HPLC injection in brief is a technique to load the sample under test into the HPLC system. Sample injection in hplc is done using a specially designed injection system as HPLC is a sensitive system with regards to its instrumentation and operation, the injection . HPLC injection is done manually by an external syringe initially at a specific position in the system. Then another inline injection system attached to the system does the actual loading of the sample. The injection system is so carefully designed to see that no handling or loading error occur during the hplc analysis.
HPLC injection has certain requirements:
1. The volume of injection for all samples under an experiment should be same.
2. The hplc injection volume is very small and is in the range of micro liters.
3. The sample injected should be free of any particulate matter.
4. The injection sample should have no air bubbles.
5.The hplc injection is always done when the mobile phase is kept under a constant flow rate through the column.
Rheodyne injector or loop injector as hplc injection system:
This is the injection system used widely in HPLC. The system has a load mode and an inject mode. In inject mode the sample from a syringe is injected into the loop. Then the injection is set to inject mode where the loaded sample is left into the flow of HPLC mobile phase.
Any excess sample is transferred to drain. The system has loops of fixed volume like 20μl, 50μl, 100μl and 200μl. So when a sample of 25 μl is injected into loop of 20μl, then only 20μl is passed into column and remaining is drained out.
The software in the computer has to be set for injection phase. Then load and inject samples are specified to record the readings of that injection.
CHECK THE VIDEO FOR LIVE DEMO……….
For theory of rheodyne injector see the video below.
External HPLC syringefor hplc injection:
Mostly syringes in micro-liter (μl) range are used. For this specifically designed Hamilton syringes are used. These syringes are to be handled carefully as they are easily prone to damage by accidental fall, particulate matter etc.