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?

• 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.

The Electromagnetic Spectrum

= c / E = h

Spectroscopy

Spectral Distribution of Radiant Energy

 Wave Number (cycles/cm)

X-Ray UV Visible IR Microwave

200nm 400nm 800nm

WAVELENGTH(nm)

Transmission and Color

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

Absorbance and Complementary Colors

Two-Component Mixture

Example of a two-component mixture with little spectral overlap

Two-Component Mixture

Example of a two-component mixture with significant spectral overlap

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%)

Absorption Spectra of Hemoglobin Derivatives

Light Sources

 UV Spectrophotometer

1. Hydrogen Gas Lamp

2. Mercury Lamp

Visible Spectrophotometer

1. Tungsten Lamp

InfraRed (IR) Spectrophotometer

1. Carborundum (SIC)

Dispersion Devices

• Non-linear dispersion• Temperature sensitive

• Linear Dispersion• Different orders

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

Photomultiplier Tube Detector

Anode

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

The Photodiode Detector

• Wide dynamic range• Very good

signal/noise at high

light levels• Solid-state device

Schematic Diagram of a Photodiode Array

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

Conventional Spectrophotometer

Schematic of a conventional single-beam spectrophotometer

Conventional Spectrophotometer

Optical system of a double-beam spectrophotometer

Conventional Spectrophotometer

Optical system of a split-beam spectrophotometer

Definition of Resolution

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

Instrumental Spectral Bandwidth

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

Natural Spectral Bandwidth

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

Transmission Characteristics of Cell Materials

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

Cells

UV Spectrophotometer

Quartz (crystalline silica)

 Visible Spectrophotometer

Glass

 IR Spectrophotometer

NaCl

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

Cell Types I

Cell Types II

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

Transmittance and Concentration The Bouguer-Lambert Law

PathlengthConsteIIT 0/

Transmittance and Path Length: Beer’s Law

ionConcentratConsteIIT 0/

Concentration

The Beer-Bouguer-Lambert Law

cbIIIITA /log/loglog 00

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.

 

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

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

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

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

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

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.

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

• 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.

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

What concentration do you think the unknown sample is?

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

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

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)

Precision and Accuracy

Precision – Precision + Precision – Precision +

Accuracy – Accuracy – Accuracy + Accuracy +