MLS 11A CLINICAL CHEMISTRY MLS 3A

MODULE 2: Analytic Techniques and Instrumentation

Lesson 1: Instrumentation (Optical Methods)

Unit Map
Instrumentation
A. Optical Methods
1. Spectrophotometry
2. Fluorometry
3. Atomic Absorption Spectrometry (AAS)
4. Flame Emission Photometry
5. Chemiluminescence
6. Turbidimetry
7. Nephelometry

B. Electrochemical Methods
1. Potentiometry
2. Amperometry
3. Couometry

C. Electromigration Methods
1. Electrophoresis
Note: - Why do we need to really study and understand this topic (Module 2)?
D. Chromatography In the modern clinical chemistry laboratory, most of the
1. Planar measurements of analytes is based on the principle of instrumentation.
2. Columnar This will give us a basic understanding on how everything works inside
the clinical chemistry laboratory. As future medical laboratory scientists,
E. Others we will play a big role later on in the diagnosis of the patient’s condition.
1. Mass spectrometry
2. Osmometry -Under instrumentation and analytic techniques, there are five lessons:
3. Point-of-care testing optical methods, electrochemical methods, electromigration methods,
4. Clinical Chemistry Automation chromatography, and others.
5. Future trends -The first topic of this module covers Colorimetry and
Spectrophotometry.

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MLS 11A CLINICAL CHEMISTRY MLS 3A
Lesson 1: Optical Methods

SPECTROPHOTOMETRY
Light energy, Wavelength and Radiant Energy Spectrum

ENERGY
 Transmitted via electromagnetic waves
 Characterized by their frequency and wavelength

WAVELENGTH
Note: -When energy is absorbed, valence electrons move to an orbital with
 Distance between two successive peaks in nanometer (nm)
a higher energy level. Following energy absorption, the excited
electron will fall back to the ground state by emitting a discrete
400-700 nm Visible Spectrum
amount of energy in the form a characteristic wavelength of radiant
<400 nm UV Region
energy.
>700 nm IR Region
PLANCK’S FORMULA
Note: - Above is energy, wavelength, and the different energy spectrum.
-The visible spectrum is the portion of the electromagnetic spectrum
that is visible to the human eye. Electromagnetic radiation in this E = hv
range of wavelength (400-700 nm) is called visible light or simply light. Where:
-A typical human eye will respond to wavelengths from about 400-700 E = energy
nm which means wavelengths which are less than 400 nm (in the h = Planck’s constant (6.626 x 10-34 erg sec)
ultraviolet region) and wavelength greater than 700 nm (in the v = frequency
infrared region) is not visible to the human eye.
Note: -The relationship between wavelength and energy is described by
Planck’s formula. Frequency is the variable.

FREQUENCY
 Number of vibrations of wave motion per second
 Lower wave frequency: longer wavelength
 Wavelength: inversely related to frequency and energy
 Shorter wavelength: higher frequency and energy and vice versa

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MLS 11A CLINICAL CHEMISTRY MLS 3A
NOMINAL WAVELENGTH Note: -In the laboratory, there are instruments or machines which use the
 Represents the wavelength in nanometers at peak transmittance principle of spectrophotometry or colorimetry.
 Slight error in wavelength adjustments: introduce a significant error -Colorimetry has its limitations because it can only be used for colored
in absorbance solutions. On the other hand, spectrophotometry can be used for both
 Wavelength accuracy colored and colorless solutions.
 indicated on the control dial
 actual wavelength of light passed by the monochromator COLORIMETRY
 Quantifies and describes physically the human color perception
 Distinguished by its interest in reducing spectra to the physical
correlates of color perception
 For identification and determination of concentrations of substances that
absorb light

Note: -Colorimetry is the science and technology used to quantify and

DIDYMIUM or HOLMIUM OXIDE FILTER
 to check wavelength accuracy (wavelength calibration)
SPECTROPHOTOMETRY
NEUTRAL DENSITY FILTERS and DICHROMATE SOLUTION
 verify absorbance accuracy on linearity
describe physically the human color perception.
-It is similar to spectrophotometry but it is distinguished by its interest in
reducing spectra to the physical correlates of color perception.
-It is used extensively for identification and determination of
concentrations of substances that absorb light.
 Quantitative measurement of the reflection or transmission properties of
a material as a function of wavelength
 To determine the concentration of the light-absorbing substances in the
solution, the amount of light transmitted by a solution is being measured

Note: -Spectrophotometry involves the measurement of the light transmitted

by a solution to determine the concentration of the light-absorbing
substances in the solution. In other words, in spectrophotometry, the
amount of light transmitted by the sample or solution is measured.
-Spectrophotometry enhances the sensitivity and precision of
colorimetric assays.
-Spectrophotometry uses monochromators instead of low-resolution
filters. That’s why spectrophotometry is suitable for measuring both
colorless and colored solutions.

Spectrophotometry Colorimetry
Colored and colorless solution Colored solution only

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MLS 11A CLINICAL CHEMISTRY MLS 3A
STRAY LIGHT
 Any wavelength outside the band transmitted by the monochromator

Note: -Stray light can be caused by diffraction, light scattering, or if the

machine needs to be repaired. All spectrophotometer has stray light.
Higher end models though have less stray light. But no machine is
perfect, which means there can still be stray light.
-In other words, stray light is light that is here but is not supposed to
be here.
Note: -Spectrophotomer is a photometer that can measure intensity as a
LINEARITY function of the light source wavelength.
 Demonstrated when a change in concentration results in a straight-line -The basic components of a spectrophotometer:
calibration curve 1. Exciter lamp
-serves as the source of light
2. Entrance slit
3. Monochromator
4. Exit slip
5. Cuvet/Cuvette
-holds the sample or solution to be measured
6. Photodetector
7. LED display
ABSORBANCE
PARTS OF A SPECTROPHOTOMETER
 Amount of light absorbed/blocked by the solution
 Proportional to the inverse log of transmittance
 Mathematically derived from %T

TRANSMITTANCE
 Ratio of the transmitted light to the incident ray

% TRANSMITTANCE
 Ratio of the radiant energy transmitted (T) divided by the radiant energy
incident on the sample (I)

1. LIGHT SOURCE
 Provides polychromatic light
 Intense beam of light is directed through the monochromator and
the sample
Note: -Solution or sample  Response to change in light must be linear
-% T: percent transmittance
-Transmittance is the ratio of the transmitted light to the incident light.
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 Tungsten bulb
VISIBLE  Tungsten iodide lamp
 Tungsten halogen lamp
 Hg arc lamp
 Deuterium lamp
UV  H vapor lamp
 Xenon arc lamp
 Tungsten iodide lamp
IR  Tungsten halogen lamp
Note: -The light source or the radiant energy source provides polychromatic
light or the radiant energy. This must generate sufficient radiant energy
or power to measure the analyte of interest.
-To give accurate absorbance measurements, throughout its
absorbance range, its response to change of light must be linear.
-Light for work in the visible and near the infrared regions is the
incandescent tungsten or tungsten iodide lamp.
-Only about 15% of radiant energy emitted falls in the visible region,
with most emitted near the infrared region.
-The lamps most commonly used for UV work are the deuterium
discharge lamp and the mercury arc lamp.
TWO TYPES OF LIGHT SOURCE
 Continuum source – emits radiation that changes in intensity
 Line source- emits limited radiation and wavelength
Note: -Above are the two types of light sources in spectrophotometry.
-Continuum light source are widely used in the laboratory. Examples
of light source under continuum source are tungsten light bulb,
deuterium lamp, and the xenon discharge lamp.
1. Tungsten light bulb is the commonly used light source in the
visible and near the infrared region.
2. Deuterium lamp is routinely used to provide UV radiation in
analytic spectrophotometers.
3. Xenon discharge lamps produce a continuum source of radiation
which covers both the UV and visible regions.
-Examples of line sources:
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1. Mercury and sodium vapor lamps – used for UV and visible
regions
2. Hollow cathode lamp – used for atomic absorption
spectrophotometry (AAS)
3. Light amplification by stimulated emission of radiation or
LASER
-Factors to be considered when choosing a light source:
1. Range
2. Spectral distribution within the range
3. Source of radiant production
4. Stability of the radiant energy
5. Temperature
2. ENTRANCE SLIT
 Minimizes stray light emitted by the lamp
 Prevents scattered light from entering the monochromator
Note: -The second component of a spectrophotometer is the entrance slit
which minimizes unwanted or stray light and prevents the entrance of
scattered light into the monochromator system. This scattered light is
the stray light.
-Stray light refers to any wavelength outside the band transmitted by
the monochromator.
 It does not originate from polychromatic light source.
 It causes absorbance error and limits the maximum
absorbance that the spectrophotometer can achieve.
 Stray light is the most common cause of loss of linearity at
high analyte concentration.
3. MONOCHROMATOR
 Used to eliminate unwanted wavelengths of light
 Isolate sharply specific wavelengths of light
 Selects amount of light
Note:-The most essential and most important function of the monochromator
is the isolation of individual wavelengths of light.
-It allows measurement of light intensity in a much narrower wavelength
or it selects the amount of light that can pass through the cuvet.
-The light source emits light of a wide variety of wavelengths. Here the
interest is in using light of only a single wavelength or at least a very
narrow band of wavelengths for the measurements. The proper band
of wavelength is selected by the monochromator.
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MLS 11A CLINICAL CHEMISTRY
-The light shines on the wavelength selector or monochromator, and
this spreads into a wide band of rays. By moving the monochromator or
by using the monochromator, we can direct the specific desired
wavelength through the sample.
KINDS OF MONOCHROMATOR
1. PRISMS
 Triangular or wedged piece transparent material
 Allows only desired wavelength to pass through an exit slit
 Visible: glass
 UV : quartz
 IR: sodium chloride and potassium bromide
2. DIFFRACTION GRATING
 Grooved piece transparent material (3000 or more per mm) that
function as prism
3. FILTERS
 Produce monochromatic light
 Light waves enter one side of the filter and reflected on the
second surface
Note: 1. Prisms are wedge-shaped pieces of glass or quartz or sodium
chloride. It can be rotated allowing only the desired wavelength to pass
through an exit slit. A narrow light focused on a prism is refracted as it
enters the more dense glass.
-In the visible region, the prism is made of glass. For UV region, it is
ideal to use quartz. The infrared region uses sodium chloride and
potassium bromide.
2. Diffraction grating – simple, least expensive but are not precise.
They are made by placing semi-transparent similar films on both sides
of the electric such as magnesium fluoride.
3. Filter – produces monochromatic light based on the principle of
constructive interference of waves. Light waves enter one side of the
filter and are reflected on the second surface.
-It usually passes a wide band of radiant energy and has a low
transmittance of the selected wavelength.
4. EXIT SLIT
 Controls the amount of emergent light that passes through the
cuvette
Note: -The exit slit controls the width of the light beam. It allows only a
narrow fraction of the spectrum to reach the sample cuvette.
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-Remember that the entrance slit minimizes stray light emitted by
lamp. It prevents scattered light from entering the monochromator. On
the other hand, the exit slit controls the amount of emergent light
that passes through the cuvette.
5. CUVETTE
 Analytical cell, sample cell, sample holder or absorption
 Holds the solution whose concentration is to be assayed
Note: -assayed or measured
-The cuvette or the sample holder is designed to pass most of the
incident light through without absorbing it.
-More sophisticated instruments use square cuvettes to minimize the
light scatter with the use of a round cuvette.
-The liquid sample or the sample to be tested is poured into the cuvette
which is then placed in the sample holder so the light can pass through
it to the detector.
KINDS OF CUVETTE
1. Glass
 Aluminosilicate glass: acidic solutions
 Borosilicate glass: alkaline solutions
2. Soft Glass
3. Quartz or plastic
 Visible and UV spectra
Note: -The composition of the cuvette is more important for proper analysis.
Although regular glass cuvettes are transparent in the visible region
and near the UV region, measurements below about 330 nm require
special quartz cuvettes that do not absorb UV light strongly. These
cuvettes are much more expensive than the glass one.
-The kind of cuvette to be used in the laboratory is dependent on the
type of solution to be measured.
-In using cuvettes, remember that the clear side of the cuvette must
always face the light source.
Considerations & Precautions
1. Free from scratches and dirt
2. Avoid prolonged standing of alkaline solution in cuvettes
3. Position properly in photometer
4. Match absorbance readings
5. Square or rounded ends
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Note: -Utmost care in the use of cuvettes must be properly observed.
-Cuvettes with scratches in their optical surface scatter light and should
be discarded. This means prior to the use of cuvettes, they must be
checked for presence of scratches or dirt.
-Alkaline solutions should not be left standing in cuvettes for prolonged
periods because alkali slowly dissolves glass which then leads to
(etching).
-Proper positioning is also essential to get accurate results (The clear
side of the cuvette must always face the light source).
6. PHOTODETECTOR
 Detector or photocell
 Measures the intensity of the emergent light form the solution
 Detects and converts transmitted light
 Detects amount of light that passes through the sample in a
cuvette
Note: -It detects and converts transmitted light into photoelectric energy.
-Any photosensitive device can be used as a detector of radiant
energy.
-The photocell and phototube are the simplest photodetectors
producing current proportional to the intensity of the light striking
them.
-The detector or the photodetector responds to light striking it. When a
transmitted light hits the photodetector, an electric signal is generated
going through the detector system to give a read-out display. The read-
out display indicates the amount of light passing through the sample.
KINDS OF PHOTODETECTOR
1. BARRIER LAYER CELL
 Photovoltaic cell
 Found in photometer with a wide bandpass
 Detecting and measuring radiation in the visible region
 Made up of selenium on a plate of iron convered with transparent
layer of silver
 The response falls off to about 10% of the maximum at 350 & 750
nm
Note: -It is the simplest photodetector, least expensive, but temperature-
sensitive. It is used in filter photometers with a wide bandpass. It is a
basic transducer that is used for detecting and measuring radiation in
the visible region.
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2. PHOTOTUBE
 Has cathode and anode (enclosed) in a glass case
 Contains photosensitive material that gives photoelectron when
light energy strikes it
 Needs an external voltage (for operation)
3. PMT
 Photomultiplier tube
 Most commonly used detector measuring visible and UV regions
 Has excellent sensitivity
 Detects very low levels of light
 Should never be exposed to room light because it will burn out
Note: -It has a rapid response because it detects very low levels of light. The
response of PMT begins when incoming photons strike a photocathode.
4. PHOTODIODE
 Has excellent linearity
 Measures light at a multitude wavelengths
 Most useful as a simultaneous multichannel detector
Note: -It is not as sensitive as PMT but it has excellent linearity.
-It has a lower dynamic range and higher noise compared to PMT.
7. METER
 Read out Device
 Converts electrical energy into readable digits and numbers
o Absorbance or concentration
 E.g. galvanometer, ammeter, LED Display
Note: -The meter or the read-out device displays the output of the detection
system. It numerically presents absorbance or percent transmittance
through conversion of electrical energy into readable digits and
numbers.
-It will show absorbance or the optical density, or the concentration
of the solution or the sample.
8. POWER SOURCE
 Provides stable energy for the spectrophotometry
Note: -In short, it is the energy source to make the spectrophotometer work.
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MAJOR CLASSES OF SPECTROPHOTOMETRIC DEVICE
SINGLE BEAM Spectrophotometer
 one measurement at a time at one specified wavelength
 absorption maximum of the analyte must be known in advance when
a single beam instrument is utilized
Note: -Single beam spectrophotometer measures the relative light intensity of
the beam before and after a test sample is inserted.
-Single beam instruments can have a larger dynamic range and are
optically simpler and more compact.
-Single beam spectrophotometer is of the simplest type.
DOUBLE BEAM Spectrophotometer
 Compares the light intensity between two light paths
Note: -In this type of spectrophotometer, before the light reaches the sample,
it is split into two separate beams. One passes through the sample.
The second one is used for reference. This gives the advantage
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because at the same time, the reference reading and the sample
reading can take place. The additional beam which is for the reference
sample, corrects for variation in light source intensity.
-In this type of spectrophotometer, the absorbance of the sample can
be recorded.
-What is the difference between the double beam spectrophotometer
and the single beam spectrophotometer? Basically, the double beam
spectrophotometer splits the monochromatic light into two components.
This makes the double beam spectrophotometer capable of
measuring the absorbance of the standard/reference sample and
the test sample all at the same time. This additional beam reflected
on the reference sample or the standard gives the double beam
spectrophotometer the ability to correct variation in light source
intensity. The absorbance of the sample can be recorded directly
as the electrical output of the sample beam.
BEER’S LAW
The concentration of a substance is directly proportional to the amount
of light absorbed or inversely proportional to the logarithm of the transmitted
light
Note: -The basic principle of spectrophotometry involves measuring the
amount of light absorbed by a solution and relating that absorption
to the solution’s concentration.
-If we shine light through a liquid material, part of the light energy is
absorbed by the molecules in the solution. This absorption of light
depends on the structure of the molecule, primarily the types of
covalent bonds present.
-The Beer’s Law explains the principle of spectrophotometry. It states
that the concentration of the unknown substance or the substance to be
measured is directly proportional to the absorbed light, and inversely
proportional to the amount of transmitted light.
-The Beer’s Law mathematically establishes the relationship between
concentration and absorbance.
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MLS 11A CLINICAL CHEMISTRY MLS 3A
Note: -Beer-Lambert’s Law is often referred to as the Beer’s Law. It
expresses the relationship between the concentration and the
absorbance.

