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UV-visible spectroscopy How They Work

UV-visible spectroscopy

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UV-visible spectroscopy. How They Work. What is Spectroscopy?. The study of molecular structure and dynamics through the absorption, emission and scattering of light. What is Light?. - PowerPoint PPT Presentation

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Page 1: UV-visible spectroscopy

UV-visible spectroscopy

How They Work

Page 2: UV-visible spectroscopy

What is Spectroscopy?

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

Page 3: UV-visible spectroscopy

What is Light?

• According to Maxwell, light is an electromagnetic field characterized by a frequency f, velocity v, and wavelength λ. Light obeys the relationship

f = v / λ.

Page 4: UV-visible spectroscopy

The Electromagnetic Spectrum

= c / E = h

Page 5: UV-visible spectroscopy

Spectroscopy

Spectral Distribution of Radiant Energy

 Wave Number (cycles/cm)

X-Ray UV Visible IR Microwave

200nm 400nm 800nm

WAVELENGTH(nm)

Page 6: UV-visible spectroscopy

Transmission and Color

The human eye sees the complementary color to that which is absorbed

Page 7: UV-visible spectroscopy

Absorbance and Complementary Colors

Page 8: UV-visible spectroscopy

Two-Component Mixture

Example of a two-component mixture with little spectral overlap

Page 9: UV-visible spectroscopy

Two-Component Mixture

Example of a two-component mixture with significant spectral overlap

Page 10: UV-visible spectroscopy

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%)

Page 11: UV-visible spectroscopy

Absorption Spectra of Hemoglobin Derivatives

Page 12: UV-visible spectroscopy

Light Sources

 UV Spectrophotometer

1. Hydrogen Gas Lamp

2. Mercury Lamp

Visible Spectrophotometer

1. Tungsten Lamp

InfraRed (IR) Spectrophotometer

1. Carborundum (SIC)

Page 13: UV-visible spectroscopy

Dispersion Devices

• Non-linear dispersion• Temperature sensitive

• Linear Dispersion• Different orders

Page 14: UV-visible spectroscopy

Dispersion of polychromatic light with a prism

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

Polychromatic Ray

Infrared

RedOrange

Yellow

Green

Blue

Violet

Ultraviolet

monochromatic Ray

SLIT

PRISM

Polychromatic Ray

Monochromatic Ray

Page 15: UV-visible spectroscopy

Photomultiplier Tube Detector

Anode

• High sensitivity at low light levels• Cathode material determines spectral sensitivity• Good signal/noise• Shock sensitive

Page 16: UV-visible spectroscopy

The Photodiode Detector

• Wide dynamic range• Very good

signal/noise at high

light levels• Solid-state device

Page 17: UV-visible spectroscopy

Schematic Diagram of a Photodiode Array

• Same characteristics as photodiodes• Solid-state device• Fast read-out cycles

Page 18: UV-visible spectroscopy

Conventional Spectrophotometer

Schematic of a conventional single-beam spectrophotometer

Page 19: UV-visible spectroscopy

Conventional Spectrophotometer

Optical system of a double-beam spectrophotometer

Page 20: UV-visible spectroscopy

Conventional Spectrophotometer

Optical system of a split-beam spectrophotometer

Page 21: UV-visible spectroscopy

Definition of Resolution

Spectral resolution is a measure of the ability of an instrument to differentiate between two adjacent wavelengths

Page 22: UV-visible spectroscopy

Instrumental Spectral Bandwidth

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

Page 23: UV-visible spectroscopy

Natural Spectral Bandwidth

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

Page 24: UV-visible spectroscopy

Transmission Characteristics of Cell Materials

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

Page 25: UV-visible spectroscopy

Cells

UV Spectrophotometer

Quartz (crystalline silica)

 Visible Spectrophotometer

Glass

 IR Spectrophotometer

NaCl

Page 26: UV-visible spectroscopy

Open-topped rectangular standard cell (a) and apertured cell (b) for limited sample volume

