What is HPLC-MS?
The full name of HPLC-MS
is high performance liquid chromatography-mass spectrometry
, which uses liquid chromatography as the separation system and mass spectrometry as the detection system. The sample is separated from the mobile phase in the mass spectrometry part, is ionized, and then the ion fragments are separated by mass number by the mass analyzer of the mass spectrometry, and the mass spectra are obtained by the detector. Liquid-liquid mass spectrometry reflects the complementary advantages of chromatography and mass spectrometry, combining the high separation ability of chromatography for complex samples with the advantages of MS with high selectivity, high sensitivity and the ability to provide relative molecular mass and structural information, and has been widely used in many fields such as drug analysis, food analysis and environmental analysis.
Application of HPLC-MS
The applications of liquid chromatography-mass spectrometry (LC-MS)
in various fields are as follows.In biochemistry
Compounds in living organisms are strongly polar, difficult to volatilize and have significant thermal instability, and at the same time, these compounds often appear in a mixture of proteins, peptides and nucleic acids, while HPLC-MS is highly capable of separating non-volatile, strongly polar, thermally unstable and high molecular weight compounds; mass spectrometry can accurately characterize compounds in complex mixtures, so liquid mass spectrometry as a biochemical analysis Therefore, liquid chromatography is gaining more and more attention as a powerful tool for biochemical analysis.In pharmaceutical analysis
In the field of drug analysis research, most pharmaceutical is more polar compounds, and the wide range of liquid chromatography analysis and the high specificity of mass spectrometry can improve more qualitative structural information and high detection sensitivity. Liquid mass spectrometry is capable of multi-stage cleavage of excimer ions, which provides relative molecular weights of compounds as well as rich fragmentation information. In the impurity study and pharmacokinetic study phases of drug development, where the blood concentration of impurities and pharmacokinetic samples are usually low and the analysis is difficult and interfering, liquid mass spectrometry can rapidly and accurately determine trace substances in drug analysis due to its high selectivity and sensitivity.In environmental monitoring
With the rapid development of industry and agriculture, various kinds of environmental pollution problems occur frequently, such as antibiotic residues in drinking water, polycyclic aromatic hydrocarbons, polychlorinated biphenyls, pesticide residues, etc. Therefore, the development of highly sensitive multi-residue reliable analytical methods has become an important strategic goal of environmental analytical chemistry. At the end of 1980s, researchers successfully applied atmospheric pressure ionization mass spectrometry in combination with liquid chromatography, and since then, LC-MS began to gradually become the most powerful tool for environmental monitoring.
HPLC-MS working principle
The principle of LC-MS instrument
is similar to that of gas chromatography, which uses liquid chromatography as the separation system and mass spectrometry as the detection system. The sample is separated from the mobile phase in the mass spectrometry section, ionized, and then the ion fragments are separated by the mass analyzer of the mass spectrometer according to the charge to mass ratio, and the mass spectrum is obtained by the detector.
Advantages of using HPLC-MS
In addition to the ability to analyze strongly polar, difficult to volatilize, and thermally unstable compounds, high-performance liquid chromatography-mass spectrometry has the following advantages:
1. With a wide analytical range, HPLC-MS can detect almost all compounds, which can easily solve the problem of analyzing thermally unstable compounds.
2. Even if the analyzed mixtures are not completely separated on the chromatogram, the characteristic ion mass chromatogram of HPLC-MS can be used to give their respective chromatograms for qualitative and quantitative purposes.
3. Reliable qualitative analysis results, LC-MS can give the molecular weight and rich structural information of each component at the same time.
4. The detection limit is low and the sensitivity of HPLC-MS is high, and the detection capacity can be increased by more than an order of magnitude by selecting the ion detection method.
5. The analysis time is fast and the liquid chromatographic column used in LC-MS is a narrow diameter column, which shortens the analysis time and improves the separation effect.
