SPECTROPHOTOMETRY Flashcards
Study that observe how radiated matter and energy interact with each
other
Spectroscopy
It involves measurement of the light transmitted by a solution to determine
the concentration of the light – absorbing substance in the solutio
Spectrophotometry
Classification of Spectroscopic Methods
- Gamma Rays
- X- rays
- Ultraviolet (UV
- Visible
- Infrared
- Microwave
- Radio – frequency (RF)
provide the most widely used tools for
the elucidation of molecular structure as well as the quantitative
and qualitative determination of both inorganic and organic
compounds
Spectrochemical Methods
is a form of energy that is transmitted through space at enormous
velocities
- described as a wave with properties of wavelength, frequency, velocity,
and amplitude
- treated as discrete packets of energy or particles called photons or
quanta
Electromagnetic Radiation
It is a vector quantity of an electromagnetic wave that provides a measure of the electric of magnetic field strength at a maximum in the wave
Amplitude
-It is the time in seconds of electromagnetic wave for successive maxima or minima to pass a point in space
Period (p)
It is the number of oscillation that occur in one second
- oscillations of the electric field vector per unit time and is equal to 1/p
Frequency (v)
- Is the linear distance between successive maxima or minima of a wave
Wavelength (λ)
Is the energy of a beam the reaches a given area per unit time
- Unit is in Watts (W
Radiant Power (P)
Is the radiant power-per-unit solid angle
Intensity
States that the relationship between wavelength and energy are
INVERSELY PROPORTIONAL.
Planck’s Law:
E = hv
Planck’s Law:
<400 nm wavelength
UV region
400 - 700 nm
visible spectrum
> 700 nm
infrared region
Samples are stimulated by applying energy
- Heat
- Electrical energy
- Light
- Particles
- Chemical reaction
Lowest Energy
Ground State
Higher energy
Excited State
Refers to the methods in which the stimulus is heat or electrical energy
Emission Spectroscopy
- Refers to excitation of the analyte by a chemical reaction
Chemiluminescence Spectroscopy
s found in the light emitted by a firefly
Chemiluminescence
Chemiluminescence involving a biological or enzyme reaction is often
termed
bioluminescence
Amount of light absorbed is measured as a function of wavelength
- Absorption measurement can give both qualitative and quantitative
information about the sample
Absorption Spectroscopy
The emission of photons is measured following absorption
Photoluminescence Spectroscopy
Forms of Photoluminescence
- Fluorescence
- Phosphorescence
States that the concentration of the unknown substance is directly
proportional to the absorbed light (absorbance or optical density) and
inversely proportional to the amount of transmitted light (%
Transmittance).
- Mathematically establishes the relationship between concentration and
absorbance
Beer’s Law
It is the amount of light absorbed
- It is proportional to the inverse log of transmittance
- Mathematically derived from %T (% transmittance)
Absorbance (A)
It is the ratio of radiant energy transmitted (T) divided by the radiant
energy incident (I) on the sample.
Percent Transmittance
Is an apparatus for measuring the
intensity of light in a part of the
spectrum, especially as transmitted
or emitted by a particular substances
Spectrophotometer
The distance between 2 successive peaks; It is expressed in
terms of nanometer (nm)
WAVELENGHT
: measurement of light intensity without
consideration of wavelength
PHOTOMETRIC MEASUREMENT
measures light intensity in a
narrower wavelength. (spectrum of light)
SPECTROPHOTOMETRIC MEASUREMENT
Parts of Spectrophotometer
- Light Source
- Entrance Slit
- Monochromator
- Cuvette/ Sample Cell/ Analytical Cell
- Exit Slit
- Photodetector
- Readout device
Provides energy that the sample will
modify or attenuate by absorption
- The light is polychromatic ( all visible
wavelength is present)
Light Source
emits radiation that changes in intensity
- most common
- Continuum Source
most commonly used light source in the
visible and near infrared region
Tungsten Light Bulb
emits limited radiation and wavelength
- limited number of discrete line or bands of radiation
- Line Source
- Mercury Arc
- Deuterium Lamp
- Hydrogen Lamp
UV Spectrum <400 nm
Minimizes stray light
- Prevent entrance of scattered light
- “GATE” only permits the needed light
Entrance Slit
Wavelength outside the band
- Can cause absorbance error
- Stray light limits the maximum
absorbance that spectrophotometer can
achieve
- Most common cause of loss of linearity
at high analyte concentration
STRAY LIGHT
Isolates specific/individual
wavelength of the light
- DEGREE OF ISOLATION is
affected the the monochomator
and the with of entance and exit
sli
Monochromator
Simple least expensive , not precise but useful
- Made by placing semi – transparent silver films on both sides of dielectric
such as magnesium fluoride produce monochromatic light based on the
principle of constructive interference of light waves.
