HPLC TRAINING COURSE

INTRODUCTION:

History of HPLC:

High-Performance Liquid Chromatography is used in many industries including in the

pharmaceutical, food, cosmetic, and environmental sectors. The history of HPLC begins with the
invention of chromatography itself, just after the turn of the 20th century. It has since evolved
to become the liquid chromatography we know and use today.

What is chromatography? Chromatography analyzes a mixture by separating its compounds.

1903: The year that Mikhail Tsvet, a botanist from Russia, is said to have invented the technique
of chromatography.

Tsvet separated plant pigments into various colored bands.

He passed the pigments through a column of calcium carbonate, after extracting them from
leaves using alcohol and ether.

Tsvet first wrote about chromatography in 1906. He wrote two pieces about chlorophyll that
appeared in a German botanical journal.

He performed a demonstration of the method in 1907, in front of the German Botanical

Society.

Tsvet’s work was the first step towards the development of HPLC, a complex and valuable
method used in today’s pharmaceutical testing careers.

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Pigments extracted from leaves were used when chromatography was invented.

Partition and Paper Chromatography as the next step:

The next step in the invention of chromatography was the development of partition
chromatography as a theory. John Porter Martin and Richard Laurence Millington Synge were
working at the Wool Industries Research Association in England when they conceptualized a
process of separation using two liquid phases.

Stationary Phase: Martin and Synge used columns of silica with water, along with a second
liquid flowing through the column (the second liquid was not miscible, or able to mix with the
other liquid).

Mobile Phase: For this phase, the pair used organic solvent chloroform.

With this method, components would be distributed between the two liquid phases, according
to their affinity for each phase. This spreading would allow for components to be collected
upon exiting the system. Partition chromatography is the basis for HPLC. In fact, Martin and
Synge would go on to win the Nobel Prize in Chemistry in 1952 for their invention.

1944: Paper chromatography is invented and used originally to identify amino acids. The
inventors credited with this are Martin once again, with two other inventors named Consden
and Gordon. It also uses stationary and mobile phases, separating colored components.

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Paper chromatography separates colored components using stationary and mobile phases.

High-Performance Liquid Chromatography for Pharmaceutical Testing:

Gas chromatography became the major technique for separating compounds in mixtures during
the 60s, but it was a limited technique and called for improvement. Matter needs to be
vaporized for this method, which has its drawbacks and reduces the number of compounds that
can be analyzed.

HPLC allows for larger as well as polar molecules to be analyzed, providing more opportunity
than gas chromatography. Liquid chromatography became interesting to researchers in the 60s
and showed promise for replacing gas methods as the leading technique.

The first commercially available HPLC system, the ALC100 HPLC, was developed by Waters
Associates (now Waters Corporation), a company offering analytic tools and systems to
scientists, in 1967. By the 1980s, HPLC was commonly used in the world of science.

Today, HPLC is widely used and because it isn’t limited by compound stability, it is a versatile
and effective analytical method. HPLC courses help graduates qualify for a range of career
opportunities, in industries that are constantly pushing the boundaries of scientific potential,
such as pharmaceuticals.

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Basics & Principles of HPLC Technique:

Meaning of HPLC:

HPLC is an abbreviation for High-Performance Liquid Chromatography. "Chromatography" is a

technique for separation, "chromatogram" is the result of chromatography, and
"chromatograph" is the instrument used to conduct chromatography.

Among the various technologies developed for chromatography, devices dedicated to

molecular separation called columns and high-performance pumps for delivering solvents at a
stable flow rate are some of the key components of chromatographs. As related technologies
become more sophisticated, the system commonly referred to as High-Performance Liquid

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Chromatography simply became referred to as "LC". Nowadays, Ultra High-Performance Liquid
Chromatography (UHPLC), capable of high-speed analysis, has also become more widespread.

Only compounds dissolved in solvents can be analyzed with HPLC. HPLC separates compounds
dissolved in a liquid sample and allows qualitative and quantitative analysis of what
components and how much of each component is contained in the sample.

The solvent used to separate components in a liquid sample for HPLC analysis is called the
mobile phase. The mobile phase is delivered to a separation column, otherwise known as the
stationary phase, and then to the detector at a stable flow rate controlled by the solvent
delivery pump. A certain amount of sample is injected into the column and the compounds
contained in the sample are separated. The compounds separated in the column are detected
by a detector downstream of the column and each compound is identified and quantified.

Overview of HPLC

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Principle of High-Performance liquid chromatography:

• The purification takes place in a separation column between a stationary and a mobile
phase.
• The stationary phase is a granular material with very small porous particles in a
separation column.
• The mobile phase, on the other hand, is a solvent or solvent mixture which is forced at
high pressure through the separation column.
• Via a valve with a connected sample loop, i.e. a small tube or a capillary made of
stainless steel, the sample is injected into the mobile phase flow from the pump to the
separation column using a syringe.
• Subsequently, the individual components of the sample migrate through the column
at different rates because they are retained to a varying degree by interactions with
the stationary phase.
• After leaving the column, the individual substances are detected by a suitable detector
and passed on as a signal to the HPLC software on the computer.
• At the end of this operation/run, a chromatogram in the HPLC software on the
computer is obtained.
• The chromatogram allows the identification and quantification of the different
substances.

Basics of HPLC:

Analyte – target compound(s) of interest for detection in an HPLC analysis.

Mobile phase – phase in motion and composed of solvent or eluents flowing from injection to
detection.

Stationary phase – the still phase where the physical separation of analytes occurs.

Flow rate – how fast the mobile phase flows with respect to time.

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Retention time – the time between sample injection and the maximum peak signal of the
analyte in a chromatogram.

Efficiency – given as the number of theoretical plates, a key metric for quantifying the
performance of separation.

Resolution – the ability to distinguish between peaks and the primary concern for any
separation.

Selectivity – the ability to separate two analytes.

Void volume – all volume within a column not occupied by the stationary phase.

Limit of detection – the smallest quantity of an analyte that can be reliably detected.

Limit of quantitation – the lower or upper quantity of an analyte that can be reliably
quantified.

Steps of HPLC:

1. The mobile phase begins to flow. The pump pushes the eluents or solvents through
the system at a specified flow rate.

2. Sample injection. Once injected into the mobile phase flow path, the sample travels
with the mobile phase from the injection point to the head of the column.

3. Compound separation. Physical separation of the compounds happens on the column

stationary phase. After elution from the column, the separated sample components
travel to the detector.

4. Analyte detection. Detection of target analytes based on an electrical signal

generated by specific properties.

5. Chromatogram generation. Translation of the detected analyte signal by the CDS into
a chromatogram of analyte signal versus time.

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 HPLC- Definition, Principle, Parts, Types, Uses, Diagram

HPLC Separations:

Factors affecting HPLC separations:

Many factors, including mobile phase composition, stationary phase chemistry, and
temperature influence HPLC separations. Successful separation only occurs if the analytes have
differing affinities for the stationary phase, so selecting the appropriate stationary phase for
your compounds is crucial. The main factors influencing the overall separation process are:

Physiochemical properties of the analyte, such as size, charge, polarity, and volatility

Physiochemical properties of the stationary phase, such as polarity, charge, and viscosity

Physiochemical properties of the mobile phase used and interaction with the analyte and
stationary phases.

