We assume because you specified enzymes that you want some kind of activity at the end of the process. The key words in HPLC are high pressure, which kills your protein—actually being under pressure isn’t as bad as the pressure drop once the eluate comes out of the orifice, exploding the structure. Denaturing conditions tolerated by small molecules that don’t depend on tertiary and quaternary structure for their activity are mortal for most enzymes, and so GPC (gel permeation chromatography) and FPLC were born. Pressure and nasty solvents are mitigated by a more complex, noninjurious bed and buffers-- and much longer flow rates. You have to think in terms of minutes (or hours) instead of seconds with these systems, if you have a touchy mixture where the target is hard to separate from an overwhelming comigrator, but there’s a lot of technical art you can fiddle with. You may want to consider the techniques suggested in literature from ÄKTA (http://www.gelifesciences.com/webapp/wcs/stores/servlet/catalog/en/GELifeSciences/applications/protein-purification-and-bioprocessing). The Nest Group is expert in all HPLC including GPC and offers terrific training (http://www.nestgrp.com/protocols/protocol.shtml#hic).

If economy is important and you can’t set up the relatively high-flow-rate GPC, then good old fashioned Sephadex is very affordable, and with patience and attention can yield great results. Also, if you have a source of purified protein and can raise antibodies to it, an antibody affinity column could be easiest of all to extract a high yield of functional enzyme.

Do talk to the Agilent guys to see whether it can be jury-rigged, but once weeks or longer are expended trying to make do, you may conclude there's good reason why nobody else is doing it. Also talk to someone who might sell you FPLC: even if you don't buy, you could save a lot of time and frustration by knowing the pitfalls in advance. It's hard enough trying to develop a process where your peak is tight, much less use a system that wasn't designed to work in that range.

yes. the presssure is not related to the system but to the column used. If your column is low pressure you might need to "add" some pressure by long capilary as some HPLC systems need some pressure for accurate work. tc

HPLC systems are meant to run with HPLC columns at higher pressure and lower flow rates. You can probably buy a HPLC column that will do the same as your FPLC column but with a lower capacity. Your Agilent system probably runs with a max flow of 4 mL /min which probably isn't enough for an FPLC column.

Thanks Warren for the reply. I checked that HPLC pump (Quaternary Pump in Agilent 1200 series HPLC system) has maximum flow rate of 10 ml/min. Can this system work with FPLC columns for protein purification?

Hi Deepak,

don't get too caught up with the flow rate, yes it's important, but it's not the only factor. I used to regularly use a FPLC column (Mono-Q) with a HPLC system. In this case I was running at 0.5 or 1 mL/min. One of the major differences between an FPLC system and HPLC system, apart from the typical flow rate used, would be that a FPLC system tends to be fully bio-compatible, whereas, your average HPLC system will have stainless steel plumbing and not fully bio-compatible. This may, or may not, be important for your application. You'll need to check if it is and you'll need to check that the buffers used in the papers you mention are compatible with your HPLC system.

In some case a SS system may not be compatible with certain buffers. If you have any doubts, go to the Agilent website and look up the system specs. Alternatively, go ask your local rep.

I'd be very surprised if you can't use your HPLC system. Remember that column flow rate will also be affected by the column you choose. Do you need a large diameter column or a smaller diameter? What about the length? This is all dependent on your application.

Good luck,

Peter

Thanks Peter for the information. It is really helpful. I will check the system specifications and talk to Agilent guys.

Probably no reason why not - does your HPLC have a fraction collector? Even if not you could do this manually (if the run isn't too long). Disconnect after detector and collect into 96 wells, tubes or whatever depending on flow rate. Get a system for relating back to detector signal (eg fractions every minute). Only collect the part of the run that is of interest.

Yes you can as long as you use the appropriate column.

You can 2 do things to be on safe side:

1. Mark the upper max pressure matching to your column specification - so that when the pressure crosses the limit, the pump will shut down.

2. under pump setting, there is a maximum stroke setting which typically is 100 mL/min - reduce this value down.

