HIGH PERFORMANCE LIQUID

CHROMATOGRAPHY

Mr. P.Prachet,
Assistant Professor,
Department of Pharmaceutical Analysis,
Chalapathi Institute of Pharmaceutical Sciences
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Principle of HPLC
The principle of separation in normal phase mode and reversed phase mode is
adsorption. When a mixture of components are introduced into a HPLC
column they travel according to their relative affinities towards the stationary
phase. The component which has more affinity towards the stationary phase
travels slower. The component which has less affinity towards the stationary
phase travels faster.
Instrumentation

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A. Solvent delivery system (Mobile phase)
• 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.
• Various types of solvents were used based on the polarity of compounds. We
use polar or non polar solvents.
• 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 5-10µ, so the resistance to the flow of solvent
is high.
• Glass and Stainless steel of SS316 grade material was used for solvent
reservoir system, but mostly we used glass material.
• Reservoir material should not interact with mobile phase nor the compound
that is being analyzed.

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B. Degasser
Degassing of mobile phase is required because bubble has property to
expand or compress. Degasser is needed to remove dissolved air.
1. By subjecting the mobile phase under vacuum.
2. By purging with fine spray of an inert gas at lower solubility such as
Argon and helium.
3. By heating and ultrasonic stirring.

C. HPLC pumps
As the name of high-pressure liquid chromatography show need to generate
the pressure. HPLC require high pressure which gives continuous and
reproducible flow of mobile phase throughout the HPLC system. Pump must
be able to take solvent from a single or more than one reservoir with pulse-
free output and different flow rates. There are three types of pumps to
provide the required pressure and flow rate as below.

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Types of HPLC pumps

1. Reciprocating pumps
• This is a common and widely used pump technique in modern
chromatography because of his precise flow rate, generate high pressure, it
should operate as an isocratic and gradient mode.
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 It consists of a small motor-driven piston which moves quickly back and
forth in a hydraulic chamber that can vary in the volume of a mobile phase.
Produce pulsing flow is the major disadvantage of a reciprocating pump.
2. Syringe pump (Displacement pump)
• This type of pump consists of a large syringe with a plunger motorized by
the electronic motor to drive used to carry a constant flow rate to the
stationary phase. It is inconvenient for the change of the mobile phase and
also for its capacity.

3. Pneumatic Pump
• In this type of pump mobile phase run into the column with the help of
pressure created from a gas cylinder, this type of column provide continuous
flow, this technique does not use as widely because have some limitations
like the low capacity of solvents, generate low pressure, pump rate varies
with viscosity.

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HPLC Injector
HPLC injection is a technique to inject the sample without disturbing the flow
rate and pressure of the HPLC system. The working pressure of an HPLC is
adequately high that we cannot inject a sample into the mobile phase by
inserting a syringe, so we need an injector that gives a reproducible result and
without interrupted the flow rate and system pressure. Nowadays HPLC
sample injector set in the different range of volume so you can inject the
specific requires a volume.

Requirements of HPLC Injector are as follows

1. Introduce the Sample with constant pressure and flow rate of the HPLC
system.
2. Introduce the sample without air bubbles.
3. The injection volumes of sample are in microliter so it should be accurate.
4. The sample must be free from any particulate matter.

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There are three types of injectors available for HPLC systems
1. Rheodyne Injector: The most commonly used injector is Rheodyne, easy to
use with high accuracy and precision. As per requirement, the analyst can set
the volume of the sample loop. Rheodyne has two positions load and inject.
The load position allows loading the sample into to loop with the help of a
syringe which is commonly used to sample load into the injector. After a load
of the sample, manually rotates of the sample injector to the inject position
allows flowing the sample onto a column without any air bubbles, without
disturbing flow rate and pressure.

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2. Septum Injector: In this system, the sample
injects through the rubber septum.
3. Stop flow Injector: In this type of system
mobile phase stopped momentarily and a
fitting at the column head is removed then the
sample is injected.
Column
There are various columns that can be used in HPLC method. They are as follows:
1. Guard Column
2. Derivatizing Column
3. Fast column
4. Analytical Column
5. Preparative Column

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 Guard Column
• Guard columns are placed anterior to the separating column.
• This protects and prolongs the life & usefulness of the separating column.
• They are dependable columns designed to filter or remove:
a. Particles that clog the separating column.
b. Compounds and ions that could ultimately cause ‘baseline drift’, decreased
resolution, decreased sensitivity and create false peaks.
• Compounds that may cause precipitation upon contact with the stationary or
mobile phase. Compounds that may co-elute and cause extraneous peaks &
interfere with the detection and quantification.
• These columns must be changed on a regular basis in order to optimize their
protectiveness.

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Derivatizing column
• Derivatization involves a chemical reaction between an analyte and a
reagent to change the chemical and physical properties of an analyte.
The four main uses of derivatization in HPLC are
• Improve detectability.
• Change the molecular structure or polarity of analyte for better
chromatography.
• Change the matrix for better separation.
• Stabilize a sensitive analyte.

