Chemistry 472
Fall 2000
UV-Vis Spectrophotometry
Introduction
This experiment is an introduction to spectrophotometry and consists of four parts. In the first part, the
components of two, disassembled spectrophotometers are examined. One is a single beam instrument, a
Bausch and Lomb Spectronic 20; the other is a Beckman DB: a double-beam-in-time spectrophotometer.
Next, the spectral response curve of a Spec 20 is determined and the Spec 20 is used to collect the
absorption spectrum of chromium(III) nitrate. For comparison, this spectrum is also collected with a
double-beam-in-time UV-Vis spectrophotometer. In the third part, a calibration curve is made and the
concentration of Cr(III) in an unknown is determined using Beer’s Law. Finally, the effect of diffraction
grating blaze density and slit width on the resolution of a spectrum is explored using Oriel optical
components and a spectral calibration lamp. Background information for this experiment is contained in
Chapters 7, 13, and 14 in Principles of Instrumental Analysis 5th Edition by Skoog, Holler, and Neiman.
NOTE: A separate station has been set up for each part of the experiment. Parts 1, 2, and 4 may be done
in any order. Part 2 must completed before starting Part 3.
Part 1. Instrumentation
In molecular absorption spectroscopy, a solution is irradiated with light. If the light is of the appropriate
wavelength, it can be absorbed by analyte molecules causing them to undergo a transition to a higher
energy state. The amount of radiation absorbed by the sample is determined by measuring the intensity
of the beam before (Po) and after (P) it passes through the sample. The transmittance T of the solution is
defined as T = P/Po. In spectrophotometry, transmittance is actually a measurement of the ratio of a
sample beam intensity to a reference beam intensity. The absorbance A is related to the transmittance by
A = -log T.
At a minimum, the instrumentation for absorption spectrometry consists of a radiation source (a tungsten
filament lamp for the visible or a deuterium lamp for the ultraviolet), a monochromator (a prism or
grating system to isolate one wavelength region), a sample cell, a photodetector, and a readout device, as
shown in Figure 13-12(a) in Skoog, Holler & Neiman. The monochromator consists of an entrance slit,
some optics, a prism or grating, and an exit slit. With this single-beam configuration, the percent
transmittance of a blank (reference sample) is adjusted to 100%T. The reference cell in then manually
replaced with the sample cell and the %T is indicated on the readout device. Anytime the wavelength is
changed, this procedure must be repeated.
Most modern spectrophotometers are double-beam instruments as shown in Figure 13-12(b & c). These
instruments are configured to allow the automatic and simultaneous (or near simultaneous) measurement
of the sample and reference beam intensities. This can be accomplished using a beam splitter to divide
the beam into the reference beam and sample beam. In a double-beam-in-time spectrophotometer (Figure
13-12(c)), the beams pass through a modulator which allows the detector to see either the reference beam
or the sample beam. Since the source beam is directed through a reference cell part of the time and
through the sample cell the rest of the time, the term "double-beam-in-time" is used.
Examination of the Spec 20
A diagram of the Spec 20 instrument used in this experiment is shown in Figure 13-17(b) in Skoog,
Holler, and Neiman. For this part of the experiment, the Spec 20 has been disassembled. Make sure that
it is unplugged, lay it on its back, and open the trap door on the bottom. Opening the door exposes the
source, filter mount, phototube, and bottom of the sample chamber. Observe the lamp but do not remove
it. It is a common 6-volt tungsten filament lamp very similar in design to an ordinary light bulb.
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Examine a phototube, noting the anode and cathode. The standard Spec 20 phototube is a type S-4
cesium-antimony tube, which is most sensitive to light in the blue region. When working with the Spec
20 in the red region, above 625 nanometers, a red sensitive phototube is used instead.
Examine the curved arm of metal at the bottom of the sample chamber. This is the occluder, which,
mounted on a pivot, opens and closes the shutter gate which allows light into the sample. Move the
occluder by hand and note its action. It is automatically pushed aside by the cuvette when a reading is
taken. When your investigation of this area is complete, close and secure the trap door, and set the
instrument upright again.
The instrument is divided into two sections. The rear contains the electrical components. These include
the power supply, the amplifier which strengthens signals coming from the phototube, and the readout
device which in this case is a voltmeter calibrated in both absorbance and percent transmittance units.
