CHE261-Chromatography_Theory_Worksheet

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CHE 261 – Instrumental Analysis 1 ©2024, Dr. Justin J. Maresh, DePaul University Chromatography Theory Worksheet Instructions Background Before attempting this activity, you must watch the three lecture videos and read the Chromatography Theory PDF document, and then pass the Chromatography Theory quiz. All of this content is on D2L. I recommend having Chromatography Theory accessible as a reference when completing this activity. Understanding how to optimize resolution provides the foundation for being able resolve complex unknown mixtures and accelerating repeated analysis of known mixtures. This exercise utilizes an open- source HPLC simulation program that will allow you to rapidly test a variety of separation conditions to help you learn how to optimize separations without using instrument time. 1 Simulation Answer all questions for this assignment on the attached worksheet. Dr. Dwight Stoll, hosts the free at https://multidlc.org/hplcsim/ . The leftmost column contains expandable tabs pertaining to the system parameters. Individual values can be edited in their windows or adjusted by clicking the arrows. The result is calculated automatically. Supplemental information detailing the simulations utilized by the HPLC Simulator is available from the Journal of Chemical Education website . 2 Note that the links mentioned in this reference are no longer active. The following list is a key to terms and constants used in the simulation: • The terms k, t R , and σ given for each compound are the same as k′ (retention factor), t r (retention time), and σ (peak variance / standard deviation) respectively from my Chromatography Theory document. • In the simulation, note that the units of t R are minutes and the units of σ are seconds. • The table of analyte parameters contains two terms I did not discuss in the background. The first is k w , the isocratic retention factor of the compound with water as the mobile phase. The second term is S , the solvent “sensitivity” factor for the analyte. The simulation uses these two terms to calculate k′ . Both terms are temperature dependent. You can ignore them is this activity. • Confusingly, the plate height H is located in the Chromatographic Properties but the reduced plate height h is found in Column Properties. • The void time t m (in units of seconds) is also located in the Column Properties. Instructions Follow the numbered instructions below and answer the questions on the attached worksheet that correspond to the instruction number.

CHE 261 – Instrumental Analysis 2 ©2024, Dr. Justin J. Maresh, DePaul University Part A – Flow Rate Optimization for Narrowest Peaks The only column available in the simulation is Agilent Zorbax SB C18. This is typical reverse-phase column that uses a stationary phase of 18-carbon alkyl chain molecules attached to silica particles. Recall that compounds are better retained if their interaction with the stationary phase is stronger as the result of intermolecular forces. The following set of parameters will be our “default” conditions for this activity. Find by expanding the appropriate tabs. Do not change any other parameters at this time. If set correctly, our two compounds are not completely resolved under these conditions. Compounds: 3-phenylpropanol and p-chlorophenol Mobile Phase Composition: Solvent A is Water and Solvent B is MeOH; isocratic mixture of 55% B Chromatographic Properties: Temperature: 30 °C; Injection: 5.0 μL; Flow Rate: 1.0 mL/min Column Properties: Length: 200 mm; Inner Diameter: 4.6 mm; Particle Size: 3 µm 1) Before you begin, answer question 1 on the worksheet. 2) Vary the flow rate from 0.05 mL/min to 8 mL/min. These are typical performance limits for an HPLC. Observe how flow rate affects the retention times, peak widths, back pressure (keep in mind that typical HPLC systems cannot run above 400 bar), reduced plate height, and resolution of the sample mixture. Answer question 2 on the worksheet. Tips: For all questions about trends in resolution, rather than calculate R s , you should visually estimate the relative resolution based on the extent of baseline resolution as shown in Figure 4 of Chromatography Theory . The relationship between theory and your observations in this question must make intuitive sense to you before you continue. If they do not, ask for help. 3) Tabulate at least five data points for plate height H at different flow rates to generate a van Deemter plot of flow rate vs. H . All of your data points should be <400 bar. Your plot should sample a wide enough range of flow rates that you can see the extremes of a U-shaped trend. The simulation gives the historical unit of cm for H . Because HPLC is so high performance, cm are an impractical unit. For HPLC van Deemter analysis, you should plot H in μm (multiply the given values in cm by 10 4 ). Perform non-linear least squares curve fitting of your data to Equation 1 to determine the flow rate that gives the lowest value for the plate height. 3 𝐻𝐻 = 𝐴𝐴 + 𝐵𝐵 𝑢𝑢 + 𝑐𝑐 · 𝑢𝑢 (Equation 1) If you do not have non-linear curve fitting software, I suggest using the GeoGebra Graphing Calculator . My short tutorial video ( https://bit.ly/vanDeemter ) demonstrates this calculation in GeoGebra. To export an image of your curve fit, first center your data in the plot window. Then select the hamburger button ( ≡) → Export Image → Download . You will find the image in your web browser’s Download folder. Attach this plot to the worksheet. It is not necessary to label it.

