AM0925 Assignment Basic chromatographic parameters and optimization in LC Introduction This is a computer exercise where you will apply a simulator of reversed phase LC to study the influence of chromatographic conditions on retention, efficiency, selectivity and resolution. The first thing you have to do is to download the HPLC simulator and become familiar with the interface. The HPLC simulator can be downloaded from http://hplcsimulator.org/simulator.php Press "Launch HPLC Simulator" Note! If the HPLC simulator is blocked by Java security settings. See solution to the problem in the appendix. Select thereafter open with Java Web Start Launcher The window should look approximately as shown in Figure 1 below. Figure 1. The HPLC Simulator In the panel to the left you can select different conditions. The conditions you need to modify during the exercise are: Mobile phase composition, Solvent B : Either methanol or acetonitrile, which in addition to water are the two most common solvents in reversed phase LC. Mobile phase composition, Solvent B fraction : Percent of mobile phase that is solvent B. Chromatographic properties, Temperature : The temperature of the column. 1
Chromatographic properties, Flow rate : The flow through the column in ml/min. Column properties, Stationary phase : Waters Acquity BEH-C18 or Agilent Zorbax SB-C18. Note that changing the column also mean changing the analytes. There are nine analytes in the example with the Waters column and five analytes in the example with the Agilent column. Column properties, Particle size: Average diameter of the particles in the column (μm) All other settings should always be kept at the default values when you do the exercise. You can reset to the default settings by choosing [File]+[Reset To Default Settings]. When you select a new column the settings will also be changed to the default values for the column. Before you start the exercise it may be a good idea to play around with different settings to get used to the user interface and see how the different parameters influence the chromatography. Part A, van Deemter curves The purpose of this part is to investigate how the flow rate and the column temperature influence the parameters in the van Deemter curve. Start by doing the following: Reset all settings to default values as explained above. Select one of the two columns. Select one of the two options (acetonitrile or methanol) for solvent B. Set the temperature to 25 C. Select any compound in the peak parameters list. Adjust solvent B fraction so that the retention factor, k (column 3 in the peak parameters list) is somewhere between 4 and 6. Thereafter you verify that the particle size is set to 3 μm and you record the following for your notes: the applied column, selected solvent B, solvent B fraction, the selected compound, and the retention factor. You should now vary the flow rate and record the following parameters: retention time, peak width and the backpressure. The backpressure is the pressure that must be applied by the pump to achieve the required flow rate and is found in the panel to the left under Chromatographic Properties. The two other values are reported in the list of peak parameters as [tr (min)] and [σ total (s)]. You should record values for the following flow rates, 0.5, 1.0, 2.0, 4.0 and 8.0 ml/min. Organize the recorded values in a spreadsheet (Excel, Calc or similar) as shown below. Flow rate [ml/min] Backpressure [Bar] Retention time, t R [min] Peak width, σ [s] 0.5......... 1......... 2......... 4......... 8......... Thereafter you increase the particle size to 5 μm and repeat the process. Increase thereafter the temperature to 75 C and repeat the process with both 3 and 5 μm particles. In the end you should have four tables (2 particle sizes 2 temperatures). When you change the temperature the retention factor will also change. Record k for 75 C in addition to the one that you have for 25 C. 2
From the recorded retention times and peak widths you thereafter calculate the plate number, N, and the plate height, H. The plate height should be calculated in mm. Note that the retention times are given in minutes and the peak widths are given in seconds. They must therefore be converted to the same scale. Also note that peak widths are reported as the standard deviation, σ, which is 1/4 of the peak width at baseline, w b. The plate number can be calculated directly from σ or from w b by the following equation: N=( t R σ ) 2 =16( t R 2 w b) The plate height, H, is the column length, L, divided by the plate number: H=L/ N To verify that you calculate correctly, you can use the following example. If the retention time is 3.22 min and the peak width reported as 1σ is 2.06 s, the plate number is 8791, and since the column length is 100 mm, the plate height is 0.0114 mm. When you have calculated the plate height for all the recorded data the next step is to solve the van Deemter equation and to evaluate how the flow rate and the temperature influence the three constants, A, B and C, which explain the multiple path effect, longitudinal diffusion and resistance to mass transfer, respectively. The van Deemter equation is given below. (1) (2) H=A+ B u +C u (3) The parameter u is the mobile phase velocity. This is the value reported as chromatographic flow velocity under Column properties in the HPLC simulator. In LC the column flow is proportional to the mobile phase velocity, and can therefore replace u. The van Deemter equation is not a standard equation that can be solved by the built-in equations in Excel or Calc, but it can be solved by a tool called "Solver", which is found in both programs (in Excel it may have to be activated, see link if necessary). In LibreOffice Calc you find the Solver under the [Tools] menu. In Excel it is usually found in the rightmost group under the [Data] menu. Depending on which spreadsheet program you use, you should download the "CURVES_LC.ods" (LibreOffice) or the "CURVES_LC.xls" (Excel) file that you find in the module for the exercise at Mitt UiB. The following example shows how you create the van Deemter curves and find the constants A, B and C in LibreOffice Calc. The spreadsheet for calculating van Deemter curves and the settings for Solver is shown in Figure 2. 3
