Laboratory Math, Instrumentation AND QA - LECTURE
SOLUTIONS and the liquid is the solvent. The solution is said to
It is a homogeneous mixture of two or more
substances. A Homogeneous mixture means that the
composition id just the same throughout. The
homogeneous solution is also known as the true
solution. The size of the dissolved particles in true
solutions is the same as that of a molecule.
COMPONENTS OF SOLUTION
SOLVENT: it is a component of the solution that is
abundant and in which other substances are
dissolved. The solvent is also the medium of the
solution. For example, in a sugar solution in water,
the component water is referred to as the solvent
SOLUTE: it is the substance that is dissolved in the
solvent to make the solution. Sugar, for example, is
referred to as a solute in a solution of sugar in water.
PROPERTIES OF SOLUTION
The important characteristic properties of a solution
are as follows:
1. A solution is a homogeneous mixture.
2. Solute particles in a solution are extremely
small. It has a diameter of less than
3. Even with a microscope, it is impossible to
see the particles of a solution.
4. The particles of a solution pass through the
filter paper. As a result, filtration cannot
separate a solution.
5. The solutions are very stable. When a
solution is kept, the solute particles do not
separate from each other.
6. A true solution does not scatter light. This is
because its particles are very, very small.
TYPES OF SOLUTIONS
SOLID-LIQUID SOLUTION
Most solids dissolve in one liquid or the other to form
homogeneous mixtures called solid-liquid solutions.
In such cases, the solid is referred to as the solute,
REVIEWER IN LAB MATH- LECTURE
be dilute when the amount of solute is much less
than the amount of solvent. If the amount of solute
is relatively greater, the solution is concentrated.
LIQUID-LIQUID SOLUTIONS
contain both solute and solvent in a liquid state.
When two liquids are mixed together, the system
can be homogeneous or not. We can have the
following liquid-liquid systems depending on their
mutual miscibility.
TYPES OF LIQUID-LIQUID SOLUTIONS
COMPLETELY MISCIBLE: Two liquids are said to be
completely miscible when they mix together in all
proportions to form homogeneous mixtures. A true
solution is obtained by mixing two completely
miscible liquids. The completely miscible liquids are
of the same type, i.e. they are either polar (for
example, alcohol and water) or non-polar (e.g.,
benzene and toluene).
PARTIALLY MISCIBLE: When some liquid pairs are
mixed together, they partially dissolve in each other,
forming two distinct layers. The first layer is the
solution of the first component into the second,
while the second layer is the solution of the second
component into the first. These liquid pairs are
known as partially miscible liquid pairs. Phenol-water
and triethylamine-water are two examples of
partially miscible liquid pairs.
IMMISCIBLE: Immiscible or nearly immiscible liquids
are those in which two liquids do not dissolve into
each other and form separate layers. Such liquid
pairs include benzene-water, carbon tetrachloride-
water, and others. When OF liquid is polar, and the
other is non-polar, the two are immiscible.
GAS-LIQUID SOLUTIONS
Gas-Liquid Solutions are solutions that have a
gaseous solute and a liquid solvent. For example, a
mixture of carbon dioxide in water, a solution of
oxygen in the water. The solubility of a gas in a liquid
MERRA CYRINE VENZUELA
 Laboratory Math, Instrumentation AND QA - LECTURE
is determined by several factors. The important ones
are: Nature of Gas, Nature of Liquid, Temperature
and Pressure
TYPES OF SOLUTION BASED ON NATURE
OF SOLVENT
AQUEOUS SOLUTION
An aqueous solution is one that is formed by
dissolving a solid in water. Aqueous solutions
include common salt solutions in water, sugar
solutions in water, and detergent solutions in
water.
NON-AQUEOUS SOLUTION
A non-aqueous solution is one that is formed by
dissolving a solid in a liquid other than water, such
as alcohol, benzene, acetone, carbon disulfide, and
so on.
CONCENTRATED SOLUTION
A concentrated solution contains a high
concentration of solute in the given solvent. Brine
solution, orange juice, and dark-colored tea are a
few examples.
DILUTED SOLUTION.
A dilute solution is made up of a small amount of
solute and a large amount of solvent. Examples
include Salt solution and light color tea.
REVIEWER IN LAB MATH- LECTURE
TYPES OF SOLUTION BASED ON
CONCENTRATION OF SOLUTE IN TWO
SOLUTIONS
ISOTONIC SOLUTION
The solute concentration in these solutions is the
same. As a result, water moves in both directions
across the cell from the solution in the beaker.
HYPERTONIC SOLUTION
Because the solution in the beaker has a larger
solute concentration, water leaks out of the cell
and into the solution in the beaker, causing the
cell to plasmolysis/shrink.
HYPOTONIC SOLUTION
Because the solution in the beaker contains a
lower concentration of solute, water enters the
cell, causing it to swell and eventually burst.
TYPES OF SOLUTION BASED ON THE
AMOUNT OF SOLUTE ADDED
UNSATURATED SOLUTION
A solution in which more quantity of solute can be
dissolved without raising its temperature, is called
an unsaturated solution. An unsaturated solution
contains less than the maximum amount of solute
that can be dissolved at that temperature. For
example, if more salt can be dissolved in an
aqueous solution of salt without raising the
temperature, the salt solution is unsaturated.
SATURATED SOLUTION
A solution in which no more solute can be
dissolved at that temperature is called a saturated
solution. For example, if no more salt can be
dissolved in an aqueous salt solution at that
temperature, the salt solution is said to be
saturated. As a result, a saturated solution
contains the most solute that can be dissolved in
it at that temperature. It is obvious that a
saturated solution contains more solute than an
unsaturated solution.
MERRA CYRINE VENZUELA
 Laboratory Math, Instrumentation AND QA - LECTURE

