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CLINICAL CHEMISTRY I
Analytic Techniques

 The four basic disciplines in analytic chemistry:

 Spectrometry – Spectrophotometry, atomic absorption and mass spectrometry
 Luminescence – fluorescence, chemiluminescence, and nephelometry
 Electroanalytic methods – electrophoresis, potentiometry, amperometry
 Chromatography – gas, liquid, and thin layer

 Miniaturization of apparatuses has enabled the development of point-of-care testing (POCT)

devices that produce results as accurate as those provided by laboratory based
instrumentation.

SPECTOPHOTOMETRY

 Electromagnetic radiation is described as photons of energy travelling in waves.

 The relationship between wavelength (the distance between successive crest or trough) and
energy can be summarized in E=hv where h is a constant (6.62 x 10-27 erg sec), E is energy,
and v is for wavelength of isolated light.
 As wavelength shortens, the more energy the ER contains and vice versa.

 The principle behind spectrophotometry is the absorption of electromagnetic radiation by the

compound of interest and then its emission of equal amount of radiant energy which will then
be measured by a detector, from which the concentration of the compound of interest can be
deduced.
 For a specific wavelength of light to be absorbed, it must have the same frequency as the
vibrational frequency of the atom or molecule it strikes.
 When a specific wavelength of light is absorbed, the valence electrons of the atom or
molecule of interest are “excited” and jump to an orbital of higher energy level.
 This “excited state” lasts only for a fraction of a millisecond, then the valence electrons
return to their “ground state”, releasing electromagnetic radiation that corresponds to the
energy of the light it absorbed.
 The ER that is released is then measured by a spectrometer (using diffraction gratings) or a
photometer (using filters). The following table shows the complementary emitted colors for
the absorbed wavelengths of light:

WAVELENGTH (μm) COLOR ABSORBED COLOR EMITTED

350 – 430 Violet Yellow-Blue
430 – 475 Blue Yellow
475 – 495 Green-Blue Orange
495 – 505 Blue-Green Red
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505 – 555 Green Purple

555 – 575 Yellow-Green Violet
575 – 600 Yellow Blue
600 – 650 Orange Green-Blue
650 – 700 Red Blue-Green

Beer-Lambert Law

 Percent of light transmitted (%T) can be calculated by multiplying the ratio of the intensity of
transmitted light (I) to the intensity of absorbed light (Io) by 100:

%T = (I / Io) x 100
 Absorbance (A), the amount of light absorbed, is inversely proportional to %T:

A 1/α log(%T)
 To compute for the absorbance, the log(%T) must be subtracted from the log of 100% (which
is equal to 2)

A = 2 – log(%T)
 Absorbance (A) can also be computed by multiplying the molar absorptivity (ε), length of
light path through the solution (b), and concentration of absorbing molecules (C):

A = εbC

 Since path length and molar absorptivity are constant for a given wavelength, it can be
deduced that absorbance is directly proportional to concentration:

A~C
 In manual spectrophotometer, only %T can be displayed by the machine whereas in digital
spectrophotometers, A can also be selected to be displayed rather than %T.
 STANDARDIZATION: To compute for the unknown concentration of the compound of
interest (Cu), the absorbance of compound of interest (Au) is divided by the absorbance of a
standard solution (Astd – the standard solution then must be tested first before the patients
sample) and then the quotient is multiplied with the concentration of the standard solution
(Cstd – this is provided by the manufacturer of the std. sol’n):

Cu = Au x Cstd
Astd
Components of a Spectrophotometer:
 Light source – Important factors like range, spectral distribution within the range, the
source of radiant production, stability of radiant energy, and
temperature.
- The most common light source for work in the visible and near infrared region
is the incandescent tungsten or tungsten-iodide lamp.
- In incandescent tungsten or tungsten-iodide lamp, only 15% of emitted ER
falls in the visible region with the rest emitted as near-infrared.
- The most common light source used for UV work are the deuterium-discharge
lamp and mercury arc lamp.
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 Monochromator – functions to isolate the

