Sensor Calibration - Materials & Procedure

The CINRG CS-APC-2 can be calibrated on-site by a laboratory technician with the correct hardware and standards in place by following the calibration procedure outlined in the CINRG CS-APC-2 manual, or by following this procedure;

Equipment & Standards Required 

In order to calibrate the CS-APC_2 particle counter for particle sizes between 4µm and 100µm the following equipment and calibration standards will be required.

1. Two lengths of PTFE delivery tubing approximately 40 cm in length and having an outside diameter of 3.2 mm (0.125”) and an inside diameter of 2.0 mm (0.08”). One end of the tubes should be flanged and contain a washer and a tube nut (preferably acetyl) and the other end should be open.
2. A magnetic stirrer, a 25 mm (1.0”) x 8 mm (0.3125”) magnetic follower and a spare magnet for magnetic follower retrieval. (A CINRG Magnetic stirrer with rare earth magnets is recommended).
3. Four clean 250 ml containers, preferably clear glass beakers.
4. 2 ml Plastic Pipette
5. XL Calibration Template for data importation.
6. Super Clean Fluid – about 500 ml (http://www.scpscience.com/en/products/details?id=150-701-003&name=partistan-super-clean-fluid)
7. 2806 Calibration fluid traceable to NIST 2806b - 450 ml bottle (https://www.scpscience.com/en/products/details?id=150-701-001&name=partistan-2806-calibration-fluid)
8. Aqueous Concentrate of nominal 40µm PSL mono-spheres (15 ml).
9. Aqueous Concentrate of nominal 70µm PSL mono-spheres (15 ml).
10. Aqueous Concentrate of nominal 100µm PSL mono-sphere (15 ml).
11. Clean Iso-Propanol or similar water masking agent.

A Note on Calibration & Required Fluids 

You can calibrate the instrument and verify the accuracy of the calibration on-site.  The biggest issue is if you purchase the standard calibration fluids the cost is approximately $4000.00 initially and then an additional $3000.00 every time you calibrate the instrument thereafter.  The 10µm, 40 µm and 70 µm PSL fluids comprise the bulk of this cost ($3000.00) and can only be purchased as a kit with one 400ml bottle of each size. The main issue here is although there is enough fluid for several calibrations, the fluids only have a shelf life of 90 days which essentially means they are only good for one calibration. 

CINRG recommends that you purchase concentrated aqueous suspensions of the PSL spheres from ThermoFisher Scientific (https://www.thermofisher.com/order/catalog/product/4270A) and make your own dilutions by adding a few drops of concentrate to 200 ml of the super clean fluid. The concentration of the spheres in the calibration fluids is not critical as they have a narrow size distribution so you are essentially only looking for the maximum value in a normal distribution and only need enough counts to be able to do this reasonably accurately. The aqueous concentrates are supplied in 15 ml bottles and have a shelf life of 2 years or so if they remain uncontaminated and are kept in a fridge.  They come with certificates stating NIST traceability. As you only need 3 or 4 drops of concentrate to make 200 ml of standard there is far more concentrate in one bottle than you will ever need in a two year period especially with only one instrument to calibrate. The PSL concentrates cost about $500.00 per size and you need a nominal 40µm and a nominal 70µm concentrate. The 100µm concentrate is only needed to determine sensor resolution and therefore not required for the sizing calibration. 

CINRG recommends following standards (item 3 is optional).  CINRG recommends 1 gallon of super clean fluid as it is can be used for several years.  The cost of these fluids is approximately $2000.00 but the fluids purchased should be good for at least three calibrations within the two year shelf life.   

1. RM 2806 secondary calibration fluid. See  - http://www.scpscience.com/en/products/details?id=150-701-001&name=partistan-2806-calibration-fluid.
2. Super clean fluid. See - http://www.scpscience.com/en/products/details?id=150-701-003&name=partistan-super-clean-fluid.
3. UFTD fluid (This is only needed if you want to verify the calibration however this is not a requirement of the sizing calibration procedure). See - http://www.scpscience.com/en/products/details?id=150-701-005&name=partistan-uftd-nist-8632-std100mgl.
4. Nominal 40µm PSL spheres (4240A). See - https://www.thermofisher.com/order/catalog/product/4240A.
5. Nominal 70µm PSL spheres (4270A). See - https://www.thermofisher.com/order/catalog/product/4270A. 

