This document provides an overview of high performance liquid chromatography (HPLC). It begins with an introduction to HPLC, describing how it works to separate mixtures by injecting a sample into a column packed with tiny particles. It then covers the instrumentation of HPLC including components like the pump, injector, column, detector, and computer system. The document discusses various types of HPLC based on stationary and mobile phases. It also outlines the operation of HPLC, describing the injection, separation, detection, and interpretation steps. Applications of HPLC in fields like pharmaceuticals, environment, and food are highlighted. Advantages like speed and accuracy are contrasted with disadvantages like cost and complexity.

1. Pokhara University
School of Health and Allied Sciences
High Performance liquid
Chromatography
Presented by : Deepa Kumari Karn
First Semester, M Pharm
School of Health and Allied Sciences
Pokhara University, Dhungepatan, Pokhara Lekhnath-30, Kaski,
Nepal

2. Pokhara University
School of Health and Allied Sciences
Contents
Introduction
General History
Classification
Instrumentation
Operation
Advantages and Disadvantages
Applications
Difference between TLC and HPLC
References
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Introduction
• Chromatographic technique used to separate a
mixture of compounds in analytical chemistry and
biochemistry with the purpose of identifying,
quantifying or purifying the individual components of
the mixture
• HPLC is a separation technique that involves:
The injection of a small volume of liquid sample into a tube
packed with tiny particles (3 to 5 micron (µm) in diameter
called the stationary phase)
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Individual components of the sample are moved down the
packed tube (column) with a liquid (mobile phase) forced
through the column by high pressure delivered by a pump
In principle, LC and HPLC work the same way except the
speed, efficiency, sensitivity and ease of operation of HPLC
is vastly superior
• Components are separated from one another by the
column packing that involves various chemical
and/or physical interactions between their molecules
and the packing particles
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• Separated components are detected at the exit of
this tube (column) by a flow-through device
(detector) that measures their amount
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General History
• The acronym HPLC, coined by the late Prof. Csaba
Horváth for his 1970 Pittcon paper, originally
indicated the fact that high pressure was used to
generate the flow required for liquid
chromatography in packed columns
• In the beginning, pumps only had a pressure
capability of 500 psi. This was called high pressure
liquid chromatography, or HPLC
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• These new HPLC instruments could develop up to
6,000 psi [400 bars] of pressure, and incorporated
improved injectors, detectors, and columns. HPLC
really began to take hold in the mid-to late-1970s
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Figure: Csaba Horváth working on pittcon paper

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Figure: Traditional HPLC Instrunment

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Classification
• Depending upon the phase system (stationary and
mobile phase) HPLC is classified into following types:
1) Normal Phase HPLC:
It has polar stationary phase and non-polar mobile phase
The stationary phase is usually silica and typical mobile
phases are hexane, methylene chloride, chloroform,
diethyl ether, and mixtures of these
Polar samples are thus retained on the polar surface of the
column packing longer than less polar materials
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2) Reverse Phase HPLC:
The stationary phase is nonpolar (hydrophobic) in nature,
while the mobile phase is a polar liquid, such as mixtures
of water and methanol or acetonitrile
It works on the principle of hydrophobic interactions hence
the more nonpolar the material is, the longer it will be
retained
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3) Size-exclusion HPLC:
The column is filled with material having precisely
controlled pore sizes, and the particles are separated
according to its their molecular size
Larger molecules are rapidly washed through the column;
smaller molecules penetrate inside the porous of the
packing particles and elute later
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4) Ion-Exchange HPLC:
The stationary phase has an ionically charged surface of
opposite charge to the sample ions
This technique is used almost exclusively with ionic or
ionizable samples
The stronger the charge on the sample, the stronger it will
be attracted to the ionic surface and thus, the longer it will
take to elute
The mobile phase is an aqueous buffer, where both pH and
ionic strength are used to control elution time
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5) Based on the mobile phase gradient:
a) Isocratic:
 Mobile phase solvent composition remains constant
with time
 Often used in quality control applications that support
and are in close proximity to a manufacturing process
b) Gradient :
 Mobile phase solvent (“B”) composition increases
with time
 Linear gradients are most popular
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Fig: mobile phase gradient representation

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• The HPLC system consists of following
components:
Solvent reservoir
Pump
Sample injector
HPLC column
Detector
Chromatogram
Instrumentation
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Figure: - Instrumentation of HPLC system
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Figure : Overall view of the HPLC

