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HPLC: A Computer Simulation of High-Performance Liquid Chromatography
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Robert C. Rittenhouse
Walla Walla College, College Place, WA 99324 |
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This issue is out of print. A Windows version of HPLC is available. Please see issue 3D, 2 for more information.
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HPLC combines emulation of a typical modern binary gradient HPLC system with sophisticated modeling of reversed-phase column behavior. The modeling, based primarily on the work of L. R. Snyder (1, 2) provides realistic chromatographic output for separations implemented over a broad range of operating conditions. The program is intended as a pedagogic aid for people wanting to learn HPLC theory and practice. As such, it provides easy, inexpensive access to many of the features of a real instrument without risk of damage, consumption of supplies, and the need for constant supervision. It can be used effectively in instrumentation courses as a training exercise prior to hands-on work with the real instrument. HPLC uses a mouse, along with graphic depictions of instrument modules, to create the "feel" of operating an instrument. The main graphic screen of the program is shown in the figure. It depicts the simulated instrument, which consists of six working modules that are described below in order of their appearance from left to right and top to bottom of the screen.
Solvent Selection. The binary solvent system consists of water and one of three organic solvents: methanol, acetonitrile, or tetrahydrofuran. These represent common solvents that provide a range of "solvent strengths" or polarity. Instrument Control Panel with Gradient Programmer. Separations are designed and may be implemented from this panel. Besides the switches and buttons and a numeric keypad (modeled loosely on the Spectra Physics series of instruments), there is a status display line that constantly reads out information on flow rate, column pressure, solvent composition, injection volume, and time (during an injection). If the gradient mode of operation is selected, then a second panel is presented that allows the operator to define a linear solvent composition gradient. Actual injection of the sample may be initiated from the instrument control panel or from the computer data system module. Chromatographic output for injections initiated from the panel may be viewed by selecting PLOTTER from the command bar at the top of the screen. Column Selection. This module presents a selection of five name brand, reversed-phase HPLC columns differing in particle size and column dimensions. Data relating to the columns may be displayed in a window by clicking the mouse on the DATA item in the command bar at the top of the screen. Detector. The UV detector can operate in either of two modes. A variable-wavelength mode measures absorbance at the selected wavelength in the 200-320 nm range. A diode-array mode measures absorbance continuously over the entire 200-320 nm range throughout a chromatogram. The sensitivity of the detector can be adjusted over a broad range. An increase in sensitivity produces the expected effect of amplifying the noise as well as the signal. Computer Data System. This module provides many of the sophisticated data-handling functions available in the real counterpart. Chromatographic data may be acquired, plotted, rescaled and replotted, integrated, stored in a file, and compared to other chromatograms. If the detector is in the diode-array mode, then it is possible to view the UV spectrum of the sample at any point of the chromatogram. There is a reference library of UV spectra for the standards available for comparison to aid in component identification. One can also examine the chromatogram at any wavelength, or generate a 3-D surface plot of absorbance versus time versus wavelength. Sample Preparation. There is a selection of nine standard compounds that can be mixed together in any proportion to create samples for separation problems. In addition, unknown samples can be created by the program to test one's analytical skills. After performing a separation and obtaining a chromatogram, it is often helpful to return to this module to spike the sample with a known amount of a particular standard. The program assumes that the user is somewhat familiar with the fundamental principles of liquid chromatography, including the concepts of elution, retention time, resolution, peak area, mobile phase. In addition, some familiarity with characteristics of reversed-phase columns is helpful. This material is commonly covered in a junior- or senior-level analytical chemistry or instrumental analysis course. Although there are HELP and DATA windows available throughout the program to assist novice users, it should not be considered a tutorial on HPLC or chromatography in general. Instead, it simulates very accurately the behavior that would occur if a student made the same choices and performed the same manipulations on a real instrument. As such it provides a wealth of firs-hand experience about the characteristics of liquid chromatographic separations. Hardware and Software Requirements HPLC runs under MS-DOS or IBM PC DOS and is supplied on a 5.25-in. disk. It can be used on a computer with either a single 5.25-in. or a single 3.5-in. floppy disk drive, or on a computer with a hard disk. At least 512K of RAM is required as is a graphics adapter (CGA, EGA, or VGA). In some cases one may wish to have a graphics printer to print copies of the chromatograms, but the program will run without a printer. HPLC requires a mouse, since most choices are made by moving a mouse cursor to one or another screen graphic that represents one or another module of the instrument. The mouse must have mouse-driver software that is compatible with the Microsoft mouse. Such software is supplied with most mice; it is not included with the HPLC disk. Literature Cited |
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Last Updated: April 27, 2001 Created: December 3, 1996 |
Created by: J. L. Holmes Comments to: jceonline@chem.wisc.edu |
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| © 1997 Division of Chemical Education, Inc., American Chemical Society. All rights reserved. | |