Advantages of In-House Generation of Nitrogen for HPLC With Evaporative Light Scattering Detection

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 phthalate-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 dissipated.