Re: [ccp4bb] HPLC vs FPLC for protein purification
Thanks everyone for your insights, I really appreciate it. Cheers, ivan On Mon, Apr 20, 2015 at 5:38 PM, Edward A. Berry wrote: > On 04/20/2015 06:44 PM, xaravich ivan wrote: > >> Hi CCP4eans, >> >> solvent based HPLC system? >> > > Do you mean like acetonitrile:water:TFA on reverse-phase columns? I think > this is > routinely used for analytical runs or to obtain material for protein > chemistry, > but the concentration of acetonitrile required to elute protein from C8 > column > is high enough to unfold the protein, so it would have to be something you > can > refold to be useful for crystallography. > (I may be wrong, I may have misunderstood, and yes I realize water is a > solvent.) > > eab > > > On 04/20/2015 06:44 PM, xaravich ivan wrote: > >> Hi CCP4eans, >> >> Do you guys have any preference in purifying a protein by SEC in FPLC >> system or using a solvent based HPLC system after the initial affinity >> column purification. Where would you prefer HPLC purification over standard >> FPLC? >> I have routinely seen HPLC based purification of organic molecules and >> small peptides but not so much of proteins. >> What in your experience are the Pros and Cons of each, in the field of >> protein purification? >> Any suggestions/insights welcome. >> >> Thanks, >> Ivan >> >
Re: [ccp4bb] HPLC vs FPLC for protein purification
On 04/20/2015 06:44 PM, xaravich ivan wrote: Hi CCP4eans, solvent based HPLC system? Do you mean like acetonitrile:water:TFA on reverse-phase columns? I think this is routinely used for analytical runs or to obtain material for protein chemistry, but the concentration of acetonitrile required to elute protein from C8 column is high enough to unfold the protein, so it would have to be something you can refold to be useful for crystallography. (I may be wrong, I may have misunderstood, and yes I realize water is a solvent.) eab On 04/20/2015 06:44 PM, xaravich ivan wrote: Hi CCP4eans, Do you guys have any preference in purifying a protein by SEC in FPLC system or using a solvent based HPLC system after the initial affinity column purification. Where would you prefer HPLC purification over standard FPLC? I have routinely seen HPLC based purification of organic molecules and small peptides but not so much of proteins. What in your experience are the Pros and Cons of each, in the field of protein purification? Any suggestions/insights welcome. Thanks, Ivan
Re: [ccp4bb] HPLC vs FPLC for protein purification
On Apr 20, 2015 8:28 PM, "Roger Rowlett" wrote: > Depends on what you want to accomplish... If you have a liter of crude > lysate, "capacity" should be one of the choices. A step gradient is fast > but low resolution; a gradient elution has more resolution but will eat > buffer and take much longer. Lower stationary phase particle size to get > more resolution and flow rates must go down, etc etc. Choices must be made > to achieve the desired goal in the desired time with available resources. > > Roger Rowlett > On Apr 20, 2015 8:14 PM, "Christopher Colbert" < > christopher.colb...@ndsu.edu> wrote: > >> Hi Roger, >> >> Which 2 do you pick? >> >> Cheers, >> >> Chris >> >> -- >> Christopher L. Colbert, Ph.D. >> Assistant Professor >> Department of Chemistry and Biochemistry >> North Dakota State University >> P.O. Box 6050 Dept. 2710 >> Fargo, ND 58108-6050 >> PH: (701) 231-7946 >> FAX: (701) 231-8324 >> >> From: Roger Rowlett >> Reply-To: Roger Rowlett >> Date: Monday, April 20, 2015 7:07 PM >> To: "CCP4BB@JISCMAIL.AC.UK" >> Subject: Re: [ccp4bb] HPLC vs FPLC for protein purification >> >> Many protein purification media have large particle sizes or will not >> mechanically stand excessive pressure without crushing. (Superdex is an >> example of the latter.) In general, smaller stationary phase particle sizes >> give rise to higher selectivity and separation efficiency at the expense of >> flow rate and possibly capacity. Higher pump pressures are required to get >> flow through columns packed with tiny particles sizes. To borrow an >> analytical chemistry maxim: speed, resolution, capacity: pick any two. >> >> Roger Rowlett >> On Apr 20, 2015 6:45 PM, "xaravich ivan" wrote: >> >>> Hi CCP4eans, >>> >>> Do you guys have any preference in purifying a protein by SEC in FPLC >>> system or using a solvent based HPLC system after the initial affinity >>> column purification. Where would you prefer HPLC purification over standard >>> FPLC? >>> I have routinely seen HPLC based purification of organic molecules and >>> small peptides but not so much of proteins. >>> What in your experience are the Pros and Cons of each, in the field of >>> protein purification? >>> Any suggestions/insights welcome. >>> >>> Thanks, >>> Ivan >>> >>
Re: [ccp4bb] HPLC vs FPLC for protein purification
Many protein purification media have large particle sizes or will not mechanically stand excessive pressure without crushing. (Superdex is an example of the latter.) In general, smaller stationary phase particle sizes give rise to higher selectivity and separation efficiency at the expense of flow rate and possibly capacity. Higher pump pressures are required to get flow through columns packed with tiny particles sizes. To borrow an analytical chemistry maxim: speed, resolution, capacity: pick any two. Roger Rowlett On Apr 20, 2015 6:45 PM, "xaravich ivan" wrote: > Hi CCP4eans, > > Do you guys have any preference in purifying a protein by SEC in FPLC > system or using a solvent based HPLC system after the initial affinity > column purification. Where would you prefer HPLC purification over standard > FPLC? > I have routinely seen HPLC based purification of organic molecules and > small peptides but not so much of proteins. > What in your experience are the Pros and Cons of each, in the field of > protein purification? > Any suggestions/insights welcome. > > Thanks, > Ivan >
[ccp4bb] HPLC vs FPLC for protein purification
Hi CCP4eans, Do you guys have any preference in purifying a protein by SEC in FPLC system or using a solvent based HPLC system after the initial affinity column purification. Where would you prefer HPLC purification over standard FPLC? I have routinely seen HPLC based purification of organic molecules and small peptides but not so much of proteins. What in your experience are the Pros and Cons of each, in the field of protein purification? Any suggestions/insights welcome. Thanks, Ivan
