1. Look at Figure 2 and discuss with your group how changes in retention time an
ID: 1053977 • Letter: 1
Question
1. Look at Figure 2 and discuss with your group how changes in retention time and the broadness of peaks A and B would in uence Rs. Write a summary of your group consensus below.
2. Resolution can be computed by dividing the difference in retention times between the two peaks by their average width along the baseline. If a value of Rs=1.50 represents baseline resolution between two peaks in time units, will the Rs value for the separation shown in Figure 2 be greater than or less than 1.50? Brie y explain your answer, and then verify it with a quick calculation by estimating these values.
3. Based on your understanding of resolution, speculate as to how a chemist might improve the resolution of two chromatographic peaks.
Explanation / Answer
Solution:
1. The resolution (Rs) in chromatographic separation is a measure of how the two components in a mixture can be well separated in a chromatographic column. A high Rs value indicates a well separation between the components from a sample mixture. Resolution (Rs) has a direct relation between retention time(t) and the broadness of solute peaks (Here, A and B). As the resolution increases, the difference between the retention times for two the different solute also increases. Resolution has a direct with the retention factor (k), as resolution increased the retention factor also increased. Here in this above chromatogram the third peak corresponds to the highest retention factor with highest retention time and therefore broad signal peak.
2. Here in this above chromatogram, let the second peak corresponds to solute A (with base line thickness of the peak= x) and the third broad peak corresponds to the solute B (with baseline thickness of the peak= y). Therefore, the resolution between the two peaks, Rs would be;
Rs = difference in retention times/average width along the baseline
Or, Rs = (14-8)/(x+y/2) = 12/(x+y) ----------------(1)
For Rs = 1.5, we have: x+y = 12/1.5 = 8
Therefore, it can be said that for a resolution of 1.5 the average base line thickness of the two peaks must be = 8 min. Here in the above chromatogram the average base line thickness = (2+4)/2 = 3 min. Therefore, Rs for the chromatogram = 12/3 = 4. Therefore, the resolution for the above chromatogram (Rs=4) is much higher than Rs=1.5.
3. The Fundamental Resolution Equation (2) indicates that resolution is affected by three important parameters: a) Selectivity (); b) Efficiency (N); c) Retention factor (k).
Rs = 1/4N x (-1)/ x k/(1+k) ----------(2)
Therefore, each of these factors contribute to the resolution of the separation.
The retention factor (k) is a measure of the retention of an analyte on the chromatographic column. A high k value indicates that the sample is highly retained and has spent a significant amount of time interacting with the stationary phase and thus increase in resolution. Altering the retention factor is the easiest way to increase the resolution. The most effective and convenient way to alter the retention factor of a peak is to adjust the ‘solvent strength’ of the chromatographic mobile phase. This is usually achieved in reversed phase chromatography by changing the amount of organic solvent in the mobile phase mixture. For example, reversed phase HPLC has a non-polar stationary phase, therefore, increasing the polarity of the mobile phase will increasingly repel the nonpolar sections of the analyte molecules into the stationary phase and the analyte will be retained for longer on the column.
The selectivity factor () is the ability of the chromatographic system to chemically distinguish between sample components. It is usually measured as a ratio of the retention factors (k) of the two peaks in question. A high values indicate good separating power and a good separation between the apex of each peak. As the selectivity of a separation is dependent upon the chemistry of the analyte, mobile, and stationary phases, all of these factors may be altered in order to change the selectivity of a chromatography column separation.
Related Questions
Navigate
Integrity-first tutoring: explanations and feedback only — we do not complete graded work. Learn more.