Liquid Chromatography/
​Mass Spectrometry Fundamentals

What is liquid chromatography mass spectrometry?

Liquid chromatography/mass spectrometry (LC/MS) is an analytical technique that combines the separation power of liquid chromatography with the direct mass measurement of a mass spectrometer as the detector. Liquid chromatography can separate a wide range of compounds, while the mass detector provides valuable information about molecular weight, structure, identity, quantity, and purity.

Read on to learn more about the typical parts of an LC/MS instrument and its basic principles of operation.

Why is LC used with mass spectrometry (MS)?

A wide variety of detector types can be integrated into an LC system. The most common are based on absorption, fluorescence, refractive index, evaporative light scattering, and mass spectrometry.

• Ultraviolet (UV) detectors are typically used to quantitate the amounts of analyte in the sample, when the analyte has significant UV absorbency.
• Mass spectrometry provides unique, valuable, and orthogonal information. How? By allowing identification of analytes while reproducibly quantitating compounds that are not amenable to UV detection due to lack of chromophores or incomplete chromatographic separation.

As shown in the image, only two peaks are detected in the UV detector, but the sample actually contains three compounds. The mass spectrometer can detect separate compounds co-eluting in the UV detector.

After compounds are separated by LC, the first component they encounter in the mass spectrometer is called the ion source.

Electrospray ionization (ESI) is a common ionization technique that generates analyte ions in solution before the analyte reaches the inlet capillary of the mass spectrometer. LC eluent is sprayed (nebulized) into a chamber at atmospheric pressure in the presence of a strong electrostatic field and heated drying gas. The heat causes further stripping of solvent from the analyte molecules.

After entering the mass spectrometer through the inlet capillary, a series of electrodes known as lenses direct the charged molecules away from the source toward the quadrupole mass analyzer (or mass filter).

A quadrupole consists of four rods to which a direct current voltage and radio frequency are applied. Various combinations of these forces ensure that only fragments of a specific mass—called a mass-to-charge ratio or m/z—will travel down the quadrupole’s electric field toward the detector at a given time. This feature dramatically decreases noise and increases sensitivity.

To produce the mass spectrum, the detector records the signal intensity from ions arriving at each given time. The pattern of this mass spectrum can be used for identification, much like a fingerprint. In addition, the response recorded for the different ion species can be calibrated for quantitative purposes.

What are the advantages of LC/MS?

LC/MS provides an additional dimension of selectivity with data collected from both a UV detector and mass detector. Analytes are separated by high performance liquid chromatography (HPLC) or ultra high performance liquid chromatography (UHPLC), creating a UV chromatogram with individual peaks for each analyte separated by retention time.

For each time point, a mass spectrum is also created and can be interrogated to identify the m/z of components eluting at that time point. With mass information, analytes can be identified without the need for a reference, and co-eluting peaks can be resolved by mass.