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6. Injection: Introduction of samples
If the system is stable: the pressure fluctuate +/- 2% in the pump, the temperatures are stable in the column oven and in the autosampler, the baseline is straight in the detector and everything is ready for the analysis, it is time to introduce the samples into the column using the injector.
Some systems' injection port is still manual, but most of them these days are equipped with an automatic injector, an autosampler.
Manual Injector (Rheodyne
or Valco)
Introduction of samples into the stream is done through a special injection port, whose engineering is complex and delicate. The sample solution is loaded onto a loop of a known precise and constant volue, so that a constant volume is injected into the column. The loop is filled with the sample by a syringe, when the knob is on "Load" position, during which the loop is NOT connected to the steam and the pressure in it is atmospheric. If the sample solution's volume in the syringe is higher than the loop's volume the access volume streams into the waste. It is recommended to use a syringe of volume double of the injection loop for a quantitative work. The syringe is equipped with a special flat tip needle, fitting especially to the HPLC injection valve.
Only special syringes with flat tip needle can be used for HPLC injection. The flat-trimmed needle is the only type that can fit an injection port, as the flat tip has to snuggly fit needle port. The trimmed tip enters through a port into a constant point on surface of graphite, a material sensitive to scratches. If an ordinary syringe is used the surface is scratched and ruined. The result is leakage from the injector.
When the syringe fills the loop in the LOAD position it is important to wash it properly with the sample solution and make sure that there are no bubble that will change the injected volume and will interfere with the separation process inside the column.
The RUN is started with the injector's knob is diverted from LOAD to INJECT. The stream enters the loop in this position and the mobile phase collects the sample and transfers it into the column. The turning of the knob must be done very quickly, not to stop the flow. The injector should be kept clean otherwise it can be clogged with dirt and colloids.
In a manual injection instruments, turning the knob will start the run, automatically starts the gradient and al other operational programs including processing and integration.
Automatic Autosampler
Most of the HPLC instruments these days are equipped with an auto-injector, or autosampler. All the process of filling of the loop and entering the sample into the stream is done automatically and controlled by the computer.
For more information:
http://www.forumsci.co.il/HPLC/supporting_waters.html#Troubleshooting