High performance liquid chromatograph HPLC

Gamal A. Hamid1

High performance liquid chromatograph

HPLC INTRODUCTION

Gamal A. Hamid2

Content

Chromatography

HPLC Chromatography

Instrument

Solvent Rack,

Pump,

Injector “ Autosampler”,

Separation column,

Detector.

Applications

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Chromatography

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Chromatography

Chromatography

Is a physical method of separation in which the components

to be separated are distributed between two phases, one of

which is stationary (stationary phase) while the other (the

mobile phase) moves in a definite direction. ( IUPAC)

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Chromatography classifications

1. Classification according to the shape of the chromatographic bed

2. Classification according to the physical state of the mobile phase

3. Classification according to the mechanism of separation

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1. Classification according to the shape of the bed

Column Chromatography

A separation technique in which the stationary

bed is within a tube.

The particles of the solid stationary phase or the

support coated with a liquid stationary phase

may fill the whole inside volume of the tube

(Packed Column) or be concentrated on or along

the inside tube wall leaving an open, unrestricted

path for the mobile phase in the middle part of

the tube (Open-Tubular Column).

Planar Chromatography

A separation technique in which the stationary

phase is present as or on a plane.

The plane can be a paper, serving as such or

impregnated by a substance as the stationary bed

(Paper Chromatography, PC) or a layer of solid

particles spread on a support, e.g., a glass plate

(Thin Layer Chromatography, TLC).

Sometimes planar chromatography is also

termed Open-Bed Chromatography.

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2. Classification according to the physical state of the mobile phase

Gas-liquid chromatography (GLC)

Gas-solid chromatography (GSC)

Liquid-liquid chromatography (LLC)

Liquid-solid chromatography (LSC)

Gas Chromatography (GC)

A separation technique in which the mobile

phase is a gas.

Liquid Chromatography (LC)

A separation technique in which the mobile

phase is a liquid.

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3. Classification according to the mechanism of separation

Adsorption Chromatography Separation is based mainly on differences between the adsorption

affinities of the sample components for the surface of an active solid.

Partition Chromatography Separation is based mainly on differences between the solubility's of the

sample components in the stationary phase (gas chromatography), or on differences between the

solubility's of the components in the mobile and stationary phases (liquid chromatography).

Ion-Exchange Chromatography Separation is based mainly on differences in the ion exchange affinities

of the sample components.

Exclusion chromatography Separation is based mainly on exclusion effects, such as differences in

molecular size and/or shape or in charge.

Affinity Chromatography This expression characterizes the particular variant of chromatography in

which the unique biological specificity of the analyte and legend interaction is utilized for the

separation.

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Specials techniques

Reversed-Phase Chromatography

Normal-Phase Chromatography

Isocratic Analysis

Gradient Elution

Stepwise Elution

Two-Dimensional Chromatography

Isothermal Chromatography

Programmed-Temperature

Chromatography

Programmed-Flow Chromatography

Programmed-pressure Chromatography

Pyrolysis-Gas Chromatography

Reaction Chromatography

Post-Column Derivatization

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HPLC Chromatography

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Liquid chromatography

Schematic of HPLC

A reservoir holds mobile phase,

A high-pressure pump is used to generate and meter a specified flow rate of mobile phase,

An injector 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.

A detector is needed to see the separated compound bands as they elute from the column .

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HPLC Separation Modes

Separation depends on the triangle relationships

among the sample, filler “ stationary phase”, and

eluent!

Polarity

Electrical Charge “ion exchange”

Molecular Size “size exclusion”

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1. Polarity Separation Modes

A molecule’s structure, activity, and physicochemical characteristics are

determined by the arrangement of its constituent atoms and the bonds

between them.

chromatographic separations based on polarity depend upon the

stronger attraction between likes and the weaker attraction between

opposites.

“Like attracts like” and opposites may be repelled.

compounds in the sample that are similar in polarity to the stationary

phase [column packing material] will be delayed because they are more

strongly attracted to the particles.

Compounds whose polarity is similar to that of the mobile phase will be

preferentially attracted to it and move faster.

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Polarity phases

Normal-Phase HPLC

An elution procedure in which the

stationary phase is more polar than the

mobile phase.

This term is used in liquid chromatography

to emphasize the contrast to reversed-

phase chromatography..

