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Overview:
This chapter introduces some of the language of chromatography, classifies chromatographic methods according to technique, basic instrumentation of high performance liquid chromatography and ultra performance liquid chromatography, advancement of chromatography and the underlying physico-chemical principles which account for the retention of sample molecules in a chromatographic system.
Chapter-1
Introduction
2
Introduction
In the modern pharmaceutical industry, high performance liquid chromatography (HPLC) and
ultra performance liquid chromatography (UPLC) is the major and integral analytical tool
applied in all stages of drug discovery, development and production.
The number of drugs introduced into the market is increasing every year. These drugs may be
either new entities or partial structural modification of the existing one. Very often there is a time
lag from the date of introduction of a drug into the market to the date of its inclusion in
pharmacopoeias. This happens because of the possible uncertainties in the continuous and wider
usage of these drugs, reports of new toxicities (resulting in their withdrawal from the market),
development of patient resistance and introduction of better drugs by competitors. Under these
conditions, standards and analytical procedures for these drugs may not be available in the
pharmacopoeias. There is a scope, therefore to develop newer analytical methods for such drugs.
Pharmaceutical analytical chemistry is an important part in monitoring the quality of
pharmaceutical products for safety and efficacy. With the advancement in synthetic organic
chemistry and other branches of chemistry including bio-analytical sciences and biotechnology,
the scope of analytical chemistry has enhanced to, much higher levels. The emphasis in current
use of analytical methods particularly involving advance analytical technology has made it
possible not only to evaluate the potency of active ingredients in dosage forms and APIs but also
to characterize, elucidate, identify and quantify important constituents like active moiety,
impurities, metabolites, isomers, chiral components and prediction of the degradations likely
impurities being generated. Pharmacopoeias rely more on instrumental techniques rather than the
classical wet chemistry method. In the present research work a modest attempt has been made to
develop validated analytical methods for the quality of pharmaceutical dosage forms. Estimation
of degradants generated during formulation and storage of finished products using a
HPLC/UPLC technique.
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Once an analytical method is developed for its intended use, it must be validated. The extent of
validation evolves with the drug development phase. Usually, a limited validation is carried out
to support an Investigational New Drug (IND) application and a more extensive validation for
New Drug Application (NDA) and Marketing Authorization Application (MAA).
1.1 HISTORY AND DEVELOPMENT OF CHROMATOGRAPHY
Liquid chromatography (LC) was originally developed by the Russian botanist, Mikhail S.
Tswett in 1903 [1] and since then there has been an enormous development of this technique. His
pioneering studies focused on separating compounds (leaf pigments), extracted from plants using
a solvent, in a column packed with particles [1].
Tswett filled an open glass column with particles. Two specific materials that he found useful
were powdered chalk (calcium carbonate) and alumina. He poured his sample (solvent extract of
homogenized plant leaves) into the column and allowed it to pass into the particle bed. This was
followed by pure solvent. As the sample passed down through the column by gravity, different
colored bands could be seen separating because some components were moving faster than
others. He related these separated, different-colored bands to the different compounds that were
originally contained in the sample [Figure 1.1].
Figure 1.1 Tswett’s experiment
Introduction
4
He had created an analytical separation of these compounds based on the differing strength of
each compound’s chemical attraction to the particles. The compounds that were more strongly
attracted to the particles slowed down, while other compounds more strongly attracted to the
solvent moved faster. This process can be described as follows: the compounds contained in the
sample distribute differently between the moving solvent, called the mobile phase, and the
particles, called the stationary phase. This causes each compound to move at a different speed,
thus creating a separation of the compounds.
Tswett coined the name chromatography (from the Greek words chroma, meaning color, and
graph, meaning writing-literally, color writing) to describe his colorful experiment. (Curiously,
the Russian name Tswett means color.) Today, liquid chromatography, in its various forms, has
become one of the most powerful tools in analytical chemistry.
The definite breakthrough for liquid chromatography of low molecular weight compounds was
the introduction of chemically modified small diameter particles (3 to 10 micrometer) e.g.
octadecyl groups bound to silica in the late 1960s [2]. The new technique rapidly became a
powerful separation tool and is today called a high performance/pressure liquid chromatography
(HPLC). The usefulness and popularity of HPLC was further increased by the possibility to
automate and computerize the systems the providing the unattended operations and high sample
capacities. Many Nobel Prize awards have been based upon the research work in which
chromatography played an important role [3]. Most recently, the 2002 Nobel Prize in chemistry
was awarded to “the development of methods for identification and structure analyses of
biological macromolecules” in which HPLC/UPLC and Mass Spectroscopy were used [4].
The International Union of Pure and Applied Chemistry has defined chromatography as: ‘A
method used primarily for the separation of components of a sample, in which the components
are distributed between two phases, one of which is stationary while the other moves. The
stationary phase may be a solid or a liquid supported on a solid, or a gel. The stationary phase
may be packed in a column spread as a layer or distributed as a film, etc. In these definitions,
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“chromatographic bed” is used as a general term to denote any of the different form which the
stationary phase may be used. The mobile phase may be gaseous or liquid’.
Chromatography is an analytical method widely used for the separation, identification, and
determination of the chemical components in complex mixtures such as pharmaceutical
formulations. No other separation method is as powerful and generally applicable as is
chromatography [5]. In the modern pharmaceutical industry, high performance liquid
chromatography is the major and integral analytical tool applied in all stages of drug discovery,
development and production.
1.2 LIQUID CHROMATOGRAPHY TECHNIQUES
Liquid chromatography (LC) can be performed using planar (Techniques 1 and 2) or column
techniques (Technique 3). Column liquid chromatography is the most powerful and has the
highest efficiency for sample. In all cases, the sample first must be dissolved (interested
compound) in a liquid that is then transported either onto, or into, the chromatographic device.
1.2.1 Technique 1
The sample is spotted onto, and then flows through, a thin layer of chromatographic particles
(stationary phase) fixed onto the surface of a glass plate [Figure 1.2].
Figure 1.2 Thin-layer chromatography
Introduction
6
The bottom edge of the plate is placed in a solvent. Flow is created by capillary action as the
solvent (mobile phase) diffuses into the dry particle layer and moves up the glass plate. This
technique is called thin-layer chromatography or TLC. [Note that the black sample is a mixture
of yellow, red and blue food dyes that has been chromatographically separated]
1.2.2 Technique 2
In Figure 1.3, samples are spotted onto paper. Solvent is then added to the center of the spot to
create an outward radial flow. This is a form of paper chromatography. In the upper image
[Figure 1.2], the same black dye sample is applied to the paper.
Figure 1.3 Paper chromatography
Notice the difference in separation power for this particular paper when compared to the TLC
plate. The green ring indicates that the paper cannot separate the yellow and blue dyes from each
other, but it could separate those dyes from the red dyes. In the bottom image, a green sample,
made up of the same yellow and blue dyes, is applied to the paper. It is predicted that the paper
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cannot separate the two dyes. In the middle, a purple sample, made up of red and blue dyes, was
applied to the paper. They are well separated from each other.
