Chromatography Forum

Hi,

The method says to use C8, 75 X 4.6mm, 3um column.
However I've got C8, 75 X 4.6mm, 5um column from the same manufacturer. Can I go for it? What would be the possible difference that I can expect? What if I use longer column (e.g. C8, 150 X 4.6mm, 5um) would that compensate the difference of particle size?

If you don't have any requirement to use *exactly* the same column, and you don't have any requirements for resolution between peaks or any peaks that elute very close together, then you should be fine. For all you know, a 3-micron may have been all they had in the drawer when the method was developed. and it may be more column than you need. Of course, it all depends on the flexibility allowed in either the method, the regulatory body which oversees the work you're doing, and the chromatographic requirements of the method.

As above. As long as you are specifically required to follows the method to a 'T' you should be fine. You will find a decrease in back pressure with a 5 over a 3 and your resolution may be slightly decreased but its unlikely any huge differences will be observed between the two. You may find peaks are not as sharp but you should be fine in the long run.

Hi,

The method says to use C8, 75 X 4.6mm, 3um column.
However I've got C8, 75 X 4.6mm, 5um column from the same manufacturer. Can I go for it? What would be the possible difference that I can expect? What if I use longer column (e.g. C8, 150 X 4.6mm, 5um) would that compensate the difference of particle size?

As already mentioned, if you're in a regulated environment (e.g. pharma) you must use the 3µm column. Increase of particle diameter is not permitted according to the pharmacopoeias.
If you're free to change the method you may use the 5µm column at the cost of a decrease in plate count (i.e. wider peaks) and thus lower resolution and signal/noise ratio.
Using a 15cm 5µm column should give you theoratically roughly the same plate count (indeed even a bit more) than the 7.5 3µm at a bit lower back pressure. Be aware that retention times will be doubled though!
You said you have a column "of the same manufacturer"? Is it exactly the same stationary phase or just the same manufacturer (i.e. a different C8 column)? Different columns may give you different selectivities even if they're coming out of the same house...

The above responses are very good.

If the method is fully validated and fixed, stick to what is stated in the method.

If it is not, then feel free to do some basic method redevelopment with a different particle size. If you are lucky it will just be a case of adjusting flow rates or phase composition to compensate for the drop in plates.

All else equal (same mobile phase program, flow rate) if you have a closely-eluting pair, the 3 micron column will improve resolution between by ~ 30%