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HPLC Separation Modes:

In general, three primary characteristics of chemical compounds can be used to create HPLC
separations. They are:

• Polarity
• Electrical Charge
• Molecular Size

First, let’s consider polarity and the two primary separation modes that exploit this
characteristic: normal-phase and reversed-phase chromatography.

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. As shown in Figure, classes of molecules can be ordered by their relative
retention into a range or spectrum of chromatographic polarity from highly polar to
highly non-polar.

Chromatographic Polarity Spectrum by Analyte Functional Group.

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Water [a small molecule with a high dipole moment] is a polar compound. Benzene [an
aromatic hydrocarbon] is a non-polar compound. Molecules with similar
chromatographic polarity tend to be attracted to each other; those with dissimilar
polarity exhibit much weaker attraction, if any, and may even repel one another. This
becomes the basis for chromatographic separation modes based on polarity.

Another way to think of this is by the familiar analogy: oil [non-polar] and water [polar]
don’t mix. Unlike in magnetism where opposite poles attract each other,
chromatographic separations based on polarity depend upon the stronger attraction
between likes and the weaker attraction between opposites. Remember, “Like attracts
like” in polarity-based chromatography.

Proper Combination of Mobile and Stationary Phases Effects Separation Based on Polarity.

To design a chromatographic separation system, we create competition for the various

compounds contained in the sample by choosing a mobile phase and a stationary phase
with different polarities. Then, compounds in the sample that are similar in polarity to
the stationary phase [column packing material] will be delayed because they are more
strongly attracted to the particles. Compounds whose polarity is like that of the mobile
phase will be preferentially attracted to it and move faster.

In this way, based upon differences in the relative attraction of each compound for each
phase, a separation is created by changing the speeds of the analytes.

Figure R-1. Mobile Phase Chromatographic Polarity Spectrum.

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 Figure R-2. Stationary Phase Particle Chromatographic Polarity Spectrum.

Figure R-3. Compound/Analyte Chromatographic Polarity Spectrum

Figures R-1, R-2, and R-3 display typical chromatographic polarity ranges for mobile phases,
stationary phases, and sample analytes, respectively. Let’s consider each, in turn, to see how a
chromatographer chooses the appropriate phases to develop the attraction competition
needed to achieve a polarity based HPLC separation. A scale, such as that shown in Figure R-1,
upon which some common solvents are placed in order of relative chromatographic polarity is
called an eluotropic series. Mobile phase molecules that compete effectively with analyte
molecules for the attractive stationary phase sites displace these analytes, causing them to
move faster through the column [weakly retained]. Water is at the polar end of the mobile-
phase-solvent scale, while hexane, an aliphatic hydrocarbon, is at the non-polar end. In
between, single solvents, as well as miscible-solvent mixtures [blended in proportions
appropriate to meet specific separation requirements], can be placed in order of elution
strength. Which end of the scale represents the ‘strongest’ mobile phase depends upon the
nature of the stationary phase surface where the competition for the analyte molecules occurs.

Silica has an active, hydrophilic [water-loving] surface containing acidic silanol [silicon-
containing analog of alcohol] functional groups. Consequently, it falls at the polar end of the
stationary-phase scale shown in Figure R-2. The activity or polarity of the silica surface may be

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modified selectively by chemically bonding to its less polar functional groups [bonded phase].
Examples shown here include, in order of decreasing polarity, cyanopropylsilyl- [CN], n-
octylsilyl- [C8], and n-octadecylsilyl- [C18, ODS] moieties on silica. The latter is a hydrophobic
[water-hating], very non-polar packing.

Figure R-3 repeats the chromatographic polarity spectrum of our sample [shown in Figure P].
After considering the polarity of both phases, then, for a given stationary phase, a
chromatographer must choose a mobile phase in which the analytes of interest are retained,
but not so strong that they cannot be eluted. Among solvents of similar strength, the
chromatographer considers which phase combination may best exploit the more subtle
differences in analyte polarity and solubility to maximize the selectivity of the chromatographic
system. Like attracts like, but, as you probably can imagine from the discussion so far, creating a
separation based on polarity involves knowledge of the sample and experience with various
kinds of analytes and retention modes. To summarize, the chromatographer will choose the
best combination of a mobile phase and particle stationary phase with appropriately opposite
polarities. Then, as the sample analytes move through the column, the rule like attracts like will
determine which analytes slow down and which proceed at a faster speed.

Normal-Phase HPLC

In his separations of plant extracts, Tswett was successful using a polar stationary phase [chalk
in a glass column; see Figure A] with a much less polar [non-polar] mobile phase. This classical
mode of chromatography became known as normal phase.

Figure S-1. Normal-Phase Chromatography.

Figure S-1 represents a normal-phase chromatographic separation of our three-dye test

mixture. The stationary phase is polar and retains the polar yellow dye most strongly. The
relatively non-polar blue dye is won in the retention competition by the mobile phase, a non-
polar solvent, and elutes quickly. Since the blue dye is most like the mobile phase [both are

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non-polar], it moves faster. It is typical for normal-phase chromatography on silica that the
mobile phase is 100% organic; no water is used.

Reversed-Phase HPLC

The term reversed-phase describes the chromatography mode that is just the opposite of the
normal phase, namely the use of a polar mobile phase and a non-polar [hydrophobic] stationary
phase. Figure S-2 illustrates the black three-dye mixture being separated using such a protocol.

Figure S-2. Reversed-Phase Chromatography.

Now the most strongly retained compound is the more non-polar blue dye, as its attraction to
the non-polar stationary phase is greatest. The polar yellow dye, being weakly retained, is won
in competition by the polar, aqueous mobile phase, moves the fastest through the bed, and
elutes earliest like attracts like.

Today, because it is more reproducible and has broad applicability, reversed-phase

chromatography is used for approximately 75% of all HPLC methods. Most of these protocols
use as the mobile phase an aqueous blend of water with a miscible, polar organic solvent, such
as acetonitrile or methanol. This typically ensures the proper interaction of analytes with the
non-polar, hydrophobic particle surface. A C18–bonded silica [sometimes called ODS] is the
most popular type of reversed-phase HPLC packing.

The table below presents a summary of the phase characteristics for the two principal HPLC
separation modes based on polarity. Remember, for these polarity-based modes, like attracts
like.

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Phase Characteristics for Separations Based on Polarity.

Hydrophilic-Interaction Chromatography [HILIC]

HILIC may be viewed as a variant of normal-phase chromatography. In normal-phase

chromatography, the mobile phase is 100% organic. Only traces of water are present in the
mobile phase and in the pores of the polar packing particles. Polar analytes bind strongly to the
polar stationary phase and may not elute.

