Instruction Manual

Manual No. 012-08914A

Spectronic 20D Kit

Model No. PS-2004

Model No. PS-2004

Table of Contents
Equipment List........................................................... 3
Introduction ............................................................. 4
Equipment Setup ........................................................ 4
Spectronic 20D Operation.............................................. 8
Adjustments and Settings .............................................. 9
Absorbance .............................................................10
Wavelength .............................................................11
Concentration...........................................................12
Scanning Absorbance versus Wavelength.............................14
Appendix A: Technical Support ....................................... 24
Appendix B: Copyright and Warranty Information .................. 24

Spectronic 20D Kit

Spectronic 20D Kit

Model No. PS-2004

Equipment List

Included Equipment
1. Cable (6-prong plug to 9-pin serial connector)

Replacement
Model Number
PS-2004

Additional Equipment Recommended

1. Spectronic 20D or 20D+

2. PC (Windows-based)
3. Datastudio V1.8.5 or later

CI-6870E

Model No. PS-2004

Introduction
The PASCO PS-2004 Spectronic 20D Kit (Spec20) consists of a cable
that connects the Spectronic 20D (Spec 20D) device to the serial port
of a Windows-based computer. The input options from the Spec20
include wavelength, transmittance, and absorbance, and respond to the
PRINT button on the Spec 20D+. The Spec20 kit allows transmittance
and absorbance to be input into DataStudio, the software program used
with all PASCO PASPORT devices and PASCO interfaces.
Measurements taken from the Spec 20D devices can be analyzed
together with input from other PASPORT devices.
The Spec 20D is the digital version of the Spectronic 20D single-beam
spectrophotometer that has been produced for student use for over 50
years. Thermo Electron Corporation currently supplies a 20D+
version of this device with a serial port on the underside of the
instrument that can be attached to a computer or a printer. The kit from
PASCO scientific enhances the function of the Spec 20D in several
ways:
By providing a connection between computer and instrument to acquire real time
data from the Spec 20D.
By acquiring and storing the data either continuously or manually
By monitoring and storing wavelength data
By maintaining the report of absorbance and transmittance regardless of the
MODE setting on the Spec 20D
By responding to the PRINT button on the Spec 20D+ when DataStudio is set up
in the Manual sample mode.

Equipment Setup
1. Attaching the cable to the Spec 20D
One end of the Spec20 cable attaches to a
connector on the bottom of the Spec 20D
with a 6-prong plug. The prongs are
asymmetric so that the plug can only be
inserted in one direction. Please consult
your Spec 20D technical documentation
for model-specific details. In some
documents, the socket on the bottom of the
Spec 20D is labeled "analog output jack" since there are plugs
assigned to analog output. (On the Spec 20D, a mixture of analog and
digital output are recognized in the software.)

Model No. PS-2004

2. Connecting to the computer

The other end of the cable with a 9-pin serial
connector attaches to a serial port on the
back of the computer. This port is accessed
from within DataStudio.
3. Setting up DataStudio
Please consult the DataStudio Installation
Guide for access and setup instruction for the
software.
4. Accessing the Spec20 in DataStudio
Access the Experiment Setup window from the main menu by pushing
the Setup button.

Select the Add Instrument option from the row of buttons in the
lower portion of the window. This opens the Instrument Selection
window. Two Spectronic options should appear: "Spectronic 20D" and
Spectronic 20D+.
When the cable is not installed these
settings can be only be browsed, not
selected, and the Data window
displays an alert symbol indicating
that the cable is not found.

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Select the appropriate model. (Please note

that the Spec 20D+ has the PRINT button.)
This activates a Data window. If the Data
window is not visible click the Summary
button at the top of the screen.
When the instrument is available, the program creates a Digits display
on the desktop.

Wavelength and transmittance are available as defaults and display

dashes until the program starts collecting data. To collect data, press
the Start button. In its default mode, DataStudio collects data
continuously. With the exception of kinetics experiments, the manual
sampling mode may be more useful in this application. Instructions on
how to change the sampling mode are provided in the following
section.
The Digits 1 display, as shown above, will cover the Experimental
Setup window area. By moving this display to the side, the Setup
window becomes accessible, and more details and selection options for
the Spec 20D become available.

