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

Factors to be considered on method optimization of peptides and proteins

Column	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
Temperature	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

Columns are selected based on molecular weights (MW) of the target substances to separate proteins and peptides. The widths at half the peak height (WHPH) were compared when peptides and proteins with molecular weight (MW) from 1,859 to 76,000 were separated using Triart Bio C18 and Triart Bio C4 with pore size 300 Å, and Triart C18 with pore size 120 Å. Good peak shape was obtained using Triart Bio C18 and Triart Bio C4, which are wide-pore columns, even in separation of proteins with high MW. On the other hand, peak broadening was observed when using Triart C18 (120 Å) on separation of proteins with MW over 10,000. Wide-pore columns with pore size 300 Å are suitable for separation of proteins with MW over 10,000.

【The effects of pore size on separation of peptides and proteins】

Column	150 X 3.0 mmI.D.
Eluent	A) water/TFA (100/0.1) B) acetonitrile/TFA (100/0.1) 10-95%B (0-15 min)
Flow rate	0.4 mL/min
Temperature	40°C
Detection	UV at 220 nm
Injection	4 µL (0.1-0.5 mg/mL)
Sample	γ-Endorphin (MW 1,859), Insulin (MW 5,733), Lysozyme (MW 14,000), β-Lactoglobulin (MW 18,363), α-Chymotrypsinogen A (MW 25,656), BSA (MW 66,000), Conalbumin (MW 76,000)

Effect of column temperature on separation

Increasing column temperature to 70°C provides selectivity change, sharper peaks, and therefore improved resolution especially for larger molecules.

Oxytocin 　　　　(MW 1,007)
Leu-Enkephalin 　 (MW 556)
β-Endorphin　　　 (MW 3,465)
Insulin 　　　　　(MW 5,733)
β-Lactoglobulin A　(MW 18,400)

Column	YMC-Triart C18 (1.9 µm, 120 Å) 50 X 2.0 mmI.D.
Eluent	A) water/TFA (100/0.1) 　 B) acetonitrile/TFA (100/0.1) 10-80%B (0-5 min)
Flow rate	0.4 mL/min
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	UV at 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.