Effect of particle size

[chichibabin]

Dear All,
I have only used 3 different RP columns and I therefore have a query.
One of the columns I use is an analytical C12 4u 90 Angstrom and another is a semi-prep C12 10u 90 Angstrom. When I try and transfer methods to the semi-prep and maintain the resolution I got from the analytical column I have to at least double the run time.
Is this due to the higher particle size of the semi-prep column or is it also related to the larger volume of the column?
Would a semi-prep column with a 4u particle size perform in an identical manner to the analytical column in terms of peak width, separation and retention time?
Cheers,
Satpal

[rhaefe]

did you adjust the flow rate accordingly? Retention times should not change if the up-scaling is done correctly.

cheers

[Uwe Neue]

To maintain resolution when you change the particle size you need to reduce the velocity.

In order to scale the gradient properly, you need to maintain a fixed ratio of gradient volume to column volume, as the previous poster has pointed out. I assume that you did this.

To answer your second question: if you maintain the same particle size and column length, and are able to scale the flow rate in proportion to the column volume, your separations should look identically on the prep column and on the analytical column.

[Victor]

The previous correspondents have given you good advice. The retention times should look identical if you have scaled the flow rates.

However, of course the resolution of the 4um column will be greater than that of the 10um column, even when operated at their optimum flow (which will be different for the two columns-i.e. slower for the 10um column, as has been pointed out). The 4um column will show less loss in performance above its optimum flow than the 10um column. It is possible, that if you have scaled the flow rates correctly, that what is happening is that you are being forced to operate the 10um column closer to its (slower) optimum flow to get closer to the resolution of the 4um column.

[chichibabin]

Thanks thats what I needed to know. Just to clarify though:

The 4u analytical column volume is 4.15cm3
10%-60% over 30 min 1ml/min
30/4.15=7.2

The 10u semi-prep column volume is 19.6cm3

Therefore for identical retention times at the possible expense of resolution I would need a 10%-60% gradient volume of 19.6 x 7.2 =141.12 (or 4.7ml/min over 30 min).

What I have been doing is running the 10u semi at 3ml/min over 60min which should give better resolution than at 4.7ml/min and still do not get resolution as good as the analytical with the above mentioned method. How can I find out what the optimal flow rate is for this semi-prep column. It's a Phenomenex Proteo 90Angstrom 10u 250mmx10mm.

If I were to get a 4u semi-prep with a volume of 19.6cm3 and run at 4.7ml/min over 30 min then the chromatogram should be identical to the 4u analytical right?

Cheers,
Sat

[Uwe Neue]

Well, 3 x 60 is not 141.12, but you would need to go to much lower flow rate for the 10 micron packing to get a match in plate count, down to maybe 1.5 mL/min, or even less. Then the run time will be silly for a matched gradient.

If you have a very tight resolution, you are better off using a matching particle size in prep and analytical.