HPLC

SCHOOL OF ELECTRICAL AND COMPUTER ENGINEERING

CONTROL AND INSTRUMENTATION ENGINEERING

ADVANCED INSTRUMENTATION ASSIGNMENT

TITLE ;HIGH PERFORMANCE LIQUID CHROMOTOGRAPH

(HPLC)

NAME GIRMA BEKELE

Sub to ass/prof N. K Kumar

 HPLC

CONTENTS PAGES

Contents
Chromatography ........................................................................................................................................... 1
History of chromatography ....................................................................................................................... 1
Chromatography terms ............................................................................................................................. 1
Liquid chromatography ................................................................................................................................. 3
High-Performance Liquid Chromatography (HPLC) ...................................................................................... 4
How Does High Performance Liquid Chromatography Work? ................................................................. 6
HPLC Operation ......................................................................................................................................... 7
What Is a Detector? ...................................................................................................................................... 8
What Is a Chromatogram? ............................................................................................................................ 8
What are the different types of contaminants in water that affect HPLC results? ...................................... 9
1. Organics ................................................................................................................................................ 9
(i) Reducing column life ........................................................................................................................ 9
(ii) Reducing sensitivity ......................................................................................................................... 9
(iii) Shift in retention time ................................................................................................................... 10
2. Ions ...................................................................................................................................................... 10
Advantages.............................................................................................................................................. 10
Disadvantages ......................................................................................................................................... 10
Conclusions ................................................................................................................................................. 11
References: ................................................................................................................................................. 12
 HPLC

Figure contents pages

Fig 1 characteristics of chromatography ………………………..2
Fig 2 liquid chromatography…………………………………….4
Fig 3 chromatography in labratory………………………………4
Fig 4 HPLC from left to righy…………………………………...5
Fig 5 HPLC system……………………………………………....6
Fig 6 a typical HPLC…………………………………………….7
Fig 7 chromatography column…………………………………...8
Fig 8 how peaks are created……………………………………...8
 HPLC

Chromatography

History of chromatography
Chromatography was first employed in Russia by the Italian-born scientist Mikhail Tsvet in
1900.[4] He continued to work with chromatography in the first decade of the 20th century,
primarily for the separation of plant pigments such as chlorophyll, carotenes, and xanthophylls.
Since these components have different colors (green, orange, and yellow, respectively) they gave
the technique its name. New types of chromatography developed during the 1930s and 1940s made
the technique useful for many separation processes.
Chromatography technique developed substantially as a result of the work of Archer John Porter
Martin and Richard Laurence Millington Synge during the 1940s and 1950s, for which they won
the 1952 Nobel Prize in Chemistry. They established the principles and basic techniques of
partition chromatography, and their work encouraged the rapid development of several
chromatographic methods: paper chromatography, gas chromatography, and what would become
known as high-performance liquid chromatography. Since then, the technology has advanced
rapidly. Researchers found that the main principles of Tsvet's chromatography could be applied in
many different ways, resulting in the different varieties of chromatography described below.
Advances are continually improving the technical performance of chromatography, allowing the
separation of increasingly similar molecules. Chromatography has also been employed as a method
to test the potency of cannabis.

Chromatography is a laboratory technique for the separation of a mixture. The mixture is

dissolved in a fluid called the mobile phase, which carries it through a structure holding another
material called the stationary phase. The various constituents of the mixture travel at different
speeds, causing them to separate. The separation is based on differential partitioning between the
mobile and stationary phases. Subtle differences in a compound's partition coefficient result in
differential retention on the stationary phase and thus affect the separation.
Chromatography may be preparative or analytical. The purpose of preparative chromatography is
to separate the components of a mixture for later use, and is thus a form of purification. Analytical
chromatography is done normally with smaller amounts of material and is for establishing the
presence or measuring the relative proportions of analytes in a mixture. The two are not mutually
exclusive.

Chromatography terms
 The analyte is the substance to be separated during chromatography. It is also normally what
is needed from the mixture.
 Analytical chromatography is used to determine the existence and possibly also the
concentration of analyte(s) in a sample.
 A bonded phase is a stationary phase that is covalently bonded to the support particles or to
the inside wall of the column tubing.

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 A chromatogram is the visual output of the chromatograph. In the case of an optimal

separation, different peaks or patterns on the chromatogram correspond to different
components of the separated mixture.