Continuous light over the entire visible spectrum emanates from the tungsten lamp. This light is
collected by the field lens and collimated (made parallel) by objective lens. Identify these components
and trace out the light path. The white light next falls upon the diffraction grating, which disperses it
horizontally into the familiar sequence of spectral colors. To perform this task, the grating is ruled at 600
lines per millimeter. The dispersed light next passes through the light control, the occluder and the exit
slit. Only when a cuvette is inserted into the sample chamber is the occluder moved out of the way. The
width of the exit slit and the spread or dispersion of the light from the diffraction grating together
determine the spectral bandwidth, which in a Spec 20 is 20 nm. Thus, for example, if the instrument is
set at 600 nm, light having wavelengths in the range 590 to 610 nm will be passed to the sample and
phototube.
Only that portion of the light dispersed by the grating which falls on the exit slit is passed to the sample.
Selection of the desired portion of the spectrum to be passed is accomplished by adjusting the angle of
incidence between the source rays and the diffraction grating. The control on top of the instrument
performs this task by physically turning the grating, and is known as the wavelength control knob.
Attached to this control is a dial calibrated in nanometers which indicates the setting.
In quantitative spectrophotometry one is interested not in the absolute intensity of light passing through a
sample, but rather in the relative intensity of such light with respect to the intensity of light passing
through a reference or blank solution. It is thus necessary to set the 0 and 100% transmittance limits
between which the transmittance of samples will be measured. The 0% T is set with no light reaching
the phototube, that is, with no cuvette in the sample chamber and all light blocked by the occluder. This
setting is performed with the amplifier control knob on the left front of the instrument. This control
adjusts the gain, or sensitivity of the amplifier, and the offset. This determines how much needle
deflection is caused by a specific intensity of light striking the phototube. The 100% T is set with the
right hand or light control knob, usually with a cuvette of water or another blank solution in the sample
chamber. Turning the light control knob causes a V-shaped slit behind the knob to move into or out of
the light beam. It is thus a purely mechanical control which simply physically blocks out more or less of
the diffracted light.
With due respect for the exposed electrical wiring, plug the instrument in and turn it on. Use a sheet
of paper or an index card to observe the light spectrum and the effect of the wavelength and light control
knobs upon the location and intensity of the dispersed light. Following this examination, turn the
instrument off and unplug it.
Examination of the Beckman DB
The Hitachi U-4001, a "double-beam-in-time" spectrophotometer, will be used in part 2 of this
experiment. You will be able to examine the structural details of an older design instrument, the
Beckman DB. With the aid of the diagram next to the instrument, trace the light path. Notice the way
the instrument switches between the tungsten and deuterium sources. Also, note that instead of a
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diffraction grating, a mirrored prism is used to disperse the light in this instrument. Finally, observe the
vibrating mirrors beneath the sample holder compartment. As these mirrors move back and forth
together, the beam alternately passes through the sample and the reference compartments. This is
another way of constructing a double-beam-in-time instrument.
Part 2. Spectral Response Curve and Chromium Nitrate Spectrum
Operation of the Spec 20
In a few brief procedures, the spectral characteristics of the Spec 20 will be examined, and the visible
spectrum of a simple substance will be recorded. The handling of cuvettes is very important. Any
variation in the cuvettes, such as stains, scratches, or changes in the curvature of the glass, will cause
varying results. Thus it is essential to follow several rules:
•
Do not handle the lower portion of the cuvette through which the light passes.
•
Always rinse a cuvette with several portions of the solution to be measured before taking the
measurement.
•
Wipe off any liquid drops or smudges with a clean tissue before placing the cuvette in the Spec
20.
•
When inserting a cuvette, always do so with the index mark facing the front of the instrument,
and after the cuvette is seated, line up the index marks exactly.
Determination of the Response Curve
NOTE: The Spec 20’s take several hours to warm up. They should be on when you come to class –
please do NOT turn them off.
1. Using the Zero Control knob (left front) adjust the 0% T with the empty sample chamber. Be
sure the chamber is firmly in place and that its cover is shut during this and all future readings to
prevent room light from leaking into the phototube.
2. Turn the Light Control knob (right front) fully counter-clockwise, and insert a cuvette filled
about one-half full with distilled water. Adjust the wavelength control to 410 nm and rotate the
Light Control knob clockwise until the meter reads about 90% T.
