Protein chromatography - an overview of affinity, ion exchange, and size exclusion chromatography

My main go-to protein cleaning tool is protein chromatography, a way to separate proteins based on differences in their properties by sending solutions of them traveling through columns filled with little beads (resin) and taking advantage of how different proteins interact differently with them.
blog form (also has static graphics): bit.ly/bbproteinchromatography
First off - something that’s always confused me - why’s it called chromatography? When I hear “chromatography” my mind jumps to “colors” - that’s what “chrom-“ means, right? And this always made sense to me because I would think back to those early paper chromatography experiments I did as a kid where you separate the different colored inks in a marker using paper chromatography. But I’m not separating proteins by their colors, so why’s it called chromatography?
The term “chromatography” did in fact come from its original uses separating colored compounds. In particular, Russian botanist Mikhail Tswett in 1903 used it to separate colored plant pigments. Etymology-wise, chromatography means “writing in color” which I think is so poetically beautiful!
But the important part - that which makes chromatography chromatography - is the separation part, not the colors part. You have 2 phases - in protein chromatography you have a solid phase which is the resin (little beads) in the column (glass or plastic cylinder) and a liquid phase which is the buffer (pH-stabilized salt water) running through. And you separate components based on which phase they’d rather hang out with.
A few common types used for proteins are affinity chromatography (AC), ion exchange chromatography (IEX) & size exclusion chromatography (SEC) and they use different resins. If the protein likes the resin (solid phase) more than it likes the liquid it came in with it’ll stick to the column. It might like the column because it’s oppositely-charged (this is the basis behind ION EXCHANGE CHROMATOGRAPHY (IEX)). The protein might also like the column more because it has some, more specific, special feature (like an engineered tag) that matches a special feature sticking off of the resin beads. This is how AFFINITY CHROMATOGRAPHY works. (note: the beads are usually porous - they have little tunnels running through them - and the affinity groups can stick out into these tunnels as well so you have more binding opportunities)
Both IEX & AC rely on the protein you want sticking to the column, while the other proteins flow through, then competing your protein off with salts and/or mimics or changing the pH to change the charge. But In SIZE EXCLUSION CHROMATOGRAPHY (SEC) you don’t want the protein to stick to the resin. Instead, you separate proteins by making smaller ones travel further because they can enter secret tunnels in the resin beads that big proteins can’t get into.
The theory behind these methods is the same whether you’re doing it with self-packed columns and gravity flow (which we still use all the time - more on this at the bottom) or the higher tech way with this fancy-dancey Fast Protein Liquid Chromatography (FPLC) machine. Ours is an AKTA, and AKTA’s kinda like the “Google” of the protein chromatography world in that it basically dominates the market and if you say AKTA other protein-purifiers know what you mean (not to make anyone feel bad if they don’t! I didn’t know until I joined the hard-core chromatography crew - and I still get confused all the time by lingo from other fields!)
The AKTA takes our protein sample and pumps it onto a column (which it’s gotten ready by flowing a bunch of buffer (pH-stable salt water) to “equilibrate” it. It then washes the column with the buffers we tell it to. We have 2 system pumps so you can use 2 different buffers that send liquid first into a mixing chamber so you can mix them if you want to make a gradient for a gradient elution to introduce the “competitor” that will push your protein off the column (e.g. have a no salt & a high salt or a no imidazole & high imidazole (for His tags) you can mix). Or you can just use 1 for an “isocratic elution” like for SEC when you don’t need to change the buffer.
The AKTA also allows us to control the flow rate (much easier than trying to twiddle with the stopcock in gravity flow). As the name implies, it can go fast, but you don’t always want it to or you’ll crush the resin in the column! Each column has different maximum flow rates. For the SEC columns I use, I typically run at ~0.7mL/min, which is actually pretty slow… And the fastest columns I run are only ~4mL/min. The times when the pumps are working their hardest is when doing pump washes. During those it’s pumping at 20mL/min, but it’s not going through any columns so you don’t have to worry about hurting them.
finished in comments

[@thebumblingbiochemist]

