High Performance Liquid Chromatography-HPLC slide.ppt

High Performance
Liquid Chromatography
(HPLC)
M. AHNAF

Introduction
• HPLC is a form of liquid chromatography used to separate
compounds that are dissolved in solution. HPLC instruments
consist of a reservoir of mobile phase, a pump, an injector, a
separation column, and a detector.
• High-performance liquid chromatography (HPLC) is an analytical
technique to separate, identify, and quantify components in a
mixture. It is the main chromatography technique used in most
laboratories worldwide.
• Compounds are separated by injecting a sample mixture onto the
column. The different component in the mixture pass through the
column at differentiates due to differences in their partition
behavior between the mobile phase and the stationary phase. The
mobile phase must be degassed to eliminate the formation of air
bubbles.

High-Performance Liquid Chromatography
(HPLC) Definition and Introduction
• HPLC is especially useful for low or non-volatile organic compounds,
which cannot be analyzed with gas chromatography.
• The technique relies on a mobile phase and a stationary phase to
separate components within a mixture. A high-pressure pump
delivers the mobile phase through the system. Compounds with a
higher affinity for the mobile phase will migrate through the column
more rapidly and interact less with the stationary phase.
• Once separated, a detector measures the concentration of the
analytes and converts them into electrical signals; the concentration
of each component is proportional to the amount that was eluted
from the column. The time taken between injection and detection –
known as the retention time – is specific for a given set of
chromatographic conditions and may be compared with a standard
for identification

How Does HPLC Work?
• HPLC in Four Steps ;
1. The sample is first dissolved in a liquid or the mobile phase.
2. This solution is then injected by means of a manual injector or an
autosampler into a continuous flow of mobile phase, being
delivered by a pump, and carried onto the LC column which
contains a stationary phase.
3. The various components of the sample travel through the column
at different speeds due to their interactions between the mobile
and stationary phases, resulting in the components separating
from one another. The different travel times are referred to as the
components’ retention time.
4. When components emerge from the column, they are carried to a
detector where a physical property of the compounds is
measured, such as absorption of light for UV detection.

FOUR TYPES OF LIQUID
CHROMATOGRAPHY
• Partition chromatography
• Adsorption, or liquid-solid
chromatography
• Ion exchange chromatography
• Size exclusion, or gel, chromatography

COMPOSITION OF A LIQUID
CHROMATOGRAPH SYSTEM
• Solvent
• Solvent Delivery System (Pump)
• Injector
• Sample
• Column
• Detectors (Diode Array)
• Waste Collector
• Recorder (Data Collection)

High Performance Liquid Chromatography-HPLC slide.ppt

HPLC consists of a variety of components, including a solvent
delivery pump, a degassing unit, a sample injector, a column
oven, a detector, and a data processor.

HPLC flow description
• As for HPLC, the pump delivers the mobile phase at a controlled flow
rate(a).
• Air can easily dissolve in the mobile phase under the standard
atmospheric pressure in which we live in. If the mobile phase
contains air bubbles and enters the delivery pump, troubles such as
flow rate fluctuations and baseline noise/drift may occur. The
degassing unit helps prevent this issue by removing air bubbles in the
mobile phase(b).
• After the dissolved air has been removed, the mobile phase is
delivered to the column. The sample injector then introduces a
standard solution or sample solution into the mobile phase (c).
• Temperature fluctuations can affect the separation of compounds in
the column. The column is placed in a column oven to keep the
temperature constant(d).
• Compounds eluted from the column are detected by a detector
which is placed downstream of the column(e).
• A workstation processes the signal from the detector to obtain a
chromatogram to identify and quantify the compounds(f).

Picture of HPLC instrument
picture of modern HPLC instrument.

