MLS 414 - CLINICAL CHEMISTRY 1 (LAB)
P2.1 ANALYTIC TECHNIQUES AND INSTRUMENTATION
Kristine Faye Dimalaluan, RMT | January 25, 2023
TOPIC OUTLINE
I.
ANALYTICAL TECHNIQUES AND
INSTRUMENTATION
LEARNING OBJECTIVES
At the end of the lecture, the students must be able
to:
Describe the principle of operation and components
parts of the following:
1. Spectrophotometry
2. Luminescence
a. Fluorometry
b. Nephelometry VS. Turbidimetry
c. Refractory
d. Osmometry
e. Densitometry
3. Chromatography
4. Electrochemistry
I.
➔
➔
➔
➔
➔
A.
➔
➔
ANALYTICAL TECHNIQUES AND
INSTRUMENTATION
One of the main principles in processing
analytes in the laboratory.
We will be able to describe the different
characteristics of our EMR.
◆ EMR: Electromagnetic Radiation
What is the Definition of Light Transmittance
and Absorbance? Relationship between the
two? Why is it important in measuring assays?
The theory of Beer-Lambert's Law.
The analytic techniques in the laboratory
fall into 4 basic areas. 4 principles that are
used in Clinical Chemistry in performing
analytic techniques and instrumentation
◆ Meaning, the principles being
followed by machines in the
laboratory to measure different asis
are divided into 4 basic areas:
◆ Spectrophotometry (Very Common)
◆ Luminescence
◆ Chromatography
◆ Electroanalytical Methods
Used by machines, glassware that's
where the magic happens.
This type of testing is made on glucose
determination.
◆ As you can see, the lighter the
solution is, the lower the concentration
in comparison doon sa pinakadark na
talaga is 1%.
◆
➔
➔
➔
B.
➔
SPECTROPHOTOMETRY
The machine we are using in the laboratory.
◆ What are the different parts?
Spectrophotometric Cuvet
C.
➔
In this slide you will see the bird’s eye view of
what we usually do in clinical chemistry.
The
colors,
light,
what
can
be
transmitted/absorbed,
what
colors
are
reflected, and so on.
◆ That is the basic principle in which our
machines are dependent whether they
may be automated or manual. Still
applies the very basis which includes
most of your EMR and the
Transmittance and Absorbance of
Light.
ELECTROMAGNETIC RADIATION (EMR)
A particle of an EMR is known as a photon.
◆ These photons actually travel in
waves. That energy travels in waves.
◆ That energy, if spread, the EMR is in
the form of electromagnetic waves.
◆ Very common form of EMR that we
receive comes from the sun. Basically
that gives us light.
◆ That energy from the sun will travel all
throughout here on Earth. And those
energy
is
in
the
form
of
Electromagnetic waves.
ELECTROMAGNETIC WAVES
Actually defined by two.
Trans Maker: Arteche, J.N., Batangon, L.B., Herrera, C.J.
Editor: Apil, J. | 1
MLS 414 - CLINICAL CHEMISTRY 1 (LAB)
P2.1 ANALYTIC TECHNIQUES AND INSTRUMENTATION
Kristine Faye Dimalaluan, RMT | January 25, 2023
◆
Wavelength (λ): Distance between
identical points (Crest to Crest; Trough
to Trough) on consecutive waves.
● Length of two peaks
Note: Always remember the relationship of the
wavelength and frequency. Your wavelength is
indirectly proportional to frequency.
Meaning, the higher the wavelength, the lower the
frequency and vice versa.
➔
➔
➔
●
◆
Another
definition
of
wavelength is defined by its
frequency. How frequent mag
appear ang wave.
Frequency (v): Number of waves that
pass through a point per unit time.
Usually, that point unit in time is fixed
into a value of 1 second.
● Fixed point at 1 second
● Example, in 1 second, how
many
wavelengths
will
appear, how frequent will the
wave pass through or how
frequent the wave will
appear.
D.
➔
➔
➔
It is considered as the high frequency because
the wave is more frequent and at the same
time you can see that the wavelength is
shorter.
Compared to low frequency, the wavelength is
longer.
With that, the higher the frequency, the
higher the value of energy. Your frequency
is directly proportional to the energy.
