Maintenance of HPLC Column

Types Of Column Used In the Laboratory and their

storage solvent

 Reversed Phase : C18, C8, C4, C2, C1,

Phenyl ( Methanol)
 Normal Phase : Silica, CN, NH2 , Diol,
Alumina (Isopropanol or Hexane)
 Ion- Exchange : DEAE, CM, SAX, SEX
 Size exclusion : Diol
Reason for column degradation
 Partially blocked/ plugged frit or column
bed.
 Initially poor packed column.
 Mechanical or thermal shocks resulting in
creation of voids (bed subsidence)
 Chemical attack on support/ stationary
phase.
 Adsorption of non-eluting compounds etc.
Symptoms of degradation
 Increase in back pressure.
 Tailing in bands
 Loss in plate number
 Decrease in retention.
Cause
 Plugged Inlet Frit : Symptoms : High
pressure, Tailing higher side, Low N
 Creation of Voids : Tailing higher side, Low N
 Adsorption of non-eluting Compounds : Variation
in Retention Time.
 Chemical Attack : Tailing higher side, Low N,
Variation in Retention Time.
Regeneration
Depending on the bonded phase being used,
various solvents are used for regeneration
(refer manufacturers leaflet supplied along
with the column)
In general following are the sequence of
solvents used for regeneration.
Regeneration
Type Solvent Remarks
Type

Silica Heptane Down the series to

wash

Si, Diol, NH2 Chloroform, Ethyl Up the series with

acetate, Ethanol, dried solvent to
Water activate
RP-18, RP-8, CN Water, Methanol, Down the series
Chloroform only.
Regeneration
 Note : Periodic washing with 0.01 M Sulphuric
Acid may be necessary to clear the surface of
Silica when simple organic and water washes are
not sufficient.
 Drying out of stationary phase during stocking and
shipping does not affect either the efficiency or
life of the column. To get whole surface activated
and to get reproducible results, its equilibrium
procedure should preferably start with pure
organic modifier such as Methanol or Acetonitrile.
Life of HPLC Column
 Life span of a well-made/ packed column depends
on how the column is used, however it must be
remembered that every column has to die
eventually. If the plate numbers (N) falls by >50%
or the resolution decreases to about 3/4th of the
original, the column should preferably be
replaced, even increased peak asymmetry is a
signal for replacement. Normally, one can easily
analyze 1500 to 2000 reasonably cleaned samples
on a column, for complex samples (biological) ,
one can hardly analyze 200- 300 samples.
HPLC column care
 Physical Changes
 Chemical Changes
 Preventive measures
 Corrective measures.
HPLC column care
 Maximum lifetime and performance for an
HPLC column is best achieved through a
proper program of care and maintenance.
 This depends mainly on Mobile Phase and
Flow Rate and also the nature of the sample
being analysed.
HPLC column care
 In general , HPLC column that is performing well offers
the following characteristics :
2. Satisfactory Peak Shapes (Minimal Tailing)
3. Satisfactory Peak widths (narrower peaks are better)
4. Reproducible retention times, assay-to-assay.
5. Reasonable operating back-pressures (lower is typically better.)
6. Satisfactory resolving power for analytes of interest (adequate
selectivity).
7. Stable detector baselines.
 Therefore, we can characterize a column that is having problems by
a change of deterioration in any of the above parameters.