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Page | 2 The experiments provided here simulate operation of both linear and reflectron TOFMS instruments for the analysis of ions formed in the gas‐phase, such as in a GC‐TOF experiment. Experiment 1: Tuning a Linear TOFMS for Maximum Resolution Initial settings: On the TOF Controls tab: Reflectron OFF Delayed extraction OFF Two‐step source On the Distances tab: Step 1: 10mm Step 2: 10mm Drift Region: 1.25m Linear Detector Acceleration Region: 2mm Linear Detector Internal Region: 2mm On the Voltages tab: G0 Voltage: 7000V G1 Voltage: 5000V Drift Voltage: 0V Linear Detector: ‐1500V On the Detection tab: Time Resolution: 1 ns/ch Delay Channels: 10000 Data Channels: 50000 On the Ion Characteristics tab: Number of masses to study: 2 Mass of Ion 1: 250 Mass of Ion 2: 251 Number of Ions: 5000 On the Distributions tab: Initial Position Distribution: X0 Mean: 5.00mm X0 St. Dev: 0.33mm Temporal Distribution: T0 Mean: 0.00ns T0 St. Dev.: 0.00ns Velocity Distribution: Select V0 St Dev button V0 Mean: 0.00m/s V0 St Dev: 0.00m/s For this experiment keep the G0 voltage constant and adjust the voltage applied to G1 to focus the ions on the detector (you are trying to “experimentally” determine the Wiley & McLaren “space‐focusing” conditions). Make small (200‐300V) changes in voltage. As you get nearer to the optimum you will need to take smaller steps in voltage. As you improve the focusing, you should note that the peak intensity increases as the peak width narrows (you see an improvement in sensitivity!) Page | 3 Questions: 1) What voltage settings give the maximum resolving power? 2) What is the maximum resolving power you are able to obtain? 3) What is the separation in TOF between the two ions of mass 250 and 251? 4) What is the width of the peak at m/z=250? 5) What is the resolution between the two peaks under the optimum conditions? 6) What do the values for “skewness” and “kurtosis” tell you? Recall that resolving power (often incorrectly called “resolution” by mass spectrometrists) is calculated using the expression: R m t m t     where m is the mass and m is the FWHM width of the peak (and the values t and t are the mean TOF and FWHM peak width in time units). The resolution (Rs) between two adjacent peaks is calculated from the expression:     2 1 2 1 2 2 s 4 t t t t R w      Where t1 and t2 are the TOF for peaks 1 and 2, respectively, and w2 is the width of peak 2 (this assumes the peak widths for peaks 1 and 2 are the same). Note also that the conversion between FWHM and standard deviation is given by: FWHM  2 2ln 2  2.355 where  is the standard deviation. Supplementary Experiments: SupplExpt 1A: Investigate how the maximum resolving power varies with the drift length of the instrument. SupplExpt 1B: Investigate how the maximum resolving power varies with the G0 voltage for a given drift length. Experiment 2: Tuning a Linear TOFMS Equipped with Delayed Extraction Using the same instrument distances and ion characteristics as the starting point for Experiment 1 and the optimum voltages found at the end of Experiment 1, turn on Delayed Extraction. Use an initial delay time setting of 100ns. Note that the resolution of the instrument can be tuned by adjusting the delay time (in ns) and the pulse voltage‐ which is determined by the difference between the voltages set for G0 and G1. For this experiment keep the delay time constant and just adjust the pulse voltage (leave G0 constant and adjust the value of G1). Did the use of delayed extraction improve the results? Why not? Don’t give up yet. On the Distributions tab set the standard deviation for the initial position to 0.0, then select Temperature Calculation for the standard deviation of the velocity and use a temperature of 300K for the first calculation. With delayed extraction turned off, repeat the simulation starting with the optimized set of voltages you identified in Experiment 1. What is the width of the m/z=250 ion peak Page | 4 observed? Why is it different than the width observed at the end of Experiment 1? Now turn on delayed extraction using an initial delay time setting of 100ns. What do you observe? Note that you will have to retune the G1 voltage to obtain the best performance. As before, make small (100‐200V) changes in voltage, making smaller changes as you get nearer to the optimum. If you notice any skewing in the peak, it might be better to measure the width of the peak using the grid lines on the oscilloscope trace rather than rely on the FWHM value given in the statistics panel. To fully appreciate the effect of delayed extraction, you should also determine how the FWHM of the peak changes with the G1 value when delayed extraction is turned off. Why can’t you simply use the plot determined in Experiment 1? Questions: Using delayed extraction with a delay time of 100ns: 1) What voltage settings give the maximum resolving power? 2) What is the resolving power of the instrument when optimized? 3) What is the separation in TOF between the two ions of mass 250 and 251? 4) What is the width of the peak at m/z=250? 