HIGH PERFORMANCE LIQUID CHROMATOGRAPHY Click to edit Master subtitle style

BY K RAKESH GUPTA

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CHROMATOGRAPHY :
Chromatography may be defined as the method of

separating a mixture of components into individual components.

This techniques is based on the differences in the

rate at which the components of a mixture move through a porous medium(stationary phase) under the influence of some solvent or gas(mobile phase).
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WHY USE HPLC?

Simultaneous analysis High resolution High sensitivity Good repeatability Moderate analysis condition Easy to fractionate and purify Not destructive

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HPLC
HPLC- It was originally referred to as High Pressure

Liquid Chromatography since high pressure is applied using a pumping system to the column.
This pressure works by forcing the mobile phase

through, at much higher rate increasing the resolution power.

Due to its high efficiency and performance High

Pressure Liquid Chromatography is referred to as High Performance Liquid Chromatography. 5/2/12

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TYPES OF LIQUID CHROMATOGRAPHY

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1.Normal Phase Chromatography:

Stationary Phase Polar nature.

Eg: SiO2,Al2O3 Mobile Phase Non-Polar nature. Eg:heptane,hexane,cyclohexane,CHCl3,CH3OH Mechanism: Polar compounds travels slower & eluted slowly due to higher affinity to st.phase Non-polar compounds travels faster & eluted 1st due to lower affinity to st.phase. This technique is not widely used in pharmaceutical separations.
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2.Reverse Phase Chromatography:

Stationary Phase Non-Polar nature.

Eg: n-octadecyl, n-octyl, ethyl, phenyl diol, hydrophobic polymers. Mobile Phase Polar nature. Eg: methanol or acetonitrile/water or buffer sometimes with additives of THF or dioxane. Mechanism: Polar compounds travels faster & eluted 1st due to lesser affinity to st.phase Non-Polar compounds travels slower & eluted slowly due to higher affinity to st.phase
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Effect of Stationary Phase

C 8 C18 (ODS) Stron g sampl e C 4 Mediu m sampl e

Wea k sampl e
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Reversed order of elution

Increasing Mob phase Polarity, Decreases Elution Time

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5.Ion-Exchange Chromatography:
It is the process by which similar charged ions

such as cations, anions can be separated.

By using the suitable ion exchange resin it can

be separated.
It exchanges the ions according to their relative

affinities.
The exchange takes place in a reversible

manner between the ions of the solution and the ions 5/2/12 present in the ion exchange resin .
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6.Molecular Exclusion Chromatography:

A mixture of components with different

molecular sizes are separated by using gels.

The gels used acts as molecular sieve & hence

a mixture of substances with different molecular sizes are separated.

Soft gels like dextran, agarose or poly

acrylamide are used.

Semi-rigid gels like polystyrene, alkyl dextran
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TYPES OF HPLC DEPENDS ON:

Molecular weight of

solute
Water solubility of

solute
Polarity of solute Ionic and non-ionic

character of solute

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Adsorption Chromatography:
The principle of separation is adsorption . Separation of compounds takes place based on

the difference in the affinity of the compunds towards stationary phase as in the normal and reverse phase.
The lesser the affinity of the sample particles

towards the stationary phase the faster the time of elution of the sample.
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Separation Mechanism Due to different interaction

Mobile Phase

between stationary phase and different sample, the molecules move at different rate, therefore separation can be done.

1
Stronger interaction Weaker interaction

Stationary Phase
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Partition chromatography:
In this the stationary phase is a liquid which is

coated on the solid support on the column.

The mobile phase is also a liquid. When solute along with the mobile phase is

passed over the stationary phase it gets dissolved to the surface of the liquid coated to the solid support.
The compounds which have more partition co5/2/12 efficient 2626 are eluted slowly when compared to

ADSORPTION CHRM:

PARTITION CHRM:

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INSTRUMENTATION OF HPLC
Solvent storage bottle Gradient controller and mixing unit De-gassing of solvents Pump Pressure gauge Pre-column Sample introduction system Column Detector Recorder
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FLOW DIAGRAM OF HPLC instrument

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Outline of LC-2010
Reservior Tray System Controller

Auto sampler Column Oven Degassing Unit Pump Unit Low pressure gradient device
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UV detector

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GRADIENT CONTROLLER

Isocratic solvents- mobile phase is prepared by

using pure solvent or mixture of solvents which has same eluting power or polarity.
Gradient solvents- in this the polarity of the

solvent is gradually increased & hence the solvent composition has to be changed.
Hence this gradient controller is used when two

or more solvent pumps are used for such

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separations.

