High Performance Liquid Chromatography (HPLC) Columns:

Equilibration, Regeneration and Maintenance

For normal reversed-phase HPLC columns, no special protection is

required as the column is washed and placed in pure methanol

(acetonitrile) or about 80% methanol (acetonitrile) in water, and then

plugged tightly at both ends of the column to avoid volatilisation of the

preserved solvent.

The maintenance of a liquid chromatography column is divided into:

equilibration, regeneration, and maintenance.

HPLC Column Equilibration

Equilibration is the process of stabilising an HPLC column by

establishing a consistent mobile phase and temperature environment prior

to sample analysis.

Steps:

Initial Equilibration:

Perform initial equilibration before starting a new analysis or installing a

new column. Allow the mobile phase to flow through the column long

enough to ensure that the column is thoroughly wetted.

Column Activation:

If the column has not been used for an extended period of time, consider

running several times the column volume of mobile phase through the

column to activate the stationary phase and ensure consistent

performance.

Mobile phase compatibility:

Use the same mobile phase during equilibration as for the actual analysis.

This ensures that the column is exposed to the same conditions as the

sample analysis.

Stable Flow Rate:

A stable and constant flow rate is maintained during equilibration to

ensure reproducibility of subsequent analyses.

Equilibration time:

Allow sufficient time for equilibration. This typically involves passing

the mobile phase through the column for 15-30 minutes.

Reversed-phase columns are factory tested and stored in

acetonitrile/water. New columns should be flushed with 10-20 times the

column volume of methanol or acetonitrile. Be sure that the mobile phase

used to analyse the sample is miscible with acetonitrile/water. By

spending enough time each day equilibrating the column with the mobile

phase, you will have maximum "compensation" for problems and the

column will last longer!

Slowly increase the flow rate at the beginning of the equilibration and

equilibrate the HPLC column with the mobile phase until a stable

baseline is obtained (equilibration takes longer if the flow rate of the

buffer salts or ion-pairing reagents is lower).

If the mobile phase used contains buffer salts, care should be taken to

"transition" with purified water, i.e., the column must be rinsed with

purified water for 30 minutes or more before the start of each day's

analysis and then equilibrated with the buffer salts mobile phase; at the

end of the analysis, it must be rinsed with purified water for 30 minutes

or more to remove the buffer salts and then the column must be rinsed

with methanol for 30 minutes to protect the column.

HPLC Column Regeneration

Regeneration is the process of cleaning and regenerating an HPLC

column when its performance has deteriorated due to contamination or

clogging.

Steps

Flush with mobile phase:

Flush the column with the mobile phase to remove loosely bound

impurities. Use the same mobile phase as for equilibration.

Solvent regeneration:

If the column is heavily contaminated, consider using a more aggressive

solvent or solvent mixture to remove impurities. Gradient or isocratic

elution can be performed using a stronger solvent or solvent mixture.

pH adjustment (if applicable):

If the stationary phase is sensitive to pH changes, adjust the pH of the

mobile phase accordingly to optimise column regeneration.

Backflush (if applicable):

Some columns can be backflushed to remove impurities from the column

bed. Consult the column manufacturer's guidelines to determine if

backflushing is appropriate for your column.

Extended regeneration time:

For high levels of contamination, consider a more aggressive regeneration

technique, such as flushing the column with an organic solvent or using a

specialised column cleaning solution.

HPLC Column Maintenance

Routine maintenance will help extend the life and performance of your

HPLC column.

Steps

Periodic Inspection:

Periodically inspect the column for signs of physical damage, such as

cracks or leaks. Check fittings and connections for tightness.

Protect the column:

Consider using a guard column to protect the analytical column from

contamination. A guard column is a disposable column that is placed

before the analytical column.

Mobile phase filtration:

The mobile phase is filtered to remove particulate matter that could

damage the column.

Sample Filtration:

The sample is filtered before injection to prevent particles and

contaminants from entering the column.

Avoid extreme conditions:

Avoid exposure to extreme pH, temperature, or pressure conditions that

could damage the column.

Backflush regularly (if applicable):

If backflushing the column is recommended, consider including it in your

routine maintenance schedule.

Record Keeping:

Keep a log of column use, regeneration, and any unusual observations.

This helps identify trends or problems that may need attention.