Hydrophilic-Interaction Chromatography

[HILIC]

Reversed-Phase HPLC

The term reversed-phase describes the

chromatography mode that is just the

opposite of normal phase, namely the use

of a polar mobile phase and a non-polar

[hydrophobic] stationary phase.

Hydrophobic-Interaction Chromatography

[HIC]

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Normal-Phase HPLC “NP-HPLC “

Adsorption strengths increase with increased

analyte polarity.

The interaction strength depends not only on the

functional groups present in the structure of the

analyte molecule, but also on steric factors.

The use of more polar solvents in the mobile

phase will decrease the retention time of analyte,

whereas more hydrophobic solvents tend to

induce slower elution (increased retention

times).

Very polar solvents such as traces of water in the

mobile phase tend to adsorb to the solid surface

of the stationary phase forming a stationary

bound (water) layer which is considered to play

an active role in retention.

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Reversed-phase HPLC (RPC)

RP-HPLC operates on the principle of

hydrophobic interactions, which originates from

the high symmetry in the dipolar water

structure .

The binding of the analyte to the stationary

phase is proportional to the contact surface area

around the non-polar segment of the analyte

molecule upon association with the ligand on the

stationary phase.

an analyte with a larger hydrophobic surface

area (C–H, C–C, and generally non-polar atomic

bonds, such as S-S and others) is retained longer

because it is non-interacting with the water

structure. On the other hand, analyte with higher

polar surface area (conferred by the presence of

polar groups, such as -OH, -NH2, COO– or -NH3+ in

their structure) are less retained as they are

better integrated into water.

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2. Electric charge Separation Modes

The rule is “Likes may repel, while opposites are

attracted to each other”.

Stationary phases for ion-exchange separations are

characterized by the nature and strength of the acidic or

basic functions on their surfaces and the types of ions

that they attract and retain.

Cation exchange is used to retain and separate positively

charged ions on a negative surface.

Conversely, anion exchange is used to retain and

separate negatively charged ions on a positive surface

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3. Molecular size Separation Modes

Rule is “Big ones come out first”

The stationary phases have been synthesized with a pore-

size distribution over a range that permits the analyte of

interest to enter, or to be excluded from, more or less of the

pore volume of the packing.

The biggest molecules may be totally excluded from pores

and pass only between the particles, eluting very quickly.

the smaller molecules travel slower [because they move

into and out of more of the pores] and elute later

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Stationary phase particles size

Smaller Particle Size Leads to

Higher plate number

Higher pressure

Shorter run time (higher sample

throughput)

Lower detection limit

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Instruments

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Instrument

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UltiMate 3000 System

The UltiMate 3000 systems are fully

modular, allowing you to choose common

system configurations or design the most

suitable system for your application needs.

RSLC nano Systems

RSLC Systems

Standard Systems

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High pressure liquid chromatograph Instrumentation

HPLC

Is a form of liquid chromatography to separate compounds to

identify and quantify each component that are dissolved

in solution.

HPLC instruments consist of:

1. Solvent rack,

2. Pump,

3. Injector,

4. Separation column,

5. Detector.

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1. Solvent Rack “SR” Instrumentation

SR-3000 Solvent Rack

without vacuum degasser typically for use with a

LPG-3400, ISO-3100BM, or HPG-3200BX

SRD-3200 Solvent Rack

with analytical 2-channel vacuum degasser typically

for use with the following pumps: - one HPG-3200

(SD or RS) - one ISO-3100SD

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SRD “Solvent Rack Degasser”


With analytical 4-channel vacuum degasser

typically for use with the following pumps: - one

HPG-3400 - two HPG-3200 (SD or RS) pumps in a

two-stack system - one HPG-3200 (SD or RS) or

ISO-3100SD if you want to degas the solvents and

the wash solution of an UltiMate 3000 series

autosampler


With analytical 6-channel vacuum degasser

typically for use with the following pumps: - one

DGP-3600 - two HPG-3200 (SD or RS) pumps in a

two-stack system - one HPG-3200 (SD or RS) and

one HPG-3400 in a two-stack system - one HPG-

3400 if you want to degas the solvents and the

wash solution of an UltiMate 3000 series

autosampler

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Effect of Eluent composition in separation

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Mobil phase

A fluid which percolates through or along the

stationary bed, in a definite direction.