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Elution Modes
Long Time Analysis Bad Separation

MeOH / H2O = 6 / 4

Isocrati c

MeOH / H2O = 8 / 2

Isocrati c
MeOH%

Volts

Gradie nt
Time

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( Column : ODS )

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Isocratic System

Injector Pump Mobile Phase

Column Ove n

Detector

Simple system with one pump and one solvent reservoir.

Data processo r

If more than one solvent is used, solvents should be premixed. 5/2/12

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High-pressure Gradient System

A

pump Mixe r pump Injector

Column Oven

Detector Data processor

pump

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Excellent gradient accuracy. 2-3 pumps required - one pump per solvent used. On-line degassing may not be critical. 3434

MIXING UNITS:

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DEGASSING OF SOLVENTS:

Several gases are soluble in organic solvents,

when high pressure is pumped, the formation of gas bubbles increases which interferes with the separation process, steady baseline & shape of the peak.
Hence de-gassing is very important and it can be

done by various ways.

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(i) Vacuum filtration: ()

De-gassing is accomplanished by applying a partial vacuum to the solvent container.

()

But it is not always reliable & complete.

(ii) Helium Purging: ()

Done by passing Helium through the solvent.

()

This is very effective but Helium is expensive.

5/2/12 (iii) Ultrasonication:

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PUMP:

The solvents or mobile phase must be passed

through a column at high pressures at up to 6000 psi(lb/in) or 414 bar.

As the particle size of stationary phase is smaller

(5 to 10) the resistance to the flow of solvent will be high.

That is, smaller the particle size of the stationary

phase the greater is the resistance to the flow of

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solvents.

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Requirements for pumps:

Generation of pressure of about 5000 psi. Pulse free output & all materials in the pump

should be chemically resistant to solvents.

Flow rates ranging from 0.1 to 10 mL/min Pumps should be capable of taking the solvent

from a single reservoir or more than one reservoir containing different solvents simultaneously.
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PUMP B

PUMP A

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DISPLACEMENT PUMPS
equipped with a plunger activated by a screw driven mechanism powered by a stepping motor.

It consists of large, syringe like chambers

So it is also called as Screw Driven Syringe Type

Pump.
Advantages:- It produces a flow that tends to be

independent of viscosity & back pressure.

Disadvantages:- It has a limited solvent 5/2/12
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To Column

Piston Drive Motor

Mobile phase reservoir Fast refill motor

Driver Control

Manual rewind

DISPLACEMENT PUMP
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RECIPROCATING PUMPS:

This pump transmits alternative pressure to the

solvent via a flexible diaphragm ,which in turn is hydraulically pumped by a reciprocating pump.
Disadvantages:-

Produces a pulsed flow which is damped because pulses appear as baseline noise on the chromatograph. This can be overcome by use of dual pump heads or elliptical cams to minimize such 5/2/12
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Plunger Reciprocating Pump

out motor and cam pump head check valve

5 - 50L

plunger plunger seal

check valve in

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Mobile phase

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 Solvent is pumped back

and forth by a motor driven piston

Two ball check valves which

open & close which controls the flow

The piston is in direct

contact with the solvent

Small internal volume 35-

400L
High output pressure up to

10,000 psi
Ready adaptability to
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Advantages:
Have small internal volume of 35-400L Higher output pressures up to 10,000 psi. Adaptability to gradient elution. Large solvent capacities & constant flow rates. Largely independent of column back pressure &

solvent viscosity.

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PNEUMATIC PUMPS:

In this pumps, the mobile phase is driven

through the column with the use of pressure produced from a gas cylinder.
It has limited capacity of solvent Due to solvent viscosity back pressure may

develop.

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COLUMN:
columns that can be used in HPLC method.

There are various

They are as follows: Guard Column Derivatizing Column Capillary Column

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GUARD COLUMN:
separating column.

Guard columns are placed anterior to the

This protects and prolongs the life &

usefulness of the separating column.

They are dependable columns designed to

filter or remove: particles that clog the separating column, compounds and ions that could ultimately

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baseline drift, decreased resolution, 5050

 Compounds that may cause precipitation

upon contact with the stationary or mobile phase.

Compounds that may co-elute and cause

extraneous peaks & interfere with the detection and quantification.

These columns must be changed on a regular

basis in order to optimize their protectiveness.