CHE 261 – Instrumental Analysis 3 ©2024, Dr. Justin J. Maresh, DePaul University 4) Set the flow rate to the optimal flow rate you calculated from van Deemter analysis. In worksheet question 4, state the optimal flow rate, retention time, peak width, back pressure, and reduced plate height of the two peaks at this flow rate. You should also calculate the resolution as 𝑅𝑅 𝑠𝑠 = 𝛥𝛥𝑡𝑡 𝑟𝑟 4 𝜎𝜎 𝑎𝑎𝑎𝑎 (Equation 2) Note that the simulation gives t R and σ in different units. The standard precision on an HPLC is 0.01 mL/min for flow rate and 0.01 min for retention time. Part B – Column Optimization for Best Resolution As with flow rate (Part A), column particle size and length can be optimized in isocratic conditions. 5) Without changing the optimal flow rate, observe how the resolution is affected by column particle size. The most common sizes are 1.8, 3, 5, and 10 μm . Answer question 5 on the worksheet. 6) Return the particle size to 3 μm and keep your optimized flow rate. Vary the column length systematically from 50 to 500 mm. Answer question 6 on the worksheet. 7) As a rule of thumb, you learned in Chromatography Theory that the k′ for your all peaks of interest should fall between 1–10. Answer question 7 on the worksheet. Part C – Mobile Phase Optimization for Separation To optimize the selectivity (relative difference in retention factors) requires adjustment of the properties of the mobile and/or stationary phases. In LC, mobile phase composition is optimized. 8) Using the parameters from the last step, determine which organic solvent gives the best resolution, methanol or acetonitrile. Answer question 8 on the worksheet. For the remainder of this activity, set the following parameters. Compounds : benzonitrile, methylbenzoate, anisole, p-nitrotoluene, toluene, and naphthalene Mobile Phase Composition: Solvent A is Water and Solvent B is MeOH Chromatographic Properties: Temperature: 30 °C; Injection: 5.0 μL; Flow Rate: 1.0 mL/min Column Properties: Length: 150 mm; Inner Diameter: 4.6 mm; Particle Size: 3 µm 9) Vary B% from 40% to 100% (note: values of %B < 40 may cause your browser to temporarily freeze since billions of data points must be calculated). Many of the remaining questions will ask you about run time. Run time must be long enough to provide sufficient baseline after the final peak to get an accurate integration. For this exercise, simply estimate the run time as one minute after the final peak has reached baseline . Answer question 9 on the worksheet.

