HPLC-MS – Info

Ultra-high performance liquid chromatography with coupled (tandem) mass spectrometry (HPLC-MS(/MS) for short)

The major advantage of HPLC-MS(/MS) analysis is the ability to detect multiple analytes in parallel from a single sample and over a wide concentration range (Köhrle and Richards 2020).

High performance liquid chromatography (HPLC)

HPLC is an instrumental analytical separation method in which analytes interact to varying degrees with a stationary phase (analytical column) while being transported by a mobile phase. The main separation mechanisms are adsorption, partitioning, ion exchange, and molecular exclusion or size exclusion (Schwedt 2008). A distinction is made between normal phase chromatography (NPC) and reversed phase chromatography (RPC). Basically, such an instrument consists of four main components: Pump, injection system, separation column and detector with evaluation system.

Ultra High Performance Liquid Chromatography (UHPLC)

Ultra High Performance Liquid Chromatography (UHPLC) represents a further development of "classical" HPLC and is more powerful (thanks to higher pressures), efficient and economical (Gey 2015). In UHPLC, columns with particle sizes of about 2 μm are used, requiring a column inlet pressure of several hundred bar. Column lengths are in the cm range and analysis times can be as short as seconds; usually only a few minutes (Gey 2015). In general, separation performance depends on many interacting factors.

The column length increases the number of theoretical plates and thus indirectly influences how long the substance can interact with the stationary phase; the column inner diameter influences, among other things, the pressure with which the HPLC is operated. The smaller the particle size of the stationary phase, the larger the surface area where the interactions take place and the greater the back pressure. The flow rate also affects how long the hydrophobic bond to the stationary phase is. If the flow velocity is high, the interaction is less strong/intense and the separation may be poorer.

Normal phase chromatography and reversed phase chromatography

NPC uses polar stationary phases, which have aluminum and oxygen molecules on their surface. These exhibit absorptive properties, which is why polar analytes interact more strongly with them and thus elute later. They form dipole-dipole interactions and induced dipoles as well as π-complex bonds. The mobile phase is classically a relatively nonpolar solvent. RPC behaves in the opposite way. In RPC, hydrophobic stationary phases are used, which carry long-chain hydrocarbons, such as C8, or C18 chains, by modification of the silanol groups (Si-OH). For steric reasons, only about half of the molecules can be modified, the remaining free silanol groups on the surface can be saturated by smaller non-polar molecules (endcapping). RP materials preferentially bind nonpolar substances through hydrophobic interactions (Gey 2015). The more nonpolar a substance is, the more intense the interactions with the stationary phase and the later it elutes. Typically, polar solvents (solvent mixtures such as methanol/water) are used.

What does a solvent gradient do?

A solvent gradient, with an increase in the amount of organic solvent and subsequent backwashing of the aqueous solvent, guarantees that all analytes elute from the column and that the column is re-equilibrated for the next measurement cycle.

What happens after chromatographic separation of the sample?

The use of an HPLC allows ionization (elution sequence) and detection of the analytes to be separated in time, often providing improved sensitivity.

Ionization methods

There are several ways to ionize the sample, distinguishing between hard and soft ionization. Ionization can be positive [M+H]+ or negative [M-H]-. To give some examples:

  • Electron impact ionization (EI), hard ionization, sample must be thermally stable and vaporizable, molecular fragments are generated by very high energy
  • Chemical ionization (CI), soft ionization, sample must be vaporizable, ion-molecule reaction, less fragmentation than EI, quasi-molecules are produced: for example [M+H]+ so molecule plus attached proton
  • Fast atom bombardment ionization (FAB), soft ionization, substance dissolved in a matrix, bombardment with fast noble gas ions, quasi-molecules are formed
  • Matrix-Assisted Laser Desorption/Ionization (MALDI), desorption, sample is provided with matrix material and fixed on a carrier, laser dissolves molecules as hot gas from the sample, release or acceptance of protons
  • Electrospray Ionization (ESI), soft ionization, ions are transferred from liquid phase to gas phase, quasi molecules are formed, in comparison few fragments are formed already in the source

What is a mass spectrometer and what is it used for?

A mass spectrometer is a device which allows to determine the mass and frequency of charged particles. The principle is based on converting molecules into charged ions, separating them according to their mass-to-charge ratio (m/z) and then detecting them. Mass spectrometry is used, for example, to elucidate structures, determine exact masses, or test ionizability (Acker and Bremer et al. n.d.).

Structure and function of a mass spectrometer

The main components of a mass spectrometer are the sample inlet system, the ion source, the separation system or analyzer (here the analytes are separated according to their m/z), the detector and a computer-based control and evaluation unit.

