1

Chapter-1

Introduction

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.

1.1 HISTORY AND DEVELOPMENT OF CHROMATOGRAPHY

Among all the different types of analytical methods, chromatography has a unique application to all

types of analytical problems. It has undergone explosive growth in the last few decades. Liquid

Chromatography is a separation method of great importance to chemical, pharmaceutical and

biotechnological industry. The principle is that a sample of a solution of the substances is

introduced into the column of a porous material (stationary phase) and a liquid (mobile phase) is

pumped through the column. The separation of substances is based on the differences in rates of

migration through the column arising from different partition of the substances between the

stationary and mobile phase. Depending on the partition behavior of the different types of

substances, these will elute at different times from the column outlet.

Liquid chromatography 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

Introduction

2

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]

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.

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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 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 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, “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

Introduction

4

chromatography is the major and integral analytical tool applied in all stages of drug discovery,

development and production.

1.2 LIQUID CHROMATOGRAPHY (LC) TECHNIQUES

Liquid chromatography 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 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]. 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.

[Figure 1.2 Thin-layer chromatography]

Note that the black sample is a mixture of yellow, red and blue food dyes that has been

chromatographically separated.

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1.2.2 Technique 2

In Figure 1.3, samples are spotted onto paper (stationary phase). Solvent (mobile phase) is then

added to the center of the spot to create an outward radial flow. This is a form of paper

chromatography (classic paper chromatography is performed in a manner similar to that of TLC

with linear flow). In the upper image [Figure 1.2], the same black dye (mixture of yellow, red and

blue) 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 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.

Introduction

6

1.2.3 Technique 3

In this, the most powerful approach, the sample passes through a column or a cartridge device

containing appropriate particles (stationary phase). These particles are called the chromatographic

packing material. Solvent (mobile phase) flows through the device. In solid-phase extraction (SPE),

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 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,

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,

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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 [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 HPLC.

[Figure 1.5 HPLC column]

1.3 HIGH PERFORMANCE 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.

High performance 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

Introduction

8

parts per trillion [ppt] may easily be identified. HPLC can be, and has been, applied to just about

any sample, such as pharmaceuticals, food, nutraceuticals, cosmetics, environmental matrices,

forensic samples, and industrial chemicals.

1.3.1 Basic instrumentation

The components of a basic high-performance 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 (called the mobile phase, because it moves). A high-pressure pump

(solvent delivery system or solvent manager) is used to generate and meter a specified flow rate of

mobile phase, usually milliliters per minute. An injector (sample manager or auto sampler) is able

to introduce (inject) the sample into the continuously flowing mobile phase stream that carries the

sample into the HPLC column. The column contains the chromatographic packing material needed

to effect the separation. This packing material is called the stationary phase because it is held in

place by the column hardware. A detector is needed to see the separated compound bands as they

elute from the HPLC column (most compounds have no color, so they cannot be seen with human

eyes). The mobile phase exits the detector and can be sent to waste, or collected, as desired. When

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the mobile phase 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. 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 [Figure 1.7]. Since sample compound characteristics

can be very different, several types of detectors have been developed. For example, if a compound

can absorb ultraviolet light, a UV-absorbance detector is used. If the compound fluoresces, a

fluorescence detector is used. If the compound does not have either of these characteristics, a more

universal type of detector is used, such as an evaporative-light-scattering detector (ELSD). The

most powerful approach is the use multiple detectors in series. For example, a UV and/or ELSD

detector may be used in combination with a mass spectrometer (MS) to analyze the results of the

chromatographic separation. This provides, from a single injection, more comprehensive

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

called LC/MS.

[Figure 1.7 A typical HPLC (Waters Alliance) system]

Introduction

10

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]

Mobile phase enters the column from the left, passes through the particle bed, and exits at the right.

Flow direction is represented by green arrows. First, consider the top image; it represents the

column at time zero (the moment of injection), when the sample enters the column and begins to

form a band. The sample shown here, a mixture of yellow, red, and blue dyes, appears at the inlet of

the column as a single black band. Many times a 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.

After a few minutes [Figure 1.8], during which mobile phase flows continuously and steadily past

the packing material particles, it can be seen that the individual dyes have moved in separate bands

at different speeds. This is because there is a competition between the mobile phase and the

stationary phase for attracting each of the dyes or analytes. Notice that the yellow dye band moves

the fastest and is about to exit the column first. The yellow dye likes (is attracted to) the mobile

phase more than the other dyes. Therefore, it moves at a faster speed, closer to that of the mobile

phase. The blue dye band likes the packing material more than the mobile phase. Its stronger

attraction to the particles causes it to move significantly slower. In other words, it is the most

retained compound in this sample mixture. The red dye band has an intermediate attraction for the

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mobile phase and therefore moves at an intermediate speed through the column. Since each dye

band moves at different speed, the dye components are separated chromatographically.

1.3.3 HPLC detector

As the separated dye bands leave the column, they pass immediately into the detector. The 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 detector has the ability to sense the presence of a

compound and send its corresponding electrical signal to a computer data station. A choice is made

among many different types of detectors, depending upon the characteristics and concentrations of

the compounds that need to be separated and analyzed, as discussed earlier.

[Figure 1.9 How peaks are created]

Detectors for HPLC are designed to take advantage of some physical or chemical attribute of either

the solute or mobile phase in the chromatographic process in one of four ways [7]:

1. A bulk property or differential measurement

2. Analyte specific properties

3. Mobile phase modification

4. Hyphenated techniques

Introduction

12

Bulk property detectors are the most universal detectors for HPLC as they measure properties

common to all analytes by measuring differences in the mobile phase with and without the sample.