ELECTROMAGNETIC SPECTRUM
Electromagnetic spectrum or the light is a wave
of alternating electric and magnetic fields.
A kind of wave that composes alternating
electric and magnetic fields.
Almost all light in the universe is visible to the
human eye.
As you can see, there is only a visible spectrum
to us around 400 - 700 nm. If ganyan lang ang
spectrum niya, ganyan lang ang frequency
niya, then that spectrum is visible to the human
eye. If it's lower than 400 or higher than 700, it
cannot be seen.
➔ Visible spectrum: 340 - 400 nm; 400 - 700
nm
➔ One of the invisible lights that are very
common, that we are using right now, is the…
◆ Infrared: Wavelength is above 700
nm
◆ Ultraviolet: wavelength falls below
the visible region (190-340 nm)
◆ We cannot see them unless with the
use
of
certain
machines
or
whatsoever. But with the human eye
only, the visible spectrum is around
400 nm - 700 nm.
Note: In chemistry, this is very important because most
of the chemicals, or the molecules, na gina measure
natin, it absorbs light and at the same time it transmits.
➔ One of the basis why light is important through
EMR, Spectrum, in the analysis of the different
body analytes that are needed to measure.
➔
➔
Other parts of wavelength are also called
Amplitude: Distance of origin and crest and
trough. The distance either from the crest or
from the trough is called an amplitude.
➔
Speed: Speed of wavelength
◆ wavelength (λ) x frequency (v)
Trans Maker: Arteche, J.N., Batangon, L.B., Herrera, C.J.
Editor: Apil, J. | 2
MLS 414 - CLINICAL CHEMISTRY 1 (LAB)
P2.1 ANALYTIC TECHNIQUES AND INSTRUMENTATION
Kristine Faye Dimalaluan, RMT | January 25, 2023
F. VISIBLE LIGHT: ROYGBIV
However, in Clinical Chemistry, we have this thing called
the light absorbed and the light emitted/transmitted.
➔ Example, red apple. Doesn’t mean na red
talaga ang color niya, it only appears to be red
because yun ang color na nag reflect sa mata
natin.
As you can see, the 400 nm appears to be violet. The
700 nm appears to be red.
➔ Below your 190nm, that's the example of a
wavelength that you will see in x-ray and
gamma radiations.
◆ These photons have the energy that
can already penetrate the flesh.
◆ Those light is sobrang baba ng
frequency,
sobrang
baba
ng
measurement, at around 190 nm lang
ang wavelength. That's why it is used
in x ray.
E.
➔
➔
➔
➔
COLORS OF VISIBLE LIGHT
Our white light is actually a composition of
the different colors, red, pink, blue, green, all
of that combined turned out to be white light.
Meaning, that white light, pag ginamitan ng
monochromator for example, will break down
into different spectrums of light.
Because again, the light is made up of all your
spectrum of different colors.
Your different colors will correspond to different
measurements of wavelengths.
WAVELENGTH
(NM)
COLOR
ABSORBED
COLOR
EMITTED/TRANS
MITTED
350-400
VIOLET
YELLOW
400-450
INDIGO
YELLOW
450-500
BLUE
ORANGE
500-550
GREEN
RED
550-600
YELLOW
INDIGO
600-650
ORANGE
BLUE
650-700
RED
GREEN
➔
➔
➔
➔
➔
➔
Cool tones: From the violet to indigo, blue to
green, the wavelengths differ.
◆ From the violet, 400nm and indigo
naga increase siya from 400 to 425 to
470 nm to 550 nm.
Remember those wavelength measurements
because having knowledge on this, being able
to know by heart, that will really help you
furthermore a better understanding on how
machines work in clinical chemistry.
Warm tones: The red light which is 665 nm
compared to violet, you can tell that the
wavelength is definitely longer.
◆ Which visible light has more energy?
It is violet.
◆ In comparison, green and orange?
Green, because it has a higher
frequency and has a shorter
wavelength.
➔
➔
➔
The complementary color of indigo and violet is
yellow.
The complementary color of red is green.
Thus, a visible color of a solution will be the
complement of the wavelength being absorbed.
If the solution is yellow, the wavelength used is
either in indigo or violet (350- 450 nm)
Example: a hemolyzed (red; bursting of RBCs)
solution containing hemoglobin will obtain
green light at 540 nm.
