Molecular Techniques and Methods

Protein Purification by Reversed Phase-HPLC

Copy Right © 2001/ Institute of Molecular Development LLC


Reversed-Phase (RP)-HPLC separates protein fragments derived from chemical cleavage or enzymatic digestion of a purified protein. However, the use of RP-HPLC for the purification of proteins is limited, because the procedure in RP-HPLC effectively denatures proteins and destroys their biological activity. The RP-HPLC separation of any peptide or protein mixture is dependent upon the strength of the hydrophobic interactions of each component in the mixture with the hydrophobic surface of the column matrix and the elution strength of the organic solvent in the mobile phase, As the concentration of the organic solvent increases, the interactions between the peptides or proteins and the column matrix are diminished, and elution of the polar species occurs first followed by the elution of nonpolar species. Peptide or protein mixtures are applied to an RP-HPLC column containing a chromatographic matrix with defined hydrophobic character. The adsorbed peptides or proteins are eluted in order of least to most strongly bound molecules by increasing the organic solvent concentration in the elution buffer.

There are a number of variables that affect the chromatographic behavior of peptides and proteins on hydrophobic supports. From the standpoint of the support itself, there is the length of the alkyl side chain (Cl, C3, C4, C8, and C18), the mean particle diameter (3-10 um), the mean pore size (60, 100, and 300 Å), and the amount of alkyl chain bonded to the silica-based or polymeric support. In general, resolution will be better on columns consisting of smaller size particles. The residual silanol groups on the silica-based supports function as sites of cationic exchange, and this can affect the separation and recovery of basic peptides or proteins. The use of polymeric supports, which are not silica-based, eliminates this problem and allows the use of pH >7 buffers, which dissolve silica-based matrices. The elution order of peptides or proteins is determined by the pH and composition of the buffer and in increasing linear gradient of an organic solvent (e.g., methanol, acetonitrile, or propanol).

In general, RP-HPLC is most widely applicable to the chromatography of peptides with <30-40 amino acids, which generally exhibit high recoveries. In contrast, proteins are denatured under reversed-phase liquid chromatographic conditions, can be difficult to recover in biologically active form, and are frequently difficult to recover with high mass yields, because of irreversible precipitation on the column.

Choice of RP-HPLC Column
Generally, peptides and proteins should be separated using columns containing C3, C4, phenyl, C8, and C18 bonded phases. For peptides derived from enzymic digestion or chemical cleavage of high-molecular-weight proteins, C8 or C18 alkyl-bonded phases should be used. Hydrophobic proteins that yield broad, tailing peaks on C8 or C18 columns should be rechromatographed on a C3 or C4 column. The loss of peptides and proteins occurring on RP-HPLC columns has been found to be proportional to the amount of support material packed into the column. Hence, columns with reduced bed volumes (e.g., .2 mm i.d. x 10 or 15 cm or, better still, 1 mm i.d. columns if a microbore HPLC apparatus is available) should be used. Hydrophobic-interaction chromatography, high-performance chromatofocusing, size-exclusion HPLC, and ion-exchange HPLC are useful alternate techniques for protein separation.

Column Specifications for Reversed-Phase HPLC of Peptides and Proteins

Peptide Purification
Protein Purification
Bond Phase
C3-C18, Phenyl
C3-C18, Phenyl
Particle Size
3-10 um
3-10 um
Pore Size
100-300 Å
300 Å
Length of Column
1-25 cm
1-25 cm
Internal Diameter of Column
1-5 mm
1-5 mm
Flow Rate
0.02-2 ml/min
0.02-2 ml/min

Choice of RP-HPLC Buffers
It is important to choose the right buffer for peptide and protein RP-HPLC. A trifluoroacetic acid (TFA) buffer provides low pH conditions necessary for amino acid side chain protonation (i.e., Asp, Glu, His, Lys, and Arg) and also provides a volatile buffer that can be removed by lyophilization. A volatile buffer can be extremely important when one is preparing on the order of a few micrograms of peptide or protein, which must be free of contaminants. Alternatively, a nonvolatile buffer can be used initially, and the peptide or protein can be desalted by a subsequent, quick RP-HPLC step using a volatile buffer (e.g., TFA or ammonium bicarbonate). The desalted peptide is eluted with 50% methanol in water, collected, and dried in a concentrator.

Column Handling
Columns should be stored in organic solvents after first flushing out all buffer salts with HPLC-grade H20. Never allow a column to dry out. Increasing column backpressure usually indicates a blockage in the column inlet frit. Column should be disconnected from the detector, reversed, and flushed with a gradient. Alternatively, the column may be disassembled, and the column inlet frit or the fitting with a pressed frit may be sonicated in 20% nitric acid followed by sonication in water or just replaced.

