Transcript No Slide Title

UV Detectors
Fixed Wavelength
Variable Wavelength
Diode array
Fixed Wavelength
Mercury vapor lamps are probably the
most common and emit intense light
at 253.7 nm (and certain other
wavelengths). Because of the limited
emission spectra, of the lamp wavelengths
are not adjustable.
Because of the intensity
of the radiation, fixed wavelength
detectors can be up to 20 times more
sensitive than variable wavelength
detectors.
High
Pressure
Mercury
Lamp
Variable Wavelength
The variable types use a deuterium or similar lamp that
produces a broad spectrum of wavelengths that are separated
by a defraction grating. The grating works like a prism but
generally the resolving power is higher for gratings
than for prisms.
Diode Array
The photo diode array detector passes a wide spectrum of light through
the sample. The spectrum of light is directed to an array of photosensitive
diodes. Each diode can measure a different wavelength which allows for
the monitoring of many wavelengths at once.
- Peak Purity
- Quantify a peak with an
interfering peak
Compound Identification
Monitor compounds with
different UV max.
Intensity of Lamp Energy
Lamp Life
As the lamps age, the light intensity drops significantly.
Therefore it is important to monitor lamp usage.
Arc lamps have an average lifetime of about 200
hours and most detectors are equipped with a timer that
keeps track of lamp usage.
Fluorescence Detectors
Fluorescence detectors are among the most sensitive of
HPLC detectors, and depending on the compound,
can be from 10-1000 times more sensitive than UV detectors
analyzing strong UV absorbing compounds.
The fluorescence detector is based on the principle that some
compounds fluoresce when bombarded with UV light.
If the compound of interest fluoresces this is a very sensitive detector.
The analyte is excited by light commonly at 253.7 nm from a low
pressure mercury lamp. The light is absorbed and the molecule
is excited and it gives off light of a different wavelength.
This wavelength is monitored by a detector which sits at right
angles to the UV light source used to excite the analyte.
Electrical Conductivity
The electrical conductivity detector was one of the first in-line
detectors developed and is still in use for detecting electrically
charged ions. It is based on the principle that the electrical
conductivity of a liquid is proportional to the ionic concentration
in the mobile phase. The design is simple, and the detector can
be made small enough for an effective sensing volume of a few
nanoliters, making this detector very useful for capillary electrophesis.
Refractive Index Detectors
In contrast to the fluorescence detector, the refractive index
detector is one of the least sensitive (about 1000 times less
sensitive than UV detectors).
Refractive index detectors are often used for the analysis of carbohydrates.
Carbohydrates are generally not ionic do not fluoresce, and do not absorb
UV or visible light to any great extent, so the refractive index is one of the
few detectors that can detect them.
Light Scattering Detectors
The flow from the column is
converted into a fine spray or mist.
The mobile phase is evaporated,
leaving tiny particles of the analytes
The particles are passed through a
laser beam and they scatter the
laser light. The scattered light
is measured at right angles to
the laser beam by a photodiode
detector.
Mass Spectrometric Detectors
For all mass spectrometer techniques, the analytes are first
ionized since the detector only measures charged particles.
The charged particles then enter the separator that separates
ions on the basis of mass using a magnetic field.
The masses of different fragments hit the detector where
they give up their charge creating an electric signal
which is recorded.
It is difficult to use a mass spectrometer with liquid chromatography because
the mobile phase must be removed before the analytes can enter the detector.
A number of interfaces have been developed to solve this problem
(ie., electrospray, ion spray, and thermospray) and LC-MS is becoming
more common.