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SPECTROMETRY

INTRODUCTION
ABSORPTIMETRIC METHODS
Analytical methods based on the
absorption of radiant energy
COLORIMETRIC METHODS
An absorptimetric methods which utilize
the visible region of the spectrum

The intensity of transmitted radiant energy
is a function of (related to) the
concentration of the chemical species
which absorbs the radiant energy
Colorimetric methods are usually used in
medicine and pharmacy in the
determination of glucose, ketone bodies,
occult blood, pH, and protein
Example Glucose determination in Urine
Urine sample paper(w/ glucose oxidase &
peroxidase enzyme and color producing
reagent)
Glucose + glucose oxidase gluconic
acid + H2O2
H2O2 + peroxidase + color producing
reagent
color change in paper
TERMINOLOGY IN
SPECTROMETRY
SPECTROMETRY
Methods of analysis which deals with the
measurement of spectra
Most common measurement: (1)
measurement of position(wavelength) in
the electromagnetic spectrum (2)
measurement of power of transmitted
fluorescent, reflected, or emitted energy
ELECTROMAGNETIC SPECTRUM
Define the complete system of
energy(radiant energy- sunlight, color,
radiowaves etc.) propagated in wave form
RADIANT ENERGY
c=
c velocity of light (3x10 cm/s)
frequency (Hz)
wavelength (cm)

Velocity of radiant energy in a given
medium is constant differences lie in
frequency and wavelength.
SPECTROPHOTOMETRY

Branch of spectrometry which embraces
the measurement of the absorption by
chemical species, of radiant energy of
definite, and narrow wavelength,
approximating monochromatic radiation
(radiation of a single wavelength




WAVELENGTH
the length of a complete wave or cycle,
from peak of one wave to the peak of the
next

UV 200 380 nm
Visible 380 780 nm
Near IR 780 3000 nm
Medium
IR
3 15 m
Far IR 15 300 m
Micrometer(m
)
10 cm
Nanometer(nm) 10 cm
Angstrom() 10 cm


WAVENUMBER
Number of waves per centimeter
1/wavelength(cm)

FREQUENCY
Number of complete cycles which pass a
given point per second(cps)
Expressed in hertz
1 Hertz = 1cps


CHROMOPHORE
Functional group which absorbs radiant
energy in the uv or visible region of the
spectrum
Ethylene, acetylene, aldehydes, ketones,
organic acids, azomethines

COLORIMETRY
Is a branch of spectrophotometry in which
the absorption measurement is made in
the visible region of the spectrum


SPECTROPHOTOMETER AND
SPECTROMETER
instrument with radiant energy dispersing
device(prism or diffraction grating) and
electronics which permit the measurement of
wavelength and radiant power
manually operated or automatic recording
instruments
COLORIMETER AND FILTER
PHOTOMETER
instrument with filter in replacement of prism
or diffraction grating and electronics to permit
measurement of radiant power
THEORIES OF RADIANT
ENERGY
FIRST THEORY
It states that energy in the
electromagnetic spectrum occurs in
bundles(photons or quanta)

E = h
E energy (erg)
frequency (Hz)
h Plancks constant (6.62x 10 erg-s)
SECOND THEORY
States that energy in electromagnetic
spectrum is propagated in wave form
c= or =c/
Equation which relates frequency or
wavelength to energy required to promote
an electron to another energy level:

E = hc/

The smaller the wavelength the larger the
energy

Initial Energy (E) = Electronic energy +
Vibrational energy + Rotational energy +
Translational energy

Translational energy is not involved in
spectroscopic studies
Electronic energy occurs in UV and visible
region
Vibrational and Rotational energy occurs in IR
region
FUNDAMENTAL LAWS
AND RELATED TERMS
OF
SPECTROPHOTOMETRY
BEERS LAW
States that the power of a transmitted radiant
beam decreases exponentially as a
concentration of the solution containing the
absorbing chemical species increases
arithmetically
LAMBERTS OR BOUGUERS LAW
States that the power of a transmitted radiant
beam decreases exponentially as a
concentration of the solution containing the
absorbing chemical species increases
arithmetically
BEER-LAMBERT OR BEER BOUGER
LAW
combination of the two laws above
TRANSMITTANCE (T)
P(solution)/P(blank)
ratio of the radiant power transmitted by
the solution to the radiant power
transmitted by the blank(solvent)
ABSORBANCE (A)
log 1/T or log [P(blank)/P(solution)]
Optical density, absorbancy, extinction
coefficient
CONCENTRATION (c)
concentration expressed in grams of
solute per liter of solvent (g/L)
ABSORPTIVITY (a)
value obtained by dividing Absorbance (A)
by the product concentration (g/L) and the
path length (cm)
MOLAR ABSORPTIVITY
value obtained by dividing Absorbance (A)
by the product concentration (moles/L)
and the path length (cm)
ABSORPTION SPECTRUM
Graph obtained when Absorbance or ay
function of absorbance is plotted against
wavelength
INSTRUMENTS USED IN
SPECTROPHOTOMETRY
Spectrophotometer and
Spectrometer have:
Radiant energy source
Radiant energy-dispersing device
Sample compartment
Associated electronics which permit
the measurement of radiant power
transmitted by the sample
Spectrophotometer and
Spectrometer
AUTOMATIC RECORDING
INSTRUMENT
Automatically scan the wavelength region
of interest and record absorbance or
some function of absorbance versus
wavelength
MANUALLY OPERATED
SPECTROPHOTOMETER
DIRECT READING
Bausch and Lomb Spectronic 20
340-950 nm (RANGE)
NULL BALANCE
Beckman DU-2 Spectrophotometer
190-1000nm (RANGE)
Beckman DU 800
Bausch and Lomb Spectronic
20
GENERAL PROCEDURE
Preparation of Spectral Absorbance
Curve (SA Curve) of the analyte
made by plotting the absorbance of
the analyte versus the wavelength
(manually operated 5nm increments
except for region of maximum 2nm
increments
Choosing the maximum wavelength
for the purpose of measuring
absorbance values of a series of
prepared reference solute
concentration(to obtain (10-75%T
accurate)
SA Curve
Choosing the wavelength at
maximum absorption(most sensitive)

Preparation of Beers Plot(plot of
absorbance values against series of
known solute concentration) to
determine unknown solute
concentration

Cunknown = (Aunknown/Astandard) Cstandard
Beers Plot