Transforming education transforming India

TERM PAPER: Bioanalytical Techniques

Topic: HPLC(High Performance Liquid
Chromatography)

SUBMITTED BY:-
SUBMITTED TO:-
Name: -Nitish Pathania Dr.
Mohit Kumar
Sec: -K7802-A06
Reg. No. : -10803694
ACKNOWLEDGEMENT

As The Saying goes

“No Work Is Done Without Cooperation”

I am extremely grateful and remain indebted to my friends and my guide Dr. Mohit
Kumar for being a source of inspiration and for their constant support in the Design,
Implementation and Evaluation of this Term Paper. I am thankful to him for their constant
constructive criticism and invaluable suggestions, which benefited me a lot while
developing this paper on topic “HPLC(High Performance Liquid Chromatography)”.
Also they provide me a constant source of inspiration and motivation for doing hard work
while preparing this term paper. Through this column, it would be my utmost pleasure to
express my warm thanks to them for their encouragement, co-operation and consent
without which I mightn’t be able to accomplish this work of Term Paper.

I also want to express my gratitude to my God and my parents those are a great source for
me of inspiration. I am again very thankful to Dr. Mohit Kumar who gave me this
chance to express my thoughts with the help of this Term paper to make my concepts very
clear and make me aware how to handle HPLC instrument and what are various
applications, components of an HPLC instrument.

Thanking You Nitish Pathania

 High Performance Liquid Chromatography:

Contents:

1. Introduction HPLC:
2. Theory of HPLC:
• Principle of chromatography:
• Qualitative and Quantitative analysis:
3. Configuration of an HPLC system:
• Pumping unit:
• Sample-injection unit:
• Separation unit:
• Detection unit:
• Data-processing unit

4. Types of HPLC:
5. Operation of the HPLC - General Procedure:
6. Functional description of the instrument:

a. Pump:
b. Injector:
c. Detector:
• Fluorescence (FL) detector:
• UV/UV-VIS detectors:
• Electrochemical detector (ECD):
d. Columns:

• Normal phase HPLC:

• Reversed phase HPLC:
• Retention time:

7. Type of separation mode

a. Ion exchange mode:

b. Size exclusion mode:
8. Applications of HPLC:

9. References:
High Performance Liquid Chromatography:

Introduction HPLC:
High Performance Liquid Chromatography (HPLC) is one mode of chromatography, one of the
most used analytical techniques. Chromatographic process can be defined as separation technique
involving mass-transfer between stationary and mobile phase.

Chromatography is based on the principal that under the same conditions, the time between
the injection of a component into the column and the elution of that component is
constant. This characteristic is used to perform qualitative or quantitative analysis.

HPLC utilizes a liquid mobile phase to separate the components of a mixture. The stationary
phase can be a liquid or a solid phase. These components are first dissolved in a solvent, and then
forced to flow through a chromatographic column under a high pressure.

In the column, the mixture separates into its components. The amount of resolution is important,
and is dependent upon the extent of interaction between the solute components and the stationary
phase. The stationary phase is defined as the immobile packing material in the column.
The interaction of the solute with mobile and stationary phases can be manipulated through
different choices of both solvents and stationary phases. As a result, HPLC has the ability to
easily separate a wide variety of chemical mixtures.

High-performance liquid chromatography (HPLC) is a form of liquid chromatography 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. Compounds are separated by
injecting a plug of the sample mixture onto the column. The different components in the mixture
pass through the column at different rates due to differences in their partitioning behavior
between the mobile liquid phase and the stationary phase.

HPLC is short for the High Performance LC. HPLC is an analysis method that yields high
performance and high speed compared with traditional column chromatography because of the
forcibly pumped mobile phase. Recently, ultrafast analysis using a high-pressure-resistant
apparatus has been attracting attention. UHPLC (Ultra High Performance LC) is becoming
established as an abbreviation for this ultrafast LC method.

