University of Dalat - Department of Chemistry

Seminar

22-12-2012

HPLC DETECTORS

Nhan L. Pham

HPLC system

Detectors

• have high sensitivity and predictable response

• respond to all solutes

• be unaffected by changes in temperature and carrier flow

• respond independently of the mobile phase

• not contribute to extra-column peak broadening

• be reliable and convenient to use

• have a response that increases linearly with the amount of solute

• be nondestructive of the solute

• provide qualitative information on the detected peak

UV/VIS detectors

- The principle: the Beer’s Law

- Wave-length range of 190–600 nm

- single wavelength: bandwidth of 5–8 nm

- analytes have UV absorbance

UV-Vis absorbance detector

Common UV Chromophores

Diode-Array Detectors (DAD) or photodiode array detectors (PAD)

Both chromatographic and spectral data

512–1,024 diodes

Three-dimensional representation

Fluorescence detector

100-fold more sensitive than UV absorption → one of the most sensitive HPLC detectors

chromatogram of a mixture of several polycyclic aromatic hydrocarbons (PAH)

roxithromycin (ROX) in human plasma

Refractive index detector (RI detector)

Refraction of light

Schematic of a refractive index (RI) detector

Characteristics of Refractive Index Detectors

-Excellent versatility; all solutes can be detected

-Moderate sensitivity

-Generally not useful for trace analyses

-Not useful for gradient elution

-Efficient heat-exchanger required

-Sensitive to temperature changes

-Reliable, fairly easy to operate

-Nondestructive

Electrochemical (amperometric) detectors

- Great selectivity for electroactive compounds

- Relying on the oxidation or reduction of compounds

- The difference of current between polarizable and reference electrodes

Schematic of an electrochemical detector

Chemiluminescent nitrogen detector (CLND)

- Nebulizing the HPLC eluent with oxygen and a carrier gas of argon or helium → pyrolyzed at 1050◦C

- Nitrogen-containing compounds (except N2) are oxidized to nitric oxide (NO)

→ mixed with ozone to form nitrogen dioxide in the excited state (NO2*)

- NO2* change to the ground state releasing a Photon → detected by a photometer.

Response of chemiluminescent nitrogen detector (CLND) for amino acids

Light-scattering detectors

Analyzing substance that are less volatile than the mobile phase

Evaporative light-scattering detector (ELSD)

ELSD Detection Principles

1. Nebulization: The mobile phase is converted into a spray of minute droplets using a gas stream.

2. Evaporation of Mobile Phase The droplets are carried into a heated drift tube where the mobile phase evaporates and the target components dry and are converted into minute particles.

3. Detection: The scattered light created by the collision of light with the minute particles that emerge from the drift tube is detected.

Comparison of ELSD-LT and RID in sugar analysis

Variation in Sensitivity for Different Detectors

Solvent peak

ELSD versus refractive index (RI) and UV at 205 nm for triglyceride sample

ELSD versus and UV at 210 nm for steroid conjugated

Condensation Nucleation Light-Scattering Detector (CNLSD)

- Growth in the particle size by condensation of the solvent added to the dried particles.

- An enhancement of the standard ELSD for improved sensitivity and linear range

1.Estrone 3-Sulphate (100ng)2.β-Estradiol 17-(β-α-Glucuronide) (100ng)3.5β-Pregnane-3α-2α-Diol Glucuronide (220ng)

Comparison of CNLSD and UV

Corona-discharge detector (CAD) or charged-aerosol detector

Schematic of the corona discharge detector.

Measuring the different oligomers found in the excipient PEG 400 (Polyethylene glycol)

Chiral detectors

1. Polarimeters (PL)

Measuring the degree of rotation of polarized light (typically in the 400–700 nm range) as it passes through the sample

2. Optical rotary dispersion (ORD) detector

Operating on a similar principle to polarimeters, but using lower wavelengths (e.g., the 365-nm mercury emission line).

3. Circular dichroism (CD) detectors

Measuring the difference in absorption of right and left circularly polarized light

Comparison of response of CD, ORD, and UV detectors for a 10μg injection of ibuprofen

Other detectors

- Radiometric Detector

- Laser Light-Scattering Detectors (LLSD) or multi-angle light-scattering, (MALS)

- Mass Spectral Detectors (MS)

- Infrared Detector

- Nuclear Magnetic Resonance (NMR)