In principle, a closed high-vacuum system is necessary for the separation and detection of ions, since scattering and collisions with hydrogen and nitrogen molecules can trigger signal superpositions. The detector would lose a lot of sensitivity due to this overload. (Cammann 2010). The vacuum system is composed of a pre-vacuum (pressure about 10-1 mbar) and a high vacuum (10-4 mbar).

The charged analytes generated in the ion source are accelerated towards the detector. There are multiple options for ionization, as described above, as well as for ion separation (Gey 2015). In ion separation, ions are separated according to their m/z using static or dynamic methods. Magnetic sector field devices separate ions by their deflection in a strong magnetic field (static method). In contrast, in the dynamic method, ion separation is effected in time-of-flight mass spectrometers, in which either the separation is based on the mass-dependent velocity of the ions. Alternatively, in quadrupole mass spectrometers, in which the ions pass through the four-cylinder poles of an alternating electric field, only ions that change direction in time with the alternating field are allowed to pass through the mass filter (Acker and Bremer et al. n.d.).

Tandem Mass Spectrometry (MS/MS)

Tandem mass spectrometry is used to study dissociations and fragmentations and thus also to perform structural elucidation; on the other hand, it is used to improve the selectivity and sensitivity of a quantification method (Mass Spectrometry n.d.). There are several ways to induce tandem mass spectrometry (MS/MS, or MS2), depending on the type of instrument (e.g. triple quadrupole or hybrid quadrupole/trap mass spectrometer) and how the sample is ionized (hard/soft).

Triple quadrupole: The target analyte is ionized with a soft ionization methdode e.g. ESI. The precursor ion is now specifically separated from the other ions in the first quadrupole (Q1) according to their m/z with a preselected ion transition specific for the molecule, isolated and passed to the second quadrupole (Q2). By collision with nitrogen or argon atoms, the precursor ion in Q2 is selectively dissociated1 (molecular fragments are formed), giving rise to product ions. The molecule-specific fragments are forwarded to the third quadrupole (Q3), where either the different ions are scanned (structure elucidation, which ions are formed?) or only one specific product ion is isolated with the previously defined m/z and subsequently detected (selective quantification). This technique is also called multiple reaction monitoring (MRM) (You and Willcox et al. 2013).

Hard ionization allows the precursor ions to fragment already in the source2 and then be further analyzed as described above, this creates a pseudo-MS3. Thus, a product ion is selectively isolated from the source in Q1, dissociated in Q2, and a new product ion is isolated and detected in Q3 starting from the product ion already formed in the source. Provided that both stages of the product ions exhibit a molecule-specific ion transition, the selectivity is increased by the further step. By observing one, two or more (MSn dissociations, the target analyte can be quantified very sensitively and selectively.

The Orbitrab as a special mass spectrometer

The Orbitrap is from Thermo Fisher Scientific represents one of the latest developments in ion trap mass spectrometers. The major components of this mass spectrometer are the quadrupole, the curved linear ion trap (C-trap), a higher-energy collisional dissociation (HCD) cell, and the Orbitrap mass analyzer. The quadrupole in this instrument serves as a mass filter for a wide or narrow mass range. It is only used to isolate the user-defined mass range and not a scan.

The Quadrupole

The quadrupole consists of four cylindrical rods arranged in parallel, each of which opposite rods carries the same electric charge (Gruber and Gruner n.d.). These charges alternate at high frequency and generate an alternating electric field. Ions can selectively pass the quadrupole by adjusting the field according to their m/z on a stabilized trajectory. This property is used for the selection of individual masses (mass filters) (Salzer et al. n.d.). In addition, a quadrupole can also scan a defined mass range and thus allow all ions of this size range to pass.

The C-trap

The C-trap is a bent-up, linear ion trap. Ions that have passed the quadrupole are collected here and energetically bundled. The C-Trap has a limited capacity for ions (3 million charges) and is protected from overfilling and space charge effects by technical regulations. It is also used to route the ions to the HCD cell (,to dissociate the ions) and/or to the Orbitrap mass analyzer (for detection) (Kaufmann and Bromirski 2018).

The HCD cell

The HCD cell induces dissociation of the ions by collisions with neutral (inert) gas particles, in this case nitrogen (Kaufmann and Bromirski 2018). The collision energy (CE) controls the acceleration of the N2 molecules in the HCD cell and thus the intensity of the collisions, directly the collision energy (Gross 2013).

The Orbitrap as a functional unit

The Orbitrap is an ion trap that does not require frequency excitation or a magnet to keep the ions inside the ion trap. Axial oscillation of the ions takes place in an inhomogeneous electrostatic field. In this process, smaller masses have a narrower trajectory than larger ones due to a stronger attraction to the electrostatic field. Ion separation can be generated. The electrical attraction to the central electrode is compensated by the centrifugal force. The Orbitrap mass analyzer has two functions: First, it serves as an analyzer, i.e., it can distinguish the different ions. On the other hand, it serves as a detector, as described above, in which it converts the time-variable signals (transients) in the voltage field into numerical values via a Fourier transformation. Basically, ions registered at the detector are converted into optical signals by means of computer calculations and finally displayed in a mass spectrum by appropriate software. Frequency detection is performed after Fourier transform of the transient signal (Gross 2013; Kaufmann and Bromirski 2018). The Orbitrap analyzers of this model are among the highest resolution mass spectrometers.