5) What is the resolution between the two peaks under the optimum conditions? Supplementary Experiments: SupplExpt 2A: Investigate how the maximum resolving power for a given mass depends on the selected value of the delay time. Note that you will have to refocus the voltage applied to G1 for each delay time. SupplExpt 2B: Investigate how the maximum resolving power of the instrument depends upon the initial velocity distribution of the ions (as given by the ion temperature)? SupplExpt 2C: Investigate how the optimum delay time depends on the m/z value of the ion under study. Experiment 3: Determining the Space Focus Curve for a Linear TOFMS Use the same instrument distances and ion characteristics as the starting point for Experiment 1 and the optimum voltages found at the end of Experiment 1. For this experiment use continuous extraction conditions (i.e., turn off the delayed extraction). First optimize the instrument resolution by adjusting the voltage applied to G1. The space focus curve can be generated by plotting ion TOF (for a particular m/z value) versus initial position in the source, x0. To most effectively calculate this, set the number of ions (on the Ion Characteristics tab) to 1. You will also want to set the standard deviation of the initial position to be equal to 0 to ensure that the TOF is calculated for the initial position value you set as the mean. Note that when only a single ion is run, only the centroid value displayed on the Flight Time tab of the Outputs panel is valid. All of the other values (FHWM, skewness and kurotisis) will register as NaN (“not a number”) as they cannot be calculated for a single ion. Questions: 1) What form does the space focus curve take? Supplementary Experiments: SupplExpt 3A: Investigate how the optimized value of the G1 voltage is affected by the initial position of ionization, x0. SupplExpt 3B: Investigate how the space focus curve changes with m/z value under study. SupplExpt 3C: Investigate how the space focus curve changes if delayed extraction is used. Page | 5 Experiment 4: Determining the Velocity Focus Curve for a Linear TOFMS Use the same instrument distances and ion characteristics as the starting point for Experiment 1 and the optimum voltages found at the end of Experiment 1. For this experiment use continuous extraction conditions (i.e., turn off the delayed extraction). First optimize the instrument resolution by adjusting the voltage applied to G1. Generate the velocity focus curve for the instrument by plotting ion TOF (for a particular m/z value) versus initial velocity of the ion, v0. You will want to set the standard deviation of the initial velocity to be equal to 0 to ensure that the TOF is calculated for the initial velocity value you set as the mean. Be sure to use a reasonable range of initial velocity values. As in Experiment 3, the most effective way to calculate this is to set the number of ions (on the Ion Characteristics tab) to 1. Questions: 1) What range of initial velocities did you study? 2) What form does the velocity focus curve take? Supplementary Experiments: SupplExpt 4A: Investigate how the velocity focus curve changes with m/z value under study. SupplExpt 4B: Investigate how the velocity focus curve changes if delayed extraction is used. Experiment 5: Mass Calibration Start with the same instrument distances and voltages used as the starting point for Experiment 1. On the Distributions tab, set the mean and standard deviation for x0 to be 5.00 and 0.05mm, respectively. Use a mean velocity of 0.0; select Temperature Calculation for the standard deviation of the velocity and use a temperature of 300K. You can leave the mean and standard deviation of the time of ionization both as 0.0. Turn on delayed extraction (use a delay time of 100ns) and optimize the operation of the instrument by adjusting the voltage applied to G1. Note how this experiment is different than those done in Experiments 1 and 2 previously. Using the optimized voltage setting for G1, run several masses through the simulation and create a calibration curve relating the measured TOF to mass. Questions: 1) What is the form of the equation that needs to be fit to the data? 2) What is both the absolute (in Da) and relative error (in ppm) for each of your calibration points? 3) What is the RMS error for the calibration, considering all of your calibration points together? 4) How does the resolving power of the instrument vary with mass? Supplementary Experiments: SupplExpt 5A: Investigate how the mass accuracy is affected by the form of the equation fit to the calibration data by calculating a TOF for a given mass, then using the calibration equation you determined in answering question 1 above to convert the measured TOF to mass. Experiment 6: Tuning a Reflectron TOFMS for Maximum Resolution Initial settings: On the TOF Controls tab: Page | 6 Reflectron ON Delayed extraction OFF Two‐step source Two‐step reflector On the Distances tab: Source step 1 (d1): 10mm Source step 2 (d2): 10mm First Drift Region (L1): 1.25m Second Drift Region (L2): 0.75m Deceleration Region (d4): 60mm Reflecting Region (d5): 100mm Reflector Detector Acceleration Region: 2mm Reflector Detector Internal Region: 2mm On the Voltages tab: G0 Voltage: 7000V G1 Voltage: 6500V G5 Voltage: 5000V G6 Voltage: 7200V Drift Voltage: 0V Linear Detector: ‐1500V On the Detection tab: Time Resolution: 1 ns/ch Delay Channels: 20000 Data Channels: 50000 On the Ion Characteristics tab: Number of masses to study: 2 Mass of Ion 1: 250 Mass of Ion 2: 251 Number of Ions: 5000 On the Distributions tab: Initial Position Distribution: X0 Mean: 5.00mm X0 St. Dev: 0.05mm Temporal Distribution: T0 Mean: 0.00ns T0 St. Dev.: 0.00ns Velocity Distribution: Select V0 St Dev button V0 Mean: 0.00m/s V0 St Dev: 200.00m/s For this experiment keep the G0, G1 and G6 voltages constant and adjust the voltage applied to G5 (the middle grid of the reflector) to focus the ions on the detector. Make small (200‐300V) changes in G5 voltage at first, and take smaller steps in voltage as you get nearer to the optimum. Questions: 1) What voltage settings give the maximum resolving power? Page | 7 2) What is the resolving power of the instrument when optimized? 