SUPERSATURATED SOLUTION Proton donor - intact (non-ionized) weak acid

A supersaturated solution is one that contains
more solute than the maximum amount that can
Be dissolved at a given temperature.
TYPES OF BUFFER SOLUTION
There are major two types of buffer solutions which
have been described as follows:
- ACIDIC BUFFER
- ALKALINE BUFFER
TYPES OF COLLOIDAL SOLUTION
The colloidal solutions have been majorly defined in
the following:
- Emulsion
- Foam
- Aerosol
HENDERSON-HASSELBACH EQUATION
Henderson-Hasselbalch Equation is used to calculate
the pH of a solution. Knowing the pH of a solution is
very important for many chemical reactions as well
as for biological systems. The Henderson-
Hasselbalch equation gives the approximate pH
value of a buffer solution.
One way to determine the pH of a buffer is by using
the Henderson-Hasselbalch equation, which is
Ph=pKa + log((A)/(HA). In this equation, IHA and IA I
refer to the equilibrium concentrations of the
conjugate acid-base pair used to create the buffer
solution.
(example - CH3COOH).
pKa value
pKa is a number that describes the acidity of a
particular molecule. It measures the strength of acid
by how tightly a proton is held by a Bronsted acid.
The lower the value of pKa, the stronger the acid and
the greater its ability to donate its protons.
PROTON DONOR
Acids are defined as proton donors. The most
common acids are aqueous solutions of HCI
(hydrochloric acid), H2S04 (sulfuric acid), HN03
(nitric acid), and H3P04 (phosphoric acid). Bases, on
the other hand, are proton acceptors.
PROTON ACCEPTOR
A proton acceptor is another name for a base, which
is the opposite of an acid. In the Broensted-Lowry
definition, a base is a negatively charged ion that will
react with, or accept a positively charged hydrogen
ion. Since a hydrogen ion is a proton, the base is
called a proton acceptor.
SAMPLE PROBLEMS:

Henderson-Hasselbalch Equation is given as:

pH = pKa + log (proton acceptor)/ (proton donor)

pH - the negative logarithm of H+ ion concentration

in the medium.