3. Rotate the cuvette in the sample chamber and note how readings can change 1% T or so if the
cuvette is not consistently positioned. Change the wavelength setting back and forth and note
how the response changes with wavelength.
4. Determine the wavelength of light to which the instrument is most sensitive. Depending on the
model of Spec 20 you are using, this wavelength may be around 410 nm or 595 nm. Adjust the
light control to give 100% T at this wavelength. Then without readjusting either control knob
further, record %T readings for the following wavelengths: 350, 375, 400, 425, 450, 475, 500,
512, 550, 575, 600, 612, and 625 nm. The resulting data give the relative response of the
instrument vs. wavelength. This is known as a spectral response curve.
5. Place a dry cuvette containing a reflecting chalk into the chamber and rotate the cuvette until the
beam reflects upward to view. Observe and record the color of the beam in 50 nm intervals from
350 to 650 nm. Adjust the light control (100%T) if necessary to see the beam, but do not allow
the meter to read off scale.
6. Adjust the wavelength to 900 nm and turn the light control fully clockwise. Note the apparent
blue-green color of "900" nm light which is actually the second order spectrum of 450 nm light.
Turn the light control back down again. Explain the need to use a red filter when working in this
region of the spectrum.
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Collection of an Absorption Spectrum
In UV-Vis spectroscopy, the characteristic manner in which an analyte absorbs different wavelengths of
radiation depends on the energies and structure of the electronic states for that species. A plot of the
absorbance vs. wavelength is called an absorption spectrum. The absorption spectrum is unique to a
particular molecule and for this reason, can be used for qualitative identification of a compound.
In this experiment, the absorption spectrum of a substance in the visible range, 375 to 625 nm, will be
recorded using the Spec 20 and the Hitachi U-4001 spectrometer. The particular species examined is the
chromium (Cr+3) cation in an aqueous solution.
Spec 20 Instructions
1. Set the Spec 20 to a wavelength of 375 nm and adjust the 0% T as before. Adjust the 100% T
with distilled water in the cuvette. Be sure to use the same cuvette filled with distilled water for
all future 100% T readings.
2. Fill a second cuvette about one half full with 0.04 M chromium nitrate solution and take a % T
reading of this solution at 375 nm.
3. Change the wavelength dial to 400 nm. Set the 0 and 100%T as done previously (no cuvette,
cuvette with distilled water). Measure the %T of the 0.04 M chromium nitrate solution..
4. Continue this procedure at wavelengths of 405, 415, 425, 440, 455, 470, 490, 500, 520, 530, 540,
550, 575, 580, 600, and 625 nm.
5. Empty solutions into the appropriate waste container. Rinse the cuvettes well with distilled
water.
Hitachi U-4001 Instructions
1. Set the scanning range from 400 – 625 nm (Scan Parameters menu), the slit width to 2 nm
(Instrument Parameters menu), and scan a background (Correct User 1) with distilled water in
both the sample and reference cuvettes. Disposable cuvettes can be used since the analysis is
conducted at wavelengths greater than 350 nm.
2. Fill the sample cuvette with the 0.04 M chromium nitrate solution and collect a spectrum.
3. Print your spectrum and then convert the file to ASCII format. This ASCII file can be opened
with EXCEL.
Part 3. Determination of Cr(III) Concentration
Quantitative Spectrophotometry
In quantitative applications, the absorbance A at a given wavelength is related to the analyte
concentration c by Beer's law: A = abc, where a is absorptivity of the analyte and b is the sample
pathlength. This is a linear equation (y = mx + b) with a slope equal to ab and a y-intercept of 0. A
calibration curve is constructed by plotting A versus c for a series of solutions of known concentration.
The equation of the best fit line is then used to determine the concentration of the analyte in unknown
solutions from the measured absorbance. It should be noted that Beer’s law will be valid and the
calibration curve linear only if the solutions are dilute. At high concentrations the analyte molecules
interact with each other causing deviations from Beer’s law. Additionally, the calibration curve is only
valid within the concentration range used to construct it. It may be necessary to dilute unknowns so that
they fall within this range.
