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Exercises for HPLC 1)Haddad and associates report the following capacity factors for the reverse-phase separation of salicylamide (k ¢sal) and caffeine (k ¢caff). %v/v methanol 30% 35% 40% 45% 50% 55% k’sal 2.4 1.6 1.6 1.0 0.7 0.7 k’caff 4.3 2.8 2.3 1.4 1.1 0.9 Explain the changes in capacity factor. What is the advantage of using a mobile phase with a smaller %v/v methanol? Are there any disadvantages? 2) Suppose that you are to separate a mixture of benzoic acid, aspartame, and caffeine in a diet soda. The following information is available to you. tr in Aqueous Mobile Phase Buffered to a pH of Compound 3.0 3.5 4.0 4.5 benzoic acid 7.4 7.0 6.9 4.4 aspartame 5.9 6.0 7.1 8.1 caffeine 3.6 3.7 4.1 4.4 (a) Explain the change in retention time for each compound. (b) Plot retention time versus pH for each compound on the same graph, and identify a pH level that will yield an acceptable separation. 3) The composition of a multivitamin tablet is conveniently determined using an HPLC with a diode array UV/Vis detector. A 5-mL standard sample containing 170 ppm vitamin C, 130 ppm niacin, 120 ppm niacinamide, 150 ppm pyridoxine, 60 ppm thiamine, 15 ppm folic acid, and 10 ppm riboflavin is injected into the HPLC, (in arbitrary units) of, respectively, 0.22, 1.35, 0.90, 1.37, 0.82, 0.36, and 0.29. The multivitamin tablet is prepared for analysis by grinding into a powder and transferring to a 125-mL Erlenmeyer flask containing 10 mL of 1% v/v NH3 in dimethyl sulfoxide. After sonicating in an ultrasonic bath for 2 min, 90 mL of 2% acetic acid is added, and the mixture is stirred for 1 min and sonicated at 40 °C for 5 min. The extract is then filtered through a 0.45-mm membrane filter. Injection of a 5-mL sample into the HPLC gives signals of 0.87 for vitamin C, 0.00 for niacin, 1.40 for niacinamide, 0.22 for pyridoxine, 0.19 for thiamine, 0.11 for folic acid, and 0.44 for riboflavin. Report the number of milligrams of each vitamin present in the tablet. 4). The amount of caffeine in an analgesic tablet was determined by HPLC using a normal calibration curve. Standard solutions of caffeine were prepared and analyzed using a 10-mL fixed-volume injection loop. Results for the standards are summarized in the following table. Concentration of Standards Signal (ppm) (arbitrary units) 50.0 8354 100.0 16925 150.0 25218 200.0 33584 250.0 42002 The sample was prepared by placing a single analgesic tablet in a small beaker and adding 10 mL of methanol. After allowing the sample to dissolve, the contents of the beaker,including the insoluble binder, were quantitatively transferred to a 25-mL volumetric flask and diluted to volume with methanol. The sample was then filtered, and a 1.00-mL aliquot was transferred to a 10-mL volumetric flask and diluted to volume with methanol. When analyzed by HPLC, the signal for the caffeine was found to be 21469. Report the number of milligrams of caffeine in the analgesic tablet. 5) Kagel and Farwell report a reverse-phase HPLC method for determining the concentration of acetylsalicylic acid (ASA) and caffeine (CAF) in analgesic tablets using salicylic acid (SA) as an internal standard. A series of standards was prepared by adding known amounts of acetylsalicylic acid and caffeine to 250-mL Erlenmeyer flasks and adding 100 mL of methanol. A 10.00-mL aliquot of a standard solution of salicylic acid was then added to each. The following results are obtained for a typical set of standard solutions. Peak Height Peak Height Milligrams Milligrams Ratio Ratio Standard ASA CAF ASA/SA CAF/SA 1 200.0 20.0 20.5 10.6 2 250.0 40.0 25.1 23.0 3 300.0 60.0 30.9 36.8 A sample of an analgesic tablet was placed in a 250-mL Erlenmeyer flask and dissolved in 100 mL of methanol. After adding a 10.00-mL portion of the internal standard, the solution was filtered. Analysis of the sample gave a peak height ratio of 23.2 for ASA and 17.9 for CAF. (a) Determine the number of milligrams ASA and CAF in the tablet. (b) Why was it necessary to filter the sample? (c) The directions indicate that approximately 100 mL of methanol is used to dissolve the standards and samples. Why is it not necessary to measure this volume more precisely? (d) In the presence of moisture, ASA decomposes to SA and acetic acid. What complication might this present for this analysis? How might you evaluate whether this is a problem? 