1.2.3 Technique 3
In this, the most powerful approach, the sample passes through a column or a cartridge device
containing appropriate particles. These particles are called the chromatographic packing material.
Solvent flows through the device. In solid-phase extraction, the sample is loaded onto the
cartridge and the solvent stream carries the sample through the device. As in Tswett’s
experiment, the compounds in the sample are then separated by traveling at different individual
speeds through the device. Here the black sample (mixture of yellow, red and blue food dyes) is
loaded onto a cartridge. Different solvents are used in each step to create the separation [Figure
1.4]. When the cartridge format is utilized, there are several ways to achieve flow. Gravity or
vacuum can be used for columns that are not designed to withstand pressure.
Figure 1.4 Column chromatography (solid-phase extraction)
Typically, the particles in this case are larger in diameter (> 50 microns) so that there is less
resistance to flow. Open glass columns (Tswett’s experiment) are an example of this. In addition,
Introduction
8
small plastic columns, typically in the shape of syringe barrels, can be filled with packing-
material particles and used to perform sample preparation. This is called solid-phase extraction
(SPE). Here, the chromatographic device, called a cartridge, is used, usually with vacuum-
assisted flow, to clean up a very complex sample before it is analyzed further.
Smaller particle sizes (<10 microns) are required to improve separation power, presented in
Figure 1.5. However, smaller particles have greater resistance to flow, so higher pressures are
needed to create the desired solvent flow rate. Pumps and columns designed to withstand high
pressure are necessary. When moderate to high pressure is used to flow the solvent through the
chromatographic column, the technique is called high pressure liquid chromatography.
Figure 1.5 HPLC column
1.3 HIGH PERFORMANCE/PRESSURE LIQUID CHROMATOGRAPHY
The acronym HPLC, coined by the late Prof. Csaba Horváth for his 1970 Pittcon paper,
originally indicated the fact that high pressure was used to generate the flow required for liquid
chromatography in packed columns [6]. In the beginning, pumps only had a pressure capability
of 500 psi [35 bar]. This was called high pressure liquid chromatography, or HPLC. The early
1970s saw a tremendous leap in technology. These new HPLC instruments could develop up to
6,000 psi [400 bar] of pressure, and incorporated improved injectors, detectors, and columns.
HPLC really began to take hold in the mid-to late-1970s. With continued advances in
performance during this time (smaller particles, even higher pressure), the acronym
HPLC remained the same, but the name was changed to high performance liquid
chromatography.
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9
High performance/pressure liquid chromatography is now one of the most powerful tools in
analytical chemistry. It has the ability to separate, identify, and quantitate the compounds that are
present in any sample that can be dissolved in a liquid. Today, compounds in trace
concentrations as low as parts per trillion [ppt] may easily be identified and can be quantified up
to parts per billion [ppb] levels. HPLC can be, and has been, applied to just about any sample,
such as pharmaceuticals, food, nutraceuticals, cosmetics, environmental matrices, petrochemical,
forensic samples, and industrial chemicals.
1.3.1 Basic instrumentation
The components of a basic high-performance/pressure liquid chromatography [HPLC] system
are shown in the simple diagram in Figure 1.6.
Figure 1.6 Scheme of a High performance liquid chromatography system
A reservoir holds the solvent. A high-pressure pump is used to generate and meter a specified
flow rate of mobile phase, usually milliliters per minute. An injector 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 (stationary phase)
needed to effect the separation. This packing material is called the stationary phase (silica)
Introduction
10
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 of the compounds have no color, so
they cannot be seen with human eyes). The mobile phase exits the detector and can be sent to
waste, or collected. When the mobile phase (MP) contains a separated compound band, HPLC
provides the ability to collect this fraction of the elute containing that purified compound for
further study. This is called preparative chromatography technique. 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.
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, scheme is presented in Figure 1.7.
Figure 1.7 A typical HPLC (Waters Alliance) system
Since sample compound characteristics can be very different, several types of detectors have
been developed in the field of analytical. 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
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detector is used, such as an evaporative-light-scattering detector (ELSD) or charged aerosol
detector (CAD). 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)
technique to analyze the results of the chromatographic separation. This provides, from a single
injection, more comprehensive information about an analyte (in mixture of solution). The
practice of coupling a mass spectrometer to an HPLC system is called LC/MS or LC-MS.
1.3.2 HPLC operation
A simple way to understand how we achieve the separation of the compounds contained in a
sample is viewed in Figure 1.8.
Figure 1.8 Understanding how a chromatographic column works – Bands
Solvent 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 sample injection), when the sample enters the column and
begins to form a band. The sample (black solution) shown here, a mixture of yellow, red, and
blue dyes, appears at the inlet of the column as a single black band. Many times sample contains
compounds that would be colorless and the column wall is opaque, a detector is needed to see the
separated compounds as they elute from the column.
After a few minutes [Figure 1.8], during which MP flows continuously and steadily past the
packing material particles, it can be seen that the individual dyes have moved in separate bands
Introduction
12
at different speeds. This is because there is a competition between the MP 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 first. The yellow dye likes 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 (stationary phase) more than the mobile phase. Blue dye stronger
attraction to the particles causes it to move significantly slower. In other words, blue 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, the dye (mixture of red, yellow and blue) components are
separated chromatographically.
1.3.3 HPLC detector
As the separated dye bands leave the column, they pass immediately into the HPLC detector. The
HPLC detector contains a flow cell that detects each separated compound band against a
background of mobile phase [Figure 1.9]. [In reality, solutions of many compounds at typical
HPLC analytical concentrations are colorless]. An appropriate HPLC 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.
Figure 1.9 How peaks are created
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13
Detectors for HPLC are designed to take advantage of some physical or chemical attribute of
either the solute or mobile phase in the liquid chromatographic process in one of four ways [7]:
A bulk property or differential measurement
Analyte specific properties
Mobile phase modification
Hyphenated techniques
Desired Detector Characteristics
High sensitivity and reproducible, predictable response.
Respond to all solutes, or have predictable specificity.
Wide linear dynamic range; Response that increases linearly with the amount of solute.
Response unaffected by changes in temperature and mobile phase flow.
Respond independently of the mobile phase.
Not contribute to extra-column band broadening.
Reliable and convenient to use.
Non destructive of the solute.
Provide qualitative and quantitative information on the detected peak.
Fast response.
Since no one detector has all of these characteristics, over time a multitude of detectors have
been used to answer one particular challenge or another.
[A] UV-Visible detectors
The UV-visible absorbance detector is the most common HPLC detector in use today since many
compounds of interest absorb in the UV (or visible) region (from 190–600 nm). Sample
concentration, output as absorbance, is determined by the fraction of light transmitted through
the detector cell by Beer’s Law:
Introduction
14
A = log (I0/I) = Єbc
Where, A is absorbance, I0 is the incident light intensity, I is the intensity of the transmitted light,
Є is the molar absoptivity of the sample, b is the path length of the cell in cm, and c is the molar
concentration of sample.
