BIO-2
Spectrophotometry and
Plotting of Calibration Curve
PURPOSE
• To understand the principles of
spectrophotometry
• To understand the structure of
spectrophotometer
• To understand the standard calibration curve
and determine the concentration of CuSO4
Questions
What is spectrophotometry?
What is the function of spectrophotometry?
Why do we use it?
How does it work?
What is spectrophotometry?
Spectrophotometry is the quantitative
measurement of the reflection or transmission
properties of a material as a function of
wavelength.
What is the function of spectrophotometer?
A spectrophotometer is employed to
measure the amount of light that a sample
absorbs.
The instrument operates by passing a
beam of light through a sample and measuring
the intensity of light reaching a detector.
Polychromatic light
light
Monochromatic light
Polychromatic light
polychromatic light composed of more
than one wavelength , having or exhibiting
many colors.
monochromatic light
Light of one color, having wavelengths
confined to an extremely narrow range.
SPECTRUM
Spectral Distribution of Radiant Energy
380nm ~ 760nm
X-Ray
UV
200nm
Visible
400nm
IR
800nm
WAVELENGTH(nm)
Ultraviolet(UV)
Infrared(IR)
Microwave
t
Ia
Ir
Why do you know some light is absorbed
by solution?
Complementary color
Colour of substance
Colour of
absorbed light
Wavelengt of
absorbed light
olivine
Yellow
Orange red
Reddish violet
violet
Blue
greenish-blue
blusih green
violet
Blue
greenish-blue
Green
Olivine
Yellow
Orange
red
380~435 nm
435~480nm
480~500nm
500~560nm
560~580nm
580~595nm
595~650nm
650~760nm
Solution can absorb light selectly.
A
Wavelength of Maximal Absorption(525nm)
λ(nm)
图1-2 KMnO4溶液的吸收曲线
Potassium permanganate
Transmittance and Absorbance
•When a ray of monochromatic
light of initial intensity (Io)
passes through a solution in a
I0
transparent vessel, some of
the light is absorbed (Ia) so
that the intensity of the
transmitted light (It) is less
than Io.
It
Ia
Transmittance = T = (It / I0) ×100%
Absorbance= A = ?
A=-lgT=lg(I0/It)
LAWS OF ABSORBTION OF LIGHT
Light
I0
II t
Glass cell filled with
concentration of solution (C)
•As the cell thickness
increases, It (transmitted
intensity of light ) decreases.
LAWS OF ABSORBTION OF LIGHT
 Lambert’s law: length-dependent
I = Io e-kL or A=kL
Where ‘k’ is a constant, e = base of natural log
length of the light path in the vessel.
 Beer’s law: concentration-dependent
I = Io e-kC or A=kC
Where ‘k’ is constant and ‘c’ = concentration
solution.
Combining both Lambert’s - Beer’s law, we
have:
I = Io e-kLC or A=kLC
L=
A =-lgT= k L C
k: extinction coefficient
L: length of the light path
C: concentration
When determinations are made, one must be sure that the
absorption produced is due to the particular substances, not
by the solvent and compounds in the reagents. The batch of
analysis must include the following solutions.
•Blank: This will help to exclude the absorption
due to reagents.
•Standard: it includes a solution of known
concentration of the substance which is going to
be determined in the test container.
•Test: it contains an unknown quantity of the
substance.
Standard contrast method
Calculate Conc. of unknown
• Let the conc. of standard = C1, and absorbance = A1
•
So,
A1 = klC1
• Let the conc. of unknown = C2, and absorbance = A2
•
So,
•
So,
•
Or,
•
A2 = klC2
A1/A2 = klC1 / klC2
C2 = [A2/A1] x C1
Ctest= [Atest/Astandard] x Cstandard
Standard Curve method
Absorbance at 280 nm
1.0
0.5
1
4
2
3
Concentration (mg/ml)
5
•There is some A vs. C where graph is linear.
•Avoid very high or low absorbencies when drawing a
standard curve.
•The best results are obtained with 0.1 < A < 1. Plot the
Absorbance vs. Concentration to get a straight line.
•NEVER extrapolate beyond point known where becomes
non-linear.
Structure of Spectrophotometer
Spectrophotometry
Spectrophotometer
Sample room
Cuvette holder
Sample Cuvette
Spectrophotometer
On/Off
Show
Wavelength
Sample
Room
Adjust
Wavelength
0%T
100%T/0A
Pull Rod of
Cuvette
Mode
How to operate Spectrophotometer ?
1. Turn on ，set wavelength ，warm-up for 20min
2. Respectively move sample solutions to cuvettes
Blank, Standard, Test
•
Height: 2/3~4/5
•
Hold the rough face ，
keep the smooth face tidy .
•
Test2
Test1
Put cuvettes into the
Standard
cuvette holder in the
Blank
proper order .
cuvette holder
Operating steps of Spectrophotometry
3. To “Blank”, mode “T” , press “100%T/0A”, Set T =100
or A=0.
4. Pull the pole once time, press “0%T”, Set T =0.
5. Repeat step “3” to “4”.
6. Change mode to “A”.
7. pull the pole second time, record A1; Third
time ,record A2; Forth time ,record A3.
Test2
Test2
Test1
Test2
Test1
Standard
Test2
Test1
Standard
Blank
Test1
Test2
Standard
Blank
Mode:
T 0% ~ 100%
A 0~∞
Standard
Test1
Blank
Standard
Blank
Determine
Blank
T
handle 0
set T 100% ： Blank, A=0
T
handle 1
set T 0% ：
A
handle2、3、4 assay A：
A=1.----Test 1、2、3
CuSO4
x% = ?
Methods:
1. Standard curve
2. Standard contrast
Reagents & Materials
•
•
•
•
•
•
•
5% CuSO4
X% CuSO4
dH2O
Test tubes
Pipettes
Spectrophotometer
Cuvette
Method
1. Standard curve method
Num
5%CuSO4 (ml)
H2O(ml)
1
2
3
1.00
2.00
3.00
4.00
3.00
2.00
4
5
4.00
5.00
1.00
0.00
6
X%CuSO4
5.00
0.00
C(%)
A
•Mix the contents of each tube, measure the
absorbance(A) of each tube at 650nm , setting zero
with distilled water.
A5
A4
A3
A2
A1
C1
C2 C3 C4 C5
Standard curve
2. Cuso4 (x%) 5ml , Ax
Ax
Cx
X% CuSO4: Ax=? Cx=?
2. Standard contrast method
Calculation:
5%CuSO4 --- the standard,
x% CuSO4,
As=?
Ax=?
As = k LCs
Ax = k LCx
Cx=?
Discussion
• Compare the two methods and the results,
which one is better? why?