LC/MS liquid chromatography-mass spectrometry experience and spectral analysis


lcms analysis

Liquid use experience and contraindications

1. Acidic substances are suitable for negative ion detection, so the mobile phase is more alkaline to promote its dissociation, and alkaline substances are suitable for positive ion detection. Add acid to the mobile phase to promote the formation of positive ions. Add some sodium acetate (or ammonium acetate) to form a sodium plus cation or an ammonium plus cation.

2. When glycosides are used for FAB and ESI+, the [M+Na] peak is often stronger than the [M+H] peak. This is empirical, and the reason is only speculated that it may be related to the extraction process of natural products; salt compounds such as salt Acid salts and sulfates generally do not appear in the acid part of the mass spectrum; dicarboxylates (esi negative ion mode), in addition to the molecular ion peak, will appear two consecutive peaks of 44, which are ions that lose carboxylate. This peak is very characteristic, but will be affected by the cone voltage, and the spectrum will be more beautiful if you lower the voltage.

3. When amine substances are used for ESI mass spectrometry, it is necessary to pay attention to the small amount of injection, because it is easy to ionize and difficult to wash, which will affect the determination of subsequent samples. Like triethylamine, it cannot be used to adjust the pH of the mobile phase in LC/MS. If triethylamine is accidentally introduced, a strong 102 peak ([M+H] for triethylamine) will always appear in positive ion detection.

4. Purified water is generally good for mass spectrometry; methanol and acetonitrile are used for mass spectrometry. After switching to many brands, it is found that Merck’s is slightly better; the nitrogen used by Finnigan does not necessarily need to use liquid nitrogen bottles, but ordinary The cylinder gas is enough, and it may save money; it is recommended that you buy a better flashlight and a magnifying glass. The flashlight is used to look inside the source, and the magnifying glass is used to see that the capillary you cut is flat.

5. The baseline of mass spectrometry is actually the same as that of liquid-phase UV detectors and fluorescence detectors. The reasons for the high baseline are nothing more than internal and external reasons.

1) The mobile phase you choose has a higher response in the mass spectrometer. For example, when there is more water, the noise is higher; and if the salt content is higher, the noise is higher.

2) When the sensitivity of the detector is higher, the noise should be higher. If the contamination of the mass spectrum is serious, the baseline must be relatively high. For example, ion trap detectors, when used for a long time, the ions in the trap will increase, which reduces the sensitivity of mass spectrometry on the one hand, and increases the baseline noise on the other hand.

3) The baseline of the mass spectrum is often related to the ion width you choose. For example, when you do a selected ion scan, the baseline is lower. When you do a selective response scan, the ion width should not be too wide, and the noise will be higher if it is too wide.

4) Multistage mass spectrometry is generally used for secondary or tertiary mass spectrometry, and the baseline noise is much lower.

6. Exchange of mass spectrometer maintenance experience: before sampling – check nitrogen, mobile phase, vacuum degree of mass spectrometer, and capillary temperature.

1) It is best not to inject the sample directly (it is easy to contaminate the ion source);

2) It is best to split when used in combination (a can use conventional columns, b shorten the analysis time, c prolong the life of the mass analyzer);

3) It is best to use an online switching valve, and cut the mobile phase 1-2 minutes before and after each sample into the waste liquid (to prevent the salt in the sample from entering the mass spectrometer, you can cut the mobile phase of the equilibrium column into the waste liquid when doing Sequence);

4) Before starting the coupling, run the mass spectrometer directly for a few minutes, you can first heat the temperature (capillary temperature and ion source temperature (APCI)) to the preset value (if it is APCI source, you can also avoid burning the heater, too expensive. , it is best not to burn);

5) Set the switching valve to waste when in standby to avoid injecting the mobile phase into the ion source when the liquid phase is just opened;

6) Before the shutdown, the temperature of the capillary is lowered first, and after a period of stability, the power is turned off, so as to prevent the heat from the periphery of the capillary from diffusing inward after the fan stops rotating, which will easily cause the aging of internal circuits and electronic components to accelerate;

7) Clean the outside of the capillary mouth every day, scrub it clean, pay attention to cleaning the Skimmer every time it stops, use dust-free wipes, the kind of kimberly;

8) If you use steel cylinders and make samples every day, connect the two cylinders in parallel, of course, if you don’t do it once a month, forget it;

9) Pay attention to the specific position of the ion source spray needle when doing quantification, otherwise the standard curve cannot be used;

10) Do not perform quantitative analysis without column separation, the results are unreliable (competitive inhibition of target molecule ionization);

11) If it is negative ion detection, a small amount of isopropanol can be added to the mobile phase;

12) Do not use non-volatile salt, if use volatile salt, but the concentration should not exceed 20mmol/L;

13) Formic acid, acetic acid and trifluoroacetic acid can be used when acid is required, but TFA should not be used when formic acid or acetic acid can be used;

7. In theory, it is forbidden to use any non-volatile buffer salts for LC/MS. If you need to use volatile salts such as ammonium acetate, the concentration should not exceed 20mmol/L.

For non-volatile buffers, you can also use purge and trap if your instrument has it, but be careful. Don’t use it as a last resort. First of all, if there are non-volatile salts, you can’t get a good ion flow, and secondly, the salts remain in the mass spectrometer and are difficult to remove. Unless the machine is shut down for cleaning, it will always affect the analysis of other samples.