Fixed wavelength detectors that rely on distinct wavelengths, and variable and photodiode array
detectors that rely on one or more wavelengths generated from a broad spectrum lamp. A
schematic instrumentation for a variable wavelength detector is presented in Figure 1.10.
Figure 1.10 Variable wavelength UV detector schematic
Photodiode array detectors (PDAs) have an optical path similar to variable wavelength detectors
except the light passes through the flow cell prior to hitting the grating, allowing it to spread the
spectrum across an array of photodiodes, as illustrated in Figure 1.11. PDAs extend the utility of
UV detection by providing spectra of eluting peaks that can be used to aid in peak identification,
and to monitor for co-elution (peak homogeneity), is helpful during method development. The
spectra collected at the chromatographic peak apex can be used to create a library that can in turn
be used to compare subsequent spectra for identification purposes, and spectra collected across
the peak at each data point can be compared to evaluate peak homogeneity or purity (required for
forced degradation study).
Chapter-1
15
Figure 1.11 PDA detector schematic
List of other chromatography detector
[B] Fluorescence detector (FD)
[C] Electrochemical detector (ED)
[D] Radioactivity detector (RD)
[E] Conductivity detector (CD)
[F] Chemiluminescent nitrogen detector (CND)
[G] Chiral detector (ChD)
[H] Refractive index detector (RID)
[I] Evaporative Light scattering detector (ELSD)
[J] Corona discharge detector (CDD)/ Corona charged aerosol detector (CAD)
A simplified schematic of how the CAD/CDD works is illustrated in Figure 1.12.
Introduction
16
Figure 1.12 A simplified schematic of a corona charged aerosol detector
1.3.4 HPLC chromatogram
A chromatogram is a graphical representation of the separation that has chromatographically
occurred in the HPLC system. A series of peaks rising from a baseline is drawn on a time axis
and, each peak represents the detector response for a different compound. The chromatogram is
plotted by the computer data station is shown in Figure 1.9.
1.3.5 Identification and quantitation of compounds
By comparing each peak’s retention time (tR) with that of injected reference standards in the
same chromatographic system, each compound is identified. In the chromatogram shown in
Figure 1.13, the chromatographer knew that, under these liquid chromatography system
conditions, the analyte, acrylamide, would be separated and elute from the column at 2.85
minutes (tR). Whenever a new sample, which happened to contain acrylamide, was injected into
Chapter-1
17
the LC system under the same conditions, a peak would be present at 2.85 minutes [unknown
sample B in Figure 1.14]. Once identity is established, the next piece of important information is
how much of each compound was present in the sample. The chromatogram and the related data
from the detector help us to calculate the concentration of each (interested) compound.
Figure 1.13 Chromatographic identification
Figure 1.14 Identification and quantitation
In chromatograms for Samples A and B, on the same time scale, are stacked one above the other,
presented in Figure 1.14. Both chromatograms display a peak at a retention time of 2.85 minutes,
indicating that each sample contains acrylamide substance. In this example, the peak for
acrylamide in Sample A has 10 times the area of that for Sample B. Using reference standards or
Introduction
18
working standard, it can be determined that Sample A contains 10 picograms of acrylamide,
which is ten times the amount in Sample B (1 picogram).
1.4 ISOCRATIC AND GRADIENT HPLC SYSTEMS
Two basic elution modes are used in HPLC, the first is called isocratic elution. In isocratic mode,
the mobile phase, either a pure solvent or a mixture, remains the same throughout the run. A
typical system is presented in Figure 1.15.
Figure 1.15 Isocratic LC system
The second type is called gradient elution, wherein, as its name implies, the mobile phase
composition changes during the chromatographic separation. Gradient mode is useful for
samples that contain compounds that span a wide range of chromatography. A typical system is
presented in Figure 1.16. In this figure, the mixer is downstream of the pumps; thus the gradient
is created under high pressure. Other HPLC systems are designed to mix multiple streams of
solvents under low pressure, ahead of a single pump, presented in Figure 1.17. A gradient
proportioning valve selects from the four different solvent bottles, changing the strength of the
mobile phase over time.
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Figure 1.16 High-pressure gradient system
Figure 1.17 Low-pressure gradient system
Introduction
20
1.4.1 HPLC scales [Analytical, Preparative, and Process]
HPLC can be used to purify and collect desired amounts of each compound (related substances),
using a fraction collector downstream of the detector flow cell. This process is called preparative
chromatography, typical system is presented in Figure 1.18.
Figure 1.18 HPLC systems for purification: preparative chromatography
In general, as the sample size increases, the size of the HPLC column will become larger and the
pump will need higher volume- and higher flow-rate capacity. Determining the capacity of an
preparative HPLC system is called selecting the HPLC scale. Various HPLC scales and their
chromatographic objectives are listed in Table 1.1 and figure of HPLC column dimensions are
presented in Figure 1.19.
Table 1.1 Chromatography scale
Scale Chromatographic Objective
Analytical Information [compound ID and concentration]
Semi-preparative Data and a small amount of purified compound [< 0.5 gram]
Preparative Large amounts of purified compound [> 0.5 gram]
Process [Industrial] Manufacturing quantities [gram to kilograms]
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Figure 1.19 HPLC column dimensions
Some simple guidelines on selecting the column i.d. and particle size (micron) range
recommended for each scale of chromatography is presented in Table 1.2.
Table 1.2 Chromatography scale vs. column diameter and particle size
Scale 1-8 mm
Column Diameter
10-40 mm
Column Diameter
50-100 mm
Column Diameter
>100 mm
Column Diameter
Particle Size
micron
Analytical X 1.7-10
Semi-prep. √ 5-15
Preparative X 15-100
Process X 100+
1.5 HPLC COLUMN HARDWARE
A column tube and fittings must contain the chromatographic packing material (stationary phase)
that is used to effect a separation and also it must withstand backpressure created both during
manufacture and in use (during analysis). Also, it must provide a well-controlled (leak-free,
minimum-volume, and zero-dead-volume) flow path for the sample at its inlet, and analyte bands
at its outlet, and be chemically inert relative to the sample, solvent and stationary phases. Most
Introduction
22
columns are constructed of stainless steel for highest pressure resistance and for less pressure
tolerant, PEEK™ (engineered plastic) and glass type of columns are available. It may be used
when inert surfaces are required for special chemical or biological applications; different types of
column are presented in Figure 1.20.
Figure 1.20 Column hardware examples
Different color dyes separation can be obtained and visualize using glass column, which is
presented in Figure 1.21.
Figure 1.21 A look inside a column
1.5.1 Separation performance - Resolution
The degree to which two compounds are separated is called chromatographic resolution (RS),
which means selectivity is a measure of chemical separation power.
