Can I use a mobile phase containing 5M guanidine HCl on a BEH SEC column? - WKB86190

ENVIRONMENT

• Guanidine HCl
• Bridged ethylene hybrid (BEH)
• Size exclusion chromatography (SEC)
• BEH SEC column

ANSWER

Yes

ADDITIONAL INFORMATION

Things to keep in mind include the following:

1. Diol-bonded silica-based columns have been used for many years with denaturing guanidine and urea mobile phases. (BEH is hybrid, as opposed to pure silica.)

2. The back pressures are higher when using guanidine HCl.

3. A reduced flow rate is needed in order to prevent the back pressure from exceeding the column limits.

4. Mobile phase should be filtered.

5. Keep the flow going in the system at all times. 

6. Under these conditions, the protein will be larger in radius, so it may be useful to evaluate the 450A column.

7. Guanidine HCl is very harsh on LC-based components and hardware. Consult an Instrument technical specialist for guidance on system compatibility with this mobile phase.

8. Consider solubilizing the sample in 5M guanidine HCl, and then use a far more dilute concentration of guanidine HCl in the SEC eluent.

9. The pH of the mobile phase with 5M guanidine HCl may be near neutral (you need to measure pH).

10. If the mobile phase pH is near 7.0, guanidine HCl is more of a salt (chloride counter ion). 

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