Getting Started in HPLC

Section 1C. HPLC Instruments

   
LC can be (and has been) carried out using a glass tube hand-packed with powder through which a solvent is allowed to gravity-flow. So why do we need all the complicated high-tech equipment? There are many aspects to the answer:

Speed. A single analysis by "Classical" LC can take anywhere from 2 to 12 hours to carry out. HPLC allows an equivalent analysis to be done in 2 to 12 minutes.

Reproducibility. A classical column must be freshly packed for each analysis, increasing the chance of errors. A single HPLC column can be used for hundreds or thousands of samples.

Quantitation. Classical LC was (and still is) an excellent technique for sample preparation or purification, but requires additional collection and analysis steps to be useful as a tool for quantitative analysis. HPLC uses on-line detectors that provide quantitative information during the course of the separation.

Sensitivity. Classical LC used relatively large columns and correspondingly large volumes of mobile phase solvents. The resulting dilution limited the sensitivity of the technique. HPLC uses miniaturized columns to keep sample dilution to a minimum.


 
  • The starting point is the column. Decreasing the average particle size of the column packing minimizes band broadening. A corresponding decrease in the size of the column minimizes solvent consumption and dilution of the sample.
  • Gravity alone cannot make liquid flow at a reasonable velocity through a column packed with very small particles, so we must use a constant-flow pump.
  • Flow resistance in the column can generate a considerable back-pressure, so the entire system, including the connecting tubing and fittings must be made of pressure-resistant material such as stainless steel or specially formulated plastics.
  • A reservoir must be provided to assure a consistent supply of mobile phase.
  • Because the system is pressurized, some provision must be made for introducing samples from the outside world into the system. This is most commonly done using a rotary injection valve.
  • Collecting and hand-analyzing fractions is tedious, so some type of flow-through detector is used to monitor for the presence of sample compounds in the column effluent.
  • Finally, a data system is required to collect and interpret the results. In the past, this was often a simple strip-chart recorder or a stand-alone integrator. Today, the data system is more likely to be a dedicated PC-type computer.
 
The end result is an HPLC system composed of six basic modules (reservoir, pump, injector, column, detector, and data system) connected by appropriate tubing and fittings. Although systems from different manufacturers can look quite different externally (see below for some examples) they all follow the same basic pattern illustrated in the figure at the right.


   

     
The output of an HPLC instrument is not physical fractions containing the analytes, but rather a "chromatogram": a plot of detector response as a function of time. The relationship between this chromatogram and the on-column separation is illustrated below.
 
   

As you watch this animation, you will see two compounds separate as they are carried through the column at the left. As each band leaves the column, it passes through the detector. The display at the left shows the detector response as a function of time. You can see that the response is low (and constant) unless a sample band is passing through the detector.


 
 

 


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Last revised: March 30, 2001.