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1.5.2 Mechanical separation power – Efficiency
If a column bed is stable and uniformly packed, its mechanical separation power is determined
by the column length (L) and the particle size (micron) and also called efficiency, is often
measured and compared by a plate number (symbolized by N). Shorter column lengths minimize
all variables i.e. longer chromatographic run times, greater solvent consumption, and higher back
pressure, but also reduce mechanical separation power, as shown in Figure 1.22.
Figure 1.22 Column Length and mechanical separating power (same particle size)
Figure 1.23 Particle size and mechanical separating power (same column length)
For a given particle chemistry, mobile phase, and flow rate, as shown in Figure 1.23, a column of
the same length and i.d., but with a smaller particle size, will deliver more mechanical separation
(chromatographic separation) power in the same time. However, smaller particle size has higher
backpressure.
Introduction
24
1.5.3 Chemical separation power – selectivity
The choice of a combination of particle chemistry (stationary phase) and mobile-phase
composition-the separation system-will determine the degree of chemical separation power and
to create a separation of any two specified compounds, a scientist may choose among a
multiplicity of phase combinations (stationary phase and mobile phase) and retention
mechanisms (modes of chromatography).
1.6 HPLC SEPARATION MECHANISMS
A useful classification of the various liquid chromatography techniques (HPLC/UPLC) is based
on the type of distribution (or equilibrium) that is responsible for the separation. The common
interaction mechanisms encountered in liquid chromatography techniques are classified as
adsorption, partition, ion-exchange, gel permeation or size exclusion, and chiral interaction. In
practice, most liquid chromatography technique separations are the result of mixed mechanisms.
A description for each of the separation mechanisms is as follow.
1.6.1 Adsorption
When the stationary phase in HPLC is a solid, the type of equilibrium between this phase and the
liquid mobile phase/solvent is termed as adsorption. All of the pioneering work in
chromatography was based upon adsorption methods, in which the stationary phase is a finely
divided polar solid that contains surface sites for retention of analytes.
1.6.2 Partition
The equilibrium between the mobile phase/solvent and a stationary phase comprising of either a
liquid adsorbed on a solid or an organic species bonded to a solid is described as partition. The
predominant type of separation in HPLC/UPLC today is based on partition using bonded
stationary phases. Bonded stationary phases are prepared by reaction of organochlorosilane with
the reactive hydroxyl groups on silica and the organic functional group is often a straight chain
Chapter-1
25
octyl (C-8) or octyldecyl (C-18); in some cases a polar functional group such as cyano, diol, or
amino may be part of the siloxane structure. Two types of partition chromatography may be
distinguished, based on the relative polarities of the phases, normal-phase chromatography and
reversed-phase chromatography.
When the stationary phase is polar and the mobile phase/solvent relatively less polar (n-hexane,
ethyl ether, chloroform), this type of chromatography technique is referred to as normal-phase
chromatography. When the mobile phase/solvent is more polar than the stationary phase (which
may be a C-8 or C-18 bonded phase), this type of chromatography technique is called reversed-
phase chromatography.
1.6.3 Ion-exchange
Ion-exchange separations are carried out using a stationary phase that is an ion-exchange resin, in
this technique packing materials (stationary phases) are based either on chemically modified
silica or on styrene-divinylbenzene copolymers, onto which ionic side groups are introduced.
Examples of the ionic groups include (I) Strong cation exchanger; sulfonic acid, (II) Weak cation
exchanger; carboxylic acid, (III) Strong anion exchanger; quaternary ammonium groups, and
(IV) Weak anion exchanger; tertiary amine group. The most important parameters that govern
the retention are the type of counter-ion, the ionic strength, pH of the mobile phase/solvent, and
temperature. Ion chromatography technique is the term applied for the chromatographic
separation of inorganic anions/cations, low molecular weight organic acids, drugs, serums,
preservatives, vitamins, sugars, ionic chelates, and certain organometallic compounds.
The separation can be based on ion-exchange, ion-exclusion effects, or ion pairing, which is
presented in Figure 1.24. Conductivity detectors in ion chromatography provide universal and
sensitive detection of charged species. The employment of some form of ion-suppression
immediately after the analytical column eliminates the limitation of high background signal from
the mobile phase in conductivity detection.
Introduction
26
Figure 1.24 Ion-Exchange chromatography
1.6.4 Size exclusion
High molecular weight solutes (>10,000) are typically separated using size exclusion
chromatography/gel filtration or gel permeation. In size-exclusion liquid chromatography, the
components of a mixture are separated according to their ability to penetrate into the pores of the
stationary phase material. Packing materials used are wide-pore silica gel, polysaccharides, and
synthetic polymers (polyacrylamide or styrene-divinylbenzene type of copolymer). In gel
filtration the mobile phase is aqueous and the packing material is hydrophilic. In gel permeation
an organic mobile phase is used and the stationary phase is hydrophobic. Size-exclusion
applications include the separation of large molecular weight biomolecules, and molecular
weight distribution studies of large polymers and natural products. For a homologous series of
oligomers, the retention time (volume) can be related to the logarithm of the molecular mass.
1.6.5 Chiral interaction
Chiral compounds or enantiomers have identical molecular structures that are non superposable
mirror images of each other, resolution of enantiomers is a challenge in the field of
pharmaceuticals and drug discovery. A chiral stationary phase contains one form of an
enantiomeric compound immobilized on the surface of the support material. A chiral separation
is based on differing degrees of stereochemical interaction between the components of an
enantiomeric sample mixture and the stationary phase.
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1.7 ULTRA PERFORMANCE LIQUID CHROMATOGRAPHY
Advancement of high performance liquid chromatography is continuously encouraged to
improve the efficacy of any one or more aspects of chromatographic analysis. Ultra performance
liquid chromatography (UPLC)/ Ultra fast liquid chromatography (UFLC)/ Rapid resolution
liquid chromatography (RRLC) improves the chromatographic analysis in three aspects, namely,
chromatographic resolution, speed and sensitivity analysis. In UPLC/UFLC/RRLC, a column
composed of fine particles, a pump with higher pressure and a detector with higher sensitivity
than they are used in HPLC. Therefore UPLC/UFLC/RRLC analysis saves time and reduces
solvent consumption [8-11]. An underlying principle of high performance/pressure liquid
chromatography states that as column packing particle size decreases, efficiency and thus
resolution increases. As particle size decreases to less than 2.5μm/2.0μm, there is a significant
gain in efficiency and it doesn’t diminish at increased linear velocities or flow rates according to
the common Van Deemter equation [12]. The terms Ultra Performance Liquid Chromatography,
Rapid Resolution Liquid Chromatography and Ultra fast liquid chromatography evolves from
HPLC.
By using smaller particles, speed and peak capacity (number of peaks resolved per unit time) can
be extended to new limits which is known as ultra performance or Rapid resolution. The classic
separation method is of HPLC (High Performance/Pressure Liquid Chromatography) with many
advantages like robustness, ease of use, good selectivity and adjustable sensitivity. Its main
limitation is the lack of efficiency compared to other chromatography technique like gas
chromatography or the capillary electrophoresis [13, 14]
due to low diffusion coefficients in
liquid phase, involving slow diffusion of analytes in the stationary phase. The Van Deemter
equation shows that efficiency increases with the use of smaller size particles but this leads to a
rapid increase in backpressure, while most of the liquid chromatography system can operate only
up to 400 or 450 bar.