BEER – LAMBERT’S FORMULA

A = abc = 2 – log %T
Where:
A: absorbance
a: absorptivity value
Note: -The figure above shows a beam of monochromatic light entering a b: light path of the solution
solution. Some of the light is absorbed. The remainder passes through, c: concentration of substance
strikes a light detector, and is converted to an electric signal.
 %T is inversely proportional and logarithmically related to the
-Percent transmittance is the ratio of the radiant energy transmitted (T)
concentration of the solution.
divided by the radiant energy incident on the sample (I).
-All light absorbed or blocked results in 0% T. A level of 100% T is  Absorbance is directly proportional to the path length.
obtained if no light is absorbed. In practice, the solvent without the
Note: -Light path of the solution/ path length is the length of the cuvette in
constituent of interest is placed in the light path, as in Figure B. Most of
centimeters
the light is transmitted, but a small amount is absorbed by the solvent
and cuvette or is reflected away from the detector. The electrical readout
% TRANSMITTANCE
of the instrument is set arbitrarily at 100% T, while the light is passing
through a “blank” or reference.  ratio of the radiant energy transmitted (T) divided by the radiant
-The sample containing absorbing molecules to be measured is placed energy incident on the sample (I)
in the light path. The difference in amount of light transmitted by the
blank and that transmitted by the sample is due only to the presence
of the compound being measured. The %T measured by commercial
𝑰𝒕
𝒙 𝟏𝟎𝟎
spectrophotometers is the ratio of the sample transmitted beam divided 𝑰𝒐
by the blank transmitted beam. Where:
𝐼𝑡 : transmitted light thru the sample
-Incident light refers to the amount of light entering the solution. 𝐼𝑜 : intensity of light striking the sample
-Transmitted light refers to the amount of light passing through the
solution without being absorbed.
-By comparing the incident light and the transmitted light, we can
calculate the concentration of the material or the analyte in the sample Application of Beer – Lambert’s Law
or solution. 1. Monochromatic incident radiation on the substance of interest
2. Solvent absorption is insignificant compared to solute absorption
3. Solute concentration is within linear limits
SPECTROPHOTOMETRY 4. Quenching phenomenon does not occur
BEER – LAMBERT’S LAW
 Concentration of a substance is directly proportional to the Note: -It must be noted that Beer’s law may only be applied in accurate
amount of light absorbed quantitative analysis by light absorption, if the following requisites are
met:
 Inversely proportional to the logarithm of the transmitted light
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MLS 11A CLINICAL CHEMISTRY
1. Incident radiation on the substance of interest is monochromatic.
Monochromatic means that the light is of single wavelength.
2. Solvent absorption is insignificant compared to solute absorption.
3. Solute concentration is within “linear limits.” It should be within the
detection limit of the spectrophotometer.
4. Quenching phenomenon does not occur.
-Quenching phenomenon is a chemical reaction between the
molecule of interest and another solute or solvent molecule.
-If these 4 requisites are met, we can ensure that we can acquire
accurate results or accurate measurements of the concentration of the
solution.
Deviations from Beer – Lambert’s Law
1. Simultaneous absorption at multiple wavelengths
2. Absorption of light by other species
3. Transmission of light by other mechanism
Note: -Deviations or the sources of inaccurate results
-We need to have monochromatic light or single wavelength.
-Transmission of light by other mechanism such as that of the quenching
phenomenon
Calculation of the Concentration of the Unknown from Absorbance
Measurements
1. Use of standard curve
2. Ratio of standard to unknown
3. Use of molar absorptivity values
Note: -There are three major methods for calculation of the concentration of
the unknown from absorbance measurements.
-The selection of a method depends on several factors.
-In some instances, the absorption value for the standard varies
significantly from one day to the next. A standard curve then needs to
be prepared every time a measurement is performed. If we know the
linear range or the linear limit of the assay, we can use one standard
within that range to compare with our unknown sample.
-If the standard curve is stable for an extended time, only when such
curve needs to be run at some predetermined interval, unknown values
can be determined from this curve.
-The use of molar absorptivity requires the knowledge of the “a”
specific value for the compound of interest. In the clinical lab, the
only material commonly measured by this means is reduced
nicotinamide adenine dinucleotide (NADH). It is a co-enzyme.
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Calculation of the Unknown Concentration
Ratio of Standard to Unknown
 Used if the linear limits of the assay is known
 One standard vs unknown sample Calculation of the Unknown
Concentration
 Used if the linear limits of the assay is known
 One standard vs unknown sample
𝑨𝒖
𝑪𝒖 = 𝒙 𝑪𝒔
𝑨𝒔
Note: -The simplest type of concentration measurement involves
determination of the absorbance values of the standard and absorbance
values of the unknown. The standard has a known concentration of
the compound of interest. On the other hand, the unknown is now
found on the patient’s sample.
-Cu represents the concentration of the unknown. Au represents the
absorbance of the unknown. As represents the absorbance of the
standard. Cs represents the concentration of the standard.
-In solving for the concentration of the unknown, the formula for that is
the optical density or the absorbance of the sample/unknown divided
by the optical density/absorbance of the standard, multiplied by the
concentration of the standard.
-The concentration of the standard is dependent on the analyte
being tested and on the unit being used.
Sample Problem:
Based on the given values, solve for BILIRUBIN concentration:
Au = 0.428
As = 0.372
Cs = 5.0 mg/dL
0.428
𝐶𝑢 𝑥 5.0 𝑚𝑔/𝑑𝐿
0.372
Cu (Bilirubin concentration) = 5.75 mg/dL
Note: -In the sample problem, the absorbance of the unknown was done at
450 nm.
-The 5.0 mg/dL concentration of the substance (in the sample problem)
may vary depending on the analyte to be measured and the unit to be
used, which means the unit can change (e.g. mmol/L).
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MLS 11A CLINICAL CHEMISTRY
-Sample Problem: The aim is to know the concentration of the unknown.
The unknown is the bilirubin level.
-Answer: The concentration of bilirubin is 5.75 mg/dL.
Use of Molar Absorptivity Values
 Used if the absorptivity value (“a”) is known
Use of Standard Curve
 measurement of the absorbance of a standard of known concentration
& examination of the acquired data
 After the concentration of each standard is determined and corrected
for the blank, a standard curve can be plotted with concentration in the
y axis and the absorbance reading on the x axis
Note: -Standard curve defines properly the limits of the assay.
-These standard solutions of known concentration are measured in the
same way that a sample would be treated.
Note: -After the concentration of the standard is measured and corrected for
the blank, the standard curve can be plotted with the concentration
found in the y axis and the absorbance on the x axis.
Factor Involved in Making Absorbance Measurements
 Blank Correction
o REAGENT BLANK
 Contains only the reagent
 Corrects OD (optical density or absorbance) readings
due to other materials in the reagent
1. Set the absorbance to zero using distilled
H2O
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Take OD of reagent blank and
sample
 Sample – reagent blank
2. Zero the instrument using the reagent
blank
 Automatic subtraction
o SAMPLE BLANK
 Contains only the sample
 Correct non-specific absorbance of other
substances in the sample
 Separate blank for every sample
 Same absorbance with bilirubin & hemoglobin
Note: -Blank correction or simply blanking. There are two types. One is
reagent blanking and the other is sample blanking.
-The use of reagent blank and sample blank is one of the most
neglected aspects of training in analytical techniques.
-Even when using very automated equipment, awareness of problems
associated with spurious/false absorbance of light can quickly solve
a problem
-Sample Blank:
 The only disadvantage is because there must be a separate blank
for every sample.
 A sample blank has the same absorbance with bilirubin and
hemoglobin. That is why, utmost care must be taken in examining
for the presence of hemolysis especially for those specimens with
higher bilirubin levels.
FLUOROMETRY
 Molecular Luminescence Spectophotometry
 measures analytes which have the ability to absorb light of lower
wavelength and transmit it at a higher wavelength
 Amount of light the compound emits is roughly proportional to the
concentration of compound.
Note: -Increasingly powerful tool in the analytical field is the use of
fluorescence measurements for identification and quantitative
measurements. In many instances, more sensitive assays were
developed like fluorometric techniques than spectrophotometry. This
change in approach often allows the use of smaller samples and less
reagents and permits analysis in less time.
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MLS 11A CLINICAL CHEMISTRY MLS 3A
-Frequently, problems with interferences seen in spectrophotometric -In clinical chemistry laboratory, the following analytes can be measured
measurements can be eliminated and decreased with fluorometric by fluorometry namely: porphyrins, Mg, Ca, catecholamines.
assays. However, problems can still arise.
-Fluorometry is otherwise known as Molecular luminescence FLUOROMETER
spectrophotometry. - uses 2 monochromators (filters, prisms, gratings)
-Fluorescence measurement involves interaction of light to a chemical - primary filter (Excitation filter)
compound. In this case, the chemical compound emits light in response -secondary filter (emission filter)
to the light striking it. The emitted light of compound is usually of a -1000x more sensitive
longer wavelength. This emitted light is detected by a phototube that -Affected by quenching
determines the intensity. The amount of light the compound emits  pH and temperature changes
is roughly proportional to the concentration of compound in most  Chemical contaminants
cases.  UV light changes

Luminescence- Production of light without the production of heat.
Fluorescence- electrons within a chemical are induced to absorb light and
become excited using high energy radiation.
Note: -Luminescence is a general term to describe the process in which the
material absorbs energy from an external source and reemits the energy
in a form of visible light.
-Fluorescence is actually a form of luminescence. This one occurs
when electrons give off light as they drop from their excited state back
from their ground state level within the molecule.
PRINCIPLE OF FLUOROMETRY
Principle: determines the amount of light emitted by a molecule after
excitation by electromagnetic radiation
Light Source: Hg Arc lamp or Xenon Lamp
Light Detector: PMT or phototube
Use: porphyrins, Mg, Ca++, catecholamines
- It is based on the principle that certain compounds absorb
electromagnetic radiation, become excited, and return to an energy
level lower than of equal to the original level. Emitted energy is less
than the absorbed energy, so that the wavelength of emitted light is
longer than absorbed light.
Note: -Fluorometry measures the amount of light intensity present over a
zero background. This principle is on the determination of the
amount of light emitted by a molecule after excitation by
electromagnetic radiation.
-The light sources are mercury arc lamp or xenon lamp.
-The light detectors are photomultiplier tube or phototube.
Note: -Fluorometer uses 2 monochromators. It can either be filters, prisms
or gratings.
-We have discussed the function of monochromator, and that is, it is
responsible in the selection of light that will pass thorugh the solution in
a cuvette. These monochromators can either be primary filter or
secondary filter. The wavelength that is best absorbed by the
solution to be measured is selected by primary filter. On the other
hand, the incident light is prevented from striking the photo
detector by secondary filter.
-In short, primary filter is the excitation filter, and secondary filter is
the emission filter.
-Fluorometer is 1000x more sensitive than spectrophotometer.
Nevertheless, it is affected by quenching. Recall that quenching is a
chemical reaction occurring between the molecule of interest and
another solute or solvent molecule, which means fluorometry is
sensitive to pH and temperature changes, sensitive to chemical
contaminants, and lastly, UV light changes.
COMPONENTS OF A FLUOROMETER
 Light Source
 Entrance Slit
 Primary monochromator
 Cuvette
 Secondary monochromator
 Exit slit
 Detector
Note: -So we have here the components of fluorometer. We have the light
source, the entrance slit, the primary monochromator, the cuvette, the
secondary monochromator, the exit slit and the detector.

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MLS 11A CLINICAL CHEMISTRY
-Note that fluorometer has two monochromators. The secondary
monochromator is placed at 90 degrees angle to the exciter lamp
but in line with photo detector. The purpose of this is to ensure that
the only light emitted from the excited sample reaches the
photodetector.
LIGHT SOURCE
-exciter lamp
-provides radiant energy that will excite the fluorogenic substance
-emits short-wavelength high-energy excitation light
Note: -The most common light source in fluorometry is a xenon lamp which
gives of here a fairly intense light output over a wide range of
wavelengths. The xenon lamp has continuous spectrum in the UV
region with the most useful wavelength range between approx. 300 to
550 nm.
PRIMARY MONOCHROMATOR
-excitation monochromator
-placed between the radiation source and the sample
-Selects the wavelength that is best absorbed by the solution to be
measured
Note: -The excitation monochromator is very similar to the
spectrophotometer which selects out a defined group of excitation
wavelengths which are then directed into a sample. In short, light of
wavelengths above and below their region of interest is blocked by this
primary monochromators. This filter only allows the light of the desired
wavelength range to strike the sample.
CUVETTE
-Holds the sample
- the fluorescing sample in the cuvette emits radiant energy in all
direction.
Note: -The cuvette functions to hold the sample to be examined. The
fluorescing sample in the cuvette emits radiant energy in all directions.
-In most situations, it is best to use glass cuvettes for fluorometric
measurements. Remember that cuvettes must be kept from
fingerprints, spots and scratches. When using in the laboratory,
please be careful not to break the cuvettes because they cost a lot
especially the glass or quartz type.
MLS 3A
SECNDARY MONOCHROMATOR
-emission monochromator
-removes most of the excitation light before being measured by a
detector photodetector
-emitted light from the fluorescent sample is detected at a right angle
to the incident light
Note: -Another is the secondary monochromators or emission
monochromator. This filter is responsible in selecting the emission
wavelength of interest as well as removing most of the excitation
light before being measured by a photo detector.
-The emitted light from the fluorescent sample is detected at a right
angle (90 degrees) to the incident light, for the purpose of
elimination of potential interference by excitation or in short, this
filter is responsible for selecting or screening out unwanted higher
wavelengths.
PHOTODETECTOR
-detects the emitted light from the fluorescent sample
-requires phototubes or photomultiplier tubes (PMT) for fluorescence
measurements because the signals are generally of low intensity.
Note: -The photodetector is responsible for detection of emitted light from the
fluorescent sample. The photodetectors for fluorometry require
phototubes or photomultiplier tubes (PMTs) for fluorescence
measurements because the signals are generally of low intensity.
The fluorescence emitted by the sample strikes the photomultiplier
tube which then converts light energy into an electrical signal.
QUANTITATION USING FLUORESCENCE MEASUREMENT
- Fluorescence concentration measurements are related to molar
absorptivity of the compound, intensity of the incident radiation, quantum
efficiency of the energy emitted per quantum absorbed, and length of the light
path.
-In dilute solutions with instrument parameters held constant,
fluorescence is DIRECTLY PROPORTIONAL to concentration.
Note: -The most common method of quantitation with fluorometry involves the
use of standard curve or single standard. It is very difficult to
determine the fluorometric equivalent of the molar absorptivity
coefficient, so reference must be made to some standard measurement
performed at the same time using the same condition.
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MLS 11A CLINICAL CHEMISTRY MLS 3A
-In dilute solutions with instrument parameters held constant, energy input is provided by atomizing a solution containing the ion of
fluorescence is directly proportional to concentration. interest and burning it in a flame.
-We have to remember that atoms when excited by heat given off by a
FLAME EMISSION SPECTROSCOPY flame, emits energy in a form of a colored light. FES measures the
-Used to measure intensity of light emitted when an element is exposed light emitted by a single atom burned in a flame. As mentioned
to a flame. earlier, FES is used to measure excited ions such as Na and K, which
-Atoms when excited by heat given off by a flame, emit energy in the are electrolytes. But we have to remember that flickering lights in FES
form of a colored light. indicate changes in the fuel reading of the instrument.

-Atoms when excited by heat given off by a a flame, emit energy in the PRINCIPLES OF FES
form of a colored light Principle: excitation of electrons from lower to higher energy state.
-Measures the light emitted by a single atom burned in a flame Light source: Flame
-Quantitative measurement of excited ions (e.g. electrolytes) Method: Indirect Internal Standard Method
-Flickering lights: changes in the fuel reading of the instrument. Internal Standard: Lithium/Cesium- corrects variations in flame and
atomizer characters.
Note: -Light intensity = number of atoms emitting the energy= concentration
of substances
-FES involves the excitation of electrons from lower to higher
energy. Basically, it is based on excitation of electrons in an atom by
heat energy of a flame. The light source is the flame itself which also
serves as the cuvette or the sample holder. The method for this one
is Indirect Internal Standard Method.
-Internal standard uses Lithium and Cesium which corrects
variations in flames and atomizer characteristics. Therefore, in FES,
the light intensity is equal to the number of atoms emitting the
energy which is also equal to the concentration of the substance.
Thus, the greater the number of atoms emitting the energy, the
greater the concentration of the substance, and vice versa.
Some FES Analytes and their characteristic colors:
Name of Element Emitted wavelength Observed Color of
range (mm) the flame
Potassium (K) 466 Violet
Lithium (Li) 670 Red
Calcium (Ca) 622 Orange
Sodium (Na) 589 Yellow
Note: -One major difference between FES and Fluorometry is the source of Barium (Ba) 554 Lime Green
excitation energy. For fluorescence, light from the xenon lamp
provides energy for electron transitions. However, in FES, the
excitation energy comes from heating the ion.
-In FES, by raising the temperature of the material sufficiently, electrons
in atoms such as Na and K can be excited so that, detectable amounts
of energy are released as they return back to their ground state. This
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MLS 11A CLINICAL CHEMISTRY MLS 3A
Note: -We have here the components of FES: the nebulizer/atomizer, the
mixing chamber, the flame, the monochromator, and the detector.
-In FES, we have the burner assembly that holds the aspirator, the
atomizer or burner and the flame.
 The aspirator draws sample into the flame through vacuum and
gravitational feed aspirators.
 The atomizers or burners breaks up solution into fine droplets
Note: -Take note of the different analytes particularly the electrolytes which or facilitates excitation by spraying the sample into fine mists or
are being measured using FES and their emitted wavelength range and droplets; and the
observable color flame. Here we have additional analytes and their color  Flame, which is the source of energy or heat for excitation and
characteristics when measured with FES. can either be conventional or a mixture of oxygen and
hydrogen, natural gas methane or acetylene or propylene
COMPONENTS OF FES with oxidant.
1. Nebulizer/Atomizer
2. Mixing Chamber NEBULIZER
3. Flame -Breaks up the sample into atoms
4. Monochromator -Facilitates excitation by spraying the sample into fine mists or droplets
5. Detector
MIXING CHAMBER
-mixes the relevant solutioin with the sample

FLAME
- a burner that is within the instrument needed for temperature control
-heats up the sample

Note: -Nebulizer or atomizer breaks up the sample into atoms and facilitates
excitation by spraying the sample into fine mists or droplets.
-The mixing chamber is necessary for mixing the relevant solution with
the sample.
-The flame is a burner found within the instrument needed for
temperature control. This is responsible for heating up the sample and
the source of energy for the excitation of atoms.

BURNER ASSEMBLY
a. Aspirator
 Draws sample into the flame (vacuum & gravitational feed aspirators)

b. Atomizer or burner
 Breaks up solution into fine droplets
 Facilitates excitation by spraying the sample into fine mists or droplets
➢Total consumption burner
 Provides energy or heat for excitation

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MLS 11A CLINICAL CHEMISTRY MLS 3A
c.Flame variation in flame and atomizer characteristics, and of course does
1. Conventional (mixture of oxygen & hydrogen) not present interferences.
2. Natural gas methane -This is the process of FES.
3. Acetylene or propylene with oxidant  The aspirator draws the sample into the flame.
 The atomizer or burner breaks up the solution into fine droplets
OPTICAL SYSTEM so that the atoms will absorb heat energy and become excited.
Optical system  The flame will provide the energy for excitation.
-Convex Mirror  The optical system or optical filter is comprised of three parts:
-Lens the monochromator, the lens, and the entrance and exit slits.
- Filter  The photodetectors will detect the emitted light and measure the
Photo Detector intensity of radiation emitted by the flame. That is, the emitted
radiation is converted to an electrical signal with the help of
METHODS OF FES photodetector. The produced electrical signals are directly
DIRECT METHOD proportional to the intensity of light.
 Standard used: Sodium and Potassium  The output device which interprets and represents the actual
 Aspirated directly into the flame then compared directly with the data the detector measures.
unknown
INDIRECT METHOD or INTERNAL TYPE INTERFERENCES
 Lithium as internal standard  two elements produce spectra and both emit radiation at some particular
 Cesium as alternative wavelength
1. Radiation buffer to prevent mutual co-excitation  spectral lines of two or more elements which are close but their spectra
2. Absent in body fluids do not overlap
3. Does not present interferences  Organic solvents

Spectral
 incomplete isolation of the radiation emitted or absorbed by the analyte
from other radiation detected by the instrument.
• First type- interferent and the analyte exhibit spectra, which
partially overlap, and both emit radiation at some particular
wavelength.
• Second type- spectral lines of two or more elements which are
close but their spectra do no overlap.
• Third type- presence of continuous background due to high salt
concentrations.

