Chromatography Forum

I just bought a new Prep Nova-Pak HR C18 Column, 6 µm, 7.8 x 300 mm. I use this column to separate NADPH, beta-NAD, ATP, and some other small compound. I can separate those compound using an analytical Agilent ZORBAX Eclipse XDB-C18 HPLC column. Buffer A was 10 mM triethylamine in water (pH 5.6) while buffer B was 100% acetonitrile. I using the same buffer system. However, ATP and NADPH can not be eluted out. Only beta-NAD can be eluted out. Is there huge difference between Nova-Pak HR C18 Column and Agilent ZORBAX Eclipse XDB-C18 HPLC column. Have anyone used Prep Nova-Pak HR C18 Column to separate similar compound? I also tried pure water and methanol as eluent.

Is there huge difference between Nova-Pak HR C18 Column and Agilent ZORBAX Eclipse XDB-C18 HPLC column.

Actually, there is. In fact, those two columns are not even close in selectivity.

I scrolled through the PQRI column selectivity database on the USP website (http://www.usp.org/USPNF/columnsDB.html). Looking at similarity to the Eclipse XDB C18 (with bases present, pH = 5.6), the Novapak column was number 320 (out of 476 columns in the database).

When scaling from an analytical to prep separation, it is always best to use the exact same packing.

Thanks a lot. Now I just bought the column last week and I will not have another new column so quickly. Does anyone know the binding specificity of Nova-Pak HR C18 Column? Or the usual solvent used in this kind of column.

NovaPak columns have been around for a looooong time (over 20 years if memory serves). Your separation can *probably* be done on that column, but you will have to start over from the beginning in developing the conditions, and there is no guarantee.

Today, NovaPak would be classed as a "type A" columns, which means that the underlying silica is fairly acidic. What you need to do to improve recovery and peak shape problems with type A columns is to minimize the ionization of residual silanols on the stationary phase surface. Generally this involves using a higher concentration of buffer in the mobile phase (anywhere up to about 50 mM) and going to a lower pH.

Gradient with A) 100% ACN, B) H2O with 10% ACN and 0,1% TFA, start with 0%A up to 40 % A would be a good start with the Nova-Pak HR C18 preparative column. I found that at a similar method and hope it helps. I agree with Tom and can recommend to check the table of hydrophobicity which should be provided by the manufacturers of HPLC columns. That will help to find a similar column, or a totally different column, what ever is needed. Good luck

Thanks, both. I will try as you suggested.
I am just very curious why NADH can be eluted out as a good peak and NADPH can not in the same condition? There is only one more phosphate group on NADPH.