Case Sharing – Take You To Understand The Separation Of Peptides

1. Properties of peptides

Polypeptide is a compound formed by connecting multiple amino acids in sequence. Amino acids are amphoteric compounds with the characteristics of zwitterions.

2. Factors Affecting Peptide Separation

Column packing pore size (120Å, 300Å)
Additives and concentrations (formic acid, ion pair reagents)
pH
Elution solvent (acetonitrile, methanol, isopropanol)
Gradient
Temperature
Flow rate

Pore size refers to the diameter of the pores on the silica ball. Molecules enter the pores and are adsorbed by the bound alkyl groups in the pores to achieve the effect of separation. The pore size is small, the porosity is high, the specific surface area is large, and the carbon load is high. Molecules are separated in the pore, the pore size must match the size of the molecule, and the molecule must be able to enter the pore. Generally, 80-120 Å is used for small molecules, and 300 Å is usually used for large molecules. In order to achieve the best separation, the pore diameter is generally required to be the diameter of the molecule. 3 times or more.

Low pH reduces the dissociation of acidic side chains, ion-pairing reagents (such as trifluoroacetic acid) interact with the basic side chains of peptide chains, neutralize their electrical properties, and the reduction in charge enhances the hydrophobic interaction of peptides, and the basic side chains of polypeptides There is no adsorption with silanol groups, but ion-pairing reagents can reduce the ionization efficiency of MS.

3. Project case

Separation in liquid phase:

Column: Welch Ultisil® XB-C18 (2.1×100mm, 5μm 300Å)

Mobile phase: Gradient A (0.1% trifluoroacetic acid) B (0.1% formic acid acetonitrile)

Column temperature: 40℃

UV detector: 220nm

Flow rate: 0.3mL/min

Injection volume: 5 μL

Discussion:

The molecular weight of the peptide compound is about 2000. The 120Å and 300Å pore diameters can be selected to achieve effective retention. During the experiment, it was found that the peak shape of 120Å was poor. The analysis may be that the molecular volume of the substance in this system is large, so the 300Å column The top peak shape has been significantly improved. It can be seen that the molecular weight can be used as a reference for selecting the pore size of the filler, but it is not absolute. When making peptides, it is recommended to try both the conventional pore size 120Å and the large pore size 300Å chromatographic column to choose a more suitable pore size;

When formic acid is used as an additive in the mobile phase, there is no retention. When trifluoroacetic acid with ion-pairing effect is selected, the retention is good. This system is the conventional system of choice when detecting peptides, and then adjust according to the results of this system.