Solver found here G13 (SSE) is the target cell Minimize target Your calculated plate heights Cells C6:E6 (A,B,C) can be changed van Deemter curve Figure 2. The CURVES_LC.odsˮ file and the settings for Solver in LibreOffice. The seven columns in the table are the following: A) The column flow. B) The observed plate heights, H obs. Your calculated values should be entered here. Use [Paste Special] and [Paste as numbers] when you copy the data into the sheet. C) The A term. D) The constant for the B term (blue) and the effect of the constant at the applied flow (B/flow). E) The constant for the C term (blue) and the effect of the constant at the applied flow (C flow). F) The calculated plate height, H calc. When the model is fitted, H calc should be similar to H obs. The values in this column are the sum of the values in columns C-E. G) Squared errors, SE. This is the squared value of the difference between observed and predicted plate height (H calc H obs ) 2. The sum of the squared errors (SSE) is given at the bottom of the table and this value explains how well the model fits the observed data. The value should be as low as possible. For each of the four sets of experiments you should calculate the van Deemter curve and record the values of the three constants, A, B, and C. You should also copy the figures of the van Deemter curves for your report. When you paste your values into the first column the model will no longer fit the data and it must therefore be updated. You can try to adjust the three constants manually to get a better fit, but in the end you should apply the Solver using the following procedure: Select the cell with the value for SSE Go to the [Tools] menu and select [Solver] ([Data] menu in Excel). In Solver, verify that the target cell is G13 or change it. Select Optimize result to minimum Put the cursor in the field next to "by changing cells" and select the three cells with blue text in the spreadsheet. 4
Thereafter you press the [Solve] button. Press [OK] and [Keep result]. In LibreOffice it can be an idea to press [Continue] a few times before pressing [OK] until you see that you have a stable result. Your observed values should now be approximately at the red curve and SSE should be zero, which show that the model now explains the observations. Record the values for the three constants and copy the plot before you continue with the next data set. Reporting Introduction Give a brief explanation of the purpose of the exercise and the theory of band broadening in packed columns (approximately 1/2 page). The van Deemter equation and the equations for plate number, plate height and optimal velocity/flow rate should be given and explained. Explain that flow were used instead of velocity and that these two parameters are proportional in LC. Explain that the HPLC simulator was applied and how the van Deemter equation was solved. Experimental Under experimental, report the applied conditions: Results The selected column Column dimensions (length, diameter, particle size) The selected compound Solvent A Solvent B Solvent B fraction The retention factor at 25 and 75 C Explain that the parameters you varied were the temperature and the column flow. Include the van Deemter plots and a table of the A, B and C constants for the four experiments. Make a figure that illustrates how the constants vary with particle size and the temperature. The mobile phase velocity (or flow rate in this case) that minimizes the plate height, and therefore maximizes the plate number, is often referred to as the optimal mobile phase velocity, u opt. Since this is a minimum in the curve, it is found where the derivative of the van Deemter equation is zero, and it can be calculated by Equation 4. u opt = B C (4) Use this equation to calculate the optimal flow rate for all four experiments. Thereafter insert u opt in the van Deemter equation and calculate the plate heights, H, at these flow rates. Verify that your calculations are in accordance with the plots. The calculated values should be included in the report. Discuss how the particle size and the temperature affect the three constants of the van Deemter equations, and how this also affects u opt and the plate height at u opt. As long as the retention factor, k, is constant, the time it takes to elute a compound is inversely proportional to the flow. Make a plot of retention time (y-axis) against flow rate (x-axis) for the two experiments with 3 μm particles (plots for 5 μm particles will be identical). Discuss what this relationship means for the time it takes to perform the separation. A power function (ax b ) can be fitted through the data points. 5
