HEL Turbidity Probe Manual

HEL Turbidity Probe Manual

Contents 1. Overview... 3 2. Set up of the reflection probe... 3 3. Calibration... 4 3.1. Calibrating with the analogue electronics... 4 4. Operation... 6 5. Data Analysis... 6 6. Typical data... 11 6.1 The recrystallisation of an indole from aqueous propan-2-ol... 11 6.2 The recrystallisation of 4-acetylbiphenyl from toluene... 12 6.3 The recrystallisation of L-Glutamic Acid in water... 14 6.4 The recrystallisation of L-Glutamic Acid in water using a Side Entry Port... 16 7. Frequently Asked Questions (FAQ)... 18 2

1. Overview A standard reflection probe is dipped into the reaction medium and changes in the amount of reflected light are monitored as the experiment progresses. In this way the probe can be used to detect changes in turbidity and its primary application is in detecting the on-set points of crystallization. In simple terms turbidity is a measure of relative sample clarity. Light is transmitted via a bundle of six fibres to the probe end where it crosses the gap to the reflector. The back reflected light returns into a seventh read fibre. The amount of reflected light is converted to a voltage signal and recorded by the WinISO software. When the light beam passes through a clear solution the light passes through relatively unobstructed (except for absorption i.e. colour) to the probe reflector and is reflected back into the read fibre of the probe. However in the presence of suspended particulates the light is scattered in all directions and the intensity of the light beam is reduced. This change in intensity and hence voltage signal is detected in the WinISO software. Absorption from dissolved substances can also reduce the intensity of the light beam and hence where possible colour should be taken into consideration during the calibration procedure. Please note that the calibration procedure, described below, can take into account the presence of colour although it will not be able to take into account significant changes in colour. However, any signal output changes due to changes in colour will generally be much less than those signal output changes due to changes in turbidity. 2. Set up of the reflection probe The gap length between the probe tip and the reflector should typically be 4 to 5mm. 3

3. Calibration Before every recrystallisation experiment is run the reflection probe must first be adequately calibrated. Calibration is necessary to ensure a satisfactory baseline and that there is an adequate change in voltage with change in turbidity. After the electronics have been calibrated the experiment can then be run either using the raw voltage output signal as the measure of turbidity (this is acceptable for a single channel, eg. Auto-LAB and Simular) or more commonly the semi-automatic calibration option in the software can be used for example, to set the turbidity over a 0 to 100 range. In this case go to the Analogue Channel Calibration window, select the turbidity channel followed by the Smart Calibration icon. Use the Smart Calibration facility and the two samples prepared previously (the neat solvent(s) as the zero level and the slurry as the 100 level) to calculate the gain and offset for the desired range. NOTE: Using the Smart Calibration facility is covered in detail in the control software manual. This equipment has been shown to operate with a wide variety of common recrystallisation solvents. However, it may be possible for an operator to be using a solvent that appears ultra clear and no stabile signal can be achieved. In this case try reducing the path length to the probe reflector. If still unsuccessful please consult the staff at HEL Ltd. 3.1. Calibrating with the analogue electronics Select the calibrations option from the Setup menu followed by the Inputs option to display the Analogue Channel Calibration window. Highlight the required turbidity channel and ensure an Offset of zero and a Gain of one. This is done manually via the keyboard. This allows the turbidity value to be displayed as a raw voltage value. The voltage signal operates over a 0 5 V range and hence saturation at the top end will be achieved at ~5.2 V. Saturation 4

at the bottom end results in the signal dropping to below zero. At this point the displayed voltage will rise rapidly to 9 10 V. Once the Offset and Gain have been set close the Analogue Channel Calibration window. Prepare two samples for calibration, one the neat recrystallisation solvent(s) and the other the recrystallisation solvent(s) and solid slurry. Immerse the turbidity probe in the neat solvent(s) ensuring that the gap between probe tip and reflector is completely covered. Allow the signal to stabilize. NOTE: It can be useful to log the calibration for future reference and this is done by selecting the logging data icon in the Main Mimic window. Select the display graph icon also and display the turbidity signal. Using the turbidity calibration dial on the electronics rack adjust the signal to approximately 1.0 V, allowing a short time for the signal to stabilize. Further adjustments of the dial will not be necessary. Next immerse the turbidity probe in the recrystallisation slurry and turn on the agitator to give good mixing. This is a check only, to ensure that the output signal increases from the baseline level set above. 5

