP pH. The retention increases as the mobile phase pH increase. I believe i can not use lower pH. I dont know where to start for the other degradant i have mentioned in my original post. I would appreciate some help. The active is an amide group and its MW is about 200.

I like the ideas from Orlovsky and Petritis. Please suggest what kind of IP reagents i can start with and at what concentration, pH? I would like to start with Ion pair coz all other chromatography from C18 for the active and other degradant is fine. If i dont have any other choise i can change entirely. Also like to know what PEG column i could try? Is the waters Symmetry Shield C18 is a good start?

[mohan_2008]

I apologize for my earlier suggestion, using 80% Methanol. I might be sleeping at that time.

I meant to say: 80% Water + 20% Methanol in 0.1%TFA which will have a lower eluting strength as compared to,

80% Water + 20% Acetonitrile.

everything else should be O.K. -

Using a Phenyl column can hold onto the compound by pi-pi interactions.
Ambient temp should slow down elution as compared to 30C.
Low flow rate also slows down the elution.

Is it o.k. Kostas.

[mohan_2008]

Further,

I agree with you Kostas.

However, Vikmurush is asking only for a 3 min RT elution.
Doing this tweaking might hit to this mark maybe not any better.

I agree that ion pairing is good option.

We had a similar situation and my proposed scheme worked for me.
I gained 2 minutes elution time.

But trying Polar embed column is a very good idea.

Thanks Kostas.

[Uwe Neue]

This is a very polar compound, without ionic functions to ion pair. You need to run it in 100% water on the most retentive RP phase that you can get for this purpose, and this is Atlantis T3. Ion pairing is a waste of time, but if you want some really good ion-pairing reagents, you should look at the Waters catalogue.
I personally would go for HILIC using Atlantis silica HILIC with an acetonitrile-based mobile phase.
You will have trouble seeing this thing; 210 nm might do OK, if you use acetonitrile/water. Alternatively use RI in isocratic mode or ELSD.

PS: I rarely do commercials, but with all the nonsense recommended above, a commercial is justified. The recommendations in these commercials will do exactly what I said...

[vikmurush]

Thanks Uwe for your suggestion. I tried up to 90% aqueous on C18 but it did not retained. I will try Atlantis T3 and see what i get. Thanks again

[JA]

We have been told this compound elutes at approximately 1 min on a 250 mm (assuming 4.6 mm dia.) Zorbax Eclipse C18 at (assuming) 1 ml/min. This is with pH 6.0 phosphate buffer/ACN mixtures.

It's clearly unretained to the point it elutes before t0, perhaps indicating one of the following:
- ion exclusion
- pore exclusion
- peak mis-identification

I find it disingenious that someone could suggest a flow rate of 0.5 ml/min with the aim of achieving 3 min retention on a 250 column where even t0 would be at approximately 5 mins.

Personally I feel the chromatographic mode should be fundamentally changed with evaluation of, as suggested by others, NP, HILIC or IEC (if it is indeed ionised) with consideration of detection interferences where applicable.

[mohan_2008]

I had a similar problem with an analyte eluting early. I tried using HILIC with Waters Atlantis C18.

Problem is: Waters Atlantis C18 column brochure does not even indicate an operable pH range.

In other words - Is it Ok to use pH 2 (and no idea!).

Apparently the Waters Atlantis column manufacturer assumes that the analysts can take a good guess!

Well, I took a good guess and used the HILIC at pH 2.5 and it was Toast!

Beware of Atlantis Hilics.

[Uwe Neue]

Mohan: why did you use the "Atlantis C18" in HILIC mode?
All information about all Atlantis columns, including the pH stability, can be found in the Atlantis brochure at http://www.waters.com/webassets/cms/sup ... 000640.pdf