Finally you should make a plot of the recorded backpressures (y-axis) against flow rates (x-axis) for all four experiments. Conventional HPLC pumps can typically deliver pressures up to 300-600 bar, while pumps for UHPLC (Ultra High Performance LC) typically can deliver pressures up to 1000-1200 bar. However, it is usually a good advice to have good margins, and to keep the pressure below 50% of the maximum allowed pressure when a method is developed. Discuss which limitations the different particle sizes and temperatures set on the flow rates that can be applied. Part B, Method optimization challenge In part B of the exercise you should try to develop a good method for the separation of the five compounds in the problem with the Agilent Zorbax column. Start by resetting to default settings ([File] menu) and select this column. Your goal is that the retention time of the last compound should be as low as possible, the resolution, R s, between all peaks should be at least 1.5, and the backpressure should not exceed 300 bar. Solvent B should be set to acetonitrile. The resolution is calculated by Equation 5. R s =2 t R (B) t R (A) w b (B) +w b (A) =0.5 t R (B) t R (A) σ (B) +σ (A) (5) w b is peak widths at baseline (4σ) and t R is retention times. A and B denote respectively the first and the last of the two peaks. You are allowed to vary the following parameters: The solvent B fraction. The temperature, but only in the range 30 to 60 C. The flow rate. The particle size can be set to 3, 4 or 5 μm. All other parameters should be kept at the default values for the Agilent Zorbax column. Note that the choice of solvent B, the solvent B fraction and the temperature all have large effects on the selectivity and can change the elution order of the compounds. Also pay attention to what part A of the exercise tells about the effects of the particle size and temperature on the constants in the van Deemter equation, the optimal flow rate and the backpressure. It may also help to consider what the Purnell equation tells about the three factors that are necessary to achieve separation, efficiency, selectivity and retention. The equation is given below. R s = N B 4 (α 1 α )( k B (6) k B +1) α is the separation factor that explains the selectivity, and this is the ratio of the two retention factors, k B /k A, where A and B refer to the first and last of the two peaks, respectively. If N (efficiency), α (selectivity), or k (retention) is zero, there will be no separation. This can for instance be illustrated by setting the solvent B fraction to 100%; the retention factors drops to nearly zero and all peaks are overlapping. The equation also tells that if the selectivity is high, it may still be possible to get sufficient separation even if the efficiency and the retention is fairly low. In a system where the parameters have large influence on the selectivity (as in this case) it is often a good idea to first find a combination of solvent composition and temperature that gives a good selectivity. When you have found the solution you are satisfied with, record the values of all the parameters you have varied. Record t R of the last eluting compound and the backpressure. For the two peaks with the poorest resolution you record the retention time (t R ), the retention factor (k) and the peak 6
width (σ). If there are three peaks that seem equally spaced you record these values for all three. Finally, you press the [Copy] button under the chromatogram, and paste the chromatogram into a document. Before you do all this, remember to verify that you have the required resolution by using Equation 5. To help you determine visually whether you have enough resolution, examples of peak resolutions of 1.0, 1.5 and 2 are given in Figure 3. R s = 1.0 R s = 1.5 R s = 2.0 Figure 3: Examples of different resolutions Reporting Introduction Give a brief explanation of the problem and explain the strategy that you followed to solve it. List the applied equations for calculation of resolution (Equations 5 and 6) and explain them. Experimental Report the following parameters for the solution that you found: Results The choice of solvent B (methanol or acetonitrile). The solvent B fraction. The temperature The flow rate The particle size Include the copy of the chromatogram for the optimized conditions and report the retention time for the last compound. For the two most closely eluting peaks you should report the following parameters: Retention time Peak width (σ or w b ) Retention factor Calculated plate number, N For these two peaks you should prove that the criterion of R s 1.5 is fulfilled by applying Equation 5. Report the value with two decimals. You should also report the backpressure to prove that the other criterion is fulfilled. In addition you should calculate the resolution for the two peaks by Equation 6. For the plate number, N, you apply the average for the two peaks. The Purnell equation is an estimate that assumes for instance equal peak widths of the two peaks, while equation 5 defines resolution. You may get slightly different values from the two equations and it is therefore acceptable that R s calculated by equation 6 is slightly below 1.5, as long as Equation 5 gives an acceptable value. 7
Appendix Circumventing that application is blocked by Java When starting the HPLC simulator you may get an error message saying that the application is blocked by Java Security. See Figure 4. Figure 4: Warning message from Java stating that Java Bindings for OpenGL is blocked To get around this problem, do the following: On Windows: Go to the Windows start menu, select [All programs], find the 'Java' folder, open it and run 'Configure Java'. Alternatively, go to the start button, type 'java' in the 'search programs and files' field and start the 'Configure Java' application. On Mac: Open System preferences. Select Java. In the 'Java control panel', choose 'Security'. You should get a window similar to the one below. Ensure that Security level is set to 'High' Press [Edit Site List] nex to the list of exceptions. Press [Add] in the new window. Add http://jogamp.org and http://www.hplcsimulator.org to the list and press [OK] followed by [Continue]. Ensure that the web adresses are added to the exception list, as shown in Figure 5. Press [Apply] followed by [OK]. Try to launch the HPLC simulator again. 8
Figure 5: http://jogamp.org and http://www.hplcsimulator.org added to the exception site list.. 9