A yellow bilirubin solution absorbs either indigo
or violet at 400-430 nm.
G. BEER-LAMBERT’S LAW
➔ Describes the relationship between the
absorption of light and the concentration of
the solution.
◆ How much light is absorbed and what
is
its
relationship
with
the
concentration of the sample.
◆ Gaano kadami ang glucose for
example.
➔ States that the concentration of a substance is
directly proportional to the amount of light
Trans Maker: Arteche, J.N., Batangon, L.B., Herrera, C.J.
Editor: Apil, J. | 3
MLS 414 - CLINICAL CHEMISTRY 1 (LAB)
P2.1 ANALYTIC TECHNIQUES AND INSTRUMENTATION
Kristine Faye Dimalaluan, RMT | January 25, 2023
absorbed and inversely proportional to the
logarithm of the light transmitted.
➔
The relative amount of light passing through
the sample - Transmittance
The ratio of radiant light transmitted divided by
the radiant energy incident of the sample Percent Transmittance
H.
ABSORBANCE
➔
A= ebc
➔
➔
➔
➔
➔
➔
➔
Lets say, this is your cuvette. You have your
solution here, of course you have your analytes
there, different molecules, of course di mo
makita, in clinical chemistry daw, since we are
primarily relying on light, that cuvettes ay
iniilawan.
That is called the Incident Light or ang light
source lang.
◆ Pag mailawan ang molecules inside
the solution, they have the ability to
absorbed the light.
◆ So, ang light saan mapunta? Sa mga
different molecules.
◆ If konti lang ang molecules, meaning,
konti lang din ang ma-absorb na light.
◆ So what will happen to the excess
light? It will be transmitted.
● The higher the concentration,
the lower the light that is
being
emitted/transmitted
because na-block na siya
na-absorb naman siya ng
molecules.
◆ What if madami na masyado ang
molecules?
● Lower transmission, because
the light has already been
blocked and absorbed by
your molecules.
That is why malaman mo pag konti nalang
gane ang light na mag pass through, then that
will give you an idea that the concentration is
higher
This is applicable to the example, the 1%
concentration is much darker than the 0.0625%
because it has a higher concentration.
➔
e = Molar Absorptivity
◆ Refers to the capability of your sample
to absorb the dye or light
b = optical path
◆ Refers to the distance that light must
pass through the sample
c = analyte concentration
◆ Amount of molecules capable of
absorbing light
A = Absorbance
◆ Relative amount of light absorbed by
the sample
◆ Only computed through transmittance
I.
PERCENT TRANSMITTANCE
➔
The ratio of transmitted light divided by incident
light times 100
◆ The light source is the incident light
(Io)
The light transmitted is defined as “ I ”
The % transmittance formula will give you an
idea of the absorbance
➔
➔
◆
J.
HOW TO DERIVE ABSORBANCE FROM
PERCENT TRANSMITTANCE
𝐼
𝐼𝑜
%𝑇 =
𝐴 = 𝑙𝑜𝑔10(
𝐴=
− 𝑙𝑜𝑔(
𝐼
𝐼𝑜
𝐼𝑜
𝐼
𝑥 100
) = 𝑙𝑜𝑔10 (
100%
1%
)
) = 𝑙𝑜𝑔(100%) − 𝑙𝑜𝑔%𝑇
𝐴 = 2 − 𝑙𝑜𝑔%𝑇
K.
HOW TO SOLVE FOR UNKNOWN
CONCENTRATION
𝑎=
𝐴
𝑏𝑐
●
●
●
⇒
𝐴1
𝐶1
=
𝐴2
𝐶2
A1 and C1 = Standard (STD)
A2 and C2 = Unknown (u)
Eliminate b (light path)
because that is already fixed.
So di mo na kailangan
icompute
Trans Maker: Arteche, J.N., Batangon, L.B., Herrera, C.J.
Editor: Apil, J. | 4
MLS 414 - CLINICAL CHEMISTRY 1 (LAB)
P2.1 ANALYTIC TECHNIQUES AND INSTRUMENTATION
Kristine Faye Dimalaluan, RMT | January 25, 2023
𝐴𝑆𝑇𝐷
𝐶𝑆𝑇𝐷
➔
➔
➔
𝐴𝑈
𝐶𝑈
N.
