LC-MS

LC-MS (Liquid Chromatography-Mass Spectrometry)

LC-MS is an analytical technique that combines the separating power of liquid chromatography (LC) with the detecting capability of mass spectrometry (MS). 

In LC, a sample is injected into a column and is separated into individual compounds (molecules) based on their chemical properties as it passes (drips) through a chromatography column over time.

These compounds then enter the mass spectrometer, where they are ionized, and a mass spectrum is generated to measure their mass-to-charge ratio (m/z) and the signal intensity (kind of concentration) for each m/z value. The mass spectrum is generated continuously and aggregated into one single chromatogram which shows the total intensity of ions at different retention times.

From the mass spectrum, it tells the concentration of each compound (each m/z value) at a retention time.

From the chromatogram, it tells the total concentration of all compounds at a retention time.

The "m/z" is the mass-to-charge ratio of an ion.

When molecules are ionized in the mass spectrometer, they may gain or lose electrons, resulting in charged species (ions).

E.g. an ion with a mass of 100 Da (Dalton, unit mass for atom) and a charge of +1 will have an m/z of 100. If the same ion has a charge of +2, the m/z will be 50.

In LC-MS, the liquid chromatography (LC) part helps separate compounds in a sample

The mass spectrometry (MS) part analyzes the ions that are produced. It helps to determine the molecular structure. i.e. what they are

m/z is also used to measure molecules mass. However, as the charge can be different from molecule to molecule, two molecules of the same mass would have different m/z values if they are charged differently (e.g. singly or multiply). In mass spectrometry (MS), whether a molecule is singly or multiply charged depends on the ionization process, While you can't directly control the charge state of each molecule,  you can influence the ionization conditions to favor singly charged ions for most molecules.

The "response" is the signal intensity detected by the mass spectrometer for a particular analyte (molecule or ion) in the sample.

It correlates to the concentration of the analyte / molecule.  A calibration curve is usually constructed using standards of known concentration to correlate 

the response to the analyte's actual concentration.

Mass Spectrum

The response is typically displayed as a peak in the mass spectrum, where the height or area of the peak is proportional to the concentration of the analyte.

The mass spectrum has m/z as the x axis, and y axis is the intensity (or abundance) of the ions.

The peaks (responses) on the spectrum can be measured by the Peak Height, or the Peak area.

Peak Height: The height of the peak at a specific m/z value represents the intensity of the ions detected at that exact m/z value.  It’s often used for qualitative analysis when you just want to know which ions are present.

Peak Area: The area under the peak (i.e., the entire area covering the peak from the base to the apex) represents the integrated signal over the range of m/z values that the peak spans. 

The area is proportional to the total number of ions detected at those m/z value range, which makes it a better indicator for quantitative analysis. In practice, the area under the peak provides a more accurate measure of the total ion count associated with that analyte, especially when the ion signal might fluctuate or spread over a small range of m/z values.

Total Ion Chromatogram

The Total Ion Chromatogram (TIC) is a type of chromatogram in which the intensity of all ions detected by the mass spectrometer is summed across ALL m/z values for each time point during the chromatography run. In other words, it shows the total ion signal over the ENTIRE mass spectrum for each point in the chromatographic separation.

X-axis: Retention time (time at which a compound elutes from the chromatographic column).

Y-axis: Intensity (or response), representing the sum of all ion intensities across the m/z range for each retention time point.

The TIC gives you an overview of the sample’s complexity and the total amount of ionizable material present. 

It allows you to observe the overall elution profile of the analytes in the sample during chromatography.

It’s particularly useful when you want to look at the total signal response, without focusing on specific m/z values or individual peaks. 

In an LC-MS experiment, you might see a TIC chromatogram where large peaks correspond to major components in your sample that elute at certain times during the chromatographic process. In a chromatogram, sometimes people use 1:TIC as the y axis to indicate the first dimension of the data or the primary m/z range.

Retention Time

The time it takes for a compound to elute from the column and be detected by the mass spectrometer.

As the sample moves through the column, different compounds interact differently, causing them to elute at different times.  The mass spectrometer detects each of these compounds as they elute.

Summary of whole process

In LC-MS, 

The liquid chromatography (LC) step separate different compounds. As different compounds have different chemical properties, it reacts differently as the sample travels  through the column (filled by gel-like material). This causes each compound to travel through the column at a different speed, resulting in the compounds eluting (coming out) of the  column at different times (i.e., different retention times).

At each retention time, a mass spectrum is generated by the Mass Spectrometry (MS).

The mass spectrum indicates the intensity (concentration) for each m/z value, while the m/z value (range) help identify the compound. The total intensity (total ion count) across all m/z value indicates the total amount of compounds elute at that time.

This total intensity becomes a dot on the Total Ion Chromatogram (y axis: TIC, x axis retention time). As different compounds elute at different times (time ranges), each peak in the chromatogram generally corresponds to a dominant compound (or sometimes a group of compounds) that has eluted from the column at that specific retention time (range). You can look at the corresponding mass spectrum(s) at that time for the intensity of each compound of the group.

An example of Chromatogram

The total counts / abundances is the y axis. Different molecules typically elute at different retention times (RT times).

Normally peop

An example of Mass Spectrum

The counts / concentration / signal intensity is the y axis.