“If you want to increase your success rate, double your failure rate”
— Thomas Watson, Sr. founder of IBM
Understanding of injectors is as important as that of pumps in the last session. Injectors serve to introduce required sample volume accurately into the HPLC system.
Sample injection into the moving mobile phase stream in HPLC is quite different from injection into a gas stream in Gas Chromatography as precise injection is required against high back pressure. In such a situation it is not possible to simply inject using a syringe alone.
Manual injection(Rheodyne/Valco injectors)
Injection is done through specially designed 6-port rotary injection valve. The sample is introduced at atmospheric pressure by a syringe into a constant volume loop. In the LOAD position the loop is not in the path of the mobile phase. By rotating to the INJECT position the sample in the loop is moved by the mobile phase stream into the column. It is important to allow some sample to flow into waste from loop so as to ensure there are no air bubbles in the loop and previously used sample is completely washed out to prevent memory effects.
Automatic injection improves laboratory productivity and also eliminates personal errors. Present day advanced HPLC systems are equipped with an auto injector along with an auto sampler. The software programmes filling of the loop and delivery of the sample to the column. The computer also controls the sequence of samples for injection from vials kept in numbered positions of the auto sampler. It is important to adopt precautions to ensure consistency of results and also prolong the service life of the automated system.
- Prime injector with solvents to be used but it should be ensured that solvent is compatible with solvent used earlier.
- Needle wash between samples will prevent carry over between injections.
- Before start and at end of analysis ensure tubing is completely washed of buffers or previously used solvents.
- Do not forget to feed the vial number correctly on auto sampler rack and list out the sequence correctly in the computer.
We have now gained an understanding of HPLC and its systems. The next session will discuss some application areas particularly in analysis of Pharmaceuticals and Foods.
Tip of the Day
Injector tubes and loops should be rinsed sufficiently before subsequent analysis to prevent cross contamination.
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