How to improve purine peak efficency

[carls]

alemaggot: thanks for the update

[alemaggot]

Hi guys!

Finally today I've perfmorm other test for my method. But I don't lead good notices. I write my problem:

1- extraneous peak given by gradient course. And I can't understand who is the font ( There is my other post dedicate to it)

2- There is one unknow impurity that is revelated only with pure water mobile phase or with 50mM phospate buffer. If I use only 20mM buffer it isn't see by detectir. How is possible?

3- At pH 3.00 the silaniols effect are completely deactivated? I ask it because now Inosine peak tailing. At flow 1.5 and 1.2 ml/min there is not difference. It tailing. And the resolution from other unkonw impurity is less. I must to regenerate the column? Guardcolumn is new.

Thanks so much!

[Vlad Orlovsky]

1. You always have gradient artifacts. If you use pure solvents and salts it is less pronounced. You can run blank and subtract blank from your run.
2. There is no way that impurity elutes with water and 50 mmol buffer and does not elute with 20 mmol. I would expect that if impurity is basic and you have retention on residual silanols that retention is proportional to buffer concentration. Please make sure that whatever you see is not eluting from previous injection and it is not randomly appearing at different places. It is always a good idea to do at least two injections in the row to see if they are identical.
3. I don't expect to see silanol cation-exchange for your compound on the column you use, at least not the way you describe. When we used even much stronger mixed-mode column there was limited ion-exchange. Your tailing also might come from guard/column connection. Inosine does not retain too long in your case and you might see effect of poor connection.

[Vlad Orlovsky]

Just though that can I post it here, since we developed a method for inosine and deoxyinosine for one of our customers. We used our new SHARC 1 hydrogen-bonding column:


retention is base on hydrogen bonding and elution order can be predicted by PSA number and accessibility of sites for hydrogen bonding:
http://www.molinspiration.com/services/psa.html

[carls]

What does this separation look like on a Primesep column with similar mobile phase?

[Vlad Orlovsky]

Primesep is a mixed-mode columns, both of the compounds are neutral and hydrophilic. Here is what we got on Primesep (K' is very low, by my standards):
http://www.sielc.com/HPLC%20Analysis%20 ... 0%20Column
and deoxy has even shorter retention. We are running this in HILIC and RP mode for demonstration purposes

[alemaggot]

Can I reproduce this Inosine retention with any ion pair reagent? Shark 1 have high Hydrogen proton donator characteristics?

[alemaggot]

I've read an article on sigma aldrich for separation of nucletides. But it's inapplicabile for me this way. They've used tetrabutylammonium sulphate such as IPC reagent. The cost of it is about 200€ for 10 g bottles. It's too expensive for me.

I must found another way.
Yesterday one person has said to me that PFP columns, if she work with CH3CN at 80 % in mobile phase, change in HILIC mode. It' right?

I think that I must resolve my extra peaks problems before. And use a phospate buffer with gradient course.

[Vlad Orlovsky]

Your compound will not benefit from ion-pairing since it is not ionic. We tried our strongest cation-exchange column and did not observe too much retention coming from cation-exchange mechanism. The best retention was achieved on Sharc 1 column (chromatogram above). We tried various approaches (HILIC, RP, mixed-mode) and only hydrogen-bonding produces reasonable retention.

P.S. If you calculate how much time (money) you spent developing this it would probably cover buying at least one column which works (Sharc 1)

[alemaggot]

If I use this column, what happen at 4-Acetamidobenozic acid salt?

[Vlad Orlovsky]

4-Acetamidobenozic acid will elute faster, you will need to use ACN/MeOH to dissolve your compounds

[alemaggot]

Ok. Can you send to me more column information? I ask to you an offer also.
In this forum there insn't the possibility to use private message?

[Vlad Orlovsky]

Here is brochure on Sharc 1 hydrogen-bonding column:
http://www.sielc.com/upload/file/pdf/SHARC_1.pdf

and here is price and part number builder:
http://www.sielc.com/Products_PriceBuilder.html

you can place order through the website or by mail. I thing that you should be okay with 100 mm column.

[alemaggot]

Vlad if I send to you my product powder, can you made any test on SHARC 1 column?

[Vlad Orlovsky]

sure. Please fill out this form that I know want do you need exactly:
http://www.sielc.com/Services_MethodDev ... tForm.html

We can set up something once we get your sample. Our turnaround time is usually less than 48 hours, unless the task is more complicated.

P.S. It's been only 8.5 months since you published your original request