Summary: HPLC Manual
Krystyna Brzezinska (email@example.com)
All samples must be filtered.
It is highly recommended that you check the separation by TLC and/or column chromatography
before attempting an HPLC run. If running UV detection on your sample, run a UV-VIS spectrum
first to see where the maximum absorbance is.
Concentration for the HPLC is best around 25-50% w/v (g/mL). The more concentrated the
samples are, the better (even neat), AS LONG AS THE SAMPLES ARE NOT TOO VISCOUS
OR HAVE SOLID PARTICLES IN THEM.
The analytical column can run 1-4 mg of sample per run. After filtration through 0.45 Ám filter
transfer solution into the auto-sampler vial (3/4 of the vial must be filled or use small inserts for
vials (150 and 300 ÁL).
It is the best to play with the HPLC variables such as mobile phase and flow rate to get the best
After you finish flush the column with polar solvent to make sure that all species have been
removed from the column. Afterwards run non-polar mobile phase (hexanes) through the column
for storage. For column longevity, it is best to store non-polar solvents in the column and HPLC