Product Information

Luteolin is a natural flavonoid compound, and the following are some common detection methods:

High performance liquid chromatography (HPLC)

Principle:

High performance liquid chromatography is based on the difference in distribution coefficients of different substances between the stationary phase and the mobile phase for separation. The sample is injected into a mobile phase (usually a solvent or mixed solvent), which carries the sample through a chromatographic column filled with a stationary phase (such as silica gel). Different compounds move at different speeds in the column to achieve separation. Finally, luteolin was detected using UV Vis detectors, as it has a specific UV absorption wavelength.

Operation steps:

Sample preparation: Extract and dissolve plant extracts or other samples containing luteolin in appropriate solvents (such as methanol, ethanol, etc.), and perform pre-treatment steps such as filtration and centrifugation to remove impurities.

Chromatographic condition setting: Select a suitable chromatographic column (such as C18 reverse phase chromatographic column), and the mobile phase can be a methanol water or acetonitrile water system. Add an appropriate amount of acid (such as formic acid, phosphoric acid) according to the actual situation to improve the separation effect. The flow rate is generally between 0.8-1.5 mL/min, and the column temperature is usually set at room temperature or at a suitable constant temperature (such as 30-40 ℃) as needed. The detection wavelength is generally set at around 350nm, the maximum absorption wavelength of luteolin.

-Injection and analysis: Inject the processed sample into the injection port of the chromatograph, record the chromatogram through computer software, and perform qualitative and quantitative analysis of luteolin based on retention time and peak area.

-Advantages:

-High separation efficiency can effectively separate luteolin from other impurities in complex samples.

-High detection sensitivity, capable of detecting low concentrations of luteolin.

-The results are accurate, have good reproducibility, and are suitable for quantitative analysis.

-Disadvantages:

-The instruments and equipment are expensive and require professional maintenance and operation skills.

-The analysis time is relatively long, especially for the separation of complex samples.

Liquid chromatography-mass spectrometry (LC-MS)

-Principle:

-Combining the separation ability of liquid chromatography with the high sensitivity and selectivity of mass spectrometry for identification. The sample is first separated by liquid chromatography and then enters the mass spectrometer. The mass spectrometer ionizes molecules and measures the mass to charge ratio (m/z) of ions to determine the mass of the molecule, thereby identifying luteolin. Meanwhile, quantitative analysis can be conducted based on the strength of ions.

-Operation steps:

-Sample pretreatment: Similar to HPLC, the sample needs to be extracted and purified to remove interfering substances.

-Instrument parameter settings: The parameter settings for the liquid chromatography section are basically the same as HPLC, but the selection of the mobile phase needs to consider compatibility with mass spectrometry. The mass spectrometer needs to set parameters such as ionization mode (such as electrospray ionization ESI or atmospheric pressure chemical ionization APCI), scanning range, detection mode (positive ion or negative ion mode), etc. For luteolin, good detection results can be obtained in ESI negative ion mode.

-Analysis and identification: Inject the sample into the instrument, first separate it by liquid chromatography, and then obtain the mass spectrum in the mass spectrometer. By comparing with known mass spectrometry databases of magnolol and combining information such as retention time, the presence of magnolol is determined, and quantification is performed based on the intensity of mass spectrometry peaks.

-Advantages:

-It has high sensitivity and selectivity, and can accurately identify the structure of luteolin.

-Trace amounts of luteolin in complex samples can also be effectively detected.

-Disadvantages:

-The instrument has high cost and complex operation, requiring professional personnel to operate and analyze data.

-The analysis of mass spectrometry data requires a certain level of professional knowledge and experience.

UV Vis spectrophotometry

-Principle:

-The conjugated system in the molecular structure of luteolin can absorb specific wavelength ranges of ultraviolet visible light. According to Lambert Beer's law (A=ε bc, where A is the absorbance, ε is the molar absorptivity, b is the optical path length, and c is the sample concentration), within a certain concentration range, the absorbance is proportional to the concentration of luteolin in the sample. The content of luteolin can be determined by measuring the absorbance.

-Operation steps:

-Standard curve drawing: Prepare a series of standard solutions of different concentrations using a known concentration of luteolin standard, and measure the absorbance at the maximum absorption wavelength of luteolin (such as 350nm) using a UV visible spectrophotometer. Draw a standard curve with concentration as the x-axis and absorbance as the y-axis.

-Sample determination: Dilute the extract of the test sample appropriately, measure the absorbance at the same wavelength, and calculate the content of luteolin in the sample based on the standard curve.

-Advantages:

-The instrument is simple, easy to operate, and cost-effective.

-For samples with high content of luteolin, rapid detection can be performed.

-Disadvantages:

-Poor selectivity and susceptibility to interference from other substances with similar absorption wavelengths in the sample.

-Cannot effectively distinguish between luteolin and its structural analogues.

Thin layer chromatography (TLC)

-Principle:

-Place the sample on a thin layer plate coated with an adsorbent (such as silica gel) and develop it on the plate using a suitable developing agent (such as a mixed solvent of toluene ethyl acetate formic acid). Different compounds have different adsorption desorption abilities between the adsorbent and the developing agent, thus moving different distances on the thin-layer plate to achieve separation. Osmanthus extract can be qualitatively analyzed by comparing its Rf value with standard samples, or semi quantitatively analyzed by the intensity of spots after color reaction.

-Operation steps:

-Thin layer board preparation or purchase: You can prepare your own silicone thin layer board, or purchase commercially available thin layer boards.

-Sample sampling: Dissolve the sample in an appropriate solvent and use a microsyringe or capillary to spot the sample on the starting line of the thin-layer plate.

-Unfolding and color development: Place the thin-layer plate into a developing cylinder containing a developing agent and unfold it. After the front edge of the developing agent rises to a certain distance, remove the thin-layer plate and air dry it. Then, appropriate color reagents (such as aluminum trichloride reagent) are used for color development, and the reaction between luteolin and aluminum trichloride will result in fluorescent spots under UV light. The spot position and intensity are observed under UV light.

-Advantages:

-The equipment is simple, the operation is fast, and the cost is low.

-Multiple samples can be preliminarily screened and separated simultaneously.

-Disadvantages:

-Inaccurate quantification, mainly used for qualitative or semi quantitative analysis.

-The separation effect is relatively poor, and it may not be possible to completely separate luteolin and other components from complex samples.