Can a HPLC function as an FPLC (sample preparation) easily

[johncarrigan]

Hello,
I am currently in the position whereby i have a complex protein extract and I use an akta to carry out partial purification of several mgs for bioassays etc.
Thing is, I really miss having the DAD detectors and other aspects of the HPLC

I am borrowing both equipment but i have been offered enough money to buy one. Originally i thought I would just get a HPLC, get a large loading loop, attach a frac collector to waste and bobs your uncle. But some people have told me that my proteins for ion exchange etc could stick to the metal parts etc. Any opinions?

FInally, can someone recommend a decent FPLC, one I might buy second hand?

Regards,

John

[Andy Alpert]

Regarding the prospective loss of protein on metal components:
This could conceivably be a concern if you had, say, a microgram of a protein that's particularly susceptible to interaction with metal, such as transferrin. Since you'll be working in the milligram range, then all potential binding sites of this sort in the column + flow lines will be saturated several thousand times over and you'll have no problem.

HPLC of proteins was introduced in the late 1970's. At that time a certain company dominated the field of protein purification using low-pressure media. This company reacted by introducing its own version of HPLC which it called FPLC in an attempt to make it appear to be something other than HPLC. It further distinguished its system by using borosilicate glass columns and titanium metal components and then instituted a sustained marketing campaign - with no evidence - that stainless steel was somehow bad for protein work. Just to make sure that the customers didn't use conditions that would break the borosilicate glass columns, the company gratuitously handicapped its HPLC - pardon me, FPLC - so that it couldn't pump above pressures of 1000 psi or so. I believe that they're up to 2000 psi now.

I agree with your sentiment about the ease of use of the current generation of FPLC's. It makes it convenient to use mediocre media (= large particle, low pressure, low efficiency) for protein chromatography.

[carlo.annaratone]

I used a HPLC as a medium scale prep system for small molecules - with most pumps limited to 5 or 10 ml/min you cannot use too large columns or the durationwill be prohibitive. Only risk I see, is to use too concentrated buffers or salts (e.g. NaCl) which might mess with the pump seals or corrode steel.

[bunnahabhain]

http://www.knauer.net/en/products/produ ... nents.html

[Hollow]

just an input, don't know if it's feasible or if there will be other pitfalls
Neither do I have experience with akta and proteins, but:

why not split the flow from the äkta so that a small part will go to an additional DAD? Maybe coupled with an additional (isocratic) pump attached to the splitter to dilute the fraction going to the detector (kinda "Make-up" solvent for detection, like it can be found for LC/MS setups)?

This way you can probably stick with normal detector cells and HPLC instrumentations (analytical vs. prep)

[lmh]

Caveat: I know absolutely nothing about proteins. There are, for those who worry about binding of peptides to stainless steel, "proper" HPLC manufacturers who can avoid steel. For example, Thermo/Dionex's Vanquish range is available, I believe, in biocompatible titanium.
Enjoyed your post, Andy Alpert!

[Andy Alpert]

Thanks, lmh!

We did switch from stainless steel to titanium frits after we started selling columns for use with phosphopeptides.