Fig characteristics of chromotography

Plotted on the x-axis is the retention time and plotted on the y-axis a signal (for example
obtained by a spectrophotometer, mass spectrometer or a variety of other detectors)
corresponding to the response created by the analytes exiting the system. In the case of an
optimal system the signal is proportional to the concentration of the specific analyte
separated.

 A chromatograph is equipment that enables a sophisticated separation, e.g. gas

chromatographic or liquid chromatographic separation.
 Chromatography is a physical method of separation that distributes components to
separate between two phases, one stationary (stationary phase), the other (the mobile
phase) moving in a definite direction.
 The eluate is the mobile phase leaving the column. This is also called effluent.
 The eluent is the solvent that carries the analyte.
 The eluite is the analyte, the eluted solute.
 An eluotropic series is a list of solvents ranked according to their eluting power.
 An immobilized phase is a stationary phase that is immobilized on the support
particles, or on the inner wall of the column tubing.
 The mobile phase is the phase that moves in a definite direction. It may be a liquid
(LC and Capillary Electrochromatography (CEC)), a gas (GC), or a supercritical fluid
(supercritical-fluid chromatography, SFC). The mobile phase consists of the sample
being separated/analyzed and the solvent that moves the sample through the column.
In the case of HPLC the mobile phase consists of a non-polar solvent(s) such as hexane
in normal phase or a polar solvent such as methanol in reverse phase chromatography
and the sample being separated. The mobile phase moves through the chromatography
column (the stationary phase) where the sample interacts with the stationary phase and
is separated.
 Preparative chromatography is used to purify sufficient quantities of a substance for
further use, rather than analysis.
 The retention time is the characteristic time it takes for a particular analyte to pass
through the system (from the column inlet to the detector) under set conditions. See
also:Kovats' retention index
 The sample is the matter analyzed in chromatography. It may consist of a single
component or it may be a mixture of components. When the sample is treated in the
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course of an analysis, the phase or the phases containing the analytes of interest is/are
referred to as the sample whereas everything out of interest separated from the sample
before or in the course of the analysis is referred to as waste.
 The solute refers to the sample components in partition chromatography.
 The solvent refers to any substance capable of solubilizing another substance, and
especially the liquid mobile phase in liquid chromatography.
 The stationary phase is the substance fixed in place for the chromatography
procedure. Examples include the silica layer in thin layer chromatography
 The detector refers to the instrument used for qualitative and quantitative detection of
analytes after separation.
Chromatography is based on the concept of partition coefficient. Any solute partitions between
two immiscible solvents. When we make one solvent immobile (by adsorption on a solid support
matrix) and another mobile it results in most common applications of chromatography. If the
matrix support, or stationary phase, is polar (e.g. paper, silica etc.) it is forward phase
chromatography, and if it is non-polar (C-18) it is reverse phase.
There are different type of chromatography. From those liquid chromatography is one type
of it.
Liquid chromatography
Liquid chromatography (LC) is a separation technique in which the mobile phase is a liquid. It can
be carried out either in a column or a plane. Present day liquid chromatography that generally
utilizes very small packing particles and a relatively high pressure is referred to as high-
performance liquid chromatography (HPLC).
In HPLC the sample is forced by a liquid at high pressure (the mobile phase) through a column
that is packed with a stationary phase composed of irregularly or spherically shaped particles,
a porous monolithic layer, or a porous membrane. HPLC is historically divided into two different
sub-classes based on the polarity of the mobile and stationary phases. Methods in which the
stationary phase is more polar than the mobile phase (e.g., toluene as the mobile phase, silica as
the stationary phase) are termed normal phase liquid chromatography (NPLC) and the opposite
(e.g., water-methanol mixture as the mobile phase and C18 (octadecylsilyl) as the stationary phase)
is termed reversed phase liquid chromatography (RPLC).
Specific techniques under this broad heading are listed below.

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Fig 2 liquid chromatography

Liquid chromatography, which encompasses methods such as High Performance Liquid
Chromatography (HPLC) and Ion Chromatography, is a separation technique. It is used to identify,
quantify and purify individual components in a mixture.

Fig 3 chromatography in laboratory

Chromatography is one of the most powerful tools in analytical chemistry and is one of the most
commonly found instruments in the laboratory. In this technique, the mobile phase is a liquid.

