H-ESI is a soft gas-phase ionization technique that is typically preferred for polar compounds. The H-ESI source transfers ions in solution to the gas-phase. H-ESI can ionize many samples that previously were not suitable for mass spectrometry analysis (for example, heat-labile compounds or high molecular mass compounds). You can use H-ESI to ionize any polar compound that is an ion in solution, including adduct ions. Included in this class of compounds are biological polymers (such as proteins, peptides, glycoproteins, and nucleotides), pharmaceuticals and their metabolites, and industrial polymers. For example, you might analyze polyethylene glycols from a solution containing ammonium acetate because of adduct formation between NH4+ ions in the solution and oxygen atoms in the polymer.

The H-ESI source can produce multiply charged ions, depending on the structure of the analyte and the solvent. For example, the mass spectrum of a protein or peptide typically consists of a distribution of multiply charged analyte ions. You can mathematically manipulate this mass spectrum to determine the molecular mass of the sample. With H-ESI, the range of molecular masses that the MS can analyze can exceed 50,000 Da if there are multiple charges on the analyte(s).

Use H-ESI in either positive or negative ion polarity mode. The polarity of the ions in solution determines the ion polarity mode: acidic molecules form negative ions in high pH solution and basic molecules form positive ions in low pH solution. The installed H-ESI spray insert can be either positively or negatively charged. When it is positively charged, it generates positive ions. When it is negatively charged, it generates negative ions.

The range for the Current LC Flow in the application is 0–3,000 µL/min. However, for optimum H-ESI performance, set the flow rate1) into the MS over a range of 1–1,000 µL/min, and configure the H-ESI spray insert for the high-flow range as applicable.

NOTE

For the lower range of 1–50 μL/min, you can install the ESI spray insert, which contains a low-flow metal needle insert. When the ESI spray insert is installed, the vaporizer heater is not available.

In H-ESI, because both the buffer type and buffer concentration have a noticeable effect on sensitivity, you must choose these variables correctly.

Large droplets with high surface tension, low volatility, low surface charge, strong ion solvation, and high conductivity negatively affect the H-ESI process. Therefore, H-ESI favors small droplets with low surface tension, high volatility, high surface charge, weak ion solvation, and low conductivity.

Mixed organic–aqueous solvent systems that include organic solvents, such as methanol, acetonitrile, and isopropyl alcohol, are superior to water alone for H-ESI. Volatile acids and bases are good, but for best results do not use salts above 10 mM. Be aware that strong mineral acids and bases are extremely detrimental to the instrument.

IMPORTANT

To obtain good H-ESI results, follow these guidelines:

  • Keep nonvolatile salts and buffers out of the solvent system. For example, avoid the use of phosphates and salts that contain potassium or sodium. Use acetate or ammonium salts instead. Do not use strong mineral acids and bases—they can damage the instrument.
  • Use organic/aqueous solvent systems and volatile acids and bases. Avoid the use of 100 percent aqueous solvents.
  • If possible, optimize the pH of the solvent system for the analyte. For example, if the analyte contains a primary or secondary amine, aim for a slight acidic mobile phase (pH 2–5). The acidic pH tends to keep positive ions in solution.