Evaporative light scattering detection (ELSD) is a universal detection technique for HPLC that provides a number of distinct advantages over other commonly employed detection methods such as UV-VIS absorbance, fluorescence, and electrochemical measurements. The technique allows for the trace-level detection of essentially all compounds in the sample and does not require the presence of a chromophoric group for detection (required for UV-VIS absorption or fluorescence detection) or an electroactive group (required for electrochemical methods such as coulometry). While other universal techniques for HPLC exist (e.g., refractive index measurements), they provide poor sensitivity and are generally restricted to isocratic elution.
Detection of the components of a sample via an ELSD includes three distinct stages (Figure 1): a) nebulization of the effluent, in which the mobile phase is transformed into a stream of small droplets by the addition of a pressurized stream of nitrogen gas through a narrow orifice such as a needle; b) evaporation of the mobile phase, where the droplets are passed through a drift tube in which the mobile phase is evaporated; and c) measurement of the scattered light, in which the particles resulting from the evaporation of the mobile phase pass through a light scattering detector.
Figure 1 - Steps in ELSD (courtesy of Grace Davison Discovery Sciences). a) Nebulization: The column effluent passes through a needle and is mixed with high-pressure nitrogen to form a dispersion of droplets. b) Evaporation: The droplets pass through a heated drift tube to evaporate the mobile phase and form a fine mist of dried sample particles in solvent vapor. c) Detection: The sample particles pass through a cell and scatter light from a laser beam. The scattered light generates a signal that is monitored to provide the chromatogram.
The nebulization step requires a supply of clean dry nitrogen from 98 to 99% purity that is regulated from 65 to 80 psig. In many laboratories, the nitrogen is provided by the evaporation of liquid nitrogen from a high-pressure liquid nitrogen tank, a Dewar flask, or a high-pressure gas cylinder. While these methods are satisfactory, the use of an in-house generator to provide the nitrogen for ELSD offers a number of significant benefits. This paper describes how an in-house nitrogen generator operates, and discusses the safety, convenience, and cost benefits of this approach.
Generation of nitrogen for ELSD using an in-house generator
Nitrogen for ELSD nebulization can be
generated from ambient air by removal
of oxygen, water vapor, and particulate
matter. The heart of an in-house nitrogen
generator is a hollow-fiber membrane that
permits oxygen and water vapor to permeate
the membrane and escape through
the sweep port while the nitrogen flows
through the tube (Figure 2). While each
individual fiber membrane has a small
internal diameter, a large number of fibers
are bundled together (Figure 3) to provide
an extremely large surface area for the permeation
of oxygen and water.
Figure 2 - Operation of hollow fiber membrane (courtesy of Parker Hannifin Corp.)
The schematic of a typical nitrogen generator
(model N2-04, Parker Hannifin Corp., Haverhill,
MA) is shown in Figure 4. Compressed air
is filtered using a high-efficiency activated carbon
filter to remove hydrocarbons, and a prefiltration
system is used to remove particulate
matter (all particles >0.01 µm are removed).
The air is then delivered to the fiber membrane
bundle, where the oxygen and water permeate
the membrane, and is vented while the purified
nitrogen is passed through another membrane
filter and then delivered to the ELSD.
Figure 3 - Hollow-fiber membrane bundle
(courtesy of Parker Hannifin Corp.).
The N2-04 nitrogen generator can produce
up to 99.5% pure nitrogen on a
continuous basis, which is more than satisfactory
for ELSD measurements. The
purity of the nitrogen is dependent on the
operating pressure and the desired flow
rate; as an example, 3 L/min of nitrogen
gas can be generated using an operating
pressure of 80 psi at room temperature,
sufficient to supply the gas that is
required for an ELSD system. The gas that
is delivered to the LC-MS by the N2-04
has an atmospheric dewpoint of -58 oF
(-50 oC) and contains no particulate
matter greater than 0.01 µm and no suspended
liquids. In addition, the nitrogen
gas is hydrocarbon-free
and is commercially sterile.
Benefits of an in-house
makeup gas generator
An in-house nitrogen generator provides a
number of significant benefits to the operator
of an HPLC with an ELSD, including a
dramatic improvement in safety, a significant
increase in convenience, and a lowering
of the cost of supplying the gas.
Minimizing safety hazards
Figure - 4 Schematic diagram of N2-04 nitrogen generator (courtesy of Parker Hannifin Corp.).
Perhaps the most important benefits of
an in-house nitrogen generator are the
safety considerations that the generator
provides. For example, when an in-house
nitrogen generator is employed,
only a small amount of the gas is present
at a low pressure at a given time, and
the gas is ported directly to the detector.
The N2-04 generates a maximum
of 11 L/min of nitrogen at a maximum
pressure of 145 psig. If a nitrogen leak were to occur with the generator, there would be a small change in the composition
of the laboratory air, and a small
quantity of nitrogen gas would be harmlessly