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Abs = 0.51
Before the prac class begins
experimental work.
The spectrophotometer actually measures
transmittance: %T = (IT/I0)*100.
This is the intensity of light transmitted
through the solution (IT) divided by the
intensity of light entering the solution, the
incident light (I0). The problem with this
measurement is that as the solution becomes more
concentrated the %T decreases. The relationship is also
not linear.
Absorbance is actually log10I0/IT
Relationship between %Transmittance and
light path length and concentration
100 100
80 80
60 60
%T %T
40 40
20 20
0 length 0 concentration
Ln (I0/IT) = acl
Converting to Log10
Log10(I0/IT) = ecl
Hence the Beer-Lambert Law
A = ecl
Before the prac class begins
experimental work.
Absorbance increases linearly with
concentration as predicted by the Beer-
Lambert Law
A = ecl
Explain why the working range of a
spectrophotometer is 0.1 1.0. Remember
Abs is a log scale. An absorbance of 1.6 is 2%
light transmitted while an absorbance of 2 is 1%
light transmitted. The class specs can not
accurately distinguish 1% from 2%. An
absorbance of 1.0 is 10% transmitted light.
Before the prac class begins
experimental work.
For the mathematically minded:
Transmittance = IT/I0*100
Absorbance = log10(I0/IT)
Converting Transmittance to Absorbance
%T/100 = IT/I0 100/%T = I0/IT
Taking logs on both sides
Log 100 log %T = log I0/IT
2 log%T = Absorbance
Before the prac class begins
experimental work.
Going back the other way
Transmittance = IT/I0*100
Absorbance = log10(I0/IT)
Converting Absorbance to Transmittance
Abs = log(I0/IT)
10Abs = (I0/IT), inverting
10-Abs = (IT/I0),
100* 10-Abs = 100*(IT/I0),
102-Abs = %T
Experiment 1: Identifying a
compound by spectrophotometry
If a compound absorbs light its absorption
spectrum is a unique property of that
compound.
The molecular structure is responsible for
the absorption properties
The most common feature of absorbing
compounds are conjugated double bonds,
often as an aromatic ring
Experiment 1: Identifying a
compound by spectrophotometry
Conjugated double bonds result in pi
electrons above and below the ring or
chain and these electrons can be moved
to higher levels by photons of light.
As the electrons are promoted to higher
levels allowed by the molecular structure
they absorb light of a specific wavelength,
based on the energy required for the
transition (DE).
Experiment 1: Identifying a
compound by spectrophotometry
H 3C N
N
NH NH 2
N N
N O H N
H
Thymine Adenine
N O
H
Cytosine
Nucleic Acid Absorption Properties
Base lmax e (mM-1cm-1)
(nm)
Guanine 275 8.0
Adenine 260 12.9
Cytosine 265 5.8
Thymine 258 8.0
Nucleic Acid Absorption Properties
Absorption Spectrum: Guanine Absorption Spectrum: Adenine
0.5 1.0
0.4 0.8
Absorbance
Absorbance
0.3 0.6
0.2 0.4
0.1 0.2
0 0.0
220 240 260 280 300 320 220 240 260 280 300 320
Wavelength (nm ) Wavelength (nm )
0.5 0.6
0.4 0.5
Absorbance
Absorbance
0.4
0.3
0.3
0.2
0.2
0.1 0.1
0.0 0.0
220 240 260 280 300 320 220 240 260 280 300 320
Wavelength (nm ) Wavelength (nm )
Amino Acid absorption Properties
O
O
O
H2N CH C OH H2N CH C OH
H2N CH C OH
CH2 CH 2
CH2
HN NH
OH Histidine
Tryptophan
O Tyrosine O
H2N CH C OH H2N CH C OH
CH2 CH 2
SH
Cysteine
Phenylanine
The Dyes
The dyes chosen for this experiment are
different colours (A F)
Each pair of students will be assigned a
dye by the demonstrator. They use this
dye for the whole practical.
Students should take note of the colour of
their dye and record the concentration
(mM) on the bottle
Obtaining a Spectrum for the dye.
Using the Shimadzu spectrophotometers
in spectrum mode (mode 2 on main menu)
place a 1 mL plastic cuvette full of H2O in
the holder and obtain a baseline correction
(F1). This will take some time.
Then, using the same cuvette, fill it with
the dye solution and obtain a spectrum.
Find the peaks. If you are unclear how to
do this practise beforehand.
Obtaining a Spectrum for the dye.
