What is FPLC ? Is it HPLC with a Fluorescent Detector??? Just give me a few pointers ..
thanks

Fast Performance Liquid Chromatography

It uses smaller particle size n small column eg. 3 cm column n 3 micron particle size.

FPLC stands for Fast Protein Liquid Chromatograph(y). This was an instrument marketed by the company Pharmacia (maybe they still market it!) specifically for the separation of proteins. It was made of "protein friendly" constituents such as plastic and glass, and had a lower maximum operating pressure than typical stainless steel HPLC systems. Most of the separations run on this equipment are/were ion-exchange or gel filtration type of separations using 10 micron particle size stationary phases which have a lower operating pressure than 5 micron phases, and were thus more compatible with the limits of the system.

The whole idea of "protein friendly" systems is an interesting and contentious issue. Stainless steel at one time was thought to denature proteins, hence the FPLC system. Another idea was that metal impurities from the system could poison the ion-exchange columns frequently used. I'd be interested in any comments on the idea of stainless steel systems and protein denaturation-has this idea been debunked? Also, maybe Chris Pohl from Dionex has some useful input on the question of metal impurities in protein or ion chromatography separations?

Oh, I forgot of course that many ion exchange separations of proteins involve salt gradients (eg NaCl). These conditions could result in attack of the stainless steel in conventional systems. Any comments on this question would also be interesting.

The Pharamcia FPLC and similar systems had/have no problems with salt gradients.

Hello all, once a well known protein chemist told me, that FPLC was the best marketing trick Pharmacia ever did. The real problem is, that FPLC needs glass columns, can not handle high back pressure etc., and on an FPLC system you only can do FPLC, but on an HPLC system you can do both. And the metal impurities??? It's like the British and their umbrellar! Perhaps it will be raining! In some cases it will rain, but in most....!?! So, there are some resons to go with such an FPLC system, and if you realy need one, than go for it. Have a nice week, and perhaps we will see us at Analytica in Munich. Gerhard

Pharmacia is now Amersham Biosciences. Just checked their catalog, they still have FPLC in the index, couldnīt find much else on it. They seem to be on to other things?
On proteins and Fe ions: We have done several antibodies, also some peptides, and never saw an effect that could be attributed to iron ions. One can imagine that some of these substances may complex with iron ions, but how much ions can there be? But! Some say that each protein is an individual regarding analysis and chemistry, we tend to support this.
On NaCl and stainless. After that chain on this subject I thought I better check carefully on my use of phosphate buffered saline (PBS), even though this was never more than a working day in contact with the HPLC. PBS (pH 7.2, NaCl concentration 0.15M)has now been in contact with stainless (the test is described in that earlier chain)since the 20th of March: No attack. Its all a matter of conditions, at higher conc. it may attack, certainly at low pH it will (HCl).

HW-nice to see someone with some real results on this question, rather than the usual speculation!
Just to add another couple of points-ion-exchange separations of proteins often involve salt gradients from 0.1M to about 1.0M NaCl. Most people reading this site are, I think, more interested in analysis where protein degradation is not that important if it is sufficiently reproducible, although some are interested in preparative separations of proteins/enzymes. Here, it is often necessary to retain the activity of the enzymes. I have seen few formal studies where enzyme activity is measured before and after separation, comparing stainless steel and "FPLC type" instruments. Does anyone know of such a study? There are so many complications in such an argument too-for instance there's no point in such a study if the plastic column/plastic HPLC system uses a plastic column with metal frits...

Such a study would interest me also, though predictably, it is going to involve one protein or a narrow class of proteins. The lit. is full of contradictions like that hypothesized in your last sentence, David.
Checking "Protein Purification", J-C Janson, L Ryden, VCH, 1989, particularily an article therein by MTW Hearn, I found nothing on iron ions. On RP-HPLC Hearn mentions four major denaturing "scenarios" (for instance denaturing by the hydrophobic C-18 surface) which can usually all be counteracted. If not, one often has the option to renature (also. stabilize) proteins with polyethers, cyclodextrins, etc. Even if Fe ions should have a negative effect, that can probably also be counteracted.

Hans

"I have a current job opening within one of the Bio-Tech facilities that I staff in South Eastern PA. I am looking for a Chemist who has FPLC experience. I do understand that this technique is not as widely used as HPLC and I was hoping that someone can refer me to anyone who maybe out of work. I can be reached @ 800-964-1586 X4604. Thanks, Joe Filachek"