method from analytical to prep

[mk12]

I have analytical method on waters xbridge C18,4.6x150mm,5 micron column with flow rate of 0.9ml/min..my peaks are more than minute apart..i am injecting 5 ul of about 100 ug/ml solution..but when i transferred the same method to semiprep waters xbridge C18,10x150mm,5 micron colum with flow rate of 4,2ml/min and i am injecting about 100ug sample in 100 meoh inj at a time my peak are not even separated..what could be the reason..we have just checked our prep..machine is fine..flow rate test is fine..both columns are same just the radius is different..then what is the resaon for my peak not to separate on prep column...any advise?

[Uwe Neue]

You did not provide complete information, but my guess is that the issue is that you are injecting the sample dissolved in methanol, while your mobile phase is something completely different.

There are multiple ways in which this problem can be solved. The simplest is to try to dissolve your analyte in mobile phase, or a solvent slightly more polar than the mobile phase. This may not work at the desired concentration due to limited solubility.

You may dissolve the sample in a more polar solvent than the mobile phase or starting mobile phase of your gradient. You can then inject very large quantities of sample, and your limitation is the volume of your injection loop.

You can work with a technique called at-column dilution, where you deliver your sample dissolved in mehtanol to the column via a second pump. This gets around ALL problems of sample solubility, independent of the cause.

I can advise you more, once you give me more details about the mobile phase, the mobile phase conditions, whether you are using a gradient or not etc.

[mk12]

my mobile phase is gradient of 5 to 8% acetonitrile (0.1%formicacid) in 30 min.
Yes i am dissolving the sample in methanol and injection volume is 100 ul per injection...
I can try to dissolve the sample in acn/water mixture..dont know yet it will dissolve or not...

[Uwe Neue]

You dissolve the sample in methanol, while your starting mobile phase in the gradient contains only 5% organic. This is the primary source of your problem.
Try to dissolve your sample in the starting mobile phase of your gradient first. We will think about further improvements of the method later.
Keep us informed!

[danko]

Hi Mk12,

Some additional thoughts/remarks: You should be aware of the fact that you haven’t up-scaled the injection volume correctly. The procedure is exactly the same as for the flow rate.

So, you should have started up with ca. 25 µL injection volume, which would have corresponded to the injection volume you found suitable in the analytical column context. Only after a confirmation of the feasibility of the transfer from analytical to preparative configuration, you should have scaled the injection volume further up to the desired value.
Alternatively you could’ve done some up-scaling studies on the analytical column, before jumping over to the preparative work.

As Uwe mentioned optimisation of the sample solvent is quite necessary in the given situation, but there is yet another matter you need to think about and that is the flow cell. It might just be too little for the applied flow rate and sample load.

Did you observe any significant retention time shift by the way? Also, did the peak get much, much broader?

Best Regards

[mk12]

Changing injeciton solution to 50% Meoh did help atleast i got baseline speartion for two peak in prep column,.but not too good resolution still but i was able to get couple of mg pure for NMR......i can not go lower on meoh becasue of solubility issue..i will definilty try lowering down injeciton volume...My flowcell is suitable for flow rate up to 20 ml per min...

tahnks for all your advise

[Uwe Neue]

I am glad that you had reasonable success.

To get around the issues that you still encounter, the technique of at-column dilution was developed. Essentially, you dissolve the analyte in an environment that favors solubility, and you change this environment just before the sample hits the column. I can give you references or direct advice, if you contact me.