Note: -There are 2 methods for FES and can either be direct or indirect
method (Internal type).
-For direct method, the standard used is Na and K, and are aspirated
directly into the flame then compared with the unknown.
-For indirect method, internal standard used is lithium or cesium as
alternative. Lithium is used as an internal standard as it acts as a
radiation buffer to prevent mutual co-excitation. It also corrects
Note: -First, spectral are incomplete isolation of the radiation emitted or
absorbed by the analyte from other radiation detected by the
instrument. It has three types:
 The first type is interferent and the analyte exhibit spectra, which
partially overlap, and both emit radiation at some particular
wavelength
 Second type are spectral lines of two or more elements which
are close but their spectra do not overlap; and
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MLS 11A CLINICAL CHEMISTRY
 The third type which have presence of continuous background
due to high salt concentrations.
Note: -Additional sources of interferences include:
 Ionization, wherein high temperature of the flame may cause
some of these analytes to become ionized;
 Chemical, wherein it can cause interaction between different
inferents and the analyte;
 Cation-anion characterized by presence of certain anions which
can affect the intensity of radiation;
 Cation-cation which reduces signal intensity and masks or hide
the analyte; and
 Oxide formation which also reduces signal intensity and affects
alkaline earth metals.
APPLICATION
 Trace metals in a liquid sample
 common metals (Alkali, Alkaline Earths, Iron, Manganese, Copper
and Zinc)
 industrial analyses(agricultural and environmental analysis)
 measure sodium, potassium ions in body fluids, muscles and heart.
 measurement of lithium, calcium, and barium levels in the body
ADVANTAGES
 a simple quantitative analytical test
 performed easily with most reliable and convenient methods
 quite quick, convenient, selective and sensitive to even parts per
million (ppm) to parts per billion (ppb) range
MLS 3A
DISADVANTAGES
 metal ion concentration present in the solution cannot be measured
accurately
 requires standard solution with known molarities for determining the
concentration of the ions which will correspond to the emission
spectra
 difficult to obtain accurate results of ions with elevated concentration
 unable to determine the information about the molecular structure of
the compound being assayed
 unable to detect elements with non- radiating nature.
ATOMIC ABSORPTION SPECTROPHOTOMETRY
 it measures the light absorbed by atoms dissociated by heat
 Dissociation of chemical bonds as free atoms in the neutral ground
state
 Atoms absorb radiation equal to the energy they will emit if they have
been excited
AAS: PRINCIPLE
The ionic form of the element is not excited in the flame but is
dissociated from its chemical bonds and is placed in the atomic ground state
by attracting free electrons produced by the combustion process. In this form,
it is capable of absorbing light at the specific wavelengths of its line spectrum
Note: -Atomic Absorption Spectrophotometry (AAS) is the inverse of Flame
Emission Spectroscopy (FES) because it is ideal for metals that are
easily excited by heat. All atoms can be converted to dissociated form.
-AAS is used for measurement of unexcited trace metals such as
calcium and magnesium.
-This is more sensitive than Flame Emission Photometry. It is precise,
accurate and very specific. Other than that, AAS does not need
internal standard.
-The disadvantage is the inability of the flame to dissociate samples
into free atoms or smaller droplets.
-AAS is based on the fact that there is no excitation of the electron
but merely dissociation of their chemical bonds as free atoms in the
neutral ground state.
 Just a quick review of what dissociation means, it means
breaking up of a compound into smaller constituents. After
dissociation process, these free atoms are capable of absorbing
radiation equal to the energy of these atoms, which will emit if
they have been excited.
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MLS 11A CLINICAL CHEMISTRY MLS 3A
-In AAS, merely all atoms can be converted into dissociated form. -Nevertheless, we have lanthanum or strontium which are being
added to the samples. These two can be added to the samples to form
COMPONENTS OF AAS stable complexes with the phosphates.
1. Light source -The hollow-cathode lamp produces wavelength of light specific for the
2. Chopper metal in the cathode. It can be neon, argon, quartz or special glass.
3. Flame/sample holder -The light source consists of an evacuated gas-tight chamber. In this
4. Monochromator gas-tight chamber, it contains an anode, a cylindrical cathode, and an
5. Detector inert gas such as helium or argon.
 What happens here inside the gas-tight chamber is when a
voltage is applied the filler gas is ionized, ions attracted to the
cathode collide with the metal, knock atoms off, and cause the
metal atoms to be excited. When they return to the ground state,
light energy is emitted that is characteristic of the metal in the
cathode.
-Generally, a separate lamp is required for each metal.

CHOPPER
 breaks the steady light from the light source into pulsating light
 chops the light leaving the light source so that when the incident beam
hits the chopper at the solid surface, the beam will be blocked and the
detector will only read the emitted signal from the flame.
 Burner, Graphite furnace or Nebulizer
 Atomizer that sprays sample directlyinto the flame

Note: -In addition, we have the chopper. It is responsible for breaking the
steady light from the light source into pulsating light.
LIGHT SOURCE -It is a rotating wheel placed between the flame and the light source.
 Hollow-cathode lamp -It chops the light leaving the light source so that when the incident beam
 INTERFERENCE: chemical, matrix (difference in viscosity) & ionization hits the chopper at the solid surface, the beam will be blocked, and the
 Lanthanum or strontium is added to samples detector will only read the emitted signal from the flame.
 Produces wavelength of light specific for the metal in the cathode -Choppers can be also in the form of a burner, graphite furnace, or
o Neon: reddish orange glow nebulizer. These are the atomizers that spray sample directly into the
o Argon: purple glow, filter flame. Atomizers are used to convert ions to atoms. In short, chopper
o Quartz or special glass: window is used to modulate the light source.
 consists of an evacuated gas-tight chamber containing:
o an anode SAMPLE CHAMBER
o a cylindrical cathode  Flameless system
o an inert gas  Carbon rod (graphite furnace), tantalum or platinum

Note: -The light source for AAS is hollow-cathode lamp.
-One disadvantage of this type of light source is it is sensitive to
chemical matrix or the difference in viscosity. Other than that, it is
sensitive to ionization.
Note: -In AAS, the sample chamber is a flameless system. So, it can be either
made up of carbon rod, tantalum, or platinum.
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MLS 11A CLINICAL CHEMISTRY MLS 3A
AAS TYPES OF AAS
 Detector will not detect emitted light from the atoms in the ground state 1. Flame atomic absorption spectrophotometer
 Amount of light measured: indirectly proportional to the concentration
of the analyte

Note: -We must remember that detector in AAS will not detect emitted light
from the atoms in the ground state. So, in AAS, the amount of light
measured is indirectly proportional to the concentration of the
analyte.

ATOMIC ABSORPTION SPECTROPHOTOMETRY

Concentration measurements are usually determined from a working 2. Graphite furnace atomic absorption spectrophotometer
curve after calibrating the instrument with standards of known
concentration.

Interferences in AAS

CHEMICAL INTERFERENCE
 Flame fails to dissociate sample into free atoms
 E.g calcium phosphate
 Lanthanum or strontium
 Higher temperature

IONIZATION INTERFERENCE
 Atoms become excited instead of being dissociated
 Emit energy with same wavelength as measured
 Lowering the flame

MATRIX INTERFERENCE
 Formation of solid particles from sample droplets
Note: -In working with Atomic Absorption Spectrophotometry, concentration  Evaporation of solvents >0.1 mol/
measurements are usually determined from a working curve after  Enhancement of light absorption by organic solvents
calibrating the instrument with standards of known concentration.  Refractory oxides of metals
-The elements that are highlighted in pink are the analytes which can be
detected using AAS. Note: -The factors that can interfere in the measurement of analyte
concentration using AAS:

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MLS 11A CLINICAL CHEMISTRY
1. We have chemical interference wherein the flame fails to
dissociate sample into free atoms. So we have here an example
of an analyte, which is calcium phosphate. Calcium phosphate
can be added with lanthanum or strontium so that there will be
a stable complex which will be formed. Other than the addition of
lanthanum or strontium, we can also increase the temperature.
2. Next, we have ionization interference. In this case, atoms
become excited instead of being dissociated. So they emit
energy with the same wavelength as measured. The remedy for
this is to lower the flame.
3. Other than chemical interference and ionization interference, we
have matrix interference. In this case, we have formation of solid
particles from sample droplets. This is due to enhancement of
light absorption by atoms in organic solvents. In this case,
there is formation of solid droplets as the solvent evaporates in
the flame. The remedy for this is pre-treatment of samples by
extraction. When we say pre-treatment, prior to the testing, we
need to add here organic solvents or we need to add here
oxides of metals.
DISADVANTAGES OF AAS
 Inability to dissociate sample into free atoms
 Ionization of atoms following dissociation by the flame
 only solutions can be analyzed
 relatively large sample quantities required
 problems with refractory elements
 Limitations in variety of analyte the machine can detect
Note: -Inability of AAS to dissociate sample into free atoms: means in
AAS there is a need for us to dilute.
APPLICATIONS OF AAS
 Clinical uses: analysis of metals in biological fluids
 Environmental analysis: finding out the levels of various elements in
rivers, seawater, drinking water, air, petrol and drinks
 Pharmaceutical
 Water analysis (e.g. Ca, Mg, Fe, Si, Al, Ba content)
 Food analysis
 Analysis of additives in lubricating oils and greases
 Analysis of soils
MLS 3A
Note: -So we have here the applications of AAS, particularly their clinical
applications, that is on the analysis of metals in biological fluids.
-It also has its purpose on environmental analysis on finding out the
levels of elements in the rivers, drinking water, or the oil or the drinks.
-This is also very useful in pharmaceutical companies.
-Other than that, it can also be used as a method of choice for water
analysis, food analysis, analysis of soils, and analysis of additives in
lubricating oils and greases.
REMEDY FOR INTERFERENCES IN AAS
 Anions that interfere with analysis by binding with the cations: adding
competitors to bind with this ions to prevent complex formation
 Formation of refractory elements: adding chelating or releasing agents
 Ionization of the atoms: proper flame temperature regulation
 Matrix interferences: pretreatment of the sample.
Note: -Remember that in the previous discussion, we have three factors which
can interfere with AAS: we have the chemical factor, ionization
interference, and the matrix interference. Now of course, we have the
remedy.
1. Anions that interfere with analysis by binding with the
cations, what will we do? That is we need to add
competitors to bind with this ions to prevent
complex formation.
2. Formation of refractory elements. With this case, we
need to add chelating or releasing agents that is to
remove some of these unwanted elements.
3. We have ionization of the atoms. The remedy for that
is to adjust flame temperature or to regulate the
flame temperature.
4. We have the matrix interference. To avoid matrix
interference, we need to pretreat the sample. In short,
prior to running or measuring the analyte, we need to
perform pretreatment of the sample.
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MLS 11A CLINICAL CHEMISTRY MLS 3A
DIFFERENCE BETWEEN AAS AND FES
FES AAS
✓ measures the radiation emitted ✓ measures the radiation
by the excited atoms that is absorbed by the unexcited
related to concentration atoms that are determined.
✓ flame emission intensity is ✓depends only upon the number
dependent upon the number of of unexcited atoms, the
excited atoms, is greatly absorption intensity is not
influenced by temperature directly affected by the
variations. temperature of the flame.

Both methods use atomization of a sample and therefore determine the

concentrations of elements.
FES AAS
✓ Elements are excited. ✓ Absorption of radiation of a
✓To identify the element, a rapid defined wavelength is passed
relaxation is accompanied by through a sample and the
emission of UV or visible absorption of the radiation is
radiation determined.
✓The intensity of the emitted ✓The absorption is defined by the
photon is proportional to element electronic transition for a given
concentration. element and is specific for a given
element.
✓The concentration is
proportional to the absorbed
radiation.

CHEMILUMINESCENCE
Principle of Chemiluminescence
 In chemiluminescence reactions, part of the chemical energy generated
produces excited intermediates that decay to a ground state with the
emission of photons. The emitted radiation is measured with a
photomultipliertube, and the signal is related to analyte concentration.

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MLS 11A CLINICAL CHEMISTRY MLS 3A
MODULE 2: Analytic Techniques and Instrumentation -In the first picture, the light scattered by nephelometry is shown (It is the light
Lesson 1: Instrumentation (Continuation) that is at an angle). Also shown in the picture is the light scattered by
turbidimetry.
TURBIDIMETRY AND NEPHELOMETRY
-Most of the analytes in the laboratory follow the principle of spectrophotometry.
The machine available in the university is the spectrophotometer.
-With the use of cuvette, at present, the one used in the Med. Tech. lab is made
of plastic so it’s cheaper and easy to be replaced. But if in any case that you
are going to use the glass cuvette, just be extra careful not to drop it because
it is expensive. When using the cuvettes, even the plastic ones, make sure not
to drop them or even scratch their surface because they are going to be
checked upon returning to the laboratory.

-Turbidimetry involves the determination of the amount of light blocked by the

particles which are suspended in the solution or on the sample to be measured.
-Determines the amount of light blocked by suspending particles in the Since we are measuring here the amount of light blocked, that would mean that
sample as light passes through the cuvette of spectrophotometer in turbidimetry, we are measuring the reduction of light or the amount of light
-Measures the decrease in the amount of light as it passes through a solution. reduced as the light passes through the sample.
-In addition to that, turbidimetry is used to measure abundant large particles. In
Principle: Determines the amount of light blocked by a particular matter in a turbid
particular, these particles are the proteins. For bacterial suspension, there is a
solution part in the bacteria that contains protein. Turbidimetry is very good in
Note: -Turbidimetry and nephelometry have the same principle with determination or measurement for the presence of proteins.
spectrophotometry. They also have the same components with -Principle of turbidimetry: It determines the amount of light blocked or in short,
spectrophotometry like the light source, cuvette, detector or the photodetector, the reduction of light by a particular matter.
and the monochromators/filters.  Particular matter or particulate matter are particles capable of clumping
-Review: In spectrophotometry, the amount of light absorbed is measured. If or aggregating together. It refers to the aggregation of particles or smaller
there is increased amount of light absorbed by the sample, less light is particles that aggregated. This means that the concentration of the
transmitted. This relates to the Beer’s law which states that the amount of solution is expected to be in a turbid solution.
absorbed light is directly proportional to the concentration of the solution. -Remember, before, we performed an experiment for the presence of proteins
 Spectrophotometry: Increased amount of absorbed light = increased using sulfosalicylic acid wherein the urine is added with a reagent and if there
concentration of the solution. is presence of proteins, particularly in eggs, there will be clumping formed.
What is measured? Direction of Light Machine
Uses:
 Protein measurement
Spectrophotometry Amount of light Forward Spectrophotometer
absorbed
 Detection of bacterial growth in broth culture
Turbidimetry Amount of light Forward Turbidimeter
 Antimicrobial test
reduced/blocked  Detection of clot formation
Nephelometry Amount of scattered Forward and at right Nephelometer
light angle (specifically Note: -Protein measurement: We have different kinds of proteins. In electrophoresis,
between 15-90 later on, there will also be some proteins that will be measured.
degrees) -The second to fourth uses are applicable for bacteriology.

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MLS 11A CLINICAL CHEMISTRY
-In a broth culture, how will we know that there is bacterial growth? It becomes
turbid (indicative of bacterial growth).
-Samples used for the determination of protein in turbidimetric assay: CSF and
urine
Determinants:
 Size and number of particles
 Cross-sectional area of each particle
 Depth of tube
Note: -The size and number of particles. Examples are proteins like albumin, gamma
and alpha globulin, and beta-globulin. Among these proteins, albumin is the
biggest.
-Aside from being many in number, if the size of the suspended particles in the
solution is large, that means it will block more light. The amount of light blocked
is equivalent to the concentration in the solution.
 Size and the number of particles are too large = more light is blocked =
decreased light transmitted to the detector.
 This transmittance is inversely proportional to concentration of the
solution (which is wrong) because it should be that the more the
concentration, the higher the amount of light blocked.
-Cross-sectional area of the particle is related to the size.
-Depth of the tube: In particular, it refers to the sample holder being used. In
this case, it refers to the cuvette.
 For example, we are using a 5 mL and 10 mL tube with the same amount
of analytes. If the tube is longer or has more depth (i.e. 10 mL), the more
diluent or solvent can be placed. Meaning, even if it still has the same
amount of particles in a 5 mL tube, the sample/solution will be diluted in
a longer tube (10 mL0.
 Diluted: the particles scatter more = less amount of light blocked = more
light will be transmitted and detected by the detector
 In turbidimetry, since we have less light blocked, this means it would
indicate less concentration.
 In the 5 mL tube, the amount of diluent that can be added is limited or
less which means the particles will scatter less. They will just concentrate
in one place.
 Concentrated: more light will be blocked = less light is transmitted. Result
is high concentration of the solution.
NEPHELOMETRY
- Measures the amount of light scattered by suspending particles
-Light scattered is measured at a right angle
-Amount of light scattered: related to the number and size in the light beam.
MLS 3A
Use: Measurement of Ag-Ab complexes (Proteins)
Principle: Determine the amount of scattered light by a particulate matter suspended
in a turbid solution.
Note: -If turbidimetry measures the amount of light blocked or the reduction of light is
measured, nephelometry measures the amount of light scattered by the
suspending particles or the particulate matter. The difference of nephelometry
from turbidimetry is the direction of light.
 In nephelometry, light is at an angle (15-90 degrees).
 In turbidimetry, the direction of light is forward.
-Nephelometry is more specific or more precise compared to turbidimetry.
-The uses of nephelometry particularly is on the measurement of antigen-
antibody complex. In the antigen-antibody complex, the antigen is bound to the
antibody. Remember that particulate matter are particles capable of clumping
or aggregating.
 Solution with many aggregated/agglutinated particles: Since the particles
are many, the tendency is the light will be too scattered. This means
the transmitted light going to the detector will be less. This will
indicate the solution has a high concentration.
 Solution has less particles of smaller size and low in number: Once
light passes, more light will pass through or be transmitted to the
photodetector. It is indicative of low absorbance. This is equivalent to
low concentration of the analyte in the sample.
-Example 1: In serology, the positive result is dependent on the presence of
clumping or agglutination. In serology, how will you know if the presence of
antibody is higher in that individual if the principle is based on agglutination?
 The more agglutination present in the test, the more elevated the amount
of antibody/antigen (depending on which is being measured).
-Example 2: Streptococcus pyogenes, once exposed to the body, is considered
as an antigen because it is foreign to the body. In response, the body will
produce antibody against the bacteria. The antibody will destroy the antigen by
binding to it.
 If the concentration of bacterial antigen in the body is high, it would
equate to high level of antibody produce.
 Remember that antigens are made of proteins.
 Imagine in the body, there are many antigens and corresponding
antibodies. That would result to many antigen-antibody complexes. In
that case, the sample will be turbid.
 More antigen-antibody complexes = more aggregates = high
concentration
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MLS 11A CLINICAL CHEMISTRY
-The principle of nephelometry is the measurement of the amount of light
scattered by a particulate matter suspended in a turbid solution.
MLS 3A
-In nephelometry, the relationship between wavelength and diameter
determines the angle at which the detector is located. Remember that in
nephelometry the measurement is at an angle. It’s between 15-90 degrees.
Most of the nephelometric instruments use 90 degrees.

Nephelometry and Turbidimetry

-both methods can be used to determine the concentration of a particulate
solution.

Note: - What is common between nephelometry and turbidimetry? It is used for the
determination of the concentration of a particulate solution. That particulate
solution refers to the unknown.

