Supporting Waters Customers in Israel

Care and Use Manual of Commonly Used Columns מדריכי שימוש ושמירה על קולונות

Successful Operation of HPLC System
תיפעול נכון של המערכת

Introduction

Before beginning to work with any HPLC instrument one must understand the basic principles of liquid chromatography at its various modes.

The liquid chromatograph is very complex and it consists of sophisticated technology and sometimes delicate and total ignorance in the manner it works might sabotage its parts which are very expensive. In addition, there are chemical processes of distribution between the two phases, the mobile and stationary, while the analysis goes on, and there is a need for a minimum understanding of their theoretical basis to operate the system correctly. Also, a minimum understanding of the method, in which the system performs data analysis, is needed so that no false data or results will be obtained in the analysis.

LISTS OF STEPS NEEDED BEFORE ANY RUN BY HPLC:

Filter the solvents with membranes with cutoff of 0.22-0.45 mM
Use clean and transparent reservoirs through which precipitates and colloids can be distinguished.
Make sure that the solvents will be easily mixed with the previous solvents in the same inlets (A, B, C, D). For example methanol or water should not be placed instead of hexane directly, or any organic solvent should not be placed directly instead of a buffer reservoir.
Degass the solvents and purge all the tubing that lead to the pump.
Connect the column carefully according to the flow direction marked on it (do not connect directly to the detector).
Flow the appropriate solvents through the column at a low flow-rate (0.1-0.5 ml/min) or reach the composition gradually using the appropriate gradient.
Select the appropriate wavelength (or other type of setting) in the detector and wait for stable baseline
Prepare the set of methods in the workstation: Instrument method for the control on the system, Processing method for the data processing and the Report method for the report of final results.
When the system and the methods are ready a blank run should be performed to test the system and verify that it is clean from interferences.