Hi Michelle,

you may check the links I attached, especially the second one ("method transfer calculator").

For our prep. HPLC, we use a 250x30 mm column which is approx. 176 ml stationary phase. Sample loop is 20 ml, flow rates 20-40 ml/min and we inject up to 300 mg per run. It always depends on initial purity and if the components you try to separate are more or less different in their hydrophobicity. If you go for ~ 12% or a bit less of the column volume for the sample loop, you'll be fine.

Anyway, you need to try with a 'not so precious' material, ideally with comparable chemical properties (hydrophobicity, molecular weight ...) at sequentially increasing sample amount. If you see fronting/tailing or bad separation, you'll know that this was too much :) Then inject ~20-30% less and this your maximum value you can go.

In any case, lower volume injections with higher concentrations are strongly recommended than the other way around. If you want to be on the save side for your purification, start with low sample amounts and do more runs than doing less runs with bad separation.

Hope this helps,

Marcus

http://www.sigmaaldrich.com/analytical-chromatography/hplc.html

http://www.sigmaaldrich.com/analytical-chromatography/hplc/method-transfer-calculator.html

https://www.agilent.com/cs/library/...s/Public/LC-Handbook-Complete-2.pdf

Thank you so much! That was really helpful :) I appreciate it!

I'll check the size of our column and sample loop to be sure I match the sizes right.  I don't think this HPLC can do that high a flow rate (it's not a full prep it's only a semi-prep ... but it will work perfectly for my compounds!)

Yes I had planned on practising with a "sample" compound before moving on to purifying my actual compounds - just in case! And thank you for the notes about fronting/tailing - I didn't know that. 

Thank you so much!

Hi Michelle,

that's right, please adjust the flow rate to the column specifications. It was just an example from our lab where I know it works. I guess for semi-prep it should be something around 5 ml/min.

A good peak shape is a general issue for HPLC analysis, you try to have best resolution with low time consuming. Be aware to use the mobile phase A (main solvent) to dissolve your material if possible. If you add too much B, you'll have bad separation. I found another two links which perfectly explain that. Very good descriptions, I should also go through it btw :)

Be aware - this presentations are from Agilent and they'll always write that they are the 'best'. Everyone has his own preferences, we stick to Phenomenex columns for instance; only for some special cases Agilent. Phenomenex offers a really nice column protection set up ("security guard" pre-columns with exchangable cartridges available for all stationary phases, third link). In any way I recommend you to use pre-filters or pre-columns to preserve the main column from get too much dirt, but this is the best solution for my taste since you see if the pre-filter gets dirty and change it. Usually you check the pressure. If it raised about 10-20% of the initial 'fresh column' pressure, you change the pre-column. If you do this, it is sometimes already too late since some impurities already entered the main column. No advertisement, I'm clearly not employed at Phenomenex, just my experience :)

https://www.agilent.com/cs/library/slidepresentation/Public/Tips_and_Tricks_HPLC_Troubleshooting.pdf

https://www.agilent.com/cs/library/eseminars/Public/secrets%20of%20good%20peak%20shape%20in%20hplc.pdf

http://www.phenomenex.com/products/detail/SecurityGuard%20Standard/C18

Again - thank you for the wonderful advice. When you say main solvent, what would you call the main solvent? I work on a gradient of 0-60% water to acetonitrile, and my peptides dissolve either in water only or acetonitrile only (depending on sequence) - in this case, which would you say is the "main solvent"?

I'll check if we have a pre-column and if we don't I'll try and get my hands on one - the compounds aren't very "dirty" but it's better to be safe than sorry for sure.

And yes for semi-prep it's about 5-10ml/min - I think this is what I will be working with. Starting with 5ml/min to start with. As for loading, would you recommend a particular concentration for that flow rate, with a column length of about 27cm? For the analytical I was using 1mg/ml and injection volume of 150ul - and the absorbance was about 1.25, which is apparently what one would be aiming for? What do you think about this?

Thank you so much for your advice, this has been so helpful :)

If you start with water - this is the main solvent.

Try to go as low as possible with AcN concentration, the peptide will else not interfere with the column then and you will not have any separation. Usually peptides do not dissolve in 100% AcN, you always need protic solvents in the mixture (H2O).

What we do for prep. HPLC: dissolve 200-300 mg crude peptide in 2-3 ml 50% AcN/H2O, soak into syringe, then wash vial with 5 ml 10% AcN/H2O, soak into syringe, then wash vial with 5 ml H2O.

With that, the peptide is in solution and the overall AcN content is ca. 2 ml AcN in 20 mL sample loop - total 10%, which is fine. If I would try to dissolve the peptide in 10% AcN/H2O right away, this would not be possible!

For injection amounts and flow rates, I can not give any advice, you have to try out for your own.

Thank you so much, Marcus - you really know your stuff when it comes to HPLC. I will take all of this into account when starting my preparative HPLC - I may even try your exact method on sample preparation! 

I was wondering if you would maybe be able to help me with something that happened me during some of the analytical runs I did last week - seeing as you know HPLC so well you might be able to help, if possible?

I had ran a sample of one of my peptides - water soluble, quite hydrophillic. I ran it at 1.5ml/min on a variable gradient (0-60% ACN over 60 minutes, then ramped up to 100% for 3 minutes, held at 100% for 3 minutes, then back down to water for 3 minutes) and it eluted at 25minutes. 

I then ran it again but changed the gradient to 0-100% water to ACN over 60 minutes, but the peptide eluted at 2 minutes - which seems wrong. Note that I had ran this peptide before over this same gradient and it eluted at about 20 minutes. This has happened me before on some other analytical runs of other peptides.

Do you know what might be causing this change in retention times? Has anything like this ever happened to you?

To be sure: did you rerun the collected eluent containing the pick?

Did you concentrate it?  how? Redissolved it?  how?

Take into account, that your sample's water/ACN ratio differs from that in the former run: It contains more ACN, if I understood the situation correctly.

That's why Marcus recommended dissolving the sample in the "first" or "Main" solvent.

I would recommend to use two wave-length simultaneously, if possible, and compare peak's ratios between runs. 

Hi Michelle,

for the second run, the peptide should elute earlier, but not so early. As Zvi mentioned, are you sure you dissolved the peptide in the same way and injected the same amounts? I think of too much AcN in the sample solution (or in the loop?) or a column overload. Be sure to fully flush the loop with solvent A before adding new sample (e.g. leaving the injector at the "inject" position after the run is finished and wait the time necessary for complete flushing of the loop, than go to the "load" position). For instance, we need to wait 1 min @ 20 ml/min to ensure the 20 ml loop is full of solvent A before adding any new sample.

Marcus

Hey everyone

Thank you for all of the answers - all great help!

I think what happened in the second run was that I didn't hold the wash at the end for long enough, and there may have been remaining ACN in the system. I haven't run that one again yet (we ran into mechanical difficulties with the HPLC!)  but I will run it again ensuring I maintain constant conditions - although I am almost 100% everything I did for the second run was the same. If anything was done differently, it would have been an accident. 

Again, thank you all for your help! I will run that one again ASAP.

:) 

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Struggled and found- Do believe!

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