Introduction
28
1.7.1 UPLC system
UPLC® Technology was created especially for scientists who seek proven, reliable technology
that simultaneously improves laboratory productivity, efficiency, and throughput. With its
success demonstrated by more than 500 peer-reviewed papers, 300 application notes, and
dramatic process improvements, leading companies and institutions around the world have
standardized on UPLC for measurable scientific and business benefits. A typical Ultra
Performance Liquid Chromatography system is depicted in Figure 1.25.
Figure 1.25 A typical UPLC (Waters,® Acquity UPLC®) system
1.7.2 Principle
The UPLC is based on the principle of stationary phase construction consisting of particles less
than 2 μm (while HPLC columns are typically filled with particles of 3 to 5 μm). The underlying
principle of this evolution are governed by the van Deemter equation, which is an empirical
formula that describes the relationship between linear velocity (flow rate) and plate height
Chapter-1
29
(HETP or column efficiency) [12]. The Van Deemter curve [Figure 1.26], governed by an
equation with three components shows that the usable flow range for a good efficiency with a
small diameter particles is much greater than for larger diameters [9, 15].
H
Figure 1.26 Van Deemter plots-influence of particle size
H= A + B/v + C v
Where A, B and C are constants and v is the linear velocity. The A term is independent of
velocity and represents "eddy" mixing. The value of A is lower when the packed column
particles are smaller and uniform. The B term represents axial diffusion or the natural diffusion
tendency of molecules. This effect is diminished at high flow rates and so this term is divided by
v. The C term is represents kinetic resistance to equilibrium in the separation process. The kinetic
resistance is the time lag involved in moving from the gas phase to the packing stationary phase
and back again. The greater the flow of gas, the more a molecule on the packing tends to lag
behind molecules in the mobile phase. Thus this term is proportional to v. Therefore it is possible
to increase throughput, and thus the speed of analysis without affecting the chromatographic
Introduction
30
performance. The advent of UPLC has demanded the development of a new instrumental system
for liquid chromatography, which can take advantage of the separation performance (by reducing
dead volumes) and consistent with the pressures (about 8000 to 15,000 PSI, compared with 2500
to 5000 PSI in HPLC). Efficiency is proportional to column length and inversely proportional to
the particle size. Therefore, the column can be shortened by the same factor as the particle size
without loss of resolution. The application of UPLC resulted in the detection of additional drug
metabolites, superior separation and improved spectral quality [9, 16].
1.7.3 Different type of UPLC system
ACQUITY UPLC I-Class
ACQUITY UPLC® I-Class provides the most powerful solution to the most critical need in
separation science today – successfully analyzing compounds that are limited in amount or
availability amid a complex matrix, more rapidly than ever before. Developed to produce the
most accurate and reproducible separations, you will get the most information possible and
accelerate laboratory results. Complex separation challenges require LC systems designed to
maximize the benefits of sub- 2-µm particle columns integrated in a system designed to optimize
MS performance.
The ACQUITY UPLC I-Class system:
Maximizes peak capacity to enhance MS sensitivity
Provides the lowest carryover, complementing MS sensitivity and extending MS linear
dynamic range
Has been purposefully engineered for the lowest dispersion; with an extended
pressure/flow envelope, complex separations can be accelerated without compromising
chromatographic fidelity
Whether your work involves therapeutics or combinations of medicines and dosing levels, as in
high potency medications or biotherapeutics, or your lab focuses on areas in trace analyses such
Chapter-1
31
as food safety, the ACQUITY UPLC I-Class System will enable you to solve your most complex
challenges.
ACQUITY UPLC
The ACQUITY UPLC® System will eliminate significant time
and cost per sample from your analytical process while
improving the quality of your results. By outperforming
traditional or optimized HPLC, the system allows
chromatographers to work at higher efficiencies with a much
wider range of linear velocities, flow rates, and backpressures.
UPLC® Technology, which has been adopted successfully in laboratories around the world for
the most demanding separations, is a highly robust, dependable, and reproducible system. What
differentiates the system’s holistic design is Waters’ patented sub-2-μm hybrid particle
chemistry, which offers significant benefits over today's HPLC systems equipped with standard
5-μm particle chemistries. The ACQUITY UPLC System, used on its own or paired with Waters
optical and MS detection technologies, provides unique end-to-end solutions for all industries:
ADME screening
Food safety
Bioanalysis
Clinical
Metabolite identification
Metabonomics
Method development
Open access
Introduction
32
Routine quality screens
The system also routinely handles demanding applications such as the turnkey solutions built for
amino acid and peptide analyses. It is compliant with strict regulatory guidelines for clinical
applications.
ACQUITY UPLC H-Class
HPLC familiarity with UPLC performance
If you are performing routine analyses or developing methods,
or just prefer the flexibility of multi-solvent capabilities in a
quaternary-based system, the only choice has been HPLC. Until
now. The ACQUITY UPLC H-Class is a streamlined system
that brings together the flexibility and simplicity of quaternary
solvent blending and a flow-through-needle injector to deliver
the advanced performance expected of UPLC type separations –
high resolution, sensitivity and improved throughput – while
maintaining the robustness and reliability that ACQUITY
systems are known for.
Choosing the ACQUITY UPLC H-Class enables you to continue running existing HPLC
methods on a forward-looking LC platform that allows you to confidently and seamlessly
transition to UPLC separations, when you’re ready, using integrated system tools and reliable
column kits for method transfer and method development that simplify migration.
ACQUITY UPLC H-Class Bio
The ACQUITY UPLC H-Class Bio System delivers the benefits of UPLC's resolution,
sensitivity, and throughput in a system purpose-built for the analysis of proteins, peptides,
nucleic acids, and glycans. Built on the foundation of the ACQUITY UPLC H-Class System,
Chapter-1
33
with its flow-through-needle injector, quaternary solvent delivery system, and AutoBlend Plus™
technology, the ACQUITY UPLC H-Class Bio System gives you more control than ever over
your bioseparation.
For biomolecular analysis
For laboratories that know biomolecules sometimes
need to work harder to move through a chromatographic
instrument, the biocompatible ACQUITY UPLC® H-
Class Bio System is ready. Engineered with a bio-inert
flow path made of non-stainless-steel materials, the
ACQUITY UPLC H-Class Bio System keeps large
molecules intact and on the move, for better sample
recovery and no carryover, whether the
chromatographic mode you're using is reversed phase
(RP), ion exchange (IEX), size exclusion (SEC), or
hydrophilic interaction (HILIC).