Initially, pressure was selected as the principal criterion of modern liquid chromatography and
thus the name was "high pressure liquid chromatography" or HPLC. This was, however, an
unfortunate term because it seems to indicate that the improved performance is primarily due to
the high pressure. This is, however, not true. In fact, high performance is the result of many
factors: very small particles of narrow distribution range and uniform pore size and distribution,
high pressure column slurry packing techniques, accurate low volume sample injectors, and
sensitive low volume detectors and, of course, good pumping systems. Naturally, pressure is
needed to permit a given flow rate of the mobile phase.

Theory of HPLC:
HPLC is a dynamic adsorption process. Analyte molecules, while moving through the porous
packing beads, tend to interact with the surface adsorption sites. Depending on the HPLC mode,
the different types of the adsorption forces may be included in the retention process:
Hydrophobic (non-specific) interactions are the main ones in reversed-phase (RP) separations.
Dipole-dipole (polar) interactions are dominant in normal phase (NP) (mode.
Ionic interactions are responsible for the retention in ion-exchange chromatography.
All these interactions are competitive. Analyte molecules are competing with the eluent
molecules for the adsorption sites. So, the stronger analyte molecules interact with the surface.
The weaker the eluent interaction, the longer the analyte will be retained on the surface.

Picture of an HPLC instrument

Principle of chromatography:
Chromatography is based on the principal that under the same conditions, the time between
the injection of a component into the column and the elution of that component is constant.

This characteristic is used to perform qualitative or quantitative analysis.

Chromatography is a technique by which a mixture sample is separated into components.
Although originally intended to separate and recover (isolate and purify) the components of a
sample, today, complete chromatography systems are often used to both separate and quantify
sample components.

The term, “chromatography" was coined by the Russian botanist, Tswett, who demonstrated that,
when a plant extract was carried by petroleum ether through a column consisting of a glass tube
packed with calcium carbonate powder, a number of dyes were separated, as shown in Figure. He
named this analysis method "Chromatographie" after "chroma" and "graphos", which are Greek
words meaning "color" and “to draw," respectively.
 Diagram showing Tswett's experiment

"Chromatography" represents a separation technique; whereas a "chromatograph"is a system

for performing chromatography. The chart displaying the time-dependent change in signal
intensity as a result of the separation is called a "chromatogram”.

Qualitative analysis:
A standard sample of aspartame was measured, yielding a peak at 12.5 minutes. This peak
corresponds to aspartame, itself.
Next, a sample prepared from a beverage was measured under the same conditions, yielding a
number of peaks. The peak appearing at 12.5 minutes can be regarded as that of aspartame.
The following conditions were the same: the type of filters, column sizes, column temperature,
composition of the mobile phase, and flow rate.

Example of aspartame measurement

Quantitative analysis:
The height and area of a peak are proportional to the concentration of the corresponding
component. A calibration curve is created using the standard sample. The concentration of
aspartame in the beverage can be determined from the peak area of the detected aspartame.

Configuration of an HPLC system:

An HPLC system consists of a pumping unit, sample-injection unit, separation unit, detection
unit, and data-processing unit. Each of these units is essential for performing the analysis.
 1. Pumping unit:
It is necessary to pump the eluent at a constant flow rate and pressure.
Conventional, analytical HPLC pumps are the most common type, but semi-micro and a
preparative pumps are also used depending on the range of the eluent flow rate required.
Analyses were first performed using isocratic separations in which the eluent composition
remains unchanged during the analysis.
2. Sample-injection unit:
A sample is injected into the flow path for analysis.
This is accomplished via a manual injector or an autosampler. Each type is equipped with six-
port valves, so that a sample can be injected into the flow path at continuous pressure.