Adding some water [<20%] to the organic mobile phase [typically an aprotic solvent like
acetonitrile] makes it possible to separate and elute polar compounds that are strongly
retained in the normal-phase mode [or weakly retained in the reversed-phase mode]. Water, a
very polar solvent, competes effectively with polar analytes for the stationary phase. HILIC may
be run in either isocratic or gradient elution modes. Polar compounds that are initially attracted
to the polar packing material particles can be eluted as the polarity [strength] of the mobile
phase is increased [by adding more water]. Analytes are eluted to increase hydrophilicity
[chromatographic polarity relative to water]. Buffers or salts may be added to the mobile phase
to keep ionizable analytes in a single form.

Hydrophobic-Interaction Chromatography [HIC]

HIC is a type of reversed-phase chromatography that is used to separate large biomolecules,

such as proteins. It is usually desirable to maintain these molecules intact in an aqueous
solution, avoiding contact with organic solvents or surfaces that might denature them. HIC
takes advantage of the hydrophobic interaction of large molecules with a moderately
hydrophobic stationary phase, e.g., butyl-bonded [C4], rather than octadecyl-bonded [C18],
silica. Initially, higher salt concentrations in water will encourage the proteins to be retained
[salted out] on the packing. Gradient separations are typically run by decreasing salt
concentration. In this way, biomolecules are eluted to increase hydrophobicity.

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Separations Based on Charge: Ion-Exchange Chromatography [IEC]

For separations based on polarity, like is attracted to like and opposites may be repelled. In ion
exchange chromatography and other separations based on 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]. With each type of ion exchange, there are at least two general approaches for
separation and elution.

Figure T. Ion-Exchange Chromatography.

Strong ion exchangers bear functional groups [e.g., quaternary amines or sulfonic acids] that
are always ionized. They are typically used to retain and separate weak ions. These weak ions
may be eluted by displacement with a mobile phase containing ions that are more strongly
attracted to the stationary phase sites. Alternately, weak ions may be retained on the column,
then neutralized by in situ changing the pH of the mobile phase, causing them to lose their
attraction and elute.

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Weak ion exchangers [e.g., with secondary-amine or carboxylic-acid functions] may be
neutralized above or below a certain pH value and lose their ability to retain ions by charge.
When charged, they are used to retain and separate strong ions. If these ions cannot be eluted
by displacement, then the stationary phase exchange sites may be neutralized, shutting off the
ionic attraction, and permitting the elution of the charged analytes.

Table D. Ion-Exchange Guidelines.

When weak ion exchangers are neutralized, they may retain and separate species by
hydrophobic [reversed-phase] or hydrophilic [normal-phase] interactions; in these cases,
elution strength is determined by the polarity of the mobile phase [Figure R-1]. Thus, weak ion
exchangers may be used for mixed-mode separations [separations based on both polarity and
charge].

Table D outlines guidelines for the principal categories of ion exchange. For example, to retain a
strongly basic analyte [always positively charged], use a weak-cation-exchange stationary phase

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particle at pH > 7; this assures a negatively charged particle surface. To release or elute the
strong base, lower the pH of the mobile phase below 3; this removes the surface charge and
shuts off the ion exchange retention mechanism.

Note that a pKa is the pH value at which 50% of the functional group is ionized and 50% is
neutral. To ensure an essentially neutral, or fully charged, analyte or particle surface, the pH
must be adjusted to a value at least 2 units beyond the pKa, as appropriate [indicated in Table
D].

Do not use a strong-cation exchanger to retain a strong base; both remain charged and strongly
attracted to each other, making the base nearly impossible to elute. It can only be removed by
swamping the strong cation exchanger with a competing base that exhibits even stronger
retention and displaces the compound of interest by winning the competition for the active
exchange sites. This approach is rarely practical, or safe, in HPLC and SPE. [Very strong acids and
bases are dangerous to work with, and they may be corrosive to materials of construction used
in HPLC fluidics!]

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.

All 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 bed. The biggest molecules may be totally
excluded from pores and pass only between the particles, eluting very quickly in a small

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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.

Since it is possible to correlate the molecular weight of a polymer with its size in solution, GPC
revolutionized the measurement of the molecular-weight distribution of polymers that, in turn,
determines the physical characteristics that may enhance, or detract from, polymer processing,
quality, and performance [how to tell good from bad polymer].

Main High-Performance Liquid Chromatography (HPLC) Components

The HPLC system mainly consists of an infusion pump, a sampler, a chromatographic column, a
detector, and a data recording and processing device. Among them, the infusion pump, the
chromatographic column, and the detector are key components. In addition, the gradient
elution device, online degasser, autosampler, pre-column or guard column, and column
temperature controller can also be configured as required. Modern HPLC instruments have a
microcomputer control system for automatic instrument control and data processing. The
preparative HPLC instrument is equipped with an automatic fraction collection device.

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HPLC System:

1. Infusion system

The infusion pump is one of the most important components of the HPLC system. Infusion
pumps are classified into constant pressure pumps and constant flow pumps according to the
factors of constant output liquid. The performance of the pump directly affects the quality of
the entire system and the reliability of the analysis results.

2. Degassing device

Bubbles are often seen in the mobile phase solution due to dissolved oxygen or air mixed in.
Bubbles entering the detector can result in sharp noise peaks on the chromatogram. Small
bubbles slowly accumulate and become large bubbles. When large bubbles enter the flow path
or the chromatographic column, the flow rate of the mobile phase will slow down or the flow
rate will become unstable, causing the baseline to fluctuate. It takes time to expel these

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bubbles once they enter the column. At present, the most used mobile phase degassing devices
in liquid chromatography are offline ultrasonic vibration degassing, online inert gas bubbling
purge degassing, and online vacuum degassing.

3. Gradient elution device

HPLC has two elution methods, isocratic and gradient. Isocratic elution means that the
composition of the mobile phase remains constant during the same analysis cycle, which is
suitable for samples with a small number of components and little difference in properties.
Gradient elution is a program to control the composition of the mobile phase within an analysis
cycle, such as the polarity of the solvent, ionic strength, and pH value. It is used to analyze
complex samples with many components and large differences in properties. The use of
gradient elution can shorten the analysis time, increase the resolution, improve the peak shape,
and increase the detection sensitivity, but it often causes baseline drift and reduces
reproducibility.

4. Sampling system

The six-port injection valve or autosampler is frequently used at present. This sampling device is
required to have good tightness, small dead volume, and good repeatability to ensure central
sampling, and that the pressure and flow rate of the chromatographic system during sampling
are small. It is generally used during sample analysis. There are two sampling methods for the
six-port valve, partial filling method, and complete filling method.

5. Separation system

The separation system includes a chromatographic column, a guard column, and a column
oven.

6. Detector

The detector is a device that is used to continuously monitor the composition and content
changes of the effluent separated by the chromatographic column. It is one of the three key
components of an HPLC system. The detector requires high sensitivity, low noise (that is,

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insensitive to external changes such as temperature and flow), wide linear range, good
repeatability, and wide application range.

7. Data processing system

The system can collect, store, display, print, and process test data, so that sample separation,
preparation, or identification can be carried out correctly.

Four Types of HPLC Columns:

• Normal Phase Column.

• Reverse Phase Column.
• Ion-exchange Column.
• Size Exclusion Column.

