COMPONENTS

OF

HPLC

Nishi ShardaScientist I

SMPIC

Instrumentation is required to enable the flow of mobile

phase through the stationary phase and also to convert

the separated components into meaningful information.

THE HPLC SYSTEM

Mobile Phase Reservoir Sample

Pump Injector Column

Detector

Collect / Waste

Recorder

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)

System component

Description

Mobile phase reservoir

Stores the mobile phase required for analysis

Degasser Degasses the mobile phasePump Solvent delivery system, enables the flow of mobile

phase through the systemInjector Sample delivery system, introduces the sample to the

systemColumn compartment

Controls the temperature of the column ,if required.Separates the analyte components.

Detector Detects each component in separated mixture after it elutes from column

Data processor

Converts the data from the detector into results

Waste Collection of the liquid waste

The Mobile phase is stored in glass containers. Plastic

containers are not used as the additives in the plastic may

leach in the mobile phase.

PTFE (Teflon) tubing connects the contents of the reservoir

with the HPLC system.

At the end of the tubing, which is in contact with the mobile

phase, is a filter to remove any particulate matter. Filter is made

of glass, stainless steel or PEEK (polyether ether ketone).

MOBILE PHASE RESERVOIR

Common solvents used forHPLC in order of IncreasingPolarity:

n-Hexane

Methylene chloride

Chloroform

Tetrahydrofuran

Isopropylalcohol

Acetonitrile

Methanol

Water

A blend of two (or more

solvents) of these is used as

mobile phase.

The proportion of different

solvents in blend act to adjust

the polarity of mobile phase.

Suitably combined with

stationary phase to achieve

separation of a mixture.

Binary MixtureA mixture of two solvents - common type of mobile phase.

Ternary Mixture

Blend of three solvents.

The choice of the solvents and their proportion is selected

during method development.

The most important property of the solvent is its ability to

interact with stationary phase and the analyte resulting in the

desired separation.

Solvents An ideal solvent is one readily available in high purity, relatively

inexpensive, safe to use routinely and compatible with entire HPLC

system including detector.

The two most common organic solvents which may be combined

with water to prepare mobile phase are methanol and acetonitrile.

Both are water miscible and have good properties i.e. readily

available, safe to use and compatible with HPLC system

Tetrahydrofuran (THF) is occasionally used.

Disadvantages: Degrades to peroxides. Results in high backpressure, Reacts with

PEEK fittings .

Desirable featuresDesirable features

Pressure generation:

Should be able to generate high pressure associated with modern HPLC pumps and for long periods of time.

The back pressure varies from column to column, change in flow rate and

the viscosity of the mobile phase.

Typical HPLC pumps have max. operating pressure around 500 bars (7000

psi).Flow Rate: should be accurate, reproducible and free of pulsation. Pulsatile flow can

limit sensitivity by causing baseline noise.

Should be able to provide wide range of flow rates with adequate levels of

accuracy and precision.

Flow rates in analytical HPLC varies from 0.01-10 ml/min (small bore HPLCs

with column id

PUMPS

Resistance: Should be inert to solvents, buffer salts and solutes.

For metallic parts, stainless steel and elsewhere resistant materials

like sapphire or ruby for pistons and valve components. Teflon for

gasgets and seals. Pump heads constructed from titanium.

Types of Pumps:

Constant pressureConstant Flow

Constant pressure:Utilise pneumatics or hydraulics to apply pressure required to force

mobile phase through column

Two designs of this category are: i)The pressurised coil pump ii) Pressure intensifier pump

The pressurised coil pump :The operating cost high and pressure limit 100 bars.

Flow is pulse free. But neither sufficiently accurate nor reproducible

enough.Pressure intensifier pumps:Work by applying moderate gas pressure on a piston which pushes

forward a small piston in contact with mobile phase.

Because of the way they operate, the flow produced is pulsatile in nature

and tend to be extremely noisy in use.

Constant Flow Pumps:

90 % of the analytical pumps used are of this type. Flow rate remains

constant.Types of constant flow pump :

i) Syringe pumpsii) Reciprocating piston pumps

PUMPS

Syringe Pumps

One of the first types of HPLC pump built for the purpose. Simple in

design,employ a stepper motor to drive plunger of a large syringe to

push mobile phase at a constant flow rate through the column.

Capacity of syringe upto 500 ml. Flow rate is extremely constant and

can be accurately and precisely controlled by altering the speed of the

stepper motor.

Not widely used. Some models need to be dismantled for refilling.Very

slow process.

Well suited to small bore HPLC. Because they require the very low

flow rates.

Reciprocating piston pumps:

Around 85% of the HPLC pumps in current use are of

reciprocating piston type.

The mobile phase is forced through the system by a piston

/syringe driven by a electric motor.

Inlet and outlet check-valves allow unidirectional flow of mobile

phase to enter the column head.

Piston made from ruby or sapphire.

The problem of pulsation is controlled by using multiple piston

heads, the use of pulse dampening systems and electronics control

of piston speed.

PUMPS

RECIPROCATING PISTON PUMP

The twin piston pumps with short

stroke are among the most commonly

used pumps for HPLC.

Both pump heads are switched in series,

whereby the piston in the first pump head

delivers a specific volume per stroke.

An eccentric disk presses piston 1 to the right and displaces the solvent.

The double ball saphire valves ensure that the solvent stream can flow in

only one direction.

The second piston is used to produce a nearly complete pulsation

damping.

Isocratic elution: It uses the same mobile phase composition throughout the run.

