Clinical Chemistry Laboratory – Spectrophotometer
A, RMT, IMT ♥
COLORIMETRY
- Involves the Beer’s law
- The constituent measured is colored
BEER’S LAW
- States that the CONCENTRATION of an analyte
is DIRECTLY PROPORTIONAL to the amount of
light ABSORBED
- States that the CONCENTRATION of an analyte
is INVERSELY PROPORTIONAL to the logarithm
of light TRANSMITTED
ABSORBANCE
- Amount of light blocked by the solution
TRANSMITTANCE
- Ratio of the transmitted light to the incident
light
𝟏𝟎𝟎
A = abc = −𝒍𝒐𝒈 %𝑻 = 2 – log %T
o A = absorbance/optical density
o a = coefficient of absorptivity
o b = path length of cuvet
o c = concentration of the analyte
o %T = percent transmittance
VISUAL COLORIMETRY
- The color intensity of the solution is matched
against a standard solution
- Less precise because it relies on the human
eye for interpretation
- Example: Dubowski (Duboscq) colorimeter
PHOTOELECTRIC COLORIMETRY
- Involves the use of a photoelectric device or
detector to measure light intensity
(independent of wavelength)
SPECTROPHOTOMER
- Used to measure the light transmitted by a
solution to determine the concentration of the
light-absorbing substance in the solution
COMPONENTS OF SPECTROPHOTOMETER
 Light Source – provides the radiant energy
o UV
o VIS
o IR
 Entrance Slit – minimizes stray light emitted
by the lamp and prevents scattered light
from entering the monochromator
♥ K.A.S.
 Monochromator – isolates specific
wavelengths of light
o PRISM – triangular or wedged piece
of transparent material
a. Glass prism – for VIS
b. Quartz prism – for UV
c. Sodium chloride or potassium
bromide – for IR
o GRATING – grooved piece of
transparent material which may
contain about 3000 or more grooves
per mm. Each groove functions as an
individual prism.
 Exit slit – controls the amount of light that
passes through the cuvette
 Cuvette/analytical cell/absorption cell –
holds the solution whose concentration is to
be assayed
o Aluminosilicate glass – strong acidic
solutions; resistant to etching
o Borosilicate glass – for strong
alkaline solutions
o Quartz or plastic – for UV; glass is not
recommended because it blocks the
passage of UV light
 Photodetector – measures the intensity of
the light from the solution
o Barrier layer cell – operate on the
principle that when light falls on
certain metals, electron flows in
proportion to the intensity of light
o Photomultiplier tubes – electron
tubes capable of converting light
energy into an electric signal
 Readout device – numerically presents the
absorbance or percent transmittance
BLANK CORRECTION
Blanking – ensures the accuracy of the results by
eliminating interfering substances inherent with the
sample or the reagent
1. Reagent blank – corrects for the absorbance
interference caused by the color of the
reagent
2. Sample blank – used to correct non-specific
absorbance of substances in the sample
(hemoglobin, bilirubin, lipids)
3. Water blank – sets the spectrophotometer
at zero absorbance