Molecular Techniques and Methods

Protein Purification by Size Exclusion-HPLC

Copy Right © 2001/ Institute of Molecular Development LLC


Size-Exclusion (SE)-HPLC separates proteins based on molecular size in order of large to small proteins. Protein mixtures are applied to an SE-HPLC column containing a chromatographic matrix of defined pore size. Proteins are eluted with an aqueous buffer. The chromatographic separation of a protein mixture by SE-HPLC is dependent upon molecular sizes of the components. Proteins larger than the pore size or the chromatographic matrix will be excluded (i.e., not retained or retarded) and will be eluted in the void volume of the column. Those molecules with molecular sizes smaller than the pore size of the matrix will penetrate into the pores, will follow longer flow paths, and will elute in order from large-to small-sized molecules. The elution volume of these individual proteins is related to the log10 of the individual molecular weights provided that there are no additional interactions (i.e., electrostatic or hydrophobic) between the SE-HPLC column matrix and the protein(s). The choice of the sodium acetate buffer at pH 5.6 containing NaCl provides the necessary ionic strength to reduce ionic interactions between the silica-based matrices and protonated amino acid side chains. The linear correlation between the log of molecular weights of the individual proteins and their respective elution volumes indicates that for this group of proteins there were no electrostatic interactions with the SE-HPLC column matrix.

When the components are retarded by either electrostatic or hydrophobic interactions with the surface of the SE-HPLC column matrix, the elution volumes no longer directly reflect their molecular sizes. In such cases, the estimated molecular size is often smaller by some factor. When using silica-based supports, low ionic strength buffers may result in ionic repulsion, early elution, and an overestimate of molecular size. To overcome such interactions, chromatography should be carried out under conditions which minimize these interactions. The addition of an organic solvent (e.g., 10% methanol), increasing the ionic strength, adding amine salts (e.g., 0.01 % triethylamine), and changing the pH to reduce net charge will minimize such interactions. When SE-HPLC is carried out in the presence of denaturants or detergents (guanidine-HCl, urea, or SDS), proteins will be denatured and their molecular sizes may be different from those determined in aqueous buffers without these additives. However, SE-HPLC completed under denaturing conditions can yield an accurate estimation of molecular size if the sample protein and protein standards are reductively alkylatted and if the products remain soluble.


All water and salts used for buffer preparation must be HPLC grade, and the glassware used to prepare and to store the buffer must be thoroughly cleaned. Any contaminants add to the general background at low wavelength detection.

SE Buffer (1 liter)
20 mM Sodium acetate (pH 5.6) ------------------------ 6.7 ml of 3 M Sodium acetate
150 mM NaCl ------------------------------------------ 30 ml of 5 M NaCl
Distilled H2O -------------------------------------------- 963.3 ml
  • Filter by 0.45 um millipore membrane.
  • Degas the solution.

    SE Protein Standards
    Thyroglobulin -------------------------------------------- 2 mg
    Catalase ------------------------------------------------- 4 mg
    Bovine serum albumin ------------------------------------ 3 mg
    Ovalbumin ----------------------------------------------- 3 mg
    Ribonuclease A ------------------------------------------ 4 mg
    SE Buffer ------------------------------------------------ 6 ml
  • Mix gently.
  • Proteins must be fully solubilized. Visual inspection will indicate the presence of turbidity or particulates. Magnificaiion 10-20 times is often necessary to determine that a sample at the microgram level is solubilized. All samples should be centrifuged prior to injection.


    1. Remove storage solvent (e.g., methanol) from the SE column with degassed, HPLC-grade water at a flow rate of 1 ml/min.

    2. Equilibrate the SE column (0.75 x 30 cm) at room temperature in the degassed SE Buffer at 1 ml/min for 30 min or until the baseline is stable.
  • Increased chromatographic resolution may be achieved by attaching two SE columns in series; this improves revolution by 40% but doubles the separation time.

    3. Carry out a blank run. Withdraw 100 ul SE Buffer into an HPLC syringe and a needle. Rush out air bubbles from the syringe barrel and needle, load the sample into the injection coil, and inject the sample onto the column. The detector at 210-220 nm should be set at 0.1 to 1.0 AUFS (absorbance units full scale).

    Protein Concentration
    AUFS Settings
    100 pmole
    500 pmole
    1 nmole
    2 nmole

  • Significant peaks, other than those due to pressure fluctuations caused by the rotating injection valve, suggest the presence of contaminants in the system. The chromatography should be repeated until the peaks disappear. If they persist and are present in amounts greater than 20% of full-scale deflection, then change either the column, the buffer, or both.

    4. Centrifuge the SE Protein Standards 5 min at 2,000g, withdraw 100 ul of the solution into a syringe, and repeat step 3.
  • Peaks eluting at greater than 2.5 times the void volume indicate adsorption of proteins to the SE-HPLC column.

    5. Collect each chromatographic peak into a separate polypropylene microcentrifuge tube.

    6. Desalt each fraction and use a Speedvac evaporator to remove solvent from each collected protein. (Polystyrene tubes should not be used.)
  • Desalt proteins by injecting small sample volumes (< 2 ml) with an HPLC syringe or to pump large volumes of salt solutions containing small quantities of protein directly onto a reversed-phase HPLC column by using an auxiliary pump.

    7. After using a SE-HPLC column, remove buffer salts by flushing with water at 1 ml/ min for 30 min, flush the column with 100% methanol at 1 ml/ min for 30 min, and store in 100% methanol.


  • Since molecular size is measured by retention volume, it is absolutely essential to employ an accurate, constant-volume pumping system. Clearly, the semilog plot will not represent the true hydrodynamic volumes if leaks or inaccurate deliveries occur. Buffers at pH 6.5-7.5 containing salts (0.1-0.4 M) are appropriate eluants. Column flow rates vary between 0.2 and 2.0 ml/ min.

  • The separation can be further optimized by altering flow rate or buffer composition. Generally, reducing the flow rate results in moderate increases in resolution. The sample should be injected in a dilute solution (0.01 to 0.5%) and in a volume that does not exceed 1% of the column volume.



  • Corbett RJT, Roche RS (1984) Use of high-speed size exclusion chromatography for the study of protein folding and stability. Biochemistry 23:1888-1894.

  • Kato Y (1984) Toyo Soda high-performance gel filtration columns. In "Handbook of HPLC for the Separation of Amino Acids, Peptides, and Proteins", Vol. 11 (W.S. Hancock, ed.) pp363- 369. CRC Press, Boca Raton, FL.

  • le Maire M, Aggerbeck LP, Monteilhet C, Andersen JP, Moller JV (1986) The use of high-performance liquid chromatography for the determination of size and molecular weight of proteins: A caution and a list of membrane proteins suitable as standards. Anal. Biochem. 154:525-535.

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