Sources of Error:
 Differences in the particle size between the standard and test solutions
 Settling of heavy particles
 Gum Arabic and gelatin

Note: -Differences in the particle size between the standard and the test solution.
Note: - (a) is for turbidimetry (light is in forward direction). (b) is nephelometry (light is Remember that the concentration of the standard is known and the unknown
at an angle). refers to what is to be measured. For example, in serum, the unknown can be
potassium.
Note: -The theories associated with nephelometry are the Rayleigh theory, Mie theory, -Don’t be confused that in the laboratory, the standard for FBS is 100 while in
and Rayleigh-Debye theory. the lipid profile, it is different. Remember that the standard varies and there are
-In Rayleigh theory, when the incident wavelength is much longer than the two factors on which the standard is dependent.
particle diameter, there is maximum backscatter and minimum right angle  First factor is the unit being used (e.g. mg/dL, g/L).
scatter. This means that in this theory, there is backscatter, meaning there are  Second is the analyte to be measured.
some light that return or go back.
-The concentration of the standard varies depending on the analyte (glucose,
Theories Conditions Light Scatter calcium, etc.). Remember the formula for the ratio of the standard to the
Rayleigh Wavelength of light > Symmetrical around the particle 90 unknown.
diameter of the particle degrees to the incident beam -Settling of heavy particles (e.g. gum Arabic and gelatin). When these settle in
the bottom of the cuvette and they can’t be reached by the light, they can’t be
Mie Wavelength of light < Forward owing to destructive out-
measured or their amount of light blocked or scattered can’t be measured. The
particle diameter of-phase backscatter result will be falsely decreased. It would appear that the concentration is less,
Rayleigh-Debye Wavelength of light = More light scatters in the forward but actually, the particles just settled in the bottom.
particle size direction than in other directions
-If the particle is large but not heavy, it will stay on the surface or float. If the
-The Rayleigh-Debye predicts the maximum right angle scatter when light passes through, the tendency in the nephelometer is that the light will
wavelength and particle diameter approach equality. scatter (In turbidimeter, it will be blocked.).

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MLS 11A CLINICAL CHEMISTRY MLS 3A
MODULE 2: Analytic Techniques and Instrumentation  It is important to keep the current constant because it serves as the
Lesson 2: Electrochemical Methods reference or the standard potential/voltage.

ELECTROCHEMICAL METHODS
Nernst Equation
 Potentiometry
 Amperometry
 Coulometry
 Voltametry

Note: -What are we aiming to measure with the use of electrochemical methods? In
this one, we aim to measure for the presence of ions in the solution by their
electrical properties.
-For electrochemical methods, we are measuring for the presence of ions
based on their electrochemical properties.
 Electrochemical properties involve the measurement of current or
voltage generated by the activity of specific ions.
-Under electrochemical methods, we have the four methods: potentiometry,
amperometry, coulometry, and voltametry.
-Optical methods: light source/flame is required, which is the source of energy.
-Electrochemical methods: source of energy is current or voltage applied to
generate the activity of ions.
Potentiometry
-Measures the potential between 2 electrodes in a solution
-Current is kept constant
Note: -Potentiometry measures the potential (voltage) between two electrodes in a
solution. Measurement of voltage or potential is the basis on the determination
of the concentration of the solution.
-What should be considered in potentiometry is to have a constant current.
 For example, if the testing lasts for 1 hour, for the whole duration, the
current should remain the same or constant (i.e. 200 volts).
Note: -In potentiometry, the cell potential or the voltage of the cell is being measured.
This involves ions. The cell potential is related to the concentration or molar
concentration (molarity) with the use of Nernst equation. Basically, the use of
this equation is to predict the electrochemical cell potential.
-No problems involving calculation will be given. Just take note of the symbols
and their meaning.
Types of Electrode:
A. Reference Electrode
B. Indicator Electrode
C. Ion-Selective Electrode
D. pH Electrode
E. pCO2 Electrode
Note: -The different types of electrodes which are being used in potentiometry or in
general, the electrode methods, are reference/standard electrode, indicator
electrode, ISE (ion-selective electrode), pH/hydrogen electrode, and the pCO2
electrode (gas electrode).

-We have here two electrodes that are being used in potentiometry: the A. Reference Electrode
reference/standard electrode and the detecting electrode. These are the two - Half-cell potential that is known, constant, and insensitive to the composition
electrodes under potentiometry. of the solution to be measured.
-Electrode potential refers to the voltage or the electromotive force.
 This force is involved in the activity of ions (what is measured in Qualities of a Good Reference Electrode:
potentiometry).  Conform to the Nernst equation
 Exhibit a potential that is constant with time
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MLS 11A CLINICAL CHEMISTRY
 Return to its original potential after being subjected to small currents
 Exhibit little hysteresis
Note: -Reference electrode. Its purpose is to deliver constant voltage to the meter
device.
-Qualities of a good reference electrode. These are the ideal characteristics of
a good reference electrode.
1. It must conform to the Nernst equation.
2. It should exhibit a potential that is constant with time.
3. It must be capable of returning to its original voltage or potential after
being subjected to small currents. Small currents are responsible for the
movement.
4. Exhibit little hysteresis. Hysteresis is found in the module. It refers to the
lagging, especially with the changes in temperature.
Note: -Potential/current/voltage
-Original potential/voltage is synonymous with baseline potential/voltage
-Remember that the current needs to be constant. The temperature also needs
to be maintained.
B. Indicator Electrode
-Selective response to changes in the activity of the unknown being measured.
Note: -Unknown refers to the ions in the analyte.
-The difference in the potential between the reference electrode and indicator
electrode is equal to the concentration of the analyte.
 This means that if the difference is higher, the concentration of the
analyte is also higher.
C. ISE
 Capable of responding to one specific ion
 Consists of:
1. membrane / barrier separating a reference solution
2. reference electrode from the solution being assayed
Note: -Ion-selective Electrode (ISE) is considered to be very sensitive and selective
for the ion that is aimed to be measured. Since it is capable of responding to
one specific ion, that contributes to its sensitive and selective properties.
-So far, ISE has replaced most of the methods in the quantitative measurement
of the different ions. At present, most of the principle are based on ISE.
-Nevertheless, even though they are sensitive and specific for one ion only, this
type of electrode also has its disadvantages.
MLS 3A
1. It is greatly affected by high lipid and high protein concentration. If the patient
has lipidemia or high protein levels, it contributes to the turbidity of the solution.
It would be difficult to use this electrode if the patient has lipidemia or has high
concentration or level of protein in the blood.
 High lipid or high protein present in the sample will lessen/decrease the
amount of water/solvent in the sample. From a diluted solution, it will
become concentrated because of the solute.
 Concentrated means the amount of solute is too high.
2. Another disadvantage as to why we need to consider the turbidity or the
presence of protein in the sample is because if ever this happens, the amount
of water present in the sample will be affected. This means that if there is more
lipid or more protein in the sample, the volume of water in the sample will
decrease. The sample will become now affected.
D. pH Electrode
 Measures hydrogen ion activity
 Glass electrode: has Cl - ion buffer solution
 Reference electrode: internal and external electrode
Note: -Biochemistry activity where we use the pH meter. It measures the activity of
the hydrogen ion.
 Inside the pH meter, we have the hydrogen electrode or the pH electrode.
This electrode interacts with the hydrogen ions in the sample. Once they
interact, there will be generation of electric potential. This will be
measured by the pH meter.
-The greater the potentials are; it will be equivalent to increased hydrogen ion
activity/concentration.
- During the testing, the hydrogen electrode/pH electrode will interact with the
hydrogen ions which are present in the sample.
-If the hydrogen ions interact with the hydrogen electrode, there will be
generation of electric potential or voltage, and this electric potential will be
measured by the pH meter.
 The difference in the potentials between the internal & external
electrodes is equal to the hydrogen ion activity.
-Hydrogen electrode consists of 2 electrodes, the glass electrode and the
reference electrode.
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MLS 11A CLINICAL CHEMISTRY
 Basically, the glass electrode contains the buffer solution, in particular
the chloride ion. Glass electrode has a known hydrogen concentration.
 Then we have the reference electrode in which we have 2 types or
subtypes: the internal electrode and the external electrode
-Both the components (the glass and reference electrodes) of the hydrogen
electrode are immersed/submerged in an unknown sample with unknown
hydrogen concentration.
-Glass electrode is a wire coated with silver or silver chloride, which is
surrounded and enclosed by a glass.
-The difference of the glass and reference electrode is that the glass electrode
does not have an opening to the solution whose pH is being measured. The
reference electrode is the one with the opening which means its contents can
flow out and interact with the sample.
 Internal reference electrode
 External reference electrode
 Filling solution
 Permeable outer casing
Note: -4 components of the reference electrode
1. Internal reference electrode – Usually is silver or silver chloride
2. External reference electrode – Saturated calomel electrode
3. Filling solution
4. Permeable outer casing – the said opening to the solution whose pH is being
measured. Liquid junction is formed from fitted glass which allows slow release
of solution from interior of the electrode.
MLS 3A
-Generally the purpose of the reference electrode is to provide constant voltage
to the meter.
-Permeable outer casing: meaning the liquid inside the electrode (reference
electrode) can flow out and interact with the outside environment (solution)
-How to distinguish between glass and reference electrodes? Reference
electrode has an opening.
Note: -The reference electrode is seen on the left of the picture because it is the only
one that has an opening towards the sample.
Note: -The glass electrode is on the right side which doesn’t have an opening towards
the sample and the reference electrode is the blue-green one which has an
opening for the sample.
E. pCO2 Electrode
 Contained in a plastic jacket filled with sodium bicarbonate buffer
 Has a gas-permeable membrane (Teflon or silicone) across its opening
Note: -pCO2 Electrode otherwise known as gas electrode or gas-sensing electrode.
 It is actually a pH electrode but the difference is the pCO2 electrode is
contained in a plastic jacket filled with sodium bicarbonate buffer.
 Similar with pH electrode in the sense that it also has a permeable part
- On ISE, we have chloride as buffer. Here in pCO2 electrode, we have sodium
bicarbonate as the buffer.
-Used for the measurement of blood gas, which means the specimen used here
is whole blood coming from the patient.
 Whole blood with dissolved CO2 will come in contact with the gas-
permeable membrane. Blood will enter the membrane and mix with the
buffer which is sodium bicarbonate. CO2 in the blood can then be
measured.
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MLS 11A CLINICAL CHEMISTRY MLS 3A
F. Enzyme Electrode -In coulometry and amperometry, the amount of chloride present in the sample
 Various ISEs may be covered by immobilized enzymes that can catalyze a (urine, plasma, sweat, CSF) is measured.
specific chemical reaction
Voltametry
Note: -This is used for the measurement of enzymes. But this electrode contains  Method in which a potential is applied to an electrochemical cell and the
immobilized enzymes. resulting current is measured
 These immobilized enzymes are enzymes that have restricted mobility.  High sensitivity and capability for multi-element measurements
 Remember in biochemistry, there are different phases associated with
enzymes like lag phase and mobile phase. Note: -The resulting current is what we are measuring here in voltametry.
 Since the enzymes are restricted in the enzyme electrode, the enzyme -This test has high sensitivity and is capable of multi-element measurements,
is not allowed to reach its mobile phase. which means the analytes can be detected in parts per billion range.

COULOMETRY
Coulometry
 Measures the quantity of electricity (in coulombs) needed to convert an analyte
to a different oxidation state
 An electrochemical titration where the titrant is electrochemically generated and
the endpoint is detected by Amperometry

Note: -Used to measure chloride ion in the following samples: serum, plasma, CSF,
and sweat.
-An electrochemical titration where the titrant is electrochemically generated
and the endpoint is detected by/measured by amperometry: which means
coulometry and amperometry work together.
 Coulometry is only for electrochemical titration.
 The endpoint is measured with amperometry.
 Titration: coulometry. Measurement of endpoint: amperometry.

Amperometry
 Measures of the current flow produced by an oxidation-reduction reaction
 Cotlove titration: coulometric-amperometric chemical titration process for
chloride determination

Note: -Measures the current flow produced by an oxidation-reduction reaction.

 Which means it measures the electrical current/potential at a single
applied potential or a single applied voltage.
-Remember both methods, the coulometry and amperometry, are used
together to determine the chloride in body fluids.
-Titration in coulometry is performed through Cotlove titration.
 Cotlove titration: coulometric-aperometric chemical titration process for
the chloride determination. It is used for the determination of chloride in
body fluids.
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MLS 11A CLINICAL CHEMISTRY
MODULE 2: Analytic Techniques and Instrumentation
Lesson 3: Electromigration Methods
Electrophoresis
 Any process that uses electricity to separate particles
 Migration of charged particles through a solution under the influence of an
electrical field
 Separation of charged compounds based on their electrical charge
 Acidic and basic amino acids determine the net charge on a protein
 ↑ net charges of a compound: faster movement toward the oppositely charged
electrode
 Net charge of a compound depends on the pH of a solution
 Electrophoretic separation requires high voltage
 Principle: When charged molecules are placed in an electric field, they
migrate either the positive or negative pole according to their charge
Note: -Electromigration refers to the transport of ions. Ions are electrically charged
particles (Cation & Anion).
 Cations + positively charged
 Anions – negatively charged
-Under electromigration methods, there is only one method: electrophoresis.
Electrochemical methods Electromigration methods
Measures the Measures the
activity of ions migration/mobility/movement of ions
-Same with electrochemical methods, voltage/current is needed. This will be
the source of energy in electromigration methods, particularly in electrophoresis.
The voltage will allow for the movement of the particles in the medium.
-Basis of electromigration or mobility of the ions: charge of the ion of interest
and the charge of the electrical field.
-Migration/transport of ions/electrically charged particles according to the
electrical field established between an anode and a cathode.
-Electrophoresis involves the migration of charged particles in an electric field,
which means this requires voltage.
-An anion will travel towards the anode. This is because the anion is a
negatively charged ion which will be attracted to the anode which is positively
charged. (Cation to cathode)
-Remember, opposite attracts.
MLS 3A
-Another explanation for electrophoresis: Increase net charges of compound
results to faster movement towards the oppositely-charged electrode.
-During the electrophoresis process, proteins that are negatively-charged
migrate towards the anode.
 The higher net charges of a compound have, the faster the movement
towards the opposite electrode.
-Net charges of a compound depend on the pH of the solution (the number of
H+ ions it has). And remember that, on electrophoresis, the separation (of
proteins) requires high voltage (between 50-200 V). Therefore, if the voltage or
power supply is weak, this can result to slow migration of our ions.
-It is useful for the separation of proteins on the basis of their electrical charge.
 Most of the proteins are negatively-charged.
 With amino acids, there are alkaline and acidic amino acids. Acidity and
alkalinity of their charge is the basis for their mobility or movement in the
medium.
 Since most proteins are negatively-charged (anions), their tendency is to
migrate/move to the positively-charged electrode (anode). Opposite
attracts.
-Principle of Electrophoresis:
When charged particles are placed in an electric field, they migrate
toward either on the positive or the negative pole/electrode according to their
charge.
-Note that the electric field must have power source or source of current. This
is what we have discussed earlier.
-“placed in an electric field” : applying voltage/current
 Anion, which is negatively charged, will migrate on the anode, which is
positively-charged electrode.
 The cation, which is positively charged, will migrate to the cathode, a
negatively charged electrode.
Amphoteric
 Has a net charge that can be either (+) or (-)
Note: -We have here amphoteric molecules, where the charge can either be positive
or negative depending on the pH they are exposed to.
- Amphoteric: their net charge can be changed depending on the pH of the
solution to which they are exposed
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MLS 11A CLINICAL CHEMISTRY MLS 3A
 Suppose, we have an amphoteric molecule that is negatively charged,  Chemical and physical properties of the support medium like pH and
but if we changed the pH or the environment, it is possible that it changes room temperature.
into a positively charged molecule.  Electrophoretic temperature, wherein due to high the temperature, there
will be production of heat and this will make the movement of molecules
Electroendoosmosis faster.
 Movement of buffer ions and solvent relative to the fixed supply (or energy
supply) Electrophoresis
Clinical Uses of Electrophoresis:
Iontophoresis  proteins
 Migration of small ions  erythrocytes
 tissues
Zone electrophoresis  isoenzyme
 Migration of macromolecules
Note:
Note: - Zone electrophoresis: migration of large molecules in a support medium. 1. Protein – for quantitative measurement of proteins
-Remember: Opposite Attracts! 2. RBCs – particularly for hemolysis
 Negatively charged ions will attract on positively charged electrode 3. Tissues
(Anion to Anode). 4. Isoenzymes – such as Creatine Kinase, Lactate Dehydrogenase, and Alkaline
 Positively charged ions will attract to negatively charged electrode. Phosphatase
(Cation to Cathode).
-The laboratory electrophoresis is used to determine proteins, RBCs
The velocity migration: (particularly the hemoglobin), tissues, and isoenzymes namely: Creatine
 Net charge of the particle Kinase (CK), Lactate Dehydrogenase (LDH) and Alkaline Phosphatase (ALP).
 Size and shape  ALP is involved in osteoblastic activity.
 Strength of the electric filed
 Chemical and physical properties of the supporting medium Components of Electrophoresis
 Electrophoretic temperature  Driving force
 Support medium
Note: -Strength of electric field: Remember that we need power supply or source of  Buffer
current for the ions to move.  Sample
-The rate (Velocity) of migration is directly proportional to the net charge of the  Detecting system
particle, and inversely proportional to the size and viscosity of the buffer.
-We have here the factors which affects the velocity or the movement of the Note: -Driving force or electrical power. We need electrical power for ions to move.
molecules.
 Net charge of the particles. As we mentioned, the higher the net charge
of the particles, the faster the mobility or movement.
 Size and shape of the particles, including weight. If the size of the
molecule is large which will also mean it is heavy, it will have a slower
movement towards the electrode.
 Strength of electrical field or the amount of voltage power applied on the
support medium.