The Super clean fluid has OT surfactant added to it when it is prepared and the surfactant is supposed to eliminate the effects of the water from the aqueous concentrates of the PSL spheres. In practice this takes a very long time to take effect even with vigorous stirring but if you add 8 ml of clean Isopropanol after adding the concentrate to the 200 ml of SCF the water disappears within minutes and you can run the standard almost immediately. 

Calibration requires an ultrasonic baths, a magnetic stirrer, a magnetic follower and a loose magnet in addition to the calibration fluids in order to calibrate the particle counter. The loose magnet is needed to recover the magnetic follower from calibration fluids once they have been used.  You will also need a computer with Microsoft Excel in order to determine the calibration points from the raw count data.

Sensor Calibration – Procedure 

1. Preparation of the system for calibration. 

• Remove the waste line from the sensor outlet and the delivery tube to the left hand port of the valve on the sample syringe and replace them will the 40 cm lengths of delivery tube mentioned above.
• If the system has been in use for several months or has run severely degraded samples then the sensor should be cleaned with Citrajet prior to calibration. To clean the sensor place the ends of both open ends of the delivery tubes into a small beaker containing a warm dilute solution of Citrajet (50% V/V). Then use the Gilson utilities to program the sample syringe to pump warm Citrajet solution back and forth across the sensor for several minutes or alternatively connect a manual syringe directly to the sensor inlet to do the same without software. Leave Citrajet in the sensor cell for 15 minutes before flushing it out first with water, then with Isopropanol and finally with heptane or hexane. 

2. Preparation of PSL Mono-Sphere Fluids – three fluids are required – 40µm, 70µm and 100µm.
   • Pour about 150 ml of Super Clean Fluid (SCF) into a clean clear glass 250 ml beaker and add a clean magnetic follower (Spin Bar).
   • Place on a magnetic stirrer and stir at sufficient speed to create a small shallow vortex.
   • Vigorously shake the 15 ml bottle of aqueous concentrate of the PSL mono-sphere standard by hand for about a minute or preferably mix it on a vortex shaker for half this time and then place it in an ultrasonic bath for about 10 seconds.
   • Add the standard drop wise to the super clean fluid using the table below as a guide.

• Add 2 ml of isopropanol to the fluid in the beaker with a 2 ml plastic pipette to start dissolving the water that was introduced with the PSL mono-sphere concentrate.
• Allow the fluid to stir for a few minutes and then repeat the process with additional 2 ml additions of Isopropanol until the fluid clarifies. The fluid will become a little cloudy on the first Isopropanol addition and then start to clear with further 2 ml additions (None of the fluids should not need more than a total of 12 ml of isopropanol to completely clarify).
• Once the fluid has clarified allow it to stir for a further 20 minutes before it is used as a calibration fluid. It is useful to pour some Super Clean fluid into a beaker to compare the clarity of this fluid against the freshly prepared standard and there should be no discernable difference in the appearance of the two fluid.

3. Creating Folders for the calibration Data.
   • Before starting the calibration procedure it is recommended that a convenient set of folders be created on the computer systems hard disc for the convenient storage and retrieval of data files.
   • An example of a suitable folder hierarchy is shown below.

