Making an HPLC Calibration work (Part 2)

Preparation of Calibration Standards It is normal to make up a master standard, and then prepare a series of dilutions to cover the required calibration range. Care must be taken here. Any mistakes, and all subsequent results will be incorrect.

PREPARATION OF THE MASTER STANDARD.

• Use a volume and weight which can be measured really precisely without difficulty. Using too low a mass of sample in too small a volume builds in a minimum error level into the calibration which is completely unnecessary.

• Remember that glass pipettes are usually more accurate than pipettors, and allow for volumetric errors caused by temperature variations in the lab. Volumetric glassware is accurate at 20°C. Check the balance calibration with a standard weight every day to ensure that its calibration remains as it was on the day it was calibrated.

• Check the shelf life of the master standard material, and its level of hydration. It may be necessary to subdivide it when new, and then use it from small containers to prevent changes due to degradation or absorption of water. Be sure to know the level of hydration of the material because concentration levels will be incorrect if the wrong number of water molecules of hydration are included in the calculation.

• It is a good idea to test the shelf life of the master standard solution when made up. It may be longer than expected, and can save a lot of time later.

• Remember to use spotlessly clean glassware. It is so much nicer not having extra peaks in the master standard.