High-Performance Liquid Chromatography (HPLC)

High-pressure liquid chromatography, now known as high-performance liquid chromatography
(HPLC), is a chromatographic technique used to identify, quantify, separate and purify individual
compounds present in a mixture.
It relies on pumps to pass a pressurized liquid solvent containing the sample mixture through a
column filled with a solid adsorbent material. Each component in the sample interacts slightly
differently with the adsorbent material, causing different flow rates for the different components
and leading to the separation of the components as they flow out of the column.
HPLC has been used for manufacturing (e.g., during the production process of pharmaceutical and
biological products), legal (e.g., detecting performance enhancement drugs in urine), research
(e.g., separating the components of a complex biological sample, or of similar synthetic chemicals
from each other), and medical (e.g., detecting vitamin D levels in blood serum) purposes.[1]

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Fig 4 An HPLC. From left to right

A pumping device generating a gradient of two different solvents- a steel-enforced column and a
detector for measuring the absorbance.
HPLC relies on pumps to pass a pressurized liquid and a sample mixture through a column filled
with adsorbent, leading to the separation of the sample components. The active component of the
column, the adsorbent, is typically a granular material made of solid particles (e.g., silica,
polymers, etc.), 2–50 μm in size. The components of the sample mixture are separated from each
other due to their different degrees of interaction with the adsorbent particles. The pressurized
liquid is typically a mixture of solvents (e.g., water, acetonitrile and/or methanol) and is referred
to as a "mobile phase". Its composition and temperature play a major role in the separation process
by influencing the interactions taking place between sample components and adsorbent. These
interactions are physical in nature, such as hydrophobic (dispersive), dipole–dipole and ionic, most
often a combination.
HPLC is distinguished from traditional ("low pressure") liquid chromatography because
operational pressures are significantly higher (50–350 bar), while ordinary liquid chromatography
typically relies on the force of gravity to pass the mobile phase through the column. Due to the
small sample amount separated in analytical HPLC, typical column dimensions are 2.1–4.6 mm
diameter, and 30–250 mm length. Also HPLC columns are made with smaller adsorbent particles
(2–50 μm in average particle size). This gives HPLC superior resolving power (the ability to
distinguish between compounds) when separating mixtures, which makes it a popular
chromatographic technique.
The schematic of a HPLC instrument typically includes a degasser, sampler, pumps, and a detector.
The sampler brings the sample mixture into the mobile phase stream which carries it into the
column. The pumps deliver the desired flow and composition of the mobile phase through the
column. The detector generates a signal proportional to the amount of sample component emerging
from the column, hence allowing for quantitative analysis of the sample components. A digital
microprocessor and user software control the HPLC instrument and provide data analysis. Some
models of mechanical pumps in a HPLC instrument can mix multiple solvents together in ratios
changing in time, generating a composition gradient in the mobile phase. Various detectors are in
common use, such as UV/Vis, photodiode array (PDA) or based on mass spectrometry. Most
HPLC instruments also have a column oven that allows for adjusting the temperature at which the
separation is performed.

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How Does High Performance Liquid Chromatography

Work?
The components of a basic high-performance liquid chromatography [HPLC] system are shown
in the simple diagram in Figure below.

A reservoir holds the solvent [called the mobile phase, because it moves]. A high-pressure pump
[solvent delivery system or solvent manager] is used to generate and meter a specified flow rate of
mobile phase, typically milliliters per minute. An injector [sample manager or autosampler] is able
to introduce [inject] the sample into the continuously flowing mobile phase stream that carries the
sample into the HPLC column. The column contains the chromatographic packing material needed
to effect the separation. This packing material is called the stationary phase because it is held in
place by the column hardware. A detector is needed to see the separated compound bands as they
elute from the HPLC column [most compounds have no color, so we cannot see them with our
eyes]. The mobile phase exits the detector and can be sent to waste, or collected, as desired. When
the mobile phase contains a separated compound band, HPLC provides the ability to collect this
fraction of the eluate containing that purified compound for further study. This is called preparative
chromatography [discussed in the section on HPLC Scale].

Note that high-pressure tubing and fittings are used to interconnect the pump, injector, column,
and detector components to form the conduit for the mobile phase, sample, and separated
compound bands.

Figure 5 High-Performance Liquid Chromatography [HPLC] System

The detector is wired to the computer data station, the HPLC system component that records the
electrical signal needed to generate the chromatogram on its display and to identify and quantitate
the concentration of the sample constituents (see Figure F). Since sample compound characteristics
can be very different, several types of detectors have been developed. For example, if a compound
can absorb ultraviolet light, a UV-absorbance detector is used. If the compound fluoresces, a
fluorescence detector is used. If the compound does not have either of these characteristics, a more
universal type of detector is used, such as an evaporative-light-scattering detector [ELSD]. The
most powerful approach is the use multiple detectors in series. For example, a UV and/or ELSD
detector may be used in combination with a mass spectrometer [MS] to analyze the results of the
chromatographic separation. This provides, from a single injection, more comprehensive

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information about an analyte. The practice of coupling a mass spectrometer to an HPLC system is
called LC/MS.