The wavelength chosen for quantitative measurements must be appreciably absorbed by the substance
being determined, otherwise, the measurement will not be sensitive to low concentrations. On the other
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hand if the substance has too high an absorbance the transmitted light is too weak for accurate
measurements. It can be shown mathematically that for spectrophotometers with simple meter readouts
such as a Spec 20, the best compromise between too little and too much absorption comes at about 36.8%
T (A = 0.43). This is the region where the error in reading the transmittance is least in comparison with
the value of the transmittance.
Consider a plot of the analyte absorptivity a versus wavelength (absorptivity is determined from the
solution absorbance by a rearrangement of Beer’s Law: A/cb = a). An appropriate wavelength for
analysis would lie in a region where the slope of the curve approaches zero. Since spectrophotometers
isolate a range of wavelengths, and not a single wavelength, it is important that all wavelengths within
this instrumental bandwidth exhibit the same molar absorptivity for a given sample concentration. Under
these conditions, the sample absorbance is proportional to concentration as if the incident radiation were
truly monochromatic. See Figure 13-5 in Skoog, Holler & Neiman for an illustration of some of these
points.
The basic method by which absorption spectrophotometry is applied quantitatively is to prepare a
calibration curve or Beer's law plot. This plot should illustrate the linear relationship between
absorbance A and concentration c as given by Beer's law. Preparing a calibration curve allows the
analyst to demonstrate that the Beer's law relationship is obeyed for the conditions employed
(concentration range and wavelength used). Since the calibration curve makes use of several standards, it
will be more accurate than an analysis based on a single standard. In this part of the experiment, a
calibration curve will be prepared for a series of Cr3+ standards and used to determine the concentration
of Cr3+ in a sample of unknown concentration.
1. Using the 0.200 M chromium nitrate stock solution, 100-mL volumetric flasks, and pipets
provided, prepare 6 standards with concentrations from 0.0100 M to 0.0600 M. NOTE:
Calculate the volume of the stock solution that will be pipetted for each standard BEFORE you
come to lab.
2. Using the spectrum of the chromium nitrate solution collected on the Spec 20, determine the
wavelength of maximum absorbance for Cr3+ (λmax). This is the wavelength that will be used to
prepare the calibration curve.
3. As done previously, set the 0 %T and 100 %T at λmax.
4. Obtain and record triplicate absorbance readings for each of the standards at this wavelength.
5. Make three measurements of the absorbance for the unknown chromium nitrate solution.
Part 4. Slit Width and Blaze Density
In this part of the experiment, the effect of slit width and grating on spectral resolution will be examined.
The experimental setup consists of (1) spectral calibration lamp as a light source, (2) a monochromator
with exchangable diffraction gratings and variable inlet and outlet slits, (3) a PMT detector, and (4) a
stripchart recorder as the readout device.
The ability of a monochromator to separate different wavelengths of light depends upon its dispersion.
The dispersion is detemined by the focal length F of the monochromator and the blaze (or line) density of
the diffraction grating. Reciprocal linear dispersion D-1 is often used to describe the ability of the
monochromator to disperse light and it can be calculated using the following equation:
D −1 =
d
nF
where d is the distance between the lines on the diffraction grating and n is the diffraction order. In
essence D-1 describes how “spreadout” the wavelengths of light are at the exit slit of the monochromator
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(dλ/dy). The dimensions are reported as nm/mm or Å/mm. The lower the value of D-1, the better the
monochromator disperses the light.
The dimensions of the entrance and exit slits of a monochromator play an important part in the resolving
power of the monochromator. Peaks in a spectrum are said to be fully resolved if they are separated from
adjacent peaks at the baseline. The effective bandwidth ∆λeff is the span of wavelengths that exit the
monochromator for a given wavelength setting and it is related to the reciprocal linear dispersion by the
following:
∆λeff = wD −1
where w is the slit width. Theoretically, complete resolution of two lines in a spectrum is achieved if the
slit width is adjusted so that the effective bandwidth is equal to one half the wavelength difference
between the two lines.
Most monochromators are equipped with variable slit widths so that the effective bandwidth can be
changed. Narrow slit width are used when the spectral details of narrow absorption or emission bands
need to be resolved. Unfortunately, narrowing the slit results in a decrease in signal intensity. Large slit
widths are used for quantitative analysis where signal intensity is an advantage and spectral detail is
unimportant.
