Maintenance Of Column

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 

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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 Type

Solvent

Remarks

Silica

Heptane

Down the series to wash

Si, Diol, NH2

Chloroform, Ethyl acetate, Ethanol, Water

Up the series with dried solvent to activate

RP-18, RP-8, CN

Water, Methanol, Chloroform

Down the series only.

Regeneration 

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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 :

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Satisfactory Peak Shapes (Minimal Tailing) Satisfactory Peak widths (narrower peaks are better) Reproducible retention times, assay-to-assay. Reasonable operating back-pressures (lower is typically better.) Satisfactory resolving power for analytes of interest (adequate selectivity). Stable detector baselines. Therefore, we can characterize a column that is having problems by a change of deterioration in any of the above parameters.

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