Figure 7. Isocratic Elution w/ 40% H2O/60% methanol at 270 nm The peak identities were determined using the polarity of the molecules and their interactions with the silica beads in the channel. The silica beads were used as the stationary phase of the HPLC while a mixture of methanol and water was used for the mobile phase. Since the mobile phase mixture of methanol and water is polar and the silica beads are non-polar, the most polar molecule, which was determined to be m-cresol, spent the least amount time in the stationary phase and therefore was the first molecule pulled from the column first, which is what caused the first peak. The second peak was caused by the second most polar molecule, which was determined to be methylbenzoate, since methylbenzoate was the second most polar molecule it had less …show more content…
The third and final peak was created by p-methylanisole, which was less polar than the other two molecules and therefore spent the most time in the stationary phase before being eluted after both m-cresol and methylbenzoate. Changing the elution conditions, in this case the ratio between water and methanol, does impact the chromatogram; a 40/60 water/methanol is more polar than a 30/70 water/methanol elution, therefore in a 40/60 water/methanol elution the mixture takes much longer to be completely eluded from the column. However the most polar molecule will be eluded at around the same time for both 30/70 and 40/60 mixtures, the differences in peak length and retention time didn’t start until the second most polar molecule since the 40/60 mixture is more polar the second and third most polar molecules will spend more time in the stationary phase since the molecules will have a stronger affinity to the non-polar silica beads than the more polar solution. In a practical lab application it would be best to use a 30/70 water/methanol elution since, as shown in figure 4 and figure 7, it took almost