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CHE 261 – Instrumental Analysis 4 ©2024, Dr. Justin J. Maresh, DePaul University 10) By adjusting the percent organic solvent, qualitatively find the shortest analysis time you can achieve in which all six peaks are resolved and the retention factors are 1< k′ <10. Record your conditions in worksheet question 10. 11) Switch solvent B to acetonitrile. Repeat the process of manual solvent optimization to find the shortest analysis time in which all six peaks are resolved and the retention factors are 1< k′ <10. Record your conditions in worksheet question 11. 12) Pick an intermediate %B (30–50%) and compare the k′ values for the analyte species for B as methanol versus acetonitrile. Answer worksheet question 12. 13) Use your observations to come up with a ‘rule of thumb’ for the separation of small molecules (less than 500 Da) by reversed phase HPLC by completing the sentence in worksheet question 13. Part D – Mobile Phase Optimization for Separation – Gradient Elution Isocratic conditions always provide the best resolution in LC and should be used whenever possible. But for a complex mixture, isocratic conditions that adequately resolve all peaks often have very long run times. Furthermore, the later peaks may become unacceptably broad due to longitudinal diffusion. Broadening reduces the precision of area measurement. In LC, run times can be shortened and peaks narrowed by gradient elution, the process of increasing the percent composition of organic solvent over time. Next, you will optimize a gradient method for the same analytes as Part B. 14) First, we will run a generic scouting gradient. Change the simulation to Gradient Elution Mode (keep the same parameters as Part B, with acetonitrile as solvent B). Set up a gradient of 0–100% acetonitrile increasing linearly over 30 minutes. This is a typical starting point that separates most mixtures. Next, make the gradient increasingly steeper by shortening the gradient time. Find the shortest gradient time that provides baseline resolution of all components while maintaining k’ > 1 for the earliest peak (note that the software does not calculate k’ in gradient mode, you will have to calculate this yourself from the void time). Calculate the slope of this gradient as ( %B final – %B initial )/gradient time. Record your results in worksheet question 14. 15) The previous method is obviously unoptimized since no peaks are eluting during most of the run. The first step towards optimization is to increase the %B initial so that the first peak elutes as early as possible (i.e. close to k′ = 1). In step 12 you found the isocratic condition that eluted the first analyte at k′ ≈ 1. Use Equation 3 to find the optimized initial %B and enter it into the simulation. The value 0.9 is a factor that accounts for the dwell time (the delay between the mobile phase mixer and the column) of a typical HPLC. % 𝐵𝐵 𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑎𝑎𝑖𝑖 , 𝑜𝑜𝑜𝑜𝑖𝑖𝑖𝑖𝑜𝑜𝑖𝑖𝑜𝑜𝑜𝑜𝑜𝑜 = 0.9 × % 𝐵𝐵 𝑖𝑖𝑠𝑠𝑜𝑜𝑖𝑖𝑟𝑟𝑎𝑎𝑖𝑖𝑖𝑖𝑖𝑖 , 𝑜𝑜𝑜𝑜𝑖𝑖𝑖𝑖𝑜𝑜𝑖𝑖𝑜𝑜𝑜𝑜𝑜𝑜 (Equation 3) Now adjust the gradient time slightly until all peaks are baseline resolved (keeping the final %B at 100%). Record your parameters in question 15. 16) If you programmed your current gradient into an HPLC, the run would continue until it reaches 100% B, several minutes after your last peak has eluted. To optimize the end of the run, stop the gradient one minute after the last peak has eluted. Equation 4 gives this run time.

