BIO-2
Spectrophotometry and
Plotting of Calibration Curve
PURPOSE
• To understand the principles of
spectrophotometry
• To understand the structure of
spectrophotometer
• To understand the 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
• In physics, the light refers to electromagnetic
wave, it exhibits properties of both wave nature
and particle nature.
• Spectral Distribution of Radiant Energy
380nm ~ 760nm
X-Ray
UV
200nm
Visible
400nm
IR
800nm
WAVELENGTH(nm)
Ultraviolet(UV)
Infrared(IR)
Microwave
Why solutions appear different color?
• When the white light (the polychromatic light) strikes the
object, different objects appear different color due to the
particular wavelength of light they absorbed.
• When the white light strike
a colored solution, certain
wavelengths can be
absorbed and the remainder
transmitted.
•The observed color is the
color to be transmitted.
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 selectively.
A
Wavelength of Maximal Absorption(525nm)
λ(nm)
•Absorption spectrum of potassium permanganate
(KMnO4) at four different concentrations
• The different concentration of potassium permanganate solutions
have similar spectrum and same λmax.
• The absorbance will increase if the concentration increase, which
can be used as the basis for quantitative analysis.
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)
• If all the light passes through a solution without
any absorption,
– then T=100%, and A=0.
• If all the light is absorbed without any
transmittance,
– then T=0, and A is infinite.
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=
Lambert-Beer law
A =-lgT= k L C
k: absorption coefficient
L: length of the light path
C: concentration
•The absorption coefficient is the
characteristic constant of the subjects.
The choice of conditions of
determination
• The choice of wavelength
– It is observed that at the wavelength of
maximum absorbance (λmax) the deviations
is minimal and the sensitive is higher.
– Due to this reason we generally select λmax
for a given sample to measure the
absorbance.
The choice of the range of
absorbance measurement
• It is proved by theory that when T=36.8%（or
A=0.434）the relative error of concentration is
smallest.
• To minimize the error, T must control to 20~65%
(or A= 0.2~0.7).
• So when design the experiment, you must
choose appropriate amount of sample and
proper type of cuvette to make the absorbance
among this range.
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
Sample
Room
Display
lamp
Of Mode
Show
Wavelength
Adjust
Wavelength
0%T
Pull Rod of
Cuvette
Mode (A, T)
100%T/
0A
How to operate Spectrophotometer ?
1. Turn on ，set wavelength ，warm-up for 20 min.
2. Respectively move sample solutions to cuvettes
Blank, Standard, Test
•
Height: 2/3~4/5
•
Hold the rough face ，
Test2
Test1
keep the smooth face tidy .
•
Put cuvettes into the
Standard
cuvette holder in the
Blank
proper order.
(Notice: Smooth side face to the light).
cuvette holder
Operating steps of Spectrophotometry
3. Set “Blank solution” face to the light, adjust Mode to
“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.
Determine the concentration of
unknown solution by spectophotometry
CuSO4
x% = ?
Methods:
1. Standard curve
2. Standard contrast
Reagents & Materials
•
•
•
•
•
•
•
5% CuSO4: dissolved 5 g anhydrous CuSO4
in 100 mL distilled water.
X% CuSO4
dH2O
Test tubes
Pipettes
Spectrophotometer
Cuvette
Method
1. Standard curve method
Num
5%CuSO4 (ml) dH2O(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 dH2O.
A5
A4
A3
A2
A1
Standard
C1
C2 C3 C4curve
C5
• The graph needs to be a best-fit straight line.
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?
• Why determine the absorbance of CuSO4 solution at
650 nm?
• If the absorbance of unknown CuSO4 solution above the
range of standard curve, how to determine the
concentration of this CuSO4 solution?