Liquid Chromatography Test Bank Docx Ch.15 Revised Edition

Chapter 15

Problem 15.1: Albuterol (a drug used to fight asthma) has a lipid:water K_OW (see “Profile—Other Applications of Partition Coefficients”) value of 0.019. How many grams of albuterol would remain in the aqueous phase if 0.001 grams of albuterol initially in a 100 mL aqueous solution were allowed to come into equilibrium with 100 mL of octanol?

Problem 15.2:

(a) Repeat Problem 15.1, but instead of extracting the albuterol with 100 mL of octanol, extract the albuterol solution five successive times with only 20 mL of octanol per extraction. After each step, use the albuterol remaining in the aqueous phase from the previous step as the starting quantity for the next step. (Note: the total volume of octanol is the same in both extractions).

(b) Compare the final concentration of aqueous albuterol to that obtained in Problem 15.1. Discuss how the extraction results changed by conducting five smaller extractions instead of one large one. What is the downside to the multiple smaller volume method in this exercise compared to the single larger volume method in Problem 15.1?

Problem 15.3: Albuterol has a lipid:water K_OW value of 0.019. If you were to con­duct a chromatographic separation using a polar solvent (i.e., methanol) and a nonpolar stationary phase, would the albuterol’s distribution favor the mobile phase or the sta­tionary phase? Explain your answer.

Problem 15.4: Caffeine has a lipid:water K_OW value of 0.79. If a mixture of alb­uterol and caffeine were to be separated using a polar solvent (i.e., methanol) and a nonpolar stationary phase, which component would elute first (having the shorter retention time)? Would the two components elute close to each other—or far apart? Explain.

Problem 15.5: Sometimes it is desirable to determine the retention (R) of an analyte as the difference between the observed retention time (t_r) and the void time (t_m). Using the chromatogram in Figure 15.4, determine the retention (R) of compounds A and B.

Problem 15.6: Sometimes you will see chromatographic peaks identified by the volume of mobile phase that was required to elute the sample instead of the time it took for the sample to elute. Using the retention times seen in Figure 15.4, calculate the re­tention volumes of compounds A and B assuming a flow rate of 1 mL/min. Repeat this exercise assuming a flow rate of 0.7 mL/min.

Problem 15.7: Determine the retention factors (k_r values) for analytes A and B seen in Figure 15.4.

Problem 15.8: Calculate the retention factor for each peak in the chromatograph seen in Example 15.3 and create a results table that might be used in a report.

Problem 15.9: Use the chromatogram from Example 15.3 to determine the reten­tion times and retention volumes of each of the peaks in this sample and create a results table that might be included in a report.

Problem 15.10: Use the chromatograms in panels (B) and (C) from Example 15.4 and estimate the resolution of the two-component system under each of these conditions. What challenges do you face for estimating σ for panel (B)? In terms of the value of the separation, discuss the significance of the resolution in panels (A), (B), and (C) with respect to the speed of the analysis (throughput), quantitative analysis, and qualitative analysis.

Problem 15.11: In a chromatographic experiment, two components elute with retention times of 11.52 and 12.76 minutes, respectively. The half-height widths of the peaks were determined to be 0.37 and 0.42 minutes, respectively. Assuming Gaussian peak shapes, estimate the resolution between the two peaks.

Problem 15.12: Use Equation 15.15 to determine N_t for each peak in the chromato­gram found in Example 15.4, panel (C).

Problem 15.13: Use Equation 15.16 to determine N_t for each peak in the chromato­gram found in Example 15.4, panel (A).

Problem 15.14: Relate the multistep extraction process described in Problem 15.2 to the concept of theoretical plates.

Problem 15.15: List at least three undesirable effects of band broadening.

Problem 15.16: Make a summary table, perhaps the size of an index card, that iden­tifies the equations that define K_D, R_s, N_t, k_r, and a. Be sure to define the underlying terms (like t_m, W_b, H, and s) that are used in these equations.

Problem 15.17: In the analysis of some analgesic preparations using a polar sta­tionary phase and a mobile phase composed of 10% water and 90% methanol, four compounds were seen to elute in the following order: caffeine, acetaminophen, aspirin, ibuprofen. The last two compounds were widely separated by several minutes. How might you decrease the time required for the experiment while maintaining adequate resolution?

Problem 15.18: If a nonpolar stationary phase were used in the above experiment, what might be the elution order?

Problem 15.19: When using a nonpolar stationary phase with 10% water and 90% methanol, the first two components co-elute in a single peak. How might you change the conditions of the experiment to resolve those components?

Problem 15.20: You have conducted an LC analysis of three different headache medicines. The column was a C₁₈, the mobile phase was 50:50 methanol:water, and the detector was a UV-vis spectrometer (output measured in AU or absorbance units). The detector response for four different standards and for the three samples is presented here. Construct a calibration curve, perform a linear regression, and determine the concentration of acetaminophen in each sample.

Problem 15.21: Assuming a flow rate of 1 mL/min, determine the retention volume of peak #6 from the profile “Analysis of Wine: Qualitative and Quantitative”.

Problem 15.22: Describe how a gradient method could be used to shorten the run time for the chromatograms seen in the profile “Analysis of Wine: Qualitative and Quantitative”. Be specific! Where in the chromatogram would you change the mobile phase and how would you change it?

