Mobile phases compatible for LC/MS

Liquid Chromatograph-Mass Spectrometry

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    Liquid Chromatography Mass Spectrometry (LC/MS) requires mobile phases that are volatile, MS-compatible and able to support efficient ionization. This page explains which mobile phases can be used for LC/MS, which should be avoided, and how LC conditions can be adjusted to ensure compatibility.

    As MS operates under high vacuum, it is essential that the LC parameters and mobile phases are compatible with MS. In most cases, existing LC conditions can be used for LC/MS analysis if the mobile phase is volatile.

    When involatile mobile phases such as phosphate buffer are used, adjustments may be required. These may include selecting a different column type, altering the chromatography mode, or switching to a volatile mobile phase.

    These parameters should be reviewed step-by-step to ensure smooth interfacing between LC and MS.

    What Mobile Phases are Suitable for LC/MS?

    A great starting point would be to use volatile mobile phases. A list of mobile phases suitable for API and LCMS are summarized in Table 3.

    In addition to the fundamental mobile phases such as water, methanol and acetonitrile, acetic acid is also commonly used to adjust the pH level. For buffer solutions, volatile salts such as ammonium acetate and ammonium formate are used. Also, volatile ion-pair reagents can be added to the mobile phase to facilitate the separation of polar compounds using reversed-phase LC. These reagents, which have a long hydrophobic tail and a polar ionic group, tend to attach to the stationary phase of the column with the ionic group sticking out. In the presence of ion-pair reagents, polar compounds interact with the charged ionic groups of the ion-pair reagent and gets retained and separated in the reversed-phase column. With focus on the ionization efficiency, protic solvents are essential for generating reaction ions for APCI, and polar solvents are essential for ESI since they are required to dissolve polar or ionic compounds.

     

    Table 3. Mobile phases suitable for API interface and LCMS.

     

    Fundamental Mobile Phase Solvents pH Adjusting Reagents (volatile, ≦10mM)
    a) Alcohols (e.g. methanol, ethanol, propanol)
    b) Acetonitrile (ACN)*1
    c) Water (pH adjusted, if necessary)    		 Acids
    a) Acetic acid
    b) Formic acid
    c) Trifluoroacetate (TFA)
    Base
    d) Aqueous ammonia
    Buffers
    e) Ammonium acetate
    f) Ammonium formate
    Relatively Volatile Ion Pair Reagents*2 Usable Organic Solvent*3
    To retain basic compounds
    a) Perfluorocarbonate, C2 to C8

    To retain acidic compounds
    b) Dibutylamine,
    c) Triethylamine (TEA)    		 a) Dimethylsulfoxide (DMSO)
    b) Dimethylformamide (DMF)
    c) Tetrahydrofuran (THF)
    d) Acetone
    e) Esters
    f) Chloroform
    g) Benzene
    h) Hexane
    1. *1 Acetonitrile is not compatible with APCI due to the reduction of nitrile to carbon for negative ionization. In this case, methanol should be used instead.
    2. *2 Use minimally as these substances can remain in the LC and MS system even after changing mobile phase. It is necessary to flush the LC system to remove any traces of these ion-pairing agents.
    3. *3 If a "fundamental mobile phase solvent" is present, it usually does not pose a problem if the mobile phase contains some of these organic solvents. (However, the ionization effect decreases as the concentration of usable organic solvents increases.)

    Mobile Phases to Avoid in LC/MS

    Involatile mobile phases are not compatible with LC/MS.

    Avoid:

    • Phosphate buffers and other involatile salts - these can precipitate at the ion source and reduce sensitivity.
    • Non-polar solvents (such as hexane) - these contribute very little to ionization, particularly in APCI.

     

    These issues can lead to signal suppression, contamination, and in some cases instrument discharge.

    In the event that they may be limitations to the mobile phase choices and changing may pose a challenge to the LC separation, post-column addition or modifications (prior to MS) may be a good alternative.

    How to Improve LC/MS Compatibility When Conditions Cannot Be Changed

    If existing LC conditions cannot be modified due to method requirements, the following techniques may help improve LC/MS compatibility:

    • Post-column addition to adjust the mobile phase before ionization
    • 2D-LC-MS/MS (trap-free) to inline-modify mobile phases
    • Changing to an alternative column type (C8, C4, phenyl, CN, etc.)
    • Using a gradient elution to manage peak width and retention
    • Changing chromatography mode if salt concentration must remain unaltered


    These adjustments allow LC/MS interfacing even under more restrictive LC conditions.

    Practical Tips for LC/MS Mobile Phase Preparation

    To ensure consistent LC/MS performance:

    • Use high-purity solvents and freshly prepared mobile phases.
    • Avoid detergents when cleaning glassware, as these can suppress ionisation.
    • Prevent contamination by properly storing methanol and acetonitrile, which can degrade or form polymers over time.


    In summary, this chapter covered the basic components of a LCMS system and the various ionization methods used in LCMS. The factors that limit the sensitivity of the API techniques and the need to select appropriate analytical conditions for LCMS are discussed to aid in your analyses and give a better understanding of the LCMS interface.

    FAQ

    What is a Mobile Phase in LC/MS?

    The mobile phase is the liquid solvent mixture that carries analytes through the LC column and into the ionization source of the mass spectrometer. LC/MS mobile phases must be volatile, MS-compatible, and able to support efficient ionization.

     

    What are the requirements for LC/MS Compatible Mobile Phases?

    Key Requirements

    The mobile phase is the liquid solvent mixture that carries analytes through the LC column and into the ionization source of the mass spectrometer. LC-MS mobile phases must be volatile, MS-compatible, and able to support efficient ionization.

    • Volatility: Allows efficient desolvation at the MS interface
    • Ionization support: Solvents must promote ESI or APCI ion formation
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    • Low salt load: Prevents precipitation and sensitivity loss
    • Chemical purity: Minimizes contaminants that cause ion suppression
    • Compatibility with analyte and stationary phase

     

     

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