HPLC detectors
In High-Performance Liquid Chromatography
(HPLC), detectors identify and measure analytes
in the sample after they have been separated by
the chromatography column.
Different types of HPLC detectors operate based
on specific physical or chemical properties of the
analytes. Here’s how the most common HPLC
detectors work:
1. UV-Visible (UV-Vis) Detector
• Principle: Measures the absorbance of ultraviolet (UV)
or visible light by the analyte as it passes through the
detector cell.
• How it detects analytes:
– Many compounds absorb light at specific wavelengths.
– The detector measures the intensity of light before and
after it passes through the sample.
– A decrease in light intensity indicates the presence of an
analyte.
• Common Applications: Detecting compounds with
chromophores (e.g., aromatic rings, double bonds).
2. Fluorescence Detector
• Principle: Measures the fluorescence emitted by
analytes when they are excited by a specific
wavelength of light.
• How it detects analytes:
– Analytes absorb light at one wavelength and emit light
at a longer wavelength.
– The detector measures the intensity of emitted light,
which is proportional to the analyte concentration.
• Common Applications: Highly sensitive detection
of compounds like polycyclic aromatic
hydrocarbons, amino acids, or proteins.
3. Refractive Index (RI) Detector
• Principle: Measures the change in the refractive
index of the mobile phase caused by the
presence of analytes.
• How it detects analytes:
– When the analyte elutes from the column, it changes
the refractive index of the solvent.
– The detector compares this change with a reference
signal.
• Common Applications: Suitable for compounds
without UV absorption, such as sugars or
polymers.
The refractive index (symbol: nnn) is a measure of how much light bends,
or refracts, when it travels from one medium to another.
4. Mass Spectrometric (MS) Detector
• Principle: Identifies analytes based on their mass-
to-charge ratio (m/z) after ionization.
• How it detects analytes:
– The eluate from the HPLC column is ionized (e.g., by
electrospray or atmospheric pressure ionization).
– The ions are separated and detected based on their
mass-to-charge ratio.
• Common Applications: Structural analysis and
quantification of small molecules and
biomolecules.
5. Conductivity Detector
• Principle: Measures the electrical conductivity
of the solution as analytes pass through.
• How it detects analytes:
– Ionic analytes increase the conductivity of the
mobile phase.
– The detector measures the change in conductivity.
• Common Applications: Detection of ions, such
as in ion chromatography.
6. Evaporative Light Scattering
Detector (ELSD)
• Principle: Detects analytes by scattering light off
particles formed after the mobile phase
evaporates.
• How it detects analytes:
– The mobile phase is nebulized and evaporated, leaving
behind non-volatile analyte particles.
– A light beam illuminates these particles, and the
scattered light is measured.
• Common Applications: For compounds that don’t
absorb UV or fluoresce, such as lipids or
carbohydrates.
7. Diode Array Detector (DAD)
• Principle: Similar to a UV-Vis detector but
captures absorbance data across a range of
wavelengths simultaneously.
• How it detects analytes:
– As analytes pass through, their absorbance spectrum
is recorded.
– This enables both quantification and compound
identification based on spectral patterns.
• Common Applications: Complex mixtures where
spectral data aids identification.