Figure 6 A Typical HPLC [Waters Alliance] System

HPLC Operation
A simple way to understand how we achieve the separation of the compounds contained in a
sample is to view the diagram in Figure 6.

Mobile phase enters the column from the left, passes through the particle bed, and exits at the right.
Flow direction is represented by green arrows. First, consider the top image; it represents the
column at time zero [the moment of injection], when the sample enters the column and begins to
form a band. The sample shown here, a mixture of yellow, red, and blue dyes, appears at the inlet
of the column as a single black band. [In reality, this sample could be anything that can be dissolved
in a solvent; typically the compounds would be colorless and the column wall opaque, so we would
need a detector to see the separated compounds as they elute.]

After a few minutes [lower image], during which mobile phase flows continuously and steadily
past the packing material particles, we can see that the individual dyes have moved in separate
bands at different speeds. This is because there is a competition between the mobile phase and the
stationary phase for attracting each of the dyes or analytes. Notice that the yellow dye band moves
the fastest and is about to exit the column. The yellow dye likes [is attracted to] the mobile phase
more than the other dyes. Therefore, it moves at a faster speed, closer to that of the mobile phase.
The blue dye band likes the packing material more than the mobile phase. Its stronger attraction to
the particles causes it to move significantly slower. In other words, it is the most retained
compound in this sample mixture. The red dye band has an intermediate attraction for the mobile
phase and therefore moves at an intermediate speed through the column. Since each dye band
moves at different speed, we are able to separate it chromatographically.

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Figure 7 Chromatographic Column

What Is a Detector?

As the separated dye bands leave the column, they pass immediately into the detector. The detector
contains a flow cell that sees [detects] each separated compound band against a background of
mobile phase . [In reality, solutions of many compounds at typical HPLC analytical concentrations
are colorless.] An appropriate detector has the ability to sense the presence of a compound and
send its corresponding electrical signal to a computer data station. A choice is made among many
different types of detectors, depending upon the characteristics and concentrations of the
compounds that need to be separated and analyzed, as discussed earlier.

What Is a Chromatogram?

A chromatogram is a representation of the separation that has chemically [chromatographically]

occurred in the HPLC system. A series of peaks rising from a baseline is drawn on a time axis.
Each peak represents the detector response for a different compound. The chromatogram is plotted
by the computer data station [see Figure H].

Figure 8 How Peaks Are Created

In Figure 6, the yellow band has completely passed through the detector flow cell; the electrical
signal generated has been sent to the computer data station. The resulting chromatogram has begun
to appear on screen. Note that the chromatogram begins when the sample was first injected and
starts as a straight line set near the bottom of the screen. This is called the baseline; it represents

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pure mobile phase passing through the flow cell over time. As the yellow analyte band passes
through the flow cell, a stronger signal is sent to the computer. The line curves, first upward, and
then downward, in proportion to the concentration of the yellow dye in the sample band. This
creates a peak in the chromatogram. After the yellow band passes completely out of the detector
cell, the signal level returns to the baseline; the flow cell now has, once again, only pure mobile
phase in it. Since the yellow band moves fastest, eluting first from the column, it is the first peak
drawn.

A little while later, the red band reaches the flow cell. The signal rises up from the baseline as the
red band first enters the cell, and the peak representing the red band begins to be drawn. In this
diagram, the red band has not fully passed through the flow cell. The diagram shows what the red
band and red peak would look like if we stopped the process at this moment. Since most of the red
band has passed through the cell, most of the peak has been drawn, as shown by the solid line. If
we could restart, the red band would completely pass through the flow cell and the red peak would
be completed [dotted line]. The blue band, the most strongly retained, travels at the slowest rate
and elutes after the red band. The dotted line shows you how the completed chromatogram would
appear if we had let the run continue to its conclusion. It is interesting to note that the width of the
blue peak will be the broadest because the width of the blue analyte band, while narrowest on the
column, becomes the widest as it elutes from the column. This is because it moves more slowly
through the chromatographic packing material bed and requires more time [and mobile phase
volume] to be eluted completely. Since mobile phase is continuously flowing at a fixed rate, this
means that the blue band widens and is more dilute. Since the detector responds in proportion to
the concentration of the band, the blue peak is lower in height, but larger in width.

