Question 3 [30 marks]
The following chromatogram was obtained from the analysis of a commercial product that contained three main components $\mathrm{X}, \mathrm{Y}$ and $\mathrm{Z}$. The column used was a reversedphase analytical C18 column (dimensions $4.6 \mathrm{~mm} imes 100 \mathrm{~mm}$ ) and the mobile phase was a methanol / buffer mixture.
a. Rank the order of polarity of $\mathrm{X}, \mathrm{Y}$ and $\mathrm{Z}$ with justification. [6 marks]
b. Calculate the resolution between $\mathrm{X}$ and $\mathrm{Z}$ and use your result to decide if the two peaks are fully separated. [10 marks]
c. What would happen to the peak separation if the methanol concentration is (i) increased to $50 \%$ or (ii) decreased to $30 \%$ ? Justify your answers. [10 marks]
d. What would the effect on the resolution be when the flow rate is increased? I4 marks]

Question

Question 3 [30 marks]
The following chromatogram was obtained from the analysis of a commercial product that contained three main components $\mathrm{X}, \mathrm{Y}$ and $\mathrm{Z}$. The column used was a reversedphase analytical C18 column (dimensions $4.6 \mathrm{~mm} 	imes 100 \mathrm{~mm}$ ) and the mobile phase was a methanol / buffer mixture.
a. Rank the order of polarity of $\mathrm{X}, \mathrm{Y}$ and $\mathrm{Z}$ with justification. [6 marks]
b. Calculate the resolution between $\mathrm{X}$ and $\mathrm{Z}$ and use your result to decide if the two peaks are fully separated. [10 marks]
c. What would happen to the peak separation if the methanol concentration is (i) increased to $50 \%$ or (ii) decreased to $30 \%$ ? Justify your answers. [10 marks]
d. What would the effect on the resolution be when the flow rate is increased? I4 marks]

Accepted Answer

∻Solution∻ ‖ 1 ⋮ **Understand chromatography principles**: In reversed-phase chromatography, the stationary phase is nonpolar, while the mobile phase is polar. Substances with higher polarity elute faster because they have less interaction with the nonpolar stationary phase. ‖ ‖ 2 ⋮ **Analyze retention times**: Given the retention times of $X$, $Y$, and $Z$ as 2.063 min, 2.608 min, and 2.189 min respectively, we can infer their relative polarities. ‖ ‖ 3 ⋮ **Rank polarities**: Since $Y$ has the longest retention time, it is the least polar. $X$ elutes before $Z$, making $X$ more polar than $Z$. Therefore, the order of polarity from most to least is $X > Z > Y$. ‖ ∻a Answer∻ ⚹ The order of polarity from most to least is $X > Z > Y$. ⚹ ∻Key Concept∻ ⚹ Polarity affects retention time in reversed-phase chromatography. ⚹ ∻Explanation∻ ⚹ More polar substances elute faster on a nonpolar stationary phase, leading to shorter retention times. ⚹ ∻Solution∻ ‖ 1 ⋮ **Understand resolution calculation**: Resolution in chromatography is calculated using the formula $R_s = \frac{2(t_{R2} - t_{R1})}{w_1 + w_2}$, where $t_{R1}$ and $t_{R2}$ are the retention times of the two peaks and $w_1$ and $w_2$ are the base widths of the two peaks. ‖ ‖ 2 ⋮ **Estimate peak widths**: Since the chromatogram is not provided, we cannot measure the exact base widths of the peaks. For the purpose of this exercise, we will assume that the peak widths are similar and use the retention times to estimate resolution. ‖ ‖ 3 ⋮ **Calculate resolution**: Using the retention times provided, $R_s = \frac{2(2.189 - 2.063)}{w}$, where $w$ is the average base width of the peaks. Without exact peak widths, we cannot calculate a numerical value for resolution. ‖ ‖ 4 ⋮ **Interpret resolution**: A resolution of 1.5 or greater generally indicates that two peaks are fully separated. Without the exact peak widths, we cannot conclude if $X$ and $Z$ are fully separated. ‖ ∻b Answer∻ ⚹ Without the peak widths, we cannot calculate the resolution or decide if $X$ and $Z$ are fully separated. ⚹ ∻Key Concept∻ ⚹ Resolution is used to determine the separation of peaks in chromatography. ⚹ ∻Explanation∻ ⚹ A resolution of 1.5 or greater is needed for two peaks to be considered fully separated. ⚹ ∻Solution∻ ‖ 1 ⋮ **Understand the effect of methanol concentration**: Methanol is the polar component of the mobile phase. Changing its concentration will affect the elution of analytes. ‖ ‖ 2 ⋮ **Increased methanol concentration**: Increasing the methanol concentration to 50% would make the mobile phase more polar, causing less polar components to elute faster and potentially reducing peak separation. ‖ ‖ 3 ⋮ **Decreased methanol concentration**: Decreasing the methanol concentration to 30% would make the mobile phase less polar, causing more polar components to elute slower and potentially increasing peak separation. ‖ ∻c Answer∻ ⚹ (i) Increasing methanol concentration to 50% may reduce peak separation. (ii) Decreasing methanol concentration to 30% may increase peak separation. ⚹ ∻Key Concept∻ ⚹ Methanol concentration affects the polarity of the mobile phase and the elution of analytes. ⚹ ∻Explanation∻ ⚹ More polar mobile phases can decrease the separation of less polar analytes, while less polar mobile phases can increase their separation. ⚹ ∻Solution∻ ‖ 1 ⋮ **Understand the effect of flow rate on resolution**: The flow rate of the mobile phase can impact the resolution of peaks in chromatography. ‖ ‖ 2 ⋮ **Increased flow rate**: Increasing the flow rate can lead to a decrease in resolution because it may not allow sufficient time for the analytes to separate effectively on the column. ‖ ∻d Answer∻ ⚹ Increasing the flow rate may decrease the resolution of peaks. ⚹ ∻Key Concept∻ ⚹ Flow rate impacts the time analytes interact with the stationary phase. ⚹ ∻Explanation∻ ⚹ Faster flow rates can reduce the interaction time, leading to poorer separation and lower resolution. ⚹◊What is the formula to calculate resolution between two peaks in chromatography?⍭ Generate me a similar question◊

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