Instrumentation - major
components of a HPLC

Basic HPLC System Components
• Solvent Degasser – removes air gases from the solvents as they flow
to the HPLC pump
HPLC Pump – provides solvent flow and proportioning
Autosampler – draws samples from vials and injects them into the
solvent flow provided by the pump.
Detector – responds to the separated analytes emerging from the
HPLC column and produces a signal output for the software
Column Oven – houses the HPLC column and keeps a stable
temperature for reproducible separations

Isocratic vs. Gradient Elution Modes
Isocratic Elution;
• A single composition of solvents is used for the duration of the separation
• Later eluting peaks are broader than earlier eluting peaks because of dispersion
• Steps must be taken to periodically flush the column at higher solvent strength
to clean it of intractable materials that build up from sample injections
Gradient Elution;
• The composition of solvents is changed either continuously or stepwise
• In general, peaks are sharper throughout the chromatogram when compared to
isocratic elution
• Some separations may be achieved which are not possible using isocratic elution
• Chromatogram run times may be shorter when compared to isocratic elution

Many different detectors forHPLC
• Most common detectors are ;
1. Ultraviolet/visible (UV/Vis),
2. Photodiode array (PDA),
3. Fluorescence (FL), and
4. Refractive index (RI).
• Each response plotted time, resulting in a chromatogram.

Available Detectors for HPLC
1. UV/Vis Detectors
• Responds to chromophoric analytes in the range 190 – 800nm
• Single wavelength monitoring
• Good for the majority of organic analytes
• Gradient elution compatible
• UV visible HPLC detector uses light to analyze samples. By
measuring the sample's absorption of light at different
wavelengths, the analyte can be identified. HPLC UV detectors
can be used by any lab using HPLC, including genomic, biology,
and biochemistry laboratories, to analyze nucleic acids,
proteins, and to do toxic and therapeutic drug testing.

2. Photodiode Array (PDA) Detectors
• Responds to chromophoric analytes in the range 190 – 800nm
• Multi-wavelength monitoring
• Complete spectral profiles of chromatographic peaks
• Good for the majority of organic analytes
PDA detects an entire spectrum
simultaneously. UV and VIS detectors visualize
the obtained result in two dimensions (light
intensity and time), but PDA adds the third
dimension (wavelength). This is convenient to
determine the most suitable wavelength
without repeating analyses.

3. Fluorescence Detectors
• Responds only to analytes which fluoresce naturally or can be made
to fluoresce through derivatization such as dansylchloride.
• High sensitivity for selective groups of compounds at ~femtogram (fg)
level.
• Gradient elution compatible.
• By using a specific wavelength, analyte atoms are excited and then
emit light signal (fluorescence). The intensity of this emitted light is
monitored to quantify the analyte concentration.
• Most pharmaceuticals, natural products, clinical samples, and
petroleum products have fluorescent absorbance.

RI detector
4. Refractive Index Detectors
• Suitable for detecting all components.
• Lower sensitivity compared to UV detector
• Not compatible with gradient elution
• Used for the detection of sugars and for SEC analysis.

Evaporative Light Scattering Detector (ELSD)
5. Evaporative Light Scattering
Detectors
• general-purpose universal detector
that can even detect components with
no UV absorption, such as
carbohydrates, lipids, surfactants, and
synthetic polymers.
• Good sensitivity

Evaporative Light Scattering Detector. It is
used as a quasi-universal detector for HPLC.

Mass Spectrometer
6. Mass Spectrometric Detector
• Analytes are detected based on
their Molecular Weight (MW).
• Obtained information is especially
useful for compound structure
identification.
• Highly sensitive
• Gradient elution compatible.
• Ideal for detecting heat-labile
compounds, such as proteins and
vitamins in food products. Food
contaminants are also often
quantified using HPLC-MS.

The basic components of a LC unit consist of:
(1) Pump - delivers the mobile phase at a required flow rate,
(2) Autosampler - injects the samples,
(3) Column - for separation of sample,
(4) Detector - for the analysis of the separated components in a sample.
• For a LCMS system, the instrumentation comprises of:
(1) a LC unit,
(2) an interface between the LC and MS,
(3) an ion source that ionizes samples (e.g. API unit),
(4) an ion guide (an electrostatic lens that efficiently introduces the
generated ions into the MS,
(5) a mass analyzer unit that separates the ions based on their mass-to-
charge (m/z),
(6) a detector unit that detects the separated ions.

