Chromatography Forum

G'day from DownUnder

I have a lit reference that uses a prep column for the separation of 2,4-D herbicide and metabolites, that i wish to run on the same column chemistry but with different dimensions. I've tried to work this out with the available method transfer calculators available on-line, but for the life of me i can't fully understand if what i am doing is correct.

so i've come here to seek some advice and help.......

the lit method is as follows
Column: Nova-Pak C18, 100* 8 mm, not sure of particle size as its not mentioned in the reference, but i think these are 4 um(?)......

Flow: 1.5 ml/min
Eluent A: acetonitrile with 1% acetic acid
Eluent B: Milli-q with 1% acetic acid

Gradient elution profile (using Waters pump)
Time %A %B curve
0 20 80
20 50 50 6
25 100 0 6
35 100 0 6

the column i wish to transfer this method to is also a Nova-Pak, but has dimensions of 150*3.9 mm with 4um particle size

any advice, pointers, assistance with this transfer would be immensely appreciated.

thanks
Greg

Hello

Just take the ratio of the square area of the columns - reduce flow and injection volume by this factor ( you will need same column length and packing do get similar results).
If you did prep in overloaded mode, you may reduce the loading (conecentration) of your sample in order to get good analytical data.

best regards
Chris

Thanks for your input Chris, much appreciated.

I changed flow rate according to the differences in column dimensions, but i am still not getting good match of Rt on the analytical versus the literature prep column Rt's. I am still flummoxed about how i alter the gradient conditions to match the differences in column dimensions.

I am giving the gradient conversion from the Thermo calculator (http://www.hplctransfer.com/GradientMethod.aspx) a go now, fingers crossed.

Cheers
Greg

Column dimension changing and gradients can be tricky....

Indeed, it can be tricky.

With the particulars of you system as you've presented them, I would expect a ~1.5x increase in retention based upon the longer column that you're using (150mm vs 100mm). Were I in your lab coat, I would worry less about matching retention times than about matching or improving resolution. Changing from an 8mm diam column to a 3.9mm column would require a flow rate change from 1.5 ml/min to ~0.36 ml/min, which strikes me as pretty slow for a 3.9mm diam column with an ACN based MP. Also, on a Waters system (Alliance or component), the system volume is going to be a significant factor at that flow rate when using a gradient. You would also need to multiply your gradient times by 1.5x, meaning your gradient would go from this:

Time Flow %A %B
0 1.5 20 80
20 1.5 50 50
25 1.5 0 100 (assuming this is correct - it doesn't quite make sense as an RP gradient as you've written it)


To this:

Time Flow %A %B
0 0.36 20 80
30 0.36 50 50
37.5 0.36 0 100

Looking at it, I think the system volume is going to have a tremendous effect at that flow rate.

If I was in your lab, I would redevelop the method using the MP compositions in the literature separation as a starting point. I'd probably boost the flow rate to ~1 ml/min and work with gradient times, flow rate, and column temperature to optimize the separation until I met or exceeded the resolution observed in the literature.

Don't worry too much about matching RT's. Worry about resolution.

Let us know how it goes!