CHE 261 – Instrumental Analysis 5 ©2024, Dr. Justin J. Maresh, DePaul University 𝑟𝑟𝑢𝑢𝑟𝑟𝑡𝑡𝑟𝑟𝑟𝑟𝑟𝑟 = 𝑡𝑡 𝑟𝑟 𝑖𝑖𝑎𝑎𝑠𝑠𝑖𝑖 𝑜𝑜𝑜𝑜𝑎𝑎𝑝𝑝 + 1 minute (Equation 4) Use Equation 5 to find the final %B at the end of your run. % 𝐵𝐵 𝑓𝑓𝑖𝑖𝑖𝑖𝑎𝑎𝑖𝑖 , 𝑜𝑜𝑜𝑜𝑖𝑖𝑖𝑖𝑜𝑜𝑖𝑖𝑜𝑜𝑜𝑜𝑜𝑜 = 𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑟𝑟 𝑜𝑜𝑜𝑜𝑖𝑖𝑖𝑖𝑜𝑜𝑖𝑖𝑜𝑜𝑜𝑜𝑜𝑜 × 𝑟𝑟𝑢𝑢𝑟𝑟𝑡𝑡𝑟𝑟𝑟𝑟𝑟𝑟 + % 𝐵𝐵 𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑎𝑎𝑖𝑖 , 𝑜𝑜𝑜𝑜𝑖𝑖𝑖𝑖𝑜𝑜𝑖𝑖𝑜𝑜𝑜𝑜𝑜𝑜 (Equation 5) When entering %B final, into the simulation, be sure to also set the gradient time to the value you used for 𝑟𝑟𝑢𝑢𝑟𝑟𝑡𝑡𝑟𝑟𝑟𝑟𝑟𝑟 in Equation 4. When done correctly, the chromatogram will not change, the experiment will simply end sooner. For example, if you had developed a gradient with a change in B of 28–100% over 16 min, the slope of solvent B gradient would be 4.5% min -1 . If the last peak eluted at 8.4 min under this condition, then your optimal run time would be 9.4 min and the %B final should be set according to Equation 6. % 𝐵𝐵 𝑓𝑓𝑖𝑖𝑖𝑖𝑎𝑎𝑖𝑖 = (4.5% min −1 ) × (9.4 min) + 28% = 70% ( 𝐸𝐸𝐸𝐸𝑢𝑢𝐸𝐸𝑡𝑡𝑟𝑟𝑠𝑠𝑟𝑟 6) It is sufficient to round your gradients to the nearest integer percent and your times to the nearest tenth of a minute. Thus, the optimized gradient would be 28–70% over 9.4 minutes. Finally, calculate the resolution ( R s ) of anisole and p-nitrotoluene using Equation 2. Record this value and the details of your optimized gradient method and in worksheet question 16. If the resolution is less than 1.5, your method is not optimized and you should repeat steps 15 and 16. You should also take a screenshot of your final separation and include a printout of this with your worksheet. References 1. Fasoula S, Nikitas P, & Pappa-Louisi A (2017) Teaching Simulation and Computer-Aided Separation Optimization in Liquid Chromatography by Means of Illustrative Microsoft Excel Spreadsheets. J. Chem. Educ. 94(8):1167-1173. 2. Boswell PG , et al. (2013) An Advanced, Interactive, High-Performance Liquid Chromatography Simulator and Instructor Resources. J. Chem. Educ. 90(2):198-202. 3. van Deemter JJ, Zuiderweg FJ, & Klinkenberg A (1956) Longitudinal diffusion and resistance to mass transfer as causes of nonideality in chromatography. Chem. Eng. Sci. 5(6):271-289.

CHE 261 – Instrumental Analysis 6 ©2024, Dr. Justin J. Maresh, DePaul University Chromatography Simulation Worksheet (70 points) 1. In your own words, explain why peaks broaden at a slow mobile phase flow rate. [3] In your own words, explain why peaks broaden at a fast mobile phase flow rate. [3] 2. For each parameter, circle one option. As flow rate increases… [4] • Retention times (a) decrease, (b) stay the same, (c) increase, (d) trend varies • Peak widths (a) narrow, (b) stay the same, (c) broaden, (d) trend varies • Back pressure (a) decreases, (b) stays the same, (c) increases, (d) trend varies • Resolution (a) decreases, (b) stays the same, (c) increases, (d) trend varies 3. PRINT AND ATTACH : Attach a printout of your GeoGebra van Deemter plot to this worksheet. Rubric: 1 point each for (a) the plot, (b) correct data, (c) correct formula and curve, (d) correct parameters, (e) labeled x-axis, (f) labeled y-axis (you may label axes by hand). [6] 4. Fill in your results and the values you used to calculate resolution in the tables below. [9] Optimal flow rate mL/min Retention times min min Back pressure bar Peak Widths (4σ) s s Plate height (HETP) μm Resolution Does this condition resolve to baseline? Yes No

