Handy Tips to Debug HPLC Column Issues

Peak Shape

Retention Time Variation

Undesired Peaks

Column BackPressure

Follow this general procedure for column flushing with the specific solvents mentioned below for your type of column. It is always good to check the manufacturer’s recommendations prior to flushing so that you don’t damage the column.

1.???Disconnect and reverse the column

2.???Connect the column to the pump, but not the detector

3.???Flush with 10-20 column volumes of solvent at a flow rate no higher than that used for the QC chromatogram

4.???If altering the procedures below, be sure to use miscible solvents for each??????successive step

Reversed-Phase Columns (C18, C8, C4, Phenyl, CN,)

a.???Mobile phase without buffer

b.???Methanol or Acetonitrile

If metal ions are thought to be causing contamination, flush with aqueous 0.05M EDTA, then water, followed by the above sequence. Columns which use ion-pairing reagents should be dedicated to ion-pairing applications.

Reversed-Phase Protein/Peptide Columns

c.???Mobile phase without buffer

d.???Gradient of 10-90% B; A = 0.1% TFA/water; B = 0.1% TFA/ACN

?

Unbonded Silica Columns (SIL)

e.???Isopropyl Alcohol

f.????Methanol

g.???Ethyl acetate

?

Bonded Normal-Phase Columns (CN, NH2, Diol)

h.???Chloroform

i.????Isopropyl Alcohol

j.????Methylene chloride

k.???Hexane

?

Anion-Exchange Columns (SAX, WAX)

l.????Water

m.??Methanol

n.???Chloroform

o.???Methanol

p.???Water

Cation-Exchange Columns (SCX, WCX)

q.????Water (inject 4x 200 μL DMSO during flush)

r.????THF