1) The knob is set at the "LOAD" position for sample injection, as shown in the left image. Using
a microsyringe, the sample can be injected into the sample loop, which is separated from the flow
path.
2) The knob is turned to the "INJECT" position. The eluent travels through the loop from the
pump then delivers the sample to the column. The autosampler can perform similar work
automatically, enabling unmanned continuous operation

3. Separation unit:
A column is selected to suit both the sample and the purpose of separation. The column oven is
used to maintain a constant column temperature. If the column temperature were allowed to vary
during qualitative or quantitative analysis, the elution time of the components would change, so
that an accurate analysis could not be performed. An analysis temperature between 25 and 50℃
is often selected.
 4. Detection unit:
The components eluted from the column are detected, and the detection data are converted into
an electrical signal. The detector is selected to suit the sample:

Major types of detector:

UV detector The light source is a D2 lamp. This detector is used mainly to
detect components having an absorption wavelength of 400 nm or
less in the ultraviolet region.
UV-VIS detector A D2 lamp and a W lamp are used as the light source. This detector
is effective in the detection of coloring components such as dyes
and stains because of coverage of the visible light region.

Diode array detector Data on the spectrum from the ultraviolet to visible light range is
(DAD) also collected.
Fluorescence (FL) Fluorescent substances can be detected specifically with high
detector sensitivity.
Differential refractive Change in the refractive index is detected. Components absorbing
index (RI) detector no ultraviolet light can also be detected despite low sensitivity.

Conductivity detector Mainly inorganic ions are detected by monitoring the conductivity.

5. Data-processing unit
The concentration of each detected component is calculated from the area or height of the
corresponding peak, and reported. Although previously, easy-to-use integrators were mainly
used, systems in which a PC performs both the operation of the units and the analysis of the
results have recently played a central role.

Types of HPLC:
There are many ways to classify liquid column chromatography. If this classification is based on
the nature of the stationary phase and the separation process, three modes can be specified.

a. Adsorption chromatography: the stationary phase is an adsorbent (like silica gel or any other
silica based packing) and the separation is based on repeated adsorption-desorption steps.

b. Ion-exchange chromatography: the stationary bed 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.

c. Size exclusion chromatography: the column is filled with material having precisely
controlled pore sizes, and the sample is simply screened or filtered according to its solvated
molecular size.
Larger molecules are rapidly washed through the column; smaller molecules penetrate inside the
porous of the packing particles and elute later. This technique is also called gel filtration or gel
permeation chromatography.

Comparison of HPLC with classical LC techniques:

• High resolution
• Small diameter (4.6 mm), stainless steel, glass or titanium columns;
• Column packing with very small (3, 5 and 10 μm) particles;
• Relatively high inlet pressures and controlled flow of the mobile phase;
• Continuous flow detectors capable of handling small flow rates and detecting very small
amounts;
• Rapid analysis;

Operation of the HPLC - General Procedure:

Step 1: Preparing the System Column – If the column mounted on the system is not correct, the
requisite assay, pump an appropriate storage liquid through the column and removing it. Cap the
ends and return the column to the correct drawer. Check the column's direction of flow. After
installing the correct column, tighten the connections so they do not leak.

Mobile Phase – The mobile phase must be free of dissolved gasses so that no bubbles form inside
the instrument during the run. When preparing a mobile phase with two or more components, be
aware that large amounts of heat can be generated by the mixing process. The selection of mobile
phases is based on the relative polarity of the analytes and the column packing. Adding acetic
acid to a mobile phase will increase its overall polarity and result in better separations when
running on a non polar stationary phase. Controlling the mobile phase's pH by the addition of
acid, buffer, or base will often improve reproducibility.
Step 2: Prime the pump. Open the “prime” or the “purge” valve on the pump module. Place a
beaker under the outlet. Activate the “prime” or “purge” function on the pump. If the pump does
not have this function, turn up the flow and switch it on. Run the system for a few minutes. When
the pump is properly primed, the system will deliver a smooth flow of mobile phase from the
outlet, produce a steady sound with no burping or grinding, and the inlet line will be free of air
bubbles. Turn off the pump and close the valve.

Step 3: Set the detector wavelength. Turn on the detector power and allow the unit to warm up.
Using the keypad or the control computer GUI, set the wavelength(s).

