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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.

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Introduction

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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

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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

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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

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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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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)

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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

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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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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

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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).

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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.

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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

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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

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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

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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

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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.

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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

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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.

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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.