Chromatography Assignment1) Normal phase chromatography uses a polar stationary phase and a non-polar, or sometimes slightly polar, mobile phase. 2) phenol, acetophenone, naphthalene3) The order of the peaks does not change when the mobile phase composition of B is set to 50%. The chromatogram shifts to the left and the signal increases, as the nonpolar, stationary phase has a higher concentration. The same happens for the shift 70% B. k, tr and σ decreased for all three of the molecules. This is likely due to the changing affinity for the mobile phase, as acetonitrile is less polar than water, so a greater percentage would make the entire mobile phase less polar. When acetonitrile is set to 5%, the chromatogram is shifted to the right, as the retention factor increases. The elution order remains the same, but the retention times and k values increased for each compound. At 5% acetonitrile, the mobile phase is very polar, so it does not dissolve the non-polar compound effectively. At 95% acetonitrile, the graph only shows two peaks, rather than three. The acetophenone and phenol have combined, because their k values are practically identical (0.1102 and 0.1199, respectively). The retention times of each molecule also decreased substantially. Increasing acetonitrile concentrations helps elute polar substances and improve the separation and retention times of each molecule. It is practically beneficial, as well, as a higher percentage of acetonitrile makes the mobile phase less polar, which decreases the length of the trials. Increasing the water concentration allows the substances to stay in the stationary phase longer. 4) The chromatogram shifts to the right, as each compound shows an increase in k and retention time. As methanol is more polar than acetonitrile, there is relatively more interaction of the compounds with the stationary phase, due to an increase in relative affinity compared to the mobile phase.5) The total retention time, k value, and tr for the gradient elution is significantly less than that of the 5% acetonitrile isocratic elution. The variance for each compound also decreased drastically, suggesting that the results are more precise.In the gradient elution, there were three distinct peaks compared to only two in the 95% acetonitrile isocratic elution. The tR and k values were greater for the gradient elution, but not by a very significant margin.The benefit of using gradient elution is that the peaks come out more defined and with better resolution and detectability. It allows the advantage of the favorable interactions at both high andlow polarities within one trial. 6) The benefit to a long column in chromatography is that as the length increases, the interaction time between the eluting compound and the stationary phase increases, increasing the column’s efficiency and resolution, resulting in well-separated peaks. The advantage of a shorter column isthat the time for separations is reduced because there is a shorter distance for the compounds to travel. 7) Based on my observations, smaller particle sizes create better efficiencies and less peak dispersion, or narrower peaks, suggesting less variance associated with a higher level of precision. 8)Compound Retention Time (min) Variance (σ, sec) W1/2 (min)1. Phenol 1.2946 0.6162 0.02412. acetophenone 2.4553 1.1554 0.04533. naphthalene 12.2052 5.7187 0.22409)Resolution=0.589 ∆trw12 ave∆ tr=2.4553−1.2946=1.1607w1/ 2 ave=0.0241+0.04532=0.0347resolution=0.589× 1.16070.0347= 19.70510)N =5.55 tr2w1/22H =LNCompound Number of Theoretical Plates (N)Theoretical Plate Height (mm)phenol 15969.4 0.00626acetophenone 16338.2 0.00612naphthalene 16479.9