Sephadex LH-20

Sephadex LH-20 is a size exclusion chromatography resin used for separating and purifying natural products like steroids, terpenoids, lipids, and low molecular weight peptides. It's a beaded, cross-linked dextran that has been hydroxypropylated to be both hydrophilic and lipophilic, allowing it to be used in a variety of organic solvents. Depending on the solvent, it can separate based on molecular size or by partition between the stationary and mobile phases. 

 

Key Features and Uses:

• Molecular Sizing:

  Primarily used for separating molecules based on size, particularly for natural products. 

   
• Dual Nature:

  The hydroxypropylation provides both hydrophilic and lipophilic character, allowing it to be used with a wide range of solvents. 

   
• Partition Chromatography:

  Can also be used for partition chromatography, separating compounds based on their solubility in the stationary and mobile phases. 

   
• Applications:

  Suitable for separating steroids, terpenoids, lipids, low molecular weight peptides, and even some proteins. 

   
• Preparative and Analytical Scales:

  Used for both small-scale analytical separations and larger-scale preparative purifications. 

   
• Solvent Compatibility:

  Works well with many organic solvents, making it versatile for different separation needs. 

   
• Column Packing:

  Requires careful column packing to prevent compression, especially when using organic solvents. 

   

How it Works:

Sephadex LH-20 is a type of gel filtration medium. The beads are porous, and molecules smaller than the pore size can enter the beads and take a longer path through the column, while larger molecules elute faster. 

 

Column Packing and Operation:

• Careful Packing:

  Sephadex LH-20 columns are susceptible to compression, so careful packing with slow flow rates is essential to prevent issues. 

   
• Adapter Placement:

  When packing, ensure no air bubbles are trapped under the adapter and that the adapter is properly sealed to the column wall. 

   
• Solvent Choice:

  The choice of solvent will affect the swelling of the resin and the separation mechanism (size exclusion or partition). 

   
• Flow Rate:

  Use low flow rates during packing and separation to prevent column compression. 

  Sephadex LH-20 resin is used for preparative purification of natural products such as steroids, terpenoids, lipids, and low molecular weight peptides.

  Broad stability in most aqueous mixtures and polar organic solvents. Powerful chromatographic selectivity from the matrix’s dual hydrophilic/lipophilic nature. High chemical and physical robustness. High reproducibility from batch to batch. Sephadex LH-20 is a resin specifically designed for molecular sizing of natural products in organic solvents.

  Sephadex LH-20 resin for organic solvent use

  This chromatography resin is made from cross-linked Sephadex G-25 dextran beads, hydroxypropylated to yield both hydrophilic and lipophilic characteristics.
  
  

  This resin is useful for molecular species preparation at different scales. It can be used during purification, prior to high performance ion exchange or reversed phase chromatography (RPC), or as a final polishing step during diastereomer preparation.
  
  

  Successful use in published research

  Applications of Sephadex LH-20 resin include lipid fractionation and purifying complex organic substances, such as traditional Chinese medicine (TCM), for research use. Examples of successful research use include:
  
  

  • Antioxidant activity in the stem bark of acacia confusa
  • Free radical scavenging activity in floral corn pollen and pollen found in apis mellifera hives
  • Antioxidant activity in pinus densiflora needles

  • Table 6.4. Approximate values for packed bed volumes of Sephadex® LH-20 swollen in different solvents* Containing 1% ethanol. † Containing 1% benzene. ‡ Solvents that give a bed volume of less than 2.5 mg/ml dry Sephadex® LH-20 are not generally useful.

    Solvent	Approx. bed volume (ml/g dry Sephadex® LH-20)
    Dimethyl sulfoxide	4.4 to 4.6
    Pyridine	4.2 to 4.4
    Water	4.0 to 4.4
    Dimethylformamide	4.0 to 4.4
    Methanol	3.9 to 4.3
    Saline	3.8 to 4.2
    Ethylene dichloride	3.8 to 4.1
    Chloroform*	3.8 to 4.1
    Propanol	3.7 to 4.0
    Ethanol†	3.6 to 3.9
    Isobutanol	3.6 to 3.9
    Formamide	3.6 to 3.9
    Methylene dichloride	3.6 to 3.9
    Butanol	3.5 to 3.8
    Isopropanol	3.3 to 3.6
    Tetrahydrofuran	3.3 to 3.6
    Dioxane	3.2 to 3.5
    Acetone	2.4 to 2.6
    Acetonitrile‡	2.2 to 2.4
    Carbon tetrachloride‡	1.8 to 2.2
    Benzene‡	1.6 to 2.0
    Ethyl acetate‡	1.6 to 1.8
    Toluene‡	1.5 to 1.6