It may be a liquid (Liquid Chromatography) or a gas

(Gas Chromatography) or a supercritical fluid

(Supercritical-Fluid Chromatography).

In gas chromatography the expression Carrier Gas

may be used for the mobile phase.

In elution chromatography the expression Eluent is

also used for the mobile phase.

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Reservoir of mobile phase

A water-based solvent, organic solvent, or a

mixture of the two is mainly used as the

mobile phase for HPLC.

The way that they are mixed can cause

large differences in analysis results.

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Common HPLC Solvents

THF “ Tetrahydrofuran” ( not compatible with PEEK)

IPA “ Isopropyl Alcohol “

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2. Pump Instrumentation

A device designed to deliver the mobile phase at a controlled flow-

rate to the separation system.

It is necessary to pump the eluent at a constant flow rate and pressure.

isocratic analysis in which the eluent composition remains unchanged

during the analysis. solvent must be pre-mixed

A gradient analysis allows the composition of the eluent to be changed

during the analysis.

Binary gradient pump –delivers two solvents

Quaternary gradient pump –four solvents

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Isocratic analysis

In this mode, the mobile phase, either a

pure solvent or a mixture, remains the

same throughout the run.

solvent must be pre-mixed

A typical system is outlined

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Gradient analysis

Low-pressure mixing method: (LPG Pump)

One pump is used for mixing.

The eluent to be absorbed is switched via

electromagnetic valves.

Up to four eluents can be mixed.

High-pressure mixing method: (HPG Pump)

Two pumps are used.

The eluents are mixed after pumping.

The response of the gradient is superior because of

the small volume from the mixing unit to the column.

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UltiMate 3000 pump Operating Principle

The pump is a zero-pulsation, serial dual-piston pump with electronic compressibility.

The two pump heads are connected in series.

Continuous delivery is achieved as follows: The working head delivers at the appropriate flow rate

while simultaneously filling the serially connected equilibration head.

The latter serves as a reservoir and delivers while the working head carries out the suction stroke.

Pulsation during the pre-compression phase is reduced to a minimum by velocity modulation of the

drive.

The flow rate is always kept constant in relation to the atmospheric pressure.

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Pump configurations

Pump Descriptions Options

1 ISO-3100A Isocratic pump (analytical; 1 solvent) 5035.0010

2 LPG-3400A Low-pressure gradient pump (analytical; 4 solvents) with integrated vacuum degasser and mixing chamber

Mixing ChamberExtension KitMicro Flow Kit

3 LPG-3400M Low-pressure gradient pump optimized for micro flows (4 solvents) with integrated vacuum degasser. The pump has no mixing chamber.

4 LPG-3400AB Same as LPG-3400A, but biocompatible Version Mixing ChamberMicro Flow Kit

5 LPG-3400MB Same as LPG-3400M, but biocompatible

6 DGP-3600A Dual low-pressure gradient pump version(analytical): Two separate pumps with integrated mixing chambers in one enclosure (2x3 solvents)

Mixing ChamberExtension KitMicro Flow Kit

7 DGP-3600M Dual low-pressure gradient pump optimized for micro flows: Two separate pumps are installed in one enclosure (2x3 solvents). The pumps have no mixing chambers.

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Pump Descriptions Options

8 DGP-3600AB Same as DGP-3600A, but biocompatible version Mixing Chamber Extension Kit

9 DGP-3600MB Same as DGP-3600M, but biocompatible version

10 HPG-3200A High-pressure gradient pump (analytical; 2 solvents) with integrated mixing chamber

Mixing Chamber Extension Kit

11 HPG-3200M High-pressure gradient pump optimized for micro flows (2 solvents). The pump has no mixing chamber.

12 HPG-3200P High-pressure gradient pump (semi preparative; 2 solvents) with integrated mixing chamber and mixing chamber extension


13 HPG-3400A High-pressure gradient pump (analytical) with integrated mixing chamber and "2 from 4" solvent selectors


14 HPG-3400M High-pressure gradient pump optimized for micro flows with "2 from 4" solvent selectors. The pump has no mixing chamber.

Pump configurations

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Precautions

When switching to another solvent, ensure

that the new solvent is miscible with the one

contained in the pump.

If the solvents are not miscible, the pump can

be damaged, for example, by flocculation.

Never run the pump dry.

Damage to the pistons or the piston seals

could result.