pKa - the negative logarithm of Ka of the acid (Ka is

the dissociation constant)

Proton acceptor — the ionized or deprotonated acid

(example — CH3COO)
REVIEWER IN LAB MATH- LECTURE MERRA CYRINE VENZUELA
 Laboratory Math, Instrumentation AND QA - LECTURE

pH = 9.248 + log [0.9 / 0.7]

pH = 9.248 + log 1.29
pH = 9.248 + 0.11
pH = 9.358

Using the Henderson-Hasselbalch equation,

calculate the pH of a buffer solution containing 0.50
M CH3COOH and 0.50 M NaC₂ H₃ O₂.
The Ka for HC₂ H₃ O₂ is 1.8 x 10–5.
First, let’s use the given Ka value to find the pKa…
pKa = - log Ka
pKa = - log (1.8 x 10–5)
pKa = 4.74
pH = pKa + log ([C₂ H₃ O₂ -] / [HC₂ H₃ O₂])
pH = 4.74 + log (0.50 M / 0.50 M)
pH = 4.74 + log (1) pH = 4.74

SPECTROPHOTOMETRY

INTRODUCTION TO
SPECTROPHOTOMETRY
SPECTROSCOPY
It is the study of the interaction of light & matter
SPECTROPHOTOMETER
instrument that uses electromagnetic radiation
from UV, visible or IR to analyze the absorption or
transmission of a sample
 We will use visible in our lab

Properties of Light
 Electromagnetic radiation moves in waves

Calculate the pH of a buffer that contains 0.7 M

ammonia and 0.9 M ammonium chloride. (pKa =
9.248).

Answer. By using the Henderson Hasselbalch

Equation is:

pH=pKa+log[A−][HA]
REVIEWER IN LAB MATH- LECTURE MERRA CYRINE VENZUELA
 Laboratory Math, Instrumentation AND QA - LECTURE

ELECTROMAGNETIC SPECTRUM WHAT IS COLORIMETRY?

 The solutions of many compounds have
characteristic colors.
 The intensity of such a color is proportional
to the concentration of the compound.

WHAT ARE SPECTROSCOPY AND

SPECTROPHOTOMETRY?
 Light can either be transmitted or absorbed
by dissolved substances
 Presence & concentration of dissolved
substances is analyzed by passing light
through the sample
 Spectroscopes measure electromagnetic
emission
 Spectrophotometers measure
electromagnetic absorption

Instruments of Measurement
Two most common:
1. Visible Spectrophotometer
 Spect 20, Spect 88
 Uses Xe or W lamps as light sources
 Glass cuvettes to hold the sample
2. Atomic-Absorption Spectrophotometer

What do visible spectrophotometers measure?

 Amount of light absorbed by the dissolved
substance
COLORS AND WAVELENGTH  Qualitative – color gives info about the
solution composition
VIOLET-NEAR INFRARED- VISIBLE LIGHT  Quantitative – provides numerical data for
COLOR WAVELENGTH the concentration
ULTRAVIOLET <380
VIOLET 380-435 Absorption of Light
BLUE 436 – 480
GREENISH-BLUE 481 – 490 White light
BLUISH-GREEN 491 – 500  All colors –Polychromatic light
GREEN 501 – 560
YELLOWISH-GREEN 561 – 580
YELLOW 581 – 595
ORANGE 596 – 650
RED 651 – 780
NEAR INFRARED > 780

REVIEWER IN LAB MATH- LECTURE MERRA CYRINE VENZUELA

 Laboratory Math, Instrumentation AND QA - LECTURE

Monochromatic light
 Light of one color
 Monochromator-spreads out light into its
component wavelength
 Red light is absorbed by the green solution