What are the different types of contaminants in water that affect HPLC
results?

Organics
Organic contamination of ultrapure water may affect chromatographic separation in
different ways:

(i) Reducing column life - Organic molecules that bind to the surface of the column can
slow-down access of sample and solvent molecules to the binding sites within the column
beads (stationary phase). This results in a decrease in the column’s ability to separate
compounds or loss of resolution and a shorter column life.

(ii) Reducing sensitivity - Organic molecules in the eluent water may compete with
sample molecules for binding to the column beads (stationary phase). This decreases the
number of sample molecules binding to the column, consequently reducing the number of
molecules released during the elution process.

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(iii) Shift in retention time - High levels of organics can create a new stationary phase
in the column, which can cause a shift in retention time and peak tailing. It can also lead to
back pressure increase.

2. Ions
The presence of ions in the solvent can also affect chromatographic separations. The presence of
any UV-absorbing ions, such as nitrates, nitrites, sulfates, bromides, chlorides, and fluorides, can
pass through the column and appear as a peak in the chromatogram making the data difficult to
analyze.

Among the different water contaminants that affect HPLC analysis, organics are by far the most
important determinants for water purity. Experimental evidence has strongly suggested that freshly
prepared ultrapure water should be the choice for any HPLC as other sources of water, namely
distilled water or even HPLC-grade bottled water still contain relatively high amounts of organics,
which can compromise the quality of the chromatograms and the performance of the apparatus.

Advantages
Liquid-solid column chromatography is an effective separation technique when all appropriate
parameters and equipment are used. This method is especially effective when the compounds
within the mixture are colored, as this gives the scientist the ability to see the separation of the
bands for the components in the sample solution. Even if the bands are not visible, certain
components can be observed by other visualization methods. One method that may work for some
compounds is irradiation with ultraviolet light. This makes it relatively easy to collect samples one
after another. However, if the components within the solution are not visible by any of these
methods, it can be difficult to determine the efficacy of the separation that was performed. In this
case, separate collections from the column are taken at specified time intervals. Since the human
eye is the primary detector for this procedure, it is most effective when the bands of the distinct
compounds are visible.

Liquid-solid column chromatography is also a less expensive procedure than other methods of
separation (HPLC, GC, etc.). This is because the most basic forms of column chromatography do
not require the help of expensive machinery like high pressure solvent pumps used in HPLC.

Disadvantages
In methods besides flash chromatography, the flow of the mobile phase, the detection of each
separation band, and the collection of each component, are all done manually by the scientist.
Although this introduces many potential instances of experimental error, this method of separation
can be very effective when done correctly. Also, the glass wear used for liquid-solid column
chromatography is relatively inexpensive and readily available in many laboratories. Burets are
commonly used as the separating column, which in many cases will work just as well as an
expensive pre-prepared column. For smaller scale chromatography, Pasteur pipettes are often used.
Flash chromatography has the potential to be more costly than the previous methods of separation,

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especially when sophisticated air pumps and vacuum pumps are needed. When these pieces of
machinery are not needed, however, a vacuum line can be instead connected to an aspirator2 on a
water faucet. Also, home-made pressurized air flow controllers can be made as shown previously.

Conclusions

The purity of water as a laboratory reagent is crucial for successful experiments. Highly sensitive
technologies, such as HPLC requires water of very high purity, which means that the water used
should have minimal TOC levels and be devoid of any other contaminants. Freshly prepared
ultrapure water is the choice for HPLC experiments and ELGA’s broad range of water purification
systems helps researchers globally to ensure that the water used in their experiments are of desired
purity.

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References:

[1] Jena A Kumar. HPLC: Highly Accessible Instrument in Pharmaceutical Industry for Effective
Method Development. Pharm Anal Acta 2012;3. doi:10.4172/2153-2435.1000147.

[2] Malviya R, Bansal V, Prakash Pal O, Kumar Sharma P. High performance liquid
chromatography: A short review. J Glob Pharma Technol 2010;2:22–6.

[3] Gerber, F.; Krummen, M.; Potgeter, H.; Roth, A.; Siffrin, C.; Spoendlin, C. (2004). "Practical
aspects of fast reversed-phase high-performance liquid chromatography using 3μm particle packed
columns and monolithic columns in pharmaceutical development and production working under
current good manufacturing practice". Journal of Chromatography A. 1036 (2): 127–133.
doi:10.1016/j.chroma.2004.02.056. PMID 15146913.

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