SPECTROPHOTOMETRY

TERMS:

 Energy → Transmitted via electromagnetic waves that are characterized by their wavelength.
 The relationship between wavelength and energy is described by Planck’s formula:

E=hv
Where: h= a constant 6.62x10-27 erg per second)
V= frequency (# of vibration per second)

 Wavelength→ The distance between 2 successive peaks; It is expressed in terms of nanometer

(nm)

400-750 nm = visible spectrum

<400 nm= ultraviolet region
>750 nm= infrared region
 Frequency→ the number of vibrations of wave motion per second
 The lower the wave frequency, the longer the wavelength
 The wavelength is inversely related to frequency and energy: the shorter the wavelength,
the higher the frequency and energy and vice versa.

I. COLORIMETRY
 Photoelectric colorimetry → the primary analytical utility of spectrophotometry or filter
photometry is the isolation of discreet portions of the spectrum for purposes of measurement.

a. Spectrophotometric measurement→ measurement of a light intensity in a narrower

wavelength
b. Photometric measurement→ measurement of light intensity w/o consideration of
wavelength.

PARTS OF SPECTROPHOTMETER

1. Light Source
a. A continuum source→ emits radiation that changes in intensity
b. Line source → emits limited radiation and wavelength
→ an intense beam of light is directed through the monochromator and the
sample.
 Tungsten lightbulb→ commonly used light source in the visible and near infrared region.
  Light Amplification by Stimulated Emission of Radiation (LASER) → is also used as light
source.

*To give accurate absorbance measurements throughout its absorbance range, its response to
change in light intensity must be linear.

FACTORS OF CHOOSING A LIGHT ALTERNATIVE LIGHT SOURCE:

SOURCE:
1. Mercury arc (visible UV)
1. Range 2. Deuterium lamp (165 nm)(UV)
2. Spectral distribution within range 3. Hydrogen lamp (UV)
3. Temperature 4. Xenon lamp (UV)
4. The source of radiant production 5. Merst-glower (IR)
5. Stability of radiant energy 6. Globar (silicon carbide)(IR)

2. Entrance slit→ minimizes unwanted or stray light and prevents scattered light into the
monochromator system.
 Stray light→ refers to any wavelength outside the band transmitted by the
monochromator, it cause s absorbance error.
 Stray light limits the maximum absorbance that a spectrophotometer can achieve.
 Stray light is the most common cause of loss of linearity at high analyte concentration.

3. Monochromator→ isolates specific/individual wavelength of light.

KINDS OF MONOCHROMATORS

A) Prisms → wedge-shaped pieces of glass, quartz, or sodium chloride.

 A narrow light focused on a prism is refracted as it enters more dense glass
 Can be rotated allowing only the desired wavelength to pass through exit slit.

B) Diffraction Gratings →”Most commonly used”

 Better than prism
 Made by cutting grooves (parallel grooves) or slits into an aluminized surface of a flat
piece of crown glass—wavelengths are bent as they pass a sharp corner.

C) Filters → simple least expensive, not precise but useful.

 Made by placing semi-transparent silver films on both sides of a dielectric such as
magnesium fluoride produce monochromatic light based on the principle of
constructive interference of light waves.
 Usually pass a wide band of radiant energy and have an ion transmittance of the
selected wavelength.
4. Exit slit → It controls the width of light beam (band pass). It allows only a fraction of the spectrum to
reach the sample cuvette.
 Spectral purity of the spectrophotometer is reflected by the band pass—the narrower the band
pass, the greater the resolution
 Accurate absorbance measurement requires a band pass <1/5 the natural band pass of the
spectrophotometer.
 The degree of wavelength isolation is a function of the type of device used and the width of
entrance and exit slit.
 Band pass →the range of wavelengths between points at which transmittance is one half peak
transmittance.

5. Cuvet/Cuvette →also called absorption cell/ analytical cell/ sample cell

It holds the solution whose concentration to be measured.