HPLC injection: The technique and instrumentation

HPLC injection 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.

This injection system is a separate and prominent part of hplc system.

High performance liquid chromatography is a highly sensitive system with regards to its instrumentation and operation.

The injection is done manually by an external syringe initially at a specific point in the HPLC system. Then another inline injection system connected to the pump and column 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. Also any the amount of sample injected is kept constant.

Even if we load more amount of sample by external syringe, still it injects the prefixed volume excluding the excess.

HPLC injection has certain requirements:

1. The volume of injection for all the samples under an experiment should be same.

2. The hplc injection volume is very small and should be 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.

HPLC injectionAny 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.

But most modern instruments have capability of self injection. There the system takes the exact volume of the sample from a prefixed vial and injects into the column. the software control the whole process of injection, pump pressure and detector.


For theory of rheodyne injector see the video below.

External HPLC syringe for 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.
Further before and after use, these syringes have to be cleansed with methanol and hplc grade water. This will prevent the injection from clogging and damage due to any material inside the column of syringe.
For auto injector machines, this is not required unless the instrument is not used regularly. So during HPLC method, the injection system should be checked and cleaned to see that it functions properly.

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