•  Calibration Data
  • 2018
    • 2806 Data
    • Sensor Noise Data
    • PSL Data
      • PSL40
      • PSL70
      • PSL100

4. Sensor noise determination.
   • Pour about 150 ml of super clean fluid into a glass beaker and place the open end of the delivery tube connected to the sample syringe into the fluid in the beaker.
   • In the particle counter software select the “Run Standard Fluids” button and then select the “Pre-Flush the syringe with standard Fluid” button and set the counter to “5” times before implementing a flushing cycle.
   • Close the window after the 5^th flush has completed and then select the “Testing Panel” button.
   • In the testing panel select the “Particle Counter” tab and set the measuring time to 40000msec (40Second).
   • Click on the “Start Measuring” button to implement a 40 second count on the stationary, clean fluid that is in the sensor cell.
   • When the count is complete the “Save Data” button will turn green. Click on this button so save this data to a text file.
   • Repeat the “Start Measuring” and “Save Data” steps until a total of twelve data files have been saved.
   • These 12 files are saved as text files in the main application folder with a date and time stamp as their file names. Cut and paste these files to the “Sensor Noise Data” folder.
   • Import these data files from the “Sensor Noise Data” folder into columns A to L of the “Noise” spreadsheet within the calibration workbook provided.
   • Look down column Y of this spreadsheet after the data has been imported to find the first value that is less than 1.0 and note the values in Columns Z and AA immediately adjacent to this value. These two values are the Noise threshold channel number and its value in millivolts (mV) respectively. The mV value is the current noise level of the sensor as defined by ISO 11171.
   • Record the noise channel number in cell M33 of the “PSL40GRAPH” spreadsheet as this value is needed in the moving window or half count method when determining the threshold settings for mono-sphere calibrations.
5. Determination of PSL mono-sphere calibration points.
   • Add a clean magnetic follower (Spin bar) into the beaker of freshly prepared and clarified PSL40 standard and place the beaker onto the magnetic stirrer.
   • Transfer the delivery tube that connects to the sampling syringe from the super clean fluid to the beaker of PSL40 fluid standard on the magnetic stirrer.
   • Position the open end of the delivery tube close to the side of the beaker and 2 to 3 cm from the bottom.
   • Plug the stirrer power cord into the motor socket on the back of the Motor Control Interface (MCI) unit after unplugging the cord for the stirrer on the sampling head.
   • Open the Testing panel, select the “Liquid Handling System” tab and set the stirrer speed to 1 to activate speed control through the knob on the front panel of the MCI unit.
   • Close the testing panel window with the “X” control. Closing the window with the close button will set the stirrer speed back to “0” and the stirrer will turn off.
   • Adjust the knob on the MCI unit until the stirrer creates a small vortex on the surface of the fluid in the beaker.
   • Click on the “Count Standard Fluids” button and use the “Pre-flush syringe with standard fluid” button in the window that opens to implement two flushes with the PSL standard.
   • When the second flush completes transfer rinse the outside of the waste line with clean solvent and place it into the beaker containing the PSL standard.
   • Set the sample volume to 20 ml and the repeat measurements option to 5 times and then start the measurement sequence.
   • A data file is saved in the principle folder each time a measurement is completed.
   • When the fifth measurement completes, cut and past the five data files from the principle folder to the PSL40 folder.
   • Import these data files from the PSL40 folder into columns C to G of the “PSL40DATA” spreadsheet within the calibration workbook provided.
   • Select the PSL40GRAPH spreadsheet and from the Counts vs Channel Number plot, estimate the channel number that corresponds with the apex of the peak displayed.
   • Enter this estimate into Cell E36 (40µm Threshold Channel) and note the value displayed in cell E39.
   • Adjust the estimate in cell E36 with small increments or decrements until the value of E39 is minimized. Ideally the value of E39 should be zero but in practice it will have a small positive of negative value as its minimum value. The value of E36 that yields the smallest value for E39 is the threshold setting for the PSL40 standard.
   • Before conducting any measurements on other calibration fluids flush the sensor with super clean fluid a minimum of 10 times to remove all traces of the current calibration fluid from the sensor and the delivery tubes.
   • The PSL70 and PSL100 standards are measured in the same way as the PSL40 fluid but using the spreadsheets in the calibration workbook dedicated to these two standards.
