spectrophotometer prof.dr. moustafa m. mohamed vice dean faculty of allied medical science pharos...

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.