One of the most common bulk property detectors is the refractive index detector. Given the

universal nature of bulk property detectors, they respond to all analytes, placing more emphasis on

the selectivity of the chromatographic column. The UV detector is the most common example of an

analyte specific property detector, responding to analytes that absorb UV light at a particular

wavelength. UV detectors are usually thought of as somewhat specific, responding only to

compounds with chromophores, but at low UV wavelengths (<210 nm), where just about every

organic compounds absorb, UV detectors are actually somewhat universal. Other analyte specific

detectors include fluorescence, conductivity, and electrochemical. Mobile phase modification

detectors change the mobile phase post-column to induce a change in the properties of the analyte,

for example, by creating particles suspended in a gas phase. Evaporative light scattering and corona

discharge detectors fit into this category. Hyphenated techniques refer to the coupling of a separate

independent analytical technology to an HPLC system. The most common is mass spectrometry

(LC-MS), and technologies such as infrared spectrometry (LC-IR) and nuclear magnetic resonance

(LC-NMR) have also been used. There are many characteristics must be considered when choosing

a detector, and following lists few of them.

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

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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. Ease of use, predictability, and reproducibility

are all very important characteristics; however, recently there is an increased emphasis on the flow

cell contribution to band broadening and faster detector responses. This emphasis is due to new,

low dispersion ultra high performance liquid chromatography (UHPLC) systems designed to take

full advantage of sub-two mm particle size column packings [8, 9]. Many detectors, including UV,

PDA, FL, ELSD, and CAD are commercially available in both HPLC and UHPLC version

depending upon the application.

[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:

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.

There are three different types of UV detector:

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. Fixed

wavelength detectors, the backbone of early HPLC systems, are cheap and simple, but are in

limited use today.

Introduction

14

Variable wavelength detectors can be tuned to operate at the absorbance maximum of an analyte or

at a wavelength that provides more selectivity. They can also be programmed to change

wavelengths during a chromatographic run to compensate for response of different analytes. In a

variable wavelength detector, light from a broad spectrum lamp (for UV deuterium is common,

tungsten for visible) is directed through a slit to a diffraction grating that spreads the light out into

its constituent wavelengths. The grating is then rotated to direct a single wavelength of light

through a slit, through the detector cell, to a photodiode. A schematic instrumentation for a variable

wavelength detector is shown 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. Z-path or tapered detector

cells designs are commonly used in most UV detectors for HPLC. 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 or purity), is helpful during method development. They

can also serve as a multi-wavelength UV-VIS detector. 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.

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[Figure 1.11 PDA detector schematic]

[B] Fluorescence detector

Fluorescence detectors (FL) measure the optical emission of light by solute molecules after they

have been excited at a higher energy wavelength and can be very sensitive for compounds that have

native fluorescence or that can be made to fluoresce through derivatization [10]. Schematically,

they resemble as Figure 1.10, except that the grating is replaced by a filter or monochromator at a

right angle to the incident light to simplify the optics and reduce background noise. The light source

is usually a broad spectrum deuterium or xenon flash lamp. The excitation wavelength (often close

to the UV λmax) is selected by a filter or monochromator between the lamp and the flow cell, always

at a higher energy (lower wavelength) than the emission wavelength. Laser-induced fluorescence

(LIF) detectors using lasers as the excitation source are sometimes used in micro- or capillary-LC

systems, where the higher energy of the laser provides better sensitivity in the small diameter flow

cells necessary to limit dispersion. FL detectors can be as much as 100 times more sensitive than a

UV detector, making them particularly useful for trace analyses, or in sample limited or low

concentration sample situations.

Introduction

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[C] Electrochemical detector

For compounds that can be oxidized or reduced the electrochemical (EC) detector is one of the

most sensitive and selective HPLC detector available [11, 12]. EC detectors require the use of

electrically conductive HPLC mobile phases (buffers suffice) and, when properly used and

maintained, are the standard bearer when it comes to response levels for the HPLC analysis of

compounds such as catecholamines and neurotransmitters. The USP recently added a method using

high-performance anion exchange with PAD (HPAE-PAD) to identify and quantitate the level of

organic impurities in heparin due to recent health concerns [13-15]. The new heparin USP

monograph relies on hydrolyzing the polysaccharide and determining the relative amounts of

galactosamine and glucosamine in the sample digests by analysis on an anion exchange column

with electrochemical detection.

[D] Radioactivity detector

Radioactivity detectors (sometimes referred to as radiometric or radio-flow detectors) are used to

measure radioactive analytes as they elute from the HPLC column. Most radioactivity detectors are

based on liquid scintillation technology to detect phosphors caused by the radioactive nuclides,

such as low-energy β-emitters (e.g., 35

S, 14

C, 3H, and

32P) or stronger α-, β-, and γ-emitters (e.g.,

131I,

210Po, and

125Sb); tritium and

14C being the most common. The radioactivity detector can be

very sensitive and is extremely useful for the detection of radio labelled compounds in

toxicological, metabolism, or degradation studies. Large flow cell volumes are typically used in

radioactivity detectors to increase analyte residence time, which increases the number of radioactive

decays that can be detected. The use of a large cell, column, and injection volumes generally limit

radiochemical detector use in UHPLC [16].

[E] Conductivity detector

A conductivity detector is a bulk property detector that measures the conductivity of the mobile

phase. Conductivity detectors are the detector of choice for ion chromatography or ion exchange

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separations when the analyte does not have a UV chromophore [17, 18]. In the conductivity

detector, the resistance (or strictly the impedance) between two electrodes in the flow cell is

measured. For many applications, particularly ion chromatography, where conductive buffers are

required in the mobile phase, a suppressor column is used post-analytical column (before the

detector) in order to reduce the background conductance of the mobile phase.

