My LC Blog: The LCMS Detector

Article written by guest author Anja Grüning , reviewed by Sascha Rexroth and Mel Euerby , edited by myself.

As you’ve already learned from Gesa’s HPLC Blog, compounds that are separated by LC can be identified using various LC detectors, which is great for analytes that can actually be resolved chromatographically. However, standard detectors often don’t provide sufficient sensitivity for trace analysis and with the growing demand to analyse more and more compounds in a single run, or in complex matrices, obtaining baseline separation can be challenging. For example, low levels of hundreds of pesticides have to be monitored in food safety analysis, which can look something like this ..

And this is where LCMS enters the stage. It provides high sensitivity and has the capability to distinguish even between co-eluting analytes, by their mass, or mass to charge ratio (m/z) to be correct.

Mass spectrometers measure the weight of molecules and atoms. The mass (m) determination is performed by separation of ions in electric or magnetic fields. Masses are usually reported as m/z, which signifies the mass to charge (z) ratio. For molecules that carry a single charge, which is the most common, m/z can easily be used to identify molecular mass.

Mass spectrometry provides a highly selective and sensitive detection method with the flexibility to cover applications in various fields, such as chemistry, pharma, clinical, food and environmental testing. During LC method development, it is a powerful tool to identify co-elution of unknowns and track peak movement.

LCMS was initially made possible with the introduction of API, which is short for Atmospheric pressure ionization. API offers vaporization of the LC eluent and formation of gaseous ions which can then be introduced into the MS. Therefore, API serves as an interface into the LCMS as well as ionization source. Ion formation is essential to the process, as all mass spectrometric instruments are based on the separation of ions in vacuum.

But what is an ion and how does ion formation happen?

Ions are positively or negatively charged particles, atoms or molecules. Therefore, ionization is the process of transferring either a positive or a negative charge. This charge transfer happens in the ionization source of an MS.

In LCMS we’re dealing with two major ionization techniques, namely ESI (Electrospray Ionization) and APCI (Atmospheric Pressure Chemical Ionization). The most commonly used ionization technique in LCMS is electrospray ionization, which I will briefly explain.

ESI is a soft ionization method which mainly leads to intact molecular ions, so determination of the molecular mass of compounds is easily done.

The sample is carried by the mobile phase from the LC system into a very thin capillary – the ESI pipe. A high voltage (up to +/- 5 kV) is applied to the tip of this pipe where the high electric field creates a fine mist of multiple charged droplets with the same polarity as the applied voltage. Nebulizer and heating gas flow support the solvent evaporation process. During evaporation, the surface to charge ratio is getting smaller and smaller. When the mutual repulsive force of the charges exceeds the liquid surface tension, the droplets “explode” into smaller ones. This process is repeated until droplets become small enough to facilitate the transition of analyte ions into the gas phase.

Several parameters affect the efficiency and sensitivity of the ionization process, so special attention should be paid to the LC flow rate and mobile phases. Generally, when switching from LC to LCMS analysis, LC method conditions can be used if the mobile phase and modifiers (e.g formic acid, ammonium acetate or formate) are volatile. In case of non-volatile buffers (e.g. phosphate buffer) modifications to the LC method are required.

For further information on APCI please check ?Fundamental LCMS Principle Guide (shimadzu.de)

Once the ions are formed they have to be transferred from the ion source into the mass analyzer. As a first step the ions enter a capillary which is the gate between atmospheric pressure and the vacuum. Multiple steps of so called ion optics (figure 4) are used to transmit and focus the ions into a beam and ensure that as much ions as possible can enter the mass analyzer, while non-ionic gas particles are removed.

Within the mass analyzer the m/z of the analyte ions can be determined and further treated depending on type of mass spectrometer. We differentiate between a number of mass analyzers, e.g. Magnetic Sector Instruments, Ion Trap mass spectrometer, Fourier Transform mass spectrometry, Quadrupole mass spectrometers, Time-of-Flight mass spectrometers. They all have different separating and operating principles which lead to differences in sensitivity, resolution and measuring modes. Explaining them all is far beyond the scope of this blog. In terms of cost, sensitivity and ease of operation quadrupole mass analyzers are superior; however other analyzers offer higher resolution, high mass accuracy and the option to generate more qualitative information. Which mass analyzer is best suited, ultimately depends on the analytical task. I’ll focus on Quadrupole MS as these systems are the most relevant and widely used in quantitative routine analysis.

As the name implies, a quadrupole consists of four parallel rods. Ions enter to the center axis of the quadrupole. A direct current (DC) and a high frequency alternating current (AC) are applied to the quadrupole. For a given combination of DC and AC only ions with a specific m/z ratio are able to pass through and reach the detector. Ions with other m/z values collide with the quadrupole, are discharged and cannot reach the detector.

A quadrupole offers two measuring modes: Scan and Selected Ion Monitoring (SIM). In scan mode a specified mass range is scanned sequentially for each m/z value while in SIM mode only the selected m/z is monitored as shown in figure 6. SIM provides higher sensitivity but is blind for everything else in the sample while scan mode offers more qualitative information.

At the end, I briefly like to mention that there are many MS/MS systems available, also known as tandem or hybrid MS. They consist of two mass analyzers connected in series with a collision or fragmentation cell in between. Ions are separated in the first mass analyzer (MS1), enter the collision cell and undergo fragmentation, resulting in generation of smaller ions called product ions, which are separated in the second mass analyzer (MS2) and detected. The use of MS/MS provides detailed mass information and allows for reduction of matrix interferences and background noise, resulting in superior selectivity and sensitivity. Widely used combinations are TQ (triple quadrupole MS instruments) or Q-TOF (quadrupole-time of flight MS instruments). In many analytical labs TQ instruments form the backbone for quantitative analysis.

I hope you enjoyed the very small insight into the basics of LCMS. If you want to learn more have a look at?Fundamental LCMS Principle Guide (shimadzu.de)