The reason for doing this is to find the
absorbing region of the dye. It takes a long
time to obtain a spectrum from 600 nm to
350 nm. A quick narrowing of the range is
to be encouraged.
Get students to consider the colour of the
solution and how this might give clues to
the absorption minima and maxima
The relationship between colour
and absorption
1.2
minimum in the
0.6
yellow/red region
0.4
0.2
0.0
350 400 450 500 550 600
w avelength (nm )
Obtaining the spectrum
Once the baseline is corrected and the
absorbing range determined, find the
absorbance of the dye every 10 nm within
the absorbing range. Every 50 nm will do
outside the absorbing range.
If the baseline correction is done you dont
have to zero every time you change
wavelengths. This saves heaps of time.
Obtaining the spectrum
Students must, by the next lab, plot the
spectrum. It would be a good idea to get
them to this now if there are free
computers. Otherwise get them to identify
the dye by the peaks, comparing to the
sample spectra at the back of this section
of the lab manual.
From the concentration on the bottle
estimate the extinction coefficient.
Obtaining the spectrum
Make sure you are very familiar with the
Excel chart drawing process as you will
need to help the students here. Practise
with the spectro.xls spreadsheet provided.
It has the raw data obtained for riboflavin
and the worked solution.
Check out what is expected graph-wise in
the worked solution. Students should have
practised much of this with the Excel task
in the last practical.
Calculating the Extinction
Coefficient
This comes directly from the relationship
A = ecl,
Where e is the extinction coefficient
expressed in the units of c, the
concentration. In this experiment the conc
units will be mM so e will have the units
mM-1cm-1. Round the value off to 1 dec pl.
Discussion
Predict which of the following parameters would
change with dilution? How would they change?
The number of peaks
The l max
Al1/Al2
Absorbance at l max
Extinction Coefficient
Transmittance at lmax
Can you predict what would happen to the
absorption spectrum if you diluted your dye with
another dye?
Experiment 2: The Standard Curve
Although identifying a compound by
spectro is a useful property,
spectrophometry is used more often to
measure the concentration of a
compound.
Sometimes the extinction coefficient can
be used directly. This occurs when the
compound of interest has an intrinsic
absorbance.
Experiment 2: The Standard Curve
If the compound of interest does not have its
own intrinsic absorbance then a coloured
derivative must be made by reacting it with
reagents. Then a standard curve must be
produced.
In todays practical students will gain experience
at producing and using a standard curve, even
though in this situation you would normally use
the extinction coefficient.
Experiment 2: The Standard Curve
Using the same solution as the one used
to obtain the spectrum, get the students to
dilute it so that there are at least 5 points
for the line. My suggestion is 200, 400,
600, 800, 1000 uL of dye, then make each
up to 1 mL with water.
Mix well and obtain the absorbances at the
lmax.
The standard curve
1.2 y = 0.0125x
Absorbance @ 445 nm
2
1.0 R = 0.9999
0.8
0.6
0.4
0.2
0.0
0 20 40 60 80 100
[Riboflavin] (nmol/mL)
The standard curve
1.2 y = 0.0125x
Absorbance @ 445 nm
2
1.0 R = 0.9999
Dilution factor 1 2
A445 0.829 0.417
[Riboflavin]
(nmol/mL) 66.5 33.4
Original Conc.
(nmol/mL) 66.5 66.9
Average
(nmol/mL) 66.7
From standard curve From standard curve
or using SLOPE or using
function in Excel INTERCEPT
function in Excel
Dilution factor 1 2
A445 0.829 0.417 [Riboflavin]
(nmol/mL) =
[Riboflavin] (A445-
(nmol/tube=mL) 66.5 33.4 intercept)/slope
Original Conc.
(nmol/mL) 66.5 66.9 [original] =
Average [riboflavin]*
(nmol/mL) 66.7 dilution
factor
Average of 2
values
Quick tips
You can use the extinction coefficient
obtained in the first experiment or the
standard curve. Get the students to try
both methods.
To get from the Absorbance to the
concentration you solve the equation of
the standard curve for x; you know the y
value (Abs) and you want to find out the x
value (conc.)
Why we express the
concentration in nmol/tube
?
The back calculations
40 uL 50 uL 60 uL 70 uL 80 uL
Average = 0.79 mM
For unknown C3: range 1 2 mM
The upper range:
2 mM 2 umol/mL 2 nmol/uL
So we need to add 40/2 = 20 uL