[F] Chemiluminescent nitrogen detector

The chemiluminescent nitrogen detector is an element specific detector where the column effluent

is nebulized with oxygen and a carrier gas of argon or helium and pyrolyzed at 1050°C [19-20].

Nitrogen containing compounds (except N2) are oxidized to nitric oxide, which is then mixed with

ozone to form nitrogen dioxide in the excited state. The nitrogen dioxide decays to the ground state

with the release of a photon, which is detected by a photometer. The area of the resultant signal is

directly proportional to the amount of nitrogen in the original analyte. Because of this relationship,

it is possible to calibrate the response with any compound with known nitrogen content and

quantitate the nitrogen content of unknown analytes. Care must be taken in choosing nitrogen free

mobile phases components and additives, like acetonitrile to be avoided.

[G] Chiral detector

Many compounds, particularly drugs, exist in enantiomeric forms that can possess significantly

different pharmacological properties, and chromatographic separation of enantiomers can be

complimented by the use of detectors capable of responding to the different chiral forms. Chiral

detectors in flow cell form essentially mimic their bench top counterparts; polarimeters (PL),

optical rotary dispersion (ORD), and circular dichroism detectors (CD) [21]. Polarimeters measure

the degree of rotation of polarized light as it passes through the sample. The amount of rotation is

dependent upon both the concentration and molecular structure of the analyte. ORD detectors

operate quite similar to polarimeters but at lower wavelengths. CD detectors measure the difference

in absorption of right and left circularly polarized light as an analyte flows through the detector cell.

Introduction

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Chromophores with absorption in the 200–420nm range yield the best responses. CD detectors have

typically proven to be the most sensitive. Chiral detector response can yield both positive and

negative peaks relative to a normal baseline, a requirement that should be kept in mind when

considering CDS software.

[H] Refractive index detector

The refractive index (RI) detector is a universal bulk property detector, and is the original, oldest

LC detector. RI detectors measure the difference in optical refractive index between mobile phase

and the sample; no chromophore on the solute molecule is required [16]. For this reason, RI

detection has been used very successfully for the analysis of sugars, triglycerides, and organic

acids. The most common RI detector design is the deflection refractometer where the light from a

tungsten source lamp is directed through a pair of wedge-shaped flow cells, (reference and sample).

The reference cell contains trapped or static mobile phase and the column effluent is sent through

the sample cell. As the light passes through the two detector cells it is refracted differently,

measured by a pair of photodiodes that convert the signal to a measurable output voltage. Modern

RI detectors use thermostat flow cells due to the susceptibility of RI measurements to temperature

fluctuations. Refractive index (RI) detector is being replaced in many applications by light

scattering or corona discharge detectors due to its limited sensitivity and gradient incompatibility.

[I] Corona discharge detector

Corona charged aerosol detection (CAD), sometimes referred to as corona discharge detection

(CDD) is a unique technology gaining in popularity in which the HPLC column eluent is first

nebulized with a nitrogen (or air) carrier gas to form droplets that are then dried to remove mobile

phase, producing analyte particles [16]. The primary stream of analyte particles is met by a

secondary stream of nitrogen (or air) that is positively charged as a result of having passed a high-

voltage, platinum corona wire. The charge transfers diffusionally to the opposing stream of analyte

particles and is further transferred to a collector where it is measured by a highly sensitive

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electrometer, generating a signal in direct proportion to the quantity of analyte present. A simplified

schematic of how the CAD works is illustrated in Figure 1.12.

[Figure 1.12 A simplified schematic of a corona charged aerosol detector]

[J] Light scattering detector

Recent improvements in the ability to nebulise efficiently an HPLC column effluent has lead to

increased utility of light scattering detectors. The most popular detector of this type is the

evaporative light scattering detector (ELSD). The ELSD works on the principle of evaporation

(nebulisation) of the mobile phase followed by measurement of the light scattered by the resulting

particles [16]. The column effluent is nebulized in a stream of nitrogen or air carrier gas in a heated

drift tube and any non-volatile particles are left suspended in the gas stream. Light scattered by the

particles is detected by a photocell mounted at an angle to the incident light beam. Carrier gas flow

rate and drift tube temperature must be adjusted for whatever mobile phase is used. Detector

response is related to the absolute quantity of analyte present, and while decreased sensitivity will

Introduction

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be obtained for volatile analytes. Unlike the UV detector, no chromophores are required and it has

orders of magnitude more response than the RI detector. ELSD also has the advantage over RI

detection in that the response is independent of the solvent, so it can be used with gradients, and is

not sensitive to temperature or flow rate fluctuations. Mobile phases of course must be volatile,

similar to those used for MS detection. Linearity can be limited in some applications, but is

certainly quantitative over a wide range, if properly calibrated.

1.3.4 HPLC chromatogram

A chromatogram is a 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. Each peak

represents the detector response for a different compound. The chromatogram is plotted by the

computer data station [Figure 1.9].