The ACQUITY UPLC H-Class Bio System enables you to run more chromatographic modes on
an application-inspired UPLC platform for biopharmaceutical analysis. The result: better peak
clarity and selectivity in a system that allows us to confidently, routinely, and robustly
characterize your biomolecule.
ACQUITY UPLC Automated SPE System
Sample Preparation by Online SPE
The ACQUITY UPLC Automated SPE System automates every step in SPE sample preparation
and UPLC/MS analysis. Sample preparation by SPE is fully integrated with the UPLC/MS
system, so that after sample preparation is complete, samples are eluted and applied directly to
the UPLC/MS analytical column for analysis without the need for manual intervention.
Introduction
34
Adding a New Dimension to Online
SPE
Benefits:
Enhance Overall Laboratory Productivity.
Improve UPLC/MS Assay Results.
Facilitate Quick Development of Optimized
Methods.
Designed as a fully integrated system for
sample preparation and analysis, the
ACQUITY UPLC Automated SPE System
can be used for both routine SPE sample
preparation and SPE method development
for ACQUITY UPLC/MS Assays.
Sample Preparation is controlled through an easy-to-use software interface in MassLynx™
that
controls many of the system parameters for SPE sample preparation. The system can operate in
one of four modes by changing fluidic paths and connections to an ACQUITY UPLC System
with MS depending upon the preference of the user.
Nano ACQUITY UPLC
The nanoACQUITY UltraPerformance LC®
(UPLC®
)
System is designed for nano-scale, capillary, and narrow-bore
separations to attain the highest chromatographic resolution,
sensitivity, and reproducibility.
Direct nano-flow offers significant improvements over
conventional nano-flow separations technologies. You’ll see
improved peak capacity and peak shape, and increase the
number of components that can be detected per separation.
Chapter-1
35
ACQUITY UPLC Systems with 2D Technology
For chromatographers who require additional capabilities to
increase their speed of analysis, gain sensitivity and
selectivity, and perform orthogonal separations, ACQUITY
UPLC® Systems can meet these needs by controlling
multiple valves and pumps for 2D separations.
The resolution, sensitivity, and throughput benefits of UPLC® Technology are even more
important in 2D applications. Waters’ 2D Technology solutions for the ACQUITY UPLC,
ACQUITY UPLC H-Class, and ACQUITY UPLC H-Class Bio systems are purpose-built, from
plumbing to software to valve control, to provide reproducible and consistent results for specific
applications.
Features:
Ready-to-use configurations to get you to successful 2D UPLC experiments faster, with
less troubleshooting and more confidence.
ACQUITY UPLC systems and proven columns enable best-in-class selectivity and
sensitivity.
Nano ACQUITY UPLC System with 2D Technology
Conventional 2D-LC uses ion exchange (IEX) followed by reversed- phase (RP). Any IEX
approach will use salt-containing buffers that can cause ionization background and fouling
problems if they enter a mass spectrometer (MS). Since IEX separations are based solely on the
charge of the peptide, the IEX dimension often results in poor chromatographic resolution with
peptides appearing in multiple fractions, making data interpretation difficult.
Introduction
36
The nanoACQUITY UPLC® System with 2D Technology
expands the use of sub-2-micron particles to achieve high
peak capacity separations. This innovative system
effectively uses two-dimensional (2D) UPLC for better
chromatographic resolution of complex proteomic samples
by using a dual reversed-phase (RP) approach.
This improved 2D approach uses RP at pH 10 in the first dimension, followed by RP at pH 2 in
the second dimension for results that far exceeds those of conventional IEX methodolgies.
Features:
High-resolution in both dimensions by exploiting the wide-ranging ionic and
hydrophobic structure of peptides.
Better protein identifications, quantification, and sequence coverage
Improved separation, method generation wizards, and enhanced data algorithms
PATROL UPLC Process Analyzer
The PATROL™ UPLC® Process Analyzer is a
real-time Process Analytical Technology (PAT)
system that detects and quantifies complex
multiple component manufacturing samples and
final product directly on the production floor.
Chapter-1
37
Designed with the same enabling technology that drives the ACQUITY UPLC® System,
PATROL UPLC moves existing liquid chromatography (LC) analysis from off-line Quality
Control (QC) laboratories directly to the manufacturing process, resulting in significant
improvements in production efficiency:
Delivers Real-Time LC™ analysis in step with manufacturing processes.
Provides the selectivity, sensitivity, and dynamic range of LC analysis.
UPLC’s fast resolving power quickly quantifies related and unrelated compounds.
Reduces process cycle times, so that more product can be produced with existing
resources.
Enables manufactures to produce more material that consistently meet or exceeds the
product specifications, potentially eliminating variability, failed batches, or the need to
re-work material.
Assures end-product quality, including final release testing.
The PATROL UPLC Process Analyzer is an ideal solution for pharmaceutical,
biopharmaceutical, petrochemical, and food manufacturers that are under increased internal and
external pressure to evaluate PAT programs and techniques. Global regulatory initiatives, such as
the U.S. Food and Drug Administration and European Medicines Agency Critical Path and PAT
Initiatives, and manufacturing quality-by-design programs, such as Six Sigma, are driving
corporations to assess and implement novel PAT solutions such as the PATROL UPLC System.
PATROL UPLC Laboratory Analyzer
The PATROL UPLC® Laboratory Analyzer provides real-time quantitative analysis of chemical
reactions in process development and optimization laboratories. Proven UPLC® Technology and
Real-TIME LC™ analysis have been integrated to an online analyzer that provides fast and
accurate quantitative results to characterize process methods. Spectroscopic technologies used in
process development laboratories provide identity information about the processes; however,
Introduction
38
lack the ability to simultaneously monitor multiple components at different levels and does not
provide the quantitative analysis, sensitivity, linearity/dynamic range, and resolution that UPLC
provides.
1.7.4 Sample injection
In UPLC, sample introduction is critical because, conventional injection valves, either automated
or manual, are not designed and hardened to work at extreme pressure.
1.7.5 UPLC columns
Resolution is increased in a 1.7 μm particle packed column because efficiency is better.
Separation of the components of a sample requires a bonded phase that provides both retention
and selectivity.
[A] BEH (Ethylene Bridged Hybrid) technology
The 1.7 µm Ethylene Bridged Hybrid [BEH] particle is one of the key enablers behind UPLC®
technology. It is available in three different pore sizes [130Å, 200Å and 300Å] and several
bonded phases [Figure 1.27] for reversed-phase and hydrophilic interaction chromatography and
is applicable from small molecule to large biopharmaceutical analysis.
Figure 1.27 Acquity UPLC BEH chemistries
Chapter-1
39
[B] CSH (Charged Surface Hybrid) technology
The 1.7 µm Charged Surface Hybrid (CSH™) particle is Waters third generation hybrid particle
technology [6]. Based on Waters Ethylene Bridged Hybrid [BEH] particle technology, CSH
particles incorporate a low level surface charge [Figure 1.28], designed to improve sample
loadability and peak asymmetry in low-ionic-strength mobile phases/ solvent, while maintaining
the mechanical and chemical stability inherent in BEH particle technology.
