The purity of silica gel can affect the peak shape of peptide, especially when the concentration of ion pair reagent is low
- Purity of silica gel on chromatographic column
- The separation of proteins and peptides was achieved by interaction with hydrophobic surface filled with particles in the column.
- Hydrophobic interface
- The common modification is to bond an octadecane linear fatty chain to form a C18 column or ODS column. The molecular weight of peptides produced by hydrolysis of Youdun ~ 3 000 is usually less than that produced by the column of C12.
Purity of silica gel on chromatographic column
The purity of silica gel packing used in liquid chromatography column is very important for separation performance. Metal ion impurities can cause tailing and resolution degradation, as shown in the figure (0.01% and 0.005% TFA).
Silica gel containing metal ion impurities needs to use high concentration ion pair reagents (such as TFA) to maintain a good peak shape.
The separation of proteins and peptides was achieved by interaction with hydrophobic surface filled with particles in the column.
The in column packing particles are usually based on silica gel because of its high stability and stability under most solvent conditions (except for pH greater than 6.5); in addition, silica gel can form porous spherical particles of various sizes with different diameters.
The purity of silica gel can affect the peak shape of peptide, especially when the concentration of ion pair reagent is low. The concentration of ion pair reagent for high purity silica gel is much lower than that for low purity silica gel.
Eluent: add TFA as shown in the figure, elute with 10% – 55% acetonitrile (ACN) gradient, and the elution time is 37.5 minutes.
Low concentration of TFA will lead to poor peak shape and resolution. When the silica gel purity is high (Fig. b), the low concentration TFA of 0.005% will produce a good peak shape.
This is especially important in liquid chromatography-mass spectrometry, because when TFA is used, the signal will weaken. Low concentration of TFA in liquid chromatography-mass spectrometry can obtain better detection signal.
Generally, the small hole (~ 100a) silica gel is not effective in protein separation by RP-HPLC.
The macroporous silica gel allows larger polypeptides to enter into the pores, thus fully interacting with the hydrophobic interface. Therefore, the Zui final peak shape is better and the resolution is higher.
Small molecular peptides produced by protease hydrolysis can enter the pores of porous silica gel and interact with hydrophobic interface; however, macroporous silica gel can also separate peptides with different selectivity and resolution.
The hydrophobic interface was formed by modifying silica gel with hydrocarbon molecules. Chlorosilanes containing hydrocarbon chains, such as octadecylchlorosilanes, react with silica gel (with Ji type silanol groups on the surface) to make hydrocarbons adhere to the surface of silica gel.
Due to the steric hindrance effect, the organosilane molecule Jin reacts with some silanol groups on the surface of silica gel, so a large number of Ji based silanol groups will remain on the surface of silica gel.
In the process of “end capping”, the smaller organosilanes react with Ji silanol groups in turn, thus reducing the number of polar silanol groups on the surface of silica gel. Select the separation surface. The chemical process used in the surface modification of silica gel allows a variety of organic groups to adhere to the surface of silica gel.
The common modification is to bond an octadecane linear fatty chain to form a C18 column or ODS column. The molecular weight of peptides produced by hydrolysis of Youdun ~ 3 000 is usually less than that produced by the column of C12.
There are few hydrophobic phases formed by butyl bonding to the surface of silica gel, which can be used to separate macromolecular peptides or hydrophobic peptides.
Other columns for peptide separation include phenyl column, which is similar to C4 column in hydrophobicity. It is a Ji inserted or Ji terminated column, which can enhance the interaction between peptide and the surface of silica gel particles. Therefore, it has different selectivity for peptides.
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