High Performance Liquid Chromatography

(HPLC)

RAJEEV KUMAR
M.PHARM, Ist Sem,
M.I.E.T. MEERUT
High Performance Liquid Chromatography
(HPLC)

High Pressure Liquid Chromatography (old name)

 High Performance Liquid Chromatography (HPLC) is one mode
of chromatography, the most widely used analytical technique.
Chromatographic processes can be defined as separation techniques involving
mass-transfer between stationary and mobile phases.
HPLC utilizes a liquid mobile phase to separate the components of a mixture.
These components are first dissolved in a solvent, and then forced to flow through
a chromatographic column under high pressure. In the column, the mixture is
resolved into its components. The amount of resolution is important, and is
dependent upon the extent of interaction between the solute components and the
stationary phase. The stationary phase is defined as the immobile packing material
in the column.
The interaction of the solute with mobile and stationary phases can be
manipulated through different choices of both solvents and stationary phases. As a
result, HPLC acquires a high degree of versatility not found in other
chromatographic systems and has the ability to easily separate a wide variety of
chemical mixtures.
 HPLC Instrumentations

1. Mobile phase reservoir and Solvent treatment

system
2. Pumping system
3. Sample injection system
4. Column
5. Detector
6. Recorder
Block diagram of an HPLC instrument.
Instruments – Solvent System
• Mobile phase reservoirs:
– Several reservoirs (> 500mL)

• Solvent Treatment system

– Degasser ( Gases can lead to irreproducible flow rate, band spreading and
interfere with detector performance)
1. Vacuum pump
2. Distillation system
3. Device for heating and stirring
4. System for sparging
–Filter (dust particles can damage the pump, injection system or clogging of
column)
millipore filter

A convenient way to treat solvents is to filter them through a milipore filter under
vacuum. This treatment remove both gases and suspended matter.
Instruments – Pumping System
• Pumping system requirement

1.High P (6kpsi),
2.pulse-free out put,
3.Flow rate 0.1 ~10ml/min.
4.flow reproducibility,
5.resistant to corrosion
• Two major type of pumps are :
1. Displacement pump (Screw-driven
syringe pump)
I. Adv: pulse free,
II.Dis adv: small capacity(250 ml),
no gradient elution

2. Reciprocating pump: ( motor driven piston

cylindrical pump )
I. Adv: Most widely used.
Small internal volume(35~400 μl),
high pressure (10 kpsi),
gradient elution,
constant flow rate. reciprocating pump for HPLC.
II.Dis adv :Produce pulse.
 Instruments -- Injection system
Requirement of sample Injection system
1. Volume: ≤ 500 µ L

2. Sample must be injected without

depressurizing system.
Most widely used is sampling loop injection system

Load sample (isolated from system)

Inject sample by rotating valve to connect to flow.

A sampling loop for HPLC.

Instruments – Columns

1.Analytical Columns “old” “new”

length (cm) 10 – 30 3 – 7.5

I. D. (mm) 4 – 10 1 – 4.6

packing size (µ m) 3, 5, 10 3, 5

plates/m 40,000 – 60,000 100,000

Advantage of “new”:
1. less HPLC-grade solvents needed (less expensive)
2. Columns can be coupled to increase the number of plates.

Most common “old” type:

25 cm, 4.6 mm I.D., 5 µ m packing , Columns can be thermostatted
at ≤ 150 °C.
2. Guard Columns –

1. Used to prolong life of the expensive analytical

columns
2. Removes particulates and other contaminants
3. Removes sample components that bind irreversibly to
stationary phase
4. Composition similar to analytical column composition
5. Particle size is larger to minimize pressure drop
6. Guard column is repacked or replaced when “used up”
Instruments – Detectors

characteristics of ideal detector

1.adequate sensitivity

2.Good stability and reproducability

3.A temp range from room tem to at least 400 C

4.A short response time independent of flow rate.

5.Detector should be non destructive.

6.Should have minimum internal volume.

Type of detectors

1. UV-visible absorption detector.

2. IR (a) filter instrument.

(b) FT-IR.

3. fluorescence detector

4. refractive index detector

5. evaporative light scattering detector

6. electrochemical detector

7. mass spectrometry (MS) detector

Instruments – Detectors

– UV-Vis Absorption detectors:

most widely used
-Z-shape,
-Volume- 1 ~ 10 μl,
-path length- 2 ~ 10 mm

– IR: filter instrument or FTIR

- Similar cell (volume- 1.5 ~ 10 μl and
path length -0.2 ~ 1.0 mm)
- Limit: no suitable solvent (low
transparency )

– Fluorescence detectors:
-Hg or Xe lamp
-Fluorescing species or fluorescent
derivatives
-high sensitivity
Instruments – Detectors

– Refractive index detectors (RI):

Adv
- respond to nearly all solvent
- unaffected by flow rate

DA:
- limited sensitivity (1 ng/μl),
-highly Tem dependent (0.001°C),
- no gradient elution

– Evaporative light scattering

detector:
adv
- same for all nonvolatile solutes,
- more sensitive than RI, 0.2 ng/μl
DS:
- mobile phase must be volatile
Instruments – Detector

– Electrochemical detectors:

It is based on Amperometry,
voltammetry, coulometry and
conductometry

Adv:
- simplicity,
- high sensitivity,
- convenience
- wide-spreading application
Instruments – Detector

– Mass spectrometry (MS) detector

- computer controlled
- computer re constructed chromatogram obtain
- spectra of eluted peaks
- major problem is gas phase sample is needed in mass
spectrometry
 Reference
 H.H. Willard, J.A. Dean’ L.L. Merritt “Instrumental method of analysis”

 H.H. Willard, J.A.Dean’ L.L.Merritt “A Text book of pharmaceutical

analysis”
 G.R. Chatwal, S.K. Anand “Instrumental methods of chemical analysis”

 D.A. Skoog, F.J.Holler, T.A.Nienan “Principals of instrumental analysis”

 www.orbigen.com
 www.pharma.uka.edu
 www.waters.com
 www.laboratorytalk.com
 www.chromspec.com
 www.capsandstems.com
 www.springerlink.com
THANK YOU