An electrospray LC/MS interface consists of an enclosed, atmospheric pressure chamber. The HPLC effluent enters this chamber through a capillary tube which is surrounded by a second, concentric tube through which a nebulizing gas is applied. This article refers to this assembly as the LC capillary. Opposite from or, in modern designs, orthogonal to the incoming HPLC effluent is the inlet to the mass spectrometer. This inlet is usually a capillary tube as well and will be referred to hereafter as the MS inlet.
The mobile phase pH, is adjusted appropriately to form the desired ions. At low pH basic analytes can be protonated to form [M+H]+. Similarly, acidic analytes may be depronated ([M-H]–) at high pH. These ions enter the electrospray interface in solution and must be evaporated into the gas phase prior to entering the mass spectrometer.
The electrospray process requires that an electrical field be applied across the LC capillary and the MS inlet. The amplitude of the required voltage is several thousand volts and the sign of the voltage determines whether positive or negative ion analysis will occur. Upon application of sufficient voltage, the liquid emerging from the LC capillary takes on a distinctive conical shape with concave sides (a “Taylor cone”) as shown in Figure 1. A jet of liquid is emitted from the tip of this cone. At a given threshold voltage this jet disintegrates into droplets. These droplets evaporate and ions are emitted into the gas phase from the droplet surface in a process known as ion evaporation.
The threshold electrospray voltage (also called onset voltage or V ON) is the applied voltage which destabilizes the Taylor cone and initiates the ion evaporation process. The amplitude of that voltage is determined as follows:
VON ≈ 2 × 105 (γ rc) ½ln (4d/rc)
where γ is the solvent surface tension, rc is the LC capillary outer radius, and d is the distance from the LC capillary tip to the MS inlet. Generally speaking, users have full control over only one of these parameters; i.e. solvent surface tension.
The relative percentages of aqueous and organic solvents in a reversed-phase mobile phase determine the surface tension of the solvent. Based on the equation above and observations in the laboratory, at a given electrospray voltage greater than V ON, a higher organic content in the mobile phase leads to more rapid and complete desolvation leading in turn to more efficient ion evaporation in the interface, and therefore improved signal strength.
Table 1: LC capillary in the electrospray interface above threshold voltage (VON).
A higher surface tension of the mobile phase solvent requires a higher voltage for onset of the ion evaporation process. An experiment to illustrate this was conducted with a Varian 1200 LC/MS system. With all dimensions and other voltages held constant, the VON settings shown in Table 1 were required to induce ion evaporation for various HPLC solvent compositions (γ is the solvent surface tension in newtons-per-meter). Remarkably, the 100% water experiment required nearly double the voltage needed to induce ion formation in 100% methanol.
From a practical standpoint, this means that any direct infusion or flow injection experiment should be conducted in the highest practical organic solvent percentage given the solubility limits of the sample. Electrospray users should also note that at low solvent flow rates the effect of higher percentages of water are not as dramatic as they are at higher flow rates. At very low flow, such as that used in nanoelectrospray, the effect of water in the mobile phase is reversed and higher percentages of water seem to provide better ion evaporation efficiency. This is probably because of the very small droplet size in nanoelectrospray. A higher percentage of water slows evaporation of these small droplets to a rate compatible with the ion evaporation process. As discussed in a previous article, low flow rates also improve electrospray response. Therefore, any electrospray experiment performed without a column should be conducted at low flow rates and/or with high percentages of organic solvent.
Then what about those experiments requiring a chromatography column? Increasing the amount of organic (strong) solvent will decrease both retention and resolution of the separated components. In some cases, this may be an acceptable solution, where the components have significantly different m /z values, or an MS/MS or SRM experiment is being conducted. In other cases, where the chromatographic separation is crucial to the experiment, it will be necessary to change to a more hydrophobic reversed-phase column packing material. Fluorinated stationary phases, such as pentafluorophenylpropyl (PFPP), are good choices in this case.
As a final note, clients often ask me “Why not just set the electrospray voltage to the highest possible value and operate there all the time?” Some people describe an electrospray interface as a sort of electrolysis cell. The validity of that description is seen at very high operating voltages where the LC capillary is actually consumed during the electrospray process. Metal adduct ions of the capillary material (Fe, Au, Pt), are seen in the mass spectrum and eventually the dimensions of the capillary begin to change which leads to poor spray stability. At high voltages there is also the possibility of electrical arcing through the interface which may cause damage to the instrument electronics.
An early step in the electrospray method development process must be the setting of the required operating voltage by experimental determination of V ON.
The blog article was created in collaboration with Fred Klink. Fred is a trainer and consultant to the pharmaceutical, biotech, and chemical industries as well as law enforcement and other government laboratories. Fred’s specialty is HPLC, LC/MS, and solidphase extraction technologies. Fred has been teaching highly regarded MS and LC/MS courses and providing consulting services since 1996. Fred is the author of several journal articles and book chapters including the LC/MS entry in the Wiley Encyclopedia of Analytical Chemistry. He is a member of the American Chemical Society and American Society for Mass Spectrometry