Pre or post primary column derivatization can be done.

Derivatization techniques includes – acetylation, silylation and acid hydrolysis.

Advantages: Although derivatization has drawbacks, it may still be required

to solve a specific separation or detection problem.

Disadvantages: It becomes a complex procedure and so it acts as a source of

error to analysis and increases the total analysis time.

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Fast columns
• This column also have the same internal diameter but much shorter length
than most other columns & packed with particles of 3µm in diameter.
• Increased sensitivity, decreased analysis time, decreased mobile phase usage
& increased reproducibility.

Analytical column
• This is the most important part of HPLC which decides the efficiency of
separation.
• Length- 5 to 25 cm, Internal Diameter 3 to 5mm.
• Particle size of packing material is 3 to 5µm.
• HPLC columns achieve separation by different intermolecular forces
• between the solute & the stationary phase and those between the solute &
mobile phase.

Preparative column
• Length – 10 to 15 cm, internal diameter – 4.6mm.
• Packed with particles having 5µm as diameter.
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• Columns of this time generate 10,000 plates per column.
• It consists of back pressure regulator and fraction collector.
• This back pressure regulator is placed posterior to the HPLC detector.

Detector characteristics
There are three essential detector characteristics.
• The first is the lower limit of detection, the smallest amount of solute
measured in terms of moles (mass-sensitive detectors) or moles per liter
(concentration-sensitive detectors) that can be detected; this entails
distinguishing a signal from the random noise inherent in all electronic
systems.
• A second is the sensitivity, which is the change in signal intensity per unit
change in the amount of solute.
• The third is the linear range i.e., the range of solute amount where the signal
intensity is directly proportional to the amount of solute; doubling the
amount doubles the signal intensity.

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1. UV, VIS, and PDA Detectors
• The UV, VIS, and PDA detectors are categorized as absorbance detectors.
They provide good sensitivity for light-absorbing compounds at micro level.
They are easy to operate and provide good stability.
• UV detector is a very commonly used detector for HPLC analysis. During
the analysis, sample goes through a clear color-less glass cell, called flow
cell. When UV light is irradiated on the flow cell, sample absorbs a part of
UV light. Thus, the intensity of UV light observed for the mobile phase
(without sample) and the eluent containing sample will differ. By measuring
this difference, the amount of sample can be determined. Since the UV
absorbance also differs depend on what wavelength is used, it is important to
choose an appropriate wavelength based on the type of analyte.
• A standard UV detector allows user to choose wavelength between 195 to
370 nm. Most commonly used is 254 nm. Compared to a UV detector, a VIS
detector uses longer wavelength (400 to 700 nm).
• There are detectors that provide wider wavelength selection, covering both
UV and VIS ranges (195 to 700 nm) called UV/VIS detector.

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• 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.

2. Refractive-Index Detector

• RI detector measures change in reflex index. A glass cell is divided

into two chambers (cells). The effluent from LC column flow through
the "sample cell", while other cell called "reference cell" is filled with
only mobile phase.
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• When the effluent going through the sample cell does not contain any
analyte, the solvent inside both cells are the same (Figure A). When a beam
is irradiate on the cells, the observed beam will be straight in this case.
• However, in a case the effluent contains any components other than mobile
phase; bending of the incident beam occurs due to the reflex index
difference between the two solvents (Figure B). By measuring this change,
the presence of components can be observed.
• RI detector has lower sensitivity compared to UV detector, and that's the
main reason why RI is not as commonly used as UV. However there are
some advantages over UV detector.
• It is suitable for detecting all components. For an example, samples which
do not have UV absorption, such as sugar, alcohol, or inorganic ions
obviously cannot be measured by a UV detector. In contrast, change in
reflective index occurs for all analyte, thus a RI detector can be used to
measure all analyte.
• It is applicable for the use with solvent that has UV absorbance. A UV
detector cannot be used with solvent which has UV absorbance. It provides a
direct relationship between the intensity and analyte concentration. The
amount of UV absorbed depends on each analyte, thus the intensity of UV
detector peak does not provide information on the analyte concentration. 16
While intensity observed by a RI detector is comparable to the concentration
of analyte. Because of those advantages, RI is often used for the detection of
sugars and for SEC analysis.

5. Mass Spectrometer
The analytes are detected based on their MW. The obtained information is
especially useful for compound structure identification. However, its use is
not limited to structure identification and can be used to quantify very low
detection limit of elemental and molecular components.

6. Conductivity Detector
Solutions containing ionic components will conduct electricity. Conductivity
detector measures electronic resistance and measured value is directly
proportional to the concentration of ions present in the solution. Thus it is
generally used for ion chromatography.

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7. Fluorescence Detector
The advantage of fluorescence method is its high sensitivity for selective groups
of compounds at few gram level. 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. For some compounds which do not have fluorescence absorbance or
low absorbance, they can be treated with fluorescence derivatives such as dansyl
chloride. The system is easy to operate and relatively stable.