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DERIVATIZING COLUMN

Derivatization involves a chemical reaction

between an analyte and a reagent to change the chemical and physical properties of an analyte. The four main uses of derivatization in HPLC are: Improve detectability, Change the molecular structure or polarity of analyte for better chromatography, Change the matrix for better separation, Stabilize a sensitive analyte.
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Pre or post primary column derivatization can

be done.
Derivatization techniques includes

acetylation, silylation, acid hydrolysis.

Disadvantages: It becomes a complex

procedure and so it acts as a source of error to analysis and increases the total analysis time.

Advantages: Although derivatization has

drawbacks, it may still be required to solve a
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specific separation or detection problem.

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CAPILLARY COLUMNS:

HPLC led to smaller analytical columns called

as micro-columns, capillary columns which have diameter less than a millimeter.

Sample used is in nanolitre volumes,

decreased flow rate, decreased solvent volume usage which leads to cost effectiveness.
Disadvantage:- since it is miniatured flow

rate is difficult to produce & gradient elution is

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MICROBORE and SMALLBORE columns are also

used for analytical and small volumes assay. Diameter of small-bore columns is 1-2mm. The instrument must also be modified to accommodate these smaller capacity columns.
FAST COLUMNS: This column also have the same internal

diameter but much shorter length than most other columns & packed with particles of 3m in diameter. Increased sensitivity, decreased analysis time, decreased mobile phase usage & increased 5/2/12 5555 reproducibility.

ANALYTICAL COLUMN:

This is the most important part of HPLC which

decides the efficiency of separation

Length- 5 to 25 cm ,Internal Diameter 3 to

5mm.
Particle size of packing material is 3 to 5m. LC columns achieve separation by different

intermolecular forces b/w the solute & the stationary phase and those b/w the solute & mobile 5/2/12 phase.
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PREPARATORY COLUMN:

Length 10 to 15 cm, Int. diameter 4.6mm Packed with particles having 5m as

diameter.
Columns of this time generate 10,000 plates

per column.
It consists of back pressure regulator and

fraction collector.
This back pressure regulator is placed
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posterior to the HPLC detector.

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SAMPLE INJECTOR SYSTEM:

Several injector devices are available either for

manual or auto injection of the sample. (i) Septum Injector (ii)Stop Flow Injector (iii)Rheodyne Injector

Rheodyne Manual injector

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(i)Septum Injector:
These are used for injecting the sample

through a rubber septum. This kind of injectors cannot be commonly used , since the septum has to withstand high pressures.

(ii)Stop Flow(On Line):

In this type the flow of mobile phase is

stopped for a while & the sample is injected through a valve.

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(iii)RHEODYNE INJECTOR:
It is the most popular injector and is widely used. This has a fixed volume of loop, for holding

sample until its injected into the column, like 20L, 50L or more.
Through an injector the sample is introduced into

the column.
The injector is positioned just before the inlet of

the column.
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LOAD POSITION: In this position the sample is loaded into the sample loop . INJECT POSITION: In this position the loaded sample is injected into the column by the forcful flow of the solvent into the sample loop by which the sample 5/2/12 is introduced into

select whether the purpose is for analytic al purpose or

Click icon to add picture

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6 Port Valve System

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Typical sample loop volume is 5-200 l.

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HPLC Auto Injectors

Inside of SIL-20AC

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Column temperature controller:

For obtaining better and reproducible

chromatograms constant column temperature should be maintained. Some are equipped with heaters that control temperatures to a few tenths of a degree from near ambient. Columns may also be fitted with water jackets fed from a constant temperature bath to give precise temperature control. For some applications, close control of column temperature is not necessary & columns are operated at R.T
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DETECTORS:

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DETECTORS:
Absorbance (UV/Vis or PDA) Refractive index (detects the change in

turbidity)
Fluorescence (if the analyte is fluorescent) Electrochemical (measures current flowing

through a pair of electrodes, on which a potential difference is imposed, due to oxidation or reduction of solute)
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(for ions)

Selection of Detectors
Detectors
UV-Vis & PDA RF CDD ECD RID & ELSD 5/2/12

Type of compounds can be detected Compounds with chromophores, such as aromatic rings or multiple alternating double bonds. Fluorescent compounds, usually with fused rings or highly conjugated planar system. Charged compounds, such as inorganic ions and organic acid. For easily oxidized compounds like quinones or amines. For compounds that do not show characteristics usable by the other detectors, eg. polymers, sccharides.
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ABSORBANCE DETECTORS:
monochromator so the wavelength can be selected, or scanned. is stopped long enough for the scan to take place. wavelength usually 254nm.

