Biochemical instrumental analysis-1

Dr. Maha Al-Sedik

Objectives:

Electromagnetic Radiation

White light

Beer’ s law

Spectrophotometer

Components of spectrophotometer

Types of spectrophotometer

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

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

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.

White light

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

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

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.

All colors absorbed except red color

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.

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

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

As /bs x cs=Au /bu x cu

B ….light path in cm is constant

As / cs = Au / cu

cu = Au x cs / As

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.

Spectrophotometer

Spectrophotometric techniques

Photometry: measurement of intensity of

light.

Spectrophotometry: measurement of

intensity of light at selected wave length.

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.

Q- Is spectrophotometer quantitative or

qualitative technique ?

A- Both

                                                           

Components of spectrophotometer:

Light source

Monochromator

Filter

Cuvet

Photo detector

Signal processor

Record

Components of spectrophotometer:

1-Light source:

Tungsten Filament Lamps: the most common

source of visible radiation.

Deuterium Lamps: also may be used.

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.

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

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.

4- SIGNAL PROCESSOR:

Digital signals from the blank are subtracted from

standered and uknown

5- RECORD:

Gives the net results on the monitor.

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.

Types of spectrophotometer:

Single beam spectrophotometer.

Double beam spectrophotometer.

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

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

THANK YOU