10. Electro Chemical Detector

There are several different types of ECs. The detection is based on amperometry,
polarography, coulometry, and conductrometry. They offer high sensitivity,
simplicity, convenience, and wide-spread applicability. It is especially suitable
for the use with semi- micro or capillary type system.

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 Applications: HPLC is one of the most widely applied analytical separation
techniques. Pharmaceutical: Tablet dissolution of pharmaceutical dosages. Shelf life
determinations of pharmaceutical products. Identification of counterfeit drug
products. Pharmaceutical quality control.
Pharmaceutical Research: All areas including early identification of clinically
relevant molecules to large-scale processing & purification.
• Tablet dissolution.
• Shelf life determinations.
• Pharmaceutical quality control.
• Therapeutic drug monitoring metabolites (substrates, inhibitors).
• Forensics (drugs of abuse).
• Toxicology e.g. Paracetamol poisoning.
Qualitative analysis
• HPLC is used for identification of compound: Here comparison of retention time
of test sample with the reference compound carried out.
• Checking purity of compound: Purity of compound checked by comparison of
chromatogram of test with the reference standard.
• Presence of impurities: If impurities present into sample we observed additional
peak when we compared the chromatogram of test with the reference standard.19
Quantitative analysis
• The measurement of the amount of a compound in a sample (concentration).
• For Preparation of Pure Compounds a pure substance can be prepared for
later use (e.g. organic synthesis, clinical studies, toxicology studies, etc.).
This methodology is called preparative chromatography.
• Is used for assay of many drugs like cephalosporin, furosemide. It is also
used into drug mixture determination.
• Biopharmaceutical and pharmacokinetic study.
• Stability study Purification of some compound of natural or synthetic origin.
• Investigation of biological material such as gastric content, blood, urine
sample etc. are done by HPLC.
• Many poisonous substances can also be investigated by HPLC.
• Inorganic chemistry Chromatographic separation of anion like I-, IO-, ClO4
can be done effectively by using ion exchange chromatography.

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we arrive at the Van Deemter equation for plate height;
HETP = A + B / u + C u
Where u is the average velocity of the mobile phase. A, B, and C are factors
which contribute to band broadening.

The Van Deemter Equation

• Band broadening is a phenomenon that reduces the efficiency of the
separation being carried out leading to poor resolution and chromatographic
performance.
• Measures the efficiency of column
• As a compound passes through the column it slowly diffuses away from the
initial injection band, which is the area of greatest concentration. The initial,
narrow, band that contained all of the sample becomes broader the longer the
analyte remains in the column. This band broadening increases the time
required for complete elution of a particular compound and is generally
undesirable. It must be minimized so that overly broad elution bands do not
overlap with one another.

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Causes
The degree of band broadening (loss of efficiency) naturally increases with the
age of the chromatographic column being used.
A - Eddy diffusion
• The mobile phase moves through the column which is packed with
stationary phase. Solute molecules will take different paths through the
stationary phase at random.
• Some particles flow at longer paths while others at shorter.
• This will cause broadening of the solute band, because different paths are of
different lengths.

Large particles Small particles

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Minimize Eddy Diffusion by
• Using particles with a narrow size distribution.
• Selecting well packed columns.
• Using smaller stationary phase particles.
• By increasing the velocity of mobile phase eddy diffusion can not be
affected.

B - Longitudinal diffusion
• The concentration of analyte is less at the edges of the band than at the
center. Analyte diffuses out from the center to the edges. This causes band
broadening.
• If the velocity of the mobile phase is high then the analyte spends less time
on the column, which decreases the effects of longitudinal diffusion.
• Increasing the velocity of mobile phase decreases the longitudinal diffusion.
In this analyte move from higher concentration to lower concentration.

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Minimise Longitudinal Diffusion by
• Using higher mobile phase flow rates.
• Keep system tubing short and as narrow as possible (careful with back-
pressure) (<0.12mm i.e. is ideal).
• Use correct nuts, ferrules and fittings wherever possible.

C - Resistance to mass transfer

• The analyte takes a certain amount of time to equilibrate between the
stationary and mobile phase.
• If the velocity of the mobile phase is high, and the analyte has a strong
affinity for the stationary phase, then the analyte in the mobile phase will
move ahead of the analyte in the stationary phase.
• The band of analyte is broadened. The higher the velocity of mobile phase,
the worse the broadening becomes.

Large Stationary Phase Particles

• Greater possible pore distance for analyte diffusion. Diffusion time
increased.
• Differences in diffusion times out of the pore are amplified Peak efficiency
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Small Stationary Phase Particles
• Reduced possible pore distance for analyte diffusion.
• Diffusion time decreased.
• Differences in diffusion times out of the pore are reduced Peak efficiency
increases (peak becomes narrower).
Mass transfer increases with increasing the velocity of mobile phase.

Van Deemter plots

A plot of plate height vs. average linear velocity of mobile phase.

Such plots are of considerable use in determining the optimum mobile phase
flow rate. 25
THANK
YOU

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