MEDICAL LABORATORY SCIENCE PROGRAM

CLINICAL CHEMISTRY 1

Learning Activity No. 4

SPECTROPHOTOMETRY

Specific Learning Objectives:

At the end of this laboratory exercise, the student is expected to:

a. Discuss the principle of spectrophotometry;

b. Enumerate the basic parts of the spectrophotometer and give its function;
c. Calculate transmittance and absorbance;
d. Enumerate other spectrophotometric methods and give its basic principle;
e. Manipulate properly the spectrophotometer; and,
f. Compute the concentration of the unknown substance.

Content:

Photometry is defined as the measurement of light; spectrophotometry is defined as

the measurement of the intensity of light at selected wavelengths. Spectrophotometric analysis
is a widely used method of quantitative and qualitative analysis in the chemical and biological
sciences; it is accurate and sensitive. The method depends on the light absorbing properties of
either the substance or a derivative of the substance being analyzed. The intensity of
transmitted light passing through a solution containing an absorbing substance (chromogen) is
decreased by the absorbed fraction. This fraction is then detected, measured, and used to
relate the light transmitted or absorbed to concentration of the analyte in question.

BASIC CONCEPTS

Consider an incident light beam with intensity I, passing through a square cell containing
a solution of a compound that absorbs light of a certain wavelength, λ. Given that the intensity
of the transmitted light beam IS, is less than I0, the transmittance (T) of light is defined as:

IS
T = ---------
I0

A portion of the incident light, however, may be reflected by the surface of the cell or
absorbed by the cell wall or solvent. To focus attention on the compound of interest, elimination
of these factors is necessary. This is achieved using a reference cell identical to the sample cell,
except that the compound of interest is omitted from the solvent in the reference cell. The
transmittance (T) through this reference cell is IR, divided by I0; the transmittance for the
compound in solution then is defined as IS divided by IR. In practice the reference cell is inserted
and the instrument adjusted to an arbitrary scale reading of 100 (corresponding to 100%
transmittance), after which the percent transmittance reading is made on the sample. As we
increase the concentration of the compound in solution, we find that transmittance varies
inversely and logarithmically with concentration Consequently, it is more convenient to define a
new term, absorbance (A), that will be directly proportional to concentration. Thus the amount
of light absorbed (A) as the incident light passes through the sample is equivalent to:

IS
A= log ------ = log T
IR
 Analytically, the amount of light absorbed or transmitted is related mathematically to the
concentration of the analyte in question by Beer's Law.

Beer's Law-Relationship between Transmittance, Absorbance, and Concentration

Beer's law (also known as the Beer-Lambert law) states that the concentration of a
substance is directly proportional to the amount of light absorbed or inversely proportional to the
logarithm of the transmitted light Mathematically, Beer's law is expressed as:

A= Ɛxbxc
A = Absorbance
Ɛ = Molar Absorptivity
b = Light path (in cm)
c = Concentration of absorbing compound
(usually in grams per liter)

Application of Beer's Law

In practice, the direct proportionality between absorbance and concentration must be

established experimentally for a given instrument under specified conditions. Frequently a linear
relationship exists up to a certain concentration or absorbance. When this relationship occurs,
the solution is said to obey Beer's law up to this point. Within this limitation, a calibration
constant (K) may be derived and used to calculate the concentration of an unknown solution by
comparison with a calibrating solution. Rearranging equation gives:
A
a = --------
bc

Therefore,

A1 A2
----------- = ------------
b1c1 b2c2

Because the light path (b) remains constant in a given method of analysis with a fixed
cuvet size (b1 = b2), Equation then becomes

A1 A2 AC AU
----------- = ----------- or --------- = ----------
c1 c2 cC cU

where subscripts c and u indicate the absorbance (A) and concentration (c) of calibrating and
unknown solutions, respectively.

Solving for the concentration of unknown:

AU
cU = ---------- x cC
AC
Components of a Spectrophotometer

A typical photometer or spectrophotometer contains six basic components in a single or

double-beam configuration. These components include:
(1) a stable source of radiant energy;
(2) a filter that isolates a specific region of the electromagnetic spectrum;
(3) a sample holder;
(4) a radiation detector;
(5) a signal processor, and
(6) a readout device.

Basic Components of a Single-Beam Spectrophotometer

(Photo taken from Mcpherson, Richard A. and Matthew R. Pincus. Henry’s Clinical Diagnosis and Management by
Laboratory Methods 22nd ed. Philadelphia: Elsevier Inc., 2011)

A, Exciter lamp; B, entrance slit; C, monochromator; D, exit slit; E, cuvet;

F, photodetector; G, light-emitting diode (LED) display.

