The efficiency of a size exclusion column is proportional to the number of theoretical plates (plate number). I would say that a concentrator has plates number=1, while any preparative column would have it >1, so SEC would always separate two different size objects better.
On Aug 20, 2014, at 1:44 PM, Roger Rowlett wrote: Excellent references. PEG 3350 appears to be hydrodynamically equivalent to a 20 kD globular protein. So for efficient separation, your protein needs to be significantly larger than 20 kDa on a GEC column. In a centrifugal filter (which is very inefficient--you need many exchanges and dilutions with buffer to get nearly quantitative removal) it is possible that "snaking" of linear polymer molecules through the pores might contribute to slightly more efficient removal than expected based solely on hydrodynamic radius. GEC or a desalting column is definitely the quickest way to do this, if possible. Flow rates may have to be slow (hence a typical flow rate column separation) to allow for efficient distribution of solutes in the sample solution if it has increased viscosity. Cheers, _______________________________________ Roger S. Rowlett Gordon & Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu<mailto:rrowl...@colgate.edu> On 8/20/2014 4:18 PM, Reza Khayat wrote: Hi, I managed to significantly reduce the viscosity of the PEG solution via buffer exchange using a 100kDa MWCO ultrafiltration device. The following papers have fantastic tables of solutes with their hydrodynamic radii. Definitely worth a read, followed by printing and posting of the tables on walls next to the FPLC :) http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3055910/ http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1304934/ Best wishes, Reza Reza Khayat, PhD Assistant Professor The City College of New York Department of Chemistry, MR-1135 160 Convent Avenue New York, NY 10031 Tel. (212) 650-6070 www.khayatlab.org<http://www.khayatlab.org> ---- Original message ---- Date: Wed, 20 Aug 2014 18:57:07 +0000 From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>> (on behalf of Alexander Aleshin <aales...@sanfordburnham.org<mailto:aales...@sanfordburnham.org>>) Subject: Re: [ccp4bb] Removing PEG3350 To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> I meant application of GF as an ion exchange column. Oh, my goodness! Ion exchange is something else! It should read "buffer-exchange" = desalting column. On Aug 20, 2014, at 11:48 AM, Alexander Aleshin wrote: Dear Remie, I meant application of GF as an ion exchange column. You can use special ion exchange columns, but our lab often uses preparative GF columns for this task. We just load the column, keeping sample volume < the void volume. Thus, we do not concentrate a protein before an ion exchange, only after it. But that is inevitable. When I am afraid to loose a protein during its concentrating, I concentrate shoulders of the eluted peak first, then add a central part. My point was that it might be okay to exchange buffers by concentrating a protein, but other molecules like Peg3K would not penetrate the membrane as well as water or salts do, as a result their reduction in concentration will be unreliable. Like, you do a 10 fold concentrating/delusion of a solution, but the final concentration of PEG3K will drop only by 3 fold... Alex On Aug 19, 2014, at 9:42 AM, Remie wrote: Hi Alex, I disagree with you even though GF is always the last step in my purifications. Because it involves concentration before and after the GF so during the concentration you can already be doing the buffer exchange. You use GF when you want to purify other protein impurities if they are different sizes. Of course it has other uses too. But not quite practical for just changing buffer also considering the amount of protein you could be loosing along the process. If one is careful, centripreps are best for concentrating and changing the buffer. I tell you this from experience with large hard to express proteins. Best of luck, Remie On Aug 19, 2014, at 10:45 AM, Alexander Aleshin <aales...@sanfordburnham.org<mailto:aales...@sanfordburnham.org>> wrote: Remie, Actually, concentrating of a protein solution is not the best approach to removing low MW impurities, gel filtration chromatography is more reliable and ... faster. Regards, Alex On Aug 19, 2014, at 7:03 AM, Remie Fawaz-Touma wrote: Hi Reza, I had to do this before. This protocol works for any PEG and any chemical to be removed from a solution: buffer exchange into the new buffer you want your protein to be in. There are ways to do that by 15 mL Amicon concentrators from millipore for large volumes, or if your protein is already concentrated, there are some small 0.5 mL concentrators from millipore as well. The key is to keep your spinning at low speeds (concentrators manuals will tell you) so you don’t precipitate or loose your protein. Check your protein concentration every 2 hours just to make sure you are not loosing it on concentrator surfaces and so on. Good Luck, Remie On Aug 19, 2014, at 9:55 AM, Reza Khayat <rkha...@ccny.cuny.edu<mailto:rkha...@ccny.cuny.edu>> wrote: Hi, Does anyone have a protocol for getting rid of PEG3350 from a protein sample? Best wishes, Reza Reza Khayat, PhD Assistant Professor The City College of New York Department of Chemistry, MR-1135 160 Convent Avenue New York, NY 10031 Tel. (212) 650-6070 www.khayatlab.org<http://www.khayatlab.org>
