Analysis of the Basics of LC-MS Analysis and Analysis of Common Problems

LCMS is an important analysis tool in organic synthesis, and analyzing LCMS spectra is also a basic skill.

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• NMR 1D、2D（H-NMR、C-NMR、P-NMR、F-NMR、HSQC、HMBC、COSY、NOESY, et al）
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• Chiral HPLC Analysis
• General LCMS Testing (ROI、LOD、Cl-、SO42-、mp、HM、Specific Rotation、Water Content, et al）

Basic principles and characteristics of LCMS

1) The characteristics of LCMS: It is a combination of HPLC and MS. It has the functions of both, and is it accurate.

2) Mobile phase method: Four methods of 0-30, 0-60, 10-80, 30-90 are common, 0, 10, and 30 all refer to the content of acetonitrile. The greater the content of acetonitrile, the lower the polarity of the mobile phase. The peak is more forward.

3) The positive ion source is suitable for alkaline compounds: nitrogen-containing compounds are easier to adhere to hydrogen positive ions, and molecular ion peaks are easy to appear in the positive ion source. Negative ion source is suitable for acidic compounds: acidic compounds are easier to bombard hydrogen positive ions, such as acids and phenolic compounds.

See LCMS steps

1) First look at the MS part to see if there is the desired ion peak, and to see if the compound has an MS signal and whether it covers the surrounding peaks.

2) Look at the HPLC section again to see how much the content is, and to see if the compound has a strong HPLC signal and whether it covers the surrounding peaks.

3) Combining the two, infer the extent of the reaction and the impurities produced by the reaction.

Common adduct ion peaks

[M+Na]+ = [M+23]+ add sodium ion;

[M+K]+ = [M+39]+Add potassium ion;

[M+NH4]+ = [M+18]+Add ammonium ion;

[M +H +H2O]+ = [M+19]+add water;

[M+X]+ where X refers to the cation in the solvent buffer;

[M+H+Solvent]+solvent adduct peak, such as [M+H+CH3CN]+ = [M+42 ]+ is the CH3CN adduct ion, [M+H+CH3OH]+ = [M+33 ]+ It is CH3OH adduct ion;

Peak reduction: M-56 (de-tert-butyl) and M-100 (de-Boc), M-16 (de-NH3) and M-17 (dehydration) and M+2/2 (more common), others are rare.

Isotope peak

Pay special attention to the difference between precise molecular weight and molar molecular weight

The performance of common chlorine and bromine isotopes: a chlorine peak height ratio M+2/M=1:3; a bromine is a peak height ratio M+2/M=1:1; the performance of multiple isotopes can be accurately simulated with Chemdraw.

Precautions

1. In the LCMS report, the components with strong MS response may cover up the components with weak MS response, which can be judged by extracting ion current or subtracting background. The LCMS report must combine the two parts of LC and MS to corroborate each other.

2. When not using TLC and directly using LCMS to monitor the reaction, it must be combined with other means (such as NMR) for auxiliary detection. Special reminder: To monitor the reaction, you can’t just rely on LCMS and not click on the TLC plate. The most important means of monitoring the reaction is the use of TLC, which is direct, fast, and provides a lot of information for subsequent separation and purification.

Common phenomena and explanations

1. The problem of M+ and (M+1)+: theoretically speaking, ESI will only show adduct ions (M＋H, M＋Na, etc.) in positive mode, and M+ ions may appear in APCI and APPI, but in actual work (LC－ MS), M+ ions may be encountered, if the compound is no problem, and M+ is generated during mass spectrometry. The characteristic of this type of compound is that it contains a bridged ring in the structure and a nitrogen with a H in the bridged ring. In private, I feel that under the action of an electric field, the nitrogen in the bridge ring neutrally loses protons (H), and M+ is (M-H+1)+.

2. The problem of M+Na: For some samples, there is only M+Ma, but almost no M+1. The characteristic of this type of compound is that the compound structure contains multiple O and (or) S atoms. The quality of chromatographically pure acetonitrile is nothing to say, but the chromatographically pure solvent still contains trace amounts of alkali metal ions. Even if the salt of alkali metal ions is not introduced in the sample processing, the trace amount of alkali metal ions in the solvent will cause the addition of alkali metal ions to appear on the mass spectrum. This is the reason why M+Na appears on the mass spectrum; The O and S atoms in the compound also have unpaired lone pairs of electrons, and Na ions have empty orbitals without electrons, so O and S are particularly easy to capture Na ions and form charged particles. When the sample contains multiple O and S, The probability of forming the M+Na peak increases, and the ratio of the M+H peak gradually decreases or even disappears.

3.2M peak and doubly charged ions: The general sample is M+1 (or 23). As the sample concentration increases, the 2M+1 (or 23) abundance increases. This is mainly because the sample concentration in the ESI spray increases when the sample concentration increases. It is caused by the increased probability of the last two molecules and one charge together in the subsequent Coulomb explosion and further desolvation process.

For doubly charged ions, there are mainly two protonation sites in the molecular structure, and the last two sites are both charged, and finally doubly charged ions are formed. In ESI, the spray needle and the instrument form a galvanic cell, the mobile phase and the surface of the spray needle undergo a charge transfer, so that the sprayed droplets are charged, and under the action of the countercurrent N2 in the spray process, the charged droplets generate one level The first-level Coulomb explosion (most of the droplets are removed as waste liquid and gaseous state), after the final desolvation, the detected ion is formed. Therefore, only when the sample is charged and is preferentially excited in the Coulomb explosion, can the detection ion be formed. This is mainly due to the influence of solvent chemistry, and the presence of matrix is ​​likely to inhibit the ionization of the sample (matrix effect). After the mobile phase of APCI is sprayed and dried, the sample molecules are charged through the corona discharge needle (in addition, charge exchange may occur). The parameters of the corona discharge needle have an effect on the ionization of the sample, but the matrix effect has little effect.

4. Weak polarity sample ionization problem: APCI (atmospheric pressure chemical ionization) or APPI (atmospheric pressure photoionization) source is recommended for low polarity. Generally, salt is added to the mobile phase of acidic compounds: such as ammonium formate, ammonium acetate, or alkali (ammonia) to enhance the acid of the compound to enhance the ionization of the compound, and it is generally detected by positive ion.

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