Where ions come from | MassSpec.Tips

Lesson 3 · 30 minutes
Prerequisites: Lesson 1 and lesson 2.
Mathematics: Arithmetic, m/z calculations and response ratios.
Audience: Beginner, with practitioner and expert extensions.

The source helps determine which ions become available for analysis. Choose it by the sample's properties and the evidence you need, then test that choice experimentally.

By the end, you will be able to:

describe the main ion-forming processes in EI, ESI, APCI, MALDI, and ICP;
connect sample properties to a defensible starting source choice;
distinguish molecular information from elemental information;
explain why changing a source can change a spectrum without changing the original sample.

Your route through the lesson

Choose a source for a question, predict an ion family, then design a comparison that could reveal why the first trial failed. By the end, you should be able to distinguish “the analyte is absent” from “this method has not yet shown it.”

Predict

You need to examine an intact, nonvolatile peptide in solution. You can begin with electrospray ionization or conventional gas-phase electron ionization.

A. Choose EI because removing an electron always preserves the molecule.
B. Choose ESI because the sample can enter as a solution and produce peptide ions.
C. Choose either because the analyzer determines all spectral differences.

Reveal the reasoning

B is the defensible starting choice for these constraints. A overlooks sample introduction and fragmentation. C overlooks source chemistry: the analyzer can only analyze the ions supplied to it. A starting choice is not a guarantee of adequate signal or intact-ion survival. Agilent source-compatibility guide

Explain

Three jobs that are easy to confuse

Introduce the sample. Deliver material in a form compatible with the source: vapor, liquid spray, deposited spot, or another defined introduction method.
Produce and release ions. The analyte must acquire or retain net charge and become available as a gas-phase ion. Different sources accomplish these tasks by different processes.
Transfer useful ions. The interface and ion optics must bring a fraction of those ions into the analyzer under suitable conditions. Formation does not guarantee transmission or survival.

These steps explain two apparently contradictory results: a compound can be present but weak in one method, and the same unchanged sample can give a substantially different spectrum with another source. Start with the information required, then ask which preparation and ion chemistry can preserve it.

Compare the ion-forming processes

Electron ionization — EI. Electrons interact with gas-phase species and can remove an electron. For a closed-shell neutral molecule, a simple representation is M + e⁻ → M⁺• + 2e⁻. The resulting molecular radical cation may fragment. Conventional GC-EI-MS is useful when the sample can reach the source in the gas phase without unacceptable thermal decomposition. Fragment-rich spectra can support comparison with appropriate reference spectra. IUPAC electron ionization, Thermo ion-source overview, EI

Electrospray ionization — ESI. An applied potential produces charged droplets from a solution. Desolvation and droplet evolution yield gas-phase ions. Depending on analyte and conditions, candidates include protonated, deprotonated, adducted, and multiply charged species. For large analytes, multiple charges can place ions within an analyzer's accessible m/z range. IUPAC electrospray ionization, Thermo electrospray overview

Atmospheric pressure chemical ionization — APCI. The liquid stream is nebulized and vaporized. A corona discharge initiates reagent-ion chemistry; gas-phase reactions then ionize the analyte. Protonated or deprotonated molecules are useful candidate assignments, depending on polarity and chemistry. APCI requires adequate volatility and thermal stability under the vaporization conditions. Lower polarity alone is not a sufficient reason to choose it. Exactive manual, APCI glossary, Agilent compatibility guide

Matrix-assisted laser desorption ionization — MALDI. A laser irradiates a matrix containing the analyte. The matrix absorbs energy and assists production of gas-phase ions. In a conventional spotted preparation, protonated or deprotonated analyte ions are possible products. Matrix and sample preparation are part of the experiment, not just packaging for it. IUPAC MALDI, Thermo ion-source overview, MALDI

Inductively coupled plasma — ICP. A hot plasma converts introduced material into species used for elemental measurement. In conventional solution ICP-MS, the analytical targets are usually atomic ions. Signals can also include polyatomic and multiply charged interferences. The question is typically which elements or isotopes are present and in what amounts. EPA Method 6020B, sections 2 and 4

Compare EI and ESI without the “hard versus soft” shortcut

In the EI equation, count charge on both sides: one incident electron gives total charge −1 on the left; a +1 radical cation and two electrons give −1 on the right. The dot in M⁺• denotes an unpaired electron. No proton has been attached. The molecular ion may survive or dissociate, and a weak or missing molecular-ion peak does not prevent a useful fragmentation spectrum. An appropriate EI reference comparison requires compatible experimental and processing conditions. IUPAC EI definition

