Lecture 2 Spectrophotometer

 Spectrophotometry is the basis for many of the instruments used in clinical chemistry.  The primary reasons for this are ease of measurement, satisfactory accuracy and precision, and the suitability of spectrophotometric techniques to use in automated instruments.  Spectrophotometer measures light absorption by a liquid substance at various wavelengths.  The Components of unknown material can be determined, or the concentration of a number of known substances can be measured.  Types of Spectrophotometer

 Ultraviolet (UV) Spectrophotometers. Uses ultraviolet light of wave lengths from 200 nm to 350 nm.

light) of wave lengths from 350 nm to 700 nm.

Spectrophotometer Block Diagram

a) Single-beam

b) Double-beam

Most common Spectrophotometer

1. Photodiode 2. Connection wire 3. Lamp 4. Filter/Detector

5. On/Off switch and zero transmission adjustment knob 6. Wavelength selector/Readout 7. Sample chamber 8. Transmittance/absorbance control 9. Absorbance/Transmittance scale

1. Light Sources

Tungsten lamp: Vis. near IR (320 nm~2500 nm) Deuterium arc lamp: UV (200~400 nm)  Uses a tungsten filament and anode placed on opposite sides of a nickel box structure designed to produce the best output spectrum.  Unlike tungsten lamps, the filament is not the source of light in deuterium lamps. Instead an arc is created from the filament to the anode.  The arc created excites the molecular deuterium contained within the bulb to a higher energy state. The deuterium then emits light as it transitions back to its initial state  Its continuous spectrum is only from 180 nm to 370 nm.

Light Intensity of Tungsten and Deuterium lamps

 A problem with tungsten lamps is that, during operation, the tungsten progressively vaporizes from the filaments and condenses on the glass envelope. This coating, which is generally uneven, alters the spectral characteristics of the lamp and can cause errors in determinations.

Gas-discharge lamp (Vis & UV)  Generate light by sending an electrical discharge through an ionized gas. This light can be anything between IR, visible or UV radiation  Typically, these lamps use nobble gases such as argon, neon, Helium, xenon, or a mixture of these gasses. Many lamps are also filled with additional gases like sodium and mercury.

 Each gas, depending on its atomic structure emits certain wavelengths which translates in different colors of the lamp.  Gas-discharge lamps offer long life and high efficiency.

Laser  High power  Very good for studying reactions  Narrow line width  Coherence  Can fine-tune the desired wavelength (but choice of wavelength is limited)