Technical Information

Tips for optimization of peptides and proteins separation by reversed-phase

Factors to be considered on method optimization of peptides and proteins


Combination of functional group and the pore diameter
⇒ Choose optimal combination by molecular weight and hydrophobicity of peptides and proteins.
     Generally, a column with large pore size and low surface hydrophobicity is suitable for large molecules.

Mobile phase

Gradient elution with 0.1% TFA/acetonitrile system as first choice
⇒ Change 1) concentration of TFA, 2) acid species and/or pH if a sample is mixture of compounds with various ionic characteristics.
⇒ Adjust the gradient conditions
  2-propanol might be effective for separation of large proteins


Effective for changing separation selectivity or improving peak shape. However, usable temperature range is limited by column durability.
(strongly acidic conditions + heating will accelerate the elimination of functional groups = short retention time and/or increase of unfavorable secondary interaction between the packing material and sample)

How to select reversed-phase columns

Comparison of separation on columns with different pore sizes and functional groups

The separation characteristics of proteins and peptides with molecular weights of 4,300–17,000 are compared using columns with different pore sizes and functional groups. In accordance with the figure below, the suitable column for compounds with a molecular weight within this range is C8, 200 Å. If either the pore size or functional group of the packing material is not optimized, peak broadening and poor resolution are observed. By using the most suitable column (C8, 200 Å) for the target compounds, sharp peak shapes and excellent separation are achieved.

  1. Cytochrome c           (MW 12,400)
  2. Insulin (Bovine)          (MW 5,700)
  3. Amyloid β-protein      (MW 4,300)
  4. Lysozyme                  (MW 14,300)
  5. α-Lactalbumin           (MW 14,100)
  6. Myoglobin                 (MW 17,000)
Column 5 μm, 150 X 4.6 mmI.D.
Eluent A) water/TFA (100/0.1)  
B) acetonitrile/TFA (100/0.1)
25-60%B (0-20 min)
Flow rate 1.0 mL/min
Temperature 37°C
Detection UV at 220 nm

Effect of column temperature and mobile phase (Optimization of antimicrobial peptides separation)

Structure (antimicrobial peptides)
HPLC common conditions
Column YMC-Triart C18 (1.9 μm, 120 Å), 
50 X 2.0 mmI.D.  
Flow rate 0.4 mL/min
Detection 220 nm

Effect of column temperature

Changing the column temperature will provide selectivity change as well as peak shape improvement. High durability column, Triart, can offer wider usable temperature range. Temperature can be used as a tool for method optimization.

Eluent A) water/TFA (100/0.1)
B) acetonitrile/TFA (100/0.1)
25-45%B (0-5 min)  

Effect of acid type, acid concentration and gradient conditions (at 70°C)

Selectivity change can be expected by changing acid type and/or concentration. It is effective when there is a large difference in ionic characteristics of compounds.

Eluent A) acid-containing aqueous solution
B) acid-containing acetonitrile solution
(0.1% HCOOH in B solution is 0.08%)
Temperature 70°C

Addition of 2-propanol in a mobile phase

2-propanol is added in the mobile phase, and gradient slope is optimized. Resolution between peak 1 and 2 is improved while maintaining the analysis time.

Eluent A) 0.1% formic acid in water
B) 0.08% formic acid in organic solvent
Temperature 70°C