In Figure 1.9, the yellow band has completely passed through the detector flow cell; the electrical

signal generated has been sent to the computer data station. The resulting chromatogram has begun

to appear on screen. Note that the chromatogram begins when the sample was first injected and

starts as a straight line set near the bottom of the screen. This is called the baseline; it represents

pure mobile phase passing through the flow cell over time. As the yellow analyte band passes

through the flow cell, a stronger signal is sent to the computer. The line curves, first upward, and

then downward, in proportion to the concentration of the yellow dye in the sample band. This

creates a peak in the chromatogram. After the yellow band passes completely out of the detector

cell, the signal level returns to the baseline; the flow cell now has, once again, only pure mobile

phase in it. Since the yellow band moves fastest, eluting first from the column, it is the first peak

drawn. A little while later, the red band reaches the flow cell. The signal rises up from the baseline

as the red band first enters the cell, and the peak representing the red band begins to be drawn. In

this diagram, the red band has not fully passed through the flow cell. The diagram shows what the

red band and red peak would look like if we stopped the process at this moment. Since most of the

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red band has passed through the cell, most of the peak has been drawn, as shown by the solid line.

If we could restart, the red band would completely pass through the flow cell and the red peak

would be completed (dotted line). The blue band, the most strongly retained, travels at the slowest

rate and elutes after the red band. The dotted line shows you how the completed chromatogram

would appear if we had let the run continue to its conclusion. It is interesting to note that the width

of the blue peak will be the broadest because the width of the blue analyte band, while narrowest on

the column, becomes the widest as it elutes from the column. This is because it moves more slowly

through the chromatographic packing material bed and requires more time (and mobile phase

volume) to be eluted completely. Since mobile phase is continuously flowing at a fixed rate, this

means that the blue band widens and is more dilute. Since the detector responds in proportion to the

concentration of the band, the blue peak is lower in height, but larger in width.

1.3.5 Identification and quantitation of compounds

In Figure 1.9, three dye compounds are represented by three peaks separated in time in the

chromatogram. Each elutes at a specific location, measured by the elapsed time between the

moment of injection (time zero) and the time when the peak maximum elutes. By comparing each

peak’s retention time (tR) with that of injected reference standards in the same chromatographic

system (same mobile and stationary phase), each compound is identified.

In the chromatogram shown in Figure 1.13, the chromatographer knew that, under these LC system

conditions, the analyte, acrylamide, would be separated and elute from the column at 2.85 minutes

(retention time). Whenever a new sample, which happened to contain acrylamide, was injected into

the LC system under the same conditions, a peak would be present at 2.85 minutes [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 compound. The detector basically responds

to the concentration of the compound band as it passes through the flow cell. The more

concentrated it is, the stronger the signal; this is seen as a greater peak height above the baseline.

Introduction

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[Figure 1.13 Chromatographic identification]

[Figure 1.14 Identification and quantitation]

In Figure 1.14, chromatograms for Samples A and B, on the same time scale, are stacked one above

the other. The same volume of sample was injected in both runs. Both chromatograms display a

peak at a retention time (tR) of 2.85 minutes, indicating that each sample contains acrylamide.

However, Sample A displays a much bigger peak for acrylamide. The area under a peak (peak area

count) is a measure of the concentration of the compound it represents. This area value is integrated

and calculated automatically by the computer data station. In this example, the peak for acrylamide

in Sample A has 10 times the area of that for Sample B. Using reference standards, it can be

determined that Sample A contains 10 picograms of acrylamide, which is ten times the amount in

Sample B (1 picogram). Note there is another peak (not identified) that elutes at 1.8 minutes in both

Chapter-1

23

samples. Since the area counts for this peak in both samples are about the same, this unknown

compound may have the same concentration in both samples.

1.4 ISOCRATIC AND GRADIENT HPLC SYSTEMS

Two basic elution modes are used in HPLC. The first is called isocratic elution. In this mode, the

mobile phase, either a pure solvent or a mixture, remains the same throughout the run. A typical

system is outlined 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 separation. This mode is useful for samples that contain

compounds that span a wide range of chromatography. As the separation proceeds, the elution

strength of the mobile phase is increased to elute the more strongly retained sample components. In

the simplest case, shown in Figure 1.16, there are two bottles of solvents and two pumps. The speed

of each pump is managed by the gradient controller to deliver more or less of each solvent over the

course of the separation. The two streams are combined in the mixer to create the actual mobile

phase composition that is delivered to the column over time. At the beginning, the mobile phase

contains a higher proportion of the weaker solvent (Solvent A). Over time, the proportion of the

stronger solvent (Solvent B) is increased, according to a predetermined timetable. Note that in

Figure 1.16, the mixer is downstream of the pumps; thus the gradient is created under high

Introduction

24

pressure. Other HPLC systems are designed to mix multiple streams of solvents under low

pressure, ahead of a single pump. A gradient proportioning valve selects from the four solvent

bottles, changing the strength of the mobile phase over time [Figure 1.17].

[Figure 1.16 High-pressure gradient system]

[Figure 1.17 Low-pressure gradient system]

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25

1.4.1 HPLC scales [Analytical, Preparative, and Process]

HPLC can be used to purify and collect desired amounts of each compound, using a fraction

collector downstream of the detector flow cell. This process is called preparative chromatography

[Figure 1.18]. In preparative chromatography, the analyst is able to collect the individual analytes

as they elute from the column (e.g., in this figure: yellow, then red, then blue). The fraction

collector selectively collects the eluate that now contains a purified analyte, for a specified length of

time. The vessels are moved so that each collects only a single analyte peak.

[Figure 1.18 HPLC systems for purification: preparative chromatography]

A analyst determines goals for purity level and amount. Coupled with knowledge of the complexity

of the sample and the nature and concentration of the desired analytes relative to that of the matrix

constituents, these goals, in turn, determine the amount of sample that needs to be processed and

the required capacity of the HPLC system. In general, as the sample size increases, the size of the

HPLC column will become larger and the pump will need higher volume-flow-rate capacity.

