Introduction to LC-MS Part4

This page discusses analytical conditions used for atmospheric pressure ionization (API) and particularly about the mobile phase, which is important for separation and ionization.

Analytical Conditions for LC-MS

For LC analysis, a variety of separation modes, such as partition (normal or reversed phase), size exclusion, and ion exchange, are available for use depending on the properties of target components. The type of stationary phase and mobile phase (water, organic solvent, pH-adjusting reagent, or buffer solution) are selected based on sample characteristics, the desired level of separation and other analytical objectives.
In contrast, LC-MS API is used to analyze organic compounds with either low-to-medium polarity (atmospheric pressure ionization - APCI) or medium-to-high polarity (electrospray ionization - ESI). Therefore, the reversed-phase mode, which is suited to separating and ionizing such compounds, is frequently used.

Since API involves spraying the sample, a volatile mobile phase must be used to ensure reliable analysis. The mobile phases used for API are summarized below. In addition to the fundamental mobile phases of water, methanol, and acetonitrile, acetic acid is also commonly used to adjust the pH level. For buffer solutions, volatile salts ammonium acetate and ammonium formate are used. In addition, protic solvents are essential for generating reaction ions for APCI, and polar solvents are considered also essential for API since polar solvents are required to dissolve polar or ionic compounds for ESI, .

Mobile Phases Suitable for API

Fundamental Mobile Phase Solvents
• Alcohols, such as methanol and ethanol
• Acetonitrile
• Water (pH adjusted, if necessary)

pH Adjusting Reagents (volatile, up to about 10 mM)
• Acetic acid, formic acid, and TFA (trifluoroacetate) (acidic)
• Aqueous ammonia (basic)
• Ammonium acetate and ammonium formate (buffer solution)

Relatively Volatile Ion Pair Reagents*
• Perfluorocarbonate (C2 to C8) (to retain basic compounds)
• Dibutylamine, triethylamine, etc. (to retain acidic compounds)
* Requires particular care, as this can remain in system even after changing mobile phases

Usable Organic Solvents*
• DMSO, DMF, THF, acetone, esters, chloroform, benzene, and hexane
* If a "fundamental mobile phase solvent" is present, it usually not a problem if the mobile phase contains some of these organic solvents. (However, the ionization effect decreases as the concentration increases.)

In contrast, phosphate buffer solution, which is commonly used for LC analysis, can precipitate its non-volatile salts at the interface, which can cause mechanical damage. This can cause physical damage, such as contamination on the needle electrode for APCI or interference with creation of fine charged droplets and reduced sensitivity for ESI.

Also, nonpolar solvents, such as hexane, contribute very little to ionizing sample molecules using APCI. Therefore, analytical conditions that use such mobile phases cannot be used without modification.

Changing from LC Analysis to LC-MS Analysis

Normally, to change from LC to LC-MS analysis, use the current LC mobile phase conditions as a reference, then investigate making changes, such as changing to a volatile salt without changing the pH level or mixing polar solvents with nonpolar solvents.

Since there are a limited number of mobile phases suitable for LC-MS, reconsider the type of column used as well.

For example, assuming the C18 (octadecyl) column for reversed-phase mode as the standard, try using C30 to increase retention or C8 or C4 to reduce retention, or phenyl or CN to increase separation selectivity. If the difference in elution times is too great or peaks are too broad, use gradient elution.
If the mobile phase and salt concentration are suitable for API, then size exclusion or ion exchange modes can be used as well.

Detecting with Good Sensitivity

ESI is an ionization method that extracts compounds existing as ions in solution via a gas phase, by spraying the solution into a high voltage electric field. To achieve high sensitivity, it is important to liberate the ions from the droplets efficiently, such as by obtaining droplets that are as fine as possible, reducing droplet surface tension, and optimizing droplet pH. In particular, the mobile phase pH affects sensitivity. For example, if a basic compound is detected as positive ions, adding an acidic reagent (AH) shifts the equilibrium in the following equation to the right, which should increase sensitivity. In general, a desired mobile phase has a pH value 1 or 2 lower than the pKa value of the sample. M-NH₂ + AH →[M-NH₃]⁺ + A^-  Conversely, for acidic compounds, add a basic reagent (B) or use a mobile phase with a pH 1 or 2 higher than the sample pKa value. M-COOH + B →[M-COO]^- + BH⁺ In the case of neutral compounds without an ionic functional group, adding a volatile salt such as ammonium acetate can sometimes increase ionization efficiency as adduct ions. M + BH⁺ →[M+BH]⁺ M + A^- →[M+A]^-  In addition, it can also be effective to increase the proportion of organic solvent to accelerate solvent evaporation. If adding these items affects LC separation, the solution can be added to the post-column as well. APCI is a technique that ionizes sample molecules (M) by transferring protons between M and reaction ions (BH+) generated from the mobile phase solvent by a corona discharge. If the proton affinity of M is greater than the proton affinity of B, then a proton is transferred from B to M. This difference in proton affinity between the mobile phase and sample influences sensitivity. M + BH⁺ →MH⁺ + B The positive ion mode is appropriate for compounds with amino groups, amides, or carbonyls, which have a strong proton affinity, whereas the negative ion mode is appropriate for compounds with carboxyl groups or phenolic hydroxyl groups. As mentioned above, APCI requires a protic solvent, where methanol/water solutions tend to provide higher ionization efficiency than acetonitrile/water solutions. APCI can be used in normal phase and size exclusion modes, and it allows ionization in the 0.2 to 2 mL/min flowrate range. Furthermore, one of its benefits is that it is less affected by salts than the ESI method.
As described above, for LC-MS, it is important to select appropriate analytical conditions by carefully considering ionization and LC separation of target components.