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Spectrophotometry and Plott
ing of Calibration Curve
BIO-2
PURPOSE
• To understand the principles of spectrophotometry
• To understand the structure of spectrophotometer
• To understand the calibration curve and determine the concentration of CuSO4
What is the function of spectrophotometry?
What is spectrophotometry?
Why do we use it?
How does it work?
Questions
Spectrophotometry is the quantitative measurement of the reflection or transmission properties of a material as a function of wavelength.
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.
What is spectrophotometry?
What is the function of spectrophotometer?
Polychromatic light polychromatic light composed of more
than one wavelength , having or exhibiting
many colors.
Polychromatic light
Monochromatic light
light
monochromatic light Light of one color, having wavelengths
confined to an extremely narrow range.
SPECTRUM
• Spectral Distribution of Radiant Energy
X-Ray UV Visible IR Microwave
200nm 400nm 800nm
WAVELENGTH(nm)
380nm ~ 760nm
Infrared(IR)Ultraviolet(UV)
• In physics, the light refers to electromagnetic wave, it exhibits properties of both wave nature and particle nature.
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.
•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.
A
λ(nm)
Wavelength of Maximal Absorption(525nm)
Transmittance = T = (It / I0) ×100%
Absorbance= A = ? A=-lgT=lg(I0/It)
Transmittance and Absorbance
•When a ray of monochromatic light of initial intensity (Io) passes through a solution in a transparent vessel, some of the light is absorbed (Ia) so that the intensity of the transmitted light (It) is less than Io.
It
Ia
I0
• 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.
•As the cell thickness increases, It (transmitted intensity of light ) decreases.
LAWS OF ABSORBTION OF LIGHT
Glass cell filled with concentration of solution (C)
IILight
0 It
LAWS OF ABSORBTION OF LIGHT
Lambert’s law: length-dependent
I = Io e-kL or A=kLWhere ‘k’ is a constant, e = base of natural log L= 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
A =-lgT= k L C
k: absorption coefficientL: length of the light path C: concentration
Lambert-Beer law
•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 maxim
um absorbance (λmax) the deviations is mini
mal and the sensitive is higher.
– Due to this reason we generally select λmax f
or 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.
•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.
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.
Standard Contrast method
• Let the conc. of standard = C1, and absorbance = A1
• So, A1 = klC1
• Let the conc. of unknown = C2, and absorbance = A2
• So, A2 = klC2
• So, A1/A2 = klC1 / klC2
• Or, C2 = [A2/A1] x C1
• Ctest= [Atest/Astandard] x Cstandard
Calculate Conc. of unknown
1 2 3 4 5
1.0
0.5
Concentration (mg/ml)
Absorbance at 280 nm
•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.
Standard Curve method
Structure of Spectrophotometer
Spectrophotometry
Spectrophotometer
Sample Cuvette
Sample room
Cuvette holder
Spectrophotometer
Sample Room
Pull Rod of Cuvette
Mode (A, T) 100%T/ 0
A
0%T
Adjust Wavelength
Show Wavelength
Display lamp
Of Mode
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 , keep the smooth face tidy .
• Put cuvettes into the
cuvette holder in the
proper order.Blank
Standard
Test1
Test2
cuvette holder (Notice: Smooth side face to the light).
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.
Operating steps of Spectrophotometry
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
1. Standard curve methodNum 5%CuSO4 (ml) dH2O(ml) C(%) A
1 1.00 4.00
2 2.00 3.00
3 3.00 2.00
4 4.00 1.00
5 5.00 0.00
6X%CuSO4
5.00 0.00
Method
•Mix the contents of each tube, measure the absorbance (A) of each tube at 650nm , setting zero with dH2O.
C1 C2 C3 C4 C5
A1
A2
A3
A4
A5
Standard curve
• 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
As = k LCs
Ax = k LCx
x% CuSO4, Ax=?
5%CuSO4 --- the standard, As=?
Cx=?
Calculation:
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?