No thanks! I would like to know more about CHROMacademy

 Over 3000 E-Learning topics / 5000 Articles & Applications
Avoiding LC-MS Signal Suppression

Some practical insights into optimizing LC-MS eluents and parameters to avoid signal suppression.

The following Ask the Expert question regarding the suppression of MS signals got the CHROMacademy team thinking.  Therefore, we have put together some information on the causes of signal suppression in LC-MS and how to avoid it. 

Members Question:

What is the best alternative to use instead of TFA (0.1%) which suppress MS signal? Analyte: Peptide.


You could try 0.1% formic acid. Making any change to the acid will likely affect the retention of the peptide.


I have tried with 0.1% formic acid but I obtained very bad results with important peaks tailing. Does HFBA (heptafluorobutyric acid) suppress the signal less than TFA?


Any compounds with ion pairing ability are likely to suppress signal in ESI, but this effect is compound dependent. All you can do is give the HFBA a try and see if it gives you an acceptable signal.

Alternatives would be to continue to use TFA, but try negative ion ESI. Positive ion APCI is also a possibility, but you won't get multiple charging so it depends on the mass of your compound vs. the maximum mass for your instrument. Have you tried manually tuning the instrument to see if this improves sensitivity?

If neither of these are options, then you'd need to consider redeveloping your chromatographic conditions using formic.


LC-MS applications require special consideration to optimize the mobile phase and achieve sensitive MS detection of analytes.

Electrospray Ionization (ESI)
The solvent should support ions in solution, i.e. a solvent with some dipole moment.  Solvents that are more viscous are less volatile and will reduce sensitivity.  A higher percentage of organic modifier gives better sensitivity due to the decreased surface tension and lower solvation energies for polar analytes.  Reversed phase solvents are suitable as they are often polar, whereas, normal phase solvents are not.

Atmospheric Pressure Chemical Ionization (APCI)
Most solvents are compatible.  It is preferable to have neutral molecules.

Other components of the mobile phase should also be considered.  For example non-volatile additives will crystallize and block the ion source so these are best avoided e.g. surfactants such as triton and sodium dodecyl sulfate.  Compounds that reduce ionization/ion formation (DMSO, TRIS, glycerol) are also not recommended for use with
LC-MS applications.  Corrosive reagents such as inorganic acids (H2SO4, H3PO4) or alkali metal bases (NaOH, KOH) will damage equipment. 


Choosing a Buffer

With LC-MS applications there are special considerations that must be taken into account when choosing a buffer.  For ESI volatile buffers are required to avoid fouling of the API interface.  In both positive and negative ESI an increase in buffer concentration can lead to a decrease in the analyte signal, however, this effect is compound dependent with some analytes showing only a small loss of response (Figure 1). 

At higher buffer concentrations the competition for surface sites in the electrosprayed droplet favors the higher concentration species and the analyte species become depleted.

In positive ion mode it has been seen that analyte response will decrease with eluent additive (10 mM eluent solutions) in the order:
Formic acid (100%) > ammonium formate (60-85%) > TFA (30-50%).1


Content on this page requires a newer version of Adobe Flash Player.

Get Adobe Flash player

Figure 1:  Effect of buffer concentration on ESI-MS response.


Several factors must be considered when choosing a buffer for APCI-MS.  In general non-volatile buffer species should be avoided.  Non-volatile buffers reduce the sensitivity of the interface through coating of the crucial interface surfaces in exactly the same way as for ESI-MS.  Other considerations when using non-volatile buffer species include:

  • The effect of the buffer species on the volatility of the eluent
  • The effect of the buffer on critical proton transfer reactions in the gas phase at the corona electrode

The effect of using different eluent systems on APCI-MS is detailed in Figure 2.  As with ESI-MS, buffer concentration should be kept to a minimum to avoid signal suppression via disruption of proton transfer process or reduced droplet or analyte volatility.


Content on this page requires a newer version of Adobe Flash Player.

Get Adobe Flash player

Figure 2: Effect of eluent systems on APCI-MS.  C8 2.1 x 50 mm, 5 μm column at 30 °C.


Additives and Ion-Paring Reagents

TFA is not a buffer and has no useful buffering capacity in the pH range usually associated with reversed phase HPLC. Instead it is used to adjust the mobile phase pH well away from the pKa of the analytes such that small changes in pH that occur will not affect the chromatographic retention or selectivity. 

