spec basics 2008

8/3/2019 Spec Basics 2008

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UV-visible spectroscopy

How They Work


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What is Spectroscopy?

The study of molecular structure anddynamics through the absorption,emission and scattering of light.


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What is Light?

According toMaxwell, light is anelectromagnetic field

characterized by afrequency f, velocityv, and wavelength .Light obeys therelationship

f = v / .


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The Electromagnetic

Spectrum

n = c /lE = hn


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Spectroscopy

Spectral Distribution of Radiant Energy

Wave Number (cycles/cm)

X-Ray UV Visible IR Microwave

200nm 400nm 800nm

WAVELENGTH(nm)


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Transmission and Color

The human eye sees the complementary color to that which is

absorbed


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Absorbance and

Complementary Colors


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Two-Component Mixture

Example of a two-component mixture with little spectraloverlap


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Two-Component Mixture

Example of a two-component mixture with significantspectral overlap


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Influence of 10%

Random Error

Influence on the calculated concentrations Little spectral overlap: 10% Error Significant spectral overlap: Depends on similarity, can be much higher (e.g. 100%)


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Absorption Spectra of

Hemoglobin Derivatives


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Light Sources

UV Spectrophotometer

1. Hydrogen Gas Lamp

2. Mercury Lamp

Visible Spectrophotometer

1. Tungsten Lamp

InfraRed (IR) Spectrophotometer

1. Carborundum (SIC)


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Dispersion Devices

Non-linear dispersion Temperature sensitive

Linear Dispersion Different orders


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Dispersion ofpolychromatic light with aprism

Prism - spray out the spectrum and choose the certainwavelength (l) that you want by moving the slit.

Polychromatic

Ray

Infrared

Red

OrangeYellow

Green

Blue

Violet

Ultraviolet

monochromatic

Ray

SLIT

PRISM

Polychromatic Ray Monochromatic Ray


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Photomultiplier Tube

Detector

Anode

High sensitivity atlow light levels

Cathode material

determines spectralsensitivity

Good signal/noise

Shock sensitive


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The Photodiode Detector

Wide dynamic range Very good

signal/noise at high

light levels

Solid-state device


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Schematic Diagram of a

Photodiode Array

Same characteristics

as photodiodes Solid-state device

Fast read-out cycles


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Conventional

Spectrophotometer

Schematic of a conventional single-beam spectrophotometer


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Conventional

Spectrophotometer

Optical system of a double-beam spectrophotometer


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Conventional

Spectrophotometer

Optical system of a split-beam spectrophotometer


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Instrumental Spectral

Bandwidth

The SBW is defined as the width, at half the maximum intensity, ofthe band of light leaving the monochromator


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Natural Spectral

Bandwidth

The NBW is the width of the sample absorption band at half theabsorption maximum


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Transmission

Characteristics of Cell

Materials

Note that all materials exhibit at least approximately 10% loss in

transmittance at all wavelengths


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Cells

UV Spectrophotometer

Quartz (crystalline silica)

Visible Spectrophotometer

Glass

IR Spectrophotometer

NaCl


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Cell Types II

Micro cell (a) for very small volumes and flow-through cell (b)for automated applications


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Transmittance and

Concentration

The Bouguer-Lambert Law

PathlengthConst

eIIT

0/


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Transmittance and Path

Length: Beers Law

ionConcentratConsteIIT

0/

Concentration


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The Beer-Bouguer-

Lambert Law

cbIIIITA /log/loglog 00


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BEER LAMBERT LAW

Glass cell filled with

concentration of solution (C)

IILight0

As the cell thickness increases, the intensity of I(transmitted intensity of light ) decreases.


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A CL = KCL by definition and it is calledthe Beer Lambert Law.

A = KCL

K = Specific Extinction Coefficient ---- 1 g of

solute per liter of solution

A = ECL

E = Molar Extinction Coefficient ----Extinction Coefficient of a solution containing1g molecule of solute per 1 liter of solution


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E =Absorbance x Liter

Moles x cm

E differs from K (Specific extinction Coefficient) bya factor of molecular weight.

UNITS

A = ECL

A = No unit (numerical number only)

E =Liter

Cm x Mole


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L = Cm

C = Moles/Liter

A = KCL

A = No unit C = Gram/Liter L = Cm

A = ECL = ( LiterCm x Mole

) x Mole

Literx Cm

K=

Liter

Cm Gram

A = KLC = (Liter

Cm x Gram

Gram

Literx Cm) x


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STEPS IN DEVELOPING ASPECTROPHOTOMETRICANALYTICAL METHOD1. Run the sample for

spectrum

2. Obtain a monochromaticwavelength for themaximum absorptionwavelength.

3. Calculate the concentrationof your sample using BeerLambert Equation: A = KCL

Wavelength (nm)

Absorbance

0.0

2.0

200 250 300 350 400 450


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Slope of Standard Curve = A

C

1 2 3 4 5

1.0

0.5

Concentration (mg/ml)

Absorbance at 280 nm

There is some A vs. C where graph is linear.

NEVER extrapolate beyond point known wherebecomes non-linear.


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SPECTROMETRIC ANALYSIS USING

STANDARD CURVE

1 2 3 4

0.4

0.8

1.2

Absorbance at 540 nm

Conc entration (g/l) glucose

Avoid very high or low absorbencies when drawing astandard curve. The best results are obtained with 0.1 < A

< 1. Plot the Absorbance vs. Concentration to get astrai ht line


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Every instrument has a useful range for aparticular analyte.

Often, you must determine that rangeexperimentally.

This is done by making a dilution series of

the known solution. These dilutions are used to make a

working curve.


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In this graph, values above A=1.0 are not linear. If we

use readings above A=1.0, graph isnt accurate.


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The best range of this spectrophotometer is A=0.1 to

A=1.0, because of lower errors. A=0.4 is best.


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Relating Absorbance and

Transmittance

Absorbance rises linearly withconcentration. Absorbance ismeasured in units.

Transmittance decreases in a non-linear fashion.

Transmittance is measured as a %.

Absorbance = log10

(100/% transmittance)


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Precision and Accuracy

Precision Precision + Precision Precision +

Accuracy Accuracy Accuracy + Accuracy +

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