Clinical Chemistry– Laboratory 09/08/2023

Spectrophotometry Prelims

OUTLINE
I. Spectrophotometry IV. Parts of
II. Standard Curves Spectrophotometer
III. Spectrophotometric V. Spectrophotometry
Measurements Quality Assurance
VI. Components
VII. Sources of Error

SPECTROPHOTOMETRY
• Light is electromagnetic radiation that travels in waves
• Wavelength is measured in nanometers (10^-9 meters)
• Wavelength is the distance between adjacent peaks or troughs in
a continuous wave
• Visible light = 400 to 700nm
• Ultraviolet light = 200 to 400nm
• Infrared light = 700 to 800 nm
• Two primary considerations for Colorimetry
® Quality of Color
® Intensity
• Colorimetry
® Visual
§ Depends on the eye to compare intensity of an unk subs.
To a standard
® Photoelectric
§ Uses filters of specific wavelengths
§ Example: Spectrophotometry
§ Measures transmitted light by a solution to determine the
concentration of analyte
§ Transmitted light registers on a photoelectric cell, which
is recorded as either %T or Absorbance (O.D.)
STANDARD CURVES
• If the colorimetric analysis of a standard solutions of a given
compound gives a straight line when plotted on a graph, the
reaction FOLLOWS the BEER’S LAW
• Obtained by plotting the concentration of known solutions against
respective readings
• When following a Beer’s Law (Straight Line), absorbance is
plotted vs concentration on linear graph paper
• % Transmittance is plotted versus concentration on semi log
graph paper
• if white light is shone through a substance that absorbs yellow light,
the complementary color of blue will be transmitted through the
substance. Blue color is observed because the substance absorbs
the yellow color
SPECTROPHOTOMETRIC MEASUREMENTS
• Detection and quantification of energy that is transmitted after
passing a beam of light through the solution being analyzed
• Spectrophotometers differ from photometers in that they use
prisms or diffraction gratings to form monochromatic light.
• %T = P/P0
® P = radiant energy (light) transmitted
® P0 = intensity of incident light (original)
• If all of the incident light is absorbed, then no light is transmitted
and the %T = zero
• If none of the incident light is absorbed, all of the light is transmitted
and the %T is 100%
• Percent transmittance is often expressed as Absorbance (A) for
ease of use in practice because %T is not a linear relationship
• Absorbance is directly proportional to the concentration of the
absorbing substance
• Beer’s Law Equation
® A = 2-log%T
• Beer-Lambert’s Law
® Absorbance is directly proportional to the length if the light
path through the sample, which is 1.0 cm in most proportional
® A= absorbance
® a= absorptivity constant in moles/cm2
® b= path length in cm
® c= concentration
§ a and b are constant, absorption has a directly
proportional linear relationship with concentration
• Concentration unknown = (Aunk/Astd) x (Conc.std)
• 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 differentiated

PARTS OF SPECTROPHOTOMETER
• Light Source
Clinical Chemistry – Laboratory Spectrophotometry Page 1 of 2
 ® Vary according to need, but must be a constant beam, cool
and orderly
® Tungsten Lamp – most common source of light for Visible
and near IR region
§ Incandescent light (400 nm to 700 nm)
® Deuterium or Mercury Arc Lamp – most common source of
light for used for UV region
§ Range: 160 to 375 nm
• Monochromators
® Purpose: promote spectral isolation, provides increased
sensitivity and specificity
® Isolates – individual wavelengths of light
® Bandpass/Bandwidth – measures the success of the
monochromator, defines the width of the segment on the
spectrum
§ Types of Monochromator:
o Colored-glass Filters – least expensive; simple but
NOT precise
o Interference Filters – functions based on the
principle of constructive interference of waves
o Prism – a beam of light focused on a prism is
refracted as it enters the more dense glass
o Diffraction Gratings – MOST commonly used;
consists of many parallel grooves etched
• Sample Cell (CUVETTE) – made of high quality glass or quartz
® Borosilicate Cuvettes/Glass – used in the visible region
® Quartz Cuvettes – UV Radiation
® Shape
§ Round cuvettes – cheaper but light refraction and
distortion occur
§ Square cuvettes – less light refraction but usually more
costly
® Optically clean
§ No inconsistencies in composition
§ No marks, scratches, or finger prints
® Positioning
§ Orientation and placement into the instrument important
§ Each time must be the same so light passes through the
cuvette at the same place
• Photodetectors
® Converts transmitted radiant energy into electrical energy
§ Types of Photodetectors:
o Barrier – layer or Photocell – least expensive but
temperature-sensitive
o Phototube – requires an outside voltage for
operation
o Photomultiplier tube – MOST COMMON, detects
& amplifies-radiant energy; very sensitive
o Photodiode – lacks internal amplification and not
sensitive
• Readout devices
® Purpose: to convert the electrical signal from the detector to
a usable form
® Types: Meters/Galvanometer, Recorders, Digital Readout
® Most are digital
SPECTROPHOTOMETRY QUALITY ASSURANCE
• Wavelength Accuracy
Subject – Laboratory/Lecture Spectrophotometry
® Wavelength indicated on the control dial is the actual
wavelength of light passed by the monochromator
® Checked by using special filters with known peak
transmission
® Must be done when the light source has changed or if the
instrument has been moved
• Didymium or Holmium Oxide
® In glass are stable and frequently used as filters
• Stray Light
® Refers to any wavelength OUTSIDE the band transmitted by
the monochromator
® Most common causes: Reflection of light from scratches or
dust particles in light path
® Detecting by using “Cut-off Filters” which eliminate all
radiation at desired wavelength
• Linearity
® Made by reading the absorbance of a set of standards
solutions (obtain commercially) at specified wavelengths
SOURCES OF ERROR
• Lamp burnout/Light source
® Most frequent source of error
® Erratic control/patient results
® Hours of use can be logged by system
® Indicator/s of a failing light source
§ Lamps turns dark or smoky in color
• Monochromator error
® Poor resolution due to wide bandpass
® Results in decreased linearity and sensitivity
• Cuvette errors/Sample Cell
® Dirt, scratches, loose cuvette holder – all cause stray light
• Air bubbles in specimen
• Reagent make-up
® Some test procedures make a product that easily foams
• Volume too low for light path
• Electrical static (noise)
• Dark current
® From the detector. Leakage of electrons when no light
passing through
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