How to find the perfect stationary phase for your protein purification

Peptide or protein purification is one of the most performed methods in any life science lab. It is a pure wonder that I’ve never addressed this topic before, but I’d like to correct the error of my ways. In this post, I explain the role of chromatography in protein purification and give you tips on how to select the right stationary phase for protein applications.

The weather has been very mild last week, so my wife and I have been going for afternoon walks in the park. One of the places that is always quite lively is the open-gym area, where people are free to exercise in open air. Mind you, most of the people are pretty fit and are doing some amazing things with their muscles. And it did catch my eye that besides a boom box and towels, one of the most common items in their belongings was the protein shake.

So naturally my mind drifted to the wonderful world of proteins. I mean proteins and peptides, the smaller version of proteins, are responsible for so many life-essential functions, such as signal transduction, heart rate regulation, food intake and growth. Besides their importance in body building, they have applications in biotechnology, bioengineering, drug discovery and delivery, cosmetics and nutrition. So why on earth’s sake, after I’ve written about cosmetics and cannabis , have I never before dedicated a post to protein purification?

This is about to change right now.

Proteins can be separated by crystallization, filtration and precipitation but chromatography has emerged as one of the most common methods for protein purification. The types of chromatography used in protein applications include:

Type of liquid chromatographyMode of separation based on:Applications & remarksPros & cons
Ion chromatographyChargePeptides
Mild conditions, no denaturation of the compounds, but low resolution
Affinity chromatographySpecific binding interactionProteinsHigh purity but tag interferes with structure of the protein
Size exclusion chromatographyMolecular sizesProteinsSimple and high preservation of the compound's activity, but high dilution and low resolution
Adsorption chromatography (reversed-phase)HydrophobicityPeptides
Proteins (small and stable)
High purity, but denaturation of the compounds

Out of these methods, reversed-phase chromatography is a clear favorite for small protein purification and peptide separation. This is largely due to the high separation power of the technique, which enables polypeptides that differ by a single amino acid to still be efficiently separated. In this method, polar organic solvents are used as mobile phases. Organic solvents create stringent conditions, limiting the suitability of the technique to peptides and small, stable proteins that can spontaneously refold after purification. Reversed-phase chromatography remains widely used for detecting impurities during investigative studies or to remove unwanted compounds during large scale purification of therapeutic drugs.

Should you choose to perform reversed-phase chromatography for your protein purification, you can decide between flash chromatography or prep HPLC (high-pressure liquid chromatography).

Flash chromatography is frequently used as a pre-purification step to purify large sample quantities at a sufficient resolution. Prep HPLC is used to obtain the highest resolution or purity under the limitation of lower loading capacities.

The two techniques differ in the material used for the stationary phase as the particle size varies, the dimensions of the cartridge or column and the flow rate of the mobile phase. These differences are summarized in the table below:

FlashPrep HPLC
Particle size15 - 63 µm5 - 15 µm
Column ID12 - 115 mm10 - 70 mm
Flow rate15 - 250 mL/min5 - 100 mL/min
Loading capacity< 300 g< 10 g
Max pressure50 bar300 bar

Whether you choose a flash chromatography or prep HPLC system for your protein purification depends on your sample and specific needs. Most biochemists use flash chromatography as a first step to get as much of the target compound pre-purified as possible. Then they purify the collected fractions, as material at reduced volume and free of most contaminants, via prep HPLC to achieve the needed high resolution.

How can you find the ideal stationary phase for your protein purification?

The stationary phase you select needs to retain the target compound, but neither too strongly nor too weakly. If the stationary phase binds too weakly, the compound will run with the mobile phase through the column and will likely fail to separate from the other compounds in the mixture. If the stationary phase binds too strongly, it will take too much time and solvent to elute the compound of interest.

There are six parameters that affect retention and I will discuss them one by one with you now. You can check out one of my previous blog posts on retention for more details.

1. Polarity – If a compound does not interact with the stationary phase, it cannot be separated. The compound needs to be compatible with the solvents used for the phase. Take a look at the table below for a list of common polar and unpolar stationary and mobile phases.

Type of adsorption chromatographyPolarity stationary phasePolarity solventsCommon solvents
Normal phase (unbonded silica)PolarUnpolarhexane/ethyl acetate or dichloromethane/methanol
Reversed phase (bonded silica, such as C18, C8, C4)UnpolarPolarmixtures of water and a second water-miscible solvent, such as methanol, acetonitrile or ethanol

2. Particle size – Smaller silica particle sizes in a cartridge or column result in a higher total surface area, which in turn increases the number of possible adsorption and desorption steps. This leads to better column efficiency, higher resolution and purity.

3. Particle shape – Spherical particles, which are more uniformly packed in the column or cartridge , help achieve a more homogenous flow than particles that are irregular in shape. Spherical particles lead to sharper peaks and higher resolution.

4. Carbon content – This property refers to the covalent attachment of hydrocarbons to a silica gel. Higher carbon content results in higher resolution, but also in longer run times.

5. Pore size – As a rule of thumb, the pore size should be at least three times the diameter of the molecule. If diffusion of the target compound into the pore structure is impeded, the resolution decreases.

6. Surface area – The surface area of silica particles is equal to the sum of their internal and external surface area. This property is affected by the particle size and pore size. The higher the surface area, the higher the resolution.

For protein purification, you need to focus mostly on polarity of the phase, particle, and pore size.

I would recommend small particle sizes, as they allow separations of even highly similar compounds. The stationary phase of choice is usually a bonded phase, such as C18, C8 or C4, which differ in the lengths of alkyl chains bonded to the silanol chains. C18 is more unipolar or hydrophobic than C8 and C4, hence peptides and proteins with a higher hydrophobicity interact stronger with this stationary phase. Polar molecules are better retained by C4 phases.

Typical solvents used for protein purification include water and a less polar solvent such as acetonitrile, isopropanol or ethanol.

Acetonitrile is the most frequently used solvent because it is volatile, can be easily removed from the collected fraction, it has a low viscosity and low UV absorption. Traditionally, trifluoroacetic acid (TFA) is used in the mobile phases for peptide separations at concentrations of about 0.1%. I’d recommend that you form a gradient, where you slowly increase the concentration of the less polar solvent to obtain sharp peaks at highest resolution.

Keep in mind that the more polar molecules will elute first.

Regarding the stationary phase, the standard silica pore sizes of 60-100Å only work for smaller molecules. Bigger proteins cannot enter the smaller pores, reducing the surface area for separation and resulting in low resolution. Bigger proteins need larger pore sizes in the range of 200-300Å. Take a look at the figure below to further help you select the stationary phase based on the polarity and molecular weight of your target protein and peptide:

protein, peptide, protein purification, flash chromatography, prep HPLC, stationary phase

Protein purification happens to be an interesting topic, so I think I might have gotten a bit carried away with my explanations. I hope this post was more of a protein shake for your brain, if not for your muscles and you found it useful. I am sure I will revisit this subject in the future. Do you think it’s a good idea? Leave me a comment below! And if you are looking to build up even more knowledge, check out my blog posts on stationary phase and mobile phase selection sooner rather than later.

Till next time,

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