Cell Types I

Page 27: UV-visible spectroscopy

Cell Types II

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

Page 28: UV-visible spectroscopy

Transmittance and Concentration The Bouguer-Lambert Law

PathlengthConsteIIT 0/

Page 29: UV-visible spectroscopy

Transmittance and Path Length: Beer’s Law

ionConcentratConsteIIT 0/

Concentration

Page 30: UV-visible spectroscopy

The Beer-Bouguer-Lambert Law

cbIIIITA /log/loglog 00

Page 31: UV-visible spectroscopy

BEER LAMBERT LAW

Glass cell filled with concentration of solution (C)

IILight

0

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

Page 32: UV-visible spectroscopy

 

R- Transmittance

R = I0 - original light intensity

I- transmitted light intensity

 

% Transmittance = 100 x

Absorbance (A) or optical density (OD) = Log

= Log = 2 - Log%T

Log is proportional to C (concentration of solution) and is also proportional to L (length of light path

through the solution).

I

I0

I

I0

I0

I

1

T

I

I0

Page 33: UV-visible spectroscopy

A CL = KCL by definition and it is called the 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 containing 1g molecule of solute per 1 liter of solution

Page 34: UV-visible spectroscopy

E =Absorbance x Liter

Moles x cm

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

UNITS

  A = ECL

A = No unit (numerical number only)

E =

Liter

Cm x Mole

Page 35: UV-visible spectroscopy

L = Cm

C = Moles/Liter

A = KCL

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

A = ECL = (Liter

Cm x Mole) x

Mole

Literx Cm

K=Liter

Cm Gram

A = KLC = (Liter

Cm x Gram

Gram

Literx Cm) x

Page 36: UV-visible spectroscopy

STEPS IN DEVELOPING A SPECTROPHOTOMETRIC ANALYTICAL METHOD

1. Run the sample for spectrum

2. Obtain a monochromatic wavelength for the maximum absorption wavelength.

3. Calculate the concentration of your sample using Beer Lambert Equation: A = KCL

Wavelength (nm)

Absorbance

0.0

2.0

200 250 300 350 400 450

Page 37: UV-visible spectroscopy

Slope of Standard Curve = AC

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 where becomes non-linear.

Page 38: UV-visible spectroscopy

SPECTROMETRIC ANALYSIS USING STANDARD CURVE

1 2 3 4

0.4

0.8

1.2Absorbance at 540 nm

Concentration (g/l) glucose

Avoid very high or low absorbencies when drawing a standard curve. The best results are obtained with 0.1 < A < 1. Plot the Absorbance vs. Concentration to get a straight line

Page 39: UV-visible spectroscopy

• Every instrument has a useful range for a particular analyte.

• Often, you must determine that range experimentally.

• This is done by making a dilution series of the known solution.  

• These dilutions are used to make a working curve.

Page 40: UV-visible spectroscopy

Make a dilution series of a known quantity of analyte and measure the Absorbance. Plot concentrations v. Absorbance.

Page 41: UV-visible spectroscopy

What concentration do you think the unknown sample is?

Page 42: UV-visible spectroscopy

In this graph, values above A=1.0 are not linear. If we use readings above A=1.0, graph isn’t accurate.

Page 43: UV-visible spectroscopy

The best range of this spectrophotometer is A=0.1 to A=1.0, because of lower errors. A=0.4 is best.

Page 44: UV-visible spectroscopy

Relating Absorbance and Transmittance• Absorbance rises linearly with

concentration. Absorbance is measured in units.

• Transmittance decreases in a non-linear fashion.

• Transmittance is measured as a %.

• Absorbance = log10 – (100/% transmittance)

Page 45: UV-visible spectroscopy
Page 46: UV-visible spectroscopy

Precision and Accuracy

Precision – Precision + Precision – Precision +

Accuracy – Accuracy – Accuracy + Accuracy +