Procedure
In order to judge the resolving power of the monochromator, a region of the lamp spectrum that contains
closely spaced lines must be examined. The source that you will use is an argon spectral calibration lamp
and closely spaced lines occur in the region between 417 nm – 421 nm. Choose the smallest step size
possible.
For two of the gratings (1200 L/mm and either: 1800 L/mm, and 400 L/mm), determine the maximum slit
width that can be used and still fully resolve 2 peaks which are about 1 nm apart. The slit widths should
always be the same for both the entrance and exit slits, so change them together. Collect data with at
least 3 different slit widths for each grating.
Operating Instructions: Oriel Components
IMPORTANT:
Unlike the commercial spectrophotometers that you will use in the other experiments in this class, these
components are easily damaged if you are careless about the way that you handle them. In addition, the
safety features built into commercial instruments are not present. Please be aware of the following and
act accordingly. Failure to follow these guidelines will result in a grade of zero for this experiment.
1. Always wear safety goggles and NEVER look directly at a light source. Many of the
light sources emit UV radiation which can damage your eyes.
2. Never put your fingers on the surface of the diffraction grating, handle it by the
handle and edges only. Gratings are very expensive and will be permanently damaged by
scratches and fingerprints.
3. Do not make connections to a power supply unless it is OFF. This includes the lamps
and the PMT, otherwise they will be destroyed.
4. Always check with a TA before powering up a source or turning on the PMT power
supply.
5. Be gentle with all components: Carefully turn the micrometer when adjusting the slit
width -- do not overcrank it. The monochromator has a manual crank but do not attempt
to operate the manual crank while the stepper motor is in operation.
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Grating Installation
The first time that you do this, you must have TA supervision. Make sure that the shutter to the PMT is
closed. Open the monochromator compartment and turn the bar above the grating so that is is parallel to
the grating. Remember to be extremely careful with the gratings and never allow anything to come in
contact with their surfaces!!! The grating should be loose in the mount (if not, DO NOT proceed – see
your TA). Carefully lift out the grating with its handles and put its cover on securely. Install grating by
carefully placing it in the grating holder while the bar is in the parallel orientation. Then rotate the bar
back to the perpendicular direction (point should be directed toward the grating) to hold the grating
securely. Close the cover of the monochromator.
Monochromator Drive Software
Boot the drive program “Mono”. You can control the monochromator from this window. The stepper
interface must be turned on (the toggle is on the left back corner of the interface box; when the outer side
of the switch is pressed down, the interface is on).
Under the “Mono” menu, select Configure Gratings. The available gratings should be listed there. Make
sure that the grating currently installed has been selected. Close this window and check the wavelength
calibration. The value in the box at the top of the software window is the wavelength that the software
thinks the monochromator is set at. The wavelength value displayed on the monochromator is for a 1200
L/mm grating. If you are using a different grating, the actual wavelength is:
1200 L / mm
× wavelength displayed = actual wavelength
groove spacing ( L / mm ) of grating in use
If the actual wavelength is different than the one displayed by the software, double click on the
calibration wavelength box and enter the actual wavelength.
The monochromator drive has been set to step in 0.01 nm intervals with a 1200 L/mm grating. This is
considered by the software to correspond to a 10:1 drive ratio. Be sure that this value and not 1:1 is
showing in the lower right hand corner of the software window.
Scanning range and intervals are set by entering values in the boxes below “Scan” on the left side of the
screen. “Pause” can be set at 0.00 and the software will scan at its maximum rate or you can set a value
(in seconds) and the software will scan more slowly.
It is suggested that you set the start wavelength in the “Goto” box before doing a scan. That way you can
easily activate the chart recorder when the scan begins.
Signal Transduction and Readout
The voltage to the PMT should not be greater than 500 V. Turn the dial on the PMT power supply fully
counterclockwise before turning it on. Power it up and then turn the dial until the reading is at –400 V.
Turn on the pre-amp and the chart recorder. Initially, set the pre-amp to a sensitivity of 10-6 amp/V.
With the PMT shutter closed, adjust the offset so that 0.000 is displayed. Set the chart recorder to have
full scale corresponding to 2 V. Once you start collecting spectra, adjustments to these setting will have
to be made.