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• Solvent reservoir: - It holds the solvent called mobile
phase which moves through the various parts of the
system
• Pump: - The role of the pump is to force a liquid
(called the mobile phase) through the liquid
chromatography at a specific flow rate, expressed in
milliliters per min (ml/min)
Normal flow rates in HPLC are in the 1to 2mL/min range
Typical pumps can reach pressures in the range of 6000-
9000 psi (400to 600bar)
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During the chromatographic experiment, a pump can
deliver a constant mobile phase composition (isocratic) or
an increasing mobile phase composition (gradient)
• Sample injector: - The injector serves to introduce
the liquid sample into the flow stream of the mobile
phase
Typical sample volumes are 5-to 20-microliters (μL)
The injector must also be able to withstand the high
pressures of the liquid system
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• HPLC column: - Considered the “heart of the
chromatograph” the column’s stationary phase
separates the sample components of interest using
various physical and chemical parameters
The small particles inside the column cause the high back
pressure at normal flow rates
The pump must push hard to move the mobile phase
through the column and this resistance causes a high
pressure within the chromatography
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• Detector: - The detector can see (detect) the
individual molecules that come out (elute) from the
column
A detector serves to measure the amount of those
molecules so that we can quantitatively analyze the
sample components
The detector provides an output to a recorder or computer
that result in the liquid chromatogram (i.e., the graph of
the detector response)
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• Computer and Chromatogram: -
 Also 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)
 Acquisition software is installed in computer. This
software collects data from Detector and will draw a graph
(Called Chromatogram)
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Operations
• Overall process of HPLC can be described by the
following flow diagram that involves following
process:-
1. Injection of sample
2. Measurement of retention time
3. Detection
4. Interpreting the output from the detector
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Figure: Various components of the HPLC

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1) Injection of sample: -
It may be manual sample injector or the auto
sampler for the injection of the sample in the HPLC
Manual injector manually load the sample into the
injector using the syringe and then turns the handle
to inject sample into the flowing mobile phase
May 29, 2017 25Manual sample injector

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In the auto sampler user loads vials filled with
sample solution into the auto sampler tray (100
samples) and the auto sampler automatically
measures the appropriate sample volume and injects
the sample
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Auto sampler

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• The loop is connected with the high pressure mobile
phase which carries the sample onto the column
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2) Measurement of 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)
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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
we are using retention times as a way of identifying
compounds.
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3) Detection: -
There are many detection principles used to detect the
compounds eluting from an HPLC column
The most common are:
Spectroscopic detection ( UV absorption and
Mass spectroscopy)
Refractive index detection
Fluorescence detection
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4) Interpreting the output from the detector: -
The output will be recorded as a series of peaks - each one
representing a compound in the mixture passing through
the detector
The output is shown as a graph of various peak in a
chromatogram
It looks like:
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In above chromatogram, there are three peaks detected.
As per Peak table; Peak RT (Retention Time), Area, Area
Percentage, Height and Height Percentage detail is given.
From chromatogram and peak table HPLC we can
determine the substances detail, substance is present or
not and if present, then how much is the concentration of
that substance
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Figure: HPLC chromatogram of mixture of compound

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Problems with the Chromatogram
1. Peak Tailing
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Possible Causes Solution
Blocked frit Reverse flush column (if it is
allowed) or replace frit (if it is
allowed) or replace column
Column void Fill void
Interfering peak Use longer column or change
mobile phase and/or column
selectivity
Wrong mobile phase pH Adjust pH
Sample reacting with active
sites
Add ion pair reagent or
volatile basic modifier or
change column

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2. Peak Fronting:
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Possible causes Solution
Low temperature Increase column temperature
Wrong sample solvent Use mobile phase for injection
solvent
Sample overload Decrease sample
concentration
Bad column Reverse flush column (if it is
allowed) or replace inlet frit (if
it is aalowed) or replace the
column

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3. Split Peaks
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Possible causes Solution
Contamination on guard or
analytical column inlet
Remove guard column and
attempt analysis. replace
guard column if necessary. If
analytical column is
obstructed, reverse and flush.
If problem persists, column
may be fouled with strongly
retained contaminants. Use
appropriate restoration
procedure. If problem persists,
inlet is probably plugged.
Change frit or replace column
Sample solvent incompatible
with mobile phase
Change solvent. whenever
possible, inject samples in
mobile phase.

