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APPI Dopants - Solvents and Post-Column Additives for Photo-Ionization

By: Rudolf Koehling, Analytix Volume 2007 Article 5

R&D Chemist, LC/MS Applications

APPI(atmospheric pressure photoionization) dopants enable and enhance ionization in the APPI source. They include substances like toluene, acetone, anisole and chlorobenzene. High gas phase concentrations of the dopants are introduced into the ionization cavity of the APPI source. There, UV radiation readily ionizes the dopant molecules forming a large number of free radicals and molecular ions. Subsequently, other molecules are ionized by the dopants through electron or proton transfer.

Compounds with higher ionization energy than the energy level of the emitted photons from the UV lamp require dopants, otherwise charge is not generated and these compounds will not drift into the mass spectrometer. However, molecules that can be analyzed directly in the APPI source also benefit from the use of dopants. The intensity and sensitivity can be improved because the dopants increase the number of ionized analyte molecules1– 4.

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Using dopants to enhance APPI signal

A typical, yet impressive, application for dopants is the ionization of benzaldehyde dissolved in toluene. Benzaldehyde cannot be ionized directly by ESI and gives poor results with APCI and APPI without dopant. Thus, the use of a dopant in combination with an APPI source represents the only suitable way to analyze benzaldehyde. To demonstrate the improvement, a benzaldehyde solution was infused via syringe directly into the APPI source. At a low fl ow rate (240 μL/hour) positively charged toluene radicals ([M(C7H8)]+• = 92.06 amu) are formed, as the upper calculated and observed mass spectra in Figure 1 demonstrate. Increasing the flow rate to 6 mL/hour causes the formation of uncharged toluene radicals, which are not detectable by MS, and indirectly ionizing benzaldehyde through proton transfer. This in turn leads to the lower calculated and observed mass spectra in Figure 1. APPI experiments are optimized in terms of dopant (single components or mixtures), percent dopant in the mobile phase and nebulizer gas (for direct injection into the APPI source). Specific requirements depend on the manufacturer of the mass spectrometer and the specific source design.

Figure 1. Mass spectra of an acetaldehyde solution in toluene at 2 different flow rates

Figure 1.Mass spectra of an acetaldehyde solution in toluene at 2 different flow rates

Mass spectra of an acetaldehyde solution in toluene at 2 different flow rates. The solution is directly infused into the APPI source with a syringe pump. The upper spectrum was obtained at a flow rate of 240 μL/h. The primary mass of m/z=92.0 amu agrees with a charged toluene radical. Increasing the flow rate to the maximum of 6 mL/h changes the mass spectrum significantly as there is only the protonated benzaldehyde molecule observed. High flow rates support the proton transfer reaction of the charged toluene radical to benzaldehyde and other molecules decreasing the number of the charged toluene molecules nearly to zero.

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Dopant purity requirements

Dopants can enhance signals of analytes in mass spectra, but they also intensify unwanted impurities when present. Small amounts of impurities in a dopant can result in excessive noise or a suppression of the analyte signal. Figure 2 shows a benzaldehyde solution in suitably pure toluene and in contaminated anisole.

Figure 2. Spectra of benzaldehyde dissolved in toluene (top) and anisole (bottom)

Figure 2.Spectra of benzaldehyde dissolved in toluene (top) and anisole (bottom)

Mass spectra of benzaldehyde dissolved in toluene (top) and anisole (bottom). The concentration is 100 μL/mL. In both cases the syringe pump operated at 6 mL/hour, resulting in comparable ionization of benzaldehyde. In the case of anisole, the mass spectrum shows additional signal from contaminants in the dopant.

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Benefits of dopants in normal phase LC-MS analysis

Electrospray ionization (ESI) is the method of choice for most polar compounds, like drugs and metabolites, and is very sensitive under reversed-phase conditions. However, if normal phase is employed, the ion source must be switched from ESI to APCI or APPI because analyte ionization, a key requirement of ESI, does not occur in non-polar organic solvents. Charge on the analyte must be generated by corona discharge or UV radiation. When normal phase in conjunction with MS detection is used, like in the separation of warfarin enantiomers on the P-CAP-DP chiral stationary phase shown in Figure 3, the combination of the APPI source with toluene dopant can significantly enhance the signal.

Figure 3.Separation of warfarin under normal phase conditions

Figure 3.Separation of warfarin under normal phase conditions

Column: Astec P-CAP DP, 25 cm x 4.6 mm, 5 μm packing (Cat. No. 35024AST
Mobile phase: heptane:ethanol (0.1% ammonium acetate, formic acid), 95:5 
Flow rate: 0.8 mL/min. 
Temperature: 50°C 
Sample: 3 μL, warfarin 1 mg/mL

Astec P-CAP DP (4.6 –250 mm, 5 μm) heptane/ethanol (0.1% ammonium acetate, formic acid), 95:5, isocratic, T=50°C conz. 1mg/mL, inject. vol. 3 μL

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  1. Robb, D.; Covey, T.; Bruins, A. Anal. Chem. 2000, 72 (15), 3653–3659.
  2. Kauppila, T.; Kostiainen, R.; Bruins, A. Rapid Comm. Mass Spectrom. 2004, 18, 808–815.
  3. Cai, S.; Hanold, K.; Syage, J. Anal. Chem., 2007, 79 (6), 2491–2498.
  4. Syage, J.; Hanold, K.; Lynn, T.; Horner, J.; Thakur, R. J.Chromatogr. A 2004, 1050, 137–149.


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