Liquid Chromatography-Nuclear Magnetic Resonance (LC-NMR)

High performance liquid chromatography (HPLC) is the most powerful tool in the separation of complex mixtures.

Liquid Chromatography-Nuclear Magnetic Resonance (LC-NMR)

High performance liquid chromatography (HPLC) is the most powerful tool in the separation of complex mixtures. By combining HPLC with various detectors such as ultraviolet absorption detectors, fluorescence detectors, electrochemical detectors, etc., structural information of some compounds can be obtained, but the amount of information is very limited. Therefore, some detection techniques with richer structural information, such as IR, MS, NMR, etc., are also needed. Among them, nuclear magnetic resonance (NMR) is a powerful tool for obtaining detailed structural information of organic substances.

NMR can provide a large amount of structural information and is one of the most powerful spectroscopy techniques for analyzing unknown structures. NMR can provide a wealth of compound structure information, including stereochemical information (in terms of configuration and optical isomers), and is simple, accurate, highly specific, and does not damage the sample. However, the samples analyzed by NMR are pure substances. Because the NMR signals of each component of a complex mixture will cover each other and influence each other, it is usually difficult to obtain the analysis results of the mixture by using NMR. Therefore, it is necessary to separate and purify the mixed sample before using NMR detection. Among them, HPLC has been widely used for the separation of complex samples. By adjusting the chromatographic conditions, it can be used to separate different samples. If chromatographic technology is combined with NMR, the separation and detection process of samples can be greatly simplified.

The attempt of liquid chromatography-nuclear magnetic resonance (LC-NMR) technology began in the 1970s, and it was not developed until the mid-to-late 1990s and was widely used. At present, HPLC-NMR has become a relatively mature analysis method, which is widely used in the structural identification of mixtures, especially unknowns.

Principle of LC-NMR

The general configuration of LC-NMR includes a conventional HPLC system, coupling interface and NMR system equipped with a flow probe. The liquid chromatograph of the LC-NMR system is responsible for the separation of the components in the sample and can be regarded as the NMR sampling device. The interface injects the components flowing out of the liquid chromatography into the NMR instrument, which plays a role in adapting HPLC and NMR. NMR analyzes the components sequentially introduced into the interface and can be regarded as a detector of HPLC.

The sample is injected into the high-performance liquid chromatography system, and the components are separated under the impetus of the high-pressure pump. The separated components sequentially pass through a conventional detector (such as a DAD detector). The effluent is then injected into the NMR instrument through a suitable interface device. Therefore, the combined use of LC-NMR is realized.

LC-NMR instrument structure diagramLC-NMR instrument structure diagram

The direct connection of HPLC and NMR will encounter a series of problems related to hardware and software, such as the suppression of NMR signal peaks by the elution solvent of LC, and the low detection sensitivity of NMR. With the improvement of the magnetic field intensity of NMR instruments, the improvement of NMR probe design, and the development of pulse sequence technology, many problems have been basically solved, making the LC-NMR combined technology more practical and widely used.

Operation mode of LC-NMR

At present, HPLC-NMR has two main operating modes to choose from, namely continuous flow mode and stopped flow mode.

  • Continuous flow mode
  • Continuous flow mode, also called on-flow mode. When using the continuous flow mode, the eluent from the HPLC column is directly sent to the NMR. The NMR spectrum is obtained when the eluent flows through the NMR probe rapidly. NMR information of all components can be obtained in one analysis. The flow of HPLC is continuous and is not affected by NMR sampling. This mode can quickly get the test results, and this mode is suitable for analyzing high-concentration samples to determine the more abundant components in complex mixtures.

  • Stopped flow mode
  • Stopped flow mode is to stop the flow of eluent when the sample reaches the NMR tube, so the component to be tested stays in the NMR tube to be detected. When using the stopped-flow mode, NMR no longer scans each sample peak in real time with the continuous flow of the eluent, but performs a single, longer-time scan of the component to be tested in different ways. The NMR signal obtained in this mode is much stronger than the signal obtained in the continuous flow mode, so this mode is suitable for analyzing low-concentration samples and using in 2D NMR.

Application of LC-NMR

  • Natural product analysis
  • The composition of natural products is very complex, and most of the effective substances are several kinds. Using traditional separation methods to purify natural products is time-consuming and laborious, but LC-NMR can simplify this process. The LC-NMR combined technology shows the advantages of high efficiency, rapidity and small amount in the analysis of natural products. The combined LC-NMR technology can obtain preliminary information of complex extracts, which can well characterize the differences in chemical composition characteristics and relative contents. Since this technology can avoid unnecessary separation steps, it is particularly suitable for the study of plant extracts with more ingredients. LC-NMR can quickly identify known compounds in the early stages of analysis and focus on the structural research of new compounds. The combination of LC-NMR and LC-MS can obtain more comprehensive data and improve the analysis efficiency of natural products.

  • Isomer analysis
  • LC-NMR still plays an important role in determining the structure, concept and optical isomers. Using LC-NMR technology for the separation and determination of stereoisomers, the detection concentration can be accurate to nanograms, which fully reflects the stereo discrimination ability of NMR.

  • Metabolite analysis
  • Metabolite analysis plays an extremely important role in new drug research and clinical drug monitoring. Because drugs and their metabolites are distributed in a large number of media, there are endogenous interfering substances, the sampling amount is small, and the samples are not repeatable. Therefore, it is necessary to choose a simple and continuous analysis method with high sensitivity and good resolution. LC-NMR is very suitable for the detection of complex metabolites in biological samples and the identification of potential markers. LC-NMR can study metabolites at the molecular level, and sequential experiments can identify multiple metabolites at the same time.

  • Polymer analysis
  • The synthesized polymer is a highly complex multi-component substance composed of macromolecules with different chain lengths, different chemical components and different structures. Using LC-NMR technology can not only separate polymers according to their molecular weight, but also distinguish polymers with the same molecular weight but different branches.

  • Environmental monitoring
  • Using LC-NMR technology to analyze environmental samples can accurately determine the types and quantities of various trace substances and even ultra-trace substances in environmental samples.


  1. Zhang H., et al. Advances of liquid chromatography coupled with nuclear magnetic resonance spectrometer. Chemical analysis and meterage. 2017, 26(3): 117-122.
  2. Yang T.T., et al. Application of LC-NMR in pharmaceutical analysis. Drugs & Clinic. 2012, 27(6): 635-641.

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