Traditional HPLC columns are packed with
porous silica particles

Considerations When Choosing a Column
• The decision of a “best” stationary phase for a separation should be
based on sample solubility and the chemical differences between
the sample constituents.
• Soluble in organic or aqueous solvents?
• Knowledge of constituent functional groups? -OH; -COOH; -CONH2; -
C6H6, etc.
• The decision about the dimensions of the column should be based
on the goals for the chromatography.
• Resolution of a critical pair of constituents? – longer column bed
• Fast analysis? – shorter column bed
• Maximum resolution for all? – smaller particles
• Minimize solvent consumption? – narrower inner diameter

The Chemistry of Bonded Phases

Through chemical reaction,
attachment of the primary bonded
phase is achieved.
Manufacturers refer to the amount
of bonded phase coverage in terms
of %Carbon load.
Much of the silica surface remains
unreacted – some surface silanol
groups are still exposed.

Frequently, a secondary bonding step
intruduces smaller “end-cap”
molecules between the primary
bonded phase strands. These
endcaps may be polar or non-polar.
Different columns of the same
bonded phase type will differ in
silanol exposure and end-capping,
resulting in a range of different
overall polarities and different
separating ability.

NON POLAR COLUMN
C18 C8
Phenyl Alkyl Amide

•Four Types of HPLC Columns:
1. Normal Phase Column.
2. Reverse Phase Column.
3. Ion-exchange Column.
4. Size Exclusion Column.

What is Normal Phase Chromatography
• Normal phase chromatography is a type of HPLC technique. It separates
analytes based on the degree of interaction towards the absorbent, which is
polar silica. Therefore, the stationary phase of this type of chromatography is
hydrophilic. It can also make hydrophilic interactions with the hydrophilic
molecules in the sample mixture. Generally, these interactions include
hydrogen bonding, dipole-dipole interactions, etc. Therefore, more non-
polar analytes stay longer in the stationary phase, increasing the retention
time.
• Furthermore, the mobile phase in the normal phase chromatography is non-
polar and non-aqueous. Therefore, non-polar or hydrophobic analytes in the
mixture wash out effectively with the mobile phase at the beginning of the
process. Meanwhile, the retention time of analytes reduces with the
increasing polarity of the mobile phase. Furthermore, the poor
reproducibility of the retention time is the major drawback of normal phase
chromatography. Basically, this occurs due to the presence of a layer of
water or protic organic solvents on the surface of silica. However, this is
eliminated in the reverse phase chromatography.

What is Reverse Phase Chromatography
• Reverse-phase chromatography is a type of recent HPLC. It has an increased
reproducibility of the retention time when compared to normal phase
chromatography. Basically, this increase of the reproducibility is achieved by
making the stationary phase non-polar. To do that, the surface of the silica
stationary phase is modified as RMe2SiCl, where R is a straight-chain alkyl
group such as C18H37 or C8H17. However, due to the non-polar nature of
the stationary phase, less polar analytes in the sample mixture tend to have
a higher retention time in contrast to the normal phase chromatography.
• Moreover, one can increase the retention time by adding more water to the
mobile phase, which, in turn, increases the hydrophobic interactions
between the non-polar analytes and the stationary phase. Also, the mobile
phase of the reverse phase chromatography is polar, washing out polar
analytes in the sample mixture. This facilitates the separation of the non-
polar analytes in the sample mixture. Furthermore, the surface tension of
the mobile phase, as well as its pH, have effects on the retention time.

Subject / content Normal phase HPLC Reverse phase HPLC
Definition an adsorptive mechanism, is used for
the analysis of solutes readily soluble
in organic solvents, based on their
polar differences such as amines,
acids, metal complexes
partition mechanism, is typically
used for separations by non-
polar differences.
Mobile phase Non-polar
Example; organic solvents such as
hexane, isopropyl ether, ethyl acetate
Polar
Example; water + organic
solvent such as acetonitrile,
methanol or acetone, THF
Stationary phase Polar
Example; silica, alumina or chemically
modified silica such as triethylene
glycol supported by silica particles
Non polar
Example; phenyl ligands bonded
with silica surface, octadecyl,
C18
Octyl C8. octadecyl (C18 or ODS)
which have octadecyl groups
bonded to silica gel and octyl
(C8) that have octyl groups
bonded to silica gel.
Sample movement/
elution order
Least polar first then most polar last Most polar first, least polar last
Separation based on Different polarities Different hydrocarbon content