Gradient elution: In this type of elution the composition of the mobile phase changes during the run e.g. from 5 to 100% Methanol.Isocratic elution is preferred over gradient elution because

Gradient equipment is not available in some laboratories Gradient elution can not be used with some HPLC detectors (e.g. RI

detector) Gradient runs take longer because of the need for column equilibration

after each run Gradient methods do not always transfer well, because differences in

equipment can cause changes in separation Baseline problems are more common with gradient elution

Applications of gradient elution: Samples with a wide k range Samples containing late eluting interferences that can either foul or

overlap in subsequent chromatograms

SOLVENT PROGRAMING

GRADIENT HPLC

Convex Program

Concave Program

Linear Program

Time

Con

cent

rato

n of

solv

ent A

Different forms of solvent gradients

SOLVENT PROGRAMERS

Computer programmer

Solvent

Solvent

Solvent

Mixing valves Pump

To sample valve

Low pressure solvent programmer

SOLVENT PROGRAMMERS

Computer programmer

Solvent

Solvent

Solvent

Mixing T To sample valve

High pressure solvent programmer

Pump

Pump

Pump

To obtain maximum chromatographic efficiency, the sample should be introduced onto the column head as an extremely narrow band.

Originally, injection onto the HPLC column was made through a septum using sharp needle

Drawbacks---Short septum life and blockage of the column with the small pieces of septum by needle

Valve type injectors:

Several types available but features are common. Loop controls the

volume of the sample injected and to hold it prior to the introduction

onto the column. The valve allows the loop either to be isolated from

the stream of eluent from the pump (Load Position) or to be

positioned in it (the Inject position).

INJECTORS

Advantages:- Excellent precision- Compatibility with pressure encountered in HPLC- Lack of septa - Easily adaptable for automation

Disadvantages:- Seals of the valves if fail, can cause leakage- Small particles from the sample can scratch the disc to cause mobile phase leak

- Source of artifact peaks in chromatogram- Contaminated loop can result in extra peaks from the previous injection

Automated sample injectors, Autosamplers act as simple robots.

VALVE TYPE INJECTORS

INJECTOR

Six-port Rheodyne valve in which the sample fills an external loop

A clockwise rotation of the valve rotor places the sample-filled loop into the mobile-phase stream, with subsequent injection of the sample onto the top of the column

It is the hub of the HPLC system, where all the chromatographic

separation happens

Generally columns are between 2.5 to 25 cm in length with few

mm i.d. If the column is too short, then the column will not have

enough resolving power to achieve the separation

If the column is too long, then analysis time is needlessly

extended.

A fine grained chromatographic material (e.g. silica gel or RP-

18), serves as stationary phase with particle size of 5-10 m

(analytical separations) and 10-50 m (preparative separations).

COLUMN

HPLC COLUMN

C18 4.6 x 250 mm 5m 300oA

Stationary Phase

Dimension

Particle Size

Pore Size

Water/Methanol

Column dimension (size), particle size and pore size, stationary phase

Parameters to describe a HPLC columnParameter Description

Packing / Matrix

The finely divided material with which the column is packed is generally silica

Bonded Phase

The stationary phase is chemically bonded to the packing matrix

Particle size The size of particles in the column ( measured in microns)

Pore size Size of pores in the particles usually measured in angstroms

Length Length of the column usually measured in cm or mm

Diameter i.d. of column usually measured in mm

Hardware The material used to construct the external tubing and end fittings of the column

Manufacture The name of manufacturer of the column

Particle size Column ID Sample Load

Analytical 3 5 m 0.3 - 4.6 mm ng mg

Semi-prep 10 m 8 10 mm 1 100 mg

Preparative 10 30 m 200 mm gram scale

Types of Detectors:

UV / Visible detector

Diode array detector

Fluorescence detector

Electro-chemical detector

Refractive index detector

Evaporative light scattering detectors (ELSD)

Mass spectrometry (MS)

DETECTORS

These should not chemically interact with the solutes or the mobile phase.

Should not contribute excessively to the dead volume in the system.

In between pump and injector, the id of the tubing is 0.5 mm.

In between the injector and column and column and detector it is 0.15 mm.

Problems:

The narrow bore tubing is prone to be blocked by small particles.

These are prone to block while bending it.

Difficulty in cutting the tubing. These problems can be overcome by using tubing made from the flexible polymer, PEEK.

CONNECTING TUBINGS

PEEK is an inert biocompatible polymer which when used to

prepare a tubing is able to withstand the pressures

encountered in HPLC, and can be easily cut with a blade. It

is highly flexible. It is used with removable finger tight

fittings.

Disadvantages: It is sensitive to few of the solvents e.g. THF, DCM etc.

CONNECTING TUBINGS

The tubings are connected to column ends or other parts by threaded nuts.

Generally these are made of stainless steel.

A seal is formed using a ferrule.

Ferrule is pear shaped with a hole running in the centre.

The wider end of the ferrule is kept towards the nut.

NUTS AND FERRULES

NUTS AND FERRULES

Slide Number 1Slide Number 2Slide Number 3Slide Number 4Slide Number 5Slide Number 6Slide Number 7Slide Number 8Slide Number 9Slide Number 10Slide Number 11Slide Number 12Slide Number 13Slide Number 14Slide Number 15Slide Number 16GRADIENT HPLCSOLVENT PROGRAMERSSOLVENT PROGRAMMERSSlide Number 20 Advantages:- Excellent precision- Compatibility with pressure encountered in HPLC- Lack of septa - Easily adaptable for automation Disadvantages:- Seals of the valves if fail, can cause leakage- Small particles from the sample can scratch the disc to cause mobile phase leak- Source of artifact peaks in chromatogram- Contaminated loop can result in extra peaks from the previous injectionAutomated sample injectors, Autosamplers act as simple robots.INJECTORSlide Number 23HPLC COLUMNParameters to describe a HPLC columnSlide Number 26Slide Number 27Slide Number 28Slide Number 29Slide Number 30Slide Number 31Slide Number 32