You can find mass spectrometer friendly conditions for LC/MS, for example, the chromatographic conditions are water/acetonitrile mobile phase with 20mM phosphate, when doing LC/MS, you can use ammonium acetate instead, and then use acetic acid to adjust the pH to be consistent with phosphate That’s it.

In addition to the less volatile salts, triethylamine, surfactants, and high concentrations (>0.5%) of TFA are not good for mass spectrometry and should be avoided in the mobile phase of LC/MS.

Quality quantitative analysis experience

1. Fragments of the target ions should be used for quantification, which has strong characteristics and eliminates interference;

2. In the method setting of quantitative analysis, increase the scan rate as much as possible, improve the accuracy and repeatability (you can increase the scan times of the target peak by reducing the range of the scan mass number, or divide the entire analysis time of a sample into n segments, set the scan mode separately for the target ions);

3. Quantitative analysis must be carried out after separation by chromatographic column to avoid the existence of competitive ions affecting the ionization efficiency of target ions; if the target molecule is not completely separated from the competitive molecule, the ionization efficiency of the target molecule will be caused during the ionization process. If it decreases, the quantitative results of sample molecules are low. Of course, samples with standard concentration should also be analyzed by the same method.

4. If the samples are pure, they can be directly injected and analyzed without going through the chromatographic column, including the samples for the standard curve (although direct injection and analysis are not recommended).

5. If the position of the needle of the ion source used is movable, be sure to remember its position when making the standard curve, otherwise the ion flux entering the mass spectrometer will change under the same conditions after the position is moved, and the standard curve cannot be used! Do not change the flow rates of shealth gas and aux gas for the called mass spectrometry method, otherwise it will affect the amount of sample entering the mass spectrometer.

6. If you want to reuse the established standard curve after one month, check the standard curve with QC samples!

7. Do not make any changes in the scanning range, mobile phase composition, gradient or flow rate of the established analytical method, otherwise, the standard curve should be redone. The scanning range changes the scanning times of the target peak, the composition of the mobile phase changes the ionization efficiency, and the flow rate changes the retention time and peak width of the chromatographic peak.

8. The strength of the ion trap is multi-level – qualitative, and the strength of the quadrupole is quantitative;

9. For samples with poor thermal stability, the repeatability of quantitative analysis can be ensured by increasing the gas velocity and reducing the capillary temperature; once the method is fixed, do not change it easily.

Analytical map

1. Analyze the molecular ion region (1) Mark the mass-to-charge ratio of each peak, especially the peaks in the high mass-to-charge ratio region.

(2) Identify molecular ion peaks. Firstly, a molecular ion peak is assumed in the high mass-to-charge ratio region, and it is judged whether the relationship between the assumed molecular ion peak and the adjacent fragment ion peak is reasonable.

(3) Analyze the relative intensity ratio of isotopic peak clusters and the Dm value between peaks to determine whether the compound contains elements such as C1, Br, S, Si, and elements without isotopes such as F, P, and I.

(4) Derive the molecular formula and calculate the degree of unsaturation. The molecular formula is calculated from the exact molecular weight measured by a high resolution mass spectrometer or from the relative intensities of clusters of isotopic peaks. If both are difficult to achieve, it can be deduced from the missing fragments and main fragment ions of the molecular ion peak, or combined with other methods.

(5) The information of molecular structure can be obtained from the relative intensity of molecular ion peaks. The relative intensity of the molecular ion peak is determined by the structure of the molecule. The greater the structural stability, the greater the relative intensity. For a compound with a molecular weight of about 200, if the molecular ion peak is a base peak or a strong bee, there are few fragment ions in the spectrum, indicating that the compound is a highly stable molecule, possibly an aromatic hydrocarbon or a condensed ring compound.

For example, the naphthalene molecular ion peak m/z 128 is the base peak, and the anthraquinone molecular ion peak m/z 208 is also the base peak.

Molecular ion peaks are weak or absent, and the compounds may be multi-branched hydrocarbons, alcohols, acids, etc.

2. Analyze fragment ions

(1) Obtain possible structural information from characteristic ion peaks and missing neutral fragments.

If a series of CnH2n+1 peaks appear in the mass spectrum, the compound may contain long-chain alkyl groups. If weak fragment ions such as m/z 77, 66, 65, 51, 40, 39 appear or partially appear, it indicates that the compound contains a phenyl group. If m/z 91 or 105 is a base peak or a strong peak, it indicates that the compound contains a benzyl or benzoyl group. If the base peak or strong peak appears in the middle of the mass-to-charge ratio in the mass spectrum, and there are few other fragment ion peaks, the compound may be more stable from two parts, which are connected by weak bonds that are easy to break.

(2) Comprehensively analyze all the information obtained above, combine the molecular formula and degree of unsaturation, and propose the possible structure of the compound.

(3) Analyze the cracking mechanism of the deduced possible structure to see if it is consistent with the mass spectrum, determine its structure, and further explain the mass spectrum, or compare it with the standard spectrum, or compare it with other spectra (1H NMR, 13C NMR, IR) Cooperate, confirm the structure.