The Spectrophotometer

FD&C Blue 1
 A solution containing this dye is blue in white
light
 The colors absorbed by solution are
complementary to the transmitted color n
Blue solution absorbs yellow, orange, & red
light
 So expect dye solution to peak at 580 – 650
nm n Optimum wavelength is determined
Success of spectrophotometry from wavelength of max. absorption λmax =
Requires sample to absorb light differently to the 630 nm for Blue 1
other chemicals in the solution  This is given for the blue solution but you will
How is the correct wavelength selected? have to calculate this for the red
 The amount of light absorbed depends on
the energy difference between 2 electron
energy levels
 Optimum wavelength for
spectrophotometric analysis is selected by
measuring the visible spectrum of the
substance
 This is done by plotting absorbance (A)
versus wavelength (λ)

Food Dyes
 Only 7 dyes are approved by the FDA for
use in foods, drugs & cosmetics
 All artificial food colors are mixtures of
these 7 dyes
 We will be using FD&C Blue in this lab

REVIEWER IN LAB MATH- LECTURE MERRA CYRINE VENZUELA

 Laboratory Math, Instrumentation AND QA - LECTURE

Wavelength of light absorbed: BEER LAMBERT’S LAW

 Is related to electronic structure of  The intensity of a ray of
substance monochromatic light
 Intensity of light absorbed depends on the decreases exponentially
concentration of solution as the concentration of
 More concentrated, the more intense color the absorbing medium
& the greater intensity of light absorbed increases
 When light is absorbed, the radiant power  More dissolved substance
(P) of light beam decreases = more absorption and
less transmittance
Transmittance (T)  ε = molar absorptivity
 This is the fraction of incident light (P/Po) coefficient and is constant
that passes through the sample for a substance
 T = P/Po  %T = Tx100 = P/P0x100%
 Po = intensity of “blank”  A = - log T
 Blank – is solution identical to sample but  A=εbc
without solute

Definitions & Symbols

 Intensity (I)
Spectral Transmission Curve
 Transmittance (T) –It’s also referred to as  Optimum wavelength
%T or T x 100
o T = P/Po
o Where Po is the intensity of the
blank
o Can also use I = Intensity instead of
Power
o T = I / Io
Graphical Relationship
 % transmission and % absorption are not
linearly related to concentration
 For a graph to be useful, a straight line is
needed
 ABSORBANCE = log(1/T) = -log(T)

The amount of light absorbed depends upon:

 Concentration (c)
 Path length of sample cell (b) thru which
light passes
 Defined by Beer’s Law

