#reverse phase

The ability to purify samples and isolate specific proteins is an important first step for studying proteins. If you want to get the 3D structure of a protein or an amino acid sequence or gain any understanding of how a protein interacts with something, then a basic requirement is having a pure sample. But let's face it your sample has a bunch of other undesirable junk, because nothing can be too convenient in science. Even if you chemically synthesize the protein instead of stealing it from the original source (like a dead anemone) you still have to purify the sample and get your target protein.

How do you do this?

There are a variety of liquid chromatography techniques used for purifying proteins such as anion exchange chromatography (separates proteins based on charge), affinity chromatography (this is really specific since the column will only bind to the antibody or ligand you choose, not just any randy that passes by), Size Exclusion Chromatography (separates proteins based on size) and Reverse-phase chromatography. These techniques exploit a specific property of the protein such as charge, size, isoelectric points etc. to separate proteins. Reverse-phase liquid chromatography (RP-HPLC) is one the most important and commonly used forms of liquid chromatography. It separates proteins based on how hydrophobic (water hating) or hydrophilic (water loving) a protein is. 

There are two phases in all types of chromatography. The stationary phase (or the lazy phase which clings to things) and the mobile phase, which slowly seeps through the stationary phase. For Reverse-phase the stationary phase is non-polar (or hydrophobic). The interaction between the stationary phase and analyte is mostly hydrophobic. So, if your protein is really hydrophobic (i.e. it has a lot of valine and alanine resides) then it will cling to stationary phase for its dear life until it is eluted by something more hydrophobic. Typically, the stationary phase in Reverse-phase columns have little beads made of silica gel and you have alkyl groups (hexyl, butyl, or ethyl groups) covalently linked to the beads. These little extensions are non-polar and they play a part in the hydrophobic interaction with your sample. The types of alkyl groups attached vary depending on the type of column you’re using and the kind of protein you're purifying. Of course, there are other types of beads like polymer based beads, which are better if you have the pH of your sample or solvent is above 7. BUT they are not as efficient at separating things.

Figure 1: Silica resin with C18 chains. 

Source: http://chem-net.blogspot.com/2013/11/reversed-phase-chromatography.html

The second player is the mobile phase or the hydrophobic solvent you will use to elute your protein. Usually as reverse phase happens you increase the hydrophobicity of the solvent and over time the proteins are eluted based on hydrophobic they are. The hydrophilic proteins come out first and the most hydrophobic proteins come out last because you need a higher concentration of your non-polar solvent. Most of the time, a mix of acetonitrile and trifluoroacetic acid (TFA) is used for the mobile phase. 

Acetonitrile (ACN) is a non-polar solvent. If you want to make the mobile phase more hydrophobic to elute the more hydrophobic proteins, then you increase the concentration of ACN. TFA is a weak hydrophobic ion-pairing reagent which helps maintain a low pH for our silica resin and helps reduce ionic interactions between the peptide/protein and the stationary phase (because there is always a small chance of something annoying like this happening). You can use other non-polar solvents like methanol, ethanol and isopropanol. Isopropanol works better for ridiculously large and hydrophobic proteins but it places a lot of pressure on the column. If the pressure is too high, your incredibly expensive column will die a horrible and resin-crushing death (Pro-tip: Always check the manual for the maximum pressure limit!).

You can tell how well your purification went based on the chromatogram. A chromatogram shows the UV absorbance for your sample against the retention time. Acetonitrile absorbs UV light very well which is why it's a great solvent for reverse phase. The peptide bonds in proteins absorb UV light at 215 nm whereas, aromatic amino acids like tryptophan absorb UV light at 280nm.  UV absorbance can be used to find the concertation of your protein or it can help determine the resolution for your feeble attempts at purification. 

If you have ‘peaks’ or ‘mini mountains’ that are well separated then your peptides are probably separated pretty well. But if your all of peaks on the chromatogram merge into an indistinguishable and horrifying blob—you have very poor resolution and the eluted sample you get at the end won't be that pure. You can take all of the eluted samples and try purifying them with even more reverse phase or other methods, but you will lose a bit of protein at each purification step. Or you can tweak your method to improve the resolution for reverse phase. 

Figure 2: What a chromatogram looks like in an ideal world where the experiment actually works. The “peaks” are sharp, narrow and distinct. 

Figure 3: A blob monster of my own making. I have probably spent more time doing Reverse phase than socialising at this point and I still get chromatograms like this. 

Stuff you need to worry about:

What kind of column your using: Sometimes you want to purify lots of your proteins in one go so you have to pick a column that can handle a high load. Other times you are analysing the purity of your sample and using a sensitive analytical column makes more sense.  If you want to isolate a really hydrophilic protein then using a c18 column is a solid option because it has more alkyl chains that can bind better to proteins that are not so hydrophobic. BUT since you are introducing a really strong hydrophobic interaction it can mess up the structure of your protein to the point where it loses its bioactivity. If you want to protect the bioactivity of your protein using a C4 or C5 column is a decent option.

Pore size: Each column will have its own pore size (the size of the gap between the beads) and you have pick the pore size based on the size of the target protein. Small pore sizes work for smaller proteins (<10kDa) but it most cases a pore size 300 Å works for a wide variety of protein sizes.

Temperature: A high temperature can speed up chromatography and give you better resolution (AKA the sharp and sexy peaks). All of this will come at the cost of your protein being denatured—of course.

pH: Proteins are pH sensitive since the amine, carboxylic and R groups of the amino acids can change charge depending on the pH.  Silica columns have a pH of 2-7, so the basic amino acids exist as positive ions. You need a hydrophobic anionic (negatively charged) ion-pairing reagent (like TFA) that will pair up with the positively charged basic amino acids, since you don't want any electrostatic interaction between the column and the peptide. (remember we only want to separate the proteins based on how hydrophobc they are)

Flow rate: A faster flow rate will allow a better separation. (Pro-Tip: Check the manual for the maximum flow rate. Usually the big, thick columns tend to have a higher flow rate than the thin, small and dainty columns)

Gradient: This is the most important factor which impacts the resolution. If you have a gradual increase in ACN concentration or hydrophobicity of your solvent (AKA a shallower gradient), you tend to get better resolution and higher peaks. However, if the run time is too long then your protein can get damaged since it’s being exposed to harsh chemicals (e.g. Acetonitrile) for a longer period of time.

There are other applications for Reverse-phase aside from protein purification. It isn't just used to purify and analyse proteins but it can work for drugs, metabolites, fatty acids, aldehydes and ketones. I hope this was a helpful introduction to Reverse-phase for anyone who is interested in the technique or actually has to use this technique. 

Next time, I’ll dive into Size Exclusion Chromatography (SEC).

Primary sources:

Dorsey, J. G. & Dill, K. A. The Molecular Mechanism of Retention in Reversed-Phase Liquid Chromatography. Chem. Rev. 89, 331–346 (1989).

Nagy, K. & Vékey, K. Separation methods. Med. Appl. Mass Spectrom. 61–92 (2008). doi:10.1016/B978-044451980-1.50007-0

Recommended readings:

Chromatography, R. Reverse-Phase Chromatography. Proteins 26–34 (2009). doi:10.1201/NOE1420084597.ch409