I'm trying to purify tagged ssDNA aptamer (26-mer, ~8.5 kDa) to remove un-tagged sequences. On HPLC I found a reverse phase procedure in the literature that works quite well: 0.1 M triethyl ammonium acetate at 0.9 mL/min with 2% / min gradient of MeCN (SolvB = 0.1 M TEAA with 25% MeCN).
The HPLC column I used is: YMC brand C18, 50x4.6 mm ID, S-5um, 8nm.
Now I want to scale-up and collect the purified product. The column I have for our CombiFlash is: Agela brand C18, 20g, 40-60 um, 6 nm.
Can anyone advise on how I can adapt the HPLC method to CombiFlash?
You will have to play around. The Combiflash only has 1 wavelength set at 254 nm. Begin by using the same mobile phases as you currently have. The difference in retention times will be mainly due to your void volume in the Combiflash (there are other factors too).
What is combiflash. Are flash chromatography columns not Thin layer chromatography or its equivalent?. Do they use some special material like aluminium. THe Question is very interesting.
Your Combiflash column will be thicker, so you peaks will be wider. Do you have a baseline separation requirement between peaks?
Thanks Bruce, I figured that I'd have to play around with the method a bit, but it always helps to start by asking for expert advice.
I think that our CombiFlash system has multiwavelength detection, so it should be easy to detect tagged vs untagged aptamer.
The HPLC method separated the products with ~12 min difference in retention time, so the peaks should stay well-resolved in the flash column. I don't have a requirement for peak separation, but ideally I would like to keep the tagged-aptamer peak narrow to collect the target in a concentrated aliquot.
I work for Teledyne Isco, which makes the Combiflash system, and I've done some work on scaling up from analytical to preparative and flash chromatography.
The peaks will be wider due to the generally lower resolution of a flash system (larger particle size, larger dead volume in tubing compared to a preparative system). I find that I achieve better resolution on the flash if I reduce the gradient end points by about 5% of the strong solvent. I am assuming you are running a focused gradient, as I didn't see that listed in the question. It seems that the HPLC resolution I saw in your reply above will allow good resolution in the flash system.
However, I note that the HPLC column is YMC and the flash column is made by Agela. Please remember there may be a selectivity difference because the C18's aren't quite the same. With 12 minutes resolution on the HPLC, it should still work well.
Which CombiFlash system? They have had multi-wavelength detection for about 10 years (or more). There is also All-wavelength Collection, and options for ELSD and mass spectrometer detection.
Jack (or anyone), could you provide any recommendations for starting conditions on the flash column? On HPLC I was able to achieve rapid separation by flowing only solvent B (0.1 M TEAA with 25% MeCN) at 1 mL/min. I tried 10 mL/min and 20 mL/min on the CombiFlash with the 20g C18 column and didn't see any response at 260 nm - even after a 30 min run. On HPLC the peaks from a 20 uL injection were 20 mAU - for flash column I added 500 uL because the LoD is 20 mAU. Do you think I need to add more sample, run longer, faster flow rate? Any advice would be greatly appreciated. Thanks!
This could be due to many factors.
1. Your injection volume should be just a factor of the respective column volumes;
Combiflash IL = (volume of Combiflash column/volume of analytical column) x injection volume of the analytical column
2. Because of the difference in the packing you made need a stronger composition of your mobile phase. Increase your ACN concentration intermittently from 25% in 5% increments until the protein comes off the column. If this doesn't work use IPA instead of ACN. Your flowrate looks OK.
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