Dr. Maha Al-Sedik. Objectives: Electromagnetic Radiation White light Beer’ s law ...

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Biochemical instrumental analysis-1 Dr. Maha Al-Sedik

Transcript of Dr. Maha Al-Sedik. Objectives: Electromagnetic Radiation White light Beer’ s law ...

Page 1: Dr. Maha Al-Sedik. Objectives:  Electromagnetic Radiation  White light  Beer’ s law  Spectrophotometer  Components of spectrophotometer  Types.

Biochemical instrumental analysis-1

Dr. Maha Al-Sedik

Page 2: Dr. Maha Al-Sedik. Objectives:  Electromagnetic Radiation  White light  Beer’ s law  Spectrophotometer  Components of spectrophotometer  Types.
Page 3: Dr. Maha Al-Sedik. Objectives:  Electromagnetic Radiation  White light  Beer’ s law  Spectrophotometer  Components of spectrophotometer  Types.

Objectives:

Electromagnetic Radiation

White light

Beer’ s law

Spectrophotometer

Components of spectrophotometer

Types of spectrophotometer

Page 4: Dr. Maha Al-Sedik. Objectives:  Electromagnetic Radiation  White light  Beer’ s law  Spectrophotometer  Components of spectrophotometer  Types.

Light is the most important example of

electromagnetic radiation.

Light moves in waves.

different types of light have different wavelengths.

Wave length: distance between 2 peaks and is

expressed by nanometers.

Electromagnetic Radiation

Page 5: Dr. Maha Al-Sedik. Objectives:  Electromagnetic Radiation  White light  Beer’ s law  Spectrophotometer  Components of spectrophotometer  Types.
Page 6: Dr. Maha Al-Sedik. Objectives:  Electromagnetic Radiation  White light  Beer’ s law  Spectrophotometer  Components of spectrophotometer  Types.

Do you think (a) or (b) has longer wave length and lowest amplitude?

Page 7: Dr. Maha Al-Sedik. Objectives:  Electromagnetic Radiation  White light  Beer’ s law  Spectrophotometer  Components of spectrophotometer  Types.

The human eye responds to radiant energy

between 380 and 750.

Sun light is mixture of radiant energy of

different wavelengths that eye recognize it as

white.

Page 8: Dr. Maha Al-Sedik. Objectives:  Electromagnetic Radiation  White light  Beer’ s law  Spectrophotometer  Components of spectrophotometer  Types.
Page 9: Dr. Maha Al-Sedik. Objectives:  Electromagnetic Radiation  White light  Beer’ s law  Spectrophotometer  Components of spectrophotometer  Types.

White light

Page 10: Dr. Maha Al-Sedik. Objectives:  Electromagnetic Radiation  White light  Beer’ s law  Spectrophotometer  Components of spectrophotometer  Types.

Colors & WavelengthsCOLOR WAVELENGTH (λ in nm)

Ultraviolet < 380

Violet 380 – 435

Blue 436 – 480

Greenish-blue 481 – 490

Bluish-green 491 – 500

Green 501 – 560

Yellowish-green 561 – 580

Yellow 581 – 595

Orange 596 – 650

Red 651 – 780

Near Infrared > 780

Vis

ible

Lig

ht

Page 11: Dr. Maha Al-Sedik. Objectives:  Electromagnetic Radiation  White light  Beer’ s law  Spectrophotometer  Components of spectrophotometer  Types.

Each object when exposed to light , It absorbs some of lights and transmit some others.

Page 12: Dr. Maha Al-Sedik. Objectives:  Electromagnetic Radiation  White light  Beer’ s law  Spectrophotometer  Components of spectrophotometer  Types.

Q- When do we see solution with green light?

A- if it transmits light maximally between 500 -560 ( green wave length) and absorbs light at other wave length.

Page 13: Dr. Maha Al-Sedik. Objectives:  Electromagnetic Radiation  White light  Beer’ s law  Spectrophotometer  Components of spectrophotometer  Types.

All colors absorbed except red color

Page 14: Dr. Maha Al-Sedik. Objectives:  Electromagnetic Radiation  White light  Beer’ s law  Spectrophotometer  Components of spectrophotometer  Types.

Beer’ s law

Beer’s Law talks about the relationship between

color and concentration.

Beer’s Law states that the concentration of

substance in solution is directly proportional to

the light absorbed by the solution or inversely

proportional to light transmitted within limits.

Page 15: Dr. Maha Al-Sedik. Objectives:  Electromagnetic Radiation  White light  Beer’ s law  Spectrophotometer  Components of spectrophotometer  Types.
Page 16: Dr. Maha Al-Sedik. Objectives:  Electromagnetic Radiation  White light  Beer’ s law  Spectrophotometer  Components of spectrophotometer  Types.
Page 17: Dr. Maha Al-Sedik. Objectives:  Electromagnetic Radiation  White light  Beer’ s law  Spectrophotometer  Components of spectrophotometer  Types.

A α b x c

A : absorbance.α : directly proportional .b : light path in cm .c : concentration of substance.© : constant

A=© x b x

c

Page 18: Dr. Maha Al-Sedik. Objectives:  Electromagnetic Radiation  White light  Beer’ s law  Spectrophotometer  Components of spectrophotometer  Types.

If we suppose that I have two solution one with known concentration we will name it standard (s) . The second with unknown concentration and we will name it unknown (u).

As = © x bs x csAu = © x bu x cu

©= As /bs x cs©= Au /bu x cu

As /bs x cs=Au /bu x cu

Page 19: Dr. Maha Al-Sedik. Objectives:  Electromagnetic Radiation  White light  Beer’ s law  Spectrophotometer  Components of spectrophotometer  Types.