Detection of Proteins
Detection should be at 210 to 220 nm where the peptide bond absorption occurs. The absorption is proportional to the number of peptide bonds in a peptide or protein. Detection at 280 nm is useful for peptides or proteins containing chromophores (e.g., Tyr or Trp). Detection can be achieved using fixed or variable wavelength or photodiode array detectors. A fixed wavelength detector at 210 to 220 nm will suffice for most peptide protein separations. Photodiode array detectors have the advantage of recording a complete spectrum at each time point, but are 10-fold less sensitive than other conventional detectors.

Isocratic versus Gradient Elution
Although isocratic elution (i.e., constant percentage of TFA/ Aectonitrile Buffer) can result in high- resolution separations, the isocratic conditions must be established empirically. A linear gradient of 0.5 to 0.8% TFA/ Acetonitrile per min at flow rates of 1-2 ml/ min is useful for separations of precious samples.

Buffers for Reversed-Phase HPLC of Peptides and Proteins

pH of Buffer
Commonly Used Buffers for RP-HPLC
pH 2-3
0.1% Trifluoroacetic acid (TFA)
0.1% H3PO4
0.1% Heptafluorobutyric acid (HFBA)
10 mM HCl
5-60% Formic acid
pH 4-6
10 mM Ammonium acetate
10 mM Triethylammonium acetate (pH 4.5)
100 mM NH4H2PO4 (pH 4.5)
pH > 6
100 mM Sodium acetate (pH 7.5)
10 mM KH2PO4 (pH 6-8)
20 mM Tris-HCl (pH 7-8)
50 mM NaH2PO4 (pH 7)
50 mM NH4HCO3 (pH 8)


HPLC Buffer Preparation
All buffer salts, organic solvents, and water must be HPLC grade. The UV cutoff (i.e., the wavelength at which the absorbance becomes high) of acetonitrile or propanol should be the lowest obtainable. Laboratory water should be filtered through a 0.45-um nylon filter and degassed using a trapped vacuum pump. The chemicals used in RP-HPLC are toxic, especially TFA, heptafluorobutyric acid, formic acid, and acetonitrile, and should be used in a well-ventilated laboratory.

TriFluoroacetic Acid (TFA) Buffer (1 liter)
0.1 % (v/v) TFA ----------------------------------------- 1 ml of TFA
Distilled H20 --------------------------------------------- 999 ml
  • Filter through 0.45 um millipore membrane.
  • Degas the solution.

    TFA/ Acelonitrile Buffer (1 liter)
    0.085% (v/v) TriFluoroacetic Acid (TFA) -------------- 0.85 ml of TFA
    70% (v/v) acetonitrile ---------------------------------- 700 ml
    Distilled H20 ------------------------------------------- 300 ml
  • Filter through 0.45 um millipore membrane.
  • Degas the solution.

    Preparaiton of Protein and Peptide Sample
    Proteins and peptides must be fully solubilizd. If visual inspection indicates turbidity or particulates, use TFA/ Guanidine Buffer to solubilize most mixtures of protein fragments. The samples should be centrifuged prior to injection in order to remove insoluble particulates.

    TFA/ Guanidine Buffer (1 liter)
    6 M Guanidine-HCl ------------------------------------ 573.2 g
    Add TFA Buffer to make a final volume of ------------ 1 liter
  • Filter through 0.45 um millipore membrane.
  • Degas the solution.

    RP-Peptide Standards (1 ml)
    alpha-Lactoglobulin A ----------------------------------- 1 mg
    0.1 M Ammonium bicarbonate -------------------------- 1 ml
    1 mg/ml Trypsin ----------------------------------------- 20 ul
  • Incubate 1-24 hr at room temperature.
  • Stop the enzymic digestion by adding 10 ul TFA buffer.
  • Following cessation of CO2 formation as evidenced by tiny bubble formation, check that the pH is < 3 by spotting a tiny drop onto pH paper.

    RP-Protein Standards (1 ml)
    Insulin --------------------------------------------------- 1 mg
    Cytochrome c ------------------------------------------- 1 mg
    alpha-Lactalbumin --------------------------------------- 1 mg
    Carbonic anhydrase ------------------------------------- 1 mg
    Ovalbumin ---------------------------------------------- 1 mg
    TFA Buffer --------------------------------------------- 1 ml
  • Mix gently.