Your TA will show you how to operate the strip chart recorder. You should mark start and stop
wavelength for each scan on the chart paper. Adjust the full scale value so that all the peaks are on scale
but as large as possible. Always write down all the parameters used for each scan on the chart paper
and/or your notebook. PLEASE put the cap back on the pen at the end of the lab period.
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At this point, you should be ready to collect data. Turn on the light source (MAKE SURE THAT YOUR
GOGGLES ARE ON), set the scan parameters, slit widths, open the shutter, and go! Remember to close
the shutter to the PMT when you are changing grating and when you are done with the analyses.
Data Analysis
Using a computer, plot the relative overall response of your Spec 20 vs. wavelength, using the data
obtained in the response curve section of this experiment. On the same graph plot a second curve
representing the relative response of the phototube vs. wavelength, using the data supplied to you at the
end of this section. Along the top of the plot indicate the colors observed for various wavelengths.
The relative emission intensity of the lamp (RLI) can be calculated from the two curves plotted as
follows. For each wavelength studied, divide the value for the overall instrumental response (from your
data) by that for the relative phototube response (from Table I). This gives a series of numbers, the
largest of which, will be about three. To convert these numbers to a scale of 100, multiply each of them
by the factor 100/3. Thus:
Instrument response 100
RLI λ =
×
Phototube response
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Finally, plot a third curve on the same graph, representing the relative intensity of the lamp vs.
wavelength. Now note how the net effect of the combination of lamp and phototube responses leads to
the overall instrument response. This change in response with wavelength is the reason why 0 and 100 %
T must be adjusted each time the wavelength of the Spec 20 is changed. Double beam
spectrophotometers like the Hitachi are capable of automatic compensation for this change in response
with wavelength.
The Relative Response of the Phototube
The phototube consists of a light sensitive cathode (cesium-antimony type photoemissive cell) and an
anode to collect the photoelectrons. The relative response of the phototube to a beam of monochromatic
light of constant intensity is given in Table I. Thus it is seen that the phototube is much more sensitive to
light of wavelength 400 nm than to light of wavelength 600 nm. This means that the phototube will
require a greater intensity of 600 nm monochromatic light, in order for the same % T reading to be
registered upon the colorimeter dial than for 400 nm light.
Table I Relative Response of a Spec 20 Phototube
Wavelength (nm)
350
375
400
425
450
475
500
Relative Response (%)
90
98
100
98
91
81
68
Wavelength (nm)
512
525
550
575
600
612
625
Relative Response (%)
61
53
37
21
10
7
5
Determine which wavelength of light the Spec 20 most responsive to, which wavelength of light is
emitted most strongly by the tungsten lamp, and which wavelength of light the phototube is most
responsive to.
Plot the absorption spectrum of the 0.04 M chromium nitrate solution using the data collected using the
Spec 20. On the same plot, plot the spectrum obtained using the Hitachi spectrometer. Locate and record
the wavelengths at which absorption maxima occur in both spectra.
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Using the spectrophotometric data obtained with the Spec 20 for the series of chromium nitrate
standards, prepare a plot of absorbance versus concentration. The plot should include error bars
indicating the uncertainty in your data points. Report the mean and standard deviation of the unknown
concentration.
For each diffraction grating you used, calculate the theoretical slit width necessary to fully resolve the
two peaks in your argon spectra.
Questions
Comment on the similarities and differences between the spectra of chromium nitrate collected on the
Spec 20 and the Hitachi instruments. What might be the origin(s) for these differences? Discuss this in
terms of what you know about the different configurations that these two instruments.
Discuss the results of the determination of Cr(III) concentration in your unknown. What are possible
sources of error in this measurement?
Discuss advantages and disadvantages of using a Spec 20 or a Hitachi U-4001 for collecting an
adsorption spectrum. Also discuss advantages and disadvantages of these instruments for measuring the
concentration of an unknown. (NOTE: the Hitachi, used in photometry mode, measures absorbance at a
selected wavelength).
Typically when a double-beam instrument is used to collect a spectrum, a background is collected first.
Why is this necessary? Would a background need to be collected with the Beckman DB? Why or why
not?
How does the slit width and grating blaze density affect peak intensity and the resolution?
For each diffraction grating, compare the theoretical slit width necessary to fully resolve two adjacent
peaks in your spectrum with the slit width determined experimentally for each. Discuss any differences.
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