Hobbies And Interests
Flow Rate & Pressure
The pump must be able to maintain accurate flow rates. Testing the flow rate is quite straightforward and merely entails measuring the amount of time required to collect an arbitrarily chosen amount of solvent from the column at different flow rate settings. Pump pressure can be tested by plugging the pump outlet with a dead-nut and connecting a recorder to measure pump-head pressure. The pressure should increase quickly to about 3,000 pounds per square inch (PSI), then increase more gradually to a higher pressure, at which point the pump will automatically switch off. If the pressure fluctuates at 3,000 PSI or decreases rapidly after the pump switches off, it usually implies a malfunction, like a leak in the system.
Gradient
The pump must also be able to accurately vary the composition of the solvent or mobile phase, thereby eluting or flushing from the column increasingly nonpolar compounds as the percentage of the less-polar solvent increases. Gradient accuracy is tested using two channels at a time, one of them providing pure solvent and the other a solvent with a dissolved tracer chemical, like caffeine. As the concentration of the second solution increases, you can measure the ultraviolet (UV) absorbance of the column output and use this data to verify gradient accuracy.
Injector Module
The injector module's precision can be tested by performing six replicate injections with a single sample; significant discrepancies in your results suggest you have a problem. Some experiments involve variable injection volumes, so it's also important to test the injector's ability to draw the appropriate amount of sample. By carrying out injections across the 0 to 100 microliter range, a chemist can obtain and plot data and determine the correlation between the results and the sample concentration. A weak correlation suggests a malfunction.
Detector Module
The detector module records absorbance for solute and solvent eluting or flowing from the bottom of the column, so its accuracy is especially critical to ensuring good results. Many HPLC instruments include built-in wavelength verification capability to ensure the wavelength the detector uses is the same as the one you have selected. Another test involves using a series of samples that contain an increasing concentration of a compound like caffeine. The chemist can then plot the results versus the test solution concentration and determine the strength of the correlation. A weak correlation suggests a malfunction.