Spectrophotometer

Prof.Dr. Moustafa M. MohamedVice Dean

Faculty of Allied Medical SciencePharos University in Alexandria,

EGYPT

Absorption Spectroscopy

Sample

M

MM

M

M

MM

M

M

M

reflection scattering

h

in out

h

****

Absorption SpectroscopyGeneric Instrument

DetectorLightSource

Mono-chromator

Sample

P0 P

P0 = intensity of light into sampleP = intensity of light out of sample

Absorption Spectroscopy

• The more photons the sample absorbs, the lower the intensity (transmission) at the detector.

• Transmittance (T)

• 0 T 1• T is independent of P0• %T = T x 100

0P

PT

Absorption Spectroscopy

P0 = 1.00 P = 0.50 P = 0.25 P = 0.125

• sample with T = 50%

Absorption Spectroscopy

0

0.5

1

Concentration or Path Length

T

Absorption Spectroscopy

• Absorbance (A)

TlogT

1log

P

PlogA 0

TAT

T %log2 ;100

%

0 A

Absorption Spectroscopy

Concentration or Path Length

A

Beer’s Law

A = bcBeer’s Law

Light sources

• Different light sources for different regions of the spectrum

• UV/Vis– Tungsten Lamp 320-2,500 nm - run an

electrical current through a wire in vacuum– Deuterium arc lamp, 200-400 nm - electrical

discharge in D2

– Laser source

Monochromators• Mono - one; chromatic - color• Prisms and gratings - disperse (spread out)

light according to wavelength

Prism

h

Monochromators• n = d(sin I + sin r)

I = constant; therefore r

Ir

d

Sample Holders• Cuvettes

– flat surface best - better reproducibility– avoid fingerprints, dust, etc. on surface– must be transparent in region of interest

Sample Holders

• UV - quartz• Visible - glass, quartz• IR - NaCl

Detectors

• Detector converts incident light to an electrical signal that we can measure and process

Detectors

• Ideal Characteristics– sensitive – linear– flat response v. – stable– fast

Detectors - photo tube

• Good for UV and Visibleh

e-

-Vdynode

amplifier

Detectors - photo multiplier• Characteristics

– sensitive (single photons)– linear– flat response v. within limitations– stable w/ time (sensitive decreases over

time, weeks to months)– fast

Detectors - Array• Channel plate (similar to multiplier)• photodiode array (less sensitive than

multiplier)• charge coupled device (CCD) (more

sensitive than multiplier)• Do not need a monochromator

Colorimeter

• An optical electronic device that measures the color concentration of a substance in solution.

• Optical color filters are used to select a narrow wavelength.

• Basic colorimeter analysis involves the precise measurement of light intensity.

• Transmittance is defined as:I1= T * I0

I0 = Initial light intensity and I1= attenuated light intensity

• The results are displayed in percent optical color transmittance or absorption to indicate hemoglobin concentration.

percentxI

IT

o

1001

Single Beam Instrument

DetectorLightSource

(TungstenLamp)

Filter, Mono-

Chromator, Grating

Sample

QuartzCuvette

Photo-multiplier

Slit

Double Beam Instrument

Slit

Beam Chopper

Reference

Mirror Mirror

Semi-transparentMirror

TungstenLamp

Grating Photo-multiplier

Quartz Cuvette

Sample

Mirror

Colorimeter- filter photometer

• The concentration of the unknown solution can be found from the following relationship:

Cu= Cs Au/As

where

Cu= unknown concentration

Cs= standard concentration (for calibration)

Au= unknown absorbance

As= standard absorbance

• Light passes through an optical color filter, is focused by lenses on the reference and sample cuvettes and falls on the reference and sample photodetectors.

• The difference in voltage between the two detectors is increased by a dc amplifier and applied to a meter.

A calibration procedure is as follows:

1- Ground the amplifier input (V1) and adjust potentiometer (R1) for 0 volt

2- Remove the ground and place reference concentration in cuvettes 1 an 2

3- Adjust potentiometer R1 for 0 V4- Leave the reference concentration in cuvette

1 and replace cuvette 2 with a cuvette containing the sample

5-Read the unbalanced voltage on the meter in percent transmittance or absorbance units.