Spectrophotometry Flashcards by pola ry

-Study that observe how radiated matter and energy interact with each other

Spectroscopy

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It involves measurement of the light transmitted by a solution to determine the concentration of the light–absorbing substance in the solution

Spectrophotometry

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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)

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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

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-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

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-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

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-It is the time in seconds of electromagnetic wave for successive maxima or minima to pass a point in space

Period (p)

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-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)

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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

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Is the linear distance between successive maxima or minima of a wave

Wavelength (λ)

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5 Wave Characteristics

amplitude
period, p
frequency, v
wavelength
velocity

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Radiation velocity and wavelength both _______ as the radiation passes from a vacuum or from air to a denser medium. Frequency remains ________.

decrease, constant

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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

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Wavelength Units for Various Spectral Regions

Region: X-ray
Unit:
Definition:

Angstrom unit, Å
10^-10 m

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Wavelength Units for Various Spectral Regions

Region: Ultraviolet/visible
Unit:
Definition:

Nanometer, nm
10^-9 m

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Wavelength Units for Various Spectral Regions

Region: Infrared
Unit:
Definition:

Micrometer, µm
10^-6 m

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-Is the energy of a beam the reaches a given area per unit time
-Unit is in Watts (W)

Radiant Power (P)

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-Is the radiant power-per-unit solid angle

Intensity

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Both quantities are proportional to the square of the amplitude of the electric field

Intensity, Radiant Power (P)

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States that the relationship between wavelength and energy are INVERSELY PROPORTIONAL.

Planck’s Law

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Planck’s Law

E = hv

Where:
h =
V =

6.63 x 10^-34 J.s (constant)
frequency

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Interaction of Radiation and Matter

•interesting and useful interactions in spectroscopy are those in which __________ occur between different energy levels of chemical species

transitions

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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

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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

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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

-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

1. A broad spectrum light (halogen, incandescent) is shone through a sample. 2. Some colors are absorbed more than others depending on its composition 3. Diffraction gratings splits light into colors so they can be measured separately. 4. 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

Spectrophotometry Flashcards

(90 cards)