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5. Setting Manual Sampling

In this window, the default input option settings can be checked, and
the Sample Mode can be changed from Continuous to Manual (see the
pull down menu in the screen). The example below shows that
Wavelength and Transmittance are selected, and that the Sample Mode
is Manual.

The Sample Mode for the Spec 20D+ indicates that the software will
respond to the PRINT button. If the PRINT button is not available,
such as on the Spec 20D, return to the Options settings by pushing the
Options button and check the Keep data values only when
commanded option.

Close the Sampling Options setup window and the Experiment Setup
window. As soon as the Start button is pressed, it will change into a
Keep button. Pressing Keep stores a single data point. When the red
box button is checked the Sampling mode is stopped.

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Spectronic 20D Operation

The instrument:
The Spec 20D is a single-beam instrument with five basic parts: a light
source, a grating, a compartment for a sample cuvette, a filter, and a
detector.
The light source:
The Spec 20D is a single-beam spectrometer, which means that there is
a single path of light from the source to the detector, and only one
signal is monitored during the experiment. This signal can drift due to
changes in lamp intensity, for example. In a double-beam instrument,
the light beam is split so that one beam sees the sample and a second
beam avoids the sample and gives a second signal. This could be a
second detector, or one detector can be used with a chopper that
creates a signal oscillating between the two. This way any changes in
the lamp intensity over time or with varying wavelength can be
corrected.
The Spec 20D needs to be warmed up so that lamp reaches a stable
temperature, and it also needs to be checked regularly to see if there is
any shift (drift: 2% Trans-mittance per hour). The difference in lamp
intensity versus wavelength is demonstrated in the section "Scanning
absorbance vs. wavelength".
The Grating:
The grating acts as a prism and produces a rainbow of colors. The
wavelength knob on top of the instrument changes the position of the
grating where different colors of light (indicated by the dial) are
positioned to pass through the slits to the sample cuvette. The spectral
bandwidth is reported to be 20 nm with an accuracy of +/- 2.5 nm.
Two filters are mounted on a lever between the sample and the
detector. One is to be used from 340 nm to 599 nm and the other from
600 nm to 950 nm. This range is due to the intensity increase of the
tungsten lamp at longer wavelengths. The filters selectively reduce the
intensity of the parts of the spectrum to maximize the performance of
the detector.
The detector
The detector converts the intensity of light to a digital signal that can
be displayed in several modes on the front of the Spec 20D and be sent
via cable to the computer. The differences in the detector response with
temperature and wavelength are small compared to the differences in

Model No. PS-2004

the lamp. See the lamp section for suggestions on mediating

temperature and wavelength effects on detector response

Adjustments and Settings

There are two knobs on the front of the Spec 20D that need to be set
before any samples are put into the instrument for measurement.
0%T adjustment
The knob labeled "0%T" acts as an on/off switch and is used to tell the
digital sensor what should be considered "complete darkness". This
setting provides an adjustment of the background voltage or electrical
bias in the detector
Allow the instrument to warm up for at least 15 minutes to allow the
circuitry to stabilize. When there is no cuvette in the instrument, a flap
falls into place to cover the opening to the detector. In this state, with
the instrument in TRANSMITTANCE mode, dial the "0%T" knob
until the TRANSMITTANCE is at 0%. If the dial does not adjust to
that setting, check that the flap is falling into place as it should. A
failed lamp will not affect this adjustment.
100%T / 0A adjustment
The amplification of the detector is adjusted with the 100 %T knob
when there is an appropriate "blank" in the sample compartment. This
reading is very sensitive to the output of the lamp and should be
repeated at least every hour during experiments to correct for drift due
to changes in lamp temperature.
The blank should be the same cuvette and background mixture (all but
the chemical of interest), and the orientation of the cuvette should be
kept the same since most cuvettes reflect light differently at different
angles. If this 100 %T knob is set too high, or if the intensity of the
light exceeds what was set with the blank, the detector will reach a
maximum and flatline at about 200 %T. On the other extreme, the
calculation of absorbance can go over 2.0 A if the intensity reaching
the detector yields transmittance less than 1 %T. If you check the
specifications of the Spec 20D, you can see that readings over
2 A are outside its range. Many experimenters recommend keeping the
Spec 20D absorbance measurements between 0 and 1 A (10%T).