We assume because you specified enzymes that you want some kind of activity at the end of the process. The key words in HPLC are high pressure, which kills your protein—actually being under pressure isn’t as bad as the pressure drop once the eluate comes out of the orifice, exploding the structure. Denaturing conditions tolerated by small molecules that don’t depend on tertiary and quaternary structure for their activity are mortal for most enzymes, and so GPC (gel permeation chromatography) and FPLC were born. Pressure and nasty solvents are mitigated by a more complex, noninjurious bed and buffers-- and much longer flow rates. You have to think in terms of minutes (or hours) instead of seconds with these systems, if you have a touchy mixture where the target is hard to separate from an overwhelming comigrator, but there’s a lot of technical art you can fiddle with. You may want to consider the techniques suggested in literature from ÄKTA (http://www.gelifesciences.com/webapp/wcs/stores/servlet/catalog/en/GELifeSciences/applications/protein-purification-and-bioprocessing). The Nest Group is expert in all HPLC including GPC and offers terrific training (http://www.nestgrp.com/protocols/protocol.shtml#hic).

If economy is important and you can’t set up the relatively high-flow-rate GPC, then good old fashioned Sephadex is very affordable, and with patience and attention can yield great results. Also, if you have a source of purified protein and can raise antibodies to it, an antibody affinity column could be easiest of all to extract a high yield of functional enzyme.

Do talk to the Agilent guys to see whether it can be jury-rigged, but once weeks or longer are expended trying to make do, you may conclude there's good reason why nobody else is doing it. Also talk to someone who might sell you FPLC: even if you don't buy, you could save a lot of time and frustration by knowing the pitfalls in advance. It's hard enough trying to develop a process where your peak is tight, much less use a system that wasn't designed to work in that range.

Thanks Kristine and Shukor!!! Your inputs are really helpful.

Most important is going to be the response of YOUR particular enzymes to any procedure: some are hardy, some are not. Why not expose the enzyme(s) to the reagents and conditions of pressure and pressure drop of your proposed system, and see whether activity is harmed? It isn't hard to purify a "dead" protein...

You should not, reason the two technologies are develop differently to address different purposes,

NOTE:  FPLC is s trademark (IT IS MARKETING).  A long time ago, Pharmacia wanted to distinguish their LC systems from everything else on the market so they started using the term FPLC (fast protein liquid chromatograph).  There is no such thing, it is a pump, a detector, an injector and a column.  It is a Liquid Chromatograph.  You can use Agilent, Waters, Hitachi, Shimadzu, Chinese Mfgr, any LC that will pump at the required flow rates.  Another thing to remember, a High  Pressure LC can run at Low Pressure.  The pressure range is from 0 to xxx.  So, even though you normally use it at 1000 or greater psi, it will also work at 10psi...It is a pump that pumps fluid! 

it is in essence HPLC after all.with its basic components. In case of macromolecules you may need special columns or stationary phases and also low pressure that you suite the chemical nature of the proteins and m=other macromlecules. 

Another thing to note...STAINLESS STEEL IS COMPATIBLE WITH WATER AND BUFFERS USED IN PROTEIN PURIFICATION.  Anything FROM THE INJECTOR DOWN should use "biocompatible" materials and not stainless steel so that your protein doesn't chelate metals.  But using a SS or PEEK pump will make little if any difference..If you worry, passivate it!  Remember most of the proteins were originally purified with metal pumps, we didn't start using "plastic" pumps until the 80s.  Dionex and Beckman came out with PEEK pumps and they were laughed at the time and referred to "plastic pump people".  The first FPLC came out in the early 90s and used glass syringe pumps...remember!  And the syringes were metal barrels.  The Akta referred to earlier...technically is an HPLC pump.  It will withstand up to 4,000 psi!

Pressure is proportional to particle size, bed height and flow rate.  Low pressure materials typically are 15 micron and larger particle sizes, remember you can use a 100 micron particle and end up with high pressure by ramping up the flow rate! There is a formula that shows that pressure is inversely proportional to the square of the particle size...So, as you go from 5 micron to 10 micron, the pressure goes up 25X.

http://www.chromacademy.com/chromatography-HPLC-Column-Dimensions.html