ESI begins with a liquid solution and charged droplets rather than requiring the intact analyte to be thermally vaporized first. Solvent removal and droplet evolution are essential parts of creating the gas-phase ion population. A peptide can carry several charges, placing a large neutral mass at a lower m/z. This is experimentally established behavior; neither a unique charge state nor an exact distribution follows from the word “ESI.” Fenn et al., electrospray of large biomolecules

Do not picture ESI as adding one identical proton to every dissolved molecule. The sample, solvent, existing ionic species, adduct chemistry, and source conditions matter. Conversely, “soft” does not mean chemically unselective: a softly formed ion can be strong, weak, or absent under different conditions.

What information survives?

EI fragmentation can preserve useful structural relationships through its product ions. ESI and MALDI can provide intact-ion information, with adducts and fragments also possible. Conventional solution ICP-MS deliberately moves toward atomic-ion measurement; that is well matched to an elemental question but sacrifices the intact molecular relationship sought in the peptide example.

These are different analytical objectives, not a ranking from good sources to bad sources. For example, finding an element in a digested sample does not reveal which original compound contained it. A speciation experiment requires a preparation and separation strategy designed to preserve and distinguish the relevant species.

Keep the source distinct from the analyzer

ESI, APCI, and the ICP source operate at atmospheric pressure; ions then pass through an interface into vacuum. Conventional EI and vacuum MALDI form ions in evacuated regions. Atmospheric-pressure MALDI also exists. Do not assume that every instrument has the same atmospheric-pressure inlet sequence. Thermo source-pressure overview, EPA Method 6020B

Use soft ionization as a comparison

“Soft” describes a tendency toward less fragmentation under suitable conditions. It does not promise that every analyte remains intact. An ion may dissociate during transfer or deliberate activation after leaving the source. Exactive manual, source CID and HCD

A source-selection decision you can explain

Name the evidence you need. Intact molecular mass, characteristic fragmentation, or an elemental/isotopic result?
Check introduction constraints. Can the analyte enter as vapor without unacceptable decomposition? Is it soluble in an appropriate liquid? Can it be prepared in a suitable matrix-containing spot?
Propose the actual ions. Choose polarity and plausible charge/adduct forms. Confirm that their predicted m/z values fall within the proposed acquisition range.
Test in the real sample context. Check standards and relevant blanks, then examine matrix samples. Response in a clean standard is only one part of suitability.

The source labels narrow the options. They do not replace sample preparation, hardware compatibility, an instrument procedure, or method validation. In particular, APCI's need for vaporization and ESI's solution compatibility are constraints to reason with, not a universal polarity-based lookup rule. Agilent source/application compatibility

Manipulate

Change one sample constraint

A small organic analyte gives inadequate ESI response. It is sufficiently volatile and thermally stable for the available APCI source.

Prediction: Is APCI a reasonable next source to evaluate?

Compare your prediction

Yes, as a hypothesis to test with standards and matrix samples. Now change just one condition: the analyte decomposes before adequate vaporization. APCI becomes a poor starting option even if the analyte has low polarity. Sample compatibility comes before a simple polarity rule. Agilent compatibility guide

Change the selected ion form

Return to hypothetical peptide P, with a teaching neutral mass of 1000.0000 Da. Suppose one experiment produces [P+2H]²⁺ and another produces [P+H]⁺. Predict the two m/z values before calculating.

Compare your prediction

They are 501.0073 and 1001.0073, respectively, using the proton constant from NIST CODATA 2022. This comparison illustrates the consequences of assigned ion forms. It does not assert that a particular source must produce either charge state.

Apply

Worked example choosing a source for a peptide

Question: Is the intact peptide present at a mass consistent with P?

Known constraints: P is in solution, is nonvolatile under suitable handling conditions, and must retain its molecular information. Available options are ESI, conventional GC-EI, and solution ICP-MS.

Start with ESI. Its solution introduction and peptide compatibility fit the question. This is a method-development choice, not an observed result.
Propose ions to search for. For a candidate [P+2H]²⁺ ion, let h = 1.0072764665789 and calculate x = (1000.0000 + 2h)/2 = 501.0072764665789. Search near m/z 501.0073, within a tolerance justified by the method.
Test the assignment. A resolved envelope with approximately 0.5017 spacing would support charge magnitude 2 under the carbon-spacing model. Check the monoisotopic assignment and consider competing ion compositions.
State the limit. The result could support an intact-mass match. It would not establish the peptide sequence by itself.

