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Unformatted text preview: y the selected wavelength to pass.
This wavelength is then projected upon the sample. The light that is able to pass through the sample
(i.e. that which is transmitted) is collected by a phototube that converts the light energy into an
electrical signal that is proportional to the amount of light that it received. This signal is then
transmitted to a processor that quantifies the amount of light received. The output from the processor
then sends to results to an output display on the front of the instrument. The results may be reported as
either percent transmittance (%T) or absorbance (A). It is important to realize that both of these values
are related to the difference in the amount entering (Io) and leaving (I) the sample.
Collimator or I
I0 %T A log Output display.
(analog or digital) 100 I0
2 I0 I 3 Monochromator
diffraction grating 4 selector (slit) Cuvette
(with sample) Phototube Figure 2. Essential parts of a spectrophotometer. In spectrophotometry and colorimetry it is necessary to “zero” the instrument at each wavelength
to be measured with a tube known as a “blank”. This tube contains the same solvent or solution that
was used to dissolve the compound that you wish to measure - less that compound. The blank is
important because it may contain a component that absorbs light at the same wavelength as the
particular compound to be measured. In which case, the absorbance of this component would be
detected in the sample, in addition to that of the compound of interest. This would result in an
inaccurate (higher) absorbance value for the sample you are measuring. The possible contribution of
the solvent or solvent component to the total absorbance reading is eliminated by “zero-ing” the
instrument with the blank before a measurement is made. This is done by adjusting the absorbance
display to read zero with the blank tube in the instrument. Once this is done, the sample tube can be
measured. Again, this process must be repeated for each different wavelength to be used.
Specific instructions for this operation as they relate to one of the instruments used in Biology
5LA (the Spectronic – 20 D+) follow: Biology 05LA – Fall Quarter 2012 Lab 2 – page 3 Zero-ing the Spectronic – 20 D+:
1. If the machine is not already on, it can be turned on with a clockwise rotation of the knob on
the left side of the lower front panel at least 15 minutes before use.
2. Set the desired wavelength with the knob on the right side of the horizontal surface.
3. Make sure that the filter selection lever, found in the lower left corner of the lower front panel,
is in the correct position for the selected wavelength.
4. Set mode to transmittance with the mode button; the left-most button on the upper front panel.
Verify your selection by observing the red light next to “Transmittance” on the display panel.
5. With no tube in the instrument, set ZERO transmittance using the knob on the left side of the
lower front panel. (clockwise rotation increases value and counter clockwise rotation
decreases value) HANDLE THIS KNOB WITH LIGHT FINGERTIP PRESSURE ONLY – otherwise
you might not feel the “stop” at the end of the limited range of rotation of this knob and thus
damage the instrument.
6. Insert the blanking tube into the tube chamber covered by the hinged lid on the left side of the
horizontal surface. NOTE: Before inserting the blanking tube into the instrument, you should make
a small mark on the lip of the tube with an indelible marker. When the tube is inserted into the
instrument, this mark should be always be positioned toward the front of the instrument. This will insure
consistent readings each time the tube is inserted. This precaution should be taken for any tube that
will be inserted into the instrument repeatedly. 7. Set mode to absorbance with the mode button used above.
8. Set absorbance to ZERO using the knob on the right side of the lower front panel. Once again
use only LIGHT FINGERTIP PRESSURE – rotation is also limited by “stops” for this knob. Here,
counter clockwise rotation increases value and clockwise rotation decreases value. NOTE: If the
display shows a reading of –1.999, several turns in the CCW direction will be required to ZERO the
instrument. The machine is now ready for insertion and measurement of the sample at the selected . 9. Steps 2 – 8 must be repeated for each wavelength to be measured. Remember that
transmittance is set with no tube in the instrument and that the correct filter position range must
be selected in accordance with the selected wavelength.
10. At the end of the session, remove the last tube measured from the instrument. If yours is the
last lab of the day, turn the instrument off with counter clockwise rotation of the knob on the
left side of the lower front panel. Otherwise, leave it on. ****
In the first of today's exercises, the absorption spectrum of potassium ferricyanide, K3Fe(CN)6,
will be determined. The second of today's exercises will demonstrate that the absorption of light at a
specific wavelength can be used for determining the concentration of a chemical in solution.
At this time, it is necessary to warn you that K3Fe(CN)6 is a POTENTIALLY
HARMFUL SUBSTANCE IF SWALLOWED OR EXPOSED TO HOT,
In order to minimize the health risk of handling this substance, keep in mind the
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- Fall '12