COURSE: Clinical Chemistry 1 (Laboratory) DOCUMENT NO.

: MLS 221L_P06
TOPIC: Study of the Principle of Spectrophotometry PREPARED BY: Sam Jeffrey Tiongco

[Ref: Bishop, M.L., Fody, E.P., & Schoeff, L.E. (2013). Clinical Chemistry Principles, Techniques, and Correlations Seventh Edition. Philadelphia:
Lippincott Williams & Wilkins; Burtis, C.A., Ashwood, E.R., & Bruns, D.E. (2013). Tietz Textbook of Clinical Chemistry and Molecular Diagnostics – 5th
Edition. Philippines: Elsevier]

Quantitative determination of concentration of analytes in clinical chemistry involves measurement of their activities in terms of their
products produced in the chemical reactions, or in the amount of the substrates they act upon (such as in the case of enzyme analytes),
or in their electrochemical activities (like in the case of ions). Most of the determinations in chemistry, however, involve measurement of
light, either absorbed, transmitted, emitted or reflected.

Photometry refers to the measurement of light intensity. A narrow range of the incident light from a source lamp is isolated by filters (used
in photometers), prisms or gratings (used in spectrophotometers) before passing through a solution which contains the analyte. The
analyte, depending on its concentration and nature, absorbs this incident. The amount of light not absorbed is transmitted to the detector
system, which translates the light (either absorbed or transmitted) into an electrical signal. Beer’s law is applied to correlate light absorbed
or transmitted to the concentration of the analyte. Factors that lead to deviation from Beer’s law would lead to erroneous results.

Many of the spectrophotometers available are automated or semiautomated. Some come out with digitalized readings on an LCD screen,
apart from the classic readings on a meter. Some instrumentations have come up with measurement of fluorescent lights emitted by the
analyte, or chemiluminescent compounds activated by a chemical reaction. Nevertheless, despite of these technological advancements,
the classic principle of light measurement remains.

• SPECTROSCOPY – The study of how light interacts with matter or the use of light to study matter.
o All molecules absorb and emit light depending upon the types of bonds.
• COLORIMETRY – The study of the determination of solute concentration after reactants are converted into colored products.
• SPECTROPHOTOMETER – It is a device used to measure the amount of light that a sample absorbs.

• It is a measure of how much a chemical substance absorbs light as a beam of light passes through sample solution
• Spectrophotometer:
o Amount of light reflected from a sample object
o Amount of light absorbed by the sample object

Figure 1. Principle of Spectrophotometry

• Basic Components of Spectrophotometer:

o LAMP – the light source
▪ Usually tungsten (W) lamps in the form of incandescent tungsten or tungsten-iodide lamp which is perfect
for observing visible region.
o ENTRANCE SLIT – reduces stray light or prevent scattered light
o MONOCHROMATOR – which selects a narrow beam of light appropriate to the absorption of the analyte (380-700
nm). This is needed for the isolation of individual wavelength of light.
o EXIST SLIT
o ANALYTICAL CELL/CUVETTE – contains solution of the analyte
o PHOTODETECTOR – translates the light absorbed or transmitted into an electrical signal
o METER

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COURSE: Clinical Chemistry 1 (Laboratory) DOCUMENT NO.: MLS 221L_P06
TOPIC: Study of the Principle of Spectrophotometry PREPARED BY: Sam Jeffrey Tiongco

Transmittance Absorbance
𝐼
%𝑇 = 𝐼 × 100 𝐴 = − log(𝐼0 ) 𝑇 → 𝐴 = 2 − 𝑙𝑜𝑔%𝑇
0
• As %T goes up, concentration decreases.
• As concentration increases, %T goes down. • As absorbance increase, concentration decreases.
• CONCLUSION: INVERSE RELATIONSHIP • CONCLUSION: DIRECT RELATIONSHIP (LINEAR)
(NONLINEAR)

• Variables:
o A = absorbance (nm)
o a (epsilon) = molar absorptivity
o b = path length (cm)
o c = concentration (mol/L or
mg/dL)

• Absorbance is measured by comparing the intensity of the beam of light as it enters the sample and the intensity as it leaves
the sample, and strikes the detector.
o Absorbance is directly proportional to concentration.
• Molar Absorptivity is a measure of how well a compound absorbs a given wavelength of light.
• Concentration = the more concentrated, the more absorbance that will occur.

Correlation with Slope Equation

• Since A is a linear function of c (Y=mx+B)

o A = (ab)C + B
▪ B = 0; there is no y-intercept.
▪ X values are represented by concentration values.
▪ The slope (m) is represented by the product of absorbance and path length.

Functional Equation of Beer’s Law

• Interpretation: Concentration of the substance is directly proportional to the amount of light absorbed or indirectly proportional
to the logarithm of the transmitted light.

Au
Cu = × Cs
As
• Correlates the amount of light absorbed or transmitted by the analyte into its concentration.
• Beer’s law tells us that absorption is proportional to concentration
o A1/A2 = C1/C2
o Au/As = Cu/Cs

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COURSE: Clinical Chemistry 1 (Laboratory) DOCUMENT NO.: MLS 221L_P06
TOPIC: Study of the Principle of Spectrophotometry PREPARED BY: Sam Jeffrey Tiongco

• Ultraviolet Region – Visible Region (400 – 700 nm) – Infrared Region

Complementary Color

• Why things appear to be a certain color.

• White light consists of different wavelengths (essentially of the rainbow)
• Simplified Explanation:
o COLORED REFLECTED = what we perceive
o COLORED ABSORBED = what is not perceived; essentially objects the absorbs the wavelength of light that is
complementary to what we perceive.
▪ E.g. Orange solutions will absorb around 450-490 nm (wavelength of blue).
▪ Hence, we use the wavelength at which most light is absorbed λmax.

Parts of a Spectrophotometer

• Power Switch
• 100% Control (Transmittance)
• Sample Compartment
• Wavelength Selector/Control
• Incubator at 37°C
• Receipt Slot
• Digital LED for result display

• Reading calibrated against a blank solution → “zero reference”

• 100% Transmittance
• Spectrophotometer needs to be calibrated against a blank solution so that measurements after it can use the blank solution’s
absorbance as a zero reference. It is analogous to “tare” before weighing.
• Blank → used to calibrated the spectrophotometer
o Contains the same properties as the sample solution except for the solute that absorbs light.
• Before placing the tube on the statfax, wipe off first exceed fluid or dusty materials, or moist build up on the sides of the tube
with the use of clean tissue paper.

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