KHWAJA YUNUS ALI UNIVERSITY

School of Biomedical Sciences

Course Code : MBL-801
Course Title : Analytical Microbiology
Term: Summer 2023 Date: August 16, 2023 Time: 3:30 pm Room no.: 201 Activity: Lecture 10
Chapter three:
Chromatographic Techniques
Topics to be discussed: High Performance Liquid chromatography (HPLC)
•Introduction
•Principle
•Types of HPLC & Instrumentation
•Application & Advantages
Target CLO & PLO: CLO2 and PLO3
Learning Outcomes: At the end of the class the students will be able to-
•Explain the principle of HPLC.
•Explain the basic methodology of HPLC.
•Elucidate how date is interpreted in HPLC.
•Clarify the advantages and applications of HPLC.
Methods of Teaching: Mode/ Activities Time
Feedback previous lesson 10 minutes
Provide sub-topics, learning objectives 05 minutes
Interactive Lecture Presentation on Topic 40 Minutes
Group Discussion 15 Minutes
Question- Answer Session 10 minutes
Class review and informing about next class 05 Minutes
Others 05 Minutes
 Assessment Strategy: Quizzes/Class tests, Class Participation
 Sample Questions (MCQ):
• In HPLC, which of the following component is considered as heart?
A) Mobile phase B) Stationary Phase C) Injector
D) Column E) Pump
 Sample Questions (SAQ):
• Explain the principle of HPLC.
• Explain the basic methodology of HPLC.
• Elucidate how date is interpreted in HPLC.
• Clarify the advantages and applications of HPLC.
 Pre-reading:
 Principles and Techniques of Biochemistry and Molecular Biology; Keith Wilson and John Walker; 7 th
edition, chapter 11, page: 446-452
 References Book:
 Principles and Techniques of Biochemistry and Molecular Biology; Keith Wilson and John Walker; 7 th
edition, chapter 11
 Introduction
• High performance liquid chromatography or commonly known as HPLC is an
analytical technique used to separate, identify or quantify each component in
a mixture
• A form of liquid chromatography/ column chromatography used to separate
compounds that are dissolved in solution.
• The mixture is separated using the basic principle of
column chromatography and then identified and quantified by spectroscopy.
• In the 1960s the column chromatography LC with its low-pressure suitable
glass columns was further developed to the HPLC with its high-pressure
adapted metal columns.
• HPLC is thus basically a highly improved form of column liquid
chromatography. Instead of a solvent being allowed to drip through a column
under gravity, it is forced through under high pressures of up to 400
atmospheres.
 Principle of HPLC
• The main principle of separation is adsorption.
• The principle of HPLC is based on the separation of the
components of a mixture based on their physical and chemical
properties as they pass through a column containing a stationary
phase material.
• The sample is dissolved in a liquid mobile phase, which is then
pumped through the column under high pressure. The stationary
phase is typically a solid material that is packed into the column,
which interacts with the sample components as they pass through.
• The separation is based on differences in the affinity of the analyte
molecules for the stationary phase material and the mobile phase.
 Principle of HPLC
• The components of the mixture will interact differently with the stationary
phase based on their properties such as size, polarity, charge, and
hydrophobicity. They travel according to their relative affinities towards the
stationary phase. The component which has more affinity towards the
adsorbent, travels slower. The component which has less affinity towards
the stationary phase travels faster.
• As a result, they will travel through the column at different rates and be
separated into individual peaks. The retention time of each peak is used to
identify and quantify the individual components of the mixture.
• Detection of the separated components can be done through various
techniques, such as UV-Vis spectroscopy or mass spectrometry, depending on
the nature of the analytes.
• The output of an HPLC analysis is a chromatogram, which is a graph of the
detector response over time, and it provides information about the individual
components of the mixture.
A . Solvent delivery system(mobile phase)/Solvent Reservoir:
•The mobile phase in HPLC refers to the solvent being continuously applied to
the column or stationary phase.
•The mobile phase acts as a carrier to the sample solution.
•A sample solution is injected into the mobile phase of an assay through the
injector port.
•As a sample solution flows through a column with the mobile phase, the
components of that solution migrate according to the non-covalent interaction
of the compound with the column.