Conventional GC-EI conflicts with the supplied nonvolatility constraint. Conventional solution ICP-MS would answer an elemental question and would not supply the intact-peptide ion assignment needed here. Agilent source compatibility, EPA Method 6020B

Worked example: is the peptide outside the scan?

Suppose a proposed acquisition covers m/z 400–800. For the 1000.0000 Da teaching peptide, [P+H]⁺ at 1001.0073 lies above that window, [P+2H]²⁺ at 501.0073 lies inside it, and [P+3H]³⁺ at 334.3406 lies below it.

A method searching only the singly protonated position would miss the candidate 2+ signal. A method recording only 400–800 cannot assess the full charge-state family. Before concluding that P is absent, check what was actually acquired, the polarity, the plausible ion forms, and the response demonstrated by a suitable standard. This range example says nothing about which charge state is most abundant.

Use the ion explorer from lesson 2 to reproduce those positions. It predicts arithmetic for specified ions; it does not select a source, simulate solvent chemistry, or predict analytical sensitivity.

Transfer the reasoning

New question and sample	Defensible starting choice	What still needs testing
A volatile, thermally stable organic compound; compare fragmentation with a reference	GC-EI-MS	Reference-spectrum compatibility, retention evidence, and interferences
A peptide prepared as a matrix-containing spot	MALDI	Ion assignment and preparation-dependent response
Element concentrations in a prepared aqueous sample	ICP-MS	Sample preparation, spectral interferences, quantitative calibration, and QC

These are reasoned applications of the source descriptions, not validated methods for the unspecified samples.

When the sample changes the response

The matrix is the rest of the sample accompanying the analyte. In LC-MS, co-introduced material can suppress or enhance the analyte's response. A clean-solvent standard and a matrix extract can therefore give different signals for equal analyte amounts. Mechanistic studies have connected electrospray suppression in biological extracts to altered droplet properties in the presence of nonvolatile solutes; that result is not a single explanation for every weak signal. King et al., 2000

Separate that problem from recovery: loss of analyte during preparation. A useful comparison evaluates a neat standard, a blank extract spiked after extraction, and a sample spiked before extraction. This experimental logic separates matrix-related response from preparation loss when concentrations, volumes, and acquisition are appropriately matched. Matuszewski et al., 2003

Worked example: two losses hidden in one weak peak

Consider original synthetic data with matched nominal analyte amounts, final volumes, and injection volumes. Assume a selective response, suitable analyte-free matrix, blank correction, stable acquisition, and a linear response over this small comparison.

Preparation	Response, arbitrary units
A: analyte standard in neat solvent	1000
B: blank matrix extracted, then spiked	600
C: matrix spiked, then extracted	300

Matrix-related response: B/A = 600/1000 = 0.60. Under these assumptions, the post-extraction matrix gives 60% of the neat response, consistent with 40% suppression.
Preparation recovery: C/B = 300/600 = 0.50. Comparing samples with matched final matrix gives an estimated 50% recovery.
Overall process response: C/A = 300/1000 = 0.30, also 0.60 × 0.50. Calling that 30% “recovery” alone would conflate two effects.

This is a reasoning exercise, not a validated assay or an acceptance criterion. Replicates, appropriate concentration levels, representative matrices, stability, and interference checks are needed before using such estimates quantitatively. The comparison indicates where to investigate; it does not diagnose the exact suppression mechanism.

What can you claim

You can justify a source as a starting point by matching the required information to sample introduction and ion chemistry. You cannot infer experimental sensitivity from the source name alone.

An elemental result does not, by itself, identify the original molecular form of that element. To investigate speciation, preserve and separate species with an appropriate method and connect the resulting evidence to the molecular question.

Practitioner extension

Record the intended ion, polarity, sample-introduction constraints, and one observation that would make you reconsider the source. This makes a failed first trial useful rather than leaving “no signal” as the whole conclusion.

When discussing calibration, name its purpose. Mass-axis calibration establishes the relationship used to assign m/z. Response calibration relates measured signal to concentration. Follow the instrument's current procedure and the laboratory's validated SOP for tuning and calibration sequence and frequency; a general lesson cannot set one cadence for every platform. Agilent TOF/Q-TOF concepts, pages 57–70, Agilent quantitative calibration

Expert extension

Distinguish a mechanistic explanation from a source-selection rule. “ESI can produce multiply charged ions” does not predict a complete charge-state distribution. Identify which sample and source conditions are missing before making that stronger prediction.