Clin Chem Lab STANDARD CURVES:

Spectrophotometry If the colorimetric analysis of standard solutions of a

given compound gives a straight line when plotted on a
graph, the reaction FOLLOWS the BEER’S LAW.
Objectives: Standard curves are obtained by plotting the
At the end of this unit, the student will be able to: concentration of known solutions against their respective
readings.
1. Identify the regions of the electromagnetic
spectrum that are used in laboratory analysis. When the reaction follows the Beer’s Law
2. Given a color of a solution, determine what (“STRAIGHT LINE”):
wavelength range of the spectrum would be Absorbance is plotted versus conc. On LINEAR-
most appropriate to use for spectrophotometric GRAPH PAPER % Transmittance is plotted versus
analysis for that substance. conc. on SEMILOG GRAPH PAPER
3. Define percent transmittance and absorbance.
4. Identify the parts of a spectrophotometer.
5. Calculate concentration of an unknown when
given the concentration of the standard and the
absorbances of the standard and the unknown.

Light is electromagnetic radiation that travels in waves.

The wavelength ( 入 ) of light is measured in

nanometers which is 10-9 meters.

The wavelength is the distance between adjacent peaks

or troughs in a continuous wave.

Visible light falls between 400-700nm, Ultraviolet light When a beam of white light is shone through a
falls between 200- 400nm, and infrared light falls substance that absorbs yellow light, the complementary
between 700-800nm. color of blue will be transmitted through the substance.
The blue color is observed because the substance
These three regions are the most commonly used absorbs the yellow color.
regions in clinical laboratory analysis
The table below shows the wavelength of light when a
color is absorbed and what complementary color is
Two primary considerations for Colorimetry: observed.

 Quality of color Wavelength Approx. Color Color

in nm Maximum absorbed Observed
 Intensity
absorbance
Colorimetry may either be Visual or Photoelectric Wavelength
in nm
Visual Colorimetry 380-450 420 Violet Yellow-
green
 depends on the eye to compare intensity of an 450-495 470 Blue Yellow
unk. subs. to a standard 495-570 530 Green Purple
570-590 580 Yellow Blue
Photoelectric Colorimetry
590-620 620 Orange Greenish
 uses FILTERS of specific wavelengths blue
620-750 700 Red Bluish
 Examples is SPECTROPHOTOMETRY
green
 Measures transmitted light by a solution to
determine the concentration of analyte
Transmitted light registers on a Photoelectric Spectrophotometric measurements are based on
Cell, which is recorded as either %T or detection and quantification of energy that is transmitted
Absorbance (O.D.)
after passing a beam of light through the solution being When Beer’s Law is followed, we can determine the
analyzed. concentration of the unknown by using the following
equation:

Concentrationunk = (Aunk/Astd) x (Conc.std)

Transmitted energy is expressed in terms of percent

transmittance (% T) by the following equation: If you are given a color of solution, you can select the
%T = I/Io appropriate wavelength to measure the substance’s
absorbance by selecting the wavelength that
%T = P/Po X 100 corresponds to maximum absorbance of its
complementary color.
 I = radiant energy (light) transmitted
 Io = intensity of incident light (original It is best to select a wavelength for measurement in
which the absorbance for that substance is the greatest,
so that changes in concentration are easily
If all of the incident light is absorbed, then no light is differentiated.
transmitted and the %T = 0 (zero). For instance, bilirubin absorbs more violet and blue light
If none of the incident light is absorbed, all of the light is in the 430-400 nm wavelength range than other colors;
transmitted and the %T is 100%. therefore, bilirubin measurements are typically analyzed
on amniotic fluid at 450 nm.

The wavelength/color chart above helps explain why

Percent transmittance is often expressed as icteric plasma has a yellow-green tint. The figure below
absorbance (A) for ease of use in practice because %T shows the best wavelength that range for this substance
is not a linear relationship. is between 520-550 nm because the absorbance is
greater and the differences between samples is greatest
Absorbance is directly proportional to the concentration
within that range. This correlates with the information in
of the absorbing substance.
the chart above as well.

The following Beer’s Law equation is used:

Parts of Spectrophotometer
A= 2-log % T
1. LIGHT SOURCE
Beer-Lambert’s law states that absorbance is directly  Tungsten Lamp – the most common
proportional to the length of the light path through the source of light for Visible & near IR
sample, which is 1.0 cm in most spectrophotometers. region
 Mercury Arc Lamp - most common
source of light for used for UV region
The Beer-Lambert’s Law is as follows:
2. MONOCHROMATORS
 A= absorbance  ISOLATES individual wavelengths of
 a = absorptivity constant in moles/cm2 light
 b = path length in cm (ie.1.0 cm)  Reason of ROYGBIV
 c = concentration  Types of Monochromators:
Because (a) and (b) are constant, absorption has a a. Colored-glass Filters - least
directly proportional linear relationship with expensive; simple, but NOT
concentration. precise
b. Interference Filters - functions
based on the principle of
 constructive interference of  Most common causes:
waves Reflection of light from
c. Prism - a beam of light focused scratches or dust
on a prism is Refracted as it particles in light path
enters the denser glass  Detected by using "Cut-
d. Diffraction Gratings - MOST off Filters" which
COMMONLY USED; consists of eliminate all radiation at
many parallel grooves etched desired wavelength

3. SAMPLE CELL (CUVET) The National Fire Protection Association (NFPA)

 Borosilicate Cuvettes -used in the has developed the Hazards Identification system to
visible region provide common, recognizable warning signs for
 Quartz Cuvettes are used for UV chemical hazards. The system consists of four color-
radiation
coded, diamond-shaped symbols arranged to form a
larger diamond shape.
Red (top) designates flammability
Blue (left) indicates health hazards
Yellow (right) indicates reactivity-stability
White (bottom) indicates special consideration
4. PHOTODETECTORS
 Converts transmitted radiant energy into
ELECTRICAL ENERGY
 Read out machine
 Types of photodetectors:
a. Barrier-layer cell or Photocell
- least expensive but
temperature-sensitive
b. Phototube - requires an outside
voltage for operation
c. Photomultiplier tube - detects
& amplifies-radiant energy; very
sensitive
d. Photodiode - lacks internal
amplification and not sensitive
 Spectrophotometry Quality Assurance:
i. Wavelength Accuracy
 Means that the
wavelength indicated on
the control dial is the
actual wavelength of
light passed by the
monochromator.
 DIDYMIUM or
HOLMIUM OXIDE in
glass are stable &
frequently used as
filters
ii. Stray Light
 Refers to any
wavelengths OUTSIDE
the band transmitted by
the monochromator