Polyphenols in Tobacco Samples By HPLC Column

The determination of 10 plant polyphenols in tobacco samples by solid phase extraction and high-performance liquid chromatography as below:

Plant polyphenols are important substances in tobacco. The content of polyphenols in tobacco has an important influence on tobacco quality. Therefore, it is of great significance to study the plant polyphenols in tobacco and its products. The determination methods of tobacco polyphenols mainly include spectrophotometry, gas chromatography, and liquid chromatography. The photometric method has poor accuracy and can only approximately determine the total amount.

Gas chromatography requires complex derivatization; The traditional liquid chromatography requires the sample to be decreased with the organic solvent, which is difficult to operate and will cause polyphenol loss. In recent years, solid phase extraction (SPE) has been widely used in analytical chemistry.

The SPE Cartridge C18 was used for pre-separation and degreasing, and HPLC Column(3. 9 mm x 150 mm) was used for separation and determination of polyphenols in tobacco. The main polyphenols were characterized by UV spectrogram. The method can simultaneously determine 10 kinds of polyphenols in tobacco and be used for tobacco sample analysis.

Analyzing polyphenols in tobacco samples using High-Performance Liquid Chromatography (HPLC) requires a specialized column that can separate and quantify these compounds effectively. Here’s a guide to selecting an appropriate HPLC column for polyphenol analysis in tobacco:

Column Selection:

  1. Phase Type:
    • Reversed-Phase (RP): Most common for polyphenol analysis. C18 columns are a popular choice due to their compatibility with a wide range of solvents and sample types.
  2. Particle Size:
    • Smaller particle sizes (1.8 µm or smaller) offer higher resolution but may require higher backpressure. For routine analysis, 3-5 µm particles are often sufficient.
  3. Column Length:
    • Longer columns provide better resolution but may increase analysis time. A common length for HPLC columns is 150 mm.
  4. Column Diameter:
    • 4.6 mm ID columns are standard, but narrower columns (2.1 mm ID) may offer higher sensitivity.
  5. End-Capping:
    • Consider a column with end-capping to reduce unwanted secondary interactions, especially if analyzing acidic or basic compounds.
  6. Column Brand and Model:
    • Reputable brands like Agilent, Waters, Phenomenex, and Thermo Fisher Scientific offer high-quality HPLC columns suitable for polyphenol analysis.

Mobile Phase:

  1. Composition:
    • A mixture of water and organic solvent (e.g., acetonitrile or methanol) is typically used. The ratio can be adjusted to optimize separation.
  2. Additives:
    • Formic acid or acetic acid may be added to improve peak shape and enhance separation.

Sample Preparation:

  1. Extraction:
    • Polyphenols are extracted from tobacco samples using an appropriate solvent (e.g., methanol, ethanol, water).
  2. Filtration:
    • Use a suitable filter (e.g., syringe filter) to remove particulates before injection.

Chromatographic Conditions:

  1. Gradient Elution:
    • A gradient of solvents (e.g., water and acetonitrile) is often used to achieve optimal separation.
  2. Column Temperature:
    • Maintaining a stable column temperature (e.g., 25-40°C) can help improve reproducibility.


  1. UV-Vis Detector:
    • Polyphenols are commonly detected at 280 nm, but other wavelengths may be used based on the specific compounds being analyzed.
  2. Diode Array Detector (DAD):
    • Offers the advantage of spectral information for peak identification.

Data Analysis:

  1. Calibration Curve:
    • Quantify polyphenols using standard solutions of known concentrations and construct a calibration curve.
  2. Integration and Identification:
    • Integrate the chromatographic peaks for quantification. Compare retention times and UV spectra with standards for identification.

Remember to follow safety protocols and regulatory guidelines when handling tobacco samples. Additionally, consult with your lab’s chromatography expert for specific recommendations based on your equipment and samples.