Model No. PS-2004

Absorbance
The operation of a spectrophotometer is to measure the light intensity
that can be transmitted through a sample. The amount of light
transmitted can be used to solve for the concentration of light
absorbing chemicals in solution by Beers Law, which can be
summarized as a direct proportion between absorbance and
concentration:
A=kc
A is absorbance, c is concentration and k is a constant that varies with
chemical species being measured and with the path length within the
instrument. This relationship is discussed in more detail below in the
section on "Concentration", and again in the section on "Beers Law".
The absorbance is related to the intensity of light of the sample
compared to a blank:
A = - log(I/Io)
The intensity of light transmitted through the sample is "I" and the
intensity of light transmitted through the blank is Io. The blank should
have identical conditions of cuvette, solvent, wavelength setting, etc.,
with the only difference being the absence of the sample compound of
interest.
The percent transmittance displayed by the Spec 20D is defined by this
ratio:
%T = 100% * I/Io
%T

I/Io

100%

10%

.1

1%

.01

0%

It should be noted that Spec 20D measurements are only accurate

between 1-100% T and 0-2 Absorbance.

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Wavelength
Visible white light contains an even distribution of the colors of the
visible spectrum between 400 nm and 750 nm. When any one part of
that spectrum is absorbed from light, the transmitted light will be seen
as colored.

Color absorbed

Color observed

Absorbed radiation

violet

yellow-green

400-435

blue

yellow

435-480

green-blue

orange

480-490

blue-green

red

490-500

green

purple

500-560

yellow-green

violet

560-580

tellow

blue

580-595

orange

green-blue

595-605

red

blue-green

605-750

The shorter wavelengths have higher energy and the longer

wavelengths have lower energy. For example blue light (~ 450nm) is
higher energy than red light (~ 700 nm). This range is defined as the
visible spectrum because it is visible to the human eye. The human eye
is most sensitive to green light.
In order to measure a specific color, the Spec 20D uses a grating to
separate the white light into the different colors. The light passing
through the sample is the color of the light indicated on the wavelength
knob plus or minus 2 nm. It is very important to be aware that the
intensity of the lamp creating the light varies quite a bit over the range
of these wavelengths, and it is very important that the appropriate
adjustments be made during measurement or during analysis of the
data.
Cloudiness in a sample is caused by light scattering, not by the
absorbance of specific wavelengths. When a sample is cloudy, the
sample preparation needs to be reconsidered. It usually means there is
contamination or the compound is not adequately dissolved.

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Concentration
Solving the relationship between concentration and the measured
absorbance is a process of developing a set of colored standards and
measuring them with the spectrophotometer under the conditions used
for the unknowns. Three methods will be presented for applying
results from standard solutions to display the concentration of
unknowns.
1. Multipoint fit to standard curve, letting zero vary in the fit.
(DataStudio solution)
The results of the standard curve can be fit to a line and the coefficients
used to convert absorbance to concentration.
Given the line of fit:
y=mx+b

The line fit should be expected to intercept close to zero, or in this

example b=-.004. When doing this fit, it is valid to consider the
Concentration = 0.00 mM as a data point where Absorbance = 0.00.
This point was generated when the 100% T adjustment was made as
described in the previous section. The slope "m" is related, with
appropriate unit conversions, to the extinction coefficient. (See the
Beers Law example below for more details.)
When an unknown is tested, x-value can be solved by rearranging the
y = m x + b equation to:
bx = y----------m