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MLS 11A CLINICAL CHEMISTRY MLS 3A
 Leads to ↓ heat production

1. Power supply
 Current  medium  heat
 Resulting to ↑ thermal agitation of the solute
 Leading to ↓ resistance and an ↑ current
 ↑ current, ↑ heat, resulting to evaporation of water
 Thus, ↑ ionic concentration of the buffer and movement of the particles
Note: -Electrical power or power supply serves as the driving force of our ions. In this
case, we have the current our power supply, it will run through the medium (like
gel), and due to current, there will be production of heat.
-Then, the heat will result to thermal agitiation of the solute, or elevated
thermal agitation of the solute. Thermal agitation will allow movement of our
solute that is present in the sample due to the heat generated from our power
supply.
-Once there is increase in thermal agitation, there is decreased resistance,
and increased current. As there is increased current, there will be more heat
generation (increase in heat) resulting to evaporation of water in the
medium.
-Once the water evaporates, there will be increase ionic concentration of the
buffer leading to movement of the particles.
Note: -The purpose of having the buffer is that, it carries the current and other than
that, it protects the sample and maintains the pH during electrophoretic analysis.
-Once the buffer is diluted (which means there will be more solvent), there will
be generation of heat. Due to the presence of heat, this will result to increased
ionic strength. But the problem on increased ionic strength or concentration is
that, there is poor separations of the fractions.
-The increased Ionic concentration/ strength will cause increase in ionic cloud
and decreased mobility of the particles, where the particles stayed on the
start point, or they moved a little bit.
-On the other hand, increased ionic strength production sharpens protein
band separation, which leads to decreased heat production. Remember that
the heat helps for the movement of the ions or particles in the medium.
Rate of mobility (μ):
𝑄
𝜇= 𝑥 𝑟 𝑥 𝑛
𝑘
Where: Q = net charge of particle
k = constant
r = ionic radius of the particle
n = viscosity of the buffer
Note: -Formula for solving the mobility of charged particles
3. Support medium

2. Buffer
 Carrier of current, protects samples, maintains pH Cellulose Acetate
 Buffer's pH and ionic strength affects the ampholyte's charges  Easy to use, low-cost, commercially available
 ↑ ionic strength does not allow good separation of the fractions  Dry and brittle
 ↑ ionic concentration, ↑ ionic cloud  Becomes pliable when soaked in electrolyte buffer
 ↓ mobility of the particle  After electrophoresis, it can be stained and read in a densitometer
 ↑ ionic strength produces sharper protein-band separation  Long term storage possible
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MLS 11A CLINICAL CHEMISTRY
Starch Gel
 Separates proteins based on their surface charge & molecular size
Polyacrylamide gel
 Different pore sizes can be layered
 Detect 20 serum protein fraction
Note: - Support medium: where our particles are moving. We have several types of
support medium but most of it are made up of gel.
a. Agarose gel – requires small amount of sample or between 2-10
microliters only. Also remember that 1mL in laboratory is equivalent to
1000 microliters, which means 2-10 microliters is the amount that is
required on the sample of agarose gel. Also, it does not bind with
proteins. It is also considered neutral or it does not produce
electroendosmosis. This will mean that it can be stained, spun, and
examined using densitometer.
b. Cellulose Acetate – easy to use, low cost, commercially- available. Only
disadvantage is that, it can become dry and brittle, where it becomes
pliable when soaked in electrolyte buffer. The same with agarose gel,
after electrophoresis, it can also be stained and read in a densitometer.
c. Starch gel – separates proteins based on their surface charge and the
molecular size of the particle.
d. Polyacrylamide gel (SDS Page) – these are gels with different pore
sizes. This gives us a very good separation of particles depending on the
size. The principle is that the gel has different pore sizes, but in the
laboratory, the pore sizes are not visible to the human eye. Other than
that, this gives us a very good resolution. Unlike other gels used in the
laboratory, the Page (Polyacrylamide gel) can detect 20 serum protein
fractions. For some gels in a support medium, it can only detect 5
samples or protein fractions.
MLS 3A
Detecting System
 Visualization under UV light
Procedure
1. The sample is soaked in hydrated support for approximately 5 minutes.
2. The support is put into the electrophoresis chamber, which was previously filled
with the buffer. Sufficient buffer must be added to the chamber to maintain
contact with the support.
3. Electrophoresis is carried out by applying a constant voltage or constant current
for a specific time.
4. The support is then removed and placed in a fixative or rapidly dried to prevent
diffusion of the sample.
5. This is followed by staining the zones with appropriate dye. The uptake of dye
by the sample is proportional to sample concentration. After excess dye is
washed away, the supporting medium may need to be placed in a clearing
agent. Otherwise, it is completely dried.
Note: -The simplest way to detect the patterns is through observations under UV Light.
When we say UV Light, it should be read in a dark room for the fractions to be
visible.
-The procedure is listed on the module.
1. Firstly, the sample is soaked in support medium for 5 mins.
2. The support will be placed in electrophoresis chamber which was filled
previously with the buffer. Remember that the added buffer must be
enough to maintain the contact with the support. It can’t be too many or
too little of the buffer added.
3. The use of power supply is to allow the movement of the charged
particles.
4. After applying voltage or electrical current on the support medium, the
support will be removed and placed in a fixative or rapidly dried to prevent
diffusion of sample. The purpose of fixating is to prepare the sample for
staining.
5. After electrophoresis procedure, the sample can be stained. The purpose
of staining is to aid us in visualizing under UV Light.
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MLS 11A CLINICAL CHEMISTRY MLS 3A
Note: -We have here examples of electrophoretic patterns for proteins as seen on the
electrophoretogram. First is the normal serum protein electrophoresis. We have
here 5 proteins namely: Albumin, Alpha, Alpha-globulin, Beta-globulin and
Gamma-globulin.
 Remember that albumin as a higher spike for normal serum
electrophoresis.
 As we have mentioned earlier, after the electrophoresis procedure, this
can be measured using a detecting system that will show us
electrophoretogram.

Relative Percent of Protein Bands

Fraction %
Albumin 53-65
Alpha-1 globulin 2-5
Alpha-2 globulin 7-13
Beta globulin 8-14
Gamma globulin 12-20

Note: -We also have here different concentrations of proteins in the serum. These are
normal concentration of proteins in the serum of an individual.
-Remember that albumin has the highest percentage or the most abundant
protein in the blood.
-Electrophoretic patterns must be memorized.
-There are also different electrophoretic patterns that differ on the condition.

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MLS 11A CLINICAL CHEMISTRY
Note: -The blue line indicates the normal electrophoretic patterns. The shade will
indicate the amount of the protein in this condition.
 Notice in nephrotic syndrome, the pattern is albumin, alpha-1, beta and
gamma-globulin are decreased, which means they didn’t reach the
normal level indicated by the blue line. Alpha-2 is normal. Therefore, in
nephrotic syndrome, only alpha-2 globulin has normal concentration, and
the rest are decreased.
 Second is on Acute Phase Reactants, where all alpha-1 and alpha-2 are
increased, and albumin and gamma-globulin have normal
concentrations.
 For multiple myeloma, we have to remember the appearance, M-Spike.
This is due to high concentrations of albumin and gamma-globulin, where
it is noticeable that gamma globulin has high spike of concentration.
 Remember in multiple myeloma, we have spiked concentration of
gamma-globulin.
 Remember the 3 M’s: Multiple Myeloma M-spike.
 Also notice that alpha-2 has decreased.
Hemoglobin Electrophoresis
 Same principle and system as protein electrophoresis
 Solubility is an important factor
Hemoglobin Electrophoresis Patterns
Note: -Next, we have hemoglobin electrophoresis where the specimen are the RBCs.
Unfortunately, we don’t perform this in our laboratory because it is costly. But
the principle and system are similar with protein electrophoresis (determination
and separation of protein).
-It is also considered that solubility is an important factor for the mobility of the
hemoglobin proteins.
-Hemoglobin electrophoresis patterns are discussed in Hematology.
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What is an ampholyte?
 Net charge can either be positive or negative
 More ACIDIC buffer than the isoelectric point of an ampholyte, it
BINDS WITH H+, becomes POSITIVELY CHARGED and migrates
toward the CATHODE
 More BASIC buffer than the isoelectric point of an ampholyte, it
LOSES WITH H+, becomes NEGATIVELY CHARGED and migrates
toward the ANODE
Stains
 Applied in the support medium for visualization
 Identification of separated fractions once electrophoresis is completed
 E.g. Amido Black, Ponceau S, Fat Red 7B, and Sudan Black
Note: -We also have here the different stains used for electrophoresis. Remember
that fixation is done as a preparation for staining. Then stain will be applied on
the sample.
-With electrophoresis, we can use Amido Black, Ponceau S, Fat Red 7B and
Sudan Black. The color reaction doesn’t necessarily mean that if it’s Amido
Black, we can see a black stain.
-The stain is applied to support medium (gel) after electrophoresis method to
aid in visualization under UV light. After fixation, the support medium or the gel
will be stained.
-For electrophoresis, the fixative commonly used is acetic acid. Acetic acid
precipitates excess proteins in our gel. So, after fixation comes staining, and
then visualization.
Separation
1. Frontal Electrophoresis
 Uses homogenous solution without a support medium
 Convective forces prevented resolution into distinct zones
 No distinct zones are formed
2. Zonal Electrophoresis
 Use of an anticonvection support medium
 Protein fractions are separated in a discrete bands
Note: -We have here 2 processes of separation for electrophoresis.
-First is frontal electrophoresis, where it separates molecules with the use of
homogenous solution without the support medium or the gel. In this case, there
are no distinct zones to be observed.
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MLS 11A CLINICAL CHEMISTRY
 Frontal electrophoresis is used for fractionization of proteins such as
albumin, alpha-1, alpha-2, beta and gamma globulins.
-Second is zonal electrophoresis, where it uses an anti-convection support
medium. It is also used for separation of protein fractions in discrete bands.
Quantification
Turbidimetric Methods
 Acid protein precipitation
 Not specific
Colorimetric
 Biuret reaction
Densitometry
 Most reliable way for quantification
Capillary electrophoresis
 Performed in a narrow-bone fused silica capillaries
 Buffer-filled capillaries
 Fundamental concept: electro-osmotic flow
Electro-osmotic flow
 Bulk of liquid toward the cathode upon application of electric field
 Controls the amount of time solutes remain in the capillary
Advantages:
 Shorter analytical time an resolving power
 Requires microsample volumes
 Detects and quantitates protein bands without the stain
 Low reagent and labor cost
Note: -Next is the process called Quantification.
-We have turbidimetric method, where we use acid for precipitation of proteins.
Therefore, after we add acid on a solution containing proteins, there will be
formation of precipitate or clumping. The only problem is that it is non-specific,
wherein other than proteins, it can also precipitate other acid-soluble
substances in the solution which can interfere with our assay.
-We also have here colorimetric method, particularly with the use of Biuret
method. It is highly specific for proteins and peptides. What’s good in Biuret
Method is that it has minimal interferences during the assay.
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-We also have another separation method called capillary electrophoresis,
wherein the separation is performed in a narrow-bone fused silica capillary, and
the capillaries are filled with buffer. The fundamental concept is based on the
electro-osmotic flow.
 What is electro-osmotic flow? (EOF)
It is involved in the separation of molecules in the presence of high
voltage or high current. The tendency is that the bulk of liquid moves
toward the cathode upon application of electric field. This controls the
amount of time the solutes remain in the capillary.
Capillary electrophoresis
 (+) ions emerge early at the capillary outlet because of EOF and the ion
moves in the same direction
 Detectors used: optical, conductivity, electrochemical, mass spectroscopy or
radioactive detectors
Note: -At capillary electrophoresis, positive ions emerge early at the capillary outlet
due to electro-osmotic flow (EOF) and the ion moves toward the same direction.
-Remember that for capillary electrophoresis, negatively-charged ions move at
a slower rate.
-Capillary electrophoresis is fast because the separation of proteins can be in
less than 10 minutes, but we need to apply very high voltage or current.
Other factors affecting electrophoresis
1. Electroendosmosis
 Movement of buffer ions and solvent relative to the buffer support
 Support media take on a (-) charge from adsorption of hydroxyl ions
 Hydroxyl ions remain fixed while positive ions move toward the cathode
2. Hemolysis
 Produced erroneously high results
Note: -First is electroendosmosis. As discussed earlier, the movement of the buffer
ion and solvent relative to the buffer support.
 Remember that the support medium adopts the negative charged ions
from absorption of hydroxyl (OH-) ions. Therefore, OH- ions remain fixed
while positive ions move towards the cathode.
 As an example, the anions that are negatively charged migrates towards
the anodes, which are positively charged.
-Hemolysis: This is destruction of RBCs which releases a lot of proteins in the
environment which can give erroneous results.
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MLS 11A CLINICAL CHEMISTRY MLS 3A
Electrophoresis 4. The “Smile Artifact,” where samples at the center of the gel migrate
 Electrophoretic mobility further than those at the edges.
 Directly proportional: net charge
 Inversely proportional: molecular size and supporting medium’s
viscosity
 Low ionic strength
 Charged proteins carries more current
 Fast mobility
 High ionic strength
 Charged proteins carries less current
 Slow mobility
 More difference on the pH of the buffer from the isoelectric point: greater
protein net charge magnitude, faster mobility
 At pH 8.6, IgG move toward the cathode

Note: -On low ionic strength, charged proteins will carry more current which results to
faster movement of molecules.
-On high ionic strength, less current will be carried by proteins thus, resulting to
slower movement of molecules.

Precautions on Electrophoresis
 Improperly aligned electrode
 Too long electrophoresis
 Electrical circuit break
 “smile artefact”

Note: - We also have precautions to remember upon performing electrophoresis.

1. Improperly aligned electrode, which means denser current on one side
of the gel.
 Let’s say we divide the gel in 2 portions, if the electrode is
improperly aligned, chances are there is more current on the first
portion and a lesser current to the second portion. The portion with
the more current will lead to a faster movement of molecules, and
proteins will migrate further at this side due to high current.
2. Too long electrophoresis which means proteins will migrate off the gel
and into the buffer, where the proteins are lost on the gel and can lead
to erroneous results.
3. Electrical circuit break or fluctuations on the current, or no current at all.
In short, no movement of particles if there is no current.

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MLS 11A CLINICAL CHEMISTRY
MODULE 2: Analytic Techniques and Instrumentation
Lesson 4: Chromatography
Chromatography
 A technique involving the separation of soluble components in a
solution by a specific differences in physio-chemical properties of the
different constituents
 Separation is based on different interactions of the specimen
compounds with the mobile phase and with the stationary phase.
Note: -The purpose of chromatography is to separate or quantify a variety of
clinically relevant analytes, like sodium, potassium, drugs, or poisons.
That can be done through chromatographic methods.
-Chromatography is a physical process or a physical technique wherein
the soluble components or the solutes in a mixture are separated based
on their differential distribution. The soluble components are in a
solution.
 A solution is composed of two entities: solute and solvent. In
chromatography, the soluble components refer to the solute. This
means that the chromatographic procedures are done for the
fractionation, quantitation, or separation of solute from the
solution.
-In chromatography, the separation method is based on the different
interactions of the specimen compounds (which are found in the
solution) between the two phases. The interactions occur as the
components move along the column.
 The two phases in chromatography are the mobile phase and the
stationary phase.
-Purposes of performing chromatography:
 Determine the chemical composition of a sample
 Purify sufficient quantities of a substance
Basis of Separation
 Molecule’s hydrophobicity
 Ionic attraction
 Differential distribution between two immiscible liquids
 Selective separation of substances
 Differences in adsorption and desorption of solutes
 Diffusion rate
 Solubility
 Nature of the solvent
 Sample volatility or solubility
 Distribution between 2 liquid phases
 Molecular size
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Note: -The factors listed above serve as the basis for the separation of the
soluble substances from the mixture/solution.
-Ionic attraction means that the electrical charge of the ions matter.
-The manner on how solutes are separated through adsorption and
desorption also matters in this separation process (chromatography).
-Diffusion rate or the movement of the solute together with the mobile
phase along the column
-Sample volatility or solubility refers to the quality of the material which
describes how readily a substance evaporates.
-Molecular size including the molecular shape of the particular
substance.
Two (2) general types of Chromatography
1. Planar Chromatography
 Stationary phase is taking on a plane
 Paper chromatography or TLC
2. Column Chromatography
 Stationary bed is within a tube
 Gas and liquid chromatography
Note: - The difference between the two general types of chromatography is
their stationary phase.
-Planar chromatography: the stationary phase is taking place on a plane
or a paper. Under planar chromatography, there are sub-classifications
which are paper chromatography and TLC (thin layer chromatography).
 The stationary phase is coated on a sheet of paper or bound to a
solid surface (for planar chromatography).
-In column chromatography, the stationary phase is within a tube. It is
can either be:
 The entire tube is filled with the stationary phase, or
 Only the inner surface of the tube is coated with the stationary
phase.
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MLS 11A CLINICAL CHEMISTRY MLS 3A
Basic Components
1. Mobile phase
 Sample carrier
 Moves in a definite direction
 Can either be liquid (LC) or gas (GC)
 Moves through the chromatography column where the sample
interacts with the stationary phase and is separated
2. Stationary phase
 Through which the mobile phase flows
Note: -Remember that the mobile phase is the one that moves between the
 Can either be gel, solid, or liquid
two phases. As the mobile phase flows past the stationary phase, there
 Immobilized on the support particles or on the inner wall of the
are three possible outcomes that can happen to the solutes:
column tubing
1. No migration: wherever the sample was dropped or added, the
3. Separated molecules
sample just stays there for the whole process. It doesn’t move.
 Eluate
2. Migration phase: the solute resides in the mobile phase. It does
 Mobile phase leaving the column not flow along the column of the stationary phase. It is only
present in the mobile phase (it sticks to the mobile phase). It
Note: -Mobile phase serves as the sample carrier. The sample contains the doesn’t leave any prints/evidences/visible results along the
solute. The solute is what is aimed to be separated from the solution. stationary phase or the column.
-The mobile phase will carry the sample along the column or the  In the migration phase, there could be no visible separation
stationary phase. The mobile phase’s movement or direction is definite or reaction. We can only see the mobile phase moving but
which means “indi siya paliko-liko” (Gallego, 2020). If its direction is there is no visible result.
straight, it will remain straight throughout the process. 3. Differential migration: distribution of solute between the two
-The mobile phase can either be a gas (GC) or liquid (LC). phases: mobile and stationary phase. In this, the solute moves
-Stationary phase otherwise known as the immobile phase. It is where together with the mobile phase. But as it moves or flows together
the mobile phase flows along the column. The best example of the with the mobile phase, it leaves behind evidences/prints.
stationary phase is the silica layer which is intended for TLC. The -In the picture: the start point is where the sample is added. In the first
stationary phase can be added in two ways: line, it hasn’t flowed yet or the sample/solute hasn’t moved yet. If ever it
 The tube can be fully filled with it like a gel. has no polarity or affinity with the stationary phase, the tendency is it will
 It can only be present in the inner surface of the tubing. move along the column or it will flow until it reaches the endpoint (where
-The eluate are the separated substances in the sample. This is the it will stop). It spreads along the way.
solute that has been separated from the solution. It moves together with -The next picture shows an example of a chromatographic procedure
the mobile phase along the column. with a good separation. There is good separation because even if only
one sample is used, two components or compounds were extracted or
Possible outcomes during the chromatography
separated along the process.
1. No migration
2. Migration phase
Factors Affecting Chromatography
3. Differential migration
1. Intermolecular interaction between the two phases
2. Extent of dispersion of solute molecules over the stationary phase

Note: -Dispersion is the movement of the solute along the stationary phase.

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MLS 11A CLINICAL CHEMISTRY MLS 3A
Planar Chromatography Paper Chromatography
1. Paper Chromatography
 For separating and identifying mixtures that are or can be
colored
 Stationary phase: porous paper
 Mobile phase: solvent (liquid)
 Samples: added to one end of the sheet of paper and dipped
into the liquid
Note: Compounds within the solution travel faster if they are non-polar.
Note: -The first general type of chromatography is the planar chromatography
wherein there are two sub-classifications: paper and TLC.
-Paper chromatography is an analytical method or process wherein this
one is used for separating or identifying mixtures that can be colored or
are colored. If the mixture is not colored, a stain can be added to make
it colored.
-Remember that chromatography is also done to purify a substance.
That is the purpose of paper chromatography: testing the purity of
compounds and identifying the substances present in that particular
solution.
-The Whatman paper or the ordinary filter paper used in the lab is the
best example of a stationary phase for paper chromatography.
 Porous paper are specially manufactured paper.
 Whatman paper is more expensive than the filter paper.
 Other than that cellulose can also be used as the stationary
phase.
-The solvent in the mobile phase must have the ability to dissolve the
analyte.
-How is paper chromatography done?
 Samples which can be serum or from the blood are added to one
end of the filter paper and then dipped in the liquid (refers to the
solvent).
 The end where the sample is dropped is the one soaked in the
solvent or mobile phase.
 Once the porous paper is soaked in the solvent, there will be
settling of water drops in the porous paper. The settling of water
drops in the pores of the filter paper makes up the stationary liquid
phase.
-The paper chromatography’s mobile phase is water. For this type of
chromatography, this follows the principle of liquid-liquid
chromatography/adsorption because the mobile phase is generally a
mixture of non-polar organic solvent which is in liquid form.
-The sample is also in liquid form.