Similarities Between Normal Phase and
Reverse Phase Chromatography
• Normal and reverse phase chromatography are two types of
chromatographic techniques of HPLC.
• Their schematic instrumentation includes a degasser, sampler, pumps, and
a detector.
• Both operate under high pressure.
• Both separate a small volume of a sample.
• The separation is based on the different degrees of interactions of the
components of the sample with the adsorbent particles. These
interactions depend on the temperature.
• The smaller adsorbent particles (2–50 μm in average particle size) give
a high-resolution power to both types of chromatography.
• Furthermore, both types of chromatography give a quantitative analysis of
the sample components.
• They take around 2-60 mins per sample but, do not allow parallel analysis.

Difference Between Normal Phase and
1. Definition
• Normal phase chromatography refers to a separation method which
allows the distribution of components of a mixture between two
phases, one of which is a polar stationary phase while the mobile
phase is non-polar. In contrast, reverse phase chromatography refers
to the separation method, whose mobile phase is more polar than
the stationary phase.
2. Evolution
• Normal phase chromatography was evolved in the 1970s in the form
of liquid chromatography. But, reverse phase chromatography is a
recently evolved form of HPLC.

3. Stationary Phase
• Furthermore, normal phase chromatography uses a polar stationary
phase, which is mainly pure silica, while reverse phase
chromatography uses a non-polar stationary phase, which is a
modified silica substrate with long hydrophobic long chains.
4. Mobile Phase
• Normal phase chromatography uses a non-polar, non-
aqueous solvent as the mobile phase, which is mainly chloroform
while reverse phase chromatography uses a polar mobile phase,
which is mainly water, methanol or acetonitrile.

5. Types of Separation
• Moreover, normal phase chromatography separates polar analytes
with high retention time in the column, while reverse phase
chromatography separates less polar analytes, which have a high
retention time in the column.
6. Analytes in the Mobile Phase
• In the normal phase chromatography, the mobile phase carries non-
polar analytes at the beginning of the separation while in the reverse
phase chromatography, the mobile phase carries polar analytes.
7. Increasing the Retention Time
• A non-polar mobile phase increases the retention time of normal
phase chromatography while a polar mobile phase increases the
retention time of reverse phase chromatography.

8. Elution
• Analytes can be eluted by increasing the polarity of the mobile phase
in the normal phase chromatography while the analytes can be
eluted by decreasing the polarity of the mobile phase in the reverse
phase chromatography.
9. Stationary Phase Characteristics
• The stationary phase of the normal phase chromatography contains a
layer of water or protic organic solvent while the stationary phase of
the reverse phase chromatography does not contain water or a layer
of protic solvent.

10. Reproducibility of the Retention Time
• Moreover, normal phase chromatography has a
poor reproducibility of the retention time while reverse phase
chromatography has a higher reproducibility of the retention time.
11. Damage to the Column
• The column of normal phase chromatography is easy to damage
while the column of the reverse phase chromatography is difficult to
damage.

Phase Characteristics for Separations Based
on Polarity.
SEPARATION MODE STATIONARY PHASE
(PARTICLE)
MOBILE PHASE
(SOLVENT)
NORMAL PHASE POLAR NON POLAR
REVERSE PHASE NON POLAR POLAR

HPLC Separation Modes
• Three primary characteristics of chemical compounds can be used to
create HPLC separations. They are:
1. Polarity
2. Electrical Charge
3. Molecular Size

Separations Based on Polarity
• A molecule’s structure, activity, and physicochemical characteristics
are determined by the arrangement of its constituent atoms and the
bonds between them.
• Within a molecule, a specific arrangement of certain atoms that is
responsible for special properties and predictable chemical reactions
is called a functional group. This structure often determines whether
the molecule is polar or non-polar.
• Organic molecules are sorted into classes according to the principal
functional group(s) each contains. Using a separation mode based on
polarity, the relative chromatographic retention of different kinds of
molecules is largely determined by the nature and location of these
functional groups.