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This can be re-written to be:

b
concentration = absorbance
----------------------------------------m
This equation can be entered into the DataStudio Calculator, and
subsequent absorbance measurements can be viewed by looking at the
"concentration" item (in this example) in the DATA window.
2. Two point fit to standard curve: one standard, one zero.
(Spec 20D-alone solution)
The Spec 20D has a feature for analyzing concentration using a blank
and a single standard solution. The blank is set to 100 %T, as above, or
equivalently, the display could be set to Absorbance and the 100%T
knob could be adjusted so that Absorbance equals 0.000. These two
procedures are just two different views of the same setting. After this
point is set, the standard concentration sample can be placed in the
Spec 20D and the display set to concentration mode. At this time the
"increase" or "decrease" buttons can be pressed until the readout
matches the concentration of the standard that is in the device. This is
in effect a single point calibration. Subsequent samples will produce a
CONCENTRATION displayed on the Spec 20D.
3. Multi-point fit to standard curve: zero not allowed to vary in
the fit. (DataStudio creating a better quality FACTOR, Spec
20D then operates alone.)
The Spec 20D instrument has output that relates to this calculation.
The FACTOR mode allows setting of a factor inversely related to m in
the above equation. If this feature is to be used the user should fit an
equation that holds the "b" value to zero. This can be done in
DataStudio by entering a "User Defined" Fit with the equation
absorbance = concentration
--------------------------------------FACTOR

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Note the function is written x/FACTOR in the box, and the "Input" is
selected as the data that was entered into the table earlier. (The name
for that data might vary.)

This factor (FACTOR = .167) is entered into the Spec 20D in the
FACTOR mode, which sets the instrument to give appropriate
concentration readouts when returned to the CONCENTRATION
mode. The concentration readout is commonly only accurate between
0 and 1999 C with a FACTOR value between .1 and 1000.

Scanning Absorbance versus Wavelength

Using a single beam instrument can allow the students to learn the
fundamentals of ultraviolet/visible spectroscopy by not making the
corrections that are made automatically in the dual beam instruments.
Detecting a single beam demonstrates to the student the variation that
is created by the intensity of the lamp and filters within the Spec 20D,

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and using DataStudio and the Spec 20D kit allows a very convenient
spectrum of Absorbance.
Measuring a blank in the Spec 20D, the student can see the lamp
intensity increases as wavelength increases and how the filter effects it.
With the filter in the 350-600 nm position the intensity of light that
reaches the detector reach a peak at about 410 nm and then decrease. In
the 600-950 nm range there is no such peak, but there is a plateau after
800 nm.
Measuring the sample is usually very tedious when the sample has to
be re-zeroed at every wavelength. With the Spec 20D connected to a
computer by the Spec 20D kit - cable, the DataStudio software is able
to store the blank spectrum for later comparison with the sample. As
discussed earlier in the section on Absorbance, the absorbance of at
any wavelength will be the intensity of the sample over the blank at
that wavelength:
As = - log(Is/Io)
In this example, the entire spectrum of Io values (measured as %T for
the sample at each of the values of wavelength) can be stored. Later the
intensity of the sample is measured, without changing the settings for 0
%T or 100%T, and the two arrays of data can be evaluated by
DataStudio to yield the absorbance of the sample (As) over those
wavelengths. It is a feature of DataStudio that the software will
automatically pair the Is being calculated with the appropriate Io, and
if the points don't line up exactly interpolation is used to estimate the
expected point. This makes the experiment convenient and the analysis
straight forward.
1.Set up Manual Sampling.
See the Setting Manual Sampling section on page 7 of this manual.
Reminder: The Spec 20D+ responds to the PRINT button, and the
Spec 20D, responds to the Keep button in DataStudio.
2. Set 0% T knob
After the instrument has warmed up for the appropriate 15 minutes or
more, and with no cuvette in the chamber, adjust the 0% T knob on the
front of the Spec 20D so that Transmittance equals 0%.
3. Set the Filter Switch
To scan the lower wavelengths (350-600nm), set the filter switch at the
bottom left of the instrument.

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4. At 410 nm, set 100 %T.