The advantages of CSH Technology include [6]:
■ Superior peak shape for basic compounds
■ Increased loading capacity
■ Rapid column equilibration after changing mobile-phase pH
■ Improved batch-to-batch reproducibility
■ Exceptional stability at low and high pH
Figure 1.28 Charged surface hybrid chemistries
Introduction
40
[C] HSS (High Strength Silica) technology
ACQUITY UPLC® HSS T3 columns utilize Waters innovative and proprietary T3 bonding. It
has the same advanced bonding process that is behind the industry-leading polar-compound
retention, aqueous mobile-phase compatibility and ultra-low MS bleed of Atlantis® T3 HPLC
columns. T3 bonding utilizes a trifunctional C18 alkyl phase bonded at a ligand density that
promotes polar compound retention and aqueous mobile-phase compatibility and also T3
endcapping is much more effective than traditional trimethyl silane [TMS] end-capping.
[D] PST (Peptide Separation Technology)
Peptide Separation Technology provides a consistent set of chromatographic tools for peptide
isolation and analysis. Waters Peptide Separation Technology columns are based on C18 BEH
Technology™ particles, ranging from 1.7 μm to 10 μm.
[E] PrST (Protein Separation Technology)
The development and successful commercialization of protein-based biopharmaceuticals and
diagnostic reagents frequently depends on the ability to adequately characterize these complex
biomolecules. Waters ACQUITY UPLC® BEH300, C4 and C18 RP, Protein-Pak
TM Hi Res IEX,
ACQUITY UPLC BEH 200 SEC, 1.7 µm columns and associated methods can help improve
your protein separation and characterization challenges.
[F] OST (Oligonucleotide Separation Technology)
ACQUITY UPLC® OST C18, 1.7 µm columns (designed for use with an ACQUITY UPLC
System) are well suited for the analysis of detritylated oligonucleotides using ion-pair, reversed-
phase chromatography. As presented in the Figure 1.29, separations are comparable to that
obtained by capillary gel electrophoresis (CGE) in terms of component resolution, yet analyses
times are significantly decreased using Waters UPLC® Technology.
Chapter-1
41
Figure 1.29 Separation of Detritylated Oligodeoxythymidine Ladders by capillary gel
electrophoresis (CGE) vs. Ion-Pair, reversed-phase chromatography
1.7.6 UPLC detector
Half-height peak widths of less than one second are obtained with 1.7μm particles, which gives
significant challenges for the detector. In order to integrate an analyte peak accurately and
reproducibly, the detector sampling rate must be high enough to capture enough data points
across the peak [Figure 1.30].
Figure 1.30 Affect of data rate on peak shape for narrow UPLC peaks
Introduction
42
1.7.7 UPLC solvent manager
The ACQUITY UPLC System consists of a binary solvent manager, sample manager including
the column heater, detector, and optional sample organiser. The binary solvent manager uses two
individual serial flow pumps to deliver a parallel binary gradient. There are built-in solvent select
valves to choose from up to four solvents. There is a 15,000-psi pressure limit (about 1000 bar)
to take full advantage of the sub-2μm particles.
1.7.8 UPLC sample manager
The sample manager also incorporates several technology advancements, by using pressure
assisted sample introduction; low dispersion is maintained through the injection process, and a
series of pressures transducers facilitate self-monitoring and diagnostics and also it uses needle-
in-needle sampling for improved ruggedness and needle calibration sensor increases accuracy.
1.7.9 Advantages [17]
Decreases run time and increases sensitivity.
Provides the selectivity, sensitivity, and dynamic range of LC analysis.
Maintaining resolution performance.
Expands scope of multiresidue methods.
UPLC’s fast resolving power quickly quantifies related and unrelated compounds.
Faster analysis through the use of a novel separation material of very fine particle size.
Operation cost is reduced.
Less solvent consumption.
Reduces process cycle times, so that more product can be produced with existing
resources.
Increases sample throughput and enables manufacturers to produce more material that
consistently meet or exceeds the product specifications, potentially eliminating
variability, failed batches, or the need to re-work material [9, 10].
Chapter-1
43
Delivers real-time analysis in step with manufacturing processes.
Assures end-product quality, including final release testing.
1.7.10 Disadvantages
Due to increased pressure requires more maintenance and reduces the life of the columns
of this type.
So far performance similar or even higher has been demonstrated by using stationary
phases of size around 2 μm without the adverse effects of high pressure.
In addition, the phases of less than 2 μm are generally non-regenerable and thus have
limited use [18].
1.8 APPLICATIONS OF UPLC
Analysis of natural products and traditional herbal medicine
Identification of metabolite
Study of metabonomics / metabolomics
ADME (Absorption, Distribution, Metabolism, Excretion) screening
Bioanalysis / Bioequivalence studies
Dissolution testing
Forced degradation studies
Manufacturing / QA / QC
Method development / validation
Impurity profiling
Inorganic compounds
1.9 OPEN ACCESS
UPLC/UFLC/RRLC and UPLC/MS systems and software enable versatile and open operation
for medicinal chemistry labs, with easy-to-use instruments, a user-friendly software interface,
Introduction
44
and fast, robust analyses using UV or MS for nominal and exact mass measurements. System
management is just as simple, the central administrator can remotely define system users and
their privileged for operating instruments across the network.
1.10 METHOD CONVERSION FROM HPLC TO UPLC
For method conversion from HPLC to UPLC or for comparison of both the technology following
aspects needs to take in consideration [6, 19, 20].
Ratio of column length to particle size (L/dp) needs to keep constant.
i.e. 150 mm/5 µm = 30,000 is closest to 50mm/1.7 µm = 29,500
Column selection should be based on same basic column chemistry
i.e. C18 column should be replaced by C18 column
5 µm to 1.7 µm particle size leads to increase in speed of 9X along with 9X pressure
3 µm to 1.7 µm particle size leads to increase in speed of 3X along with 3X pressure
5 µm to 1.7 µm particle size leads to increase in peak height of 1.7X
3 µm to 1.7 µm particle size leads to increase in peak height of 1.3X
5 µm to 1.7 µm particle size leads to decrease in peak width of 0.6X
3 µm to 1.7 µm particle size leads to decrease in peak width of 0.8X
Column efficiency (N) is inversely proportional to dp
i.e. 5 µm to 1.7 µm particle size leads to increase in column efficiency (N) 3X but
So, resolution also increase by 1.7X
Based on above facts practically an example for chromatogram comparison against column
dimension for rune time and resolution is shown in Figure 1.31.
dpN
1
NRs
Chapter-1
45
Figure 1.31 Chromatogram comparisons against column dimension
Remark: Here, X is used to express the mathematical relation in multi fold.
e.g. pressure increased by 3X i.e. pressure increase by three times.
1.11 FASTER METHOD DEVELOPMENT WITH UPLC
Now there is requirement to develop, rapid and stability indicating method during development
to reduce the cycle time for formulation development and routine quality control analysis [21-
26].