Separations Based on Charge: Ion-Exchange
Chromatography [IEC]
• In ion-exchange chromatography and other separations based upon
electrical charge, the rule is reversed. Likes may repel, while
opposites are attracted to each other.
• Stationary phases for ion-exchange separations are characterized by
the nature and strength of the acidic or basic functions on their
surfaces and the types of ions that they attract and
retain. Cation exchange is used to retain and separate positively
charged ions on a negative surface.
• Conversely, anion exchange is used to retain and separate negatively
charged ions on a positive surface [see Figure T].

Figure T. Ion-Exchange Chromatography.

Ion-Exchange Chromatography IEC

Procedure of ion exchange chromatography
• Ion exchange separations are carried out mainly in columns packed with
an ion-exchanger.
• These ionic exchangers are commercially available. They are made up
of styrene and divinyl benzene. Example. DEAE-cellulose is an anionic
exchanger, CM-cellulose is a cationic exchanger.
• The choice of the exchanger depends upon the charge of particle to be
separated. To separate anions “Anionic exchanger” is used, to separate
cations “Cationic exchanger” is used.
• First the column is filled with ion exchanger then the sample is applied
followed by the buffer. The tris-buffer, pyridine buffer, acetate buffer,
citrate and phosphate buffers are widely used.
• The particles which have high affinity for ion exchanger will come down
the column along with buffers.
• In next step using corresponding buffer separates the tightly bound
particles.
• Then these particles are analyzed spectroscopically.

Separations Based on Size: Size-Exclusion
Chromatography [SEC] – Gel-Permeation
Chromatography [GPC]
• In the 1950s, Porath and Flodin discovered that biomolecules could
be separated based on their size, rather than on their charge or
polarity, by passing, or filtering, them through a controlled-porosity,
hydrophilic dextran polymer. This process was termed gel filtration.
Later, an analogous scheme was used to separate synthetic oligomers
and polymers using organic-polymer packings with specific pore-size
ranges. This process was called gel-permeation chromatography
[GPC]
• Similar separations done using controlled-porosity silica packings
were called size-exclusion chromatography [SEC]. Introduced in 1963,
the first commercial HPLC instruments were designed for GPC
applications.
https://www.waters.com/nextgen/us/en/educ
ation/primers/beginner-s-guide-to-liquid-
chromatography/hplc-separation-modes.html

Separations Based on Size: Size-Exclusion
Chromatography [SEC] – Gel-Permeation
Chromatography [GPC]
• All of these techniques are typically done on stationary phases that have
been synthesized with a pore-size distribution over a range that permits the
analytes of interest to enter, or to be excluded from, more or less of the
pore volume of the packing.
• Smaller molecules penetrate more of the pores on their passage through
the bed. Larger molecules may only penetrate pores above a certain size so
they spend less time in the bed. The biggest molecules may be totally
excluded from pores and pass only between the particles, eluting very
quickly in a small volume.
• Mobile phases are chosen for two reasons: first, they are good solvents for
the analytes; and, second, they may prevent any interactions [based on
polarity or charge] between the analytes and the stationary phase surface.
In this way, the larger molecules elute first, while the smaller molecules
travel slower [because they move into and out of more of the pores] and
elute later, in decreasing order of their size in solution. Hence the simple
rule: Big ones come out first.

Gel permeation chromatography GPC

Uses of High Performance Liquid
Chromatography
• High performance liquid chromatography technique is mainly used
for the separation and analysis of samples in the pharmaceutical and
pesticide industries.
• In liquid-liquid chromatography with the HPLC system, separation of
moderately polar substances is possible. We also used a combination
of ion exchange and HPLC on porous layer beads (PLB).

Uses of HPLC
• This technique is used for chemistry and biochemistry
research analyzing complex mixtures, purifying chemical
compounds, developing processes for synthesizing chemical
compounds, isolating natural products, or predicting physical
properties. It is also used in quality control to ensure the
purity of raw materials, to control and improve process
yields, to quantify assays of final products, or to evaluate
product stability and monitor degradation.
• In addition, it is used for analyzing air and water pollutants,
for monitoring materials that may jeopardize occupational
safety or health, and for monitoring pesticide levels in the
environment. Federal and state regulatory agencies use
HPLC to survey food and drug products, for identifying
confiscated narcotics or to check for adherence to label
claims.

