run interferon with C18 column

[nguyen]

I have run protein interferon injection with C18 column.
My method is:
mobile phase: A: 0.1% triflouroacetic acid(TFA) , B: 0.1% TFA water:ACN=10:90.
A:B from 52:48 to 45:55 in 45 min, to 20:80 over 2.5
min
Flow rate: 0.6mL/min
detector: UV: 214
retention time: 20 min
But i can see 1 peak at 5 min and the water valley nothings else.
I also inject solution of salt contain in the interferon injection.
there are no peaks
but i thinks the peak i have at the fifth minute is not interferon.
Could you please help me how to solve the problem
Thanks you very much

[Mark Tracy]

You may be right. A peak at 5 minutes is much too close to void for a well-designed method that lasts 45 minutes.

It is sometimes the case that proteins adsorb to components of your HPLC system, or to impurity sites on your column. One trick people sometimes use is to inject a concentrated sample of protein to cover over the sticky sites. You can either use your interferon, or something common like bovine serum albumin; make it in the initial mobile phase, and be sure it is completely soluble and well filtered. If you use interferon, keep injecting until you get a consistent peak, then try at your normal concentration.

You may also need to passivate your HPLC system. Consult the manufacturer on their recommendations, but usually it involves pumping 2-6 molar HNO3 for 30 min through everything except the column then a thorough wash with water.

[Uwe Neue]

What is the column? Is it resonably new?

[nguyen]

this is the new column. This is Kromasil C18 150*4.6 mm 5um. I have run the same method with inject milipore water there are no peaks. The peak is always at 5 min

[Uwe Neue]

The peak up front is not retained a lot, just a tiny bit. If you start your gradient at a lower organic concentration it should get retained more.

What is the pore size of your Kromasil? If it is the 100 A stuff, it may not give you much retention for your analyte. You should use a packing with a larger pore size. Even better, use one that has been designed for the separation of proteins.

[Herman]

Imtakt has a new column that separates Interferon extremely well. It is called the Intrada. This webpage shows the Intrada's application data for Interferon: http://www.imtakt.com/TecInfo/TI263E.pdf.

If you are located in North America, you can contact Imtakt’s North American partner (Silvertone Sciences) for more information. Silvertone’s website is http://www.silvertonesciences.com and their phone number is 215-966-6157.

If you are located outside of North America, you can contact Imtakt directly through http://www.imtakt.com.