UPLC screening method 7X faster than directly scaled HPLC method [Table 1.3, 1.4].
Table 1.3 Method screening
Introduction
46
Table 1.4 UPLC allows for faster method development
UPLC method development protocol Equivalent HPLC method development protocol
Column dimensions: 2.1 x 50mm x 1.7µm Column dimensions: 4.6 x 150mm x 5µm
pH 3.0/ acetonitrile Time pH 3.0/ acetonitrile Time
Flow ramp 5 min Flow ramp 5 min
Column conditioning (2 blank gradients) 11 min Column conditioning (2 blank gradients) 80 min
Sample injection (2 replicates) 11 min Sample injection (2 replicates) 80 min
pH 3.0/ methanol pH 3.0/ methanol
Flow ramp 5 min Flow ramp 5 min
Column conditioning (2 blank gradients) 11 min Column conditioning (2 blank gradients) 80 min
Sample injection (2 replicates) 11 min Sample injection (2 replicates) 80 min
Column purge 6 min Column purge 35 min
pH 10/ acetonitrile pH 10/ acetonitrile
Flow ramp 5 min Flow ramp 5 min
Column conditioning (2 blank gradients) 11 min Column conditioning (2 blank gradients) 80 min
Sample injection (2 replicates) 11 min Sample injection (2 replicates) 80 min
pH 10/ methanol pH 10/ methanol
Flow ramp 5 min Flow ramp 5 min
Column conditioning (2 blank gradients) 11 min Column conditioning (2 blank gradients) 80 min
Sample injection (2 replicates) 11 min Sample injection (2 replicates) 80 min
Column purge 6 min Column purge 35 min
120 min 730 min
Screening time: 2 hours/column x 4 columns Screening time: 12.2 hours/column x 4 columns
TOTAL SCREENING TIME 8 HOURS TOTAL SCREENING TIME 48.8 HOURS
Chapter-1
47
1.12 REFERENCES
[1] Tswett MS, Protok TR, Otd Bioi, 1930; Published, 1905; 14: 20.
[2] Skoog DA and Leary JM, “Principles of Instrumental analysis” fourth ed., Saunders
Publishing College, 1992.
[3] Horvath C, “High Performance Liquid Chromatography” Academic Press, New York,
1980.
[4] Nobel website htpp://www.nobel. se/chemistry/laureates/2002/chemadv02.pdf
[5] Heftmann E, “Fundamentals and Applications of Chromatographic and Electrophoretic
Methods” ed. New York: Elsevier, 1983.
[6] http://www.waters.com/waters/nav.htm
[7] Snyder LR, Kirkland JJ and Dolan JW, “Introduction to modern liquid
chromatography” John Wiley and Sons; New York, 2010.
[8] Swartz ME, “Ultra performance liquide chromatography: tomorrows HPLC technology
today” Lab Plus Int, 2004; 18(3): 6-9.
[9] Jerkovich AD, Mellors JS and Jorgenson JW, LCGC, 2003; 21: 600-610.
[10] Wu N, Lippert JA and Lee ML, “Practical aspects of ultra high pressure capillary liquid
chromatography” J Chrom A, 2001; 911(1): 1-12, pmid:11269586.
[11] Swartz ME, “Degradation analysis using UPLC” Pharm Formulation Quality, 2004;
6(5): 40-42.
[12] Van Deemter JJ, Zuiderweg FJ and Klinkenberg A, “Longitudinal diffusion and
resistance to mass transfer as causes of nonideality in chromatography” J Chem Eng Sci,
1956; 5(6): 271-289, doi: 10.1016/0009-2509(56)80003-1.
[13] Zhang YH, Gong XY, Zhang HM, Larock RC, Yeung ES, “Combinatorial screening of
homogeneous catalysis and reaction optimization based on multiplexed capillary
electrophoresis” J Comb Chem, 2000; 2(5): 450-452.
Introduction
48
[14] Zhou C, Jin Y, Kenseth JR, Stella M, Wehmeyer KR and Heineman WR, “Rapid pKa
estimation using vacuum-assisted multiplexed capillary electrophoresis (VAMCE) with
ultraviolet detection” J Pharm Sci, 2005; 94(3): 576-589, pmid: 15666290.
[15] MacNair JE, Lewis KC and Jorgenson JW, “Ultrahigh-pressure reversed-phase liquid
chromatography in packed capillary columns” Journal of Anal Chem, 1997; 69: 983-
989, pmid: 9075400.
[16] MacNair JE, Patel KD and Jorgenson JW, “Ultrahigh-pressure reversed-phase capillary
liquid chromatography: isocratic and gradient elution using columns packed with 1.0-
micron particles” Journal of Anal Chem, 1999; 71(3): 700-708, pmid: 9989386.
[17] Rakshit KT and Mukesh CP “Development and validation of new analytical method for
bioactive compounds” URI: http://hdl.handle.net/10603/8513.
[18] Broske AD et al., Agilent Technologies application note 5988-9251EN (2004).
[19] www.chromatographyonline.com
[20] UPLC waters seminar presentation at Singapore (2006).
[21] Harshal KT and Mukesh CP, “Development and validation of a stability-indicating RP-
UPLC method for determination of rosuvastatin and related substances in
pharmaceutical dosage form” Scientia Pharmaceutica, 2012; 80: 393-406, doi:10.3797/
scipharm.1201-09.
[22] Rakshit KT, Mukesh CP and Sushant BJ, “A rapid, stability indicating RP-UPLC
method for simultaneous determination of ambroxol hydrochloride, cetirizine
hydrochloride and antimicrobial preservatives in liquid pharmaceutical formulation”
Scientia Pharmaceutica, 2011; 79: 525-543, doi:10.3797/scipharm.1103-19.
[23] Rakshit KT and Mukesh CP, “Development of a stability indicating RP-UPLC method
for rapid determination of metaxalone and its degradation products in solid oral dosage
form” Scientia Pharmaceutica, 2012; 80: 353-366, doi:10.3797/scipharm.1112-08.
[24] Rakshit KT and Mukesh CP, “Evaluation of pharmaceutical quality of mesalamine
delayed release tablets using a new high sensitivity reversed-phase UPLC method for its
genotoxic/aniline impurity” E- Jornal of Chemistry, 2011; 8(1): 167-179. doi:10.1155/
2011/953235.
Chapter-1
49
[25] Rakshit KT, Mukesh CP and Kharkar AR, “Determination of mesalamine related
impurities from drug product by reversed phase validated UPLC method”
E- Jornal of Chemistry, 2011; 8(1): 131-148. doi:10.1155/2011/382137.
[26] Harshal KT and Mukesh CP, “Development and validation of a precise and stability
indicating LC method for the determination of benzalkonium chloride in pharmaceutical
formulation using an experimental design” E- Jornal of Chemistry, 2010; 7(4): 1514-
1522. doi:10.1155/2010/681420.