Some applications of the high performance
liquid chromatography technique;
• A mixture of sugars containing fructose, glucose, sucrose, maltose, and
lactose can be separated by the HPLC technique in presence of a
refractive index detector.
• Different types of drugs have been separated on Mercosorb SL-100
coated with 0.6M picric acid at pH 6.0 with chloroform (mobile phase) in
ion-pair chromatography. We used a UV detector to complete this
operation. The above HPLC technique works mostly on the drugs like
scopolamine, ergotamine, and hyoscyamine.
• In the cation exchange HPLC technique, a urinary extract containing
metabolite and antipyrine have been separated on zipax SCX
with borax buffer at the pressure of 100 psi.
• Protein has been separated by size exclusion HPLC technique. Proteins
can be separated on column TSK 2000SW with phosphate buffer at pH
6.5 in presence of mobile phase 0.2M sodium sulfate solution. We
commonly used a UV detector for the separation or analysis of protein
samples in size exclusion high performance liquid chromatography
(HPLC) system.

HPLC Chromatograph injectors
• The function of the injector is to place the sample into the
high-pressure flow in as narrow volume as possible so that
the sample enters the column as a homogeneous, low-
volume plug. To minimize spreading of the injected volume
during transport to the column, the shortest possible length
of tubing should be used from the injector to the column.
• When an injection is started, an air actuator rotates the
valve: solvent goes directly to the column; and the injector
needle is connected to the syringe. The air pressure lifts the
needle and the vial is moved into position beneath the
needle. Then, the needle is lowered to the vial.

Principle of manual injection
• Most important factors in injection are precision, accuracy and
carryover. They are effected by the injection technique and
equipment and for manual injection also by the user.
• In the load position a sample loop is filled with sample while the
system is equillibrating. When turning to the inject position, the
sample loop is switched to the high pressure part of the HPLC system.
The flow delivered by the pump flows through the loop and feeds the
sample onto the column.

HPLC Autosamplers
• The autosampler, an automated sample injector, introduces a precise
aliquot of a sample solution from a sample container to the high
performance liquid chromatography (HPLC) column under high-
pressure flow conditions. It is a sophisticated automation device,
capable of great precision and long-term reliability.

WHAT AFFECTS SYSTEM
Column Parameters
• Column Material
• Deactivation
• Stationary Phase
• Coating Material
Instrument Parameters
• Temperature
• Flow
• Signal
• Sample Sensitivity
• Detector

WHAT AFFECTS SYSTEM
Sample Parameters
• Concentration
• Matrix
• Solvent Effect
• Sample Effect

Selectivity Factor
• K’ values tell us where bands elute relative to the void volume. These
values are unaffected by such variables as flow rate and column
dimensions. The value tell us where two peaks elute relative to each
other. This is referred to as the selectivity factor or separation factor
(now and then as the chemistry factor).

EVALUATION PARAMETERS
• EFFICIENCY
• RESOLUTION
• INERTNESS
• RETENTION INDEX
• COLUMN BLEED
• CAPACITY FACTOR

References
• What is HPLC (High Performance Liquid Chromatography) ？
• https://www.shimadzu.com/an/service-support/technical-
support/analysis-
basics/basic/what_is_hplc.html#:~:text=HPLC%20is%20an%20abbrev
iation%20for,instrument%20used%20to%20conduct%20chromatogra
phy.
• HIGH PERFORMANCE LIQUID CHROMATOGRAPHY - HPLC
• https://www.chemguide.co.uk/analysis/chromatography/hplc.html
• HPLC basics –Principles and parameters
• https://www.knauer.net/en/Systems-Solutions/Analytical-HPLC-
UHPLC/HPLC-Basics---principles-and-parameters
• High performance liquid chromatography (HPLC)
• http://hiq.linde-
gas.com/en/analytical_methods/liquid_chromatography/high_perfor
mance_liquid_chromatography.html

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