Determining the capacity of an HPLC system is called selecting the HPLC scale. Table 1.1 lists

various HPLC scales and their chromatographic objectives.

Introduction

26

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]

The ability to maximize selectivity with a specific combination of HPLC stationary and mobile

phases, achieving the largest possible separation between two sample components of interest is

critical in determining the requirements for scaling up a separation. Capacity then becomes a matter

of scaling the column volume (Vc) to the amount of sample to be injected and choosing an

appropriate particle size. Column volume, a function of bed length (L) and internal diameter (i.d.),

determines the amount of packing material (particles) that can be contained [Figure 1.19].

[Figure 1.19 HPLC column dimensions]

In general, HPLC columns range from 20 mm to 500 mm in length (L) and 1 mm to 100 mm in

internal diameter (i.d.). As the scale of chromatography increases, so do column dimensions,

especially the cross-sectional area. To optimize throughput, mobile phase flow rates must increase

Chapter-1

27

in proportion to cross-sectional area. If a smaller particle size is desirable for more separation

power, pumps must then be designed to sustain higher mobile-phase-volume flow rates at high back

pressure. Table 1.2 represents some simple guidelines on selecting the column i.d. and particle size

range recommended for each scale of chromatography.

For example, a semi-preparative-scale application (√) would use a column with an internal diameter

of 10-40 mm containing 5-15 micron particles. Column length could then be calculated based on

how much purified compound needs to be processed during each run and on how much separation

power is required.

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. It must withstand backpressure created both during manufacture

and in use. 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 separation system (sample, mobile, and stationary phases). Most columns are

constructed of stainless steel for highest pressure resistance. PEEK™ (an engineered plastic) and

glass, while less pressure tolerant, may be used when inert surfaces are required for special

chemical or biological applications [Figure 1.20].

Introduction

28

[Figure 1.20 Column hardware examples]

A glass column wall offers a visual advantage. In Figure 1.21, flow has been stopped while the

sample bands are still in the column. It can be seen that the three dyes in the injected sample

mixture have already separated in the bed; the yellow analyte, traveling fastest, is just about to exit

the column.

[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). Two

principal factors that determine the overall separation power or resolution that can be achieved by

an HPLC column are: mechanical separation power, created by the column length, particle size, and

packed-bed uniformity, and chemical separation power, created by the physicochemical

competition for compounds between the packing material and the mobile phase. Efficiency is a

measure of mechanical separation power, while selectivity is a measure of chemical separation

power.

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29

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 and the particle size. Mechanical separation power, also called efficiency, is

often measured and compared by a plate number (symbolized by N). Smaller-particle

chromatographic beds have higher efficiency and higher backpressure. For a given particle size,

more mechanical separation power is gained by increasing column length. However, the trade-offs

are longer chromatographic run times, greater solvent consumption, and higher back pressure.

Shorter column lengths minimize all these variables 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)]

Introduction

30

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

power in the same time. However, its backpressure will be much higher.

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 (how

one can change the speed of each analyte). Optimizing selectivity is the most powerful means of

creating a separation; this may obviate the need for the brute force of the highest possible

mechanical efficiency. 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 LC techniques is based on the type of distribution (or

equilibrium) that is responsible for the separation. The common interaction mechanisms

encountered in LC are classified as adsorption, partition, ion-exchange, gel permeation or size

exclusion, and chiral interaction. In practice, most LC separations are the result of mixed

mechanisms. A brief 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 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. The composition of the mobile phase is the main

variable that affects the partitioning of analytes. Silica and alumina are the only stationary phases

used, the former being preferred for most applications. Applications of adsorption chromatography

Chapter-1

31

include the separation of relatively non-polar water-insoluble organic compounds. Because of the

polar nature of the stationary phase and the impact of slight variations in mobile phase composition

on the retention time, adsorption chromatography is very useful for the separation of isomers in a

mixture.

1.6.2 Partition

The equilibrium between the mobile phase 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 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. The organic functional group is often a straight chain 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.

When the stationary phase is polar and the mobile phase relatively less polar (n-hexane, ethyl ether,

chloroform), this type of chromatography is referred to as normal-phase chromatography. For this

reason, the use of silica as the stationary phase (as in adsorption chromatography) is also considered

to be a normal phase separation method. When the mobile phase is more polar than the stationary

phase (which may be a C-8 or C-18 bonded phase), this type of chromatography is called reversed-

phase chromatography. Reversed-phase chromatography separations are carried out using a polar

aqueous-based mobile phase mixture that contains an organic polar solvent such as methanol or

acetonitrile. Because of its versatility and wide range of applicability, reversed-phased

chromatography is the most frequently used HPLC method. Applications include non-ionic

compounds, polar compounds, and in certain cases ionic compounds.

Introduction

32

1.6.3 Ion-exchange

Ion-exchange separations are carried out using a stationary phase that is an ion-exchange resin.

Packing materials 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 (a)

Sulfonic acid (strong cation exchanger), (b) Carboxylic acid (weak cation exchanger), (c)

Quaternary ammonium groups (strong anion exchanger), and (d) Tertiary amine group (weak anion

exchanger). The most important parameters that govern the retention are the type of counter-ion,

the ionic strength, pH of the mobile phase, and temperature. Ion chromatography 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 [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.