•The chemical interaction of the mobile phase and sample, with the column,
determine the degree of migration and separation of components contained
in the sample
•The solvents or mobile phases used must be passed through the column at
high pressure at about 1000 to 3000 psi. this is because as the particle size of
stationary phase is around 5-10 μ, so the resistance to the flow of solvent is
high.
B. Pumps
•The role of the pump is to force a liquid (called the mobile phase)
through the liquid chromatograph at a specific flow rate, expressed
in milliliters per min (mL/min).
•Normal flow rates in HPLC are in the 1 to 2 mL/min range.
•Typical pumps can reach pressures in the range of 6000-9000 psi
(400 to 600 bar).
•During the chromatographic experiment, a pump can deliver a
constant mobile phase composition (isocratic) or an increasing
mobile phase composition (gradient).
C. Injector:
•An injector is placed next to the pump.
•The simplest method is to use a syringe, and the sample is
introduced to the flow of eluent.
•The most widely used injection method is based on sampling loops.
•The use of the autosampler (auto-injector) system is also widely
used that allows repeated injections in a set scheduled-timing.
D. Column
•Considered the “heart of the chromatograph” the column’s
stationary phase separates the sample components of interest
using various physical and chemical parameters.
•It is usually made of stainless steel to withstand high pressure
caused by the pump to move the mobile phase through the
column.
•The small particles inside the column are called the “packing”
what cause the high back pressure at normal flow rates.
•Column packing is usually silica gel because of its particle shape,
surface properties , and pore structure give us a good separation
D. Column (cont.……)
•Other material used include alumina, a polystyrene-divinyl
benzene synthetic or an ion-exchange resin
•The dimensions of the analytical column are usually straight,
Length(5~25 cm), diameter of column(3~5 mm), diameter of
particle(35 μm). Number (40 k~70 k plates/m).
•Guard column is used to remove particular matter and
contamination, it protect the analytical column and contains similar
packing its temperature is controlled at <150°C, 0.1°C As mention
before, columns are divided into different types according to their
functions (see types of HPLC)
Retention time
• The time taken for a particular compound to travel through the column to
the detector is known as its retention time. This time is measured from the
time at which the sample is injected to the point at which the display shows a
maximum peak height for that compound.
• Different compounds have different retention times. For a particular
compound, the retention time will vary depending on:
the pressure used (because that affects the flow rate of the solvent)
the nature of the stationary phase (not only what material it is made of, but
also particle size)
the exact composition of the solvent
the temperature of the column
• That means that conditions have to be carefully controlled if you are using
retention times as a way of identifying compounds.
E . Detector:
•The detector can detect the individual molecules that elute from
the column and convert the data into an electrical signal.
•A detector serves to measure the amount of those molecules
•The detector provides an output to a recorder or computer that
results in the liquid chromatogram
•Detector is selected based on the analyte or the sample under
detection
Commonly used detectors in HPLC
Ultraviolet (UV)
•This type of detector responds to substances that absorb light.
•The UV detector is mainly to separate and identify the principal
active components of a mixture.
•UV detectors are the most versatile, having the best sensitivity
and linearity.
•UV detectors cannot be used for testing substances that are low in
chromophores (colorless or virtually colorless) as they cannot
absorb light at low range.
•They are cost-effective and popular and are widely used in industry
Commonly used detectors in HPLC:
Fluorescence
•This is a specific detector that senses only those substances that
emit light. This detector is popular for trace analysis in
environmental science.
•As it is very sensitive, its response is only linear over a relatively
limited concentration range. As there are not many elements that
fluoresce , samples must be synthesized to make them detectable.
Mass Spectrometry
•The mass spectrometry detector coupled with HPLC is called
HPLCMS. HPLC-MS is the most powerful detector, widely used in
pharmaceutical laboratories and research and development.
•The principal benefit of HPLC-MS is that it is capable of analyzing
and providing molecular identity of a wide range of components.
Commonly used detectors in HPLC:
Refractive Index (RI) Detection
The refractive index (RI) detector uses a monochromator and is one
of the least sensitive LC detectors.
•This detector is extremely useful for detecting those compounds
that are non-ionic, do not absorb ultraviolet light and do not
fluoresce.
•e.g. sugar, alcohol, fatty acid and polymers.
F . Data processing unit (Computer)
•Frequently called the data system, the computer not only controls all the
modules of the HPLC instrument but it takes the signal from the detector and
uses it to determine the time of elution (retention time) of the sample
components (qualitative analysis) and the amount of sample (quantitative
analysis).
