What is LC-MS?
Liquid chromatography-mass spectrometry (LC-MS) is an analytical technique that combines the excellent separation power of high performance liquid chromatography (HPLC) with the high sensitivity analysis offered by mass spectrometry (MS). In LC-MS, the components of the sample mixture are first to separate with HPLC, and then analyze each component by MS.
Electrospray Ionization Technique and Its Applications in LC-MS
Electrospray ionization (ESI) was first introduced in the 1980s. It is a soft ionization technique, i.e., does not break large molecules, based on desorption. ESI uses electricity to charge the sample molecules, which are introduce into the ionization chamber in the form of a mist.
Electrospray ionization mass spectrometry (ESI-MS) is one of the main types of MS. It can be coupled with HPLC for more accurate results (the combined technique is called LC-MS/ESI or HPLC/ESI-MS.) Similar to the other types of spectrometers, ESI-MS consists of three main components: an ion source, a mass analyzer, and a detector.
Gaseous ions generate in the ionization chamber, which contains the electrospray ionization (ESI) ion source, and then move to the mass analyzer to be sorted according to their mass/charge ratio (m/z). Finally, they are conveyed to the detector to identify the ions and determine their abundance in the sample.
How LC-MS/ESI Works
In LC-MS/ESI, each component obtained from HPLC (mixed with the polar solvent, i.e., the mobile phase) infuses into the ionization chamber through a charged metal capillary at a very low flowrate (typically 1–20 μL/min and 20–50 nL/min in case of nanospray ionization) using a syringe pump. The electric field transforms the sample into a mist, droplets dry by a hot gas, which results in solvent evaporation and consequently shrinkage of the droplets.
When the droplets shrink, charged ions become more close and the electrostatic repulsion between them increases, resulting in their ejection from the solvent droplets. This technique is suitable for ionic polar compounds, and it can be of use to analyze both large and small molecules, but it is especially useful for larger molecules, e.g., proteins.
Principle Components of LC-MS/ESI
LC-MS/ESI consists of the following components:
- A pump to deliver the solvent into the HPLC column (syringe pumps are suitable in this case, because they can accommodate the very small flow rates used in LC-MS)
- An HPLC column
- A syringe or an automatic sampler for manual or automatic sample introduction, respectively, into the HPLC column
- A syringe pump to introduce the LC elute into the ESI chamber
- An ionization chamber, which contains the ESI ion source
- A mass analyzer, e.g., quadrupole mass analyzer
- A detector.
Setup and Operation of LC-MS/ESI
To setup the device, first select the injection mode (automatic or manual), and then prepare the solvent pump. Second, attach the tubing to the ESI chamber, which connects LC with MS incase of normal operation or which is used to directly introduce the sample into MS in case of direct injection.
The syringe pump used for solvent delivery should then be prepared by specifying the diameter of the syringe and the solvent flow rate. Similarly, the other syringe pump, which is used to introduce the HPLC elute into the ESI tubing, should be also set, and they both should be placed in their appropriate locations. The LC-MS machine could start operating normally when all equipment is correctly attached, and all parameters are specified in the device software.
It should be noted that LC-MS must be calibrated every three months to maintain the high sensitivity of the device. The calibration could be performed manually or automatically. In both cases the calibration solution can be directly introduced into the ESI chamber through the syringe pump or into the HPLC device through the LC solvent pump.
It is also very important to follow the manufacturer recommendations to ensure smooth and accurate performance of the machine.
When an issue occurs in the LC-MS/ESI, it is important to isolate the problem to a specific part of the LC-MS analyzer, i.e., main part, e.g., LC or MS and then to a specific part within them, such as ESI chamber, mass analyzer, and syringe pump.
Following are some mass spectrometry troubleshooting examples of the problems that could occur in HPLC:
- Air in the syringe may indicate that the syringe of the autosampler or syringe pump connection is not correct
- Air in the system could result from a faulty pump seal or valve
- Solvent contamination, which resolves by replacing the solvent
- Flow rate fluctuations, which indicate a leak in the autosampler or in one of the pumps.
On the other hand, an example of the problems that can occur in MS is unstable ESI, which may indicate a fault in the voltage connection or unclean/leaking ESI tip.
Finally, before each operation, all connections and tubing checks to ensure they are unclogged, and test rotors to confirm they are performing properly.