my developing solvent is hexane toluene acetic acid(5:5:1)nd my samples rf nd impurity rf is very close,my spots r not separating properly,so which hexane should hav to be used?n hexane or hexane fraction?is only hexane affect or other factor is there?

To determine which hexane to use in your developing solvent, you need to understand the properties of the hexane you have, as well as consider other factors that may be affecting your spots' separation. Here's how you can approach this issue:

1. Consider the purity of your hexane samples: Hexane can come in different purities, such as reagent grade or analytical grade. It is crucial to ensure that your hexane is of high purity, as impurities could affect the separation of your spots. Check the specifications or consult the supplier to determine the purity of your hexane.

2. Evaluate the hexane fraction: If you have a hexane fraction, it might indicate that the hexane sample contains impurities or components other than hexane. In such cases, it's important to assess the impurities present in your hexane fraction and consider whether they could be contributing to the poor separation of spots.

3. Analyze the solubility of your compound and impurities: Assess the solubility of your compound and impurities in both hexane and toluene. Different components may have different solubilities in these solvents, which could lead to incomplete separation. If your compound is more soluble in toluene than hexane, it could result in similar RF values for your compound and impurity spots.

4. Consider the polarity of your mixture: Acetic acid is a polar solvent, while hexane and toluene are nonpolar. The presence of acetic acid in the solvent mixture can affect the overall polarity. Depending on the polarity requirements of your samples, you may need to adjust the ratios of the solvents or consider using a different solvent system altogether.

5. Optimize the solvent system: If your spots are not separating properly, you can try modifying the ratios of hexane, toluene, and acetic acid in your developing solvent system. Making slight adjustments to the ratios may help in achieving better separation.

6. Test different combinations: Perform some test runs using different hexane samples, adjusting the solvent ratios, and evaluating the spot separation. Keep track of the changes you make and note any improvements.

Remember, it's not only the choice of hexane that can affect your spot separation, but also the overall solvent system, including the other components and their ratios. By carefully considering these factors and making adjustments, you can optimize your developing solvent for better spot separation.