REVIEWER IN LAB MATH- LECTURE MERRA CYRINE VENZUELA

 Laboratory Math, Instrumentation AND QA - LECTURE
Standardization Graph
Standards (solutions of known concentration) of
the compound of interest are made, treated, and
their absorbance (ABS) and concentration values
are used to create a Standardization Graph.
FLUOROMETRY, TURBIDIMETRY AND
NEPHELOMETRY
FLOUROMETRY
Luminescence is of 2 types:
1. FLUORESCENCE
 When a beam of light is incident on certain
materials, they emit visible light or
radiations.
 This phenomenon is known as fluorescence
and the substance showing this
phenomenon is known as fluorescent
substances.
 The phenomenon of fluorescence is
instantaneous and starts immediately after
the absorption of light and stops as soon as
the incident light is cut off.
2. PHOSPHORESCENCE
 When light radiation is incident on certain
materials, they continue to emit light even
after the incident light is cut off.
 This type of delayed fluorescence is called
phosphorescence and the substances are
called phosphorescent substances.
REVIEWER IN LAB MATH- LECTURE
 A material exhibiting fluorescence generally
re-emits excess radiation within 10-6 to 10-
4 seconds of absorption.
 On the other hand, materials exhibiting
phosphorescence re-emit excess radiation
within 10-4 to 20 seconds or longer.
Principle of Fluorimetry:
 Is used widely for automated
immunoassays
 It is approximately 1000 times more
sensitive than comparable absorbance
spectrophotometry, but background
interference due to the fluorescence of
native serum is a major problem. This
inference is minimized by:
 Careful design of the filters used for
spectral isolation
 Selection of a fluorophore with an
emission spectrum distinct from
those of interfering compounds, or
 Used of time-or phase-resolved
fluorometry
Applications of Fluorimetry:
 Determination of uranium in salts used
extensively in the field of nuclear research.
 Estimation of traces of boron in steel by
means of the complex formed with
benzene.
 Estimation of calcium by fluorimetry with a
calcium solution.
 Determination of Vitamin B (B1 thiamine
and B2 riboflavin) in the food samples like
meat, cereals, etc.
 Fluorimetry is employed to carry out both
qualitative and quantitative analyses for
various aromatic compounds present in
cigarette smoke, air-pollutant,
concentrates, and automobiles exhaust.
MERRA CYRINE VENZUELA
 Laboratory Math, Instrumentation AND QA - LECTURE
NEPHELOMETRY AND TURBIDIMETRY
INTRODUCTION
 When electromagnetic radiation (light)
strikes on a particle in solution, some of
the light will be absorbed by the particle,
some will be transmitted through the
solution and some of the light will be
scattered or reflected. The amount of
light scattered is proportional to the
concentration of insoluble particle.
Light Scattering Phenomenon:
 The blue color of the sky and the red
color of the sun at sunset result from
scattering of light of small dust particles,
H2O molecules and other gases in the
atmosphere.
 The efficiency with which light is scattered
depends on its wavelength, λ.
 The sky is blue because violet and blue
light are scattered to a greater extent than
other longer wavelengths.
THEORY
 Turbidimetry deals with measurement of
Intensity of transmitted light.
 Nephelometry deals with measurement
of Intensity of scattered light.
 Turbidometric measurements are made
at 180o from the incident light beam.
 In Nephelometry, the intensity of the
scattered light is measured, usually at
right angles to the incident light beam.
FACTORS AFFECTING ON THE SCATTERING
OF LIGHT
 Concentration of particles
 Particle size
 Wavelength
 Distance of observation
 MW of particles
REVIEWER IN LAB MATH- LECTURE
CONCENTRATION OF
PARTICLES:
TURBIDIMETRY
Concentration of
particles: At low
concentration of
particles for scattering of
light Beers Lamberts law
is applicable
S=Log10 Io/It
S=KtC=-logT
Turbidance is directly proportional to
concentration
i.e. S α C
Where;
S = Turbidance
IO= Intensity of incident light
It=Intensity of transmitted radiation
T=Turbidance
C=Concentration of solution
K=constant depend on linearity of light
Concentration of particles: Nephelometry
In Nephelometry an equation that describe the
relation between the intensity of scattered
radiation, intensity of incident radiation, and
concentration of particles
Is= Ks x IO x C
Where;
IO= Intensity of incident light
Is=Intensity of scattered radiation
Ks= It is constant which depend on suspended
particle and suspension medium.
C=Concentration of solution
Particle Size
 The fraction of light scattered at any
angle depends upon size and shape of
particles.
 The amount of scattering (S) α
proportional to square of effective radius
of the particle.
 To control the particle size and shape,
sample solutions and standards must be
prepared under identical conditions.
MERRA CYRINE VENZUELA
 Laboratory Math, Instrumentation AND QA - LECTURE

Following care must be taken: used in Turbidimeter and Semi octagonal

 Concentrations of two ions forming ppt. sample cell are used in Nephelometer.
 Ratio of concentration of the solutions.
 Order of mixing of ppt. TUBIDIMETER
 Temperature at which suspension is
prepared.