Problem 15.23: An aqueous sample contained traces of the compounds listed in Table 15.1. The octanol–water distribution coefficients are tabulated as pK_D values. Assume you analyzed an aliquot of the aqueous sample using high-performance LC with a mobile phase that was 75% methanol and 25% water on a cellulose column. Predict the elution order and explain your answer.

Problem 15.24: Based solely on molecular polarity, predict the most likely order of elution for each set of three compounds in a normal phase chromatographic experiment.

Problem 15.25: Assume that an aqueous sample contains equal trace amounts of the compounds listed in Table 15.1. If you extract the aqueous sample with an aliquot of octanol:

(a) List the compounds in order of increasing concentration in the octanol phase at equilibrium.

(b) Assume that you analyzed an aliquot of the aqueous sample using high-performance LC with a mobile phase that was 75% methanol and 25% water on a C₈ column. Make a sketch of the predicted chromatogram showing the expected elution order. Label each peak. Compare and contrast your answer with the one you gave in Problem 15.21.

Problem 15.26: Based solely on molecular polarity, predict the most likely order of elution for each set of three compounds for a reversed phase chromatographic experi­ment. Compare your answer with the one you gave in Problem 15.22.

Problem 15.27: Describe schematically how you might incorporate a detection system in the column chromatography system depicted in Figure 15.25. (There is more than one correct answer.)

Problem 15.28: Using Figure 15.29, make a plot of the instrument’s response as a func­tion of the concentration of compound A. The concentrations used to obtain the chromato­grams were 1.0, 2.0, and 3.0 mM respectively. This type of plot is called a sensitivity curve.

Problem 15.29: Using Figure 15.29 and the plot you made in Problem 15.28:

(a) Estimate the minimum detection quantity for compound A.

(b) Estimate the instrument’s response to a 4 mM sample of compound A.

(c) Estimate the concentration of a sample that exhibited a retention time of 4.25 minutes and a peak height of 0.366 AU.

Exercise 15.1: Define the following terms:

1. Exclusion limit
2. Isocratic
3. Gradient
4. Mobile phase strength
5. Distribution constant
6. Stationary phase
7. Reverse phase
8. Normal phase
9. Ion exchange
10. Size exclusion
11. Eddy diffusion
12. Selectivity factor
13. Retention time
14. Elute
15. Resolution
16. Theoretical plates
17. Plate height
18. Void volume
19. van Deemter equation
20. W_b
21. W_h
22. Column efficiency
23. Hydrodynamic volume
24. Baseline resolution

Exercise 15.2: Assume that an aqueous sample contained traces of the following compounds listed in the table and that you extracted the aqueous sample with an aliquot of octanol. The distribution coefficients are given as pK_D values.

(a) List the compounds in order of increasing concentration in the octanol phase at equilibrium.

(b) Assume that you analyzed an aliquot of the aqueous sample using HPLC with a mobile phase of 75% methanol and 25% water on a C₈ column. Predict the elution order and make a sketch of the predicted chromatogram. Label each peak.

Exercise 15.3: Assume that an aqueous sample contained traces of the following compounds listed in the table and that you extracted the aqueous sample with an aliquot of octanol. The distribution coefficients are given as pK_D values.

(a) Assume that you analyzed an aliquot of the aqueous sample using HPLC with a mobile phase of 75% methanol and 25% water on a C₈ column. Make a sketch of the predicted chromatogram and label each peak.

(b) How would the chromatogram change if you repeated the experiment using a mobile phase of 50% methanol and 50% water?

Exercise 15.4: Purification of a fragment of BRCA1 (amino acids 230–534) produced the chromatogram shown.

(a) Report retention times and retention volumes for each numbered peak assuming a flow rate of 1 mL/ min and a flow rate of 0.3 mL/min.

(b) Assuming a t_m value of 3 minutes, use the half-height method to estimate the resolution between peak 69 and 71. What inferences can you draw from this resolution value?

Exercise 15.6: Determine N_t and H for each numbered peak in the chromatogram.

Exercise 15.7: Calculate the resolution and retention factor (k_r) for each peak in the chromatogram found in Exercise 15.6.

Exercise 15.8: For each of the parameters given on the right-hand side of Equation 15.19, indicate what effect in­creasing the parameter has on the expected resolution be­tween two closely spaced peaks.

Exercise 15.9: Describe at least two advantages of a gra­dient chromatography method compared to an isocratic chromatography method.

Exercise 15.10: Use one of these manufacturer’s websites (Agilent Technologies, Amersham Pharmacia Biotech, Beckman Coulter, Bioanalytical Systems, Gilson, Hitachi, PerkinElmer, Shimadzu Scientific, Varian, Waters Corporation) to find the chemical composition and struc­ture of at least two normal phase stationary materials.

Exercise 15.11: Describe various ways one might alter the chromatographic experiment in order to improve the resolution of the two, circled peaks seen in the following chromatogram. Justify your suggestions.

Exercise 15.12: In the HPLC analysis of caffeine in an energy drink by standard addition, the sample was pre­pared by pipetting 5.00 mL of the drink into a 50 mL vol­umetric flask, adding a fixed volume of a 1.205 mg/mL caf­feine standard solution, and then diluting to volume using the HPLC mobile phase. The table summarizes the sample preparation protocol and the results obtained from the three injections:

a. Use the peak height data to estimate the original concentration of caffeine in the energy drink.

b. Use the peak area data to estimate the original concentration of caffeine in the energy drink.

c. Of the two values calculated in a and b above, which would you consider more reliable? Explain your answer.