Description of a UV-Visible Spectrophotometer

Data aprovació:
Equipment identification: UV-Visible Spectrophotometer
Brand: Hewlett Packard
Model: 8453/G1103A
Serial number: DE81801618
URV number: 002961
Provided by: Hewlett-Packard Española, S.A.
Bought in: 1998
Interface with: PC, thermostatted water bath
Location: University Rovira i Virgili, Chemical Engineering Department.
Laboratory 222
Technical Specifications:
Line Voltage
Line Frequency
Power consumption
Ambient operating temperature
Ambient non-operating temperature
Operating altitude
Non-operating altitude
Safety standards: IEC, CSA, UL
34.4 cm wide
56.0 cm deep
18.5 cm high
16.5 kg
90-264 V AC, wide ranging capability
47-63 Hz,
220 VA, maximum
<95%, at 25-40ºC, non-condensing
Up to 2000 m
Up to 4600 m for storing the instrument
Installation Category II, Pollution Degree 2
Available techniques:
Transmittance, Absorbance (Fixed wavelengths, spectrum/peaks, ration/equation,
Kinetic measurements
Thermal Denaturation
Verification and Diagnostics
Description of a UV-Visible Spectrophotometer:
Beck, Shane. Across the Spectrum: Instrumentation for UV/Vis Spectrophotometry. The
Scientist February 2, 1998.
The measuring process of a UV-Visible spectrophotometer begins with a light source that
generates light at a specific wavelength or wavelengths. Commonly, UV/Vis
spectrophotometers utilize two light sources: a deuterium arc lamp for consistent intensity in the
UV range (190 to 380 nm) and a tungsten-halogen lamp for consistent intensity in the visible
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spectrum (380 to about 800 nm). Some spectrophotometers use xenon flash lamps, which offer
decent intensity over the UV and visible regions.
The source light is then directed to a dispersion device that causes different wavelengths of light
to be dispersed at different angles. Two common dispersion devices used in UV/Vis
spectrophotometers are prisms and holographic gratings. The angle of dispersion with a prism,
however, can be nonlinear and sensitive to changes in temperature. Holographic gratings are
glass blanks with narrow ruled grooves. The grating itself is usually coated with aluminum to
create a reflecting source. Holographic gratings eliminate nonlinear dispersion and are not
temperature sensitive. They do require filters, though, since light is reflected in different orders
with overlapping wavelengths.
Once the light has been passed through the dispersion device and the sample of interest it
reaches a detector. Detectors in UV/Vis spectrophotometers come in a variety of shapes and
sizes. Photomultiplier tubes are common; they provide good sensitivity throughout the
UV/Visible spectral range and are highly sensitive at low light levels. Photodiodes have seen
increasing use as detectors in spectrophotometers, bringing to the table a wider dynamic range.
A photodiode is generally made up of a semiconductor and a capacitor to charge the
semiconductor. As light hits the semiconductor, electrons flow through it, thereby lowering the
charge on the capacitor. The intensity of light of the sample is proportional to the amount of
charge needed to recharge the capacitor at predetermined intervals. As opposed to having
single photodiodes, some spectrophotometers are composed of a photodiode array. Here,
several photodiode detectors are arranged on a silicon crystal. The advantage of an array is the
ability to do side-by-side readings, thus increasing speed.
The entrance slit, dispersion device, and exit slit are referred to as the monochromator. Light
passing through the monochromator exits as a band. The width of this band of light at half the
maximum intensity is the spectral bandwidth. Bandwidth comes in to play with regard to
accuracy, since the accuracy of any absorbance measurement is dependent on the ratio of the
spectral bandwidth to the natural bandwidth of the substance being measured. The natural
bandwidth is the width of the absorption band of the sample at half the absorption maximum. As
a rule, a ratio between spectral bandwidth and natural bandwidth of 0.1 or less will generate
absorbance measurements 99.5 percent accurate or better. Above this, accuracy deteriorates.
In a conventional spectrophotometer, polychromatic light from the monochromator is transmitted
through the sample, and the sample absorbance is determined by comparing the intensity of the
light hitting the detector with just a sample blank with the intensity of light hitting the detector
with the sample in place. With a diode array spectrophotometer, polychromatic light passes
through the sample and is focused on the entrance slit of the polychromator (the entrance slit
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and dispersion device). The light is then dispersed onto the diode array with each diode
measuring a portion of the spectrum.
Single-beam spectrophotometers (which include conventional and diode array) are highly
sensitive devices. Spectra can be acquired quickly in diode array spectrophotometers due to the
single-beam design. In conventional spectrophotometers with a single beam, the blank and the
sample are not measured simultaneously, and the interval between measurements-especially if
measuring a broad spectrum-can subject results to lamp drift. Dual-beam spectrophotometers
utilize a "chopper" that alternates the light path between the reference optical path and sample
optical path to the detector at a speed that minimizes medium- or long-term effects of lamp drift.
