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I've used HPLC (high performance liquid chromatography) before (once, so I'm barely even qualified to know what it stands for) so I was surprised when my labmate told me she would be using an alternate technique to isolate a protein.

What exactly are differences in HPLC and FPLC (fast protein liquid chromatography) instruments, and why would FPLC be a better technique than HPLC to use with proteins? What situations make HPLC preferable?

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3 Answers 3

up vote 12 down vote accepted

The only difference between FPLC and HPLC is the amount of pressure the pumps apply to the column. FPLC columns have a maximum pressure of about of 3-4 MPa, whereas HPLC columns can withstand or require much higher pressures. As a general rule, HPLC columns won't work with old FPLC equipment; FPLC columns can go on HPLCs as long as the pressure can be regulated.

Manufacturers have been marketing separate equipment to handle these different classes of columns, but the trend seems to be heading towards machines that can handle both types of columns without issue. A GE rep told me a few years ago that they've improved the pumps on the AKTAs to the point that "they're technically HPLCs now." The term "FPLC" is probably on its way out.

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There are so many differences between FLPC and HPLC....

Of course major difference is of pressure. As name shows itself, high pressure is required in HPLC, whereas pressure is of no matter in FPLC and in fact it shouldn't be high. Another difference is that in HPLC only horizontal columns have been run and in FPLC only vertical columns. HPLC is used for the detection of any compound whereas FPLC specifically for proteins. In HPLC, only solvents are used but in FPLC we have to choose only buffers of different molarities. In FPLC, temperature doesn't matter but in HPLC, temperature matters.

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Could you elaborate on the importance of column orientation and temperature? –  dd3 Apr 14 '13 at 18:18
Unfortunately, several of your points are just wrong. Anything an FPLC can do, an HPLC can do, but an HPLC can also do more. An FPLC is essentially a specialized HPLC for proteins. High pressure is not required in HPLC, but it is an option, depending on the type of column. I assume by "solvent" you mean organic solvents, but that is incorrect - an HPLC can use a salt/buffer mobile phase just as easily as an FPLC. I use vertical columns in an HPLC all the time. Temperature doesn't have to matter in an HPLC, it's all in the parameters you set for a particular experiment. –  MattDMo Apr 15 '13 at 0:57

In FPLC Analysis are not temperature controlled but in HPLC analysis are temperature controlled but they dont have any major difference apart from this. FPLC is called so because of protein purification done on FPLC. Anatomy of HPLC & FPLC are same.

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I think this is a bit disingenuous. One of the main differences is in buffer pressure, and how the flow of your protein is set up. FPLC has better scales and gentler resins that can be used, and these might have been reasons to pick FPLC. I don't have time to write a full answer, but I think this is too limiting. –  Atl LED Oct 21 '13 at 19:04

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