An HPLC column is capable of giving highly reproducible performance run after run for months together. However, it has a finite lifetime and over a period of time its performance begins to deteriorate as evidenced by distortion of the peak shapes and resolution between them, erratic increase in column back pressure and appearance of ghost peaks. Such abnormalities can be avoided by adoption of recommended restoration practices and care of HPLC columns.
A column can be restored if it becomes fouled due to overloading or use of incompatible mobile phases but a damaged column or one that has out lived its useful life cannot be restored and would require to be replaced.
Column Restoration Procedures
Reverse phase columns
Reverse phase separation is the main operational mode used in majority of HPLC separations. The packings mainly comprise of C18, C4, CN,NH2 and phenyl stationary phases. Eluting with 10 to 20 volumes of solvents in the sequence shown is recommended
- Mobile phase
The equilibrium time for common dimensions of HPLC columns are indicated for ready reference :
|Column Dimension||Flow rate||Time|
|250x 4.6mm||1ml/min||60 min|
Reverse Phase (Protein/ Peptide separations)
- Flush with 20 volumes of mobile phase without buffer followed by
- 0.1%TFA in water
- 0.1%TFA in acetonitrile/isopropanol(1:2)
- Finally equilibrate with the mobile phase to be used
Normal phase columns
- (Amino, diol, nitro,amino stationary phases)
- Flush with 20 volumes each of
Ion Exchange columns
Ion exchange columns comprising of both cation and anion exchangers such as SAX, SCX, WAX, WCX.
- 20 volumes of 500 mM phosphate buffer at pH 7
- 10 percent acetic acid
- 5 volumes of water
- 10 volumes of phosphate buffer at pH 7
- 5volumes of water
- 10 volumes of methanol
- 10 volumes of water
- Mobile phase
- 5 column volumes of 0.1 phosphate buffer at pH 3
- If strongly retained proteins are used then use a 60 minute gradient from water to acetonitrile to water.
After regeneration of the column is not to be used immediately then the following storage procedures are suggested :
- Overnight – can be stored in last used mobile phase
- 2-3 days to a week – flush with water to prevent microbial growth
- Long-term storage (several months) – store in aprotic solvents such as acetonitrile.
Validation Before Use
Even after regeneration it is important to validate the performance of a restored column by verifying its performance parameters such as height equivalent to a theoretical plate, asymmetry factor and retention times using standard compound injections.