Investigation 1
What is the relationship between the
concentration of a solution and the
amount of transmitted light through the
solution?
What is absorbance? Transmittance?
• Groups of 3-4 students.
• Set absorbance at 630 nm
• The molarity of the solution is 6.4X10-6M or 6.4μM
You were given the solution containing blue#1
dissolved in water with known concentration.
Safety: all solutions can be safely flushed down the
drain with copious amounts of water.
• We will explore how light, transmitted by a
solution is related to the concentration of the
solution.
• Send one person per team to get the dilution
stock ratio you are required to test.
• Use the same cuvette (test tube) for the
reference/baseline (aka auto zero)
measurement and the sample measurement.
This eliminates error due to difference in
cuvettes.
• Record the % transmittance of known
concentration in the data chart.
Sample data
Solution
Dilution ration
Molar conc
Measured
Transmittance
Measured
Transmittance
as decimal
1
10/0
2.31
49%
.49
2
8/2
1.85
58%
.58
3
6/4
1.39
65%
.65
4
4/6
0.924
77%
.77
5
3/7
0.693
81%
.81
6
2/8
0.462
87%
.87
7
1/9
0.231
95%
.95
8
0/10
0
100%
1.00
Step 5 and Step 6: Graph
• Make sure you graph the transmittance as a
decimal and concentration in μM.
• We are trying to find a straight line that goes
through zero with a positive slope.
• Your group will have two different suggestions
that I would like you to try to graph. You will
report back tomorrow your findings.
• From your data graph –log T vs. [ ]
• From sample data graph
- Log T vs [ ]
And
Log T vs [ ]
And the winner is –log T vs [ ]
• Called calibration line
• You can use this calibration line to obtain
concentration of the same absorbing species
in a solution of unknown concentration.
• HOW????
• The equation of the line displays the relationship
between concentration and –logT.
• The slope is determined and known.
• If you hold the instrument the same way and
wavelength and cuvette the same, you can measure
transmittance, manipulate data as needed and along
with the slope have enough info to determine the
concentration of absorbing species.
What is absorbance???
Transmittance???
-log T= slope [dye] which is related to
absorbance which is another mode of data
collection on the spectrophotometer.
Absorbance is defined as Abs=- log (T)
• T is transmittance ( not % T decimal number)
How does it work?
• The instrument passes a beam of light into a
solution containing the sample and then
detects how much of the light comes out of
the other side of the solution. By comparing
the amount of light transmitted by the pure
solvent to the amount transmitted when the
sample is dissolved in it we can calculate
absorbance.
Key concept for this Abs=- log (T)
relationship?
• When absorbance is 1.0 only 10% if the light
beam is reaching the detector.
• When abs is 2 only 1% of the light beam is
reaching the detector.
• When abs is 3 only 0.1% of the light is reaching
the detector.
• As a result accuracy and sensitivity of the low
cost instrument starts to suffer at absorbance
value higher than 1.5. Don’t accept measurement
higher than 1.5. Dilute the sample and retest.