HPLC Column Principles

The physical properties of the target molecules (analytes) determine the most suitable HPLC
column for a given separation. The molecular characteristics that impact HPLC column selection
include hydrophobicity/hydrophilicity, intermolecular forces (particularly dipole-dipole),
intramolecular forces (ionic), and size. HPLC column separations can often exploit multiple
differences in the molecular properties of the target molecules. Generally, the structure and
chemistry of the HPLC column packing (stationary phase) determine the analyte elution profile.

HPLC column sizes range from capillary to process scale. The internal diameter (ID) and volume
of a column determine both how many samples can be loaded onto a column and the
sensitivity of separation. The column ID can affect the separation profile, particularly when
using gradient elution, with smaller IDs yielding increased separation and detection sensitivity.

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Therefore, for analytical separations, there is typically a trade-off between sensitivity and the
sample volume loaded onto a column.

HPLC Column Packings

Many of the types of packing used for gravity or low-pressure chromatography are not able to
withstand the high pressures used in an HPLC system. Common packing materials in HPLC
columns include silica or hydroxyapatite media and polymeric resins such as polystyrene divinyl
benzene.

The use of smaller-diameter beads generally results in improved separation sensitivity due to
the increased surface area. However, column pressure increases as bead diameter is reduced
for a given flow rate, placing a practical lower limit on bead size. Media bead diameters are
typically in the range of (1.8–5) μm for an analytical HPLC column.

Early HPLC columns were packed with irregularly shaped silica particles to increase surface
area. Currently, spherical porous silica has replaced irregular silica for most uses. The spherical
shape provides increased efficiency and lower back pressure, and the porosity increases the
surface area. Polymeric resins are highly cross-linked and are particularly useful for separations
where the pH is outside the operating range of silica packing.

HPLC Column Types

A wide variety of HPLC column types are now available for various analytical applications. A few
of the most used types are described below, classified by separation mechanism.

Ion exchange HPLC columns have charged packing. An ion exchange column can be either
cationic or anionic. This type of HPLC column separates polar molecules based on their charge.
The mobile phase is an aqueous buffer. Ion exchange HPLC columns can be used to separate
many types of analytes and are commonly used for the separation of carbohydrates, amino
acids, and proteins.

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Ion exchange and ligand exchange chromatography may be combined in a column. In these
combined-mode columns, ion exchange is usually via metal ions, and the ligands are electron-
donor molecules such as hydroxyl groups or amines. This type of HPLC column is frequently
used for the separation of monosaccharides.

Reversed phase HPLC columns have nonpolar packing. They are used with aqueous and water-
miscible organic solvent mobile phases. The most common solvents are acetonitrile, methanol,
and tetrahydrofuran (THF). Both isocratic (constant concentration) and gradient (increasing
organic solvent concentration) elution are used with reversed-phase columns. Selectivity and
retention times are dependent on several parameters including the pH of the mobile phase.
The reversed-phase HPLC column is the most versatile and commonly used column type and
can be used for a wide range of different types of analytes.

Normal-phase HPLC columns have polar packing. The mobile phase is nonpolar and therefore
usually an organic solvent such as hexane or methylene chloride. This type of HPLC column
includes a type of partition chromatography using hydrophilic interaction liquid
chromatography (HILC), in which the mobile phase contains a low concentration of water. In an
ion-moderated partition HPLC column, the addition of ionic compounds such as ammonium
acetate to the mobile phase can both change the retention times of analytes and increase their
polarity. This class of HPLC column is used for small molecules such as organic acids, some
drugs, and a range of biomolecules including glycosylated proteins.

Size exclusion HPLC columns do not rely on the interaction of the analytes with the column
packing but rather utilize a sieving effect based on molecular weight. The packing contains both
mesopores and micropores. The size distribution of the pores determines the size of molecules
in the sample that can diffuse into the pores. The extent to which molecules can diffuse into the
pores determines the retention time and elution profile. Molecules that are too large to enter
the pores pass through the column rapidly, eluting as a single peak after the void volume. Size
exclusion HPLC columns are used primarily for the separation of proteins and carbohydrates.

Other types of HPLC columns include affinity, ion exclusion, and displacement chromatography
columns; a chiral HPLC column can be used to resolve racemic mixtures.

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Detectors for HPLC:

1. UV, VIS, and PDA Detectors

2. Refractive-Index Detector

3. Evaporative Light Scattering Detector

4. Multi-Angle Light Scattering Detector

5. Mass Spectrometer

6. Conductivity Detector

7. Fluorescence Detector

8. Chemiluminescence Detector

9. Optical Rotation Detector

10. Electro Chemical Detector

The actual separation of each component in the sample is carried inside a column; however,
this separation needs to be "collected" for us to be able to see it. The detectors are used for
this purpose. The separated components are monitored and expressed electronically. There is
no universal detector that can monitor all compounds and there are many detectors used for LC
analysis.

Type Common Abbreviation

Ultraviolet UV

Visible VIS

Photo Diode Array PDA

Refractive Index RI

Evaporative Light Scattering ELS

Multi-Angle Light Scattering MALS

Mass Spectrometer MS

Conductivity CD

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Fluorescence FL

Chemiluminescence CL

Optical Rotation OR

Electro Chemical EC

1. UV, VIS, and PDA Detectors

UV, VIS, and PDA detectors are categorized as absorbance detectors. They provide good
sensitivity for light-absorbing compounds at ~pg level. They are easy to operate and provide
good stability. UV detector is a very commonly used detector for HPLC analysis. During the
analysis, the sample goes through a clear color-less glass cell, called a flow cell. When UV light is
irradiated on the flow cell, the sample absorbs a part of the 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 depending on what wavelength is used, it is important to choose an
appropriate wavelength based on the type of analyte. A standard UV detector allows the user
to choose wavelengths between 195 to 370 nm. The most used is 254 nm. Compared to a UV
detector, a VIS detector uses a 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 detectors.

PDA detects an entire spectrum simultaneously. UV and VIS detectors visualize the obtained
result in two dimensions (light intensity and time), but PDA adds a third dimension
(wavelength). This is convenient to determine the most suitable wavelength without repeating
analyses.

25
a typical Diode Array UV detector used in HPLC systems.

2. Refractive-Index Detector

RI detector measures the change in reflex index. A glass cell is divided into two chambers (cells).
The eluent from the LC column flows through the "sample cell", while another cell called the
"reference cell" is filled with only the mobile phase. When the eluent going through the sample
cell does not contain any analyte, the solvent inside both cells is the same (Figure 1A). When a
beam is irradiated on the cells, the observed beam will be straight in this case. However, in a
case the effluent contains any components other than the mobile phase; bending of the
incident beam occurs due to the reflex index difference between the two solvents (Figure 1B).
By measuring this change, the presence of components can be observed.

Figure 1

26
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 detectors.

It is suitable for detecting all components. For example, samples that 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 analytes, thus a RI detector can be
used to measure all analytes.

It is applicable for the use of a solvent that has UV absorbance. A UV detector cannot be used
with a solvent that has UV absorbance. Sometimes the organic solvent used for GPC analysis
absorbs UV, and thus UV detector cannot be used.