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MLS 11A CLINICAL CHEMISTRY
Note: - The picture is a diagram of how paper chromatography is done in the
laboratory.
Step 1. In the filter or porous paper that hasn’t yet been dipped in
the solvent, draw a horizontal line in one end using a pencil. The
line should be about 1.5 cm from the bottom edge.
 When labeling and drawing the horizontal line, a pencil
must be used instead of pens. Because the ink of the pens
once they are wet by liquid has a possibility of spreading.
The ink are colored because they are somewhat dyes.
Once the ink is wet by the liquid solvent and it spreads, it
leads to erroneous results or outcomes.
Step 2. The laboratorian is holding a capillary tube. Fill the
capillary tube with the sample you want to separate. The purpose
of the tube is to hold the sample and to drop the sample into the
filter paper. The samples are dropped along the horizontal lines.
The samples have equal distance from each other to prevent
overlap with each other later on.
Step 3. Label the samples using a pencil. The label can be
through numbers or letters.
Step 4. The porous paper is then dipped in the solvent. The
solvent should be enough or sufficient. It should not be over the
drawn horizontal line. It should be just below the horizontal line or
below the drops of the sample applied on the porous paper.
 In the picture, 2, 3, 4, 5 represents the samples
added/applied to the filter paper. After that, it was soaked
in the solvent. The samples represent the unknown.
 The X represents the standard. The purpose of the
standard is to serve as reference.
Step 5. After dipping in the liquid solvent, we need to seal the
chamber with the lid. The purpose to enclose the chamber is to
avoid the solvent from evaporating and to avoid any
contamination.
 Once the paper is dipped in the solvent, it is expected that
over time, the solvent slowly rises up along the porous
paper or the filter paper.
Step 6. If ever the mobile phase or solvent is near the other end
of the paper, remove the filter/porous paper from the solvent.
Don’t wait that the entire paper is soaked. Once it almost or nearly
reaches the other end/edge of the paper, remove the filter/porous
paper from the solvent.
Step 7. During observation, (in the picture it is good since there
is a colored reaction), if ever there is no visible reaction/result,
you can observe the bands formed under the UV light because it
can be reflected by the light.
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-At present, between paper chromatography and TLC, TLC is more
preferred. The sole purpose of paper chromatography is as a teaching
tool to show the principle of TLC.
 TLC and paper chromatography have the same principle. They
only differ in the stationary or mobile phases.
-Cellulose has an embedded polar substance. Filter paper is a polar
substance.
 For example, the added sample is polar. Do not expect that the
sample will move. If the paper and the sample are both polar, it
will not move. It will just stay where it was added.
 If ever the paper is polar and the sample contains non-polar
substance, you expect that there will be migration along the
stationary phase or the porous paper until it reaches something
like the 3rd picture.
-Polar attracts polar.
Paper Chromatography
Advantages Disadvantages
 Simple, easy to perform and  Limited to qualitative
inexpensive measurement
 Gives off graphic, clear results  Unable of separating complex
mixtures
 Cannot be used for large quantity
of analytes
Note: -Graphic and clear results since paper chromatography can be used for
substances which are colored or can be colored. If it is non-colored,
there is an option to stain them to see the movement of the solutes.
-Does not measure the analyte quantitatively. It cannot give the specific
concentration. It can only give the distance that the solute traveled
without any numerical equivalent (short or long distance, etc.).
-The things that can be separated or isolated using paper
chromatography are limited.
2. Thin–layer Chromatography (TLC)
 Glass or plastic plates with a thin layer of adsorbent material
 Mobile phase: solvent
 Stationary phase: alumina, silica gel, cellulose, or cross-linked
dextran
 Mobile phase travels upward through the stationary phase
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MLS 11A CLINICAL CHEMISTRY
 Solvent travels up the thin plate soaked with the solvent
 Also drives the mixture priorly
dropped on the lower parts of the
plate with a pipette upwards with
different flow rates
 Colorless molecules in the sample
 Use fluorescence,
radioactivity or a specific
chemical substance to
produce a visible colored
reactive product helps to
identify the positions of the
molecules in a sample on
the chromatogram
Note: -TLC is an analytical technique which can be used to monitor reactions
and just like paper chromatography, it can give us qualitative analysis
of a complex mixture. What’s common between the two is that they both
purely give qualitative analysis but in paper chromatography, limited
compounds can be separated unlike in TLC which can be used to
separate complex mixtures.
-TLC is also used to separate non-volatile mixtures/substances (these
are substances that does not readily evaporate into gas).
-TLC is same with paper chromatography but they differ in their
stationary and mobile phases. For TLC, the stationary phase is glass or
plastic with a thin layer of adsorbent material.
 Adsorbent material can be alumina, gel, cellulose or dextran.
These are embedded in the glass or plastic plates.
-TLC is solid-liquid adsorption because the stationary phase is
adsorbent material in solid form. Liquid, because the solvent is in liquid
form. Remember that the solvent has the capacity to dissolve the solute
that needs to be separated from the mixture/solution.
-After the stationary phase is soaked towards the mobile phase or the
liquid solvent, expect the same as what happens in paper
chromatography wherein the mobile phase will travel upward through
the stationary phase.
 In TLC, the solvent travels up the thin plate soaked with solvent
by means of capillary action.
-Why is there movement of the mixture upward? Why does the mixture
travel upward? Why does the solute travel upward?
 Because the mobile phase carries the solute to the target or
where it should move or travel.
-In TLC, there are instances wherein there will be no visible results when
using the eyes only. In that case, the formation of visible color can only
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be observed by using UV light or under a room light. Between the two,
UV light is better because this can give fluorescence.
TLC
Chromatogram
- An output representing the separation given by a chromatograph
Retention Time
- Time takes for a particular analyte to pass through the system
under set conditions.
Note: -The chromatogram is a graphical representation of the detector’s
response (signal). The chromatogram shows the concentration of the
analyte in the eluent.
-The chromatogram represents the separation given by the
chromatograph.
-In the example of a chromatogram, there are two peaks/patterns. The
pattern or the peaks in the chromatogram correspond to the different
components of the separated mixtures. Meaning in this specific
chromatogram, two compounds were separated because there are two
peaks.
 The number of peaks is equivalent to the number of different
components which were separated from the mixture.
-System refers to the time from the sample was added, as it runs
through or moves along the column or the stationary phase, until it
reaches the detector.
-In the chromatogram, the Y axis gives us the signal or the detector’s
response. The X axis is the retention time.
Relative Mobility or Retention Factor (Rf)
- Relative distance of migration from the point of application
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MLS 11A CLINICAL CHEMISTRY
 Rf = 0, solute remains at the stationary phase
 Rf = 1, solute has no affinity for the stationary phase and travels with
the solvent front.
 Better separation of components and has better resolution but
requires the Rf values to be known beforehand
Note: Otherwise known as retention factor of the solute (Rf).
-When we observed under room light or UV light, the position of the
molecules can be observed in the mixture. How do we measure it?
 The distance can be measured.
 The visible results, for example, for our paper chromatography or
the gel in our TLC shows how far did our solute traveled.
-The relative mobility or retention factor (Rf) is the ratio between the
distances traveled by our molecule (solute) and the solvent. The solvent
here is the mobile phase.
 Rf is the distance traveled by our solute over the total distance
traveled by the solvent.
-Even if there is a value on our relative mobility or retention factor (Rf),
still the TLC is a qualitative description of the molecule. We can’t say
that for example, the distance traveled was 0.5, therefore the
concentration is 0.5. But we can just say that the distance traveled by
the molecule (solute) is near or far.
-(Refer to picture) We have here an example of TLC that has a standard
and an unknown. So, each sample component or retention factor is
compared with the standard retention factor.
 Going back, the standard serves as the reference.
 In this case, we have 3 standards: Standards A, B and C. We
need to remember that the standard used in spectrophotometry
is the same, but the standard in TLC has a known value. In this
case, the distance traveled by standard A is 0.4, standard B is
0.6, and standard C is 0.8, in centimeters (cm).
 The purpose of the standard is to compare the movement of the
molecule to our solute.
 We have here 2 unknown samples: Unknown 1 and 2. What we
do in TLC is that we put a sample on Unknown 1 and 2. As I
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mentioned earlier, TLC can only show us qualitative description.
Unknown 1 has two separated components (refer to the dots
below Unk 1).
 If we compared both components to the standards, we can
say that: the first isolated component is the same with
Standard A having 0.4 cm of the distance traveled. Say for
example, the 0.4 cm of standard A represents glucose, the
0.6 cm of standard B is cholesterol, and 0.8 cm of standard
C is creatinine. This means that, the unknown sample
aligned with the standard A (0.4 cm) is glucose as they
traveled the same distance. But we can’t know the
concentration, because it is only qualitative description.
 Next, the unknown component was aligned with standard
C (0.8 cm), therefore it is creatinine. Thus, we can say the
blood of the patient from Unknown 1 contains glucose and
creatinine.
 On the other hand, Unknown 2 aligns with standard B (0.6
cm), therefore we can say that Unknown 2 has lipids
(cholesterol).
-Just remember that the standards are used as reference for testing.
-We also have the term solvent front which indicates the distance or
the height of the solvent, or the distance or the height reached by our
mobile phase (solvent).
-Let me refresh your minds on spectrophotometry. We have mentioned
that the standard varies depending on the unit used, and the analyte
being tested.
 For example, we prepared a standard for glucose. The visible
standard result in the laboratory is pink. Then we run 2 tests for 2
patients: Serum of Patient 1 and Serum of Patient 2 but with
added reagent.
 Results were: for patient 1, dark pink in color; and for patient 2, it
was light pink.
 Say for example, the value for our standard prepared earlier
(pink) is 100 mg/dL. And it appeared that patient 1 has dark pink
in color. So we can say that patient 1 has higher concentration
than the standard (Patient 1 > 100 mg/dL of standard), but we
can’t quantify it because it is darker in color, or say it is 500 mg/dL
because it has higher concentration than the standard (100
mg/dL). Same goes with patient 2. We can say that it has lower
concentration (Patient 2 < 100 mg/dL of standard).
-As we know in the laboratory, the principle of spectrophotometry says
that the amount of light absorbed is directly proportional to the
concentration of the sample. Thus, the darker the color of the solution
is, the higher the light absorbed, and therefore the higher the
concentration of the solution.
6
MLS 11A CLINICAL CHEMISTRY MLS 3A
-Going back to the TLC, if ever the Unknown 1 has 3 isolated value of standard is 100 mL, the standard ran on the
components, and it traveled in between standard B (0.6 cm) and spectrophotometer must be 100 mL. If ever the amount of
Standard C (0.8 cm). Therefore, we can’t say that it traveled 0.7 cm as standard you prepared is different with the value of standard
it traveled in between both standards because it is not a quantitative stated on the reagent kit, this will imply an error on the procedure.
test. We can just say that it is greater than 0.6 cm (Standard B) and It may be insufficiency on the standard added, or insufficiency in
lesser than 0.8 cm (Standard C). Just don’t say it is 0.7 cm. Just do the reagent, or absence of reagent. Just be careful upon
remember that the purpose of the standard is to serve as basis for preparing the standard as what we say earlier, it is the basis for
comparison. our comparison.
-There are times in TLC that we use uncolored solutes, or absence of -As we said earlier, TLC is qualitative. But sometimes it can be semi-
dye. It will surely overlap the components of the unknowns due to quantitative. Qualitative in a sense that we described if the distance
absence of color. What we do is, we stain the gel on the stationary traveled is near or far, and semi-quantitative, in a sense that we say it
phase, make it dry, and compare with the standard. This is done if ever is greater than 0.6 cm but it is not exactly 0.6 or it traveled near 0.4 cm
there is absence in color. but it is not exactly 0.4.
-It’s possible for us to have a retention factor (Rf) of 0 and 1. But what
Components of TLC
is its significance?
1. TLC plates
 Remember that the relative mobility (retention factor) is the
 Stable and chemically inert plates
distance traveled by the molecule (solute) over the total distance
traveled by the solvent. Remember that the stationary phase in  Stationary phase on the plates is of uniform thickness and
TLC is polar. consists of fine particle size
 If ever that the solute is also polar, expect that the Rf is 0,
or it did not travel or moved at all. Thus, we can say that
the solute has no movement or stayed on the stationary
phase.
 On the other hand, if the Rf is 1, it indicates that the solute
has no affinity for the stationary phase and travels with the
solvent front. This means that if the stationary phase is 2. TLC Chamber
polar, and the Rf is 1, therefore, the solute is non polar, or  Maintains uniform environment inside for proper
it is not attracted with the stationary phase. development of spots
 This implies that: if the stationary phase and the solute are alike,
 Prevents the evaporation of solvents and keep the
it will stay and will have no movement, and if they are not alike,
process dust free
movement will be observed; and if the Rf is 0, it has high affinity
for the stationary phase, and if Rf is 1, low affinity for the
stationary phase.

Note: Lower affinity solutes separate from solutes having greater

affinities with the stationary phase. Therefore, lower affinity solutes
travel and have movements, while higher affinity solute stays “(Sana all
ga stay)”.
-TLC has better separation of components and has better resolution but
requires the Rf values to be known before the tests are performed. Just 3. Stationary phase
note that the standard is known. On spectrophotometry, when the  Alumina, cellulose, cross-linked dextran and silica gel
standards are made by reagent companies, the Rf is usually stated on
the inserts and are incorporated with procedures. 4. Mobile Phase
 Say for example, you prepared 1 mL standard solution and 1 mL  Comprises of a solvent or solvent mixture
of reagent and mixed. If the reagent kit states that the expected  Should be particulate-free

7
MLS 11A CLINICAL CHEMISTRY MLS 3A
 Of highest purity for proper TLC development Visualization Techniques for Colorless Solutes
 Recommended solvents: chemically inert with the sample 1. UV Light
2. Iodine Staining
Note: -TLC plates are ready made together with stationary phase, which 3. KMnO4 stain (organic molecules)
means TLC plates holds the stationary phase. For example, if our 4. Ninhydrin reagent
stationary phase is a gel, our TLC plate may be the support, or the TLC
plate has already the gel in it. Note:-These are used if there are no visible results.
-The TLC Chamber has lid or cover. Its purpose is to protect the sample -UV light: When observing you must be in a dark room
and to protect our set-up. Why does it have lid or cover? -Iodine staining (betadine solution): very useful in detecting
 To avoid evaporation of solvents and to avoid contamination. carbohydrates. Whenever carbohydrates are exposed to iodine
Therefore, it avoids erroneous results. We have to remember that staining, it will be colored black.
during testing, it must be particulate-free. -Potassium Permanganate (KMnO4) is used for organic molecules
 Since it was stated that we need the lid on the TLC chamber in -Ninhydrin Reagent: for proteins and amino acid.
order for it to be particulate-free, then TLC has the highest purity
where it doesn’t have other particles or solute present. Application of TLC
-We also have the stationary phase. It can be in the form of alumina, 1. Check purity of given samples
cellulose, cross-linked dextran and silica gel. It is possible that the 2. Identification of compounds
stationary phase is embedded on the TLC plate, where it acts as a 3. Evaluate reaction process by assessment of intermediates, reaction
support on your stationary phase. course etc.
-The mobile phase comprises a solvent or solvent mixture and it 4. Purify samples
depends on what type of analyte we are trying to separate. 5. Check of other separation processes
-Since we stated that our mobile phase is the solvent, we have to 6. Drug screening test
remember that the solvent must be chemically inert with the sample or 7. Sample components are identified by comparison with standards on the
the stationary phase. same plate
 Chemically inert: It means that it must not be chemically reactive, 8. Extraction of the drugs is pH dependent
where, as we expose our sample or stationary phase to the 9. Migration of all drug spots including the standards: incorrect aqueous to
mobile phase or solvent, it must be free of chemical reactions. nonaqueous mixture

Interferences for TLC Note: -Identification of compounds, whether its alcohol, acid, protein,
1. Impurities of the solution alkaloid,amineor antibiotic.
2. Blow drying the chromatograph -TLC involves purification process. It is because they separate solutes,
3. Presence of elongated stains and when the other solutes are removed, it is already considered
4. Mixing of the sample purified.
-Drug screening test, can only be either positive or negative. Cannot
Note: -The sources of errors for TLC: be used for confirmatory test.
 Impurities of the solution. Remember that the aim is to isolate the -We need to adjust pH in order to reduce solubility of the drug.
wanted solute, so if there are impurities, it could give us false
results. Column Chromatography
 Blow drying the chromatograph. The chromatograph is the  Separate substances based on differential adsorption of compounds
machine used for chromatography. So NO BLOW DRYING. to the adsorbent
 Presence of elongated stains, whenever the stain is elongated,
the stain will represent our sample and it will be read as the solute Note: -Is either the tube is filled with stationary phase or the stationary phase
has traveled far, but actually it didn’t. It was just the stain that has is only in the inner walls of our tube.
elongated producing false results. -Adsorbent is the stationary phase for our column chromatography.

8
MLS 11A CLINICAL CHEMISTRY MLS 3A
Separation Mechanisms in Column Chromatography -Liquid-liquid chromatography: this means both the mobile phase and
1. Adsorption column chromatography (ACC) stationary phase are in liquid form.
 Basis of Separation: differences between the adsorption and -Each solute partitions itself between the stationary and mobile phase.
desorption of the solutes at the surface of an adsorbent
 In GC: utilized to separate low molecular weight compounds and 3. Gel column chromatography
compounds that are normally gases at RT  Basis of separation: molecular size and shape of solute
 Also referred to as:
1. Liquid-solid chromatography
2. Size-exclusion chromatography
3. Gel-filtration chromatography
4. Gel-permeation chromatography
5. Steric exclusion chromatography
Note: - There are 4 separation mechanisms in the column chromatography. 6. Molecular exclusion chromatography
The basis here is the chemical and physical mechanism to separate the 7. Molecular-sieve chromatography
solute.
-The adsorbent is the solute. The solid particle is our adsorbent and our
stationary phase.
 Solute: the component aimed to be separated in this process
-ACC is otherwise known as liquid-solid chromatography. This
separation mechanism, the solute in the sample and our mobile phase
which is in liquid form, compete with each other in the adsorptive site in
our adsorbent/stationary phase. Note: -Separation of molecules based on their size (size-exclusion
-Factors which affects the adsorption of the solute chromatography)
 Electrostatic property -Gel permeation involving the property of the solutes to permeate the
 Hydrogen bonding gel.
 Dispersive/spread and interaction of the molecules, particularly  Separation takes place on a column packed with gel.
with the aid of physical forces  Gel has different pore sizes (Very small pores not visible to the
-In gas chromatography, the ACC is used to separate low molecular naked eye)
weight compounds and these compounds are not normally gases at
room temperature. 4. Ion-Exchange column chromatography
2. Partition column chromatography  Basis of separation: exchange of ions between a charged
stationary phase and ions of the opposite charge in the mobile
 Basis of separation: distribution of solutes between two
phase
immiscible liquids
 Stationary phase: resin consisting of large polymers of
 Based on the solute’s relative solubility with organic solvent
substituted benzene, silicates, or cellulose
and an aqueous solvent
Note: -The stationary phase is charged, can either be positive or negative.
-The mobile phase is charged but the charge of the mobile phase and
the stationary phase are not the same. Their charges are always
opposite.
-Solute are separated by their ionic charges or electrical charges. Other
than that, magnitude of their ionic charges.
Note: -The separation of solute here is based on their relative solubility with
organic solvent or polar solvent and the relative solubility with aqueous
or non-polar solvent.