However, it has been seen that this approach is often not feasible and produces poor chromatographic results.  Furthermore, a significant disadvantage of TFA is its ion-pairing capability and its tendency to ion pair with ionized analyte molecules in the gas phase within the API interface and potentially drastically reduce MS sensitivity for certain analytes. TFA is best avoided unless one knows something about the interaction of TFA with the analytes under investigation.

Formic acid can be used in preference to TFA, for while it has ion-pairing capability, the ion pair strength is low enough that when the associated pair moves from the condensed phase into the gas phase within the API interface it will dissociate, allowing the gas phase charged analyte to be successfully detected by the mass spectrometer. 

However, additives such as triethyl- and trimethylamine may enhance ion formation in negative mode. 

Some acids, bases, and buffers that can be used for LC-MS applications are listed below.


In APCI, due to possible charge exchange reactions, methanol is usually a better choice of organic solvent than acetonitrile (which tends to reduce sensitivity).  Samples with lower proton affinities than ammonia or triethylamine will often lose a proton, become neutralized or form adducts in the gas phase and, therefore, should be carefully controlled.

One common problem with gas phase acid-base chemistry in negative APCI is signal quenching by acidic mobile phase constituents (Figure 3).  The extent of quenching (or signal suppression) is directly correlated to gas phase acidities and basicities.  In general positive ion APCI is preferred over negative ion mode whenever possible.


Content on this page requires a newer version of Adobe Flash Player.

Get Adobe Flash player

Figure 3: Signal quenching in negative mode APCI.


Electrospray Voltage

In ESI the threshold electrospray voltage (also called the onset voltage or VON) is the applied voltage which destabilizes the Taylor cone (which is formed at the capillary tip) and initiates the ion evaporation process (Figure 4). 


Content on this page requires a newer version of Adobe Flash Player.

Get Adobe Flash player

Figure 4: Formation of the Taylor cone.


This voltage can be estimated using Equation 1.  For the most part users only have full control over the solvent surface tension parameter.  In reversed phase HPLC the percentage of aqueous and organic solvents determines the surface tension of the eluent.  From Equation 1 at a given electrospray voltage greater than VON a higher organic content in the mobile phase will result in more rapid and complete desolvation which gives more efficient ion evaporation at the interface and an improved MS signal.  If the surface tension of the eluent is higher, a higher threshold voltage will be required to initiate the ion evaporation process (Table 1).



γ = solvent surface tension

rc = LC capillary outer radius

d = distance from the LC capillary tip to the MS inlet



γ (N/m)

Capillary Voltage (kV)










Table 1: Threshold electrospray voltages.2


Finally, a great rule of thumb when working with most LC-MS parameters is...
“if a little bit works, a little bit less probably works better”.



  1. Yilmaz, H. J. Phys. 2002, 26, 243-246.
  2. Klink, F.; //

For more information see the following CHROMacademy pages:

Ion Suppression Principles
Ion Suppression - Competing Ions
Ion Suppression in Practice
Signal Suppression by Additives in APCI
ESI Ion Pair Reagents - Overview
ESI Ion Pair Reagents - Considerations


CHROMacademy Forum

loading data
loading data
loading data
loading data
loading data

group  subsCHROMacademy can deliver to corporate clients on a multi-user subscription basis.
Served up from secure servers to the corporate intranet or individual desktops.

  • Microsite - your own learning site powered by CHROMacademy
  • Your Landing Pages -with your logo and branding
  • Customized Assessments - Based on content agreed upon Certificate of Completion
  • Certification Programs - Offer your learners a goal to strive towards
  • LMS : Connect - Our Learning Management System is S.C.O.R.M. compliant and will connect to your system
  • Engagement Package - E-newsletter stimulation program derived from your content and ours
  • Full archive of Essential Guide webcasts & tutorials
  • 1000’s of eLearning topics - HPLC / GC / Sample Prep / Mass Spec
  • Ask the Expert - our experts will answer your chromatography questions within 24 hrs.
  • Assessments - test your knowledge
  • Application notes & LCGC articles
  • Troubleshooting and virtual lab tools

Request a quote


 Home | About UsContact Us | SubscribeTerms and Conditions | Advertise | Privacy Policy 

loading data

loading data

loading data


loading data

loading data