May 26, 2005   |   Volume 2, Number 5
 
 

Welcome to the May edition of LCGC Electronic
Water-Only HPLC: A Greener Chemistry Method for the Analysis of Challenging Compounds-By Jody Clark
Analysis of Methyl Mercury in Water and Soil by HPLC-ICP-MS-By Dengyun Chen, Miao Jing, and Xiaoru Wang
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Manual Interpretation of Electrospray Ionization Mass Spectra-By the HPLC Innovations Team at Restek Corporation
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Manual Interpretation of Electrospray Ionization Mass Spectra
Tip of the Month
Manual Interpretation of Electrospray Ionization Mass Spectra

HPLC Innovations Team, Restek Corporation, Bellefonte, Pennsylvania.

Relative to electron impact or chemical ionization, “soft”-ionization techniques like electrospray ionization and atmospheric-pressure chemical ionization require different skills for interpreting mass spectra. Important points are presented here.

E
lectron impact ionization (EI) is considered a “hard” ionization process that imparts a large quantity of energy into molecules. It is very good for producing fragments that generate information about the structure of the compound. Often, the molecular ion does not appear in the EI spectrum or is a smaller peak, but if the molecular ion is observed, it will be the highest mass in the spectrum. Chemical ionization (CI) also causes fragmentation, but to a lesser extent than EI. Relatively new “soft” ionization techniques like electrospray ionization (ESI) and atmospheric-pressure chemical ionization (APCI) call for different skills for interpreting mass spectra, which possibly are unfamiliar to those trained to analyze EI or CI spectra.

With an EI or CI spectrum, the typical approach is to identify the molecular ion and fragment ions. Fragmentation is studied and general rules are followed based upon functional groups, bond energetics, and rearrangements. EI mass spectral patterns are reproducible, and fragmentation patterns are used as “fingerprints.” EI mass spectra of many compounds have been published, and these can be used to identify unknowns.

In interpreting ESI mass spectra of larger molecules, it is important to consider that the soft ESI process causes less fragmentation. The ESI spectrum will contain multiply charged molecules, and adduct formation and noncovalent -mers (dimer, trimer) add to the complexity of the spectrum.

It is important to understand the ESI process. Droplets are generated when a high voltage is applied to a liquid stream. In ESI, larger droplets explode into smaller droplets, and the process is repeated until the analyte enters the gas phase in ion form. Consequently, ESI of a single molecular species usually produces a population of multiply charged molecules that is reflected in the intensity of the peaks in the spectrum. The number of positive charges that a molecule can carry is related commonly to the number of basic sites on the molecule. To promote positive-ion formation from a positive-ion-mode analyte, dissolve the analyte in a low pH medium. Negative-ion analysis normally is conducted above the molecule’s isoelectric point, to deprotonate the molecule.

Concepts to remember about the general spectrum form, and when examining ESI spectra, include the following:
  • The y-axis typically is intensity and is relative to the tallest peak in the spectrum (also known as the “base peak”) with the tallest peak set to 100%.
  • The x-axis is the mass divided by the charge m/z. Here, m is the ion mass, not the molecular mass, and z is the number of charges per ion. This is important because often, multiply charged ions are seen. Initially, this can be unfamiliar, because with EI, only singly charged ions are formed, and one becomes accustomed to thinking of the x-axis as representing mass.
  • Similarly, it is common to use the term “molecular ion” when referring to an ESI peak, but molecular ions per se are not observed commonly in ESI. A molecular ion is formed through the loss of an electron, but in the ESI process, ionization is accomplished through the loss or gain of a proton (hydrogen).
Steps to interpreting ESI mass spectra and determining the mass of a compound:
  • Find the tallest peak in the spectrum above a 200–300 m/z value. There will be considerable solvent noise at the low end of the spectrum.
  • Look for peaks that are roughly double or half the mass; this will reveal whether multiply charged species are present. Calculation of multiply charged peaks is an essential skill (Figure 1).
  • A single charge is mass + 1 proton (M + 1); m/z is then (M + 1)/1.
  • A double-charge is mass + 2 protons (M + 2); m/z is then (M + 2)/2.
  • To convert from m/z to mass, assume a tripe-charged peak (M + 3) = Q.
  • To determine mass, reverse the math: (Q x 3) – 3 = mass. (continued)


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