HPLC Detectors:

PRADEEP JAISWALM.SC.PART- 1

SEM-1(2015-2016)ROLL NO. 6

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cuvette

Tungsten Filament (vis)

slit

Diode array detector328 individual detectors

monochromator

Deuterium lampFilament (UV)

slit

mirror

• Principle of HPLC.• Instrumentation.• Detectors.1. Characterisation.2. Types.• ECD.1. Principle.2. Type.3. Working.4. Advantages.5. Disadvantages6. Application.

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Contents :

Principle :• High Performance Liquid Chromatography (HPLC), is

a method of separating a mixture of compounds based on their differing physical properties, such as polarity, charge and size.

• The compounds are partitioned between a liquid mobile phase and a bonded or stationary phase packed within a column .

• Separation is based on the analyte’s relative solubility between two liquid phases

Instrumentation:

The components of a basic HPLC system are shown in the simple diagram below :

What is detector• A device or instrument designed to detect the

presence of a particular object or substance.

• Sensitive universal detector for HPLC has not devised yet.

• Selection of detector is on the basis of analyte or sample under detection.

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Characterization of detector

• Adequate sensitivity • Stability and reproducibility• Wide linear dynamic range• Short response time• Minimum volume detected easily to reduce zero• Similarity in response towards all analytes• Non-destructive• Universality• Ease of use• Qualitative information• Reliability• Linearity• Selectivity

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• BULK PROPERTY DETECTORS: Compare an over all change in physical property of mobile phase

with or without an eluting solute. These types of detectors tend to be relatively insensitive and require temperature control.

e.g. Refractive index detector.• SOLUTE PROPERTY DETECTORS: They respond to a physical property of the solute that is not

exhibited by the pure mobile phase. These detectors are more sensitive, detect the sample in nanograms quantity.

e.g. uv visible detector , electrochemical detector, fluorescence detector.

Type of detectors:

Detectors for HPLC:

• UV-VIS1. Diode Array2. Fixed Wavelength3. Variable Wavelength

• PDA- Photodiode Array detector

• RF - Fluorescence detector

• CDD - Conductivity detector

• RID -Refractive Index detector

• ECD - Electrochemical detector

• ELSD- Evaporative light scattering detector

• MS - Mass spectrometer detector

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Principle of ECD Detection:• It measure either the conductance of the eluent or

the current associated with the oxidation or reduction of solutes.

• To be capable of detection using the first method the solutes must be ionic, and using the second method the solutes must be relatively easy to oxidize or reduce.

e-

A

R O + H+

ElectrodeGlassy Carbon (GC)Pt, Ag, Au

[ Applications ]GC : phenol compounds general usePt : H2O2

Ag : halogen ionAu : sugar analysis

Types of ECD:

1.CONDUCTIVITY DETECTOR.Used for the detection of inorganic or organic ions , usually after separation by ion exchange chromatography.

2.Amperometric detectors:It measure the current associated with oxidation or reduction of solutes.The term EC refers to these types of detectors.

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Working :• 3 electrodes are employed in ECD: Working electrode. Reference electrode. Auxiliary electrode.• When current passes through a solution , reaction occur at each

electrode in which electron exchange take place between electrode and substance in the solution .

• At cathode substance gain electron and anode lose the electron .• A cathode becomes a stronger reducing agent as its potential become

more negative • Anode become a stronger oxidizing agent as its potential become more

positive • A substance that can be electrochemically oxidized or reduced is said to

be electro-active.

Advantages:1) Measures a specific property.2) Highly Selective (depends on reduction potential).3) High sensitivity (LOD ≈ 0.01 ng).

Disadvantages :1) Not Universal.2) Must have electrolyte in mobile phase.3) Mobile phase must be aqueous.4) Gradients not possible.

Applications of ELSD:

　

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ELCD

FOOD(Saccharide

s , Fatty acid)

PHARMACETICAL)

( impurities)

CHEMICAL INDUSTRY(Polymers, Surfactants

)

REFERENCES

• Chatwal R.Gurdeep, Anand K.Sham, Instrumental method of analysis 2.625-2.626

• Williards,Merritt,Dean,Settle Instrumental method of analysis 600-608

• Beckett A.H., Stenlake J.B. Practical Pharmaceutical chemistry 4th ed.,157& 247

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