6. Highly automated, LC-MS has a high degree of automation.
Types of HPLC-MS
Along with the development of HPLC-MS interface technology, the liquid mass spectrometer instrument itself is also evolving, with the emergence of several types of mass spectrometry detectors. The more commonly used liquid mass spectrometers are:
Quadrupole mass spectrometer
Quadrupole mass spectrometry is still the most widely used, and the selective reaction monitoring (SRM) mode of the three-stage quadrupole mass spectrometer is suitable for routine and high-throughput bioanalysis. Improvements in the quadrupole mass spectrometer process and the strong stability of RF have greatly improved the resolution of the mass spectra, resolving mass number widths up to 0.1 Da and improving the selectivity of the analyzed compounds. Among all mass spectrometry analyzers, the quadrupole mass spectrometer provides the best accuracy and precision of quantitative analysis results.
Time of flight mass spectrometer (TOF)
With the advent of matrix-assisted laser desorption ionization and the development of computers, TOP was rapidly developed in the 1990s. Currently, the best time-of-flight mass spectrometers are capable of resolutions up to 20,000 Da and measure the mass numbers of molecules with very high accuracy. Time-of-flight mass spectrometers have largely replaced high-resolution dual-focus magnetic fan analyzers, but they cannot effectively utilize selected ion monitoring modes for analysis.
Fourier-transform ion-cyclotron resonance mass spectrometry(FT-ICR-MS)
has become an effective tool in the basic study of gas-phase ion-molecular reactions. This mass spectrometry is widely used in the study of biological macromolecules when coupled with an ESI ion source, and is able to take full advantage of its high resolution and accuracy.
HPLC-MS specifications and features
Type: Quadrupole mass spectrometer
1. Quadrupole mass spectrometer provides the sensitivity, performance, and overall system stability needed for quantitative analysis.
2. Quadrupole mass spectrometers are suitable for quantitative analysis applications in research, biomarker validation, studies, food testing, and environmental analysis.
3. Quadrupole mass spectrometers deliver efficiency, performance, and value tailored to your analytical needs.
Effect of contaminants on HPLC-MS
The sensitivity, selectivity, reproducibility, and separation of LC-MS analyses are affected by contaminants such as metal ions, phthalates, polyethylene glycol (PEG), slip enhancers, water, and particles entering the system from various sources, including:
* Reagents and solvents
* Water used to prepare buffers
* Chemicals leached from glassware
* Microcentrifuge tubes
* Inlet filters
* Solvent lines
* Instrument components, such as pump seals
* Gases for desolventizing eluate in the source and collision chamber
*The sample itself
Thus, contaminants can interfere with the analysis by:
a. Inhibiting or enhancing the ionization of the analyte in the source
b. Forming adducts with the analyte
c. Masking analyte peaks and/or appearing as ghost peaks in the chromatogram
d. Making the baseline noisy
e. Scaling systems and columns, requiring frequent maintenance and replacement parts
To minimize contamination
a. The mobile phase should be prepared using high purity solvents, water, and reagents.
b. Freshly prepared mobile phases must be used to minimize the possibility of microbial contamination of aqueous mobile phases and acetonitrile (ACN) polymerization.
c. Cleaning glassware with soap or detergents should be avoided as they can be difficult to remove and can cause interference during the analysis.
d. High purity gases must be used (e.g., commonly used nitrogen of >95% purity).
e. The nitrogen generator must be well maintained and the gas cylinder must be replaced when the pressure drops below acceptable levels.
f. Analytes must be extracted from the sample matrix and chromatographic parameters must be optimized to improve the separation of analyte peaks from interfering peaks.
How to use HPLC-MS?
Verify the initial state of the HPLC-MS
1. Verify that the tubing between the column outlet of the liquid chromatograph and the population entering the ion source of the mass spectrometer is connected and disconnected if it is.
2. Whether the main power switch of the liquid chromatograph and the power switch of the autosampler are off, and if not, turn them off.
3. Whether the main power switch of the mass spectrometer and the power switch of the main board are off, and if not, turn them off.
4. Check the color of the pump oil and the oil level of the low vacuum pump of the mass spectrometer
5. Open the main valve of the liquid nitrogen tank and check the pressure of the N2 gas source, which should be greater than 1MPa, and open the booster valve if it does not meet the requirements. Check whether the indication on the pressure gauge reaches the required working pressure. If you use nitrogen machine, turn on the machine according to the operation procedure and check whether the pressure of N2 tank is normal.