As /bs x cs=Au /bu x cu

B ….light path in cm is constant

As / cs = Au / cu

cu = Au x cs / As

Page 20: Dr. Maha Al-Sedik. Objectives:  Electromagnetic Radiation  White light  Beer’ s law  Spectrophotometer  Components of spectrophotometer  Types.
Page 21: Dr. Maha Al-Sedik. Objectives:  Electromagnetic Radiation  White light  Beer’ s law  Spectrophotometer  Components of spectrophotometer  Types.

Beer’ s law is followed only if the following

conditions are met :

The light must be monochromatic.

The solute concentration is within given limits.

A chemical reaction does not occur between the

molecule of interest and another solute.

Page 22: Dr. Maha Al-Sedik. Objectives:  Electromagnetic Radiation  White light  Beer’ s law  Spectrophotometer  Components of spectrophotometer  Types.

Spectrophotometer

Page 23: Dr. Maha Al-Sedik. Objectives:  Electromagnetic Radiation  White light  Beer’ s law  Spectrophotometer  Components of spectrophotometer  Types.

Spectrophotometric techniques

Photometry: measurement of intensity of

light.

Spectrophotometry: measurement of

intensity of light at selected wave length.

Page 24: Dr. Maha Al-Sedik. Objectives:  Electromagnetic Radiation  White light  Beer’ s law  Spectrophotometer  Components of spectrophotometer  Types.

Principle:

The solutions of many compounds have

characteristic colors.

The intensity of a color is directly proportional to the

concentration of the compound.

Light is absorbed by dissolved substances.

The concentration of the substance is directly

proportional to the amount of light absorbed.

Page 25: Dr. Maha Al-Sedik. Objectives:  Electromagnetic Radiation  White light  Beer’ s law  Spectrophotometer  Components of spectrophotometer  Types.

Q- Is spectrophotometer quantitative or

qualitative technique ?

A- Both

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Page 27: Dr. Maha Al-Sedik. Objectives:  Electromagnetic Radiation  White light  Beer’ s law  Spectrophotometer  Components of spectrophotometer  Types.

Components of spectrophotometer:

Light source

Monochromator

Filter

Cuvet

Photo detector

Signal processor

Record

Page 28: Dr. Maha Al-Sedik. Objectives:  Electromagnetic Radiation  White light  Beer’ s law  Spectrophotometer  Components of spectrophotometer  Types.

Components of spectrophotometer:

Page 29: Dr. Maha Al-Sedik. Objectives:  Electromagnetic Radiation  White light  Beer’ s law  Spectrophotometer  Components of spectrophotometer  Types.

1-Light source:

Tungsten Filament Lamps: the most common

source of visible radiation.

Deuterium Lamps: also may be used.

Page 30: Dr. Maha Al-Sedik. Objectives:  Electromagnetic Radiation  White light  Beer’ s law  Spectrophotometer  Components of spectrophotometer  Types.

2-Monochromator:

Used as a filter: the monochromator will select a

narrow portion of the spectrum of a given source.

Monochromator may be filters or prism and grating.

Page 31: Dr. Maha Al-Sedik. Objectives:  Electromagnetic Radiation  White light  Beer’ s law  Spectrophotometer  Components of spectrophotometer  Types.

3-Cuvet:

It may be Glass or plastic.

It may be round, square or

rectangular.

It must be clean and clear.

The most popular is 1 cm light

path.

Cleaned in solution of

concentrated HCL : water :

ethanol = 1 :3 :4

Page 32: Dr. Maha Al-Sedik. Objectives:  Electromagnetic Radiation  White light  Beer’ s law  Spectrophotometer  Components of spectrophotometer  Types.

3- Photodetector:

Photodetector transforms light to electrical

impulses.

When light strikes the photodetector , electrons are

exited in the semiconducting material .

The electricity is directly proportional to the

intensity of light.

Page 33: Dr. Maha Al-Sedik. Objectives:  Electromagnetic Radiation  White light  Beer’ s law  Spectrophotometer  Components of spectrophotometer  Types.

4- SIGNAL PROCESSOR:

Digital signals from the blank are subtracted from

standered and uknown

5- RECORD:

Gives the net results on the monitor.

Page 34: Dr. Maha Al-Sedik. Objectives:  Electromagnetic Radiation  White light  Beer’ s law  Spectrophotometer  Components of spectrophotometer  Types.

General rules and precautions in spectrophotometer:

A blank should be used for each set of determinations.

Cuvet should be clean, clear and dry.

For each size of cuvet there is a specified minimum

volume.

The solution must be free of air.

Cuvet must be put with the same side facing light

every time.

Cuvet must be covered before each reading.

Page 35: Dr. Maha Al-Sedik. Objectives:  Electromagnetic Radiation  White light  Beer’ s law  Spectrophotometer  Components of spectrophotometer  Types.

Types of spectrophotometer:

Single beam spectrophotometer.

Double beam spectrophotometer.

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Page 37: Dr. Maha Al-Sedik. Objectives:  Electromagnetic Radiation  White light  Beer’ s law  Spectrophotometer  Components of spectrophotometer  Types.
Page 38: Dr. Maha Al-Sedik. Objectives:  Electromagnetic Radiation  White light  Beer’ s law  Spectrophotometer  Components of spectrophotometer  Types.

http://www.youtube.com/watch?v=pxC6F7bK8CU

http://www.youtube.com/watch?v=QdufRwbkeKo

Page 39: Dr. Maha Al-Sedik. Objectives:  Electromagnetic Radiation  White light  Beer’ s law  Spectrophotometer  Components of spectrophotometer  Types.

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