Before you start operating the pump, check

the seal wash reservoir level and refill as

needed.

After turning on the pump, wait until the

wash solution has passed all pump heads.

If a leak occurs, turn off the pump and remedy

the situation immediately.

Some components are made of PEEK™.

This polymer has superb chemical resistance to

most organic solvents. However, it tends to

swell when in contact with trichlormethane

(CHCl3), dimethyl sulfoxide (DMSO), or

Tetrahydrofuran (THF).

In addition, it is attacked by concentrated acids,

such as, sulfuric acid and nitric acid or a mixture

of hexane, ethyl acetate, and methanol. In both

cases, capillaries may start leaking or they can

burst. Swelling or attack by concentrated acids

is not a problem with brief flushing procedures.

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SD and SDN pumps

ISO-3100SD,

LPG-3400SD(N),

DGP-3600SD(N),

HPG-3200 SD,

HPG-3400SD

SD “ Standard”

No Description

1 Peristaltic pump

2 Detector of the rear seal wash system

3 Capillary guides

4 Pump head with working cylinder and equilibration cylinder

5 Leak sensor

6 Inline filter

7 Pump unit without purge valve and pressure transducer for the system pressure

8 Pump lights

9 Pump block status LED

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ISO 3400 SD Operating Principle

No. Description

1 pump head withWorking cylinder ( no. 1a) and equilibrium cylinder (no. 1B)

2 Purge unit with Purge valve knob ( no.2a) and outlet nozzle (no. 2b)

3 Inline filter

4 Pump outlet

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RS pumps

LPG-3400RS,

DGP-3600RS,

HPG-3200RS,

HPG-3400RS

RS “ Rapid Separation”

No Description1 Peristaltic pump


3 Capillary guides


5 Leak sensor

6 LPG-3400SD and RS: Capillary mixer LPG-3400BM: Capillary from purge unit to inline filter

7 4-channel vacuum degasser

8 Pump lights

9 LPG-3400SD and RS: Static mixer LPG-3400BM: Inline filter

10 Purge unit with purge valve and pressure transducer for the system pressure


12 4-channel proportioning valve

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LPG 3400 RS operating principle

No. Description

1 Inbuilt vacuum degasser

2 Proportioning valve

3 Pump head withWorking cylinder ( no. 3a)and outlet nozzle ( no.4)

4 Purge unit withPurge valve knob (no. 4a) and outlet nozzle ( no. 4b)

5 LPG – 3400 SD and LPG – 3400 RSTwo steps mixing system with capillary mixer( no.5) and static mixer (no. 6)LPG 3400 BMCapillary from purge unit to inline filter( no. 5) and filter ( no.6)

6

7 Pump outlet

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BM pumps

ISO-3100BM,

LPG-3400BM,

DGP-3600BM

No Description1 Peristaltic pump


3 Capillary guides


5 Leak sensor

6 Pulse damper

7 Inline filter


9 Pump light


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ISO 3100 BM Operating Principle

No. Description

1 pump head withWorking cylinder ( no. 1a) and equilibrium cylinder (no. 1b)

2 Purge unit with Purge valve knob ( no.2a) and outlet nozzle (no. 2b)

3 Pulse damper

4 Inline filter

5 Pump outlet

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BX pump

HPG-3200BX No Description1 Peristaltic pump


3 Capillary guides


5 Leak sensor


7 Pump lights


9 Capillary mixer (HPG – 3200 SD and RS) or capillary from purge unit to static mixer (HPG -3200 BX)

10 Static mixer

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HPG 3200 BX Operating Principle

No Element NO Element

11a1b

Left pump head with working cylinderEquilibrium cylinder

4+5 HPG-3200SD & HPG 3200 RSTwo- step mixing system with Capillary mixer (no.4) and Static mixer (no. 5)

22a2b

Right pump head with working cylinderEquilibrium cylinder

HPG 3200 BXCapillary from purge unit to static mixer ( no. 5)

33a3b

Purge unit withPurge valve knobOutlet nozzle

6 Pump outlet

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Visual Inspection

When a problem occurs, it is advisable to

perform a quick visual check of the

instrument and column.

This will pick up leaks, loose or

disconnected tubing, changes in

instrument settings etc.

These problems are easy to rectify and will

save time.

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Pump Chromeleon page

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3. Injector Instrumentation

A device by which a liquid, solid or gaseous

sample is introduced into the mobile phase

or the chromatographic bed.

