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HPLC Simulator Exercises In this computer exercise, you will simulate HPLC separations using a Windows-based computer program. As you become familiar with the HPLC simulator, you will find that it is very detailed and realistic. Your objective is to complete the exercises described below. The first thing you should do is get familiar with how the program works. Note: although it is in your best interests to keep a record of how you developed the separation for each exercise (so that you don't inadvertently repeat an experiment), I only want the requested information (column, detector, wavelength, mobile phase, flow rate, tR's, peak areas, etc.) for your final separation. One of the many advantages of this simulator is that you can explore many variations in the experimental parameters in a minimum of time. Use this to your advantage by getting an understanding of how such things as the identity and percentage of an organic solvent in the mobile phase, flow rate, injection volume, and differences in selectivity among columns affect qualitative and/or quantitative aspects of the separation. columns 1. DuPont C18, 23 x 0.46 cm, 6 μm dp 2. DuPont C8, 23 x 0.46 cm, 6 μm dp 3. Waters C18, 30 x 0.39 cm, 10 μm dp 4. Merck C8, 25 x 0.46 cm, 10 μm dp 5. Hypercil C18, 16 x 0.46 cm, 6 μm dp organic solvents for this reversed phase simulation: methanol, acetonitrile, tetrahydrofuran compounds (analytes) in simulation: acetophenone, anisole, benzene, p-cresol, methyl benzoate, p-nitrophenol, phenol, phenetole (ethoxybenzene), toluene, 2,5-xylenol 1. The first analysis will be of some benzene derivatives. You should learn how to make up samples and then do an analysis. After you name the sample, choose anisole, toluene, and phenol as analytes and enter concentrations between 8 and 15 mg/mL. Then after setting up the instrument with the variable wavelength detector at 254 nm, your assigned column (above), flow rate 1.0 mL/min, and 100% methanol (MeOH) as the mobile phase, inject 25 μL of sample. Do you observe 3 well-separated peaks in the chromatogram? If not, reduce the percentage of MeOH by increments of 10% (i.e., 100%, 90%, 80%, 70%, etc.) until you can get baseline resolution of the 3 compounds in less than 15 minutes. If no combination of methanol and water turns out to be a satisfactory mobile phase, switch from MeOH to acetonitrile (MeCN) as the organic solvent and repeat the strategy you employed for the MeOH/water mobile phase (100%, 90%, etc.). (You may also change other parameters if you like just to see how the separation is affected). As soon as you obtain a satisfactory separation, integrate the chromatogram and obtain retention times (tR) and peak areas. Next, analyze the appropriate qualitative standards to identify the peaks*. Report, in a table, the identity of each of the compounds along with its retention time (tR) and peak area. *Strictly speaking, matching a retention time to identify an unknown peak in an unknown sample is not acceptable. Additional information (UV, IR, mass spectra, etc.) is nearly always needed to confirm the chemical composition of such a peak. But for our purposes and in most "real world" samples in which the identities (but not the concentrations) of the analytes are known, matching retention times is OK. 2. Now that you are an HPLC "expert", your next task will be to identifiy an unknown mixture. You will have a choice of unknowns, the data files are chm431_A, chm431_B, chm431_C, chm431_D, choose one. First you will need to qualitatively determine the compounds in your unknown mixture. Choose a column, solvent, solvent %, flow rate, and detector wavelength in order to obtain the fastest separation with baseline resolution of all of the compounds in your unknown. Once you have qualitatively determined the identity of the compounds in your unknown, now you can quantitatively analyze them in step 3. **Note: The flow rate for your optimized separation should be such that the pressure drop across the column does not exceed 3500 psi. Columns usually "die" quickly when their pressure limits are exceeded. Although a computer-simulated column cannot die, it is important to perform the simulations according to the realistic limitations of the experiment if it were actually done in the lab. 3. Quantitative Analysis Construct calibration curves for the compounds qualitatively determined in your unknowns using either a standard calibration curve or by an internal standard. Determine the quantitiy of each unknown. 