You might try amine columns running as HILIC, dissolving the sample in DMSO:

https://www.teledynelabs.com/en-us/resources/Documents/Application-Notes/Purification-of-simple-carbohydrates-with-flash-chromatography.pdf

This reference: https://www.sciencedirect.com/science/article/abs/pii/S0003269712001029

used Graphitized carbon chromatography

To isolate oligosaccharide compounds from the crude ethanol extract of medicinal plants using column chromatography, here’s a detailed protocol that addresses your specific points of interest:

1. Suitable Stationery and Mobile Phases

• Stationary Phase:
  • Sephadex G-25 or G-50: These are commonly used for gel filtration chromatography to separate oligosaccharides based on size. They allow for the effective separation of smaller oligosaccharides from larger molecules like proteins and polysaccharides.
  • Silica Gel: Silica gel can be used for normal phase chromatography, especially when separating oligosaccharides based on polarity.
• Mobile Phase:
  • Water and Acetonitrile: A gradient of water and acetonitrile can be effective for normal phase chromatography. Start with a high percentage of water and gradually increase acetonitrile to elute oligosaccharides based on their polarity.
  • Buffer Solutions: For ion-exchange chromatography, buffers like phosphate or acetate can maintain pH and ionic strength, crucial for separating charged oligosaccharides.

2. TLC Visualization Techniques

• Orcinol-Sulfuric Acid: This reagent is effective for visualizing oligosaccharides on TLC plates. After developing the plate, spraying it with orcinol-sulfuric acid, and heating it, will produce colored spots corresponding to oligosaccharides.
• Aniline-diphenylamine: This reagent can also be used to detect oligosaccharides. It provides a different colorimetric response and can help differentiate between oligosaccharide structures.

3. Guidance on Gradient Selection for Column Chromatography

• Gradient Elution: Start with a low organic solvent concentration (e.g., acetonitrile) and gradually increase it. For example, you might begin with 10% acetonitrile and rise to 50% over a series of fractions. This allows for the gradual elution of oligosaccharides based on their hydrophobicity.
• Monitoring: Collect fractions during the gradient and monitor them using TLC to determine when oligosaccharides elute.

4. Tips for Identifying and Pooling Fractions Post-TLC

• TLC Analysis: Visualize the spots using the chosen reagent after running TLC. Compare the Rf values of the spots to identify which fractions contain oligosaccharides.
• Pooling Fractions: Combine fractions with similar Rf values that are visually identical on the TLC plate. This helps ensure you pool fractions that likely contain the same oligosaccharide species.
• Further Analysis: After pooling, consider using methods like HPLC or mass spectrometry to further characterize the pooled fractions and confirm the presence of specific oligosaccharides.

Mehrab Dashtdar

"Mobile Phase:

• Water and Acetonitrile: A gradient of water and acetonitrile can be effective for normal phase chromatography. Start with a high percentage of water and gradually increase acetonitrile to elute oligosaccharides based on their polarity."

Are you sure about this? Silica is normal phase, so the gradient should go from non-polar (acetonitrile) to polar (water). This gradient is used for HILIC purifications (see the amine column link in the earlier post). That comment seems to directly contradict your later comment:

"3. Guidance on Gradient Selection for Column Chromatography

• Gradient Elution: Start with a low organic solvent concentration (e.g., acetonitrile) and gradually increase it. For example, you might begin with 10% acetonitrile and rise to 50% over a series of fractions. This allows for the gradual elution of oligosaccharides based on their hydrophobicity."

Jack Silver:

In normal phase chromatography, such as silica, the gradient should start with a less polar solvent, like acetonitrile, and gradually increase the polar solvent, typically water.

In the case of Hydrophilic Interaction Liquid Chromatography (HILIC), the opposite gradient is used: starting with a high percentage of water and gradually increasing acetonitrile to elute more polar compounds.

Thank you for pointing out the contradiction! Your understanding aligns with the principles of chromatography.

Mehrab Dashtdar HILIC is a normal phase technique and so starts with a less polar solvent (such as acetonitrile) and increases to a more polar solvent (more water). The organic solvent is typically spiked with a little water.

See Figure 3 here: https://www.teledynelabs.com/en-us/resources/Documents/Application-Notes/RediSep-Gold-Silica-and-Highly-Polar-Solvents.pdf

Also see here: https://www.agilent.com/cs/library/technicaloverviews/public/5991-9271EN_HILIC_method_development_TechOverview.pdf

The gradient described in your post is good for reverse phase.

Thank you, Jack Silver and Mehrab Dashtdar , for your valuable insights and detailed responses.

@Mehrab – your breakdown of the protocol is very helpful, especially the use of Sephadex for size-based separation and the visualization methods like orcinol-sulfuric acid and aniline-diphenylamine. These TLC techniques are exactly what I was looking for to confirm oligosaccharide presence in fractions.

@Jack – thank you for pointing out the important clarification regarding the solvent gradient in normal phase chromatography. Your explanation on HILIC using amine columns and the references shared (Teledyne and Agilent) are highly useful. I now understand that in HILIC, the mobile phase starts with high acetonitrile and increases in water content to elute polar compounds like oligosaccharides.

I'm now considering testing both silica-based normal phase and HILIC-based amine columns to see which suits my oligosaccharide profile better. I’ll also explore the graphitized carbon approach for finer separation, especially if I face challenges with isomeric separation.

I truly appreciate both your contributions they’ve greatly enhanced my approach and understanding of carbohydrate chromatography!