3) What is the separation in TOF between the two ions of mass 250 and 251? 4) What is the width of the peak at m/z=250? 5) What is the resolution between the two peaks under the optimum conditions? Supplementary Experiments: SupplExpt 6A: Investigate how using delayed extraction (start with a delay time of 100ns) affects the required voltage settings and instrument performance (i.e., the maximum resolving power and peak resolution). SupplExpt 6B: Investigate how the delayed extraction delay time affects the required voltage settings and instrument performance (i.e., the maximum resolving power and peak resolution). Experiment 7: Determining the Space Focus Curve for a Reflectron TOFMS Use the same instrument distances and ion characteristics as the starting point for Experiment 6 and the optimum voltages found at the end of Experiment 6. Note that for this experiment we are using continuous extraction conditions (i.e., delayed extraction is turned off) and the instrument has been optimized by adjusting the voltage applied to G5. As in Experiment 3, the space focus curve can be generated by plotting ion TOF (for a particular m/z value) versus initial position in the source, x0. To most effectively calculate this, set the number of ions (on the Ion Characteristics tab) to 1. You will also want to set the standard deviation of the initial position to be equal to 0 to ensure that the TOF is calculated for the initial position value you set as the mean. Questions: 1) What form does the space focus curve take? 2) How does this differ from the space focus curve for a linear TOFMS? Supplementary Experiments: SupplExpt 7A: Investigate how the space focus curve changes with m/z value under study. SupplExpt 7B: Investigate how the space focus curve changes if delayed extraction is used. Experiment 8: Determining the Velocity Focus Curve for a Reflectron TOFMS Use the same instrument distances and ion characteristics as the starting point for Experiment 6 and the optimum voltages found at the end of Experiment 6. Note that for this experiment we are using continuous extraction conditions (i.e., delayed extraction is turned off) and the instrument has been optimized by adjusting the voltage applied to G5. Generate the velocity focus curve for the instrument by plotting ion TOF (for a particular m/z value) versus initial velocity of the ion, v0 (again, as you did above in Experiment 4 for the linear instrument). You will want to set the standard deviation of the initial velocity to be equal to 0 to ensure that the TOF is calculated for the initial velocity value you set as the mean. Be sure to use a reasonable range of initial velocity values. As in Experiment 7, the most effective way to calculate this is to set the number of ions (on the Ion Characteristics tab) to 1. Questions: 1) What range of initial velocities did you study? 2) What form does the velocity focus curve take? 3) How does this differ from the velocity focus curve for a linear TOFMS? Supplementary Experiments: SupplExpt 8A: Investigate how the velocity focus curve changes with m/z value under study. SupplExpt 8B: Investigate how the velocity focus curve changes if delayed extraction is used. Caveat Utilitor (let the user beware!): If it hasn’t become apparent yet, working with the reflectron in Experiment 6 should really have you questioning whether you have determined the global optimum instrument conditions corresponding to the best instrument performance, or simply just a local optimum. Even in doing the supplementary experiments, note how many instrument variables are kept constant while working to optimize the performance. Even making the assumption that distances remain constant (as they are set when an instrument is constructed) and that only the voltages (and of course, the delayed extraction delay time) can be varied, questions should arise as to whether there are different combinations of voltages and delay times that might give better performance. Varying the distances (or more fundamentally, whether a one‐ two‐ or three‐step source or reflectron is employed) as well during a true instrument optimization adds an even higher degree of complication‐ particularly if there are statistically significant interactions between the various instrument parameters (which there are). It would be a good idea to follow some sort of a design‐of‐experiments or simplex optimization approach to efficiently search for the best instrument conditions. An advantage of using simulations such as TOFSim is that you can save significant amounts of both time and money not having to construct multiple instrument geometries as you attempt to develop improved instrumentation. Also note that you can investigate the effect of different ion characteristics on the performance of the TOFMS instrument with a given geometry and voltage settings. Any changes to the ionization process that result in changes in the initial time or time spread of ionization, initial position or spatial spread, or the initial mean velocity or velocity spread of the ions can and will affect the performance of the instrument. Recall that the initial conditions used here simulate the creation of ions formed in a gasphase electron impact or laser ionization experiment. The desorption of ions from a surface using laser desorption or the matrix‐assisted laser desorption ionization (MALDI) technique can also be modeled using the appropriate ion distribution parameters. See the TOFSim User’s Manual for additional calculational details. Page | 8

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