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Y1-2ND SEM
ANCH 111
SPECTROPHOTOMETRY
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Spectrophotometry
a technique used in chemistry and biology to measure the concentration of substances in a sample
measures the amount of light absorbed or transmitted by the sample at different wavelengths
HOW DOES IT WORK?
1.)
Spectrophotometers
use a light source to generate a beam of light, which passes through a sample.
HOW DOES IT WORK?
2.) The amount of light absorbed or transmitted by the sample at different wavelengths is detected by a
photodetector.
HOW DOES IT WORK?
3.) A spectrophotometer then produces a spectrum or graph of
absorbance
or
transmittance
versus wavelength.
Absorbance
measure of the amount of light absorbed by the sample
Absorbance
A=
-log
(
T
)
The absorbance is
directly
proportional to the concentration of the absorbing substance in the samples .
Transmittance
measure of the amount of light that passes through a sample and reaches the detector
Transmittance
T= 100%
-
A
Transmittance
is the complement of absorbance.
BEER'S LAW
A = ε x c x l
A =
Absorbance
ε =
molar
absorptivity
c =
concentration
of
sample
l =
path
length
BEER’S
LAW
this states that the concentration of a substance is directly proportional to the amount of light absorbed
Light
Source
Source of Light
Monochromator
Isolation of individual wavelengths of light
Sample
Cell
/
Cuvet
Holds samples, designed to ensure that light beam passes through the sample evenly
Photodetector
(
Photomultiplier
Tube
)
Convert the transmitted radiant energy into an equivalent amount of electrical energy
Display
/
Data
Output
Shows the measurement results, including absorbance or transmittance values at the selected wavelength
Entrance
Slit
prevents stray lights from entering
controlled light
Exit
Slit
isolates the desired wavelength from the monochromator
Blank
used in spectrophotometry to account for background signals or interferences
Blank
Used to measure background signal from solvent, reagents, or sample matrix
Blank
Distilled water
is the most commonly used
Blank
Reagent
Blank
- a solution that contains all the reagents used in the analysis, but no sample
Standard
solution that contains a known concentration of the analyte of interest
Concentration of Atom = (
Absorbance
of
Unknown
/
Absorbance
of
Standard
) x (
Concentration
of
Standard
)
CONCENTRATION -
Directly
Proportional - ABSORBANTS
CONCENTRATION & ABSORBANTS -
Inversely
Proportional - TRANSMITTANCE