Wavelength
 The intensity of scattered radiation
depends upon wavelength of the
incident light.
 Shorter wavelength are scattered to
greater extent than the longer one.
 Turbidity coefficient depends on
wavelengths of incident light
 K=Sxλ-1 (where s= constant for system)
 Wavelength of light is chosen in such a
way that analyte solution does not
absorbs strongly.
 Turbimetric & Nephelometric
measurements are carried using white
light.
Instrumentation
The instrument called as Turbidimeter and
Nephelometer.
The Basic components of Instruments are
Radiation->source-> Sample cell-> Detector->
Read out device

CHOICE OF THE METHOD

Radiation source: Ordinary tungston filament
lamp or mercury arc lamp can be used as
source of radiation.
Sample cell: The cells made from glass or
plastic are used for study. Rectangular cell are
Choice Of The Method depends upon the
amount of light scattered by suspended
particles present in solution.
 TURBIDIMETRY - high concentrated
suspensions.
 NEPHELOMETRY - low concentrated
suspensions - more accurate results
APPLICATIONS:
 Quantitative analysis of inorganic
species
 Air and water pollution
 Turbidimetric titrations
 Phase titrations
 Determination of molecular weight

VIDEO AS FOLLOWS: IN YOUTUBE

https://youtu.be/5lYveFIegzY
https://youtu.be/Y2PBe_va7I4
https://youtu.be/6XoaGLcjz4E

REVIEWER IN LAB MATH- LECTURE MERRA CYRINE VENZUELA

 Laboratory Math, Instrumentation AND QA - LECTURE
METHODS OF INSTRUMENTATION
ULTRACENTRIFUGATION
 Commonly used to purify, as well as
characterize, low-molecular-weight
polymers up to multi-megaDalton
protein complexes and organelles
 The basis of ultracentrifugation is the
same as normal centrifugation: to
separate the components of a solution
based on their size and density, and the
density (viscosity) of the medium
(solvent)
CHEMILUMINISCENCE
 describes the emission of light that
occurs as a result of certain chemical
reactions that produce high amounts of
energy lost in the form of photons when
electronically excited product molecules
relax to their stable ground state.
 CHEMILUMINESCENT
IMMUNOASSAY is a technique used for
disease diagnosis, drug development,
chemical reaction monitoring, and many
other applications. The protein or
antigen to be identified in these assays
is referred to as an analyte
ELECTROCHEMISTRY
 the study of electron movement in an
oxidation or reduction reaction at a
polarized electrode surface. Each
analyte is oxidized or reduced at a
specific potential and the current
measured is proportional to
concentration. This technique is a
powerful methodology towards
bioanalysis.
 Electrochemical techniques, including
potentiometry, amperometry,
coulometry, and voltammetry, are used
in chemical research to measure redox
potentials, characterize reversibility of a
chemical process, assemble or
synthesize materials, and analyze the
efficiency and function of catalysts,
among many other uses.
IMMUNOCHEMISTRY
 Used to help diagnose disease, such as
cancer. It may be also be used to help
REVIEWER IN LAB MATH- LECTURE
tell the difference between different
types of cancer.
DRY CHEMISTRY
 refers to the use of strips impregnated
with dry reagents to which the specimen
is added. This assessment focusses on
quantitative analysis of the chemical
reactions by desktop analysers.
 there is no conventional liquid utilized. It
is a reflected light photometry whereas
in conventional chemistry, it is
transmitted light photometry.
AUTOMATED MACHINE IN LABORATORY
ADVANTAGES
 reducing the use of manpower
 faster results
 efficiency
 eliminating human error
 lower operating costs
 safer working conditions
 increased productivity
 saves time
 gives more precise and more accurate
results
DISADVANTAGES
 very expensive
 worker displacement
 Space requirement and infrastructure
constraints
 Skills of worker are not growing
 Increased costs for supplies
 Higher risk of downtime
 Higher risk of system failures
 Dependent on the automated machines
MERRA CYRINE VENZUELA
 Laboratory Math, Instrumentation AND QA - LECTURE