4. Operation Once the calibration has been performed the experiment can then be run in the normal way using a WinISO experimental plan ensuring that turbidity has been selected as one of the parameters in the Output file. Note that the probe will respond to any variable that will cause a change in the intensity of the light beam. Thus, not only will the probe respond to changes in sample turbidity it will also respond to other physical effects such as bubbles in solution (this can be evident in aqueous systems) or absorption effects due to colour changes. 5. Data Analysis After an experiment the data file can be analysed in the normal way using HEL s iq software. However, there is a special extension to WinISO that enables the user to quickly evaluate meta-stable zone widths (MSZW) and solubility curves from the data. To access the turbidity analyser, start iq and load the data file. Then, from the menu bar, select Data and then Analyse Turbidity. The turbidity analyser screen will then be shown. The user will be asked to supply information to the analyser in order that the data can be processed properly. To move forward or backwards through the analyser the next and back buttons can be pressed or simply select the tab at the top of the window. Note that more tabs become available as more information is supplied. 6

Select Data Channels The first screen is the Select data channels tab. Available channels from the left can be dragged into the grid on the right for analysis (however, normally this will pick the correct channels by default). Also on the grid are entries that enable the user to specify specific regions of the experimental step that will be analysed: Min Time: This is a time from the beginning of the step that can be left out from the analysis in case the data in this region is noisy. Leave this as zero in order to analyse the whole step. Min / Max Temp: The temperature window in which the analysis will be done. At the bottom of the screen is the level of filtering that will be applied to the turbidity channel. Sometimes the turbidity channel needs to be filtered in order to prevent false positives. 7

Select Steps Here the user selects which steps in the experiment will be analysed. Simply drag from the left to the right or double-click on one of the steps to place it in the Selected Steps window. Under analysis mode, select the appropriate analysis method: Turbidity: Analyses the turbidity signal, when the threshold is exceeded then an event has occurred. The threshold can be set in the threshold box. ROC Change: Analyses the ROC (rate of change of turbidity) signal, when the threshold is exceeded then an event has occurred. The threshold can be set in the threshold box. ROC Peak: Analyses the ROC (rate of change of turbidity) signal and looks for the minimum or maximum value. At this point, it is deemed that an event has occurred. Turbidity Results 8

This shows the individual results for each reactor by selecting the appropriate tab. When an event has occurred in a step, the resultant temperature at that moment is displayed in the grid at the bottom left of the page along with information on whether the event was a dissolve or a crystallisation. By double-clicking on the temperature, the graph display is zoomed into the appropriate region of the event. The cross-hair will then show the point at which the event has taken place. This point can be edited by dragging the cross-hair to a different position, doing this will update the temperature in the grid accordingly. In the grid is an entry for the concentration. If a value of the concentration of the mixture in that step is entered into this box then the next screen becomes available. Solubility Curves and MSZW s This displays the solubility curve for each of the reactors along with MSZW s shown in the grid. 9

By pressing the Hide Fit button on the graph, the best fit line can be removed. The bottom right window shows the equation of the graph (y = mx + c) and also the correlation coefficient. The correlation coefficient shows how well the best fit line fits the actual data, a value of 1 would indicate a perfect fit. Pressing save allows this data to be saved as a text or csv file and pressing print will print a report to an attached printer. Curve Fit Report Shows the overall best fit and correlation coefficients for all of the reactors that have been analysed. Again, reports can be saved and printed by pressing the appropriate buttons. 10

6. Typical data 6.1 The recrystallisation of an indole from aqueous propan-2-ol The graph shown below (graph 1) plots the data for three different recrystallisation experiments. In each case the reflection probe was calibrated as described above and then the slurry heated to 70 o C, held for 5 minutes and then cooled. As the solid began to dissolve at approximately 35 minutes so the turbidity value begins to drop until the entire solid dissolves after approximately 45 minutes. After approximately 75 minutes solid begins to precipitate out and the turbidity value rises steeply. It should be noted that the resolution of the reflection probe was observed to be high in that the probe observed changes in turbidity before the change was visible to the naked eye. As can be seen from the graph, the reflection probe gives good reproducibility of results. However, the table below (Table 1) does show that the data gives some variation in the detected on-set temperature for crystallisation. For this system it was found that the detected on-set temperatures for the crystallisation varied over a 3 o C band. 11

