Are you aware of any FPLC vs HPLC comparison studies of a given protein, evaluated under virtually the same conditions to see the final stability or quantification of the protein assessed (and which was more effective)?
Generally, HPLC provides higher resolution; hence it is used for high quality analysis of a protein. On the other hand, it is not used to purify proteins in higher quantities. FPLC is used to purify proteins in higher amounts.
If you want to purify partially proteins from whole extract you must use FPLC as primary method. This is because columns used for FPLC have particles larger than particles in HPLC columns. In chromatography FPLC columns have less "separations plates" that HPLC and for this reason separations in HPLC are more accurate but you only can separate too little ammount of sample in comparison of FPLC. One disadvantage of HPLC is you only must use samples that have purified previously, for example with FPLC. Everything depends on the analysis you are going to make the protein.
Thank you, thank you Jose! You've boiled it down to theoretical plate numbers and chromatography ... finally a concise answer! I've been looking through so many research papers, and this finally makes sense, so you can assure me that the particle size of FPLC columns is usually larger than HPLC columns? I know due to manufacturing differences, there is no absolute number, but can you please give me an estimated range of particle diameter for HPLC and FPLC columns?
Hello again. The particle size for FPLC resins is on the order of 40-160 microns while the stationary phase HPLC column is generally 5 microns. If you have a smaller particle, you have more surface that interacts with the sample and therefore you improve the separation (by increasing the number of theorical plates).
José is right, that FPLC is used usually for purification of proteins simply because of their capacity. When you look at the dimensions of HPLC columns (max 5 mm ID and length 50-150 mm mostly), the volume (and thus capacity) is low. Unless you use semi-prep HPLC.
Tomas, I'm still having trouble understanding how it could be advantageous for FPLC to have a lower number of theoretical plates than HPLC. I mean, if the desired protein(s) moving through the mobile phase spend less time interacting with the slightly larger beads in the column, the analysis time for FPLC should be faster than HPLC, right? What's the advantage? Analysis time? Or in HPLC is a lot of sample simply lost in the pores of the small stationary beads in the column?
Please correct if I'm wrong, or tell me what I should be imagining.
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