Sample Injection System:

It is a limiting factor in the precision of HPLC measurement because of reproducibility with which samples may be introduced onto the column packing. Insertion of the sample into the column must be through a narrow plug so that peak broadening is minimized and the system should have no dead volume by itself. Generally, samples are dissolved in a mobile phase solvent to avoid solvent peak and 10 to 50 µL is introduced through micro sampling valves. These devices form an integral part of liquid chromatography equipment having interchangeable loops with a choice of sample size from 5 to 500 µL. The most hugely used method of introduction is based on sampling loop as shown in Figure. It is filled by thoroughly flushing it using a sample solution by means of a microsyringe at pressures up to 7000 psi. A rotation of the valve rotor places the sample filled loop into the high pressure mobile phase stream whereby the sample is sent to the column. The system can be located within a temperature controlled oven if handling at elevated temperatures is required. Many HPLC instruments incorporate an auto sampler with an automatic injector that can inject variable volumes as per needs. Within stopped flow injection method, a pump is turned off till atmospheric pressure is attained, syringe is inserted and the sample injected. The flow of sample can be brought to zero and rapidly resumed by diverting the mobile phase using a three way valve placed before the injector. This method is especially very useful for very high pressures. For best output, a two to fivefold excess of sample should be passed by the loop to ensure in which previous sample has been purged thoroughly.

Figure:  Schematic of injector valve with external sample loop in a microvolume sampler