9
MLS 11A CLINICAL CHEMISTRY MLS 3A
Components of a Chromatograph:
 Carrier Gas and Regulator
-Gas transports the sample components through the column
-Hydrogen, Helium, Carbon Dioxide, Nitrous Oxide, NH3,
Nitrogen

Note: -The mobile phase are the beads with negative charge/ the gray ones.
-The negatively charged molecules are not attracted because of the
principle opposite attracts. Since they are the same charge, they will not
attract each other.
Note: -The components of a chromatograph:
-The pink molecules which are negatively charged will not stick to our
negatively charged beads.  Gas cylinder which contains the gas or the carrier gas which has
-The tendency of the pink molecules when the sample is poured into the a regulator.
tube, the pink will continuously flow down.  Sample injector or the sample syringe
-On the other hand, the blue beads which are positively charged ions  Chromatographic column which is found inside the column oven.
will stick to the negatively charged beads and won’t be able to flow The column oven is protecting the column maintaining the
down. temperature.
 Detector
Column Chromatographic Methods  Recorder, including the chromatogram
 Gas chromatography
 Liquid Chromatography -The regulator controls the flow of gas going to the column.
 Ion-exchange chromatography -The purpose of the carrier gas is that it is the mobile phase.
 Size Exclusion Chromatography -The carrier gas is dependent on the detector and column used.
 Chiral Chromatography -The carrier gas should be chemically inert chemicals.
 Hydrogen, helium and nitrogen are considered as chemically inert
Note: -These are methods which employ the principles of chromatography. substances.
1. Gas chromatography (GC)  We can also use compounds that produce steam like C02, N2O
andNH3.
 For compounds that are naturally volatile or can be easily
converted into a volatile form
Sample Injector
 Mobile phase: gas/carrier gas
-injects sample through the septum
 Two categories: Gas-solid Chromatography and Gas-liquid -temperature of the injection portion must be above boiling points of
Chromatography compounds
Note: -In the Gas-solid chromatography, the mobile phase is in the form of
gas, and the adsorbent or the stationary phase is in the form of solid.
-In the Gas-liquid chromatography, the mobile phase is in the form of
gas, and the adsorbent or the stationary phase is in the liquid coated on
a solid support.

10
MLS 11A CLINICAL CHEMISTRY MLS 3A
Note: -It is still a typical syringe with a barrel and a needle. The purpose is to Detector
deliver sample into the column. -Detects separated analytes as they elute from the column
-But before the sample goes to the column, the syringe’s needle must  Flame ionization detector (FID)
pass through the septum (rubber cap of the tube).  Thermal conductivity detector (TCD)
-Pipette or micro syringe can also be used.  Nitrogen-phosphorus detector
-This means that the sample can be introduced to the column manually.  Electron capture detector
-One requirement of the injection port is that it must have a higher  Flame photometric detector
temperature (Must be above the boiling point). This is to allow  Mass spectometric detector
evaporation of samples upon injection as long as it is volatile.
Note: -The function is to detect the separated analytes as they elute from the
column.
Chromatographic Column
-FIDs is the most common detector used in the lab may it be for organic
-where fundamental separation takes place
or inorganic compound.
-enclosed in a temperature-controlled oven
-One requirement of the FID is that it can measure ions as long as we
-Two types:
heat it with hydrogen air flame.
Capillary Column
-TCD contains wire or filaments that raises the temperature of the
Packed Column
detector. One of the earliest detectors and is now obsolete because it
has a very low sensitivity.

Recorder/Computer Data System

Note: -The part of the chromatograph where the separation takes place. It is
enclosed by the oven that controls the temperature of the column.
-It is used because we need to maintain high temperature in gas
chromatography.
-Two types of column are: capillary and packed. Difference:
-Computer regulates various parameters:
 Mobile phase composition and flow rate
 Column back pressure
 Column & detector temperature
 Sample injection
 Detector selection and operation
 Various timing steps that command the operation of the system
Note: -provides us system control and data processing functions
-it can control a variety of processes that we can do with our
chromatograph. The processes are listed above.
-Sample injection: can control the amount of sample that will be used
Sources of Interference in Gas Chromatography

Capillary Packed Temperature of injector:

Length 5-100 meters 1-5 meters -Ensures evaporation of sample but do not decompose it (200 - 300°C)
Diameter 0.1-0.8 meters 2-4 meters
Advantages
Location of Inner wall of the Fills the whole column -Fast analysis
stationary phase column -Small samples
-Reliable, simple, cheap
-Columns with thicker film can retain more analyte. -Sensitive detectors
-Suitable for routine analysis
Column Oven
-Maintains good temperature control of the column (houses the column)

11
MLS 11A CLINICAL CHEMISTRY MLS 3A
Disadvantages 3. One or more pumps to force the liquid mobile phase through the
-For volatile samples only system
-Some samples may require intensive preparation, may react with column 4. An injector to introduce an aliquot sample into the column
-Requires MS spectroscopy to confirm result 5. Online detector(s) to detect the separated analytes as they elute from
the column
Note: -Gas chromatography requires higher temperature in order for the solute 6. A Computer that controls the system and process data
to evaporate.
 Higher temperature means above boiling point (between 200 to Note: -Solvent: the difference is here, the solvent has a reservoir while in GC,
300 degrees). The temperature should remain constant within there’s a tank but of course, it’s different because that tank contains gas
that range. If ever you go below that, then of course, you will have which is the mobile phase in GC.
problems with the movement or the mobility of our solute. -The purpose of the pump/s is to be the source of pressure in order for
-One advantage of GC is that you don’t need to be very skillful when the solute to move.
you are to use this machine because you can learn this as it is very easy
Types of Liquid Chromatography
to operate.
1. High Performance Liquid Chromatography (HPLC)
-The disadvantage is that gas chromatography is not intended or not
 Uses pressure for fast separation, controlled temperature, in-line
suitable for those unstable samples or thermally unstable samples.
detectors and gradient elution technique
 Thermally unstable samples are samples whose properties
change quickly if exposed to different temperatures or changes in  Enable the dissection of complex mixture into individual
temperature. constituents
 Separation in a densely packed column
2. Liquid chromatography
 Use lower temperatures for separation Components of HPLC
 Pumps
 Gives better separation of thermolabile compounds
 Columns
 Mobile phase can be removed
 Sample Injector
 Sample can be processed further or reanalyzed
 Detector
 Recorder
Note: -What’s the difference between liquid and gas chromatography?  Injectors- introduce the liquid sample into the flow stream of the
 Gas chromatography requires high temperature while in liquid
mobile phase
chromatography, low temperature is required.  Column- the “heart of chromatograph”
 Gas chromatography is not suitable for what we call as
thermolabile compounds (thermally unstable). Liquid Advantages
chromatography can give better separation for those substances
-Rapid purification or assay method
which are easily affected by any changes in the temperature.
-identify and quantify many components of a complex mixture
 These two factors, low temperature and can be used for
-allows unanticipated components to be detected
thermolabile substances, are the edge of liquid chromatography
over gas chromatography. Disadvantages
-With liquid chromatography, we can easily recover sample. For -Cannot be used to analyze large quantities of samples
example, you recovered a sample and you need to repeat the test. You
-Initial cost for automated apparatus may be high
can reanalyze the sample. You can repeat the test using the same -Sensitive to interference by sample or solvent contaminants
sample. In GC, remember that the sample evaporated so that is why the
test can’t be repeated on the same sample anymore. Clinical Applications of HPLC
 Measures glycated hemoglobin
Components of Liquid Chromatography
1. A chromatographic column to separate the solutes  Investigation of many congenital metabolic metabolic disorders or
diseases
2. A solvent reservoir to hold the mobile phase
 Identifying and quantifying individual urinary porphyrins
12
MLS 11A CLINICAL CHEMISTRY
 TDM
 Identify causes of poisoning
 Optimum for the separation of chemical and biological non volatile
compounds
Note: -Between the two types, HPLC is the most common type of liquid
chromatography. This one uses small rigid support and special
mechanical pumps.
 These mechanical pumps are the ones which will produce the
high pressure needed to pass the mobile phase to the column,
which means the high pressure produced by the pumps will assist
the solute when it passes through as it is carried by the mobile
phase along the column.
 That means HPLC has high pressure requirement
-HPLC separates and quantifies compounds that have been dissolved
in the solution. It has this ability to identify specific amount of compound
in a solution.
-The pump can deliver constant mobile phase composition or increasing
mobile phase composition.
-The required or ideal rate of flow of the pressure (force) from the pump
is 1 to 2 mL per minute.
-Advantage with liquid chromatography: This requires only very small
amount of sample, as small as 5 to 20 microliters.
-Remember in GC, the requirement for the injector is that it can
withstand high temperature. In liquid chromatography, the requirement
for the injector is to withstand high pressure.
-The column is called as the “heart of the chromatograph” because it is
where the separation takes place.
-We have here clinical application of HPLC particularly for the
measurement of glycated hemoglobin or that is hemoglobin A1c.
-It can also be used to investigate for congenital metabolic disorders or
the ones included in newborn screening.
-TDM (therapeutic drug monitoring) or to monitor the level of therapeutic
drug.
1. Liquid Chromatography – Mass Spectroscopy (LC-MS)
 Detecting nonvolatile substances in body fluids
 Utilized to confirm positive results
 Complementary method to GC-MS
 Uses: TDM, toxicology, and drug metabolite studies
Note: -This is the second type of liquid chromatography. It is very good in
detecting non-volatile substances which are present in the body fluids
and this one can be used to confirm positive results from screening illicit
drugs. This means, if you were screened, for example, for the presence
of drug using TLC, you can seek for confirmatory testing using LC-MS.
MLS 3A
-LC-MS is a complementary method of GC-MS (gas chromatography-
mass spectroscopy or spectrometry).
-It can be used in TDM (therapeutic drug monitoring) but for LC-MS, it
requires interface methods. The purpose of interface methods it to
convert non-volatile compounds into volatile compounds.
2. Ion-Exchange chromatography
 for separation of ions and molecules that can be easily ionized
 the electrostatic interactions between the analytes, mobile phase,
and stationary phase, contribute to the separation of ions in the
sample
Note: -Ion-exchange columns are used to separate ions and molecules that
can be easily ionized. This separation is dependent on the ion’s affinity
for the stationary phase which means electrostatic interactions are very
important in this type of chromatographic method.
3. Size Exclusion chromatography
4. Chiral chromatography
 Separation of enantiomers
 these columns have a stationary phase that selectively
interacts with one enentiomer over the other
Note: -Chiral chromatography is used to separate enantiomers.
 Enantiomers compounds or molecules that are mirrored images
of each other. [“Please review on that it is still biochem
ha?,”(Gallego, 2020].
-For chiral chromatography, the separation of molecules or compounds
are based upon their stereochemical properties (stereochemistry).
-What happens in chiral chromatography is that there is a column which
contains the stationary phase that selectively interacts with one
enantiomer over the other, which means if there are two enantiomers, it
will only react with one. It will choose only one enantiomer with which it
will react.
-Chiral chromatography is very useful in separating racemic mixtures.
 Racemic: mixtures with dextrorotatory and levorotatory forms of
a compound, which are equal in proportion.
“Ta? CC?”
CLINICAL CHEMISTRY now, COCKTAIL CRUISER later
Padayon, RMT
13
MLS 11A CLINICAL CHEMISTRY
MODULE 2: Analytic Techniques and Instrumentation
Lesson 5: Mass Spectrometry
MASS SPECTROMETRY
-fragmentation and ionization of molecules using a suitable source of
energy
-detects structural information and determination of molecular weight.
Major steps in Mass Spectrometry
1. Conversion of the parent molecule into a stream of ions.
2. Separation of the ions by mass/charge ratio (m/z)
3. Counting of the number of ions of each type or measurement of current
produced when the ions strike a transducer.
Note: -This is based on the fragmentation and ionization of molecules using a
suitable source of energy.
 Fragmentation: from a big molecule to a smaller molecule through
ionization.
-For mass spectrometry, the resulting fragment masses and the relative
abundance yield a characteristic mass spectrum of the current
molecule.
-Mass spectrometry can also detect structural information and
determination of molecular weight.
-One thing we need to remember with mass spectrometry is that before
fragmentation or detection or quantification using this method, there
should first be separation or isolation of the molecules using either gas
chromatography or HPLC.
-Major steps involved in mass spectrometry:
1. The principle of mass spectrometry is based on the
fragmentation and ionization of molecules which means in the
first step, from a parent molecule, it will be fragmented or
converted into a stream of ions. Usually, ions are in singles.
2. After fragmentation, the ions will be separated from each other.
The separation of ions is based on their mass and charge ratio.
3. After separating the ions, the number of ions of each type or
measurement is counted. The machine will do the counting. The
machine will count the number of ions of each type or the
measurement of current produced when the ions strike a
transducer.
 Particularly for mass spectrometry, the transducer for MS
converts the beam of ions into an electrical signal that can
be processed and stored in the computer memory, and
presented in graphical form (chromatograph).
MLS 3A
Components of Mass Spectrometry
-Ion source
-Vacuum system
-Mass analyzer
-detector
-computer
 Ion Source
- maintained at high temperature and vacuum
-e.g. beam of electrons produced by a heated filament
-MALDI and SELDI
 Mass Analyzer
- sorts the parent molecular ions and their fragment ions
 Ion Detectors
-Electron multiplier:
 Ions strike the detector’s first dynode, which triggers
the release of secondary electrons
 Amplification of about a millionfold of electron
-Ion-photon conversion detector
 Ions strike a phosphor that emits a photon for each
corresponding ion
Note: -The requirement of the ion source unit is that it should have high
temperature and high vacuum pressure. That is to provide adequate
conditions for ionizing the vaporized sample molecules.
-Ionized: there is fragmentation involved.
-Several types of energy sources that are available for fragmentation or
ionization of sample molecules in mass spectrometry:
1. Commonly used ion source for MS is the beam of electrons
which are producing a heat, or which are produced by a heated
element.
2. MALDI source
3. SELDI.
-“So, I will be leaving to you kung ano na siya meaning sang MALDI,
ano ang meaning sang SELDI and then kung ano ang function nila,
ano ang characteristic nila, so kamo nalng magread sina. I have seen
this sa Bishop so please take note on that.” – Gallego, 2020
-The mass analyzer is the part of the spectrometer which isolates or
sorts parent molecular ions and their fragment ions based on their mass
charge ratio. So, sorting is the function of the mass analyzer.
1
MLS 11A CLINICAL CHEMISTRY
-Before the fragmented ions reach the mass analyzer, from the ouput of
the ion source, there are already positive or negative gaseous ions
produced.
-After that from the fragmentation using the ion source, those ions will
be accelerated going to the mass analyzer.
-Once they reach the mass analyzer, they will be segregated according
to their mass charge ratio.
-There are two types or commonly used ion detectors.
1. Electron multiplier: The secondary electrons will be amplified
and there will be production of millionfold of electrons.
2. Ion-photon conversion detector:
 Phosphor is a chemical compound that emits light when it
is exposed to a light of a different wavelength.
GAS CHROMATOGRAPHY- MASS SPECTROSCOPY (GC-MS)
-Uses an electron beam to split drug emerging from the column into its
component ions
-quantitative measurement of drug can be performed by a selective ion
monitoring
Note: -Gas chromatography-mass spectroscopy or GC-MS is the gold
standard for drug testing.
-The fragmentation of the parent molecules is with the use of electron
beam. The electron beam will split the drug coming from the column into
component ions.
-GC-MS can give us quantitative measurement of the drug through
selective ion monitoring.
-It can be used for xenobiotics, anabolic steroids, or for pesticides.
TANDEM MASS SPECTROSCOPY (MS-MS)
-can detect 20 inborn errors of metabolism from a single blood spot
Note: -It is called MS-MS because it is TANDEM spectroscopy or mass
specstroscopy.
-This is capable of detecting 20 inborn errors of metabolism from a
single blood spot. This refers to the newborn screening.
-The components are found in the module.
MLS 3A
MODULE 2: Analytic Techniques and Instrumentation
Lesson 6: Osmometry
OSMOMETRY
-Addition of active osmotic pressure: osmolality increases and four other
properties of the solution are affected
-as the osmolality of a solution increases the following reaction occurs:
 Osmotic pressure and boiling point: INCREASES
 Vapor pressure and freezing point: DECREASES
Note: -Basically, in osmometry, the addition is the osmotic pressure.
-While adding the osmotic pressure, the other four colligative properties
of the solution are also affected.
 Osmotic pressure
 Boiling point
 Vapor pressure
 Freezing point.
-Addition of active osmotic pressure: The reactions already have
osmotic pressured but more is added.
 Osmotic pressure increases, the boiling point is elevated.
 Vapor pressure and the freezing point are decreased or
depressed.
-That is the effect of osmometry or if active osmotic pressure is added.
-If osmolality is increased, the four other colligative properties of
the solution are also affected.
“Ta? CC?”
CLINICAL CHEMISTRY now, COCKTAIL CRUISER later
Padayon, RMT
2
MLS 11A CLINICAL CHEMISTRY
MODULE 2: Analytic Techniques and Instrumentation
Lesson 7: POCT
POCT
-Point-of-care-testing
-laboratory testing that is performed outside the central or core
laboratory
 Physician office testing
 ER
 OR
 ICU
Note: -POCT stands for point-of-care testing. It is a laboratory testing that is
performed outside the central or core laboratory. It can be performed in
the doctor’s clinic, ER/emergency room, OR/operating room, and
ICU/intensive care unit. It can even be performed at home.
-POCT is performed at the site of clinical care or in short, bedside
testing. The results can be available in a matter of seconds.
-In the physician’s office, it can be that the doctor is the one performing
the test. But at home, anyone can perform the test.
-In most settings, POCT is performed by clinical staff rather than
laboratorians. Laboratorians refer to RMTs. Clinical staff include nurses,
doctor’s secretary, or if at home, anybody can perform POCT.
-Example of POCT being used at home: Glucometer for the
measurement of glucose.
 The glucometer measures/examines random blood sugar or it
can also be fasting blood sugar.
 The difference with the POCT machine for glucose or the
glucometer is the required sample is whole blood.
-Another example of POCT is the one that measures cholesterol, uric
acid, or triglycerides. These can be used at home.
-There are also POCTs used for coagulation testing. The sample used
is plasma.
Advantages:
-Decreased TAT
-Decreased time to treatment
-Collaboration between laboratory and clinical staff
-Decreased length of stay
-Fewer follow-up visits or calls
-Improved patient and provider satisfaction
-Less traumatic sample collection
Note: -The test is performed at bedside meaning beside the patient. In a few
seconds, the result is ready.
MLS 3A
-The pros (advantages) and cons (disadvantages) of POCT depend on
the clinical setting (where it is performed) and on the need of the patient.
 For example, when it comes to glucose testing, there are times
when it is not necessary for the patient to go to the laboratory to
have his/her glucose level checked.
 There are nurses who take whole blood from the tip of the
patient’s finger (meaning it is capillary blood) and they monitor
the blood sugar routinely. If the doctor sees that the CBG or the
capillary blood glucose of the patient is normal, the doctor does
not need to request further for FBS because it is already normal
based on the POCT testing.
-It can also lessen the workload of MLS and nurses. In the Philippines,
the practice is that it is the MT that extracts blood but in some other
countries, the role of MT is to process the blood in the laboratory which
means there is a phlebotomist. Remember that it is not necessary for a
phlebotomist to be a MT. It can be a nurse.
 When there is a POCT machine, there is no need for the nurse to
collect the blood and deliver it to the laboratory. When using the
POCT, the blood can be collected and immediately testing can be
done.
-If the purpose is only to measure glucose, there is no need to extract
that much blood like 5 mL or 10 mL. The POCT only requires for a small
amount of blood. One drop is enough. This means the patient is
prevented from having iatrogenic anemia that is anemia caused by
excessive or too much blood extraction.
-Improved patient satisfaction. For example, if you are a diabetic and
you have a glucometer at home, you don’t need to go to the lab anymore
to monitor your glucose level. Diabetic patients take their glucose level
every morning to be monitored.
-Less traumatic sample collection because since for glucometer for
example, the sample needed is capillary blood so only a finger prick is
needed. When going to the laboratory, 3-5 mL of blood is usually
extracted. Add to that if the MT has to take the blood twice.
Disadvantages:
-Results may not correlate with central laboratory
-increased cost
-Increased regulatory burden
-Increased work for clinical staff
-Increased oversight for laboratory
-Greater potential for error
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MLS 11A CLINICAL CHEMISTRY MLS 3A
Note: -Greater potential for error. What can contribute? Either false negative Security access Different levels of users and
or false positive. managers
 When using POCT, this does not require well-trained or skillful
Reference ranges May transmit to LIS
staff/personnel. For glucose, anyone can do that even if the
maintenance or janitor personnel of a hospital can perform it Quality control ranges
because it only needs a finger prick. But these people are not Bar code scanner For patient identification, reagent
trained to do that. and quality control identification
 Remember that when performing the finger prick method, the first Data Management Patient results, quality control
drop of blood needs to be wiped. These people don’t know this
records, maintenance and
so the tendency is when they perform POCT testing, the first drop
of blood is included in the sample being read by the machine. calibration documentation
That would mean that the blood is diluted so the glucose Connectivity Data manager or middleware, LIS
concentration will be falsely decreased.
-Increased regulatory burden because there are some POCT machines Note: -With the use of POCT machines or instruments, there is a device
that require the laboratory or hospital to be certified that they will be software present.
performing those specific tests outside of the laboratory. For example,  System lockouts: it is included in the system of the POCT
if it is to be performed in the ICU, they have to be accredited/certified machine.
first.  Quality control/out of range/not performed: there are machines
that require to run QC. If you can’t run QC, you cannot perform
the test. The machine will prompt you that you did not run QC.
 Security access. Some machines require password/s.
 Reference range: the POCT machine can be connected to a
computer so that the results can be directly transmitted to the LIS.
 Barcode scanner is the unique identification for the patient. That
means there is no need to label the tubes/specimen of the patient
because there is already a barcode.
 Data management: it can store results. For example, it can store
results for 1 month and then auto-delete after that.
 Connectivity: it can be connected to the computer to directly
transmit results.
Note: -This is an example of a glucometer. The blue one is the strip. When
you turn on the POCT machine, just insert the strip. Prick the patient’s
finger. Wipe the first drop of blood. Allow the blood to be absorbed by
this part of the glucose strip.
-The other picture is a coagulation machine. The patient is in the ICU. It
is done bedside.