39. Pokhara University
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Advantages and Disadvantages
Advantages:
Both qualitative as well as quantative analysis
can be performed
Separate and identify a multitude of
compounds in low concentration in a complex
mixture with very little assay optimization
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Gives result in a few minutes
Gives high resolution, speed and accuracy
Results are reproducible
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 Disadvantages: -
 Expensive and not portable
Costlier technique as compared to
conventional chromatographic technique
Operation is complex so trained person is
required to operate HPLC
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Applications
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Fig: Various fields for the applications of HPLC

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1) Pharmaceutical Applications:-
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S. N. Applications
1 Pharmaceutical quality control, drug stability,
2 Shelf life determination of the pharmaceutical products
3 Identification of the counterfeit drug products
4 Separation of complex molecules
5 Determination of concentration of drugs in the blood after certain intervals
of administration
6 Detection of alkyl sulfuric acid (used as catalyst, solvent and blocking
agents in the synthesis of organic compounds and various pharmaceutical
drugs)
7 Detection and separations of sugars, amino acids, polymers and others
organic acids
8 Separation of antibiotics, analgesic, sedatives, steroids etc

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2) Environmental Applications: -
Detection of phenolic compounds in drinking water
Bio-monitoring of pollutants
3) Food and Flavour:-
Measurement of Quality of soft drinks and water
Sugar analysis in fruit juices
Analysis of polycyclic compounds in vegetables
Preservative analysis
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4) Applications in Forensics: -
Quantification of drugs in biological samples
Identification of steroids in blood, urine etc
Forensic analysis of textile dyes
Determination of cocaine and other drugs of abuse in
blood, urine
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5) Applications in Clinical Tests: -
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S.N. Applications
1 Analysis of Urine , antibiotics in blood, bilirubin, biliverdin in hepatic
disorders
2 Detection of endogenous neuropeptides in extracellular fluid of brain
3 Diagnosis of many disorders related to body metabolism, those related to
endocrine and exocrine gland secretion, alteration in body fluids
4 Separation of bile acids, drugs metabolites, estrogen, urine extracts etc

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6) Application in the Scientific Research :
 It is a mandatory tool in most of the labs involved in
research which include medical, biological, chemical,
biochemical, phytochemical (plant chemical research)
Components with similar chemistry and properties can be
easily distinguished
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Difference between TLC and HPLC
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TLC HPLC
Type of Analysis Qualitative Both qualitative and
quantitative
Instrumentation minimal Much with many
adjustable parameter
Stationary Phase 2- dimensional column 3-dimensional column
Sample Application spotting injection
Mobile Phase Movement Capillary action High pressure
Visualization of Result UV lightbox On-line detection
Form of Results Spots, retention factor Peaks, retention times

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Additional Things
Theoretical Plate
– In many separation process there is a hypothetical zone or
stage in which two phases, such as the liquid and vapor
phases of a substance, establish an equilibrium with each
other
– The theory assume that in the column chromatography
column is divided into the a number of zone called
theoretical plates
– Such equilibrium stages may also be referred to as
an equilibrium stage, ideal stage, or a theoretical tray
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• The number of the theoretical plates in the column is
used as measure of the efficiency of the column to
separate the compounds
• The number of the theoretical plates can be
calculated by using the formula:
• Where, N= no of theoretical plate, tr =retention time
W= base width of the peak
•
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References
1) Skoog, Holler and Crouch (2007) Principle of Instrumental Analysis (6th edition), Thomos
Brooks/Cole (762-782)
2) Giri D (2012) High Performance Liquid Chromatography (HPLC): Principle, Types,
Instrumentation and Applications. Laboratoryinfo.com
3) Dr NR and Pacakova V (2009) Chromatography in biochemistry. Institute of Medical
Biochemistry,Charles University in Parague.
4) Gupta A (2017) Pharma Loksewa, Goodwill Publication Pvt.Ltd, Bagbazar, 64-70.
5) Theory of HPLC (www.chromacademy.com) (Accessed on Feb 10, 2017)
6) http://chemguide.co.uk/analysis/chromatography/hplc.html (Accessed on Feb 12,2017)
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Thank You
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