[Figure 1.24 Ion-Exchange chromatography]

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33

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 LC, 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

like polyacrylamide or styrene-divinylbenzene copolymer. In gel filtration the mobile phase is

aqueous and the packing material is hydrophilic, while 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. Rapid and accurate stereochemical 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. Typically,

derivatives of optically active polysaccharides that are chemically bonded to silica form the packing

material. A chiral separation is based on differing degrees of stereochemical interaction between the

components of an enantiomeric sample mixture and the stationary phase.

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 improves the chromatographic analysis in three aspects, namely, chromatographic

resolution, speed and sensitivity analysis. In UPLC, a column composed of fine particles, a pump

Introduction

34

with higher pressure and a detector with higher sensitivity than they are used in HPLC. Therefore

UPLC analysis saves time and reduces solvent consumption [22-25]. An underlying principle of

HPLC states that as column packing particle size decreases, efficiency and thus resolution

increases. As particle size decreases to less than 2.5μm, 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 [26]. The terms Ultra Performance Liquid Chromatography and Rapid

Resolution Liquid Chromatography (RRLC) 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. The classic separation method is of

HPLC (High Performance 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 gas chromatography or the capillary electrophoresis [27, 28]

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 HPLC system can operate only up

to 400 bar. That is why short columns filled with particles of about 2μm are used with these

systems, to accelerate the analysis without loss of efficiency, while maintaining an acceptable loss

of load.

1.7.1 UPLC system

Elevated-temperature chromatography also allows for high flow rates by lowering the viscosity of

the mobile phase, which significantly reduces the column backpressure [29, 30]. Monolithic

columns contain a polymerized porous material that provides lower flow resistances than

conventional particle-packed columns [31, 32]. A typical UPLC system is depicted in Figure 1.25.

Chapter-1

35

[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 (HETP

or column efficiency) [26]. 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 [23, 34].

H

Introduction

36

[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 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

Chapter-1

37

resolution. The application of UPLC resulted in the detection of additional drug metabolites,

superior separation and improved spectral quality [24, 35].

1.7.3 Sample injection

In UPLC, sample introduction is critical. Conventional injection valves, either automated or

manual, are not designed and hardened to work at extreme pressure. To protect the column from

extreme pressure fluctuations, the injection process must be relatively pulse-free and the swept

volume of the device also needs to be minimal to reduce potential band spreading. A fast injection

cycle time is needed to fully capitalise on the speed afforded by UPLC, which in turn requires a

high sample capacity. Low volume injections with minimal carryover are also required to increase

sensitivity. There are also direct injection approaches for biological samples [36, 37].

1.7.4 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.

The ACQUITY UPLC column family continues to evolve and expand, now including 5 particle

substrates [BEH 130Å, BEH 200Å, BEH 300Å, HSS and CSH] and 14 chemistries which are

scalable between HPLC and UPLC particle sizes. Additionally, 6 application-directed UPLC

chemistries for SEC, amino acids, proteins, peptides, oligonucleotides and glycan analyses are

available [6].

[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. Due to the intrinsic chemical

stability of hybrid particle technology, a wider usable pH range [pH 1-12] can be employed,

Introduction

38

enabling a versatile and robust separation technology for method development. BEH particle

technology is available in HPLC particle sizes [2.5, 3.5, 5 and 10 µm] in the XBridge™ family of

HPLC columns, enabling seamless transfer between HPLC and UPLC technology platforms. An

internal dimension (ID) of 2.1 mm column is used. For maximum resolution, choose a 100 mm

length and for faster analysis, and higher sample throughput, choose 50 mm column.

[Figure 1.27 Acquity UPLC BEH chemistries]

[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, 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

Chapter-1

39

■ 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]

CSH particle technology is also available in HPLC particle sizes [3.5 and 5 µm] in the XSelect™

family of HPLC columns enabling seamless transfer between HPLC and UPLC® Technology

platforms. CSH Technology dramatically improves virtually all facets of LC column performance

in acidic, low ionic strength mobile phases that are commonly used in the chromatographic

laboratory.

[C] HSS (High Strength Silica) technology

ACQUITY UPLC® HSS T3 columns utilize Waters innovative and proprietary T3 bonding. This is

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. The T3 endcapping is much more

effective than traditional trimethyl silane [TMS] end-capping. This unique combination of bonding

Introduction

40

and endcapping provides superior polar compound retention and aqueous compatibility while also

enhancing column performance, lifetime, peak shape and stability. Although the rugged and

efficient ACQUITY UPLC BEH particle provides a wide pH range and superior peak shapes, its

hydrophilic nature does not promote polar compound retention. ACQUITY UPLC HSS T3 columns

are designed to retain and separate polar organic compounds in reversed-phase UPLC® separations.

When compared to ACQUITY UPLC BEH C18 columns, most compounds are more strongly

retained on ACQUITY UPLC HSS T3 columns.

[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.

Peptide Separation Technology provides:

Improved separation of a wide range of peptides in both TFA and Formic Acid eluents

Narrow, symmetrical peaks for best resolution

Good peak shape and retention

Unique column chemistry with the benefits of sub-2 μm particles for ultimate resolution

QC-tested with a peptide map, ensuring stability of well-developed peptide separation methods

[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.

Chapter-1

41

[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. The ability to resolve large

oligonucleotide sequences (e.g., N from N-1) is possible due to the enhanced resolving power

obtained using sub-3 µm, BEH Technology™ particles. In addition, quantitation with molecular

weight characterization of the separated target oligonucleotide product form failure sequences is

possible using Waters OST columns with hyphenated-Mass Spectrometry methods and MS friendly

eluents.

[Figure 1.29 Separation of Detritylated Oligodeoxythymidine Ladders by capillary gel

electrophoresis (CGE) vs. Ion-Pair, reversed-phase chromatography]

1.7.5 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

Introduction

42

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]

The detector cell must have minimal dispersion (volume) to preserve separation efficiency.

