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?