The injector serves to introduce the liquid

sample into the flow stream of the mobile

phase.

The injector must also be able to withstand

the high pressures of the liquid system.

A sample is injected into the flow path for

analysis.

Each type is equipped with six-port valves, so

that a sample can be injected into the flow

path at continuous pressure.

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Autosampler

Excellent retention time and gradient precision.

Pulled-loop injection principle (full- and partial-

loop injections).

Fast and stable column thermostatting between

5 °C above ambient and 50 °C.

High detection sensitivity with low detector

noise and drift.

Robust, dependable performance at low cost of

ownership.

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Pressure troubleshooting

System pressure is affected by a number of

variables including the viscosity of the

solvent used, column variables, flow rate

and temperature.

Pressure problems fall into one of three

categories: high, low or fluctuating

pressure.

They can occur suddenly or be a gradual

process.

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High Pressure troubleshooting

Has the ambient temperature changed?

Is the flow rate correct?

Is the eluent viscous?

Is the pressure transducer operating

correctly?

Loosen detector waste outlet fitting.

Loosen detector inlet fitting.

Loosen column outlet fitting.

Loosen column inlet fitting.

Loosen fitting at guard or in-line filter.

Loosen injector outlet fitting.

Loosen pump outlet fittings.

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Low Pressure troubleshooting

Is the pump fuse in working order?

Is the pump on?

Is there solvent flow?

Is there solvent in the reservoir?

Is the low pressure cut-off higher than the

operating pressure?

Does solvent flow out of the purge valve

when opened?

Was the pump primed?

Is air visible in the solvent lines?

Are the pump heads functioning correctly?

Is the flow rate set correctly?

Is the column temperature constant?

Are there any leaks?

Is the correct solvent being used?

Was the purge valve closed after priming?

Is the auto injector in prime mode?

Is the flow rate delivered same as the rate

entered?

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Fluctuating Pressure Reading

Is the pressure functioning correctly?

Was the pump primed properly?

Are you performing a gradient analysis?

Are the pump heads functioning correctly?

Are all the solvents degassed?

Are all solvents miscible?

Are the solvents volatile?

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Autosampler Chromeleon page

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4. Column Instrumentation

Is The tube and the stationary phase contained

within, through which the mobile phase passes.

The heart of the chromatograph, the column’s

stationary phase separates the sample

components of interest using various physical and

chemical parameters.

The pump push hard to move the mobile phase

through the column and this resistance causes a

high pressure.

A column is selected to suit both the sample and

the purpose of separation.

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Column compartment

Freely-configurable and user-interchangeable high-pressure

switching valves

Accommodation of up to 12 columns

Temperatures from 5 to 110 °C with the Rapid Separation

Thermostatted Column Compartment

Short equilibration times for temperature step gradients and

fast application switching

Low-dispersion eluent preconditioned for better peak shapes

at elevated column temperature

Homogeneous temperature distribution via a fan-based,

forced-air design

Column identification system and comprehensive system

wellness features

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Separation power principals

Efficiency

Mechanical separation power,

created by the column length,

particle size, and packed-bed

uniformity,

Efficiency is a measure of

mechanical separation power.

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Separation power principals

Selectivity

Chemical separation power, created by the

physicochemical competition for

compounds between the packing material

and the mobile phase.

Selectivity is a measure of chemical

separation power.

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Column heating

Reproducibility

Retention in HPLC is temperature-dependent

If temperature varies, then it is difficult to assign “peaks” to

specific compounds in the chromatogram and the peak

areas/heights may vary

Solubility

Certain chemical compounds may have low solubility in the HPLC

mobile phase

If they are injected into the flow stream they may precipitate or

other difficulties may arise

Stability

Certain chemical compounds, especially biological compounds

such as enzymes or proteins, may not be stable at room

temperature or higher

The temperature needs to be much lower down to 4°C

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5. Detector Instrumentation

A device that measures the change in the

composition of the eluent by measuring

physical or chemical properties.

The desirable features of a detector are:

Sensitivity towards solute over mobile phase

Low cell volumes to minimize memory effects

Low detector noise

Low detection limits

Large linear dynamic range

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UV/UV-VIS Detectors (variable λ detector)

Wavelength range of 190–900 nm with

combined use of deuterium and tungsten

lamp on one optical axis.