Step 4: Start the mobile phase flow. Use the pump controller, to set the flow rate for the mobile
phase. Restart the pump. Watch the system's pressure indicator or gauges to see that it does not
exceed the maximum allowed for the various components. If pressures become too high, slow
down the flow rate.

If pressure continues to rise, turn off the pump, and perform the following procedures:

a. If the system has a guard column, replace it.

b. The analytical column may need to be back flushed. Remove the column and reverse it so that
mobile phase flows through it in the wrong direction. Monitor the detector output, when the
signal is stable, begin running the samples.

Step 5: Inject the samples.

• Before injecting a sample, check the needle's tip. HPLC Needles have a smooth or blunt
tip. Remember this is a two-step process; syringe injection followed by turning the valve
from "load" to "inject."
• Open the injection valves by turning them to “load.” Insert needle into the plastic needle
guide as far as it will go. Smoothly inject the appropriate amount of sample.
• After the syringe is completely empty, quickly and smoothly turn the valve to “inject.” It
is safe to leave the syringe in place.
• Start the data system recording.
• When the injector is set to the 'load" position this loop is isolated from the mobile phase
flow. Turning the valve to "inject" diverts the mobile phase flow through the sample loop
and sweeps the sample onto the column.

Step 6: After the run. Stop the data station. Return the injector to the "load" position and remove
the syringe.

Step 7: Cleanup. Turn off the detector, pump, and any other components. Rinse any sample
residues from the syringe. Record the sample date and initials in the appropriate notebook. Label
the chromatogram with:
1. The users name, date, and sample ID.
2. The column, mobile phase, flow rate, and detector wavelength.
3. The system number and its calibration date.

Step 8: Is this data any good? Evaluate the chromatogram, look for:

1. Good separation. Is the first analyte of interest sufficiently separated from the solvent
front? Are all analytes separated or are peaks running into each other?
2. Peak broadening and tailing. Broad peaks are difficult to quantify. Excessive tailing can
hide minor peaks.
3. Irregular retention times. Retention times should not shift from one run to the next
although small shifts caused by operator variability are normal.

Functional description of the instrument:

1. Pump:
High pressure pumps are needed to force solvents through packed stationary phase beds. Smaller
bed particles require higher pressures. There are many advantages to using smaller particles, but
they may not be essential for all separations.
The most important advantages are: higher resolution, faster analyses, and increased sample load
capacity. However, only the most demanding separations require these advances in significant
amounts. Many separation problems can be resolved with larger particle packing’s that require
less pressure.
An additional feature found on the more elaborate pumps is external electronic control. Although
it adds to the expense of the pump, external electronic control is a very desirable feature when
automation or electronically controlled gradients are to be run.
Modern pumps have the following parameters:
Flow rate range: 0.01 to 5 mL/min
Flow rate stability: not more than 1%
For SEC flow rate stability should be less than 0.2%
Maximum pressure: up to 300 hPa.
It is desirable to have an integrated degassing system, either helium purging, or membrane
filtering.

2. Injector:
Sample introduction can be accomplished in various ways. The simplest method is to use an
injection valve. In more sophisticated LC systems, automatic sampling devices are incorporated
where the sample is introduced with the help of autosamplers and microprocessors.
It is always best to remove particles from the sample by filtering over a 5 μm filter, or
centrifuging, since continuous injections of particulate material will eventually cause blockages
in injection devices or columns.
 3. Detector:
Optical detectors are used most frequently in liquid chromatographic systems. These detectors
pass a beam of light through the flowing column effluent as it passes through a low volume (~ 10
μl) flow cell. The variations in light intensity caused by UV absorption, fluorescence emission or
change in refractive index, from the sample components passing through the cell, are monitored
as changes in the output voltage. These voltage changes are recorded on a strip chart recorder and
frequently are fed into a computer to provide retention time and peak area data.

Other detectors in common use include: Photo Diode Array UV detector (PAD), refractive index
(RI), fluorescence (FLU), electrochemical (EC). The RI detector is universal but also the less
sensitive one.