•The concentration of each detected component is calculated from the area or
height of the corresponding peak and reported.
•In older days, the pen (paper)-chart recorder was popularly used. Nowadays, a
computer-based data processor (integrator) is more common.
•There are various types of data processors; from a simple system consisting of
the in-built printer and word processor while those with software that are
specifically designed for an LC system which not only data acquisition but
features like peak-fitting, baseline correction, automatic concentration
calculation, molecular weight determination, etc.
Degasser
•The eluent used for LC analysis may contain gases such as oxygen
that are non-visible to our eyes.
•When gas is present in the eluent, this is detected as noise and
causes an unstable baseline.
•Degasser uses special polymer membrane tubing to remove gases.
•The numerous very small pores on the surface of the polymer tube
allow the air to go through while preventing any liquid to go
through the pore.
Column Heater
•The LC separation is often largely influenced by the column
temperature.
•In order to obtain repeatable results, it is important to keep
consistent temperature conditions.
•Also for some analysis, such as sugar and organic acid, better
resolutions can be obtained at elevated temperatures (50 to 80°C).
•Thus columns are generally kept inside the column oven (column
heater).
Application of HPLC:
The HPLC has developed into a universally applicable method so that it finds its use
in almost all areas of chemistry, biochemistry, and pharmacy.
•Analysis of drugs
•Analysis of synthetic polymers
•Analysis of pollutants in environmental analytics
•Determination of drugs in biological matrices
•Isolation of valuable products
•Product purity and quality control of industrial products and fine chemicals
•Separation and purification of biopolymers such as enzymes or nucleic acids
•Water purification
•Pre-concentration of trace components
•Determination of proteins, carbohydrates and oligosaccharides
•Applications in Clinical Tests: Urine analysis, antibiotics analysis in blood, bilirubin,
biliverdin, Detection of endogenous Neuropeptides in extracellular fluid of brain
etc.
 ADVANTAGES OF HPLC:
HPLC, or high-performance liquid chromatography, is a powerful analytical technique used to separate,
identify, and quantify components of a mixture. Some of the advantages of HPLC include:
High resolution: HPLC can separate components of a mixture with high resolution, even when they are
present in very low concentrations.
Versatility: HPLC can be used to separate a wide range of compounds, including small and large molecules,
polar and nonpolar compounds, and acidic and basic compounds.
Sensitivity: HPLC can detect and quantify components at very low concentrations, making it a powerful
tool for analyzing complex mixtures.
Speed: HPLC analyses can be completed relatively quickly, with run times typically ranging from 10 to 60
minutes.
Automation: HPLC can be automated, which improves the reproducibility and accuracy of the analysis.
Quantitative: HPLC can provide quantitative data on the amount of each component in a mixture, which is
useful for quality control and for determining the efficacy of drugs and other compounds.
Non-destructive: HPLC is a non-destructive analytical technique, which means that the sample can be
recovered and used for further analysis or other purposes.
Sample compatibility: HPLC is compatible with a wide range of sample types, including biological fluids,
environmental samples, and pharmaceuticals.
Limitations of HPLC
While HPLC is a powerful analytical technique, there are some limitations that should be considered:
1.Cost: HPLC equipment and consumables can be expensive, which may limit its accessibility to some
labs.
2.Complexity: HPLC requires a high level of expertise to operate, and the method development
process can be time-consuming and challenging.
3.Sensitivity to sample preparation: HPLC results can be affected by factors such as sample
preparation, matrix effects, and contamination, which can impact the accuracy and reproducibility of
the analysis.
4.Limited resolution: Although HPLC can provide high resolution, it may not be able to separate all
components in a complex mixture, particularly when they have similar physical and chemical
properties.
5.Limited detection range: HPLC may not be able to detect compounds that are present at very low
concentrations or compounds that have low UV absorbance.
6.Risk of column damage: HPLC columns can be fragile and may be damaged by factors such as high
pressure, temperature, or pH, which can affect the quality of the results and increase the need for
column replacement.
7.Limited versatility: While HPLC can be used to separate a wide range of compounds, some
compounds may require specialized columns or detectors, which can limit the versatility of the
technique.
 • Assignment on:
Comparison between gas chromatography and HPLC.