Chromatography Forum

If you measure peak area without column you will know how much response you got. When you inject you compound with the column sum of peak areas for all your peaks should match.
Injecting without column will give you indication on UV activity of your sample (NOT COMPOUND!!).
If you have CAD try ACN/water/TFA=30/70/0.1 with Primesep A column. Expect retention of 10-15 minutes (or even more)-it is hard to predict. If retention of your compound is too long, increase conc. of TFA (0.2%). If it is too low decrease concentration of ACN and TFA (5% and 0.05% respectively)


Regards,

Vlad

Do you know 100% aqueous handles the Primesep A column? and what is the typical cleaning procedure for this column?

By the way i have some luck with this column and still working with the method.

Thanks

Primesep A can handle ny amount of water or ACN. Stay away from MeOH as you can esterify the column (partially) and change properties of stationary phase. My gues thta using ACN/water with 5-30% ACN and 0.0.5 to 0.15% TFA will do the trick.
For washing column use higher ACN (50%) and 0.2% of TFA.

i have injected bis.. amine using primesep 100 with 90ACN 10% of 0.1% TFA and i am not getting any response from CAD or UV.

any help appreciated.

you need 10% ACN not 90% for this application. try 10% ACN with 0.1% of TFA, if this is not enough try 0.25% TFA.

My mistake i have used 10% ACN not 90% ACN. My question is i am not seeing the compound i have listed by UV or CAD with primesep. We are planning to get ELSD next week.

The compound of interest is one of the imp/deg for the formulation i am working. All of the other imp/degs found using primesep100, but not the bis.. amine.

If you have any suggestions on getting this compund by UV/CAD i will appreciate.

Bis-amine (two amino groups??) will retain strongly on Primesep 100 column and is not eluting from the column with your mobile phase due to strong ion-exchange interaction. You have few choices:

• Use much shorter Primesep 100 column (25 or 50 mm). Primesep 100 column has pKa value of 1
• Use a weaker Primesep column-Primesep 200. Primesep 200 has pKa value of 2.
• Increase concentration of acid in the mobile phase (to 0.3% TFA)

Here is application on similar mixture (several basic groups). Please notice number of amines in compounds and length of Primesep 200 column-it is only 10 mm for compounds with several amino groups:

http://www.sielc.com/compound_140.html

I am not sure that Primesep C will work for you due to the fact that you need to use ammonium acetate or formate. AmFm or AmAc does not produce non-volatile salt as in case of TFA. TFA will suppress ion properties of Primesep C column (pKa of the column is 3).

Thank you for the information. The compound i am trying to see by UV/CAD is Bis(2-chloroethyl) amine HCl.

I tried with 0.3% TFA on primesep 100 and nothing has seen.

Thanks