MLS 221 CHAPTER III

INSTRUMENTATION BEER LAMBERT LAW

SPECTROMETRY ü Conc. is directly related to absorbance
Light Energy ü Conc. is inversely related to log of transmission
- Electromagnetic radiation PERCENT TRANSMITTANCE
o Photons of energy traveling in waves ü Light transmitted device by the 𝑻
- Characterize by waves and frequency % T = 𝐱𝟏𝟎𝟎
𝑰
incident light on sample
ü Sample transmitted beam divided by
FREQUENCY
blank transmitted beam
- # of vibrations of wave per second
- Blank: use to remove any color
- Direct relationship energy interference from any substance that is not 𝒔𝒂𝒎𝒑𝒍𝒆 𝒃𝒆𝒂𝒎 𝒔𝒊𝒈𝒏𝒂𝒍
- Planck’s Formula the analyte of interest (e.g.
% T = 𝐱𝟏𝟎𝟎
𝒃𝒍𝒂𝒏𝒌 𝒃𝒆𝒂𝒎 𝒔𝒊𝒈𝒏𝒂𝒍
E= hv distilled/deionized water)
E= energy - Reagent Blank: use to remove any
color interference contributed by reagent
h = constant
itself
v = frequency
ABSORBANCE
WAVELENGTH
- Distance between two successive peaks in nanometers ü Amt. of light absorbed by substance Mathematically derived from % T
- Inverse relationship with energy A= 2 -log % T
- Low WV = High Energy A = abc
a = molar absorptivity
ELECTROMAGNETIC SPECTRUM b = length of light path through
HIGH frequency = LOW WV the solution (1 cm)
• Cosmic c = conc. of absorbing
substance
• Gamma Rays
Note:
• X-rays
ü a & b are constants
• UV region ü A~ C
• Visible light 𝑨𝒖 𝑪𝒖
• Infrared region =
𝑨𝒔 𝑪𝒔
• Microwaves
𝑨𝒖
= 𝑪𝒔 = 𝑪𝒖
UV SPECTRUM VISIBLE LIGHT 𝑨𝒔
<400 nm 400-700 nm
colorless Diff. colors
PHOTOMETRY VS. SPECTROPHOMETRY
ü Both uses radiant energy
VISIBLE LIGHT PHOTOMETRY
Wavelength Color Absorbed Color Observed - Measurement of light’s luminous intensity
- Didn’t consider WV
380-440 Violet Yellow
- Conventional
440-500 Blue Orange o No isolation of WV of light
500-580 Green Red - Modern
580-600 Yellow Violet o Use filters for isolation of a narrow WV range
600-620 Orange Blue SPECTROPHOTOMETRY
620-750 Red Green - Measurement of light’s light intensity at selected WV
- Uses prisms and gratings
INFRARED REGION o for isolation of a narrow WV range
ü WV: >700
800-2500 Near IR SINGLE BEAM SPECTROPHOTOMETER
2500-15, 000 Mid IR
15,000 – 1, 000, 000 Far IR
ü Not used in CC
ü Colorless

0883|CLINICAL CHEMISTRY (LEC)|BSMLS 2-E| ROBIANES, SHANNEN YNEZ D. 1

 MLS 221 CHAPTER III

Light Source
Incandescent lamps
Tungsten light - The most common source of light for work in the
source visible and near-infrared regions is the
incandescent tungsten or tungsten-iodide lamp.
- Only about 15% of radiant energy emitted falls in
the visible region, with most emitted as near-
infrared.
- Often, a heat-absorbing filter is inserted between
the lamp and the sample to absorb the infrared
radiation.
- Presence of iodine / bromine vapor increase to
make the lifetime of tungsten longer
- Does not supply sufficient radiant energy for
meas. Below 320 nm
Deuterium - Most commonly used for ultraviolet (UV) work are
discharge the deuterium discharge lamp and the mercury
lamp arc lamp
- Provides continuous emission/spectra down to
165 nm.
Hydrogen - Measurement in UV region
lamp - Less stable & shorter life than deuterium-
discharge
Mercury-arc - Measurement in UV region
lamp - 3 forms:
ü Low pressure mercury lamp - emit a
sharp line spectrum with both UV and
visible lines
ü Medium-pressure mercury lamp
ü High pressure mercury lamp – both
medium and high emits a continuum
from UV to the mid-visible region
Xenon lamp - Measurement in UV region
- Xenon lamp is a discharge light source with xenon
gas sealed in a bulb

Laser Source
- Light Amplification by Stimulated Emission of Radiation
- Transform light at extremely intense, focused & non-divergent
beam of monochromatic light
Argon fluoride 193 nm / 248 nm
Helium- Cadmium 325 nm / 442 nm
Nitrogen 337 nm
Argon (blue) 488
Argon (green) 514 nm
Helium-neon (green) 543 nm
Helium-neon (red) 633 nm
The most important factors for a light source:
ü range
ü spectral distribution within the range
ü source of radiant production
ü stability of the radiant energy
ü temperature.

0883|CLINICAL CHEMISTRY (LEC)|BSMLS 2-E| ROBIANES, SHANNEN YNEZ D. 2