➔
Therefore…
𝐶𝑢 =
L.
➔
=
𝐴𝑈
𝐴𝑆𝑇𝐷
𝑥 𝐶𝑆𝑇𝐷
SPECTROPHOTOMETRY
The measurement of the light transmitted by a
solution is determined by the concentration of
the light-absorbing substance in the solution
Again, spectrophotometry is dependent on
beer-lambert's law because the higher the
concentration is in the solution, the lower the
amount of light transmitted.
● The light being transmitted is the one
being measure by spectrophotometers
External components
➔
➔
EXIT SLIT
Eliminates light spectrum that are not needed
● If we need violet, the exit slit will only
project violet
Monochromators are movable
● If we have ROYGBIV light, the
monochromator can be adjusted or
modified to the desired color in the
spectrum. For instance, our desired
color is red, we adjust it to red, then,
the monochromator will adjust and
project the red color
Difference between Entrance Slit and Exit Slit
● Entrance slit will not allow any stray
light to enter into the monochromator
● Exit slit eliminates unnecessary
spectrum or wavelength
○ We want red, it will project
red only
O. SAMPLE CUVETTE
➔ After we obtained our desired wavelength of
light, then it will go into the sample cuvette
P.
➔
Internal components
M. LUMINESCENCE
I.
➔
Purpose:
Ex. White Light
● The
monochromator
is
now
responsible to breaking out the
separation of the different lights
depending on the wavelength
● The prism or diamond is lighted by the
white light, it will release different
colors, it is the same with the
monochromator
● The lights are being separated
depending on the measurement of
their wavelength, this is the purpose of
the monochromator.
● After the light is broken down, it will
now go now to the exit slit
PHOTOMULTIPLIER or PHOTODETECTOR or
PHOTOREADERS or PM tube
➔ Photomultiplier or the photodetector or
photoreaders will now measure the transmitted
light
➔ The measured result of the photomultipliers is
in the computation of the absorbance, and it
will now give the concentration
➔ The purpose of the detector is to convert the
transmitted radiant energy into an equivalent
amount of electrical energy.
➔ The least expensive of the devices is known as
a barrier-layer cell, or photocell.
● The photocell is composed of a film of
light-sensitive material, frequently
selenium, on a plate of iron.
Q. LIGHT SOURCE
➔ Also called as exciter lamp
◆ It excites energy or release energy
➔ Light source
◆ A form of our energy source
◆ It is a source of our electromagnetic
spectrum
◆ The
electromagnetic
graduation
provides a visible, infrared or UV light
● Visible
colors
are
ROYGBIV that are visible to
the human eye
● Invisible - Infrared light and
UV light
➔ The Light source used in spectrophotometry
may be visible or not visible
Trans Maker: Arteche, J.N., Batangon, L.B., Herrera, C.J.
Editor: Apil, J. | 5
MLS 414 - CLINICAL CHEMISTRY 1 (LAB)
P2.1 ANALYTIC TECHNIQUES AND INSTRUMENTATION
Kristine Faye Dimalaluan, RMT | January 25, 2023
TYPES OF LIGHT SOURCE
Incandescent
● Most common light at visible,
tungsten/Tungst
near UV and near IR
en-iodide lamp
● Commonly used if the
or
Halogen
machine needs visible light,
Quartz Lamp
we can assume that the light
used is a tungsten or
tungsten-iodide
lamp
or
halogen quartz lamp or
halogen lamp
● The light that is being
transmitted by the halogen
tungsten lamp is visible
Mercury/Hydrog
● Fluorometry
en Lamp/ Xenon
● Often
used
when
the
arc
principle
used
by
the
machine is Fluorometry
Hollow cathode
● The principle used by the
lamp
machine
is
Atomic
Absorption
Spectrophotometry (AAS)
Deuterium
● UV range: continuous light
emission down to 165 nm
INFRARED LIGHT
Nernst Glower
●
Globar
●
I.
Uses an electrically heated
rod of rare earth element
oxides
Uses silicon carbide heated
to 1200 degrees celsius
Conclusion:
➔ The different kinds of light source
corresponds to different types of
principles of how the analytes are
being measured
○ It is important to know these
so we will have the idea how
to measure the analytes of
the machine
II.