6) Bohman and colleagues described a reverse-phase HPLC method for the quantitative analysis of vitamin A in food using the method of standard additions. In a typical example, a 10.067-g sample of cereal is placed in a 250-mL Erlenmeyer flask along with 1 g of sodium ascorbate, 40 mL of ethanol, and 10 mL of 50% w/v KOH. After refluxing for 30 min, 60 mL of ethanol is added, and the solution is cooled to room temperature. Vitamin A is extracted using three 100-mL portions of hexane. The combined portions of hexane are evaporated, and the residue containing vitamin A is transferred to a 5-mL volumetric flask and diluted to volume with methanol. A standard addition is prepared in a similar manner using a 10.093-g sample of the cereal and spiking it with 0.0200 mg of vitamin A. Injecting the sample and standard addition into the HPLC gives peak areas of 6.77 ´ 103 and 1.32 ´ 104, respectively. Report the vitamin A content of the sample in milligrams/100 g cereal. Determination of Fluoxetine in Serum Description of Method. Fluoxetine is another name for the antidepressant drug Prozac. The determination of fluoxetine and its metabolite norfluoxetine, in serum is an important part of monitoring its therapeutic use. The analysis is complicated by the complex matrix of serum samples. A solid-phase extraction followed by an HPLC analysis using a fluorescence detector provides the necessary selectivity and detection limits. Procedure. A known amount of the antidepressant protriptyline is added to a serum sample, serving as an internal standard. A 0.5-mL aliquot of the serum is passed through a solid-phase extraction cartridge containing silica particles with a bonded C18 phase. After washing to remove interfering constituents from the sample matrix, the remaining constituents, including both analytes and the internal standard, are removed by washing the cartridge with 0.25 mL of a 25:75 v/v mixture of 0.1 M HClO4 and acetonitrile. A 20-mL aliquot is injected onto a 15-cm ´ 4.6-mm column packed with a 5-mm C8-bonded stationary phase. An isocratic mobile-phase mixture of 37.5:62.5 v/v acetonitrile and water (containing 1.5 g of etramethylammonium perchlorate and 0.1 mL of 70% v/v HClO4) is used to elute the sample. Detection is with a fluorescence detector using an excitation wavelength of 235 nm and an emission wavelength of 310 nm. Questions 1. What is the purpose of including an initial solid-phase extraction? A direct injection of serum is not advisable since the presence of particulate material in the sample matrix may clog the column. In addition, some of the sample’s constituents may absorb too strongly to the stationary phase, thus degrading the column’s performance. Finally, although an HPLC is capable of separating and analyzing complex mixtures, an analysis may still be difficult if there are too many constituents to provide adequate resolution of the analytes. The solid-phase extraction serves the purpose of cleaning up the sample before using the HPLC. 2. One advantage of an HPLC analysis is that a loop injector often eliminates the need for an internal standard. Why is an internal standard used in this analysis? What assumption(s) must we make about the internal standard? An internal standard is used because of difficulties introduced in the solid-phase extraction. For example, the volume of serum taken for the solid-phase extraction and the volume of solvent used to remove the analyte and internal standard are quite small (0.5 mL and 0.25 mL, respectively). The precision and accuracy with which these volumes can be measured are not as good as when using larger volumes. Using an internal standard compensates for any variations in these volumes. To be useful, the analytes and internal standard must be assumed to be retained to the same extent during the initial loading and washing of the cartridge, and they must be assumed to be extracted to the same extent during the final elution. 3. If the peaks for fluoxetine and protriptyline are insufficiently resolved, how might you alter the mobile phase to improve their separation? Decreasing the amount of acetonitrile and increasing the amount of water in the mobile will increase retention times, thereby providing a better resolution. Aspartame Caffein salicylamide