It provides a direct relationship between the intensity and analyte concentration. The amount
of UV absorbed depends on each analyte, thus the intensity of the UV detector peak does not
provide information on the analyte concentration. While the intensity observed by an RI
detector is comparable to the concentration of the analyte.

Because of those advantages, RI is often used for the detection of sugars and for SEC analysis.

3. Evaporative Light Scattering Detector

ELSD provides good sensitivity for non-volatile analytes at the ng level. The column effluent is
nebulized and then evaporated to make it form fine particles. The analyte is then radiated with
a laser beam and the scattered radiation is detected. The target sample includes lipids, sugar,
and high molecular weight analytes. It is used in a similar way to an RI detector but can provide
more sensitive detection with a stable baseline. Another advantage is that ELSD can be used for
the gradient method whereas RI cannot.

4. Multi-Angle Light Scattering Detector

For the SEC analysis, the MW of the analyte is estimated from the calibration curve drawn using
a set of known standards. However, by using MALS, MW can be determined directly without
the need for a calibration curve. Also, MALS can provide an absolute MW of the analyte with a
very low detection limit.

27
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 limits of elemental and molecular components.

6. Conductivity Detector

Solutions containing ionic components will conduct electricity. A conductivity detector

measures electronic resistance, and the measured value is directly proportional to the
concentration of ions present in the solution. Thus, it is generally used for ion chromatography.

7. Fluorescence Detector

The advantage of the fluorescence method is its high sensitivity for selective groups of
compounds at ~fg level. By using a specific wavelength, analyte atoms are excited and then
emit a 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.

8. Chemiluminescence Detector

Like FL, but instead of using a light source to excite the analyte atoms, the excitation is initiated
by a chemical reaction. Since it does not rely on the external excitation source, the noise is
small, resulting in a high signal-to-noise ratio, i.e., it provides even higher sensitivity than FL.

9. Optical Rotation Detector

Specific for the optical isomer measurement. The column can separate R- and L- type optical
isomers, but the general detectors (e.g., UV) cannot distinguish which is R or L. OR detector
provides this information.

28
10. Electro Chemical Detector

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

fluorescence detector in HPLC

HPLC data software:

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29
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HPLC Troubleshooting:

Potential Sources of Chromatographic Problems

• Mobile Phase
• Injector
• In-Line Filter
• Pump
• Guard Column
• In-Line Filter
• Connecting Tubing
• Column
• Detector
• Connecting Tubing and Fittings
• Integrator/Recorder

30
System flush (no column in-line)

• Check for little or no back pressure.

• Inject blank – No baseline problems.
• Compare chromatograms.
• Inspect baseline.

Baseline Troubleshooting

Noisy baseline

• Gas in the mobile phase → Degas mobile phase

• Leaks → Find leak and repair.
• Electronic noise → Remove source. Shield cables
• Weak detector lamp → Replace the lamp.
• Sensitivity too high → Lower sensitivity
• Detector cell dirty → Flush with 6N nitric acid.

Synchronous Noise

ALMOST ALWAYS CAUSED BY THE PUMP

• Air in the pump head → Prime pump and degas solvent.

31
 • Valve problem → Clean or replace.
• Broken plunger → Replace.
• Mixing Problem → Increase system volume.
• Electrical noise → Remove source.

Asynchronous Noise

• Bubbles → Degas mobile phase.

• Gas caught in detector → Degas mobile phase. Put back pressure on the cell.
• Leaks → Find leak and repair.
• Mixing problems → Increase system volume.
• Plugged lines → Remove plug–flush system.
• Electrical problems → Remove source.

Baseline Drift

• Gradient – Solvent B absorbs more than Solvent A → Try a new mobile phase. Use
baseline subtraction.
• Compounds eluting off column → Run strong solvent until the baseline is stable.
• Solvent composition change (e.g., Evaporation) → Ensure solvents are enclosed.
• Solvent leaks → Tighten, replace fittings.
• Backpressure changes → Filter solvents and samples. Samples may be too viscous.
• Mixing problems → Increase system volume.

32
Cyclic Baseline

• Temperature fluctuations → Thermally insulate. Move away from ventilation.

Increase cell temperature.
• Mixing problems → Increase system volume.
• Gas in mobile phase → Degas solvents.
• Electrical problems → Remove source.
• Erratic pump → Repair.
• Plug → Remove obstruction – flush system.

Spikes
• Bubbles → Degas solvent.
• Poor electrical connection, loose wiring → Clean and tighten detector leads, check
wiring.
• Lamp relay trying to fire a dead lamp → Replace the lamp.
• Electrical noise → Remove source. Common sources – Switching valves,
compressors, muffle furnaces, fraction collectors, power conditioners, lighting.

33
 No Peaks
1- Injector not injecting.
2- Pump not pumping.
3- Dead detector.
4- Integrator/recorder/PC not connected correctly.
5- Gain setting too low.
6- Leaks.
7- Column retaining all compounds.
8- Bad or incorrect mobile phase.
9- Bad or incorrect standard or sample.
10- Incorrect guard column.

To solve: INJECT ACETONE SOLUTION TO MAKE A PEAK

Negative & Positive Peaks

• Some eluting compounds absorb less than solvent → Use a different or cleaner
solvent.
• Air bubbles passing through cell → Degas mobile phase.
• All peaks are negative → Change detector polarity.
• Negative peaks with RI detector → May be normal (peak direction is the function of
RI differential from the mobile phase.

34
 Poor Peak Shape
• Contaminated in-line filter → Replace frit.
• Column dying → Replace column.
• System void volume → Check system tubing.
• Contaminated guard column → Replace.
• Incorrect or wrong solvent → Make new mobile phase. Consider ion
pairing/suppression.
• Column destroyed → pH < 2 washes off the functional group.

→ pH >8 dissolves silica base.

35
Column Troubleshooting

Common Problems

• Peak shape.
• Retention time
• Other.

Chromatographic problems may be related to:

• Instrument
• Sample
• Column

Two Major Problems are:

• Peak Shape/Width.
• Retention Time Changes.

Peak Shape Problems

Most Common Problem in HPLC:
Distorted peaks will cause integration or resolution problems.

The indication that optimal column performance is not being attained.

36
 Peak Shape Problems
• Column Destroyed
• Secondary Interactions
• Incorrect Sample Solvent
• Column Overload
1) Mass Overload
2) Volume Overload
• Other Extra-Column Effects
1) Sampling Rate
2) Time Constant

All Peaks Affected

• COLUMN
1) Connection.
2) Replace frit double peaks fronting peaks.
3) Regenerate or replace the column.

• COLUMN DESTROYED
1) pH < 2 washes off functional groups.
2) pH >8 dissolves silica base.

Column Collapse
well packed column

37
Voided column

38
Column Protection:

The major cause of column deterioration is contamination.

The use of guard columns may increase column lifetime to > 10,000 analyses.

Guard columns should be regarded as a cost-effective sacrifice to extend the analytical column
lifetime.

Should contain IDENTICAL packing material as the analytical column.

e.g., using a different C18, with different retention properties could destroy the separation or
impair protection.

Well-designed, well-packed guard columns will IMPROVE analytical separation efficiency.