    1. Remove the organic solvent (e.g., acetonitrile, methanol, or propanol) from the RP-HPLC column with degassed water using a gradient from 100 % organic solvent to 100 % water over 15 min at 1 ml/ min.

    2. Blank run the RP-HPLC column at 1 ml/ min as followings.

    Distilled H2O
    15 min
    TFA Buffer
    15 min
    linear gradient
    TFA Buffer
    5 min
    TFA/ Acetonitrile Buffer
    45 min
    linear gradient
    TFA/ Acetonitrile Buffer
    5 min
    TFA Buffer
    15 min
    linear gradient
    TFA Buffer
    15 min

  • Detection settings should be 0.1 AUFS (Absorption Units Full Scale) at 210 to 220 nm for 50 to 200 pmole peptide.
  • Convenient AUFS settings are 0.3 for 500 pmol, 0.5 for 1 nmol, and 1.0 for 2 nmole.

    3. Centrifuge the RP-Peptide Standards or a digestion mixture of peptides from a protein of interest for 5 min at 5,000g.
  • Withdraw an aliquot of the solution into an HPLC syringe that is thoroughly rinsed with TFA buffer through a blunt-end needle compatible with the HPLC injector.

    4. Load the injection loop while taking care not to inject air bubbles (e.g., 10 ul of the RP peptide standards). For a digestion mixture of peptides from a protein of interest, the injection volume can be considerably larger. Inject the mixture.

    5. Run the RP-HPLC column at 1 ml/ min as in step 2.


  • Although ambient temperature is used routinely for HPLC of peptides and proteins, there are reports that elevated temperature may improve the recovery of proteins.

  • Since many organic and inorganic compounds absorb light in the far ultraviolet (210 to 220 nm), it is necessary to use the highest purity solvents and buffers to minimize such contaminants.

  • The glassware used to prepare and store HPLC buffers should be detergent-free, since trace amounts of detergent can lead to significant contamination.

  • The presence of any significantly sized peaks in the blank run other than those due to pressure fluctuations caused by injection at the beginning of the chromatogram suggests the presence of contaminants from water, buffers, or column. If spurious peaks are observed, then the chromatography should be repeated.
  • If peaks persist, replace TFA Buffer with water and TFA/ Acetonitrile buffer with pure acetonitrile. If the spurious peaks disappear, the buffer was the source of contamination.
  • If the peaks persist, then replace laboratory water with commercially available, HPLC-grade water. If the peaks disappear, the laboratory water was as the source of the spurious peaks.
  • If the peaks persist, pump only pure acetonitrile. If there is a noisy or high background, the organic solvent was the source of contamination. Replace TFA/ Acetonitrile buffer with a previously unopened bottle of organic solvent with a low UV cutoff, and pump this pure acetonitrile.
  • If spurious peaks persist, the peaks may be due to material leaching from a fouled column. Change the column. The contaminating peaks will probably disappear.

  • Occasionally, contamination comes from detergents used to solubilize a protein prior to enzymic digestion. Triton X-100 and Lubrol may contribute multiple contamination peaks. Octylglucoside, Zwittergent 3-14, and SDS are relatively free of UV impurities. If detergents are used, it is important to account for their UV impurities with a suitable control.

  • Spurious peaks, eluting from a previously used column, is referred to as "ghosting" and is due to precipitated peptide/ protein from a previous chromatographic separation being eluted during a subsequent separation. The occurrence of ghosting may be minimized by using shallow gradients (although recoveries of certain proteins may decrease with shalow gradients) and a high flow rate or complete gradient cycles from 0 to 100 % TFA/ Acetonitrile Buffer (60 min) followed by 100 to 0% TFA/ Acetonitrile Buffer (60 min). Columns can also be "scrubbed" by pumping 50% dimethyl sulfoxide or 4 M urea through the column. A new column should be purchased for any crucial protein-separation to eliminate the possibility of cross-contamination. For this purpose, short 3-cm columns are most economical.

  • When the column is equilibrated at some isocratic condition, there should be no variation in column backpressure or UV absorbance. If the column backpressure is not stable, the problem is usually pump relateted: check valves are sticking, pump seals are worn, or there is air in a pump.



  • Hancock WS (ed.) (1984) Handbook of HPLC for the Separation of Amino Acids, Peptides and Proteins, Vols. I and II. CRC Press, Boca Raton, FL.

  • Hughes GJ, Wilson KJ (1983) High-performance liquid chromatography: Analytic and preparative applications in protein structure determination. Meth. Biochem. Anal. 29.59-135.

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