4. Optimization of detection wavelength for several compounds in a sample. Assuming a range of 215 to 320 nm, use the diode array detector to estimate what wavelength gives the best overall sensitivity for all compounds in your unknown sample. 5. Bonus: Using the Merck C8 column and acetonitrile, measure the retention volume (VR = tR • F, where F = flow rate in mL/min.) as a function of %B (in 10% increments starting at 100%) for each of 9 analytes. After plotting the results for all analytes on the same graph (log10 VR vs. %B), answer the following questions: a. Is there a mobile phase composition at which all components are baseline resolved in less than 25 mL of mobile phase? If yes, please report the conditions and include a chromatogram if possible. b. Is there a mobile phase composition at which the following components are baseline resolved in less than 25 mL of mobile phase: (a) phenol, (b) methyl benzoate, (c) anisole, (d) benzene, and (e) toluene)? If yes, please report the conditions and include a chromatogram if possible. High Performance Liquid Chromatography Part B: Soft Drink Analysis I. Qualitative Analysis of Artificial Sweeteners and Additives in Soft Drinks II. Quantitation of the Caffeine Content of Soft Drinks In this experiment you will be analyzing a set of soft drink samples as described in the J. Chem Ed article by Bidlingmeyer and Schmitz [1]. Soft Drink Samples Available: Pepsi Diet Pepsi Coke Diet Coke Mountain Dew I. Qualitative Analysis of Artificial Sweeteners and Additives in Soft Drinks You will prepare a set of individual samples of each of the four analytes aspartame, benzoic acid, caffeine, and saccharine in the mobile phase mixture (a 75:25 v/v mixture of 1M acetic acid in water/methanol). These qualitative standards should be prepared at concentrations of approximately 2 mg/mL for aspartame, 0.5 mg/mL for benzoic acid, 0.25 mg/mL for caffeine, and 0.5 mg/mL for saccharine. Note that these standards are qualitative, and you will need only ~1 mL of solution for the HPLC analysis. You should use the UV/visible spectra of these qualitative analysis standards (provided to you here on Bb Learn) to select the best wavelength for analysis. As TotalChrom allows you to collect data at two wavelengths simultaneously, you should also collect data at a wavelength of 254nm for comparison. Run each of the qualitative standards as well as a known four-component mixture that you prepare on the HPLC. Determine the retention time of each of the individual components under study. Compare the UV/visible spectra provided here to reference spectra that you can find on-line. Identify the peaks in the chromatogram. II. Quantitation of the Caffeine Content of Soft Drinks Using the 500 ppm stock solution of caffeine available in lab, create a set of calibration standards over the range of 10-100 ppm. The soft drink samples should be prepared by first decarbonating them using the ultrasonic cleaner; as better chromatographic results are obtained when the composition of the sample more closely matches that of the mobile phase in use, the samples should be diluted 1:1 using the mobile phase. This will also ensure that the caffeine concentrations are within the range of the calibration curve that you prepared. Analyze at least three of the soft drink samples provided to you. Replicate injections of the standards and soft drink samples should be performed. Data Analysis Prepare a calibration curve using the data collected and quantitate the amount of caffeine present in each of the samples. Note: The samples should be filtered using the syringe filters before they are placed into the auto-sampler vials. Questions: 1. Why do the soft drink samples need to be filtered prior to analysis? 2. Why is the analysis also conducted at a wavelength of 254nm (i.e., what is special about this wavelength)? 3. Why can we not quantify the amount of sugar in the non-diet sodas using the instrument setup available in the lab? References: 1. B.A. Bidlingmeyer, S. Schmitz, “The Analysis of Artificial Sweeteners and Additives in Beverages by HPLC”, J. Chem. Educ., 68, A195-200, (1991).

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Data analysis for part B:
Based on the calibration curve given in excel file:
pk_ht@254 = 228.15*[caffeine];
pk_ar@254 = 2610.36*[caffeine];
pk_ht@280 = 439.04*[caffeine];
pk_ar@280 = 4973.86*[caffeine];
where pk_ht = peak height, pk_ar = peak area and [caffeine] = concentration of caffeine.
Now for unknown C1:
Assuming the replicated values of peak height and peak area at retention time of 3.20 min have a Gaussian distribution, the average peak height and peak area at 254 nm and 280 nm can be calculated using “AVERAGE” and “STDEV”...

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