HPLC-MS cold start
1. Turn on the main power switch of LC-MS instrument self-test; however, the basic method is similar. That is, first start .
2. The procedure of starting the vacuum system will be different for different LC-MS instruments, but the basic method is similar. That is, first start the mechanical pump, and after reaching the low vacuum requirement or automatically start the molecular turbine pump, or first turn on the host switch of the mass spectrometer and let the molecular pump start pumping high vacuum.
3. Turn on the computer power and click the corresponding software logo on the screen to enter the home page of the liquid mass spectrometer. Only when the high vacuum reaches the predetermined requirements, the self-test of the mass spectrometer can be completed, and then the functional parts of the mass spectrometer can be operated. Some instruments require to turn on the power of the main board of the mass spectrometer to achieve the above operation.
4. When the self-test of liquid chromatograph and mass spectrometer is passed, the power-up phase of the instrument is completed.
5. Activate the software to ensure that the system's software connects the liquid chromatography with the mass spectrometer.
HPLC-MS hot start
When you are ready to leave the instrument overnight after daily work, you should enter the following state:
1. First disconnect the connection line between LC and MS, switch the flow valve of I.C. so that the 1C effluent is fed into the waste reservoir.
2. Turn off the multiplier voltage on the computer screen and allow the heating components of the mass spectrometer (if any) to cool down to below the specified temperature, and then exit the corresponding working interface.
3. Turn off all working gas lines, except for some working gas lines that require N. to maintain the required gas flow during overnight standby, and turn off the booster valve of the liquid nitrogen tank.
4. Leave the vacuum system of the mass spectrometer in operation.
5. If the mobile phase contains acids, bases, or salts before turning off the LC pump, the plunger pump and column will be cleaned with pre-prepared mobile phase without these additives (if the LC pump is used at low flow rates overnight, the LC pump does not need to be turned off and the mobile phase with acids, bases, or salts does not need to be replaced). Clean the autosampler with the specified cleaning solution.
Start up the LC-MS instrument the next day after the above mentioned overnight condition, which is called a hot start.
How to maintain HPLC-MS?
1. When using HPLC-MS, use a regulated power supply UPS to ensure that the instrument power supply voltage is stable and continuous. Especially the impact of power failure on the liquid chromatography-mass spectrometer.
2. Flush the LC-MS system and clean the nebulizer chamber with isopropyl alcohol solution daily, and open the shock valve regularly to shock the air. For ESI sources, do this at least once a week. For the APCI source, do it once a day to ensure the capillary is clean.
3. The flow rate of the mobile phase should not be too high, not more than 0.5ml/min for the ESI ion source, and not more than 1.0ml/min for APCI.
4. Replace the vacuum pump oil off the liquid chromatography-mass spectrometer once a year or half a year.
5. Control the sample running environment, including temperature and humidity.
6. Clean the injection needle and injection valve of the liquid chromatograph-mass spectrometer after the experiment is finished. After using the mobile phase containing acid, the column and ion source should be rinsed with methanol or water.
7. Periodically clean the sample cone hole of the liquid chromatography-mass spectrometer, close the isolation valve, and take off the sample cone hole, firstly, use methanol: water: formic acid (45:45:10) solution to ultrasonically clean for 10 minutes, and then ultrasonically clean each solution with ultrapure water and methanol for 10 minutes respectively, and then install it to the instrument after drying.
8. The mobile phase should be ready to use, preferably not overnight, and must be fresh.
9. Replace the electron multiplier of the liquid chromatograph-mass spectrometer regularly.
10. It is better to split the flow when doing the coupling, a to use the conventional column, b to shorten the analysis time, and c to extend the life of the mass analyzer.
11. Turn off the capillary temperature before changing the liquid chromatograph-mass spectrometer down, and stabilize for some time before turning off the power, to avoid the fan to stop the rotation of the capillary periphery of the heat diffusion inward, easy to cause the internal circuit and electronic components aging accelerated.
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