Conceptually, the sensitivity increase for UPLC detection should be 2-3 times higher than HPLC

separations, depending on the detection technique. MS detection is significantly enhanced by

UPLC; an increased peak concentration with reduced chromatographic dispersion at lower flow

rates promotes increased source ionization efficiencies. For UPLC detection, the tunable

UV/Visible detector is used which includes new electronics and firmware to support Ethernet

communications at the high data rates. Conventional absorbance-based optical detectors are

concentration sensitive detectors, and for UPLC use, the flow cell volume would have to be

reduced in standard UV/Visible detectors to maintain concentration and signal. According to Beer’s

Law, smaller volume conventional flow cells would also reduce the path length upon which the

signal strength depends. A reduction in cross-section means the light path is reduced, and

transmission drops with increasing noise. Therefore, if a conventional HPLC flow cell were used,

UPLC sensitivity would be compromised. The ACQUITY tunable UV/Visible detector cell consists

of a light guided flow cell equivalent to an optical fibre. Light is efficiently transferred down the

flow cell in an internal reflectance mode that still maintains a 10mm flow cell path length with a

volume of only 500 mL. Tubing and connections in the system are efficiently routed to maintain

Chapter-1

43

low dispersion and to take advantage of leak detectors that interact with the software to alert the

user to potential problems.

1.7.6 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.

1.7.7 UPLC sample manager

The sample manager also incorporates several technology advancements. Using pressure assisted

sample introduction, low dispersion is maintained through the injection process, and a series of

pressures transducers facilitate self-monitoring and diagnostics. It uses needle-in-needle sampling

for improved ruggedness and needle calibration sensor increases accuracy. Injection cycle time is

25 seconds without a wash and 60 sec with a dual wash used to further decrease carry over. A

variety of microliter plate formats (deep well, mid height, or vials) can also be accommodated in a

thermostatically controlled environment. Using the optional sample organiser, the sample manager

can inject from up to 22 microliter plates. The sample manager also controls the column heater.

Column temperatures up to 65°C can be attained. To minimise sample dispersion, a “pivot out”

design allows the column outlet to be placed in closer proximity to the source inlet of an MS

detector.

1.7.8 Advantages

Decreases run time and increases sensitivity

Provides the selectivity, sensitivity, and dynamic range of LC analysis

Maintaining resolution performance.

Expands scope of multiresidue methods

Introduction

44

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 [23, 24]

Delivers real-time analysis in step with manufacturing processes

Assures end-product quality, including final release testing

1.7.9 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 [38].

1.8 APPLICATIONS OF UPLC

1.8.1 Analysis of natural products and traditional herbal medicine

UPLC is widely used for analysis of natural products and herbal medicines. For traditional herbal

medicines, also known as natural products or traditional Chinese medicines, analytical laboratories

need to expand their understanding of their pharmacology to provide evidence-based validation of

their effectiveness as medicines and to establish safety parameters for their production.

Chapter-1

45

1.8.2 Identification of metabolite

Biotransformation of new chemical entities (NCE) is necessary for drug discovery. When a

compound reaches the development stage, metabolite identification becomes a regulated process. It

is of the utmost importance for laboratory to successfully detect and identify all circulating

metabolites of a candidate drug. Discovery studies are generally carried out in vitro to identify

major metabolites so that metabolic weak spots on the drug candidate molecule can be recognized

and protected by changing the compound structure. Key for analysts in metabolite identification is

maintaining high sample throughput and providing results to medicinal chemists as soon as they are

available. UPLC/MS/MS addresses the complex analytical requirements of biomarker discovery by

offering unmatched sensitivity, resolution, dynamic range, and mass accuracy.

1.8.3 Study of metabonomics / metabolomics

Metabonomics studies are carried out in laboratories to accelerate the development of new

medicines. The ability to compare and contrast large sample groups provides insight into the

biochemical changes that occur when a biological system is exposed to a new chemical entity

(NCE). Metabonomics provides a rapid and robust method for detecting these changes, improves

understanding of potential toxicity, and allows monitoring the efficacy. The correct implementation

of metabonomic and metabolomic information helps similar discovery, development, and

manufacturing processes in the biotechnology and chemical industry companies. With these

studies, scientists are better able to visualize and identify fundamental differences in sample sets.

The UPLC/MS system combines the benefits of UPLC analyses, high resolution exact mass MS,

and specialized application managers to rapidly generate and interpret information-rich data,

allowing rapid and informed decisions to be made.

1.8.4 ADME (Absorption, Distribution, Metabolism, Excretion) screening

Pharmacokinetics includes studies of ADME (Absorption, Distribution, Metabolism and

Excretion). ADME studies measure physical and biochemical properties – absorption, distribution,

Introduction

46

metabolism, elimination, and toxicity of drugs where such compounds exhibit activity against the

target disease. A significant number of candidate medicines fall out of the development process due

to toxicity. If toxic reactions or any side effect occurs in the discovery/development process, then it

becomes more costly. It is difficult to evaluate candidate drugs for possible toxicity, drug-drug

interactions, inhibition, and/or induction of metabolizing enzymes in the body. Failure to properly

identify these potential toxic events can cause a compound to be withdrawn from the market. The

high resolution of UPLC enables accurate detection and integration of peaks in complex matrices

and extra sensitivity allows peak detection for samples generated by lower concentration

incubations and sample pooling. These are important for automated generic methods as they reduce

failed sample analyses and save time.