The UV/Vis source usually comes from a

If wavelength scanning is desired, the flow

Fixed wavelength-measures at single Variable wavelength-measures at single

wavelength at a time but can detect over a 5/2/12 6969 wide range of wavelengths simultaneously.

Refractive Index (RI) detector:

Nearly universal but poor detection limit.

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Detection occurs when the light is bent

due to samples eluting from the columns, and this is read as a disparity b/w the two channels.
It is not much used for analytical

applications because of low sensitivity & specificity.

When a solute is in the sample

compartment, refractive index changes will

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shift the light beam from the detector.

7171

Refractive Index Detector

Photodiod e

Reference

Refractio n

W Lamp Sample

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FLUORIMETRIC DETECTORS:

It is based on the fluorescent radiation emitted

by some compounds. The excitation source passes through the flow cell to a photodetector while a monochromator measures the emission wavelengths. More sensitive and specific. The disadvantge is that most compounds are not fluorescent in nature.

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Fluorescence of Compounds
Fluorescence is a type of luminescence in which the light energy is released in the form of a photon in nanoseconds to microseconds
S 1 T 1 Light absorption Non-radiation transition Non-radiation transition

Fluorescence
Phosphorescence S 0

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DIAGRAM OF FLUORESCENCE DETECTORS:

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Fluorescenc e Detector
Advantage Sensitivity is higher than UV-Vis detector Selectivity is high because relatively few compounds fluorescence Compatible with gradient elution Disadvantage Difficult to predict fluorescence Greatly affected by environment Solvent pH Temperature Viscosity Ionic strength Dissolved gas
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AMPEROMETRIC DETECTOR:
reducing and oxidizing property of the sample when a potential is applied.

Amperometric detectors works based on the

The diffusion current recorded is directly

proportional to the concentration of the compound recorded.

DISADVANTAGE: This detector is applicable only

when the functional groups present in the sample can be either oxidized or reduced.
5/2/12 ADVANTAGE: Highly sensitive detector. 7777

AMPEROMETRIC DETECTOR:

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Instrumentation of UVVis Detector

Gratin g M1 Ei n M2 Ei n
Light source D2 / W lamp 5/2/12

Sample Cell Eou t

Photodiod e

Eou t Reference Cell

Photodiod e

7979

Ultraviolet / Visible Detector

Advantage: Sensitivity is high Relative robust to temperature and flow rate change Compatible with gradient elution Disadvantage: Only compounds with UV or visible absorption could be detected. Additional Functions Dual Wavelength mode Wavelength Time Program mode Wavelength Scan mode
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PHOTODIODE ARRAY DETECTORS:

UV detector which operates from 190-600nm.

This is a recent detector which is similar to

Radiations of all wavelength fall on the

detector simultaneously.
The resulting spectra is a three dimensional

plot of Response Vs Time Vs Wavelength.

ADVANTAGE: The wavelength need not be

selected but detector detects the responses

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of all compounds.

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PHOTODIODE ARRAY DETECTORS:

Sample Cell D2 / W lamp Gratin g
One element detects one absorbance at one wavelength.

512 Elements Photodiode Array

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RECORDERS AND INTEGRATORS:

obtained from the detectors after amplification,if necessary.

Recorders are used to record responses

They record the baseline & all the peaks

obtained with respect tot ime.

Retention time can be found out from this

recordings,but area under curve cannot be determined.

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INTEGRATORS:

These are improved versions of recorders with

some data processing capabilities. They can record the individual peaks with retention time,height,width of peaks,peak area,percentage area,etc. Integrators provides more information on peaks than recorders. In recent days computers and printers are used for recording and processing the obtained data & for controlling several operations.
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Photodiode Array Detector (3-D Data) Spectr

a Chromatogra m
h

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Tim e

n gt

el

A b s or b a n c e

8686

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PARAMETERS:

Retention time(Rt) Retention volume(Vr) Separation factor(S) Resolution Theoritical plates Column efficiency Assymetry factor

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REFERENCE:

Gurdeep R. Chatwal, Sham K. Anand,

Instrumental Method Of Chemical Analysis, Himalaya Publishing House, 2003, p. 2.624 to 2.638 P.D Sethi, Quantitative Analysis Of Pharmaceutical Preparations . Douglas A. Skoog, Instrumental Analysis, Brooks/Cole, 2007, p. 897 to 899 Dr. S Ravi Shankar, Textbook Of Pharmaceutical Analysis, Rx Publications, 2005, p. 18-1 to 1811 Robert D. Braun, Introduction Instrumental Analysis, Pharma Book Syndicate, 2006, p. 860 5/2/12 8989

THANK U
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