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 Beers Law
- Wave-length range of 190600 nm
- single wavelength: bandwidth of 58 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







5121,024 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 N
2
) are oxidized to nitric oxide
(NO)
mixed with ozone to form nitrogen dioxide in the excited state (NO
2
*)
- NO
2
* 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.-Estradiol 17-(--Glucuronide) (100ng)
3.5-Pregnane-3-2-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 400700
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 10g
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)