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 solution
Spectrophotometry
7 Classifications of Spectroscopic Methods
-Classified according to the region of the electromagnetic spectrum
1.Gamma Rays
2.X- rays
3.Ultraviolet (UV
4.Visible
5.Infrared
6.Microwave
7.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 determined by the source that emits it and remains constant regardless of the medium traversed
frequency of a light wave or any wave of electromagnetic radiation
- Is the linear distance between successive maxima or minima of a wave
Wavelength (λ)
5 Wave Characteristics
amplitude
period, p
frequency, v
wavelength
velocity
Radiation velocity and wavelength both _______ as the radiation passes from a vacuum or from air to a denser medium. Frequency remains ________.
decrease, constant
The amplitude of the wave is the ______ of the electric field vector at the wave maximum, while the wavelength is the _______________ successive maxima.
length, distance between
Wavelength Units for Various Spectral Regions
Region: X-ray
Unit:
Definition:
Angstrom unit, Å
10^-10 m
Wavelength Units for Various Spectral Regions
Region: Ultraviolet/visible
Unit:
Definition:
Nanometer, nm
10^-9 m
Wavelength Units for Various Spectral Regions
Region: Infrared
Unit:
Definition:
Micrometer, µm
10^-6 m
-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
Both quantities are proportional to the square of the amplitude of the electric field
Intensity, Radiant Power (P)
States that the relationship between wavelength and energy are INVERSELY PROPORTIONAL.
Planck’s Law
Planck’s Law
E = hv
Where:
h =
V =
6.63 x 10^-34 J.s (constant)
frequency
Interaction of Radiation and Matter
•interesting and useful interactions in spectroscopy are those in which __________ occur between different energy levels of chemical species
transitions
Interaction of Radiation and Matter
•interactions, such as reflection, refraction, elastic scattering, interference, and diffraction, are often related to the ________________________ rather than to the unique energy levels of specific molecules or atoms
bulk properties of materials
Interaction of Radiation and Matter
•the specific types of interactions observed depend strongly on the _______ of the radiation used and the
energy, mode of detection
Electromagnetic Spectrum
Region
UV (ultraviolet) region
Wavelengths?
Wavelength
< 400 nm
400 – 700 nm
>700 nm
Spectroscopic Measurement
Samples are stimulated by applying energy (5)
1.Heat
2.Electrical energy
3.Light
4.Particles
5.Chemical reaction
Ground State -
Excited State -
Lowest Energy
Higher energy
Emission or chemilumi-nescence processes.
the sample is excited by applying _______________________________________ No radiant energy is used to produce excited states, and so, these are called non-radiative processes.
thermal, electrical, or chemical energy.
-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
both measures the radiant power emitted that can give the analytes’ identity and concentration
Emission Spectroscopy
Chemiluminescence Spectroscopy
Chemiluminescence is found in the _________________________.
An enzyme luciferase catalyzes the oxidative phosphorylation reaction of luciferin with adenosine triphosphate (ATP) to produce oxyluciferin, carbon dioxide, adenosine monophosphate (AMP), and light.
light emitted by a firefly
Chemiluminescence involving a biological or enzyme reaction is often termed _______________.
bioluminescence
is another familiar example of chemiluminescence.
The popular light stick
-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
1.Fluorescence
2.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)
Absorbance (A)
A = abc = 2 – log%T
Where:
A = Absorbance
a = molar absorptivity; absorptivity of the compound under standard conditions
b = length of light through the solution
c = concentration of absorbing molecules/solution
-It is the ratio of radiant energy transmitted (T) divided by the radiant energy incident (I) on the sample.
Percent Transmittance
%T = It/Io x 100
Where:
It= Transmitted light thru the sample Io= Intensity of light striking the sample
•The % T measured by commercial spectrophotometers is the ratio of the sample transmitted beam divided by the blank transmitted beam.
% T = sample beam signal / blank beam signal x 100
In actual practice, the light transmitted by blank is substituted for
Io
-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
FREQUENCY: The lower the wave frequency, the longer the __________
The wavelength is inversely related to frequency and energy: the shorter the wavelength, the ______ the frequency and energy and vice versa.
wavelength
higher
Analytical Technique
PHOTOMETRIC MEASUREMENT
SPECTROPHOTOMETRIC MEASUREMENT
Analytical Technique
measurement of light intensity without consideration of wavelength.
PHOTOMETRIC MEASUREMENT
Analytical Technique
: measures light intensity in a narrower wavelength. (spectrum of light).