POCT Device Software features

Feature Details
System Lockouts Quality control out of range or not
performed, no patient ID, no
operator training
“Ta? CC?”
Calibration CLINICAL CHEMISTRY now, COCKTAIL CRUISER later

Padayon, RMT

2
MLS 11A CLINICAL CHEMISTRY MLS 3A
MODULE 2: Analytic Techniques and Instrumentation from one container to another, but now, we have the use of
Lesson 8: Automation in Clinical Chemistry aspirator or micropipettes.
 On mouth pipetting, in order to transfer the liquid, the
Automation in Clinical Chemistry laboratorian must suck the liquid from the mouthpiece to enable
 process by which an instrument automatically performs tasks that the transfer of liquid. This process however is not allowed today
would otherwise have to be done manually by laboratory staff due to imposed harms on the laboratory staff. One example is
 process wherein multiple devices are capable of automating pre- contamination of the mouth due to excessive sucking of the
analytic, analytic, and post-analytic stages of testing liquid, either urine, semen, blood, serum or plasma.
 Nowadays, glass pipettes are used with rubber aspirators to
transfer liquid onto the pipette, and to the container.
 Single channel micropipettes or Eppendorf pipettes are also
used, wherein we just attached something on the pipette tip and
there is no need for rubber aspirators. Just push the plunger,
place the pipette on the solution, release the plunger, and the
fluid will be automatically sucked in. This also includes the
volume indicator, where we can measure multiple amounts
using the micropipette.
 Multichannel pipettes are also used, where it can hold 8
samples or more than that. We can aspirate multiple samples in
one setting.
 In some countries, there is also a robotic pipette called Andrew.
It has single channel and multiple channel pipettes. Just
program the machine and it will automatically choose what
pipette tip is to be used.

Rationale for Laboratory Automation

1. Human performance varies over the course of time.
2. Repetitiveness of many lab tasks.
3. Shortage of trained laboratory personnel.
Note: -There has been a revolutionary change from performing manual
methods in the laboratory up to automatic methods. At present, most
Note: -These are the reasons why we need advancements on the processes
areas of the laboratory have automation. For example, there is a
and instruments in the laboratory.
machine in hematology that makes a blood smear.
-Firstly, human performance varies over the course of time. Say for
-Laboratory automation enabled broader processes over the years. It
example, our physical energy and mental alertness depletes or
covers three phases or stages of testing: pre-analytic, analytic and
changes over time. Thus, as we get tired, we have difficulty in
post-analytic phases. This also includes specimen sorting.
maintaining accuracy on work. Instruments on the other hand, can do
 For example, we have different samples on the phlebotomy
the job with the same high degree of reliability at the course of time.
area, the machine will automatically transfer the samples based
-Next is repetitiveness of many lab tasks. For example, if you pipetted
on its required area (Hematology, Clin. Microscopy, etc.)
100 samples, it can be tiresome for the part of the laboratory staff,
 Other than specimen sorting, it will also introduce the sample to
resulting to inaccuracy and lots of errors in testing and release of
the machine, will sort, and will release the results.
results. But if machines are used, they can’t be bored or tired.
 Kindly refer to the module for the advantages and
-Next is shortage of trained laboratory personnel. Here in the PH, we
disadvantages of automation.
have abundant medical laboratory staff, but on some countries, there
-Laboratory automation has been present in all areas of the laboratory.
is growing shortage of laboratory personnel, therefore laboratory
 For example, the use of the pipettes. In the past, mouth
pipetting has been the mechanism for pipetting to transfer liquid

1
MLS 11A CLINICAL CHEMISTRY MLS 3A
machines are needed. This will fasten the assays done even at Process Control Software Functions
multiple samples. 1. Calculate number of aliquots and volume needed for each
workstation
 Turnaround time (TAT) demands 2. Route samples to analyzers in most optimal way
 Specimen integrity issues 3. Recap samples
 Staff shortages 4. Autoretrieval of samples for reflex or repeat testing
 Economic factors 5. Autoverification of results
 Need to lower maintenance
 Need to reduce calibration Note: -These are some of the functions that can be performed with the
 Need to reduce downtime automation in the laboratory.
-Firstly, calculate number of aliquots and volume needed for each
 Faster start-up times
workstation.
 24/7 upbme Throughput
 There are times that patients have multiple samples with
 Computer and software technology different tests. Say for example, the patient is difficult to be
 Primary tube sampling extracted and you only extracted 5 mL of blood. Good thing
 Increasing the number of different analytes on one system about this is that, for example in hematology, we have
 Increasing the number of different methods on one system microtainers which can hold volumes like 0.5 or 1 mL. Instead of
 Reducing laboratory errors placing the blood in 2 mL of tube, you can proportion the
 Number of specimens amount of blood to each tube if you happened to extract
 Types of fluids insufficient volumes.
 Safety  Another is that, the machine can be programmed to adjust the
 Environmental concerns (i.e., biohazard risks) volume of suction depending on the amount of the sample
collected. Say for example, in a 2.5 mL of blood from the
Note: -What we mentioned about the rationale for laboratory automation patient, the serum collected would be half of that or 1mL.
focuses on the staffing. This one focuses on the processes done in the Suppose we will run 10 tests and we need 200 µL per test. If we
laboratory why laboratory automation developments became a thing. have 10 tests, we need 2 mL of serum. We can program the
-First is turnaround time (TAT) demands. Laboratory automation machine to adjust the amount to be aspirated from 200 µL to
shortens the laboratory processes to save the life of the patient. 100 µL. In this case, we can still run the test even if there is
-Another is number of specimens, where it can process multiple minimal sample used.
samples in a much efficient time. -Another is route samples to analyzers in most optimal way. One
example is on Metro Iloilo Hospital. They have tubing from the nurse
Factors Contributing to Advances in Automation station where they just drop the sample in a carrier and program it to
1. Robotics be delivered on the designated area for testing. The sample carrier will
2. Computers and microprocessors automatically deliver the sample on to the desired area. It will save the
3. Sensors for monitoring reactions nurse’s time in delivering the sample manually.
-Another is the recapping of sample. Say for example we have the
Note: -These three major areas greatly contribute to our ability to automate SST Tube. The machine will automatically remove the cap on the tube,
laboratory processes. obtain the sample, and recap the tube.
-Robotics, wherein these are machines that automatically performs -Next is autoretrieval of samples for reflex or repeat testing. There is a
laboratory procedures. reference range for samples in the laboratory. For example, patient 1
-Sensors for monitoring reactions. For example, we can’t process has increased glucose, therefore, the collection must be repeated. We
samples that are hemolyzed, the machines have the ability to detect just need to press a button on the machine, and it will repeat the
for presence of hemolyzed, icterous, or lipemic samples. testing.
-Finally, autoverification of results. When results are released by the
machine, it prompts to indicate whether the results are high or low.

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MLS 11A CLINICAL CHEMISTRY MLS 3A
-As we mentioned earlier, the three phases of testings can be Laboratory Automation RFP
improved due to laboratory automation. What is good on laboratory 90th percentile for test completion: general them try
automation is that, it has been able to address two important phase of Laboratory (30 minutes), immunoassay (45 minutes), CBC (20
analysis: the pre-analytic and post-analytic phase particularly on the turnaround time minutes), basic coagulation (30 minutes)
sample identification and specimen integrity issues. The machine can
Ability to prioritize stats
detect defects in specimen integrity.
 For example, if we have coagulation test and the sample obtain During peak periods, throughput adequate to meet
has presence of clots, this will result to a false decrease on Throughput TAT and grow business by 20% with existing
clotting time. The machines at present that alarms if it aspirates configuration
with a clot. Proposed test menu and (current) volume with ability
Method
to add open (user-defined) channels
Automation: Pre-analytic and Post-analytic Steps
Operator alerts for sample integrity issues (like
 Primary tube ID/labeling (if not done remotely)
Specimen hemolysis, icterus), insufficient sample, label error
 LIS Receiving
 Sorting (and prioritizing stats) management System should be able to store, locate, and retrieve
 Centrituging sample for add-on or repeat tests
 Decapping Seamless and bidirectio4 interface
LIS Needs
 Secondary tube labeling Can operate independent of TLA
 Volume checks/clot detection Budget neutral; with reallocation of staff
 Aliquoting Financial impact Contract length; fixed reagent and consumable
 Destination sorting into analyzer racks and contract pricing over contract period versus annual
 Recapping terms adjustment
 Sample storage and retrieval
Service included for life of contract versus initial 2
Analytic Automation years
 Sample introduction and transport to cuvet or dilution cup Environment and Will not generate excessive heat or noise
 Addition of reagent safety System will minimize workplace exposure to hazards
 Mixing of sample and reagent Dedicated service engineer for first 2 months
Downtime and
 Incubation 24/7 oh-call service thereafter with 2-hour response
 Detection service
time Minimal downtime
 Calculations Contact information for at least two current
 Readout and result reporting References customers using the proposed or a similar
configuration
Note: -All fully-automated machines, particularly on clinical chemistry, have
the ability of sampling directly from the collecting tube. This means it
can aspirate directly from the tube by puncturing or removing the cap, Note: -Request for Proposal (RPF) is necessary if the laboratory aims for
obtain the sample, and run for tests. total laboratory automation. RFP is forwarded to the supplier company
-At present, barcodes are unique identifiers that are used to easily of the machines. The RFP will contain data to be included by the
program what tests are to be run by the machine. company which will be presented in the laboratory.
-The request for proposal contains the necessary information that we
need to know from the distributors. We have here Laboratory
turnaround time.
 Say for example, we request for automation for clinical
chemistry. How long will the processing of results be with the
machine?

3
MLS 11A CLINICAL CHEMISTRY MLS 3A
 Ideally, in clinical chemistry, the results must be up for 30
mins, particularly in immunoassays
 For the determination of antibodies, it must be up to 45
mins.
 For general chemistry, it must be up to 30 mins.
 For CBC, it must be 20 mins
 For basic coagulation, it must be 30 mins.
 There are also tests in the laboratory that can release
results for just 30 seconds. In particular, it is the
determination of electrolytes (Na & K). That’s why, in the
laboratory, when it’s just the electrolytes to be tested, the
patients are often ask to just wait for the results after
collection, because we don’t need to follow the protocol
that the release of results must be 2 hrs. after collection.
In this case, there is no need to prolong the waiting time
of patients for hours if we know we can release the results Note: -This type of analysis describes that the chemical and the sample
already within 5 minutes. travels through the instrument/system.
-What is presented in RFP to the distributors are the needs and goals, -The unique thing about this analysis is that it uses air bubbles in the
and the terms to be considered in the laboratory. In return, the sample and the reagent strips.
distributors will present to us the RFP. -As the sample and air bubbles are pushed through the system, they
-In short, RFP can be considered as the agreement between the will meet with the reagent and they will go to the incubator and
supplier and the customer upon making requests for automation. The reaction detector. This means there is a sequential passing of the
request for proposal to the distributors is a part of our total laboratory sample and the reagent going to the analytical pathway. There is only
automation. We can’t just proceed with TLA without knowing the one analytical pathway in which the bubbles force the sample to meet
advantages or disadvantages that the machine can give us. with the reagent and then into the reaction detector for the continuous
-For references, contact information for at least two current customers flow of the analysis.
using the proposed or similar configuration. -The air bubbles minimize the diffusion of the reagents and mixing
between samples. The air bubbles preserve the integrity of each
Basic Approaches in Automation individual reaction.
1. Continuous Flow -The reaction is when the sample and the reagent is mixed already.
2. Centrifugal Analysis
3. Discrete Analysis Centrifugal Flow Analysis
• Sample and reagent: placed in separate chambers in a rotor
Continuous Flow Analysis • Capable of running multiple samples, one test at a time, in a batch
• Reagents and samples travel through the instrument
• Unique characteristic: use of air bubbles 1. Application of centrifugal force results to spinning of the rotor
• Air is injected into each stream as a series of small bubbles which spins
travel along with the reaction system 2. Flowing of two components into the reaction chamber
Major drawback: 3. Product solution then moves to a cuvette
• Significant carry-over problems 4. Determination of light absorbance for each sample by a
• Wasteful use of continuously-flowing reagents (Use too much phototube
reagents)

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MLS 11A CLINICAL CHEMISTRY MLS 3A

Note: - Can run multiple samples, one test at a time.

Note: -Advantage of this is that it has the ability to run multiple tests one
-Application of centrifugal force. That will result to the spinning of the
sample at a time or multiple samples, one test at a time.
rotors. The centrifugal force will pull the 2 components (Sample and
-Comparing with the centrifugal, which can run multiple sample but can
Reagent) to the reaction chamber/cuvette.
only have one test at a time, here in discrete analysis, it can do either
-When the sample and the reagent meet, it is already called a solution.
that of the centrifugal or it can run multiple test but with one sample at
-The rotor is found within the analyzer. It is where the mixing, reaction,
a time.
and analysis take place.
-Cuvette is found on the outer rim of the rotor.
-2 types of discrete analyzers: Direct read discrete analyzer or Indirect
-After the solution is already in the cuvette, a light will pass through the
read discrete analyzer
cuvette and the absorbance of light of the solution is measured by the
 The difference is that the direct uses only one cuvette for each
detector (Phototube) and is displayed by the computer or the data
sample, while the indirect uses 2 cuvettes. And on the indirect
processor or the signal processor.
read analyzer, all samples pass through the flow cell in order for
-The principle used is of spectrophotometry, or could be nephelometry
the absorbance to be read.
or turbidimetry depending on what we are measuring: the amount of
light that passed through (Spectrophotometry), the amount of light
Summary of Chemistry Analyzer Operations
blocked (Turbidimetry), or the amount of light scattered
(Nephelometry). IDENTIFICATION AND PREPARATION

1. Sample identification This is usually done by reading the

Discrete Analysis
bar code. This information can be
• Separation of each sample and accompanying reagents in a
separate container entered manually.
• Can run multiple tests one sample at a time or multiple samples one 2. Determine test(s) to perform The LIS communicates to the
test at a time analyzer which test(s) have been
ordered.
CHEMICAL REACTION

3. Reagent systems and One or more reagents can be

delivery dispensed into the reaction cuvet.

5
MLS 11A CLINICAL CHEMISTRY
4. Specimen measurement and
delivery
5. Chemical reaction phase
DATA COLLECTION AND ANALYSIS
6. Measurement phase
7. Signal processing and data
handling
8 Send result(s) to LIS
A small aliquot of the sample is
introduced into the reaction cuvet.
The sample and reagents are mixed
and incubated.
Optical readings may be initiated
before or after all reagents have
been added.
The analyte concentration is
estimated from a calibration curve
that is stored in the analyzer.
The analyzer communicates results
for the ordered WAS to the LIS.
MLS 3A
Note: Process:
 The Medtech loads the sample into the TLA (Total Laboratory
Automation System) then the transport system sorts the sample
into which test it would go to.
 They have an automated centrifuge which prepares the sample
for testing. The medtech won’t check if there is hemolysis or if
the sample needs re-spinning. The machine will take care of
that.
 The refrigerated storage manager takes care of samples that
needs refrigeration or low temperature when testing.
 Recapper/Uncapper removes and returns the cap of the sample.
 Specimen sorter- sorts the sample to whichever test they are
needed.
 Automated aliquoter – when serum needs to be separated, this
is the part of the system that does the separation.
-Sysmex – name of machine used for hema and/or clin chem.
-Even if the laboratory is fully automated, the medtechs are still
needed in the laboratory.

Note: -Sample identification is with the use of barcode reader. Some

laboratories gives the barcode to the patient once they have already
paid. Through that barcode, they will know the tests to be run ordered
by the doctor.
-If before, large amounts of samples are used in performing laboratory
tests, now only a small aliquot of sample is introduced into a reaction
cuvette.
-Quality control are done every day in order to calibrate the machines
and get accurate results. Having outlying results during the testing
won’t allow testing of that analyte until the machine has been
recalibrated or has yielded results within the quality control standard.

Schematic of Total Laboratory Automation System

“Ta? CC?”
CLINICAL CHEMISTRY now, COCKTAIL CRUISER later

Padayon, RMT