A variant of this is the split-beam design, which uses a beam splitter rather than a chopper. This
allows light to be sent to the reference cell and sample cell simultaneously.
Description of Our UV-Vis Spectrophotometer:
The HP 8453 that we have is a diode array spectrophotometer with a graphical user interface and
built-in GLP capabilities. This instrument allows better than 2 nm resolution and less than 0.03
percent stray light. It has various capabilities for standard measurement of absorbance and
transmítanse as well as various modes of measurement including the kinetic mode, which allows us
to measure the change in absorbance or transmítanse over time for specified wavelengths. The
sample can be thermostatted.
Diode array spectrophotometers are capable of acquiring complete UV/Visible absorbance
spectra in as little as 100 msec. The key is that the grating of these instruments is fixed, and
rather than moving the grating to acquire spectra, hundreds of detectors are placed at the exit of
the monochromator. The detectors are all integrated on a single silicon chip called a photodiode
array. The diodes act as capacitors that discharge in proportion to the incident light flux. The
capacitance of each diode is converted to a binary word that is input to a computer. The HP
8452A simplifies the operation of the spectrophotometer even further by using a deuterium
discharge lamp for the full UV and visible range, rather than a deuterium lamp for the UV and a
tungsten incandescent lamp for the visible, as in done in most other instruments. The system is
a single beam instrument, which means that you first run a scan on a cuvette containing just the
solvent to determine the intensity of the lamp at each wavelength, Io(). Then you put in your
sample in the same cuvette and scan the spectrum again. The absorbance is then calculated
from the ratio of the two spectra:
A() = log ( Io() / I())
The plot of A() versus  is the spectrum of the solution.
Operation and Measurement
A. Start-up
1. Turn on the monitor, computer, and the spectrophotometer. There should not be a
cuvette in the holder when you turn on the spectrophotometer since the instrument
conducts its internal calibration at this time and calibration with a cuvette is erroneous.
2. Allow 15-20 minutes for the instrument to warm up before beginning measurement.
3. Clicking on the start menu, choose Programs, HP UV-Vis ChemStation, Instrument
Online. The Windows based program will load. The system will then start the spectrum
acquisition program and display the Main window.
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B. Measuring Absorbance or Transmittance Spectra
1. In “Mode” on the top right of screen, pull down menu and choose “Standard”.
2. on left side of screen, choose “Task” from pull down menu (fixed wavelengths,
spectrum/peaks, ratio/equation, quantification).
3. Click the “Setup” button. Specify the wavelength you wish to measure and other
parameters such as data presentation and type (transmittance, absorbance).
4. Lift the cell holder lever to open the holder. Place a cuvette with your sample´s solvent
in the sample compartment. Press the cell holder lever down to secure the cuvette.
NOTE on CUVETTES: When using the spectrophotometer, you must first decide which
type of cuvette is most suitable to your needs. If you are interested in wavelengths
<300 nm, you must use a quartz crystal cuvette, since the polystyrene and plastic
varieties have high noise in this range. At wavelengths >800 nm, plastic is more
appropriate. There are also volume considerations. If you have very little sample, use
the small quartz cuvette. When doing enzymatic assays, use the plastic 1 mL cuvettes
so as not to waste reagent with the 3 mL cuvettes.
5. Click on the "Blank" menu button on the bottom left of the screen. The screen will
change to the “Sample Spectra” window and the background scan will be displayed.
With a polystyrene cuvette, there should be noise below 300 nm and a flat line above
300nm. If you see excessive noise, repeat the blank. If this does not solve the
problem, try restarting the program and/or computer.
6. Gently clean with Kimwipe the sides of the cuvette that the light will pass through, and
place the cuvette containing your sample, in the cell holder.
7. Click on the "Sample" menu button below the blank button on the bottom left of the
screen. The spectrum will be displayed in a few seconds. The absorbance or
transmittance at each wavelength will be displayed in the window “Sample table” below
the “Sample Spectra” window. You can see a table of the absorbance or transmittance
and standard deviation at each wavelength by double clicking on the spectrum itself.
8. You can measure all your samples and the spectra will overlay in the graphic window. If
you wish to see only the current spectrum, click on the button “Last Spectrum”
underneath the spectrum window. To return to overlaying spectra, click on “Overlay
Spectra”, in the same location.
9. To save the spectrum, highlight the line containing the absorbance or transmittance
information of the spectrum in the sample window, and go to File menu, “Save
Samples As”. Choose file destination and an 8 character name for the file. Type in the
file name you wish, however this file name should follow DOS naming conventions: 8
characters or less, no spaces, no punctuation symbols (no - / \ * . & _ , etc.), and start
with a letter.