The highest signal for the region 350-600 nm is usually close to
410 nm. This is a combination of the lamp intensity, the filter, and the
detector sensitivity. Dial the wavelength knob to 410 nm, place a blank
cuvette in the instrument, make note of the direction of the mark on the
cuvette, and then dial the 100%T knob until the transmittance equals
100%. The blank cuvette should have a solution in it that contains the
same solvent and background as is in the sample, but with none of the
compound to be measured. Neither the blank nor any of the samples
should be cloudy or "milky", since that indicates light scattering which
interferes with the measurement of light-absorbing compounds.
5. Graph Transmittance vs. Wavelength
Create a graph by clicking on the %Transmittance icon in the Data
Window

and dragging it down over the Graph icon in the Display window
where it can be "dropped" by letting go of the mouse button.

A graph of Transmittance(%) vs Time(s) will be created.

Next click on the Wavelength icon and drag it over the x-axis of the
new graph display.

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Notice that the x-axis is highlighted with a dotted line in the

illustration.

Alternatively the axis labeled can be clicked on and a pull-down menu

will reveal options for that axis.
6. Scan the blank cuvette from 350 to 600 nm.
This procedure is easiest performed using a blank work space. Delete
all data runs (Experiment>Delete All Data Runs). This allows the
following instructions to match Run Numbers.
With the blank in the Spec 20D, check to see that the "Manual
Sampling" mode is selected, make the %Transmittance vs Wavelength
visible, and press the Start button to enable sampling.
Dial the wavelength to 350 nm and press PRINT. A data point should
appear on the graph. Dial the wavelength up to about 360 nm, wait
until the transmittance value stabilizes, and then press PRINT again.
Continue collecting data in regular intervals (every 10 nm for
example), until the 600 nm range is met. Press Stop at this point to end
collection of "Run #1."
The student should understand that some readings stabilize much more
quickly than others. While some transmittance values change so little
that it seems it takes no time at all for the instrument to quit drifting
and oscillate around a single value, some readings can drift for 10-20
seconds. One way to double-check for this type of error is to have the
students collect the data in the opposite direction (from high to low
transmittance) and see if the two data strings match. In general, the
larger the change in transmittance the longer the time it takes to
stabilize.
It is not critical that the wavelength is set at exactly 360 nm, or at any
other specific set of points, including duplicate settings. Choose as
many or as few points across the spectrum as desired. The calculation
on all datapoints will be performed by DataStudio. In this example, the
absorbance = -log(Is/Io), will be analyzed by lining up the data sets
and interpolating where necessary. It is recommended not to reverse
direction within a spectrum.

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7. Find a reasonable dilution of your sample

The same cuvette should be used for the sample as was used for the
blank. Empty the blank, add a small amount of sample to the tube,
rinse, then fill to the mark with sample.
The sample should not be so dark as to yield more than an absorbance
of 2 at any point. To test this, set the Spec 20D to ABSORBANCE
mode and dial the wavelength slowly from 350 nm up to 600 nm. If
any of the readings exceed 2, the sample needs to be diluted. If none of
them read more than .5 it is recommended that a more concentrated
sample be used. In the example used below the sample was green food
coloring diluted to roughly 3 drops per cup or 1 drop per 75 mL.
One more suggestion that makes this very easy is to temporarily switch
DataStudio from Manual back to Continuous Sampling, and collect a
quick stream of data over this range. Create an absorbance vs
wavelength graph and scan it to find the maximum,
Be aware that the highest absorbance might be in the 600-950 nm
range. This might create an iterative problem where it may be desirable
to re-zero the blank at 810 nm, scan through the 600-950 range with
the sample to find a maximum, then compare that maximum with the
one that was found in the 350-600 nm range. Once a decision is made
which wavelength provides the maximum absorbance, the focus can
then be switched to testing the dilutions at that wavelength alone until
the proper level is reached.
8. Scan the sample cuvette from 350 to 600 nm.
Without changing the settings, measure the sample solution over the
same spectral range as the blank. Best results are obtained when the
same cuvette is used and it is carefully oriented in the same direction
using the markings on the cuvette. Press Start in DataStudio to
generate the "Run #2" data set, and press PRINT as before, after the
instrument stabilizes at each wavelength. When reaching 600 nm,
press Stop to end collection for this set of data.