desired wavelength of light
- The degree of wavelength
isolation is a function of the
type of monochromator
used and the width of the
entrance and exit slits.
- Colored glass filters:
usually pass a wide band of
ER and have low transmittance of the selected wavelength; although not
precise, it is simple, inexpensive, and useful.
- Interference filters: produce monochromatic lights on the principle of
constructive interference of waves; very efficient but uses accessory
filters to eliminate harmonic wavelengths.
- Prism: produces monochromatic light by refracting light from source as
it enters denser glass and rotating whole body to allow only the desired
wavelength to pass.
- Diffraction grating: consists of many parallel grooves etched onto a
polished surface; produces monochromatic light based on the principle
that wavelengths bends as they pass sharp corners; those wavelengths that
are in phase reinforce each other while those that don’t cancel each other;
uses accessory filters to eliminate stray light.
 Sample Cell – also called cuvet; contains the sample to be tested for the compound ofinterest
- Made of very special, polished glass. Scratched cuvets are discarded since this
causes light scattering which can cause erroneous results.
- May be round or square (square is preferred since it causes less lens error
from lens effect, orientation of spectrophotometer, and refraction)
- Glass cuvets may be used for applications in the visible range but unsuitable
for UV application since glass absorbs UV.
- Quartz cuvets must be used for UV spectrophotometry since quartz doesn’t
absorb UV
 Photodetectors – functions to convert the transmitted ER into an equivalent amount of
electrical energy which now be measured by a meter or displayed on a
monitor
- Barrier-layer cell/Photocell: composed of a light-sensitive film
(selenium) on a plate of iron and overlayed with a transparent layer of
silver; once light hits the light-sensitive film, electrons are excited and
flow toward silver but silver has a moderate resistance that opposes the
electron flow toward the iron; this flow produces a electromotive force
which can be measured; this type of photodetector is inexpensive and
durable but is temperature sensitive and nonlinear at extreme illumination
levels.
- Phototube: consists of negatively charged cathode and a positively
charged anode enclosed in a glass case; when light hits the cathode it
releases electrons that jump toward the anode, where they are collected
and returned to a external, measurable circuit; a vacuum within the glass
case avoids light scattering caused by gas molecules.
- Photomultiplier (PM): detects and amplifies radiant energy; when light
strikes the cathode, it emits electrons that are attracted toward a dynode;
each anode in the dynode chain is made up of material that emits
secondary electrons when hit by a primary electron and have successively
increasing + voltage; the initial electron emission will trigger a cascade of
secondary electron emission which causes the PM’s ability to amplify ER.
The accumulation of electrons striking the anode produces a current
(measured and amperes) which is directly proportional to the intensity of
transmitted light; this is the most sensitive photodetector among all types
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and converts the signal into digital signals which can then be displayed as
absorbance readings.
- Photodiode: uses a reverse-biased +/- junction diode that produces a
photocurrent that is proportional to the transmitted light; though not as
sensitive as PM, its speed and small size make them useful in applications
where light levels are adequate.

Blanking – the use of a reference solution that does not contain the compound of interest; this is
for the calibration of the spectrophotometer. The steps to blanking are:
1. Water reagent blanking – the use of distilled water as the sample to be tested; if the
machine’s meter is not zeroed, then the meter is adjusted so that it points to zero. This is
to make sure that the spectrophotometer reads a 100% transmittance.
2. Reagent blanking – the use of a colored reagent that will be used as a diluting medium
for the samples with the compound of interest; the reagent is put in the cuvet and then
tested in the spectrophotometer; it is expected that there will be a reading in the meter,
this is the A or %T of the reagent – this adds to the amount of light absorbed or
transmitted by the compound of interest; to remove this, simply adjust the meter reading
until it reads zero; this functions in the same way as the “TARE” option in a electronic
balance which removes the weight of the container from the reading and adjusts the
reading back to zero and the balance will now measure only the weight of the substance
being added.

Spectrophotometer QA
 Wavelength accuracy – means the wavelength indicated on the control dial is the actual
wavelength being passed by the monochromator; this is commonly checked using standard
absorbing solutions or filters with absorbance maxima of known wavelength; the solution is
tested and the control dial is then rotated to the wavelength of maximum absorbance and, if
working properly, will result in a 0%T; if not, the optics must be adjusted to calibrate the
monochromator correctly. If the wavelength indicator control is the one with a problem, a
mercury vapour lamp is used as light source and the spectrum is scanned to locate mercury
emission lines; the wavelength indicated on the control is then compared to the known
mercury emission peaks.
 Stray light – refers to any wavelengths outside the band of the monochromator; commonly
caused by scratched and dust particles anywhere on the light path; checked using cutoff
filters which eliminate wavelengths except the one of interest; the reading in a quality
assured machine while performing this QC must be 0%T; if reading is greater, there is
presence of stray light.
 Linearity – demonstrated when a change in concentration results in a straight-line calibration
curve; checked using colored solutions with known concentration of constituents; less than
expected absorbance is an indication of stray light or bandpass that is wider than specified
(wavelength accuracy issues).

Source of Variation
 Transfer from testube into the cuvet
 Contamination of sample to be tested

FLAME PHOTOMETRY

 Used to measure concentration by detecting absorption of electromagnetic radiation by atoms

rather than molecules.
 Used to determine the concentration of ions like Na+, K+, and Li+
 This machine uses the following general steps to measure concentration:
1. Reduction of metal ions in the sample into neutral/free, unexcited atoms
2. Excitation of neutral atoms by providing it with thermal energy
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 The following are the step-by-step process in the use of a flame photometer:
1. Sample is atomized into small droplets by an atomizer
or nebulizer.
2. The droplets are mixed with gas and air in the mixing
chamber, forming an aerosol mixture.
3. The aerosol mixture is then led up to burner, where the
droplets containing the element of interest are reduced
and excited.
4. After the element’s electrons are excited, they go back
to their ground state and release ER.
5. Desired ER is filtered from other wavelengths by a
monochromator and then detected by a photodetector
which will give a reading according to the intensity of
light produced.
 The more intense the light emitted, the higher the concentration of the element of interest.