Nephlometer Turbidimeter
Definition the Light passing
measurement of through a medium
the intensity of with dispersed
scattered light particles, so the
at right angles intensity of light
to the direction transmitted is
of the incident measured.
light as a
function of the
concentration of
the dispersed
phase. lt is most
sensitive for
very dilute
suspensions
(100 mg/L).
Instrument Nephlometery spectrophotometer
used machine
Type of light Scattered light Transmitted light
measured
Arrangement measure the made in the same
of light scattered direction as the
photometer at right angle to propagation of the
the direction of light from the
the propagation source.
of light from
the source. It
could be
movable
detectors which
allow operator
to vary the
angle of
detection
Clinical uses Ag-Ab rxn, Ag-Ab rxn,
immunocomplex immunocomplex
rxn, ppts, rxn,ppts, liver dis,
lipoprotein protein in urine or
CSF

REVIEWER IN LAB MATH- LECTURE MERRA CYRINE VENZUELA

 GENERAL
TECHNIQUES AND
INSTRUMENTATION
Abigail Phee Cabigao
OBJECTIVES
◦ Know the principle, methodology, components, advantages,
limitations, and usefulness of using various analytical techniques
in the laboratory.
◦ Compare and contrast the concepts and theories of different
quantitative techniques.
◦ Relate and integrate different quantitative techniques that are
used in the clinical and industrial laboratory settings.
Analytical Method
It is a generic process combining the power of the Scientific Method with the use of the formal
process to solve any type of problem. It has these nine steps:
1. Identify the problem to solve.
2. Choose an appropriate process. (THE KEY STEP)
3. Use the process to hypothesize analysis or solution elements.
4. Design an experiment(s) to test the hypothesis.
5. Perform the experiment(s).
6. Accept, reject, or modify the hypothesis.
7. Repeat steps 3, 4, 5, and 6 until the hypothesis is accepted.
8. Implement the solution.
9. Continuously improve the process as opportunities arise.
Why is this important?

The use of the Analytical Method is critical to

solving the sustainability problem because it
appears that current processes are inadequate.
They are intuitive, simple, and based on how
activists approach everyday problems.
Colorimetry
It is a scientific technique that is used to determine the
concentration of colored compounds in solutions by the
application of the Beer-Lambert law, which states that
the concentration of a solute is proportional to the
absorbance.
Principle of Colorimetry
A colorimeter can measure the absorbency of light waves. During
color measurement, the change in the intensity of
electromagnetic radiation in the visible wavelength region of the
spectrum after transmitting or reflecting by an object or solution is
measured.
Types of Colorimeter

There are two types of colorimeters.

📌 Color densitometers: measure the density of primary colors.

📌 Color photometers: measure the color reflection and

transmission.
APPLICATION OF COLORIMETER

◦ Colorimeters are widely used to monitor the growth of a bacterial or yeast culture by
providing reliable and highly accurate results when used for the assessment of color in bird
plumage.
◦ They are used to measure and monitor the color in various foods and beverages, including
vegetable products and sugar.
◦ Colorimeters are used for basic research in chemistry laboratories.
◦ Colorimeters have many practical applications such as testing water quality by screening
chemicals such as chlorine, fluoride, cyanide, dissolved oxygen, iron, molybdenum, ,zinc
and hydrazine.
◦ Also, they are used to determine the concentrations of plant nutrients such as ammonia,
nitrat,e and phosphorus in soil or hemoglobin in the blood.
Photometry
It is the science of measuring visible light in units that are weighted according to
the sensitivity of the human eye. It is a quantitative science based on a
statistical model of the human visual response to light - that is, our perception of
light - under carefully controlled conditions.

Photometry involves the measurement of the psychophysical attributes of

electromagnetic energy that is visible to the human eye. The use of the term
'luminous', which refers to visible light, defines photometry in terms of human
perception.
Types of Photometer
Two types of photometers are used: spectrophotometer and
filter photometer.

In spectrophotometers, a monochromator (with a prism or with

grating) is used to obtain monochromatic light of one defined
wavelength.

