Getting Started in HPLC

Section 2C. HPLC Injectors

The pressure between the pump and the HPLC column is controlled by the flow velocity of the mobile phase through the column (the higher the flow, the higher the pressure), by the viscosity of the mobile phase (the higher the viscosity, the higher the pressure), and by the flow resistance of the column (smaller particle sizes, longer columns, and narrower columns mean more flow resistance and higher pressure). This "back pressure" may range from several hundred to several thousand psi. In order to introduce a sample onto the column for analysis, a special valve called the injector must be used to transfer the sample into the pressurized system.

Typical HPLC injectors made by Valco (upper left) and Rheodyne (lower right).

Injectors may look different from the outside, but internally, most are 6-port rotary valves. These valves consist of a fixed body (the "stator") plus an internal seal that rotates (the "rotor"). Three internal passages connect alternate pairs of external ports. The valves can switch between two positions, referred to as the "inject" and "load" positions, respectively. In the load position, the pump is connected to the column, and the sample inlet is connected to one end of a piece of tubing, called the sample loop. The other end of the sample loop is connected to the waste port. Rotation results in reconnecting the various lines that enter the valve, so that a sample volume can be inserted into the mobile phase that flows from the pump to the column inlet.

Let's see how the 6-port sample valve works. In the LOAD position above, the pump is connected directly to the column inlet. The syringe containing the sample solution is inserted into the sample inlet line and sample is forced out of syringe into the SAMPLE LOOP; and from the loop excess sample goes to waste. The sample loop is used to precisely measure the volume of sample solution to be injected. If the volume of the loop is 20 microliters (20 L), then by filling the loop completely the injected volume will be exactly 20 L.

Now the seal is rotated into the INJECT position. This can be done manually by turning the handle of the valve or it can be done automatically using a valve actuator or autosampler In the inject position above, the pump is now seen to connect to the sample loop, so that flow of mobile phase from the pump pushes the sample out of the loop and into the column. The syringe containing the sample solution is now connected directly to waste. While the sample is being emptied out of the loop, the syringe can be refilled with wash solution and used to clean out the injection port of the sample valve. When it is time to inject the next sample, the valve is rotated back to the load position, a new sample is injected into the loop, and the entire process is repeated.

Place your cursor over the diagram to switch the injection valve from the "LOAD" to the "INJECT" position.

For the most precise injection, the sample valve is usually used as described above. This is called FILLED-LOOP injection. If an excess of sample is used to fill the loop (usually 3 or more times the volume of the loop), then the injected volume will be very precise, and this will improve the precision of our analysis.

If we want to vary the sample volume we inject, another technique of sample injection can be used: PARTIAL-LOOP injection. In partial-loop (sometimes also called "partial-fill") injection we inject a sample volume that is smaller than the volume of the loop.

Why would we use partial-loop injection, when we know that it is less precise than filled-loop injection? We may want to deliberately vary the volume of sample injected, in order to determine the effect of sample volume on the separation. In this case it is easier to change the injected volume (using the syringe) than to change the loop on the sample valve. And we may not have all the loops of the right volume available. Most autosamplers allow us to carry out partial-loop injection, so that the sample size injected can be changed automatically - without changing the sample loop. The majority of autosamplers can inject the sample precisely even in this partial-fill mode.



2000, LC Resources Inc. All rights reserved.
Last revised: April 02, 2001.