Problem in Light source:
➔ The whole machine cannot function
without the source of the light
○ Without the incident light,
there is no possibility for you
to measure your transmitted
light
R.
➔
➔
ENTRANCE SLIT
Reduces stray light
● Stray light is light that we don’t need
for testing
Prevents scattered light from entering the
monochromator
● If the stray light is allowed to pass
through the analytical cell, it would
cause a deviation from the readings
● If
extra
light
enters
the
monochromator, then definitely the
breakdown of light is altered, we
cannot properly distinguish the colors.
Purpose:
● All the necessary light from the light
source will go inside
● Any types of stray light that are not
needed for the testing would be
eliminated
● It is where the necessary light will
pass that we need for the analysis
Without entrance slit:
● Close the laboratory, since your
machine is not functional for
measurement
I.
II.
S.
➔
➔
MONOCHROMATOR
Device that produces light of specific
wavelengths of a light source
● Isolation of individual wavelengths of
light is an important and necessary
function of a monochromator.
Monochromatic light
● This is a light radiation of a single
wavelength
● If monochromatic light is red, then that
is the type of light that comes from the
monochromator, a breakdown from
the white light or the initial light source
T.
Types of Monochromator:
1.
2.
3.
1.
Prism
➔
➔
Figure 1.1 Prism
➔
Prism
Diffraction Gratings
Interference Filters
Wedge-shaped piece of glass, quarts,
or sodium chloride or some other
material that allows transmission of
light
Based on refraction of light
The Prism type of monochromator
depends on the refraction of light
● From the light source, light
will pass through the prism,
by the refraction or bending
of light, it is able to
differentiate the different
lights in the light source
Trans Maker: Arteche, J.N., Batangon, L.B., Herrera, C.J.
Editor: Apil, J. | 6
MLS 414 - CLINICAL CHEMISTRY 1 (LAB)
P2.1 ANALYTIC TECHNIQUES AND INSTRUMENTATION
Kristine Faye Dimalaluan, RMT | January 25, 2023
●
●
●
●
When you look inside the
spectrophotometer this is
how it looks like:
Insert pic
From the incident light or
light source, it goes to the
prism
The prism is able to
breakdown the white light
into different specs
➔
2.
Diffraction grating
➔ Made up of luminized surfaces that
has been cut into tiny grooves that
can act as a prism and a slit
Figure 1.1 Diffraction grating
●
●
●
●
Diffraction
○ This
type
of
monochromator
depends on the
bending of light
○ The principle that is
being followed by
your
diffraction
gratings
Light is able to bend at a
certain length depending on
the size of the wavelength
If the light will be able to
reach the gratings of the
diffraction gratings
○ If light hits the
surface, it will bend
○ Instead of going
forward, the light will
bend
The direction of the bending
of light
○ Will differ since they
don’t have uniform
measurement
○ This is how the
diffraction gratings
are
able
to
differentiate
the
different lights since
the direction of the
➔
➔
bending of light
differs
Based on the principles that
wavelength bend as they pass a sharp
corner
● If white light enters the
diffraction
gratings,
the
gratings will be able to bend
the late through the principle
of diffraction
○ From white light, it
is now able to
produce other color
of light
○ Bending of light will
differ once it hits the
grating, since they
differ
in
measurement
○ It will bend on a
certain
angle,
depending on the
wavelength
it
possesses
The degree of bending depends on
the wavelength
Diffracted Light
● Light
bended
in
the
Diffraction Grating
● Differentiated light of the
diffraction grating from the
incident light source
3.
Interference Filter
➔ Based on the principle of constructive
interference of waves
I.
How does this filter work?
● Only light with desired
wavelength which is reflected
twice will be in phase and
come out of the filter
Figure 1.1 Interference Filter
II.
Made of:
● It is constructed by using two
parallel glass plates, which
are silvered internally and
separated by thin film of
dielectric material (CaF₂, SiO,
Trans Maker: Arteche, J.N., Batangon, L.B., Herrera, C.J.