Experiments of the Orbitrap

Basically, different experiments can be performed with the Orbitrap or combined. The so-called Full-MS is an experiment in which all ions passing the quadrupole within a defined mass range (m/z) are detected directly in the Orbitrap without dissociation in the HCD cell. Here, the quadrupole is used as a broadband mass filter covering the complete mass range of the molecules to be analyzed. Only intact precursor ions (possibly fragments due to ionization but no product ions analyzed) are measured with high resolution.

Targeted-Single Ion Monitoring (t-SIM) isolates a very narrow (e.g., 0.4 Da), user-defined mass range in the quadrupole. Thus, the Orbitrap mass analyzer detects only ions of this small mass range. By setting the quadrupole, the concentration of target analytes is much higher and it can be collected longer until the capacity is reached (time limit next to the filling amount). The higher number of target ions injected to the Orbitrap results in higher sensitivity.

Parallel Reaction Monitoring (PRM) is a special tandem mass spectrometric experiment that measures selective product ion transitions (for multiple analytes in parallel). Here, the quadrupole serves as a mass filter to isolate a precursor ion. Only the ions belonging to a narrow mass range (e.g., 1 Da) enter the C-trap. The C-trap transports the isolated precursor ions into the HCD cell, where the molecules dissociate by collision with nitrogen. These resulting product ions return to the C-trap for energetically-bundled injection into the Orbitrap mass analyzer. The PRM is experimentally comparable to the product ion scan of a triple quadrupole mass spectrometer MRM (see above), but provides much higher resolution mass spectra.

The difference to the t-SIM lies mainly in the collision-induced dissociation of the ions. The t-SIM is minimally more sensitive due to a smaller number of experiments in the instrument, but the majority of information obtained is outweighed by the product ion spectra, which is why this experiment is considered the "best" (Kaufmann and Bromirski 2018). The two most intense peaks of the product ion spectra are evaluated as quantifier and qualifier ion transitions. The required collision energy is adjusted individually for the precursor ions during method development, so that the precursor ions are eventually barely visible in the product ion spectrum and the product ions themselves are as large as possible (Gross 2013).

1Molecular fragments are formed by deliberate application of collision energy and bombardment with gas particles.

2Molecular fragments are created by high energy or ionization in the source.


Acker, Bremer, Dannecker, Däßler, Dreier: Massenspektrometrie. Hg. v. Spektrum Akademischer Verlag. Online verfügbar unter, zuletzt geprüft am 07.07.2021.

Cammann (2010). Instrumentelle Analytische Chemie: Verfahren, Anwendungen, Qualitätssicherung. 9-1. SpektrumAkademischer Verlag.

Gey (2015): Instrumentelle Analytik und Bioanalytik. Biosubstanzen, Trennmethoden, Strukturanalytik, Applikationen. 3. Aufl. Deutschland: Spinger

Gross, Jürgen H. (2013): Massenspektrometrie. Berlin, Heidelberg: Springer Berlin Heidelberg.

Gruber; Gruner: Massenspektrometrie. Hg. v. Fraunhofer-Institut für Verfahrenstechnik und Verpackung IVV. Online verfügbar unter, zuletzt geprüft am 14.01.2021.

Köhrle; Richards, Keith (2020): Mass Spectrometry-Based Determination of Thyroid Hormones and Their Metabolites in Endocrine Diagnostics and Biomedical Research -Implications for Human Serum Diagnostics. In: Experimental and clinical endocrinology & diabetes : official journal, German Society of Endocrinology [and] German Diabetes Association 128 (6-07), S. 358–374. DOI: 10.1055/a-1175-4610.

Massenspektrometrie (o.D.). Hg. Online verfügbar unter, zuletzt geprüft am 12.07.2021

Salzer; Thiele; Machill; Zuern; Bezugla; Baetz: Massenspektrometer -Der Massenanalysator -Quadrupol. Hg. v. ChemGaroo-ChemgaPedia. Online verfügbar unter, zuletzt geprüft am 14.01.2021

Schwedt (2008). Analytische Chemie : Grundlagen, Methoden und Praxis. Weinheim: Wiley-VCH

You; Willcox; Madigan; Wasinger; Schiller; Walsh;. Graham; Kearsley; Li (2013): Tear Fluid Protein Biomarkers.In: Advances in Clinical Chemistry, Elsevier. Volume 62 (4), S. 151-196. Online verfügbar unter