10. To print, highlight the spectrum window, and go to the File menu, then “Print”.
C. To conduct an Enzymatic Assay using Kinetic Mode
A spectrophotometric enzymatic assay is conducted by using a colorimetric cosubstrate of the
enzyme. This means that the cosubstrate changes color or color intensity signal once acted
upon by the enzyme, therefore changing the absorbance spectrum of the sample. To illustrate
this point, let us take as an example the enzyme Horseradish Peroxidase (HRP).
HRPactive + H2O2 → HRPinactive
HRPinactive + ABTSred → HRPactive + ABTSox
HRP is a redox enzyme. As it catalyzes the reduction of hydrogen peroxide, it is oxidized. In
order to further catalyze the reduction of hydrodrogen peroxide, it must be regenerated. The
cosubstrate does this by reducing it back to its active form. ABTS is a good spectrophotometric
enzymatic activity assay substrate because it turns color when it is oxidized by the inactive
HRP. We can measure the activity of an HRP solution by determining how quickly it converts
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reduced ABTS to oxidized ABTS, determined by measuring its change in absorbance over time.
However, we must be careful not to relate the measured activity from the enzymatic activity
assay with ABTS to the absolute activity of the enzyme, since the measured activity is also a
measure of the kinetics between the enzyme and ABTS in the limiting reaction step. These
assays are most useful for relative measurements of activity when using the same cosubstrate
across all assays.
There are several HRP cosubstrates we can use for enzymatic assays. There are also many
enzymes whose activity can be measured with a spectrophotometrically active cosubstrate.
Some examples include glucose oxidase and alkaline phosphatase, other commonly used
enzymes in our lab. One good source of enzymatic assay protocols is the Sigma-Aldrich
1. To conduct an enzymatic assay, change the mode of the instrument to “Kinetic” in pull
down menu window on the top right of the screen.
2. Blank the spectrophotometer as described above.
3. Measure a blank tailored to your assay in kinetic mode (usually contains all components
of assay except the enzyme).
4. Measure your samples following the protocol. The data of interest is the absorbance
change over the measured time. You can use this value to calculated the units of
enzyme per mL, per mg, etc.
Training Exercise
Conduct an HRP assay with a range of concentrations of peroxide to find the saturated
substrate condition.
The HP 8453 conducts a wavelength calibration during the measurement and data processing
of each sample. It does this by using the two emission lines from the deuterium lamp as
calibration parameters.
Performance Verification
For the complete version, please refer to the manual “Operational Qualification/Performance
Verification for HP 8453 UV-Visible Spectroscopy Systems”, pages 18-67.
There are 7 tests that can be conducted on the spectrophotometer to verify its performance
within the error laid out by published specifications. These tests are the photometric accuracy
test, the wavelength accuracy test, the stray light test, the resolution test, the baseline flatness
test, the photometric noise test, and the photometric stability test. The first four tests are done
through exterior standards, and the last three though the software. The complete testing
procedure can be completed within three hours. The procedure can be conducted by a qualified
professional or by a worker with sufficient expertise. For details, please refer to the manual cited
It is recommended that the OQ/PV procedure be conducted once a year, immediately after
conducting user maintenance. It is important that the instrument be allowed 2 hours to warm up
previous to the procedure. If the OQ/PV tests do not yield the published specifications, HP
should be contacted immediately.
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Maintenance and servicing:
For the complete version, please refer to the HP manual “Reference Manual”, pages 94-134.
This section of the manual describes maintenance procedures such as cleaning the
instrument, exchanging the deuterium and tungsten lamps, and cleaning lenses.
It is recommended especially that the spectrograph lens and the sources lens be
cleaned once a year. The procedure can be found on pages 101-104 of this manual.
The spectrophotometer case and sample compartment should be kept clean. Cleaning
should be done with a soft cloth slightly dampened with water or a solution of water and
a mild detergent. Do not let liquid drip into the instrument.
Exchange the deuterium or tungsten lamp when the intensity test falls below the
specified level or when one of the lamps no longer ignites.
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Data aprovació:
Kristin Horton
Data: 04/05/02
Comissió de Qualitat
Ioanis Katakis
Director del Centre
Tipus Document
HP 8453 UV-Visible Spectrophotometer Reference Handbook
Understanding Your Advanced Software
Understanding your Biochemical Analysis Software
Installing Your UV-Visible Spectroscopy System
Operational Qualification/Performance Verification for HP 8453 UVVisible Spectroscopy Systems
Understanding Your UV-Visible Spectroscopy System
Descripció revisió
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