   • When the three standards have been measured open spreadsheet “38-70-100” and adjust the values in cells B5 to B7 to the actual sizes of the mono-spheres of the three PSL calibration standards. These values have to be obtained from the certificates supplied with the aqueous concentrates.
   • Finally edit the equation in cells G5 to G7 so that it is the same as the equation displayed on the Channel # vs. Particle Size µm plot.
   • The threshold settings for 38µm, 70µm and 100µm are displayed in cells H5 to H7.
   • The 100µm setting is not normally required but 100µm data is collected to allow a PSL curve to be plotted. If only a nominal 40µm and nominal 70µm standard are measured the threshold setting for the actual 38µm and 70µm particle can only be determined from a linear plot between two points.
6. Determination of calibration points using PartiStan secondary 2806 calibration fluid.
   • Flush the sensor several times with super clean fluid.
   • Shake the calibration fluid vigorously by hand for at least two minutes and then place it upright in an ultrasonic bath briefly to degas the fluid. The sample will clarify from the base upwards and should remain in the bath only until the clarified layer reaches the surface of the fluid. Repeat this process a second time.
   • Hold the bottle on its side and roll it several times to help breakdown the large bubbles that remain on the liquid surface after removal from the ultrasonic bath.
   • Immediately after rolling the fluid, place a clean magnetic follower (Spin bar) into the bottle and place the bottle onto a magnetic stirrer and adjust the stirring speed to create a small vortex on the surface of the fluid.
   • Rinse the delivery tube that is connected to the sample syringe with clean solvent and after shaking it to remove an excess solvent, place it into the calibration fluid. It should be position to draw fluid from about 15 to 20mm above the bottom of the fluid and not interfere with the magnetic follower.
   • Use the “Measure Calibration Fluid” option in the particle counter software to flush the sensor five times with the calibration fluid.
   • When the flushing cycles have completed, rinse the outside of the waste tube from the senor outlet with clean solvent and place it into a clean bottle to collect the calibration fluid that will pass through the sensor during the collection of the calibration data. This fluid can be subsequently analyzed as a sample to check the validity of the calibration process.
   • Set the sample volume to 20 ml measurements and the repeat option to six times and then start the measurements.
   • When the sixth measurement is completed cut and paste the six data files from the application folder to the “2806 data” folder.
   • Import the data for the last 5 measurements into columns A to E in the” RM2806B_Data” spreadsheet. This data should be imported starting from the second row of each column as the first column of each row in this spreadsheet has a column heading.
   • When all the data is imported, column “S” of the spreadsheet will contain the average cumulative counts per ml for each of the 4096 channels. For particle sizes greater than 4µm, 6 µm, 14 µm and 21µm respectively match the certified counts published on the certificate for the calibration fluid with the closest count values in column “S” and note the channel number that yields this count. This can be done for both the µm(c) and µm (b) values as both sets of count data are published on the Partistan certificate for their calibration fluid. The µm (b) counts are in the first column of the table on the certificate and the µm(c) are in the last column).
   • In the spreadsheet “Calibration µm (b)” record the certified µm (b) counts for particles greater than 4µm, 6 µm, 14 µm and 21µm in cells 06 to 09 and record the channel numbers that yielded the closet counts to these values in cells B6 to B9.
   • Repeat this process in Spreadsheet “Calibration µm(c)” with the corresponding um(c) data.
   • Edit the formulas in both spreadsheets in cells C6 to C9 through to cells G6 to G9 so that the correct data is displayed in the table for the five measurements. E.g. If the channel number in cell C6 is 104 then the count data for the five measurements for this channel are in cells F105 through J105 in spreadsheet “RM2806b_Data”. The row number is one higher than the actual channel number because the data is displaced by one unit owing to the presence of the column headings.
   • The channel numbers in the µm(c) and µm (b) tables are transferred automatically to the “Summary” spreadsheet of the calibration workbook where the mV thresholds values are calculated from the channel numbers. The mV threshold values can be entered into the applicable calibration parameters of the particle counter software.
7. Calibration Verification.
   • The collected 2806 waste or some of the remaining fresh calibration fluid can be run as an undiluted sample on the particle counter to verify the newly entered calibration data.