1. Fluorescence (FL) detector:

A UV/UV-VIS detector monitors the absorption of light with a specified wavelength. Some
substances absorb light at one wavelength, and then emit light called fluorescence at another
wavelength. This is a phenomenon in which a substance absorbs light to reach a high-energy
level and then emits light to return to its original level.

Fluorescence detection is suitable for trace analysis because of generally having high sensitivity
and selectivity (not detecting impurities).
There are not many components that originally emit fluorescence (natural fluorescence).
However, amino acids, etc. can be detected as fluorescent substances, after reaction with a
fluorescence reagent. This method makes it possible to measure various components with high
sensitivity.

2. UV/UV-VIS detectors:
UV/UV-VIS detectors are most frequently used to measure components showing an absorption
spectrum in the ultraviolet or visible region.
A UV detector employs a deuterium discharge lamp (D2 lamp) as a light source, with the
wavelength of its light ranging from 190 to 380 nm.
If components are to be detected at wavelength longer than this, a UV-VIS detector is used,
which employs an additional tungsten lamp (W lamp).

A many components have an absorption in the ultraviolet or visible region. The measurement is
performed at a certain fixed wavelength.
If all of the components of a sample are to be detected with high sensitivity, the time program
function can be used to measure each component along with its maximum absorption wavelength
during the analysis.

3. Electrochemical detector (ECD):

An ECD is used to measure components displaying oxidation-reduction reactions, and detects
electric currents generated by these reactions. The ECD has high selectivity, because the
necessary voltage to cause an oxidation-reduction reaction depends on the component. The ECD
has also high sensitivity, and is often used for measurement of biogenic substances such as
catecholamine.
 4. Columns:

There are two main classes of column: "normal" and "reversed" phase. Normal phase columns
are most usually packed with silica gel; they work in the
partition/adsorption mode in the same manner as a normal silica
gel column in conventional chromatography. Reversed phase
chromatography, which is the most common form of HPLC, is a
type of partition chromatography.

Frequently, reversed phase columns are packed with a

chemically bonded octadecylsilyl coated silica; such columns are
referred to as C-18 and are very non-polar. Other popular bonded
phase columns have octasilyl, cyanopropyl, or phenylsilyl
packings. The eluent used with reversed phase columns is
relatively polar, e.g. MeOH/H2O.

Unlike normal phase chromatography, the more polar

components of a mixture elute first, since these partition relatively unfavorably on the highly
non-polar packing. Increasing the polarity of the solvent increases the retention time of a
particular component. The situation is the reverse of normal adsorption chromatography:

 Normal phase HPLC:

It is described as "normal", it isn't the most commonly used form of HPLC. The column is filled
with tiny silica particles, and the solvent is non-polar - hexane, for example. A typical column has
an internal diameter of 4.6 mm (and may be less than that), and a length of 150 to 250 mm.
Polar compounds in the mixture being passed through the column will stick longer to the polar
silica than non-polar compounds will. The non-polar ones will therefore pass more quickly
through the column.

 Reversed phase HPLC:

In this case, the column size is the same, but the silica is modified to make it non-polar by
attaching long hydrocarbon chains to its surface - typically with either 8 or 18 carbon atoms in
them. A polar solvent is used - for example, a mixture of water and an alcohol such as methanol.

In this case, there will be a strong attraction between the polar solvent and polar molecules in the
mixture being passed through the column. Non-polar compounds in the mixture will tend to form
attractions with the hydrocarbon groups because of vander Waals dispersion forces. They will
also be less soluble in the solvent because of the need to break hydrogen bonds as they squeeze in
between the water or methanol molecules.

That means that now it is the polar molecules that will travel through the column more quickly.

Reversed phase HPLC is the most commonly used form of HPLC.

Normal vs. Reversed Phase HPLC

Normal Reverse
Packing polarity High Low
Solvent polarity Low High
Elution order Non-polar first, then polar Polar first, then non-polar
Effect of increasing solvent polarity Decreases retention time Increases retention time

 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.