UPLC/MS/MS provides following advantages:

UPLC can more than double throughput with no loss in method robustness.

UPLC is also simpler and more robust than the staggered separations sometimes applied

with HPLC methods.

1.8.5 Bioanalysis / Bioequivalence studies

For pharmacokinetic, toxicity, and bioequivalence studies, quantitation of a drug in biological

samples is an important part of analytical method development programs. The drugs are generally

of low molecular weight and are tested during both preclinical and clinical studies. Several

biological matrices are used for quantitative bioanalysis, the most common being blood, plasma,

and urine [39]. The primary technique for quantitative bioanalysis is LC/MS/MS. The sensitivity

and selectivity of UPLC/MS/MS at low detection levels generates accurate and reliable data that

can be used for a variety of different purposes, including statistical pharmacokinetics (PK) analysis.

1.8.6 Dissolution testing

For quality control and release in drug manufacturing, dissolution testing is essential in the

formulation, development and production process in pharmaceutical industries. In sustained-release

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dosage formulations, testing higher potency drugs is particularly important where dissolution can

be the rate-limiting step in medicine delivery. The dissolution profile is used to demonstrate

reliability and batch-to-batch uniformity of the active ingredient. Additionally, newer and more

potent formulations require increased analytical sensitivity. UPLC provides precise and reliable

automated online sample acquisition. It automated dissolution testing, from pill drop to test start,

through data acquisition and analysis of sample aliquots, to the management of test result

publication and distribution.

1.8.7 Forced degradation studies

One of the most important factor that impact the quality and safety of pharmaceuticals is chemical

stability. The FDA and ICH require stability testing data to understand how the quality of an API

(active pharmaceutical ingredient) or a drug product changes with time under the influence of

environmental factors such as heat, light, pressure and moisture or humidity. Knowledge of these

stability characteristics defines storage conditions and shelf life, the selection of proper

formulations and protective packaging, and is required for regulatory documentation. Forced

degradation, or stress testing, is carried out under even harsher conditions than those used for

accelerated stability testing. Generally performed early in the drug development process,

laboratories cause the potential drug to degrade under a variety of conditions like peroxide

oxidation, acid and base hydrolysis, photostability, and temperature to understand resulting by

products and pathways that are necessary to develop stability indicating methods.

1.8.8 Manufacturing / QA / QC

Identity, purity, quality, safety and efficacy are the important factors to be considered while

manufacturing a drug product. The successful production of quality pharmaceutical products

requires that raw materials meet purity specifications. That manufacturing processes proceed as

designed. Those final pharmaceutical products meet, and hopefully exceed, defined release

specifications. Continued monitoring of material stability is also a component of quality assurance

and control.

Introduction

48

1.8.9 Method development / validation

According to FDA, validation is defined as establishing documented evidence that provides a high

degree of assurance that a specific process will consistently produce a product meeting its

predetermined specifications and quality attributes. Method development and validation is a time-

consuming and complicated process: laboratories need to evaluate multiple combinations of mobile

phase, pH, temperature, column chemistries, and gradient profiles to arrive at a robust, reliable

separation for every activity. UPLC help in critical laboratory function by increasing efficiency,

reducing costs, and improving opportunities for business success.

1.8.10 Impurity profiling

For the drug development and formulation process, profiling, detecting, and quantifying drug

substances and their impurities in raw materials and final product testing is an essential part.

Impurity profiling requires high-resolution chromatography capable of reliably and reproducibly

separating and detecting all of the known impurities of the active compound. Also critical is the

ability to accurately measure low-level impurities at the same time as the higher concentration

active pharmaceutical component. This activity, however, can be complicated by the presence of

excipients in the sample, often resulting in long HPLC analysis times to achieve sufficient

resolution.

1.8.10 Inorganic compounds

Recently stability indicating reversed phase high performance liquid chromatographic method is

used for the determination of inorganic salt in the pharmaceutical formulation [40].

1.9 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, 41, 42].

Ratio of column length to particle size (L/dp) needs to keep constant.

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49

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.

[Figure 1.31 Chromatogram comparison against column dimension]

dpN

1

NRs

Introduction

50

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.

1.10 FASTER METHOD DEVELOPMENT WITH UPLC

UPLC screening method 7X faster than directly scaled HPLC method [Table 1.3, 1.4].

Table 1.3 Method screening

UPLC gradient conditions Equivalent HPLC gradient conditions

Column dimensions: 2.1 x 50mm x 1.7µm

Flow rate: 0.5 mL/min

Gradient profile:

Time (min) Solvent-A (%) Solvent-B (%)

0.0 95 5

5.0 10 90

Column dimensions: 4.6 x 150mm x 5µm

Flow rate: 1.0 mL/min

Gradient profile:

Time (min) Solvent-A (%) Solvent-B (%)

0.0 95 5

35.0 10 90

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

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1.11 COMPOUND LIBRARY MAINTENANCE

Confirming the identity and purity of a candidate pharmaceutical is critical to effectively screening

chemical libraries that contain vast types of small molecules across a range of biological targets.

Chemists need to be sure they have synthesized the expected compound. In this high-throughput

screening environment, the ability to obtain information in multiple MS and UV detection modes in

a single injection is invaluable.

1.12 OPEN ACCESS

Maximum efficiency is essential for analytical laboratories that are constantly challenged to

increase throughput and deliver results to research chemists in pharmaceutical discovery. UPLC

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, and fast, robust analyses

using UV or MS for nominal and exact mass measurements. System management is just as simple.

Online, the central administrator can remotely define system users and their privileged for operating

instruments across the network.

Introduction

52

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