Editor: Apil, J. | 7
MLS 414 - CLINICAL CHEMISTRY 1 (LAB)
P2.1 ANALYTIC TECHNIQUES AND INSTRUMENTATION
Kristine Faye Dimalaluan, RMT | January 25, 2023
MgF₂) of different refractive
index
➔ If light passes through the glass plate,
if it is the desired wavelength it will be
multiplied twice (bounce) until it exits
the interference filter
➔ The light that exits the filter, will pass
through the sample
➔ What happens if the light that passes
through the interference filter is not of
desired wavelength?
● It
bounces
but
gets
destructed, it is not able to
reflect until the end
● It will not go out of the
monochromator
➔ The interference filter can only reflect
the desired wavelength, it will be
multiplied twice, such as in a manner
that it will be able to bounce all
throughout the filter and go out the
monochromator
➔ The specific wavelength range is
measured
● Ex. Incident radiation enters
the glass plate
○ If the IR is of the
desired wavelength,
at 665 nm, it will be
able to reflect or
bounce from one
plate to another, it
will also exit 665
nm.
○ If it is set on 665
nm, and a light with
400 nm passes
through the glass
plate, it will not
reach the end since
it is not the desired
wavelength
I.
Constructive and Destructive Interference
Grating
Figure 1.1 Constructive and Destructive Interference
Grating
➔
➔
Constructive Interference grating
● If you have already selected
your desired wavelength, it
will be multiplied into 2
wavelengths since those 2
wavelengths will bounce from
one plate to another
● It is set at 400 nm
○ If we have 400 nm it
will be multiplied
into 2
○ This
type
of
wavelength is now
able to go out of the
interference filter
Destructive Interference grating
● You don’t have the desired
wavelength, it is not able to
multiply and it meets halfway,
therefore,
there
is
no
bouncing of light.
Recap:
➔ Prism
● Adjust to desired wavelength
➔ Diffraction Grating
● Bending of light through a grating
➔ Interference Filter
● Filter light at desired wavelength
➔ Entrance slit
● Filters stray light
● Exclusion of the unwanted light from
entering the monochromator
➔ Exit slit
● Will only allow the exit of a specific
color
● After the monochromator is adjusted,
if you already identified your desired
wavelength, it will release the
assigned light
● Ex. Orange type of light
○ Meaning the exit slit will
release orange color
○ The light source that we
need to measure is orange
light, since it has already
been
filtered
and
differentiated by the entrance
slit, monochromator, and exit
slit.
○ The
light
emitted
is
measured
by
the
photodetectors
or
the
photomultipliers
● Allows only a selected spectrum to
pass through the cuvette
Trans Maker: Arteche, J.N., Batangon, L.B., Herrera, C.J.
Editor: Apil, J. | 8
MLS 414 - CLINICAL CHEMISTRY 1 (LAB)
P2.1 ANALYTIC TECHNIQUES AND INSTRUMENTATION
Kristine Faye Dimalaluan, RMT | January 25, 2023
U.
➔
I.
1.
2.
3.
4.
Components of the Cuvette
Cuvette
● Referred to as the analytical set or
sample holder
● This is where the sample and
reagents are pipetted
● This is used to hold the solution in the
instrument whose concentration is to
be measured
● The material used in the cuvette does
not absorb light at all
○ Ex. High silica glass
■ This type of glass is
guaranteed that the
type will not absorb
any type of light
■ This is to make sure
that all light will be
allotted
for
the
sample and not on
the cuvettes
● What if we measure blanking or
standard do we need to use cuvettes?
○ Yes,
because
standard
solutions are prepared and at
the same time, during
blanking, cuvettes are used
to run the blanking or
standard
○ Cuvettes don’t absorb light
○ You have to know that when
we use cuvette the machine
is able to measure 0
● If we run blanking and there is a result
in the measurement and absorbance:
○ It means that the cuvette is
not suitable for testing, since
it absorb light even though
there is no sample
Types of Cuvette
Borosilicate glass
➔ For alkaline solutions
Quartz or plastic
➔ For wavelength below 320 nm
➔ Disclaimer:
● Manufacturers are now able
to reproduce plastic cuvettes
that is able to measure
beyond 500 nm
● Generally, quartz or plastic
types of cuvettes measured
around 220 nm
Alumina Silica Glass
➔ Good at visible region
● 420 nm -700 nm
Soft glass
➔ Preferable for acidic solutions
Trans Maker: Arteche, J.N., Batangon, L.B., Herrera, C.J.
Editor: Apil, J. | 9