And, just in case, the system has pressure monitors at the entrance and exit of the columns. If the pre-column pressure (pressure going in) is too high it can damage the column hardware (the cylinder itself) & if the delta column pressure (difference between pressure going in & going out) is too high it could the resin in the column and/or the filter on top of the column are clogging up and generating dangerous pressure that can hurt the resin. So the AKTA will stop and alert you.
The liquid flows through lots of little tubes that offer different flow paths. Which path the liquid takes is dictated by lots and lots of valves to go with those lots and lots of little tubes. It’s kinda like a subway system that can change the tracks. So we can direct liquid into different columns and, when it comes out of the columns into the waste or a fractionater which collects it to “keep.”
We choose which fractions we actually want to keep based on the chromatogram This is where we see the evidence of our protein coming out in the form of a peak in the 280nM wavelength absorbance. Proteins (in particular tryptophan, tyrosine, and phenylalanine) absorb that type of light so you can tell when protein’s elute because they “steal” that wavelength from the light spectrum. A UV monitor on the path between the bottom of the column & the fractionater measures this. And the computer shows this to us as a peak. more here: bit.ly/2yzyi4w
FPLC looks a lot like a related technique, HPLC. HPLC stands for High Performance Liquid Chromatography. HPLC uses higher pressures but lower flow rates. It’s usually used for small chemical compounds and sturdier beads that can withstand those high pressures.
The kind of chromatography wI use is typically “preparatory” - we separate things to use them in their purer form. There’s also analytical chromatography, where you separate things just to see what’s there. this is similar to one of the differences between SEC & SDS-PAGE bit.ly/2xWH7VZ
There are many advantages to using the FPLC over relying on gravity to drip liquid through (gravity flow). My favorite reason - you don’t have to work in the cold room forever because it’s automated and, if you’re lucky, housed in a deli fridge not the cold room. When you’re working with precious proteins, you want to keep them safe. At higher temperatures, proteins can start to degrade especially if there are contaminating proteases (protein chewers) which at cold temps don’t have enough energy to chew your protein but do at higher temps.
But I don’t use the AKTA for everything. One of the nice things about gravity flow is that you can pack your own columns. AKTA pre-packed columns come in multiple sizes, but with gravity flow you can choose exactly what you want.
I prefer to use gravity flow for initial purification steps, where I’m working with “crude lysate” - the liquid you get when you break open cells (lyse them). Even though you then spin them down really fast (ultracentrifugation) to pellet the membrane pieces and other insoluble stuff and then you push it through a syringe filter, it can still be pretty viscous (syrupy) and this can clog up the AKTA lines. The AKTA’s really smart - it senses the buildup of pressure and slows down the flow rate - so it starts flowing REALLY SLOWLY. So much for that fast part… So it can be much more time-effective to go with gravity flow in these cases.
Even when it’s not crude lysate, you still need to worry about stuff clogging the tiny tubes - so we filter all of the buffers we send through them - usually using a bottle-top vacuum filter - it has a kind of “bowl” you screw on to the top of the bottle and you pour the buffer into the bowl and it gets sucked through a membrane into the bottle.
Want more details on protein purification and the various forms of chromatography? I have a whole page of posts on my blog: thebumblingbiochemist.com/lets-talk-science/protein-purification/
   
and a CZcams playlist on protein purification: czcams.com/play/PLUWsCDtjESrEtqQkEXsQpTyRfMhywBVQ7.html
as well as one specifically on chromatography: czcams.com/play/PLUWsCDtjESrEtqQkEXsQpTyRfMhywBVQ7.html
more about all sorts of things: #365DaysOfScience All (with topics listed) 👉 bit.ly/2OllAB0 or search blog: thebumblingbiochemist.com

[@MaicMayer]

You are a lifesaver!! Whenever I have free time, I always listen to your "podcast"!

[@thebumblingbiochemist]

Happy to help!

[@Val-zx7ro]

Are there more advanced videos? This helped a lot when I started but now I am looking at things like which columns to use and in what order, troubleshooting gradient elutions to better purify proteins, choosing different buffers to try and prevent dimerization etc. Is there anything like that on your channel right now? Thx!

[@thebumblingbiochemist]

I have a whole playlist of purification videos that can hopefully help: czcams.com/play/PLUWsCDtjESrEtqQkEXsQpTyRfMhywBVQ7.html