Protein chromatography is a way to separate proteins based on differences in their properties – size, charge, etc. by flowing them through columns filled with little beads (resin) that interact differently with them based on those properties. We often do this with Fast Protein Liquid Chromatography (FPLC). Here a machine like 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. And at the speeds we tell it to (FLOW RATE). When our protein comes off of the column (elutes) we see it as a peak on a UV chromatogram. So today, here’s a look at how to interpret then and a practical look at working with them on an AKTA. Including some of the “extra” things to add to the curve to monitor – like conductivity & pressure.

Here’s a little text from a past post, but the video’s the main thing today because it’s easiest to see with examples!



much more on chromatography here: http://bit.ly/proteincleaning

As the name implies, the FPLC *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.

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.”

It can collect different fraction sizes

  • for things like the flow-through in affinity chromatography, where your protein shouldn’t be there, you mainly collect the flow-through as a “just in case” so you don’t want to collect tons of tiny fractions, so we usually collect things like that in 50mL tubes
    • if you have a LOT of flow-through (like the other day when I was trying to get my protein to bind an anion exchange column but there was too much salt so I had to super dilute it and ended up having to flow almost 2L through a 5mL column) you can direct it through an “out” valve hooked up to a tube you can stick anywhere – like a giant graduated cylinder
  • when we’re doing the actual protein elution, we want to collect smaller portions for a couple reasons
    • 1) because the more concentrated it is the easier our life will be (and, as long as it’s not so concentrated that it clumps up (aggregates), proteins are usually happier when they’re more concentrated, so the next step is usually concentrating the eluted protein and the more concentrated it is to begin with, the less concentrating we’ll have to do
      • -for things like stepwise elutions, where you unstick everything in one fell swoop (e.g. go from no competitor to maximum competitor) 15mL tubes are good for this
    • 2) if proteins come off the column at different times but you collect everything that comes out in one tube, you’ve unpurified what you purified! But if you collect smaller portions, and different things are in different portions, you can keep separate the things you separate!  
      • for gradient elutions (work your way up to that max competitor concentration) depending on how tightly your protein’s stuck on, it’ll elute (get pushed off) at different concentrations – we often collect the elations in 96-well blocks. Each well can hold up to 2mL, but you can program the AKTA to do smaller fractions than that so you can be really picky about what you want 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. 

We’re able to choose the flow rate because we control it with pumps. Yes, plural. 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.

We also have another pump – a sample pump. This pump sucks up liquid through a sample line either until it reaches a volume we specify or until it senses air (hopefully because it’s sucked up all your sample and not because the line popped out…)

Another way to get a sample onto the column is through an INJECTION LOOP – kinda like people in line to get into a museum before it opens. – When you have a small volume to load, you can inject it into a loop of tubing that holds it until you want it to be let onto the column – then the injection valve opens up and lets your protein in.

FPLC can be used for tiny volumes (like when we inject samples into a 250uL loop) or huge volumes – industrial companies have huge columns that can handle liters and liters.

Another thing it has is a CONDUCTIVITY MONITOR to follow column equilibration (you want the conductivity to be stable before you put your sample on – this indicates that all the liquid in the column is the one you want your protein to meet) & salt gradient formation (as you add salt, the conductivity should increase – if it doesn’t, you have a problem somewhere…) We have a pH MONITOR too.

Our lab has 4 AKTAs! (pinch me!) and we have an online “booking system” where we can sign up for when we want to use which one. Normally it’s not a problem to book it (and we don’t get charged or anything, the system’s just to “call it first”)

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 we 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 https://bit.ly/sizeexclusionchromatographytalk

Some practical advice:

  • Before starting an AKTA run, check all the tubing for bubbles. Air in the pumps can really mess up your runs, so you can manually prime the pumps with a syringe to pull through any trapped air. If you have the AKTA set to collect fixed fraction sizes, visually check after the fact to make sure that all the wells really do have the same volume. Variability could indicate air in the system. 
  • Remember to regularly empty the waste beaker and check that it doesn’t need emptying before you start an AKTA run – also, always have paper towels handy! 
  • Make sure the lines of your AKTA don’t pop out or they’ll start sucking in air not buffer/sample. If air gets into the pumps the peaks will go haywire, the pressure will flatline and the fraction sizes will be off if the line just pops out from one pump. Tape works nicely, and so do those caps with the little slits in them (don’t know the technical name)
  • you can use remote desktop software as a sort of “baby monitor” to keep an eye on the run from another room (or even from your bed) as it goes – good time for cleaning that bench mess… At first I felt sorry for my bench – it only ever gets a few minutes of unmessiness at a time – as soon as one’s cleaned up, another seems to magically appear… but now I think of it more as me allowing it to fulfill it’s life purpose of enabling scientific exploration – like on Friends where Phoebe’s opposed to trees being cut down to use as Christmas decorations, so Joey tells Phoebe that Christmas trees were “fulfilling their life purpose, by making people happy” 

more on the AKTA: blog form: https://bit.ly/aktainaction ; video: https://youtu.be/b1xwNjfD6jI   

more on protein UV absorbance: http://bit.ly/bradforduv 

Want more details on protein purification and the various forms of chromatography? I have a whole page of posts on my blog: https://thebumblingbiochemist.com/lets-talk-science/protein-purification/ 


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