In filter photometers, optical filters are used to give

monochromatic light.
Filter Photometer
Chromatography

It is a process for separating components of a

mixture. To get the process started, the mixture is
dissolved in a substance called the mobile phase,
which carries it through a second substance
called the stationary phase.
Types of Chromatography

◦Adsorption Chromatography
◦Thin Layer Chromatography
◦Column Chromatography
◦Partition Chromatography
 Applications of Chromatography
In bioanalytical chemistry, chromatography is mainly used for the separation, isolation, and
purification of proteins from complex sample matrices. In cells, for example, proteins occur
alongside numerous other compounds such as lipids and nucleic acids. In order to be
analyzed, these proteins must be separated from all the other cell components. Then the
proteins of interest might have to be isolated from other proteins and purified further.

Chromatography is an essential part of almost any protein purification strategy. A number of

different chromatographic techniques are used for the purification and analysis of proteins.
They can be classified according to the physical principle involved in the separation process.
Typical examples include reversed-phase chromatography, ion-exchange chromatography,
affinity chromatography, and size exclusion chromatography.
 FAQ in Chromatography
What is the basic principle of chromatography?

Chromatography is based on the concept of separating molecules in a mixture added to the ground or solid and liquid stationary state (stable
phase) when traveling with the aid of a mobile phase.

What is the Rf value in chromatography?

RF stands for retention factor in paper chromatography, or the distance a fluid compound moves up a plate of chromatography. For each
particular solvent, all compounds have a common RF value, and RF values are used to equate unknown samples with known compounds.

How is RF value useful?

The distance traveled by the sample is divided by the distance traveled by the solvent. It is a component characteristic and can be used to
classify components for a given system at a known temperature.

Where is chromatography used?

Chromatography is used in industrial processes to purify materials, test trace amounts of contaminants, isolate chiral compounds, and quality
control test products. Chromatography is the physical process of separating or analyzing complex mixtures.
Electrophoresis
It is an electrokinetic process that separates charged
particles in a fluid using a field of electrical charge. It is
most often used in life sciences to separate protein
molecules or DNA and can be achieved through several
different procedures depending on the type and size of
the molecules.
How does Electrophoresis work?
An electric field is applied to molecules and as they are electrically charged themselves
it results in a force acting upon them. The greater the charge of the molecule the
greater the force applied by the electrical field and therefore the further through the
support medium the molecule will move relative to its mass.

Some example applications of electrophoresis include DNA and RNA analysis as well as
protein electrophoresis which is a medical procedure used to analyze and separate the
molecules found in a fluid sample (most commonly blood and urine samples).
Types of Electrophoresis
Polyacrylamide gel electrophoresis (PAGE) has a
clearer resolution than agarose gel making it more
suitable for quantitative analysis. This makes it
possible to identify how proteins bind to DNA. It
can also be used to develop an understanding of
how bacteria are becoming resistant to antibiotics
through plasmid analysis.

2D Electrophoresis separates molecules along an

x-axis and a y-axis – one separating them by
charge and the other by size.
 Turbidimetry
In analytical chemistry,
methods for
determining the
amount of cloudiness,
or turbidity, in a solution
are based upon
measurement of the
effect of this turbidity
upon the transmission
and scattering of light.
Why is it important?
Besides being a measure of treatment, turbidity can
affect the taste and odor of drinking water. It is essential
to reduce the turbidity of water in order to effectively
disinfect it. Turbidity can act as a shield against
pathogens and the particles that cause turbidity can
harbor bacteria and viruses
Ultracentrifugation
Ultracentrifugation is a specialized technique used to spin samples at exceptionally
high speeds. Current ultracentrifuges can spin to as many as 150 000 rotations per
minute (rpm).

Ultracentrifugation is commonly used to purify, as well as to characterize, low-

molecular-weight polymers up to multi-megaDalton protein complexes and
organelles.

There is a vast difference in the speed of the rotors of centrifuge and ultracentrifuge
as they rotate. An ultracentrifuge rotor can spin as high as 1000000 x g, while the
most common centrifuges have a relatively lesser speed.
Thank you and God bless!