OPERATING INSTRUCTIONS FOR THE SHIMADZU UV-2401PC UV-VISIBLE

SPECTROPHOTOMETER

The Shimadzu UV-2401PC UV-Visible Spectrophotometer is a double-beam instrument that

allows acquisition of UV-Visible spectra and accurate quantitative measurement of an
absorbing analyte of interest by application of a Beer’s Law Analysis. A copy of the
instrument specifications and a diagram of components, obtained from Instruction Manual:
UV-2401PC/2501PC User’s System Guide, are included with this document, as are
instructions for general use of the instrument for acquisition of spectral data. For advanced
applications such as those for which the photometric or kinetics module would prove useful,
the Instruction Manual: UVProbe Tutorial should be consulted. The basic principles of
molecular absorption spectroscopy should be well understood before operating this
instrument.

BASIC OPERATING PROCEDURES

1. Prepare your samples according to methods appropriate for the analyte of interest, and
prepare a blank for use. If you are carrying out a Beer’s Law Analysis, prepare a set of
standards within the concentration ranges appropriate for the method you have chosen.
Note that the matrix for the standards should ideally be identical to the matrix used in
your final samples.

2. Log into the User Log Book found beside the instrument.

3. Make certain that both the sample and reference holders are empty, and turn ON the
spectrophotometer. (The Power Switch is located on the left hand side of the instrument.)
In addition, turn on the CPS-Controller using the switch found on the rear panel. Allow
the instrument to warm and initialize for a period of about 5 minutes.

4. Turn on the computer, and log-in as “User.” Open the UVProbe program, and click on
the Connect button at the bottom of the screen to establish communication with the
instrument. This step will take several minutes.

5. Once the instrument has initialized, select the Spectrum Module by clicking on the prism
icon found on the menu bar. In this module, one can scan a range of wavelengths while
recording absorbance, transmittance, reflectance, or energy data. Alternatively, one can
measure at a fixed wavelength for a quantitative Beer’s Law analysis.

6. Perform a baseline correction by clicking on the Baseline button at the bottom of the
screen. When prompted, enter 700 nm as the starting wavelength and 300 nm as the
ending wavelength, and select OK. (This instrument scans from high to low
wavelengths.) During this scan, the instrument sets the background to zero over the
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 wavelength range of interest. Note that if at any time during the analysis you decide to
change the slit width, scan speed, sampling interval, etc., it is necessary to repeat the
baseline correction. You can assure that the baseline scan is complete by clicking the
Instrument History tab and verifying that the background scan is listed. (If the
Instrument History tab is not apparent, select ViewÆOutput Window.)

7. Choose the appropriate cuvettes for analysis. Some important points follow:

a. You will need to use matched cuvettes for the wavelength range of interest.
b. Use quartz when recording from 190-300 nm, plastic from 290-900 nm, and glass
from 300-900 nm.
c. Do not use plastic cuvettes with organic solvents and/or materials that will dissolve
the plastic.

8. Proceed with data collection as follows:

To obtain a spectrum:

a. Select the Edit menu at the top of the screen, and choose Method. Alternatively, you
can select the green M shortcut button from the Spectrum toolbar
b. On the measurement tab, set the desired wavelength range, scan speed, sampling
interval, and scan mode. A typical series of parameters is:

Wavelength Range (nm): Start: 700 to End 300

Scan Speed: Medium
Sampling Interval (nm): 1.0
Scan Mode: Single

c. From the instrument parameters tab, select the measuring mode (Transmittance,
Absorbance, Energy, or Reflectance) and the Slit Width in nm. Typically, one might
obtain a spectrum of absorbance versus wavelength with a slit width of 5 nm.
d. Select OK when you are satisfied with all parameters.
e. Place a cuvette containing your solution for measurement in the sample path (the
sample cell toward the front of the instrument) and a matched cuvette containing your
blank solution in the reference cell (the rear cell in the sample compartment).
f. Record your spectrum by selecting the Start Button at the bottom of the screen. Your
spectrum will be displayed on the Active tab of the display window. When prompted
with a New Data Set pop-up box, select OK to store the spectrum temporarily under
the default name. You can edit the appearance of your graph by selecting options
from the Graph menu.
g. Save the spectrum by selecting FileÆSave As. Select the appropriate folder, and
enter a file name containing 8 or fewer characters.