Sensitive detection in the UV, visible, and

near-infrared range.

Up to four wavelengths can be recorded

simultaneously, making the four-channel

mode compatible with even fast

chromatography.

61

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VWD Principle

A UV detector employs a deuterium discharge

lamp (D2 lamp) as a light source, with the

wavelength of its light ranging from 190 to

380 nm.

If components are to be detected at

wavelength longer than this, a UV-VIS

detector is used, which employs an additional

tungsten lamp (W lamp).

By monitoring the reference light divided

from the light in front of the flow cell, the

difference in light intensity can be

determined between the back and front of

the flow cell, and this is output as

absorbance.

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UV Detector Chromeleon page

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Diode array detector (DAD, PDA)

Photodiode arrays (semiconductor devices)

are used in the detection unit.

Incorporation of large number of diodes

which serve as detector elements makes

possible simultaneous monitoring of more

than one absorbing component at different

wavelengths.

This provides benefit of time saving and

cost reduction on expensive solvents.

Optical System, Single-beam, reverse optics

design with concave holographic grating

Wavelength Range 190 to 800 nm

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PDA Principle

The tungsten and deuterium lamps emits light in

UV and visible ranges (190 -800) nm.

The polychromatic beam passes the flow cell.

The grating splits up the polychromatic beam to

different wavelengths, the intensities of which are

measured by an array or photodiodes.

A photodiode is a semiconductor device that

converts light into current. The current is

generated when photons are absorbed in the

photodiode.

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Fluorescence Detector

Xenon lamp Light source for the UV to near-

infrared wavelength range.

PMT Photomultiplier tube (PMT) - Converts the

emitted light to a measureable current signal.

Wavelength range 200 – 900 nm (dual).

Fluorescence detection offers greater sensitivity

than a UV-VIS detector. However, the number of

naturally fluorescent compounds is smaller in

comparison to light absorbing compounds. This

limitation is overcome by post column

Derivatization

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FL Principle

FL is a phenomenon in which a substance absorbs light to reach a high-energy level and

then emits light to return to its original level.

Such a substance has specific wavelengths of light that it absorbs (excitation

wavelengths) and emits (emission wavelengths).

While a UV/UV-VIS detector detects light that has passed through the flow cell, an FL

detector detects fluorescence emitted in the direction orthogonal to the exciting light.

A UV/UV-VIS detector monitors the absorption of light with a specified wavelength.

However, some substances absorb light at one wavelength, and then emit light called

fluorescence at another wavelength.

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FL Detector Chromeleon page

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Refracted index Detector

The response is dependent on changes in refractive index of eluting

compounds in the mobile phase.

The mobile phase itself should have refractive

index different from the sample.

Gradient programming is not possible due to

resulting changes in refractive index of mobile phase.

Temperature control is necessary as it has

high temperature sensitivity.

Any component in the eluate can be detected;

thus, the RI detector is often called a “universal detector”.

Typical applications are in Size Exclusion Chromatography.

The detector is less sensitive than UV-VIS detector.

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RI Principle

RI detector detects components based on the

refraction of light in solution.

The reference-side cell is filled with eluate, and the

column eluate is introduced into the sample-side cell

through the changed flow channel.

When components are eluted from the column, the

chemical composition changes in the sample-side

solution, which changes its photorefractive level.

As a result, the amount of light received by the light-

receiving section changes, showing a peak which can

be detected.

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Applications

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Applications fields

Environmental applications

Food applications

Pharmaceutical applications

Bioanalytical applications

Pharmaceuticals like aspirin, ibuprofen, or acetaminophen (Tylenol)

Salts like sodium chloride and potassium phosphate

Proteins like egg white or blood protein

Organic chemicals like polymers (e.g. polystyrene, polyethylene)

Heavy hydrocarbons like asphalt or motor oil

Many natural products such as ginseng, herbal medicines, plant extracts

Thermally unstable compounds such as trinitrotoluene (TNT), enzymes

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Preparation of Pure Compounds

Preparative chromatography

By collecting the chromatographic peaks at the exit

of the detector, - and concentrating the compound

(analyte) by removing/evaporating the solvent, - a

pure substance can be prepared for later use (e.g.

organic synthesis, clinical studies, toxicology

studies

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High performance liquid chromatograph HPLC