1 | Clinical Chemistry Midterm
CLINICAL CHEMISTRY MIDTERM Parts of a Spectrophotometer
Chapter 4: Instrumentation Light Source
• Provides radiant energy which is either visible light
COLORIMETRY or non-visible light.
• Visible light is produced using a tungsten bulb (400-
• Involves Beer's Law which states that the concentration 700 nm)
of an analyte in a solution is directly proportional to • Non-visible UV light can be produced with the use
the amount of light it absorbs. of a Hg arc lamp, deuterium lamp, or H vapor
• In colorimetry, the constituent measured is colored. I.e., lamp.
it absorbs light within the visible spectrum (400-700 • Tungsten iodide lamp provides high intensity and
nm light) long-lasting visible and UV light.
• The colorimeter uses ordinary filters to screen light • Non-visible infrared (IR) light has been used for
which will strike the solution Since the resolution of analysis of the composition of renal and gallstones,
these filters is not sharp enough, what is measured is purified drugs, and toxicologic substances.
light intensity of multiple wavelengths.
Two types of Colorimetry: Entrance Slit
• Minimizes stray light emitted by the lamp and
Visual colorimetry prevents scattered light from entering the
• No machine monochromator.
• The color intensity of the solution is matched
against a standard solution.
• Since this relies much on the human eye for
interpretation, it is less precise.
• An example of this is the Dubowski (Duboscq)
colorimeter.
Photoelectric colorimetry
• measurement of light intensity (independent of
wavelength) involves the use of a photoelectric Monochromator
device or detector • Replace the filters of colorimeters.
• Has the ability to isolate sharply specific
SPECTROPHOTOMETRY wavelengths of light or to select the amount of light
that will pass through the cuvette.
• Also known as MAS (Molecular Absorption • It may either be a prism or a grating
Spectrophotometry) • A prism is a triangular or wedged piece of
• Has the same principle as colorimetry but uses transparent material capable of resolving specific
monochromators rather than filters. wavelengths. It may be made of glass (for visible
• Monochromators such as prisms and diffraction spectrophotometry), quartz (for UV
gratings are used instead of low-resolution filters to spectrophotometry), or sodium chloride or potassium
enhance the sensitivity and precision of colorimetric bromide (for infrared spectrophotometry)
assays. • On the other hand, a diffraction grating is a
• The use of monochromators allows measurement of grooved piece of transparent material that may
light intensity in a much narrower wavelength range. contain about 3000 or more grooves per mm.
• This makes the analysis suitable for both colored or Each groove functions as an individual prism.
colorless solutions. Thus, there is both visible and UV
spectrophotometers depending on the absorbance
Exit Slit
peak of the analyte.
• This controls the amount of emergent light the passes
through the cuvette.
Prepared by: Joshua S. Manong
, 2 | Clinical Chemistry Midterm
Cuvette • Transmittance is the ratio of the transmitted light to
the incident light.
• Also known as analytical cell or absorption cell
• Beer's law can be expressed in equation form as
• Holds the solution whose concentration is to be shown below:
assayed
• Must not have optical flaws and their walls must be
precisely arranged.
• Different kinds of cuvettes may be used depending
on the type of solution measured.
o Aluminosilicate glass cuvettes (resistant to
etching) - for strong acidic solutions. • where A is the absorbance, a is the absorptivity
o Borosilicate glass- for strong alkaline value, b is the light path of the solution (in cm), b is the
solutions. concentration of the substance of interest, and %T is
o Cuvettes made of quartz – for UV the percent transmittance.
spectrophotometry • A solution transmits light corresponding in wavelength
o Glasses are not suitable for UV to its color, and usually absorbs of light wavelength
spectrophotometry since they block the complementary to its color. For example, red solutions
passage of UV light. transmit light of 600-650 nm and strongly absorb
• Among the common errors in handling cuvettes are: 400-500 nm light. Green solutions transmit 500-550
(1) failure to position the analytical cell properly in nm light and absorb at 475-575.
the photometer and • %T is inversely and logarithmically related to the
(2) failure to match the absorbance readings of the concentration of the solution.
cells. • Absorbance is directly proportional to path length
i.e., doubling the path length (or diameter of the
Detector or Photocell
cuvette) results in incident light contracting twice the
• Measures the intensity of the emergent light from the number of molecules in the solution. This causes A to
solution. double, the same effect as doubling the concentration
• Some detectors are made of barrier layer cells of the molecules.
