Liquid Chromatography

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Calculations: a. 1. W = Vend − Vstart
W = 1.87 mL – 0.92 mL
W = .95 mL

Wave = W1+W22

Wave = .95 mL+ .88 mL2

Wave = .915 mL

2. Vstartave = Vstart1+Vstart22

Vstartave = .92 mL+ .90mL2
Vstartave = .910 mL

VRave = Vstartave + Wave2

VRave = .910 mL + .915 mL2
VRave = 1.37 mL

3. k’ = Vrave-VmVm

k’ = 1.37 mL-.49 mL.49 mL

k’ = 1.79

4. α = k'bluek'red

α = 4.141.79

α = 2.31

5. R = (VRaveBlue-VRaveRed).5 (Wave(Blue)+Wave(Red))
R = (2.52 mL-1.37 mL).5 (2.04 mL+.915 mL)
R = .778

d. Table #1: Calculated Bandwidths of Runs

| Red Dye| Blue Dye|
| Run #1| Run #2| Run #1| Run #2|
Bandwidth (mL)| .95| .88| 2.15| 1.92|

Table #2: Calculated Average Bandwidths of Dyes

| Red Dye| Blue Dye|
Average Bandwidth (mL)| .915| 2.04|

Table #3: Calculated Average Vstarts of Dyes

| Red Dye| Blue Dye|
Average Vstarts (mL)| .910| 1.50|

Table #4: Calculated Average VRave of Dyes

| Red Dye| Blue Dye|
Average VRave (mL)| 1.37| 2.52|

Table #5: Calculated k’ of Dyes

| Red Dye| Blue Dye|
k'| 1.79| 4.14|

Table #6: Summative Calculation of Resolution and Selectivity of Dyes

α| 2.31|
R| .778|

Post-Lab Questions: 1. What is meant by polarity of molecules? What causes differences in polarity? The polarity of molecules is the slight charge associated with a covalent molecule due to the uneven sharing of electrons. Electrons, being the source of mobile charge within atoms, can be attracted to one of a pair of atoms within a bond, due to a greater of affinity of electrons by an element compared to another. With an excess of this negative charge carriers, one side of the molecule becomes more negative than the other, thus creating two distinct “poles”: a positive end and a negative one. Differences in polarity are due to the greater affinity for electrons by certain elements contrasted with others, such as oxygen over hydrogen.

2. In discussing solubility, the rule “like dissolves like” is frequently used. What does this mean?
This term means that:
* Solvent and solute have similar/compatible structures: causes solute to readily dissolve in the solvent * Example: Water (H2O) participates in hydrogen and so does methanol CH4O; thus, methanol readily dissolves when placed in water

3. Draw the structural formula of isopropyl alcohol. Explain how it differs in polarity from water.

Within the isopropyl alcohol is less polar:
* The molecule as a whole is symmetrical about its center: there is an even distribution of hydrogen atoms on the left and right of oxygen atom * Water, on the other hand, has uneven distribution: hydrogen all on side * Charges accumulate in water collectively whereas spread out in this * Also, since O-H bonds are polar, the more that are present, the more polar the molecule is * Isopropyl alcohol only has one O-H whereas water has two (more polar)

4. For good separation of the dyes, the resolution should be greater than one. What was the value you calculated? Did the two dyes overlap as they emerged from the column, or was the separation a good one? The resolution calculated was .778 (see calculations), which made it difficult to perceive where exactly one dye began and the other ended. Thus, when the dyes were emerging from the column, they mixed and formed a blend of the two colors to form a light purple color, which made it difficult to ascertain where the red ended and to discern where the blue section began. Thus, it was not a perfect separation as the dyes mixed when emerging.

5. In the step gradient, four separate fractions were collected. How were these related to the polarities of the column and the eluting solution? The four eluting solvents decreased in polarity from one run to the next, starting water and then isopropyl alcohol starting from 5%, to 28%, to 70%. The water...
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