Hello,
I am doing disulfide bond mapping of a TNFR with a free cysteine that may be forming a covalent dimer with another protomer. After digesting the complex, I have some really large peptides with up to 6 disulfide bonds. The peptide of interest has an O-linked glycan at 60% abundance as well.
It doesn't seem like a C18 is able to elute the peptide at up to 50% B. At what size of a peptide should I switch from a C18 to a C8? Alternatively, would HILIC provide better ionization/fragmentation opportunities?
Hello, Octadecylsilane (C18) has 18 carbon and octylsilane C(8)has 8 carbon atom in column structure filled with silica. If similar component eluted in both column then it will faster in C8 and slower in C18 due to surface area density of column. C18 is denser than C8 and denser packing of column increase surface area that leads mobile phase to travel per unit of length. When a short retention time is required then C8 column is more preferred. Non polar compounds will move down the column more rapidly with C8 than with C18.
So made your decision based on above info.
Good luck!
In general (GENERAL), a C18 column has a 'higher' selectivity than C8 and is commonly used for 'challenging' matrices or low concentrations like a related substance (RS).
Adding to previous answers, do not forget to use large pore silica either for C18 or C8 if your peptide is large.
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