SPECTROPHOTOMETRIC MEASUREMENT
Parts of Spectrophotometer
1.Light Source
2.Entrance Slit
3.Monochromator
4.Cuvette/ Sample Cell/ Analytical Cell 5.Exit Slit
6.Photodetector
7.Readout device
Part of Spectrophotometer
-Provides energy that the sample will modify or attenuate by absorption -The light is polychromatic ( all visible wavelength is present)
Light Source
Part of Spectrophotometer
- emits radiation that changes in intensity
- most common
ex:
_________ Light Bulb – most commonly used light source in the visible and near infrared region
Continuum Source
Tungsten
Part of Spectrophotometer
- 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
- Mercury Arc
- Nernst Glower
- Globar
IR Spectrum >700 nm
Part of Spectrophotometer
-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
Part of Spectrophotometer
-Isolates specific/individual wavelength of the light l
-DEGREE OF ISOLATION is affected the monochomator and the width of entance and exit slit
Monochromator
Different Monochromators
•FILTERS
•PRISM
•DIFFRACTION GRATINGS
Type of 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
Type of Monochromator
-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
- A broad spectrum light (halogen, incandescent) is shone through a sample.
- Some colors are absorbed more than others depending on its composition
- Diffraction gratings splits light into colors so they can be measured separately.
- A webcam measures each color and graphs their intensities. This is compared to known samples.
Diffraction Gratings
Part of Spectrophotometer
-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
Part of Spectrophotometer
Types of Cuvette/ Sample Cell/Analytical Cell
•Glass Cuvettes – for visible range •Quartz of Fused Silica – for UV range •Borosilicate – 350 – 2000 nm
Part of Spectrophotometer
-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 Slit
– the range of wavelength between point at which transmittance is one half peak transmittance
BAND PASS
Part of Spectrophotometer
-Converts transmitted radiant an equivalent amount of electrical energy
Photodetector
Part of Spectrophotometer
Types of Photodetector
PHOTOCELL
PHOTOTUBE
PHOTOTRANSISTOR/PHOTODIODE PHOTOMULTIPLIER TUBE
Part of Spectrophotometer
Type of Photodetector
– simplest and least expensive, low sensitivity and fatigue are the downfall of this, needs frequent replacement
PHOTOCELL
Part of Spectrophotometer
Type of Photodetector
– contains anode and cathode in a tube, it gives off electron when energy strikes it •
PHOTOTUBE
Part of Spectrophotometer
Type of Photodetector
– more sensitive than vacuum phototube but less sensitive than Photomultiplier
PHOTOTRANSISTOR/PHOTODIODE
Part of Spectrophotometer
Type of Photodetector
– 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
Part of Spectrophotometer
Type of Photodetector
-It displays the output of the detection system
Read Out Device
Part of Spectrophotometer
Type of Photodetector
Kinds of Read Out Device
•Galvanometer: the more electrical energy the more the hand will move •Ammeter
•Led Display
-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
Two Types of Double – Beam Spectrophotometer
•Double – Beam in Space
•Double – Beam in Time
Type of Double – Beam Spectrophotometer
- uses 2 photodetectors (for the sample beam and reference beam)
•Double – Beam in Space
Type of Double – Beam Spectrophotometer
- uses one photodetector and alternatively passes the monochromatic light through the sample cuvette and then reference cuvette using a chopper
•Double – Beam in Time
If a solution absorbs light of a certain color (2ndcolumn) the observed color of the solution is the
complementary color
-Measures the light (wavelength) emitted by a single atom burned in flame
-Principle: Excitation of electrons from lower to higher energy state -Light Source: Flame
-Method: Indirect Internal Standard Method
-Internal Standard: Lithium/ Cesium
– to correct for variations in flame and atomizer characteristics -It is used for the measurement of excited ions (sodium and potassium) -Flickering light indicates charges in the fuel reading of the instrument
Flame Emission Photometry FEP
-Measures the light absorbed by atoms dissociated by heat
-Light Source: Hallow – Cathode Lamp
-Principle: Element is not excited by merely dissociated from its chemical bond and place in an unionized, unexcited, ground state -It is used for the measurement of unexcited trace metals (calcium and magnesium)
-More sensitive than FEP; it is accurate, precise and very specific -Internal Standard is not need
– changes in aspiration have little effect on the number of ground state atoms
-An atomizer (nebulizer/graphite furnace) is used to convert ions to atoms; a chopper is used to modulate the light source
-Lanthanum or Strontium Chloride is added to samples to form stable complexes with phosphate to avoid calcium interference
Atomic Absorption Spectrophotometry
Turbidity readings on spectrophotometer are ______ in the blue region than in the red region of the spectrum
•A slight error in wavelength adjustment can introduce a ____________ error in absorbance reading.
greater
significant
- the wavelength indicated on thee control dial is the actual wavelength of light passed by the monochromator.
Wavelength Accuracy
– 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