• They operate on the principle that when light falls • It must be noted that Beer's law may only be applied
on certain metals, electron flows in proportion to the in accurate quantitative analysis by light absorption,
intensity of light. if the following requisites are met:
• Other detectors are made of photomultiplier tubes. 1. Incident radiation on the substance of interest is
These are electron tubes capable of significantly monochromatic.
amplifying a current. They convert light energy into 2. Solvent absorption is significant compared to
an electrical signal. solute absorption,
3. Solute concentration is within "linear limits" (see
Meter or Read-out Device. below), and
4. A chemical reaction does not occur between the
• Reads the result molecule of interest and another solute or solvent
• This numerically presents absorbance or percent molecules ("quenching" phenomenon).
transmittance
• There are, however, certain deviations from Beer's
Law. In such cases, appropriate measures must be
Beer's Law (Beer-Lambert's Law) taken to ensure the accuracy of the assay. Examples
of these deviations are:
• This law states that the concentration of a substance is (1) Simultaneous absorption at multiple
directly proportional to the amount of light absorbed wavelengths.
or inversely proportional to the logarithm of the (2) Absorption of light by other species, and
transmitted light. (3) Transmission of light by other mechanism.
• The higher the concentration of substance, the more
light is absorbed. Hence, the amount of light
transmitted is reduced.
• Absorbance (optical density) is the amount of light
blocked by the solution
Prepared by: Joshua S. Manong
CLINICAL CHEMISTRY MIDTERM Parts of a Spectrophotometer
Chapter 4: Instrumentation Light Source
• Provides radiant energy which is either visible light
COLORIMETRY or non-visible light.
• Visible light is produced using a tungsten bulb (400-
• Involves Beer's Law which states that the concentration 700 nm)
of an analyte in a solution is directly proportional to • Non-visible UV light can be produced with the use
the amount of light it absorbs. of a Hg arc lamp, deuterium lamp, or H vapor
• In colorimetry, the constituent measured is colored. I.e., lamp.
it absorbs light within the visible spectrum (400-700 • Tungsten iodide lamp provides high intensity and
nm light) long-lasting visible and UV light.
• The colorimeter uses ordinary filters to screen light • Non-visible infrared (IR) light has been used for
which will strike the solution Since the resolution of analysis of the composition of renal and gallstones,
these filters is not sharp enough, what is measured is purified drugs, and toxicologic substances.
light intensity of multiple wavelengths.
Two types of Colorimetry: Entrance Slit
• Minimizes stray light emitted by the lamp and
Visual colorimetry prevents scattered light from entering the
• No machine monochromator.
• The color intensity of the solution is matched
against a standard solution.
• Since this relies much on the human eye for
interpretation, it is less precise.
• An example of this is the Dubowski (Duboscq)
colorimeter.
Photoelectric colorimetry
• measurement of light intensity (independent of
wavelength) involves the use of a photoelectric Monochromator
device or detector • Replace the filters of colorimeters.
• Has the ability to isolate sharply specific
SPECTROPHOTOMETRY wavelengths of light or to select the amount of light
that will pass through the cuvette.
