In my experience, FPLC is usually used as a preparative method for protein purification (although it can be used for purifying other things), whereas HPLC and UPLC are usually used as an analytical method. For example, an HPLC SEC-MALS system is an excellent method for measuring the molecular weight distribution of purified proteins in the native state. HPLC/UPLC systems are frequently coupled to mass spectrometers for LC-MS analytical applications. With respect to proteins, these include protein mass measurement, proteomics, amino acid sequencing, and analysis of post-translational modifications. The samples applied to these systems often are not whole proteins, but proteolytic digests, sometimes in very tiny amounts extracted from gels.

Moreover, the analytical uses of HPLC/UPLC are very broad and include uses unrelated to protein analysis and purification. In my opinion, someone who can operate an FPLC can also learn to operate an HPLC/UPLC system, but the uses to which the systems are put and the nature of the samples are different, and may involve different scientific disciplines from protein purification (phamacokinetics, ADME, QC/QA to name a few) and sophisticated analytical tools such as mass spectrometers. Thus, the experience of protein purification by FPLC may not be generalizable to the uses of an HPLC/UPLC system.

In my experience, FPLC is usually used as a preparative method for protein purification (although it can be used for purifying other things), whereas HPLC and UPLC are usually used as an analytical method. For example, an HPLC SEC-MALS system is an excellent method for measuring the molecular weight distribution of purified proteins in the native state. HPLC/UPLC systems are frequently coupled to mass spectrometers for LC-MS analytical applications. With respect to proteins, these include protein mass measurement, proteomics, amino acid sequencing, and analysis of post-translational modifications. The samples applied to these systems often are not whole proteins, but proteolytic digests, sometimes in very tiny amounts extracted from gels.

Moreover, the analytical uses of HPLC/UPLC are very broad and include uses unrelated to protein analysis and purification. In my opinion, someone who can operate an FPLC can also learn to operate an HPLC/UPLC system, but the uses to which the systems are put and the nature of the samples are different, and may involve different scientific disciplines from protein purification (phamacokinetics, ADME, QC/QA to name a few) and sophisticated analytical tools such as mass spectrometers. Thus, the experience of protein purification by FPLC may not be generalizable to the uses of an HPLC/UPLC system.

Thanks, I was aware of the difference in the applications of the techniques.

It sound like having FPLC experience is better than starting from nothing when it comes to leaning how to run HPLC, but depending on the application it may not be much better.

It's not the answer I wanted, but it's good to know.

Unfortunately, my work thus far has not included LC-MS/MS or PK/PD analysis, despite working in labs where both could have been relevant. At best, we would have sent our samples out to a core facility for such analysis.

I'm going to say it's easier.  Small molecules have fewer functions that you can exploit, so the separations are strategically simpler.  (That's why you need higher pressure -- to increase the resolution you can get out of simple chemistries).  I did HPLC for 5 years or so for different institutions and never came across anything other than a C18 column with solvent-based hydrophobic gradient.  You can find other chemistries, but their use is rare.  

Also, small molecules are tougher than proteins -- they can't denature.  You can find analytes that don't like water, o2, light and some such, but again, this kind of problem is the exception rather than the rule.  By your (protein) standards, you can really abuse them and they'll be just fine. 

So in reality, if you really understand FPLC and how and why it works, and can develop methods for diverse new proteins, then I think HPLC is going to make you feel overqualified and bored, due to its repetitive simplicity.  In practice, it's going to depend on whether your interviewer thinks so too, and they might just not believe it unless they themselves have actual experience in both methods.  Good luck. 

Dear Brian Bower ,

       I did several run of native molecular weight determination of protein/s with Amersham Biosciences (FPLC system). FPLC system could be used and only for protein purification strategies of Proteins. Based on the principle, the systemic mechanism of HPLC used wide variate molecule/s in the different system of bio-molecules as well as synthetically produced of chemical compounds and molecules.

I could tell FPLC, specific for only proteins and HPLC, specific for proteins and other than protein molecules.

Thanking you.

Yours sincerely

sakthivel

Brian, FPLC experience can serve as a good basic primer to HPLC separations. Someone with HPLC experience is also much more marketable. Experience running FPLC separations will not prepare you for all of the additional knowledge and skills you need to become proficient at HPLC method development or analysis. While the two types of chromatography share many basic concepts, the hardware, software, fittings and required/used parameters are much more detailed and sensitive to small changes. Having FPLC exposure is good; better than none at all for sure, but you may still require the same amount of training as someone w/o any chromatography experience.

I have hired many scientists over the past decades. The best ones were usually people who were very "hands-on" and had a good understanding of the fundamentals and concepts (this is where 90% disappoint). Being curious and ambitious also helps a lot. Those people do not get bored because they are constantly increasing their knowledge, experimenting, improving methods and sharing information with others. Their own interest improves products, services and methods. So, having some FPLC experience is a plus (from an employment pov), but how much value it will have will depend on what types of specific HPLC / UHPLC applications you initially start working with. There are thousands of different types of samples, HPLC applications, using many types of columns, modes, scales (nano to Prep) and specialty detectors. You could become expert in one area and still not be experienced or qualified to run other types of samples. So the field is pretty broad and there is a lot of room to specialize. If this analytical field interests you , then I do suggest you continue to pursue it. *Having FPLC and HPLC experience will also make you more marketable as a potential employee too.