Experiment On-set temperature Number for crystallisation 1 51.0 2 49.0 3 48.3 Table 1 On-set temperatures for the crystallisation of an indole 6.2 The recrystallisation of 4-acetylbiphenyl from toluene The following describes a typical recrystallisation experiment in detail: 1) Into a 50ml auto-mate reactor cell add 4-acetylbiphenyl (6.0g). 2) Into the reactor cell also add toluene (30ml) and then cover with parafilm to prevent evaporation. 3) In a clean beaker have some neat toluene available for use in calibration. 4) Go to Set up/calibrations/inputs to display the Analogue Channel Calibration window. Highlight the turbidity channel and if different enter an Offset of zero and a Gain of one via the keyboard. This allows the turbidity value to be displayed as a raw voltage value. Save the new values and close the calibration window. 5) Immerse the turbidity probe in the neat toluene ensuring that the gap between probe tip and reflector is completely covered. Allow the signal to stabilize. Using the turbidity calibration dial on the electronics rack adjust the signal to between 0.5 and 1.0 V, allowing a short time for the signal to stabilize. Further adjustments of the dial will not be necessary. 6) Immerse the turbidity probe in the recrystallisation slurry prepared above; agitation is required to give good mixing. Observe that the turbidity signal increases. This is a check only, to ensure that the output signal increases from the baseline level set above. 7) The recrystallisation experiment can be run using this raw value. However, typically the signal is then normalized (especially with multiple reactors) over 0 to 100 and this is done using the semiautomatic calibration option in the software. In this case go back to the Analogue Channel Calibration window, select the turbidity channel 12

followed by the Smart Calibration icon. Use the Smart Calibration facility and the two samples prepared previously (the neat solvent(s) as the zero level and the slurry as the 100 level) to calculate the gain and offset for the desired range. 8) Fully assemble the reactor cell into the auto-mate ready for the experiment to begin. 9) Write an experimental plan with the following steps: Reactor constant at 25 o C for 5 minutes Reactor heat ramp to 60 o C at 1oC/min Reactor constant at 60 o C for 5 minutes Reactor heat ramp to 25 o C at -1oC/min 10) Run the plan and plot the resulting temperature and turbidity data. It should look similar to the graphs below: 13

6.3 The recrystallisation of L-Glutamic Acid in water This experiment was originally taken from a study carried out using Raman s Spectrometry. The following scale was used: The reactor volume = 1000ml Amount of Liquid used = 1200ml just enough to cover the probe. Quantity of Glutamic Acid used = 50g The experiment was carried out on a 1 pot automate. The scale used was: The reactor volume = 100ml Amount of water used = 70ml more than enough to cover the probe. Quantity of Glutamic Acid that will be used = Xg Therefore for 1200ml use 50g and 70ml use Xg. 1200ml = 50g 70ml = Xg 1200mlXg = 3500 Xg = 2.9167g 14

Therefore 2.9167g of Glutamic Acid is needed. These can be discharged in three amounts of 0.972g to see the transition prior to crystallization. 3 T urbidity T est 2.dat 80 70 60 Turbidity 2 1 50 40 30 Reactor Temperature ( C) 20 Go To 70. 0 0 50 100 Crash Cool 150 200 Constant 10 250 Go To 70. Crash Cool Constant 10 300 350 400 450 500 550 Time (mins) R4:Reactor Temp Turbidity Go To 70. 600 650 700 Crash Cool 750 800 Constant 10 850 Go To 70. 900 10 0 The above graph represents 2.9167g of Glutamic Acid in 70ml of Distilled water. The plan involved taking the reactor temperature to a constant 70ºC for 2hrs, then a slow cool down at 0.1ºC/min to a reactor temperature of 10ºC. This was cycled three times in order to establish consistency at the point of recrystalisation. The lower baseline is not as consistent as the upper baseline but on the third cycle a stable lower baseline is present. 15