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CHE 261 – Instrumental Analysis 7 ©2024, Dr. Justin J. Maresh, DePaul University 5. For each parameter, circle one option. As particle size decreases… [5] • Retention times (a) decrease, (b) stay the same, (c) increase, (d) trend varies • Peak widths (a) narrow, (b) stay the same, (c) broaden, (d) trend varies • Back pressure (a) decreases, (b) stays the same, (c) increases, (d) trend varies • Resolution (a) decreases, (b) stays the same, (c) increases, (d) trend varies Which particle size would you purchase to give the best resolution (assume no pressure limits)? • Particle size: (a) 1.8 μm, (b) 3.0 μm, (c) 5.0 μm, (d) 10.0 μm 6. For each parameter, circle one option. As column length increases… [7] • Retention times (a) decrease, (b) stay the same, (c) increase, (d) trend varies • Peak widths (a) narrow, (b) stay the same, (c) broaden, (d) trend varies • Back pressure (a) decreases, (b) stays the same, (c) increases, (d) trend varies • Resolution (a) decreases, (b) stays the same, (c) increases, (d) trend varies Which column length would you purchase to give the best resolution (assume no pressure limits)? • Column length: (a) 50 mm, (b) 100 mm, (c) 150 mm, (d) 200 mm, (e) 250 mm Which particle size and column length would you purchase to give the best resolution given that your system can only handle a maximum backpressure of 300 bar? • Particle size: (a) 1.8 μm, (b) 3.0 μm, (c) 5.0 μm, (d) 10.0 μm • Column length: (a) 50 mm, (b) 100 mm, (c) 150 mm, (d) 200 mm, (e) 250 mm 7. Is the rule 1 < k′ < 10 true for your optimized flow rate and column conditions from step 6? [1] (a) yes (b) no 8. Which solvent provides better resolution? [1] (a) methanol (b) acetonitrile (c) both solvents give the same resolution 9. For each parameter, circle one option. As the percent of organic solvent B increases… [4] • Run time (a) decreases, (b) stays the same, (c) increases, (d) trend varies • Peak widths (a) narrow, (b) stay the same, (c) broaden, (d) trend varies • Back pressure (a) decreases, (b) stays the same, (c) increases, (d) trend varies • Resolution (a) decreases, (b) stays the same, (c) increases, (d) trend varies 10. Shortest run time with all peaks between 1< k′ <10 and resolved: [2] % Methanol % Run time minutes 11. Shortest run time with all peaks between 1< k′ <10 and resolved: [2] % Acetonitrile % Run time minutes

CHE 261 – Instrumental Analysis 8 ©2024, Dr. Justin J. Maresh, DePaul University 12. Which solvent is stronger (i.e. makes solutes elute faster)? [1] (a) methanol (b) acetonitrile (c) both solvents are equally strong 13. Fill in the blanks to make the following statement true. [2] “When changing from methanol to acetonitrile as the organic mobile phase solvent, approximately ___ ____ (give a number) percent _________ (more/less) acetonitrile must be used to obtain a retention factor similar to that obtained when using methanol.” 14. Results of your scouting gradient. [3] Shortest gradient time minutes Slope of this gradient Δ%B/min Run time ( t r of last peak + 1 min) minutes 15. Results of your first optimized gradient. [4] Initial %B Final %B 100% Gradient time minutes Slope of this gradient Δ%B/min Run time ( t r of last peak + 1 min) minutes 16. Results of your final optimized gradient. This gradient method should have the shortest run time, every peak should be resolved, and k′ ≈ 1 for the first peak. If this is not the case, you need to keep optimizing. [7] Initial %B Final %B Gradient time / Run time minutes Slope of this gradient Δ%B/min Retention time of first peak minutes Retention time of last peak minutes Resolution of anisole and p- nitrotolene PRINT AND ATTACH : Take a screen shot of your optimized method and include an image with your worksheet. Rubric: 5 points all peaks resolved, 1 point for all k’>1, 1 point for optimal runtime. [7]