While this is not an accurate method of compound identification, it helps when some information about the sample is known a priori. Although a wide variety of detectors of differing technologies and sensitivities have been coupled with LC for analyzing different sample types, the mass spectrometer has emerged as a selective, sensitive and universal detector.
Unlike other detectors, the LC eluent carrying the separated analytes is not allowed to flow into the mass spectrometer. While the LC system is operated at ambient pressures, the mass spectrometer is operated under vacuum and the two are coupled through an interface. As the column eluent flows into the interface, the solvent is evaporated by applying heat and the analyte molecules are vaporized and ionized.
This is a crucial step as the mass spectrometer is only capable of detecting and measuring the gas phase ions. As the analyte ions are generated at atmospheric pressure in the interface, the process is called atmospheric pressure ionization API and the interface is known as the API source. Post-separation, the ions can be collected and detected by a variety of mass detectors , 2 of which the most common one is the electron-multiplier.
When the separated ions strike the surface of the electron-multiplier a dynode , secondary electrons are released. These secondary electrons are multiplied by cascading them through a series of dynodes. The amplified current generated by the flow of the secondary electrons is measured and correlated to the ion concentrations in the mass spectrometer at any given instant in time Figure 1.
This plot displays the peak intensities of the analyte ions versus their RT. Further, each point in the chromatogram is associated with a mass spectrum. The area of the analyte peak is used for its quantification. The mass spectrometer can be operated in two modes, a scan and b selected ion monitoring SIM.
This mode is used when analyzing unknown samples or when there is no available information about the ions present in a sample. This is the preferred mode of operation for accurate quantification of known compounds in a sample.
Further improvements in sample identification and accurate quantification can be achieved by coupling two mass analyzers that are operated in series. These configurations offer several possibilities for sample analysis. The scan range of Q 3 is offset by the NL value. This mode is preferred for compound quantification due to its specificity and sensitivity. TQMS can be operated to monitor multiple precursor-to-product transitions of the same as well as different analytes. The fragmentation depends on the structure of the molecule and the experimental conditions, such as gas pressure and collision energy.
Therefore, under a specific reaction condition, the fragmentation pattern is used along with the compound RT and its accurate mass value for identification. Moreover, monitoring of specific fragment ions helps to improve the sensitivity of detection and thus enables quantification of smaller amounts of the target compounds.
The QTOF mass spectrometer has a quadrupole mass analyzer and a time-of-flight mass analyzer separated by a collision cell. The quadrupole can be used either to transmit the ions or to isolate a specific precursor ion which is then fragmented in the collision cell.
A small fraction of the ions are first pulsed into the TOF analyzer by a modulator and subsequently accelerated into the high-vacuum field-free region by applying high voltage. TOF mass analyzers offer high mass resolutions while being able to scan over large mass ranges quickly. LC-MS has been extensively applied for the analysis of both small molecules and large protein molecules in diverse matrices.
Some examples of the applications of this technology are:. Today, drug development is carried out in human subjects and animals. However, as computing power and the number of sophisticated technology platforms grow exponentially, and our knowledge of human health and disease increases, the virtualization of clinical research and development will grow steadily.
Read this article to learn more. Featured Product. Simcyp: 20 Years of Innovation. Certara Appoints James E. Contact Investors Careers Support. Tue, July 15th Knowledge Base. Contact Us. About the author. Teuscher has been involved in clinical pharmacology and pharmacometrics work since He holds a PhD in Pharmaceutical Sciences from the University of Michigan and has held leadership roles at biotechnology companies, contract research organizations, and mid-sized pharmaceutical companies.
Prior to joining Certara, Dr. At Certara, Dr. Teuscher developed the software training department, led the software development of Phoenix, and now works as a pharmacometrics consultant.
It is especially useful for low or non-volatile organic compounds, which cannot be handled with gas chromatography. In the application of HPLC, the solvent travels under high pressure obtained by means of a pump to overcome the pressure drop in the packed column, which reduces the time of separation. As will be discussed, a continuous flow syringe pump is very useful in HPLC.
Direct gas-pressure system , which is inexpensive and reliable; however, changing solvent is difficult. Syringe pumps , which can provide a pulseless continuous flow rate. Syringe pumps are reliable, very accurate, precise, and can have a large capacity. Here is an example of a syringe pump:. Chemyx Fusion X Syringe Pump.
Pneumatic intensifier , which operates under constant pressure, i. Reciprocating pumps , which are an economical solution that provides a constant flow and high pressure, but can cause pulsing. The sample injector should work within very small volumes and withstand the high pressure of the solvent.
Most devices use sample injection valves instead of direct injection because the former have superior characteristics. Other types of detectors can be of use, e. Mass spectrometry MS ionizes atoms or molecules to facilitate their separation and detection in accordance with their molecular masses and charges mass to charge ratio.
MS is used in various applications, e.
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