Getting Started in HPLC

Section 2C. HPLC Injectors

Most modern HPLC systems use autosamplers. The actual injection valve is the same as the manual valves described above, but the sample is introduced by an automated syringe drawing samples from vials in a motorized tray.

Most autosamplers work by penetrating the capped sample vial with a needle attached to a length of flexible tubing. Sample is then withdrawn from the vial into the loop of a sample injector by suction (or positive displacement in some cases). The injector then rotates into the inject position at the right time for the next sample injection into the LC. While this sample is being separated and analyzed, the needle and injector are washed out with rinse fluid, and the next sample vial on the turntable rotates into position for penetration by the needle. All of these operations (injection, rotation, etc.) are controlled by timers in either the LC or the autosampler.


Some autosamplers use suction to pull samples from vials through the injector loop.

Others use positive displacement to transfer samples from vial to injector.

Autosamplers save work, because now you can do other things while the autosampler is injecting samples. Some people say that this takes all the fun out of chromatography, but people eventually get tired of injecting samples and watching peaks come out on the recorder. Autosamplers are more reliable (they don't forget to inject the next sample) and they are usually more precise.

In many LC labs today you can't find an LC system without its own autosampler. However, things can go wrong with autosamplers, especially if we ignore certain recommendations. First, samples have to be very clean for injection by an autosampler. Otherwise the needle of the autosampler is likely to plug, which means that either no sample is injected or (worse) that an insufficient sample volume is injected. It is also much more important to keep track of all samples when using an autosampler, than when operating manually. One mistake could conceivably confuse the results for all the samples on the turntable.

Finally, remember that the samples on the turntable may sit there for quite a while. Is there any chance that the sample might be unstable under these conditions? You might have to take samples out of the refrigerator just before placing them on the turntable, so that no more than a few samples are sitting there at any one time. Also, keep in mind that frozen samples should not be placed directly on the turntable. First thaw the samples, then mix them thoroughly - because freezing often results in "layering" of different sample components in the vial.

Autosamplers differ from one brand to another. Some autosamplers are fairly simple, and they can only be operated in a limited number of ways. For example, all sample injections would have to be the same size (e.g., 100 microliters), and all runs would require that the samples be placed in sequence on the turntable. Other units are more complex and flexible, allowing for different size injections from each vial, reinjection of a calibrator from the same vial, or duplicate injections of each sample - when duplicate runs are required. Some autosamplers require a large sample, regardless of the injection volume - simply to fill the lines between the vial and the injector loop. Other autoinjectors can work with much smaller sample volumes.



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