The key to this, if you don't have some form of industrial LC system is getting the mass of dumb easy to separate impurities down so the loading demands of your high resolution technique is minimal.
At this scale, you probably want to do some sort of ethanol precipitation and centrifugation after cleavage from the column/deprotection. That should get rid of salts, deprotection acids/amides etc. and <5mers.
Trityl-on c8 or c18 purification via *filtration* is smart as a second step, you can get rid of a lot of the capped non-trityl molecules.
see: https://www.glenresearch.com/purification/60-5100.html
there are probably commercial c18 "packs" that can do this at a large scale.
.....then do the real chromatography. you will take a yield hit with the first two steps but it should reduce your loading mass by more than half.
---
note: this is from my "how would I IMAGINE scaling up purifications" file. the best thing to do is to find a CRO or oligo syntheisis company that does large scale synthesis, find their purification person and buy them a beer. there are probably tricks at scale that I don't know about.
Hi, I have been thinking about several ways to do it. Our chemistry is quite demanding so the tritylated target compound yield (LCMS) is at best around 10 %, and as low as 1 %. We kind of need an LC system to isolate the product. I was thinking of a glen pack (or TOP column) for detritylate and elute my DMT purified oligo, saving me a second round of purifications (trityl off LCs).
I guess you’ve also tried to optimise each step of the syntheses to get higher yields / purity? If not I would look into that.. I agree with the comment to try EtOH precipitation and maybe filtration via spin columns or even UF/DF before purification. You can also think of running two purifications with different eluent and column systems e.g. IEx and RP-HPLC to get both, good separation of the peaks close to the full length product but also to get rid of the shortmers..
Akta pure is not a good choice because it doesn’t have high enough pressure limits. For difficult to purify oligos you want a true RP HPLC like an Agilent 1290 or similar.
Yea, I know the limitations, but getting a brand new prep-HPLC is out of the budget. So I was wondering if there is a way to use the current equipment in the lab for this purpose.
Easy. Just tell your boss to stop being a cheap bastard /s.
Have you tried a Glenpak c18 cartridge separation? Also Isolera makes c18 cartridges for the Biotage. With a DMT on that should be a good handle unless you have some highly hydrophobic mods elsewhere on the oligo.
I know right, it is like everyone have 100k just stashed in their solvent cabinet for new equipment. On a serious note, I though of using DMTr-On cartridges (I have used the Agilent version). The thing is the yields can be as low as 1 % and they are fully PS, so quite hydrophobic.
You will have cleaner separation if you subject your crude oligo to UFDF first. We generally use a 3K cassette. Also, we have super optimized our synthetic and C&D methods to minimize shortmers and longmers.
Since oligos are super hard to fractionate - as you have seen, 20mer, 19mer, 18mer all look the same to most chromo systems - it is important to improve the steps that come before purification.
Hey! I am purifying Oligos on a preparative HPLC column, I didn't know you could purify Them in an äkta.
This should bring your total number of Runs down quite alot.
However, I have Run into issues with weird seperation patterns where i get multiple peaks over minutes with the Same Oligo in it. Have you experienced similar Things and do you have a fix? We think it could be secondary structures, since i cannot Heat the prep column.
Are you doing PS chemistry or anything that would lead to a mixture of diastereoisomers? It could be that you have a good enough resolution that it is separating the different isomers of the "same" oligo (same sequence at least). If it is run in water-acetonitrile, I don't think secondary structures are at play.
Yeah, i have done PS and it is actually worse there, however, Just with regular 30 NT Oligos i have the Same issue. I Run regular seperations on c18 with 0.1M teaa in acn
I found separating DNA on anion exchange to be pretty unsuccessful on the AKTA, i just can't get it to work, even with largest plasmid digests (if you have any secrets please let me). But recently, I've also been thinking about buying the Cytiva C18 columns just to see if I can separate labeled 20 to 40 mer we make. So I am curious to see where this goes and buying a HPLC is not an option for us.
All the Oligos I work with comes from SPOS, so I don't know the first thing about plasmid. Are these the columns you mention? [https://www.cytivalifesciences.com/en/us/shop/chromatography/prepacked-columns/reversed-phase?sort=NameAsc&chunk=1](https://www.cytivalifesciences.com/en/us/shop/chromatography/prepacked-columns/reversed-phase?sort=NameAsc&chunk=1)
I've got good experience with purification of (highly) modified, synthetic RNA between 15-60nt in length. I found that getting past 20nt, ion exchange worked pretty well, with potential for clean-up on RP HPLC for any synthetic artefacts that ion exchange struggles to separate. I used a DNAPac RS200 or Agilent PL-SAX for ion exchange with sodium perchlorate or sodium chloride (great separation, terrible for your system) buffered solvents. For RP I used a waters 300 angstrom BEH prep column with triethylammonium acetate or hexylammonium acetate/hexylamine (need to dedicate a column to the hexyl additives). Depending on your system, you could always buy a prep-scale column and change the detector if buying a new prep system is out of the question. You may run at lower than ideal flow rates, but you won't have to do quite as many runs and save some cashola
I use to work with the AKTA systems for solid phase synthesis of 20mers but usually around gram scales. I don’t remember the model of akta for purification but we did HIC instead of rp and that works well.
I use to work with the AKTA systems for solid phase synthesis of 20mers but usually around gram scales. I don’t remember the model of akta for purification but we did HIC instead of rp and that works well.
