(LEC) MODULE 3.1: UV-VIS SPECTROMETRY

UV-Visible absorption

a process where a molecule absorbs ultraviolet or visible light that excites electrons (makes them high energy). This energy causes an electronic transition from a ground state (non excited) to an excited state.

1. 100nm - 400nm

2. 400nm - 700nm

1. UV range

2. Visible light range

1. 400-420 nm

2. 420-440 nm

3. 440-490 nm

4. 480-570 nm

5. 570-585 nm

6. 585-620 nm

7. 620-780 nm

Ranges:

1. Violet

2. Indigo

3. Blue

4. Green

5. Yellow

6. Orange

7. Red

1. Continous Spectrum

2. Emission Spectrum

3. Absorption Spectrum

Types of Electromagnetic Spectrum (3)

Continous Spectrum

contains all wavelengths of light in a certain range. Hot, dense light sources like stars, for example, emit a nearly continuous spectrum of light, which travels out in all directions and interacts with other materials in space. The broad range of colors that a star emits depends on its temperature.

Emission Spectrum

inverse of an absorption spectrum. An emission spectrum is mostly dark with bright lines of color known as emission lines. Emission lines also correspond to specific atoms. Each atom has a specific pattern of colors that it emits. In fact, the wavelengths of an atom’s emission lines are exactly the same as the wavelengths of its absorption lines.

An absorption spectrum looks like a continuous spectrum, but with some colors significantly dimmer than others, or nearly missing. These missing colors appear as black lines known as absorption lines. As you might have guessed, absorption lines are caused by absorption: When starlight passes through a material—say a dense gas—atoms and molecules in the gas absorb some wavelengths.

• Inversely proportional with emission spectrum

Electronic Transitions

The absorption of light by a sample in the ultraviolet or visible region is accompanied by a change in the electronic state of the molecules in the sample.

Electronic Transitions

The energy supplied by the light will promote electrons from their ground state orbital to higher energy or excited state orbital or anti-bonding orbital.

n, π or σ or combination of these electronsParts

Any molecule has either

1. POWER SUPPLY

2. LAMP - LIGHT SOURCE

3. MONOCHROMATOR - DISPERSION DEVICE

4. CUVETTE- SAMPLE HOLDER

5. DETECTOR

6. OPTICS/ OPTICAL SYSTEM

7. READOUT DEVICE

Parts of Spectrophotometer (7)

Hydrogen Discharge Lamp

pair of electrodes is enclosed in a glass tube filled with hydrogen gas. When current is passed through these electrodes maintained at high voltage, discharge of electrons occurs which excites hydrogen molecules which in turn cause emission of UV radiation.

Deuterium Lamp

Good intensity continuum in the UV region and useful intensity in the Vis region (160–375 nm), noise from the lamp is often a limiting factor; half- life : approx. 1000 hr.

is stored under pressure. The UV- light produced by this lamp is of a greater intensity compared to hydrogen discharge lamp. 

• Since the lamp operates at a high voltage, it becomes very hot during operations and hence needs thermal insulation. 

• Emission of visible radiation also occurs along with the UV radiation. 

• Wavelength range (200 – 1000)nm.

Tungsten Halogen Lamp

It is a special class of lamp with iodine added to the normal filling gas.

• The envelope is made up of quartz to tolerate higher lamP operating temperatures. 

• Often a heat absorbing filter is inserted between the lamp and the sample holder to remove IR-radiations. 

• The glass envelope absorbs strongly below 350nm. 

• Wavelength range (350 – 3000)nm.

Monochromator - Dispersion Device

• Entrance slit

• Exit slit

  • controls the width of the light beam and allows only a narrow fraction of the spectrum to reach the sample cuvette.

Prism Monochromators

They are usually made up of glass, quartz or fused silica.

1. Refractive type

2. Reflective type

Types of Prism Monochromators (2)

Grating Monochromators

made up of glass, quartz or alkyl halides like KBr and NaBr. Back surface of the gratings are coated with aluminum to make them reflective.

1. Diffraction gratings

2. Transmission gratings

Two types of Grating Monochromators

Diffraction gratings

an optical component with a periodic structure that splits the light into various beams that travel in different directions.

Transmission gratings

disperses light into a spectrum.

1. 335 - 2500nm

2. 320 - 2500nm

3. 220 - 3800nm

4. 170 - 2700nm

In Cuvette

1. Optical Glass range

2. Special Optical Glass range

3. Quartz (Infrared) range

4. Quartz (Far-UV) range

Detector

It produces a current in response to the light impinging upon it.

Achromatic Lenses

combined multiple lenses of different glass (w/ different refractive indices) in a compound glass, free from chromatic aberrations.

Concave mirrors

free from chromatic aberrations, aluminum surface easily corroded results to loss of efficiency.

Readout Device

Measures the magnitude of the current generated by a detector.

Photocell

Also known as “barrier layer cell” or “selenide cell”

Photocell

It is the simplest of all the detectors and not sensitive and output is not readily amplified

• it consists of three layers sealed in a protective casing:

  • Bottom support layer

  • Photosensitive layer

  • Transparent conductive layer

1. Bottom support layer

2. Photosensitive layer

3. Transparent conductive layer

1. made up of a conductive metal like iron

2. made up of selenium or cadmium, it is light-sensitive and releases electrons when strike by light

3. covers the light sensitive layer

Phototube

It consists of a curved cathode of metal coated with a photosensitive material, more sensitive than the photocells.

Photomultiplier tube

It is sensitive and produce a fast response and it is required when low levels of light or quick bursts of light must be measured.

dynodes

Photomultiplier tube is consists of a photosensitive cathode and an anode with several intermediate faces called

Photomultiplier tube

Used in spectrophotometers with a narrow band pass and in instruments that must record fast changes in light emission or absorbance.

Phototransistors and Photodiodes

Newest of the light detectors, small, durable and capable of high amplification, they are constructed of two types of semiconductors joined together that resist current flows across the junction.

• multichannel photon detector, which is capable of measuring all wavelengths of dispersed radiation simultaneously.

1. The technique is non-destructive, allowing the sample to be reused or proceed to further processing or analyses.

2. Measurements can be made quickly, allowing easy integration into experimental protocols.

3. Instruments are easy to use, requiring little user training prior to use.

4. Data analysis generally requires minimal processing, again meaning little user training is required.

5. The instrument is generally inexpensive to acquire and operate, making it accessible for many laboratories.

Pros of UV-VIS (5)

1. Stray light

   • Small amount of light from a wide wavelength range may still be transmitted from the light source, possibly causing serious measurement errors.

   • Stray light may also come from the environment or a loosely fitted compartment in the instrument.

2. Light scattering

   • Caused by suspended solids in liquid sample

   • The presence of bubbles in the cuvette or sample will scatter light.

3. Interference from multiple absorbing species

   • A sample may, for example, have multiple types of the green pigment chlorophyll. The different chlorophylls will have overlapping spectra when examined together in the same sample.

4. Geometrical considerations

   • Misaligned positioning of any one of the instrument's components, especially the cuvette holding the sample, may yield irreproducible and inaccurate results.

Cons of UV-VIS (4)

1. DNA & RNA analysis

   • Verification of Concentration

2. Pharmaceutical analysis

   • Impurities

3. Bacterial culture

   • Optical density

4. Beverage analysis

Applications of UV-VIS (4)