39
Other Techniques to Protect the Column:

• In-line Filter between the Injector and Column

• Filtering of the Sample (Doesn’t Protect against Seal Shedding)
• Sample Cleanup through Solid Phase Extraction (SPE).

Column Storage:

• Store in Mobile Phase for Short Periods of Time (<72hrs.)

• Store in Shipping Solvent for Longer Periods of Time.
• Columns should be stored in a solvent that the manufacturer recommends, for
bonded phases, use an organic solvent (e.g., MeOH or ACN) Using nonaqueous
solvents minimizes hydrolysis.
• Some bonded phases (CN) become unstable in polar organic mobile.
phases.
• Storage in water or buffer is then okay.
• The worst mobile phase for the CN column is CH3CN (Acetonitrile).

Columns which may be stored in Water or Buffered Solvents:

• Ion exchangers.
• Aqueous SEC packing.

However:
Prevent microbial growth by using 0.05% sodium azide in the mobile phase.
OR
A small quantity of organic solvent (acetonitrile 5% or methanol 10%).

Columns which should be stored in Mobile Phase:

• Normal Phase
• Organic SEC (GPC)

40
Other Issues:

Tailing

What Causes Tailing?

• Mixed mode retention.

• Hydrophobic – interaction with bonded phase.
• Ion exchange– interaction with charged sites.

41
Column/Volume Overload:

A good approach to a loading study is to increase the amount injected by a factor of 2.

This is fine enough to find the point of maximum load but course enough to cover a
large concentration quickly.
It is often not necessary to perform measurements on the chromatogram. A visual
inspection is often sufficient.

42
 Mass Overload:

How to identify?
• Indicated by a rapid detector response.

How to remedy the problem?

• Dilute the sample by 50% then re-inject.
One can observe better resolution and a slightly rounded leading edge of the peak,
We then dilute the sample by 50% again until we observe a much more pronounced.
the leading edge of the peak of interest.

Summary
• Assess all potential sources.
• What is the baseline telling you?
• Treat your columns well!
• Assess the suitability of your method → Mobile phase composition etc.
• Ensure injection volume and sample concentration are suitable.

HPLC Solvents selection:

As we know, chromatography is the separation of analytes from a mixture using:

• A Stationary phase, into which the analyte partitions and is retained.
• A mobile phase, to partition the analytes from the stationary phase and transport
them through the stationary phase bed.

Analytes will have a different chemical affinity for each of the phases, those with less
affinity for the stationary phase will elute earlier and those with a greater affinity will
elute later and hence separation will be achieved. The solvents used for the mobile
phase, and the ratio in which they are used, can be tuned to change the relative
analyte affinity and hence the retention time and selectivity (chemical separating
power) of the separation.

43
Solvent and elution:
To predict or anticipate the elution order of our analytes, we need to know the type of
chromatographic column we are using, and the polarity of the mobile phase solvents
relative to that of the analyte species.
It is commonly necessary, to achieve the desired/required separation, to use a mixture
of mobile phase solvents and the first rule is that these solvents MUST be miscible.
Solvent miscibility charts can be used to determine if two solvents are miscible.

44
Solvent Polarity and Chemical Properties:

There are many factors that control the ‘separating power’ (usually called selectivity)
of the chromatographic system, and include the nature of the mobile phase solvents,
the mobile phase pH, and the nature and ionic strength of any buffers used. In
reversed-phase HPLC, for example, users would typically select acetonitrile or
methanol (or mixtures of both) with water as the other major mobile phase
constituent. Methanol and acetonitrile have fundamentally different properties in
terms of dipole moment, acidity, and basicity and therefore will produce separations
whose separating power are different. By mixing different proportions of these
solvents or by using different gradient profiles, the selectivity of separation may be
‘tuned’ to produce the best resolution between the analytes. In reversed phase HPLC
the amount of organic solvent relative to the aqueous component is typically low or
begins low and then rises during a gradient separation. In Hydrophilic Interaction
chromatography (HILIC) the reverse is true. In normal-phase chromatography, two
organic solvents are used, one of which has a higher chemical affinity for the
stationary phase than the other and is therefore used to displace analytes and acts like
the organic modifier in reversed-phase chromatography. Choosing the correct solvent
and obtaining the correct proportion of solvents is a key skill in HPLC method
development.

The next important factor to consider is how the solvent will be pumped and possibly
mixed within the system. Binary and quaternary pumps both have advantages and
disadvantages but are required to accurately mix the different mobile phase solvents
in a reproducible fashion. The online mixing of solvents in a constant (fixed) ratio
during the entire analysis is known as the Isocratic separation mode. Where one
solvent is increased in proportion to another over a pre-defined time range, this is
known as Gradient HPLC and again we need pumps capable of accurate mixing or
proportioning on the fly.

Binary pumps use two separate pump heads (effectively two separate and
independent pumps) and a mixing chamber to deliver fast, efficient, and low dwell
volume flow in your system but can only deliver two solvents at a time and they are
also more expensive.

45
 Quaternary pumps are more versatile as they can operate up to four solvents in
different proportions during a gradient but only use one pump and proportioning
valves to achieve this. The result could be a slightly less accurate mixture proportion
than a binary system.

One further issue to consider in chromatography is the potential for dissolved gases in
the mobile phase which may accumulate in the pump, column, or detector, giving rise
to fluctuating baselines and unstable pressure.

There are various things you can do to remove these such as:

• Sonication (highly effective but inconvenient).

• Filtration under vacuum (relatively effective, remove physical particulates BUT can
induce contamination).
• Online degasser (most convenient and widely used option).

To protect the HPLC system and your column from particulate matter, manufacturers
recommend filtering mobile phases prior to use. This task can be accomplished with
simple vacuum filtration devices purchased through HPLC supply catalogs. Add all
buffers and modifiers prior to filtration. A vacuum is applied to pull the solvent
through a filter –which also acts to simultaneously degas the mobile phase. Handle
filters with tweezers and make certain the filtration apparatus is always clean. Nylon
66 is a good filter material for aqueous mobile phases, while PTFE is an excellent filter
for most organic solvents. Inorganic membranes are resistant to chemical degradation
from a wide range of HPLC solvents. Be aware that Teflon filters cannot be used with
water due to the material’s nonpolar characteristics. HPLC mobile phase filters have a
pore size of around 4.5 microns.

The requirement for filtration of HPLC mobile phases is always a hotly debated topic.
Yes, it would remove physical particles, but you are risking contaminating your mobile
phase through particulates on the filter or the surface of the glassware used, also
potentially altering the pH through evaporation of volatile pH adjusting additives
(trifluoroacetic acid for example is volatile and can be lost to the atmosphere under
vacuum filtration conditions).

46
 Mobile Phase Preparation:

Mobile Phase preparation can be divided into the following major Steps:

• Measure the appropriate volume of each solvent.

• Adjust the pH of the aqueous component using buffers.
• Add any other required additives to the aqueous component of the mobile phase.
• Mix solvents (if required for isocratic operation).
• Filter mobile phase.
• Degas mobile phase.