- Usually pass a wide band of radiant energy and have an ion transmittance
of the selected wavelength
Colored Filters
Wedge – shaped pieces of glass, quarts, or sodium chloride
- A narrow light focused on a prism is refracted as it enters more dense
glass
- Can be rotated allowing only the desired wavelength to pass through exit
slit
Prisms
“Most Commonly Used”, better resolution than prism
- Made by cutting grooves ( parallel groves) or slit into an aluminized
surface of a flat pieceof crown glass - wavelengths are bent as they pass a
sharp corner
Diffraction Gratings
the breaking up of a ray of light into component
wavelengths based on the principle that WAVELENGTHS BENDS AS THEY
PASS A SHARP CORNER
DIFFFRACTION
It holds the solution whose concentration is to be measured
- It should be scratched free which can cause erroneous result
- Most common is rectangular shape, it is easier to maintain the length of
light
Cuvette/ Sample Cell/Analytical Cell
It controls the with of light beam (band pass). It only allows a fraction of
the spectrum to reach the sample cuvette.
- Spectral purity of the spectrophotometer is reflected by the band pass –
the narrower the band pass, the greater the resolution.
- Accurate absorbance measurement requires a band pass < 1/5 the natural
band pass of the spectrophotometer.
- The degree of wavelength isolation is a function of the type of device used
and the with of entrance and exit slit
Exit
– the range of wavelength between point at which
transmittance is one half peak transmittance
BAND PASS
Converts transmitted radiant energy into an equivalent amount of electrical
energy
Photodetector
– simplest and least expensive, low sensitivity and fatigue are the
downfall of this, needs frequent replacemen
PHOTOCELL
contains anode and cathode in a tube, it gives off electron when
energy strikes it
PHOTOTUBE
more sensitive than vacuum phototube
but less sensitive than Photomultiplie
PHOTOTRANSISTOR/PHOTODIODE
most common, detect wide range (Visible and UV)
- 200 x more sensitive, it amplifies radiant energy
- detect very low light energy and quick bust of light
PHOTOMULTIPLIER TUBE
It displays the output of the detection system
Read Out Device
it is an important instrument that splits the monochromatic light into two
components
- One beam passes through the sample and the other through a reference
solution of blank
- The additional beam corrects for variation in light source intensity
- The absorbance of the sample can be recorded directly as the electrical
output of the sample beam
Double – Beam Spectrophotometer
uses 2 photodetectors (for the sample beam and reference beam)
- Double – Beam in Space
uses one photodetector and alternatively passes the
monochromatic light through the sample cuvette and then reference cuvette
using a chopper
- Double – Beam in Time
Measures the light (wavelength) emitted by a single atom burned in
flame
Flame Emission Photometry FEP
Measures the light absorbed by atoms dissociated by heat
Atomic Absorption Spectrophotometry
Used to check wavelength accuracy
Didymium or Holmium Oxide Filter
Verify absorbance
accuracy on linearity
Neutral Density filters and Dichromate Solution
contain all the components of the solution to be
analyzed except to the one compound being tested
Blank Solution
corrects absorbance caused by the color of the reagents
Reagent blank
measures absorbance of the sample and reagent in the
absence of the end product and corrects the measurement for the optical
interference
Sample blank
