What type of column should be used for liquid chromatography of peptides with a molecular weight of 20,000 Da?
When performing liquid chromatography separation for peptides with a molecular weight close to 20,000 Da, larger pore size reversed-phase high-performance liquid chromatography (RP-HPLC) columns, especially C18 columns, are typically used.
1. C18 Reversed-Phase Column
C18 columns are the most commonly used columns and are suitable for the separation of peptides and proteins. For larger molecular weight peptides, the choice of pore size is particularly important. Generally, for peptides around 20,000 Da, it is recommended to use C18 columns with a pore size of 300Å (or larger), which ensures that larger molecular weight peptides can enter the pores of the stationary phase for effective separation.
2. Other column types available
1. C4 Column: When dealing with very large molecular peptides (especially those approaching the size of proteins), sometimes a C4 column can be used. It has weaker hydrophobicity compared to C18 columns, making it more suitable for larger molecules.
2. Size Exclusion Chromatography Column (SEC): If you are concerned with separation based on molecular size rather than hydrophobicity, consider using a size exclusion chromatography column (SEC). For peptides of approximately 20,000 Da, selecting an SEC column with an appropriate molecular weight range (such as Superdex 200) can also provide good separation results.
In summary, for peptides of 20,000 Da, the most commonly used column is a C18 column with a pore size of 300Å, but C4 columns and size exclusion chromatography columns may also be effective choices in certain special cases.
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