Type of separation mode - Separation depends on the relationships among the

sample, filler, and eluent:

1. Ion exchange mode:

Ionic substances, including amino acids and inorganic ions pass through an ODS column without
being separated by ODS. Such substances can be separated with an ion exchange resin. Negative
ions such as chloride (Cl-) and sulfide (SO42-) ions, and positive ions such as Na+ and Ca2+ are
separated with anion- and cation-exchange resin. Despite having both -NH2 (amino, cationic) and
-COOH (carboxyl, anionic) groups, amino acids can be separated with cation-exchange resins
using an acidic eluent to suppress the dissociation of -COOH.

2. Size exclusion mode:

Macromolecular components such as synthetic resins and proteins can be separated according to
their size, with sieve-like fillers. Small molecules can pass through the filler via these holes, but
spend much time running through mazes in the fillers. Meanwhile, large molecules unable to
enter the holes migrate among the fillers, arriving earlier at the outlet of the column.
Once a standard sample with known molecular weight is measured, a calibration curve can be
prepared, in which the vertical axis represents logarithmic molecular weight and the horizontal
axis represents elution time (volume). Using this curve, the molecular-weight distribution and
mean molecular weight of an unknown sample can be determined.
This separation mode is referred to as size exclusion (SEC:Size ExclusionChromatography) or
gel permeation (GPC: Gel Permeation Chromatography).

Applications of HPLC:
• DNA from different sources is placed into the column and treated to allow it to separate and
recombine. Two strands from the same source, termed a homoduplex, bind strongly and tend
to remain together longer. Heteroduplexes do not attract as strongly, and remain unattached
more often.

• A high-performance liquid chromatography system allows geneticists to perform molecular

research efficiently by automating necessary processes.

• Analytical HPLC where the focus is to obtain information about the sample compound. The
information that can be obtained using this HPLC includes identification, quantification, and
resolution of a compound.

• Chemical Separations can be accomplished using HPLC by utilizing the fact that certain
compounds have different migration rates given a particular column and mobile phase. Thus,
the chromatographer can separate compounds from each other using HPLC.

• HPLC of Proteins and Polynucleotide’s: Identification of compounds by HPLC is a

crucial part of any HPLC assay. In order to identify any compound by HPLC a detector must
first be selected. Once the detector is selected and is set to optimal detection settings, a
separation assay must be developed.

• The Role of HPLC in Drug Analysis: The most characteristic feature of the development in
the methodology of pharmaceutical and biomedical analysis during the past 25 years is that
HPLC became undoubtedly the most important analytical method for identification and
quantification of drugs, either in their active pharmaceutical ingredient or in their
formulations during the process of their discovery, development and manufacturing

References:

Internet websites:
 1. http://ntri.tamuk.edu/hplc/hplc.html#introduction
2. http://www.savant4training.com/clc_10.htm
3. http://www.colorado.edu/chemistry/chem5181/c1_introduction

4. http://www.hitachi-hitec.com/global/science/lc/lc_basic_2.html

5. http://www.files.chem.vt.edu/chem-ed/sep/lc/hplc.html

6. http://www.chemistry.nmsu.edu/Instrumentation/NMSU_HPLC_bkg.html

7. http://www.chemguide.co.uk/analysis/chromatography/hplc.html

Reference textbooks:
1. D.A.Skoog, D.M.West, F.J.Holler: Fundamentals of Analytical Chemistry, Saunders
College Publishing
2. A.Gratzfeld-Husgen, R.Schuster, HPLC for Food Analysis, Hewlett Packerd

3. S. Ahuja and M.W. Dong (ed), Handbook of Pharmaceutical Analysis by HPLC,

Elsevier/Academic Press, 2005.

4. Bryan Williams,Keith Wilson: Principles and Techniques of Biochemistry and Molecular

Biology, Cambridge University Press, 2006