Preparing a sample for paper chromatography primarily involves dissolving the sample in an appropriate solvent and then carefully applying it to the chromatography paper.
The Core Principle of Sample Preparation
Effective sample preparation is fundamental to successful paper chromatography. The primary step, as highlighted in the provided reference, is the dissolution of the sample in a suitable solvent. This solvent must be:
- Inert with the sample under analysis: Meaning it should not react chemically with the components of your sample.
- Used in making the mobile phase: This implies compatibility with the chromatographic system, ensuring the sample components can interact effectively with both the stationary and mobile phases. Ideally, the solvent should be volatile enough to evaporate quickly after spotting.
Step-by-Step Sample Preparation Process
The process can be broken down into distinct stages to ensure optimal separation results.
Choosing the Right Solvent
Selecting the appropriate solvent for dissolving your sample is crucial.
- Solubility: The sample must be fully soluble in the chosen solvent.
- Volatility: A solvent that evaporates quickly is preferred, as it helps create a concentrated spot on the paper without excessive spreading.
- Inertness: As mentioned, the solvent must not chemically interact with or alter the sample components.
- Compatibility: The solvent should be compatible with the mobile phase solvent system used in the chromatography run.
Dissolving and Concentrating the Sample
Once the solvent is chosen, proceed with dissolving the sample.
- Dissolution: Add a small amount of the chosen solvent to your sample and stir or shake gently to ensure complete dissolution.
- Concentration: It's important to prepare a relatively concentrated sample solution. If the sample is too dilute, the separated components might not be visible or detectable after the chromatography run. You might need to evaporate some solvent to achieve the desired concentration.
- Filtration (Optional but Recommended): If your sample contains any insoluble particles, it's advisable to filter the solution to prevent them from interfering with the chromatographic separation or clogging the paper pores.
Spotting the Sample on the Chromatography Paper
After preparing the solution, the next critical step is applying it to the paper. The reference clearly states: "Spot the sample on the paper: Samples should be spotted at a proper position on the paper by using a capillary tube."
- Baseline Marking: Draw a light pencil line (the "baseline" or "origin line") about 1-2 cm from one end of the chromatography paper. This is the proper position for spotting.
- Using a Capillary Tube: Dip a fine capillary tube into your prepared sample solution. The solution will draw up into the tube by capillary action.
- Spotting: Gently touch the tip of the capillary tube onto the marked baseline on the paper. The solution will transfer from the tube to the paper, forming a small, discrete spot.
- Small Spot: Aim for the smallest possible spot size (ideally 1-2 mm in diameter). A large spot will lead to broader, less resolved bands or spots during separation.
- Concentrated Spot: For a more concentrated spot, allow the first application to dry completely, then apply more sample to the exact same spot. Repeat this process until sufficient sample is applied.
- Drying: Ensure the spot is completely dry before placing the paper into the chromatography chamber. Residual solvent can interfere with the mobile phase front and affect separation quality. A hairdryer or air blower can be used for faster drying, ensuring no excessive heat is applied that could degrade the sample.
Key Considerations for Effective Sample Preparation
- Sample Amount: Do not overload the paper. Applying too much sample can lead to streaking and poor separation.
- Spot Size and Concentration: As mentioned, a small, concentrated spot is essential for sharp, well-separated bands.
- Purity of Reagents: Use high-purity solvents and samples to avoid contamination that could introduce spurious spots.
- Handling the Paper: Always handle the chromatography paper by its edges to avoid transferring oils or contaminants from your skin, which can affect the results.
- Multiple Samples: If running multiple samples on the same paper, ensure adequate spacing between spots to prevent overlap during separation.