Most mobile phases are a mixture of at least two solvents. The volume of each solvent
should be measured independently, and pH adjustment and additives added to the
aqueous component PRIOR to combining the solvents. This avoids problems
associated with volume change on mixing certain solvents – a 60:40 mix of water:
methanol (v: v) may be incorrect by up to 10% due to latent heat of mixing affecting
the overall volume of the mixed solution. Mixing is often carried out online by the HPC
system – this tends to overcome the problems when pre-mixing mobile phase
components, but this requires the use of more expensive binary or quaternary pumps
as described above. When using buffers in gradient analysis make sure they are
entirely soluble in the full range of expected mobile phase compositions. Less soluble
buffers may precipitate as the organic strength of the mobile phase is increased –
mobile phases containing more than 60% acetonitrile are particularly renowned for
this phenomenon.

Companies such as ROMIL have long provided an optional service whereby ready-to-
use solvent mixtures are manufactured to customer specifications. Such a service finds
favor in Quality Control departments for example, where methods are established on
defined mixtures, and which must be prepared frequently or in large volumes.
Solvents that are manufactured by a supplier can typically be controlled to a set purity
specification and tested to ensure that each batch of solvent meets the required
purity specifications.

These mixtures are available in super purity (SpS) (for UV detection) and ultra purity
(UpS) (for MS detection) grades ROMIL-SpS™ and ROMIL-UpS™ Solvent Mixes.

47
HPLC Eluents:
The purity of the solvent is extremely important as it can affect the achievable
sensitivity of the analysis. A less pure solvent can cause an increased level of baseline
noise, as well as ghost peaks. It is recommended that HPLC Grade (ROMIL SpS) or
better solvents are used. Gradient-grade solvents are much less likely to produce
ghost peaks when using UV detection. For MS detection, which is particularly
susceptible to solvent impurities, which change the dielectric constant of the mobile
phase and hence the MS detector response especially when using Electrospray
ionization mode, ‘MS Grade’ solvents (such as the ROMIL UpS product line, should be
chosen for optimum sensitivity and to avoid insidious effects such as ion suppression.

These principles should also be strictly applied to any aqueous solvents used in the
HPLC mobile phase and it is often the case that the quality of the in-house produced
water is not sufficiently controlled to maintain the quality of the eluent, and this can
result in wasted time tracking the source of ghost peaks and failed batches of sample
analysis.

That is not to say that commercial laboratory water purifiers cannot produce the
highest quality water. They can. But to do that reliably not only requires the
investment in the purification equipment itself but also an ongoing system of regular
maintenance and strict quality control of the output.

Solvent manufacturers will typically also supply a range of water for HPLC analysis to
assure the end user of the quality of all reagents used for mobile phase preparation.

ROMIL specifications for Super purity Solvents >

ROMIL specifications for Ultra purity Solvents >

48
 UV considerations:

When choosing your solvents, you also must consider the UV absorbance of the
solvents as this can vary from one type to another, depending upon the nature and
concentration of solvent impurities.

Here are some examples of UV absorbance cut-off for a range of common HPLC
solvents:

• Isooctane: 197 nm
• Cyclohexane: 200 nm
• Tetrachloromethane: 265 nm
• Propan-2-ol: 205 nm
• Ethanol: 210 nm
• Acetonitrile: 190 nm
• DMSO: 268 nm
• Methanol: 205 nm.

By working above the solvent cut-off and setting up the detector acquisition
properties correctly, one can avoid issues such as rising baselines during analysis
and/or reduced detector sensitivity.

In addition, when lower grade solvents are used, increased absorbance from the
impurities contained within the solvents or solvent containers will results in reduced
analytical performance which may manifest as compromised method reproducibility
and reduced sensitivity.

Other considerations:
Viscosity

Lower viscosity solvents will in general give narrower peaks due to improved mass
transfer of analyte in the mobile phase. Viscosity is also important when considering
system backpressure. The more viscous the solvent the higher the system
backpressure.

49
 Refractive Index

A refractive index detector works by comparing the refractive index of a reference cell
filled with a mobile phase to the refractive index of the mobile phase containing the
analyte. The greater the difference in refractive index the greater the concentration of
species present at that time and the larger the output signal. Better detection limits
will be achieved if the refractive index of the mobile phase varies greatly from that of
the sample.

Boiling Point

Important in preparative HPLC. A solvent with a low boiling point will be easier to
remove (by evaporation) from the sample.

Sample preparation:
Sample preparation is central to successful HPLC and UHPLC analyses. Examples of
sample preparation include:

• Converting solid samples into a liquid form.

• Simplifying complex mixtures.
• Removing interfering matrix.
• Concentrating or diluting.

50
Preparation method Analytical principle Application(s)

Decrease analyte, Preventing column/detector

Dilution solvent, or matrix overloading, reducing sample
concentration in the solvent elution strength
sample
Centrifugation Sedimentation based on Removing large cellular
density components from the solution
Filtration Remove particulates from Extending column lifetime,
the sample preventing clogging of fluidics
de-solubilize proteins by Removal of protein from
Protein precipitation adding salt, solvent, or solution
altering pH
Isolate sample Purifying compounds based
Liquid-liquid components based on on polarity/charge
extraction solubility differences in
two miscible solvents
Selective Isolating small molecules from
Solid phase separation/purification of biological matrices, desalting
extraction target analytes using a large biomolecules
sorbent stationary phase
Immunoaffinity Selective purification of Isolating small molecules from
capture an analyte using an biological/environmental/food
antibody and beverage matrices
Enzymatic cleavage of Generating peptides for
Protein digestion protein into peptides bottom-up
proteomics/peptide mapping
A chemical reaction to Improving analyte retention,
Derivatization alter the physicochemical stability, or detectability
properties of the analyte
Separate samples Protein buffer
Size exclusion components based on exchange/desalting,
size macromolecule removal
Break down solid sample Disruption of biological tissue,
Homogenizing structure using environmental samples for
mechanical force improved extraction

51
 Matrix effects

What is the sample matrix?

the sample matrix as anything in a sample except the analytes of interest, which
includes everything from salts to other compounds and solvents. The matrix type can
dictate the sample preparation, the mode of chromatography, and the detection
method. Understanding the sample matrix is a fundamental consideration in method
development.

What are matrix effects?

Matrix effect is a broad term describing the tendency of specific analyte matrices to
alter the detection or quantification of an analyte. This effect usually manifests itself
as a bias and results in under or overestimating the solution's existing analyte
concentration.

Matrix effects can appear in nearly any stage within an analysis, including sample
preparation, separation on the column, and detection. Here are a few general
examples:

• Co-elution of a UV-absorbing compound with the analyte of interest

• Sample pH alters the retention factor of ionizable analytes.
• Ion suppression of the analyte in electrospray ionization.

How to mitigate interfering matrix?

There are a few common ways to mitigate matrix effects. The correct choice depends
on the specifics of the analysis.

If analyte sensitivity is adequate, the most straightforward approach is to dilute the

sample in a proper injection solvent. A more dilute sample gives a more negligible
matrix effect.