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The illustration shows that the blank spectrum with higher

transmittance has a peak that reaches 100%T at 410 nm. This spectrum
is a measure of the instrument, of the lamp intensity, the filter
properties, and the response of the detector. The sample will have the
same or lower transmittance than the blank at all points.
9. Calculate Absorbance for 350-600 nm
The absorbance can now be calculated for this range. Click the
calculator button to open the calculator.
Before typing the appropriate equation in the Definition field, click on
the "New" button to avoid overwriting a previous equation. Enter the
following equation into the Definition field:
RatioAtLowNM = -log(lowSample/lowBlank)

Next, identify the lowSample and the lowBlank as being linked to the
"Data Measurement" under the heading "Transmittance vs
Wavelength" form the pull-down menu.

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Be sure to select "Run #1" and not the header above it.

A dialog box indicates that a yes response is requires to verify that

only one run is being selected.

The same procedure applies to identify and select lowBlank as Run #2.
The conceptual assumption is that the absorbance is proportional to the
ratio of the sample and the blank:

%Transmittance of Sample
Absorbance = log ----------------------------------------------------------------------
%Transmittance of Blank

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10. Create an Absorbance vs Wavelength graph.

Similar to the procedure in Step 5, drag the icon for "RatioAtLowNM"
down over a Graph icon. Then change the x-axis from time to
Wavelength.

11. Collect Transmittance for the Blank from 600 nm -950 nm.
Remove the sample from the cuvette and another prepare another
blank. This time, switch the filter to the 600-950 nm range (as descibed
in Step 3) and set the 100% at 810 nm (as described in Step 4). Place
the new blank back into the Spec 20D and bring the graph of
Transmittance vs Wavelength (produced in Step 5) to the front. Dial
the wavelength to 600 nm, press Start to begin collecting data of "Run
#3." Change the wavelength as needed and press PRINT to collect
data at each point. Be sure to press Stop when the settings reach 950
nm to end data collection for "Run #3".
It may be prudent to point out the difference in the intensity between
Run #1 and Run #3 to the student. Not only has the filter been
changed, but due to the adjustment of the knob for 100%T at 810 nm,
there is no relevant comparison between these two values. The
comparison will have meaning only after the absorbance is calculated.
12. Collect Transmittance for the Sample from 600 nm-950 nm.
Empty the sample-filled cuvette can now be emptied to prepare a new
run for the range from 600 nm to 950 nm. Be sure that the 100% T
knob is not moved, as this data will be compared directly to that
collected in Step 11. Press Start to begin collecting data of "Run #4."
PRINT colects each point, and STOP ends collection for that run.

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13. Calculate absorbance for the "high nm" section.

The calculation for this section of the curve will be as follows:
RatioAtLowNM = -log(lowSample/lowBlank)

Step 14. Include the absorbance for the high wavelengths.

Bring the graph to the top that shows "RatioAtLowNM" against
Wavelength. Drag and drop the "RatioAtHighNM" on this graph. The
resulting graph shows the data combined.

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The two spectra fit into the same graph as long as the units on the x and
y axis are the same.

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Appendix A: Technical Support

For assistance with the PS-2004 or any other PASCO products, contact PASCO as follows:
Address: PASCO scientific
10101 Foothills Blvd.
Roseville, CA 95747-7100
Phone:

(916) 786-3800

FAX:

(916) 786-3292

Web:

www.pasco.com

Email:

techsupp@pasco.com

Appendix B: Copyright and Warranty Information

Copyright Notice
The PASCO scientific 012-08914A Spectronic 20D Kit Manual is copyrighted and all rights
reserved. However, permission is granted to non-profit educational institutions for
reproduction of any part of this manual, providing the reproductions are used only for their
laboratories and are not sold for profit. Reproduction under any other circumstances, without
the written consent of PASCO scientific, is prohibited.
Limited Warranty
For a description of the product warranty, see the PASCO catalog.

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