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 To perform a quantitative Beer’s Law analysis at a fixed wavelength.

a. Set the wavelength of analysis by clicking on the Go to WL button.

b. Place a cuvette containing your blank solution into the reference cell (the rear cell in
the sample compartment).
c. Insert a matched cuvette containing your blank, standards, and samples into the
sample cell, and record the absorbance indicated on the Photometer status window.
(If it is not apparent, select ViewÆPhotometer Status.)
d. NOTE: The Photometric Module allows one to generate a standard curve
automatically and determine the concentration of unknowns from the standard curve.
(Details can be found in the UV-2401 PC Users’ Manual.) The module is particularly
useful for routine analyses when the user is already quite familiar with the method.
For a new analysis, it is wise to begin by collecting raw data as described in (a) and
(b).

9. Incorporating Spectra Into a Laboratory Report1

The Shimadzu UV-2401PC UV-Visible Spectrophotometer and its operating software,

UVProbe produce high quality plots of UV-Visible spectra, and the Report Generator
module of the UVProbe software allows great flexibility in the design of reports
containing spectral, photometric, or kinetic data. In addition to creating such reports and
printing them from the UVProbe software, you may wish to incorporate spectral data into
laboratory reports and documentation. One useful method of data transport is described
here:

Text Format. This method allows you to save the spectral data as a text file containing
two columns of numerical data. The first column lists the wavelengths, and the second,
which is separated from the first by commas, includes the corresponding absorbances.
The file can be opened in Excel, and the spectrum can be plotted as an X-Y scatter plot in
Excel. The benefit of this approach is that it allows you to edit the appearance of the plot,
perform mathematical operations such as spectral normalization, include multiple spectra,
etc. To save data in this format:

a. In the Spectrum Module of UVProbe, assure that the spectrum of interest is on the
Active display. If necessary, open the saved spectrum.
b. From the File Menu, select Save As. In the Save Spectrum File pop-up menu, select
Data Print Table (*.txt) as the file type, and enter an appropriate name with 8 or fewer
characters. Save the file to your public folder on the network or temporarily to the
appropriate folder on the computer.
c. On another computer, open the file in Excel. You will be asked to respond to a series
of questions. Indicate that the data: is delimited, starts in Row 1, originates from
Mac or Windows as is the case, has commas as the delimiter, and is in a General
format. Upon completion of the series of questions, you should see your wavelengths
and absorbances in Columns A and B, respectively, of an Excel Workbook.

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 d. At this point, you can use what you have learned previously to create an X-Y scatter
plot with a smooth line through the points rather than symbols for the individual data
points. You will wish to change the scales of axes to assure that the spectrum fills the
entire plot, remove a legend in the event of a single spectrum, add appropriate axis
labels, adjust the font size, etc., to prepare a report-ready spectrum.

SHUTDOWN PROCEDURES

10. Exit the software, close Windows, and shutdown the computer and the spectrometer.

11. Remove cuvettes from the sample and reference path, clean them carefully, and, if
obtained from the instructor, return them for storage.

12. Store samples and standards or dispose of them properly (in another laboratory).
Standards and samples should not be left in the instrument laboratory.

13. If you are the last person working in the laboratory, turn off the lights, and request that an
instructor lock the lab behind you.

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FROM USERS’ MANUAL

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Department of Chemistry & Biochemistry
Elizabethtown College Last Updated by KK 3/09
Department of Chemistry & Biochemistry
Elizabethtown College Last Updated by KK 3/09
Department of Chemistry & Biochemistry
Elizabethtown College Last Updated by KK 3/09
Department of Chemistry & Biochemistry
1
Originally written by Mary Harner, E-Town ’06.

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Department of Chemistry & Biochemistry