Other solutions include extraction before analysis, which improves the separation by
eliminating possible sources of sample contamination. Using a 2D-LC or switching to a
more selective detection method can also circumvent matrix effects.

Lastly, you can perform standard addition without changing the method. But this
technique is generally avoided due to the increased number of injections per sample.

52
 METHOD STEPS

Method development steps:

Aside from sample preparation, there are four main steps to know when creating HPLC
or UHPLC method:

1) Method scouting: Involves screening various columns and eluent conditions. The
purpose of this phase is to select the best combinations for a successful HPLC
separation. In practice, method scouting requires significant manual work for
column and mobile phase switching and instrument method creation. By
understanding the target analyte properties, scouting can be initially limited to
several of the most promising column candidates.

2) Method optimization: Includes iterative testing of various separation conditions of

the HPLC method and is performed to achieve the best possible resolution, speed,
and reproducibility. This step is the most time-consuming part of method
development and often requires expert knowledge to perfect.

53
 3) Robustness testing: Done to determine the impact of changing parameters of the
separation method. Optimizing robustness is important for many method
development and validation processes.

4) Method validation: Method validation is a formal and systematic process of

performing investigational procedures with the aim of verifying that the HPLC
method is appropriate and fit for the purpose to provide satisfactory and consistent
results within the limits being described for that method.

Sample Extraction:
Extraction is one of the most common techniques for sample preparation. It involves
extracting the analyte of interest from the sample matrix. There are various ways to
do so, from solid-phase extraction to microwave-assisted extraction.

1) Solid-phase extraction
Solid-phase extraction is a simple extraction technique where the sample is loaded
onto a cartridge with a sorbent. The sample is then loaded onto the cartridge. The
analyte will be retained on the sorbent, along with some impurities which can be
washed away. This fundamental extraction type has several variants, including:

• Solid phase microextraction

Solid-phase extraction can be performed with a small fiber or tube using a thin layer
of sorbent. In this case, it’s known as solid phase microextraction.
• MIP adsorbent
Short for molecularly imprinted polymer, MIP is a type of sorbent that can be used
for the two methods above. Produced as a molecular template in line with the target
analyte, MIP sorbents can selectively extract the target analyte from samples.
• Stir-bar sorptive extraction.
As the name suggests, stir-bar sorptive extraction involves a stir bar that is coated
with the sorbent and placed into the sample to extract the analyte. The coated stir
bar is used for a set amount of time, achieving high quantities of extraction – if not
quite as pure as alternative methods.

54
 2) Liquid-liquid extraction

Also known as Soxhlet extraction, liquid-liquid extraction uses a sample thimble above a
boiling flask of solvent, all contained within a condenser. As the solvent vapor rises and
condenses, it drips back to the sample thimble. The analyte eventually reaches the top of the
thimble and is siphoned into the flask. As the process is repeated, analytes accumulate in the
flask due to their higher boiling point compared to solvents.

3) Supercritical fluid extraction

Known as SFE, supercritical fluid extraction is a highly selective method that uses
a pressurized fluid as the solvent. The pressurizing process increases its solvating
power, which allows it to selectively isolate certain analytes.

4) Ultrasonic-assisted extraction
Ultrasonic extraction or sonication is where a probe sends high and low
ultrasonic waves into a sample, creating microscopic currents which assist in the
extraction process.

5) Microwave-assisted extraction
Microwave-assisted extraction uses microwaves to rapidly heat a sample solvent
mixture, in turn partitioning analytes. When suitable, it vastly reduces extraction
time and can be used for multiple samples simultaneously.

Which type of extraction is best?

As with practically all parts of chromatography analysis, the technique used

depends on the sample being analyzed. It may also depend on the environment
in which analysis is taking place. That’s certainly true for in-field analysis, where
sample preparation is limited due to time and location constraints.

55
HPLC Applications

What is HPLC used for?

The applications of HPLC extend from analyzing small molecules to peptides to
larger complex compounds like proteins, antibodies, and more.

1) Pharma & Biopharma:

Pharma
The pharmaceutical industry uses HPLC for research and development,
manufacturing quality control, and impurity and degradation analysis to
ensure our medications are free of unintended or harmful ingredients.
Biopharma
Biopharma companies use HPLC to characterize and identify molecular
targets, screen drug targets, and produce medicine from peptide mapping
and sequencing, analyzing antibodies, and purifying the biological actives.

2) Proteomics & metabolomics:

Researchers studying complex proteomics and metabolomics biological
samples use nano-, capillary-, and micro-flow liquid chromatography
hyphenated with high-resolution accurate-mass or triple quadrupole mass
spectrometry.

3) Clinical research:
Clinical labs utilize HPLC to analyze complex biological fluids like blood,
plasma, and serum, aiding the development of targeted precision medicines.

4) Food & beverage:

HPLC analysis helps ensure foods and beverages are unadulterated and free
of harmful toxins and carcinogens by detecting residual pesticides and
verifying the purity and authenticity of ingredients.

5) Environmental:
1) Environmental companies use HPLC to identify, monitor, and quantify
metal-free organic-based pollutants in water and soil.

56
6) Forensics & toxicology:
Forensic and toxicology labs depend on HPLC analysis to screen, quantify,
and confirm drugs associated with criminal investigations and drug
screenings.

57
1) http://www.aaps.ca-Academy of Applied pharmaceutical science.

2) Shimadzu.com.

3) ThermoFisher.com.

4) Waters.com.

5) Creative-proteomics.com.

6) Bio-rad.com.

7) Shodex.com.

8) Sigmaaldrich.com.

9) Ucd.ie.com.

10) hplc.eu/downloads/ACE.

11) Crawfordscientific.com.

12) Thermfisher.com.

13) Chromatographytoday.com.

58
Contents

1) Introduction Pages: (1-4)

a) History of HPLC P.1

b) Meaning of HPLC P.4

2) Basics and principles of HPLC Pages: (4-7)

a) Steps of HPLC P.7

3) HPLC Separations Pages: (8-18)

a) Separation modes P.9
b) Normal phase P.12
c) Reverse phase P13
d) HILIC & HIC P.14
e) IEC P. (15-16)
f) GPC P.17
g) HPLC components P. (18-19)

4) HPLC System Pages: (19-29)

a) HPLC Column P. (21-23)
b) HPLC detectors P. (24-29)
c) UV VIS-detector P.25
d) Refractive index detector P.26
e) ELSD detector P.27
f) MALS detector P.27
g) HPLC Software P.29

5) HPLC troubleshooting Pages: (30-43)

a) Baseline troubleshooting P. (31-34)
b) Peak shape P.35
c) Column Troubleshooting P. (36-38)

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 d) Column Protection P. (39-42)

6) HPLC solvent selection Pages: 43-52

a) Solvent polarity & chemical properties P.45-46
b) Mobile phase preparation P.47
c) HPLC eluents P.48
d) UV consideration P.49
e) Viscosity P.49
f) Refractive Index P.50
g) Sample preparation P.50-51
h) Matrix effects P.52

7) HPLC Method steps Pages: 53-55

a) Method validation P.53
b) Sample Extraction P.54-55

8) HPLC Applications Pages: 56-57

9) References Page: 58

60