6.4 The recrystallisation of L-Glutamic Acid in water using a Side Entry Port Using water, as a solvent does not give the best results when the turbidity probe is insert vertically. It was found that when the probe was inserted horizontally, through a side entry port, the results were much improved. Using this idea we were hoping that the reoccurring problem of bubbles sticking to the surface of the light source would be eliminated or reduce. The only problem with inserting the probe horizontally was the stirrer would be obstructing it. Therefore a slight modification was made to the length of probe ensuring enough space between the tip of the probe and the stirrer it self. The mirror attached to the probe was made certain that it would be facing vertically up in order to eradicate the possibility of bubbles sticking to the body of the mirror and therefore eventually sliding/accumulating on the probe/mirror. The results from the experiment (see turbidity with new side port) showed a great improvement on the previous attempt 4 T urbidity T est with New side port - Crash Cool + Slow Cool.dat 80 70 3 60 Turbidity 2 50 40 30 Reactor temperature ( C) 1 20 Go To 70. 0 0 Crash Cool 200 Constant 10 Go To 70. Crash Cool 400 Constant 10 Go To 70. 600 Crash Cool Constant 10 Go To 70. Crash Cool Constant 10 Go To 70. Crash Cool 800 1000 1200 Time (mins) R4:Reactor Temp Turbidity Constant 10 Go To 70. 1400 Crash Cool 1600 1800 10 0 2000 The first experiment was done for a crash/fast cool to achieve alpha L- Glutamic acid. The baselines all reached the specified set points with steady lines. The point at which they come out of solution and form the polymorph remained constant/same every time. 16

Although it was a great improvement, the fact is you can t eliminate all the bubbles; there were a couple of fluctuations in the baselines. The second experiment was to do a slow cool using this side probe, the results were reasonable but not as had hoped. 17

7. Frequently Asked Questions (FAQ) Question: The turbidity reading does not fall/rise to the same value as in a previous experiment. Answer: Many things can affect the absolute turbidity value that a probe reports. Eg. An unclean mirror or probe. When looking at the data it is important to remember that the turbidity probe is designed to detect points of solubility and onsets of crystallisation. Therefore we are looking at when a point-to-point change occurs and not the actual turbidity value that the probe goes to. A typical real-world data plot is shown below: Note that although the turbidity value when the mixture is in solution varies between cycles, the point at which the mixture enters the solution and then crystallises out is clearly seen as shown by sharp rises and falls in the turbidity value. See the plots for the question when can a solubility or crystallisation event be judged to have been completed. In a similar way, one turbidity probe cannot be directly compared to another due to variations in construction, mirror-gap length, cabling and electronics. If a comparison between probes is desired then the software needs to be setup to adjust probe signals to read-world values. See the manual for details on calibrating analogue inputs. 18

Question: The sensitivity of the probe appears to be reducing, how do I clean it? Answer: It is important to ensure that the probe tip and the mirror are correctly cleaned. It is advisable to do this before each run. Cleaning the Probe 1) Remove mirror (if used) by loosening the grub screw (if available). Do not remove the grub screw from the thread, as it can be difficult to put back on afterwards. Alternatively, some turbidity probes ship with threaded probes and mirrors; in this case, simply unscrew the mirror. 2) Rinse the probe with solvent. 3) OPTIONAL: Use a soft tissue/cloth to gently wipe the tip of the probe. 4) Leave solvent to dry out. 5) Check probe tip for a solid residue/film that was left behind by materials in the solvent solution. Repeat process as necessary. Cleaning the mirror 1) Remove mirror as outlined above. 2) Place mirrors in a beaker of suitable solvent and ultra-sonicate for 10 minutes. 3) Remove solvent and replace with clean. 4) Repeat ultra-sonication. 5) Remove mirrors and dry. Question: I am observing unusual changes in turbidity similar to what is shown in the graph below. What is causing it? 19

This can include spiking of the turbidity value over short time periods. Many things can cause what we are seeing above which is a change in the normal profile: 1) Is the experiment aqueous based? If so there could be an air-bubble lodged on the probe tip that was left there while the reactor was set-up. Try and ensure air-bubbles are removed during the set-up process simply by shaking the vial. 2) Are you using low volumes with high stirring speeds? If so, the agitator may be producing a vortex that pushes the liquid out to the side of the reactor tube. Consequently the liquid level in the middle of the tube drops which could result in the probe tip coming out of the solution. 3) The probe tip/mirror may be dirty. Follow the protocols mentioned in this FAQ for cleaning the tip and mirror. 4) The small space between the probe tip and the mirror may have become lodged with a group of crystals that have clumped together. This can be especially true of materials that have just come out of a storage container. Try crushing or milling the material before loading into the vessel or have a pre-run experiment where the mixture is heated-up so that the solid eventually goes into solution. On cooling down the crystallised material will be more homogenous. 20