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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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2 Answers 2

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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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I wouldn't say that's the only difference, but it's a large difference. Most of your HPLCs will expose the compounds to several metal components, which can denature some proteins, while FPLCs are designed to minimize metal exposure and preserve protein structure better. HPLCs usually have better detectors, often with monochromators for full wavelength selection, while basic FPLCs have simple detectors with 2 wavelengths, 254 and 280nm, which is usually fine for proteins. Reverse phase HPLC will often use organic solvents, while FPLC usually uses buffered aqueous solutions. –  user137 Aug 7 at 15:48

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
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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

protected by Chris Aug 7 at 8:45

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