The following steps outline the function of each component in any absorption-type
photometer as it detects light and provides information to the operator:

1. The light source provides the energy that the sample will modify or attenuate by
absorption. The light is polychromatic (i.e., all visible wavelengths are present).
2. A wavelength selector or filter isolates a portion of the spectrum emitted by the source
and focuses it on the sample.
3. The sample in a suitable container (e.g., a cuvet) absorbs a fraction of the incident light
and transmits the remainder.
4. The light that passes through the cuvet and sample strikes the cathode of a
photodetector and generates an electrical signal.
5. The electrical signal is processed electronically (e.g., amplified, digitized).
6. The processed signal is electronically coupled to the display unit (e.g., light-emitting
diode [LED], X-Y strip chart recorder, meter).

Types of Photometric Instruments

Several instrument designs and configurations may be used for absorption photometry.
Each has unique terminology associated with its design. The terminology is not universal among
users but is presented here as a guide.

A spectroscope is an optical instrument used for visual identification of atomic emission

lines. It has a monochromator, usually a prism or diffraction grating, in which the exit slit is
replaced by an eyepiece that can be moved along the focal plane. The wavelength of an
emission line can then be determined from the angle between the incident and dispersed beam
when the line is centered on the eyepiece.
 A colorimeter uses the human eye as the detector. The user compares the observed
color of the unknown sample against a standard or a series of colored standards of known
concentrations. Photometers consist of a light source, a filter, and a photoelectric transducer as
well as a signal processor and readout. Some manufacturers use the term colorimeter or
photoelectric colorimeters for photometers. These photometers use filters for isolation of specific
wavelengths—not gratings or prisms.

A spectrometer is an instrument that provides information about the intensity of

radiation as a function of wavelength or frequency. Spectrophotometers are spectrometers
equipped with one or more exit slits and photoelectron transducers that permit determination of
the ratio of the power of two beams as a function of wavelength, as in absorption spectroscopy.
Most spectrophotometers use a grating monochromator to break up the light into a spectrum.
Single-beam spectrophotometers are the simplest types of absorption spectrometers. These
instruments are designed to make one measurement at a time at one specified wavelength. The
absorption maximum of the analyte must be known in advance when a single-beam instrument
is used. The wavelength is then set to this value. The reference material (solvent blank) is
transferred into a suitable cuvet and is placed in the path of the monochromatic light. The
spectrometer is adjusted to read 0%T when a shutter is placed, so as to block all radiation from
the detector, and to read 100%T when the shutter is removed. After these adjustments have
been made, the sample is placed in the light path, the absorbance is measured, and the
concentration is determined using calculations based on Beer’s law or by constructing a
calibration curve for the analyte.

Other types of photometers are:

1. Atomic Emission Spectrophotometer

2. Atomic Absorption Spectrophotometer
3. Fluorometer
4. Turbidimeter
5. Nephelometer

Time Allotment:
Three (3) hours in the laboratory.

Learning Resources:
Spectrophotometer
Cuvet
Operation Manual (provided by the manufacturer of the machine)
Calculator
Chemistry books

Learning Strategies:
a. Lecture of the spectrophotometry by the instructor.
b. Individual seat work or blackboard work.
c. Describe or demonstrate how to properly operate the spectrophotometer. Follow the
instructions stipulated in the Operation Manual provided by the manufacturer of the
spectrophotometer.
d. Written quiz and examination.

References:

Bishop, Michael L. et.al. “Clinical Chemistry: Principles, Procedures, Correlation’s”, 6th

edition. Philadelphia: Lippincott Williams, Philadelphia, USA, ©2010

Burtis, Ashwood, and Bruns.“Tietz Textbook of Clinical Chemistry and Molecular

Diagnostics” 6th edition; Saunders, an imprint of Elsevier Inc. USA ©2012.

Mcpherson, Richard A. and Matthew R. Pincus. Henry’s Clinical Diagnosis and Management
by Laboratory Methods 22nd ed. Philadelphia: Elsevier Inc., 2011
 MEDICAL LABORATORY SCIENCE PROGRAM

CLINICAL CHEMISTRY 1

Name__________________________________ Points Earned:_________________________

Section ________________________________ Date ________________________________

Learning Activity No. 4

SPECTROPHOTOMETRY

General Instructions:
1. All final answers must be in capital letters.
2. Avoid erasures.

1. Briefly discuss the principle of the spectrophotometer. (10 points)

Content 6 points
Accuracy 3 points
Neatness 1 point

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2. Complete the table: (1 point each entry = 16 points)

BASIC COMPONENTS OF THE FUNCTION/S

SINGLE-BEAM SPECTROPHOTOMETER
3. Given the following data, compute the following:

a. SERUM GLUCOSE ASSAY (5 points)

Absorbance of the sample = 0.304
Absorbance of the standard = 0.299
Concentration of the standard = 5.5 mmol/L
What is the concentration of the serum glucose?

b. SERUM TRIGLYCERIDES ASSAY (5 points)

Absorbance of the sample = 0.456
Absorbance of the standard = 0.387
Concentration of the standard = 110 mg/dL
What is the concentration of the serum triglyceride?

4. Give the basic principle of each photometric methods: (5 point each = 25 points)

PHOTOMETRIC METHODS BASIC PRINCIPLE

Atomic Emission Photometry

Atomic Absorption Photometry

Fluorometry

Turbidimetry

Nephelometry