• Also known as MAS (Molecular Absorption • It may either be a prism or a grating
Spectrophotometry) • A prism is a triangular or wedged piece of
• Has the same principle as colorimetry but uses transparent material capable of resolving specific
monochromators rather than filters. wavelengths. It may be made of glass (for visible
• Monochromators such as prisms and diffraction spectrophotometry), quartz (for UV
gratings are used instead of low-resolution filters to spectrophotometry), or sodium chloride or potassium
enhance the sensitivity and precision of colorimetric bromide (for infrared spectrophotometry)
assays. • On the other hand, a diffraction grating is a
• The use of monochromators allows measurement of grooved piece of transparent material that may
light intensity in a much narrower wavelength range. contain about 3000 or more grooves per mm.
• This makes the analysis suitable for both colored or Each groove functions as an individual prism.
colorless solutions. Thus, there is both visible and UV
spectrophotometers depending on the absorbance
Exit Slit
peak of the analyte.
• This controls the amount of emergent light the passes
through the cuvette.
Prepared by: Joshua S. Manong
, 2 | Clinical Chemistry Midterm
Cuvette • Transmittance is the ratio of the transmitted light to
the incident light.
• Also known as analytical cell or absorption cell
• Beer's law can be expressed in equation form as
• Holds the solution whose concentration is to be shown below:
assayed
• Must not have optical flaws and their walls must be
precisely arranged.
• Different kinds of cuvettes may be used depending
on the type of solution measured.
o Aluminosilicate glass cuvettes (resistant to
etching) - for strong acidic solutions. • where A is the absorbance, a is the absorptivity
o Borosilicate glass- for strong alkaline value, b is the light path of the solution (in cm), b is the
solutions. concentration of the substance of interest, and %T is
o Cuvettes made of quartz – for UV the percent transmittance.
spectrophotometry • A solution transmits light corresponding in wavelength
o Glasses are not suitable for UV to its color, and usually absorbs of light wavelength
spectrophotometry since they block the complementary to its color. For example, red solutions
passage of UV light. transmit light of 600-650 nm and strongly absorb
• Among the common errors in handling cuvettes are: 400-500 nm light. Green solutions transmit 500-550
(1) failure to position the analytical cell properly in nm light and absorb at 475-575.
the photometer and • %T is inversely and logarithmically related to the
(2) failure to match the absorbance readings of the concentration of the solution.
cells. • Absorbance is directly proportional to path length
i.e., doubling the path length (or diameter of the
Detector or Photocell
cuvette) results in incident light contracting twice the
• Measures the intensity of the emergent light from the number of molecules in the solution. This causes A to
solution. double, the same effect as doubling the concentration
• Some detectors are made of barrier layer cells of the molecules.
• They operate on the principle that when light falls • It must be noted that Beer's law may only be applied
on certain metals, electron flows in proportion to the in accurate quantitative analysis by light absorption,
intensity of light. if the following requisites are met:
• Other detectors are made of photomultiplier tubes. 1. Incident radiation on the substance of interest is
These are electron tubes capable of significantly monochromatic.
amplifying a current. They convert light energy into 2. Solvent absorption is significant compared to
an electrical signal. solute absorption,
3. Solute concentration is within "linear limits" (see
Meter or Read-out Device. below), and
4. A chemical reaction does not occur between the
• Reads the result molecule of interest and another solute or solvent
• This numerically presents absorbance or percent molecules ("quenching" phenomenon).
transmittance
• There are, however, certain deviations from Beer's
Law. In such cases, appropriate measures must be
Beer's Law (Beer-Lambert's Law) taken to ensure the accuracy of the assay. Examples
of these deviations are:
• This law states that the concentration of a substance is (1) Simultaneous absorption at multiple
directly proportional to the amount of light absorbed wavelengths.
or inversely proportional to the logarithm of the (2) Absorption of light by other species, and
transmitted light. (3) Transmission of light by other mechanism.
• The higher the concentration of substance, the more
light is absorbed. Hence, the amount of light
transmitted is reduced.
• Absorbance (optical density) is the amount of light
blocked by the solution
Prepared by: Joshua S. Manong