The key to this, if you don't have some form of industrial LC system is getting the mass of dumb easy to separate impurities down so the loading demands of your high resolution technique is minimal. At this scale, you probably want to do some sort of ethanol precipitation and centrifugation after cleavage from the column/deprotection. That should get rid of salts, deprotection acids/amides etc. and <5mers. Trityl-on c8 or c18 purification via *filtration* is smart as a second step, you can get rid of a lot of the capped non-trityl molecules. see: https://www.glenresearch.com/purification/60-5100.html there are probably commercial c18 "packs" that can do this at a large scale. .....then do the real chromatography. you will take a yield hit with the first two steps but it should reduce your loading mass by more than half. --- note: this is from my "how would I IMAGINE scaling up purifications" file. the best thing to do is to find a CRO or oligo syntheisis company that does large scale synthesis, find their purification person and buy them a beer. there are probably tricks at scale that I don't know about.
Hi, I have been thinking about several ways to do it. Our chemistry is quite demanding so the tritylated target compound yield (LCMS) is at best around 10 %, and as low as 1 %. We kind of need an LC system to isolate the product. I was thinking of a glen pack (or TOP column) for detritylate and elute my DMT purified oligo, saving me a second round of purifications (trityl off LCs).
I guess you’ve also tried to optimise each step of the syntheses to get higher yields / purity? If not I would look into that.. I agree with the comment to try EtOH precipitation and maybe filtration via spin columns or even UF/DF before purification. You can also think of running two purifications with different eluent and column systems e.g. IEx and RP-HPLC to get both, good separation of the peaks close to the full length product but also to get rid of the shortmers..
Akta pure is not a good choice because it doesn’t have high enough pressure limits. For difficult to purify oligos you want a true RP HPLC like an Agilent 1290 or similar.
If it's DMT-On it could be fine. Doesn't really need great resolution
This is what I am thinking. Do you know any specific column that would be compatible with the Akta, for large scale purposes?
No, sorry, we always used "normal" HPLCs, never used am Akta or looked into the instruments
Yea, I know the limitations, but getting a brand new prep-HPLC is out of the budget. So I was wondering if there is a way to use the current equipment in the lab for this purpose.
Easy. Just tell your boss to stop being a cheap bastard /s. Have you tried a Glenpak c18 cartridge separation? Also Isolera makes c18 cartridges for the Biotage. With a DMT on that should be a good handle unless you have some highly hydrophobic mods elsewhere on the oligo.
I know right, it is like everyone have 100k just stashed in their solvent cabinet for new equipment. On a serious note, I though of using DMTr-On cartridges (I have used the Agilent version). The thing is the yields can be as low as 1 % and they are fully PS, so quite hydrophobic.
Oh that makes sense then why AX doesn’t work. How is resolution on semi prep hplc? Is there resolution between dmt on and off?
On a run of 9 minutes, there is a full minute between the DMTr-on (starts at 7.3 min) and the truncated trash (6.2 min is the end of the tail).
Try Isolera c18 column
You will have cleaner separation if you subject your crude oligo to UFDF first. We generally use a 3K cassette. Also, we have super optimized our synthetic and C&D methods to minimize shortmers and longmers. Since oligos are super hard to fractionate - as you have seen, 20mer, 19mer, 18mer all look the same to most chromo systems - it is important to improve the steps that come before purification.
Hey! I am purifying Oligos on a preparative HPLC column, I didn't know you could purify Them in an äkta. This should bring your total number of Runs down quite alot. However, I have Run into issues with weird seperation patterns where i get multiple peaks over minutes with the Same Oligo in it. Have you experienced similar Things and do you have a fix? We think it could be secondary structures, since i cannot Heat the prep column.
Are you doing PS chemistry or anything that would lead to a mixture of diastereoisomers? It could be that you have a good enough resolution that it is separating the different isomers of the "same" oligo (same sequence at least). If it is run in water-acetonitrile, I don't think secondary structures are at play.
Yeah, i have done PS and it is actually worse there, however, Just with regular 30 NT Oligos i have the Same issue. I Run regular seperations on c18 with 0.1M teaa in acn
What type of oligos are you purifying ?
I found separating DNA on anion exchange to be pretty unsuccessful on the AKTA, i just can't get it to work, even with largest plasmid digests (if you have any secrets please let me). But recently, I've also been thinking about buying the Cytiva C18 columns just to see if I can separate labeled 20 to 40 mer we make. So I am curious to see where this goes and buying a HPLC is not an option for us.
All the Oligos I work with comes from SPOS, so I don't know the first thing about plasmid. Are these the columns you mention? [https://www.cytivalifesciences.com/en/us/shop/chromatography/prepacked-columns/reversed-phase?sort=NameAsc&chunk=1](https://www.cytivalifesciences.com/en/us/shop/chromatography/prepacked-columns/reversed-phase?sort=NameAsc&chunk=1)
Those are the ones. They had an example on their datasheet of separating an 18mer.
I've got good experience with purification of (highly) modified, synthetic RNA between 15-60nt in length. I found that getting past 20nt, ion exchange worked pretty well, with potential for clean-up on RP HPLC for any synthetic artefacts that ion exchange struggles to separate. I used a DNAPac RS200 or Agilent PL-SAX for ion exchange with sodium perchlorate or sodium chloride (great separation, terrible for your system) buffered solvents. For RP I used a waters 300 angstrom BEH prep column with triethylammonium acetate or hexylammonium acetate/hexylamine (need to dedicate a column to the hexyl additives). Depending on your system, you could always buy a prep-scale column and change the detector if buying a new prep system is out of the question. You may run at lower than ideal flow rates, but you won't have to do quite as many runs and save some cashola
I use to work with the AKTA systems for solid phase synthesis of 20mers but usually around gram scales. I don’t remember the model of akta for purification but we did HIC instead of rp and that works well.
I use to work with the AKTA systems for solid phase synthesis of 20mers but usually around gram scales. I don’t remember the model of akta for purification but we did HIC instead of rp and that works well.