Preparative HPLC of peptides (13kDa) on a ACE C18 150x10.0mm

[daggio]

Hello,

I have some questions regarding purification of my peptides, its about 500 mg crude peptide.

I dont have access to full prep. system and only have an analytical cell

Thinking of using these condtions:
10-30% MeCN/0,1% TFA over 30 minutes at 4,7 ml/min with column as described in subject.

Questions:
Can I select an non-optimal UV wavelength for the analytical cell, in such way avoiding saturation of the signal. Or do I have to but a prep. cell.

Approx. how much peptide can I load per run. I was hoping to load between 30 to 50 mg. Analytical gives base line separation from impuritites.


Thank you!

[danko]

How much did you loag in the analytical set-up?

What was the analytical column's dimensions (most importantly the diameter, but the length would be relevant as well if the particle size on the prep. column is larger)

Sure you can use a non-optimal wavelength in order to reduse the absorbance, but you have to remember that absorbance isn't all you need to think about when upscaling from analytical to prep. Very small flow-cell will simply not be large enough to contain a large fraction/peak.

What is your current detection wavelenght - in analytical context?

Finally did you load more and more sample until you observed severe peak broadening and resolution decrease in the analytical set-up?

Best Regards

[daggio]

hi,

wavelength is 254 nm

analytical column is 4.6 mm id, 50 mm. 3 um

semi-prep is 5 um.

i have not determined loading for analytical.

thanks.

[Bruce Hamilton]

wavelength is 254 nm
analytical column is 4.6 mm id, 50 mm. 3 um
semi-prep is 5 um.
i have not determined loading for analytical.

I'm assuming that your analytical column brand and packing is identical to the proposed column, if not, you need to determine loading on the proposed column, not the analytical.

Assuming your impurities have similar UV profiles you can use non-optimal wavelengths, but if they have different absorbances at 254nm you have to be aware of what you are missing by using a different wavelength.

Maybe the simplest solution is to just put the proposed column into your HPLC and measure the ability to separate the peaks at different loadings to determine if 500 mg can be processed in a viable number of runs.

If the separation is difficult ( lot of closely-eluting peaks, ) your loading will be low, but if impurities are very well resolved, your loading will be much higher. Quantity loaded also depends on final product specification.

[danko]

Hi Daggio,

254 nm is not a feasible wavelength for detection of peptides/proteins, you have already chosen a non-optimal wavelength. Hope it’s adequate in your setup. I'd try 280 nm if I were you.

The length of the new/prep column is OK in the context of the larger particles compared to those in the analytical column.

So, the next – and I’d say the most important – step you should go to now, is a loadability study – either on the analytical column or directly on the prep.
For a start (if you want to go directly to the prep column) you could calculate the initial load (which most probably could be increased gradually) by finding a correction factor using the following equation: F = D^2/d^2.

F ~ factor to multiply by
D ~ the diameter of the prep column
d ~ the diameter of the analytical column

Then you multiply the load you’ve found reasonable using the analytical column by that factor and you get an initial suggestion.

Good luck

Best Regards

[daggio]

Hi Danko,

My peptide which is 1,3 kDa not 13 kDa (Sorry)

The peptide contains a tyr and trp residues that absorbes around 254 nm.

I have performed loadability study now on the semi-prep...an it seem that a maximum of 20 mg injected in about 2 ml solvent (H2O) is ok, with a gradient of 10 to 20% MeCN over 30 min at 5 ml/min.

254 nm, however, seems to work fine without overwhelming the detector completly.

Thanks.