AutoSampler for Liquid Samples

Setup and Software Instruction

At 12-ID-B, we use a in-house automatic sample changer (or autosampler) for high throughput solution/bio-SAXS/WAXS measurements. The autosampler consists of three linear slides (X-, Y- and Z-) to position the samples and the sampling needle, and a 6-valves tricontinent syinge pump to drive the liquids.

A python-based software with friendly graphic user interfaces (GUI) has been developed to control the syringe pum, XYZ slides and data collection.12-ID-C runs a different autosampler but with a similar software control.


Here are the instruction on the autosampler setup and software operation.


0. AutoSampler Setup

Fig 1. The AutoSampler at 12-ID-B, running a 96-well plate.

Fig 2. AutoSampler running 32-tube rack.


Autosampler:

  • Fig 1. The autosampler with 96-well plate.
  • Fig 2. The autosampler with 32-tube rack.
  • X-,Y--Z-slides position samples and the sampling needle.
  • The cooling base conecting to a water chiller.
  • Two type of sample racks for: standard non-skirted 96-well plates(Fig 1), and 0.5ml tubes(Fig 2).

    • Syringe Pump:

  • The syringe pump has 6 valves connecting to: valve#1. buffer; #2 flow cell; #3 water; #4 bleach; #5 TBD; #6 waste bottle.
  • Standard cleaning/washing liquids are water and 30% bleach, both connected to syringe pump.
  • Communication: RS232 cable.

    • Quartz Capillary for Flow Cell:

  • Diameter: 1.5 - 2.0 mm
  • Wall thickness: ~10um .

    • Tubing length:

  • Flow Cell end: 30cm
  • Needle end: 45 cm .
  • Draw sample volume: 450 uL.
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    1. Start AutoSampler program on Purple

    Fig 4. Start AutoSampler program.

    Fig 5. Example AutoSampler GUI screen.


    Fig 6. Example AutoSampler print out in the linx terminal.


  • [For staff] Connect motor cables, chiller water lines, syringe cable, flow cell tubing, etc.

  • Find the existing "Autosampler" terminal, or open a new linux terminal on Purple (Fig 4).
  • Enter the autosampler folder, and run "./runAutoSampler" as shown left to start the Autosampler program.
  • An Autosampler GUI will pop-up like below (Fig 5).
  • During the Autosampler operation, it will print out messages which are useful for knowing the status of the autosampler (Fig 6).
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    2. Syringe Pump Setting and Operation

    Fig 7. Syringe pump operations



    Fig 7. Syringe pump operations (duplicate)

    Parameters of Syringe Pump

  • Speed: 0 - 250 uL/s
  • Volume: 1000uL; 1000uL/cycle in Prime
  • Vavles: 6

    • Prime

  • Flow direction: Sampling needle --> flow cell --> valve#2 --> syringe --> valve#6 --> waste bottle.
  • When use: Empty(or washing) the flow cell.

    • Wash

  • Flow direction: Washing liquids(water/bleach) --> syringe --> flow cell --> sampling needle --> waste well(in washing station).
  • Automatical washing sequence per cycle: 1 mL water, 2 mL bleach, then 3 mL water.
  • Buffer can be used to wash the flow cell after N cycles of water-bleach-water washing.
  • When use: Washing the flow cell
  • Setting(speed/cycles/buffer) of automatic washing will be used when automatical sample running.
  • !!!Make sure the sampling needle at the washing station!!!

    • Running Samples

  • Here set the parameters of operation sequence for running one sample.
  • Setting here will be used for all samples when run multiple samples automatically from Sample Rack page.
  • Draw Air to seperate the sample and rest liquid in the tubing.
  • Draw Sample to the flow cell.
  • Shake/flow sample to reduce radiation damage. 8-9 sec / repeat for shaking 40uL at 10uL/s.
  • Auto/M Return automatically or manually return specified amount samples to the sample tube.
  • Manual Return manually return specified amount samples to the sample tube.
  • Collect Data? if ON, data will be collected with the command in the box, e.g."sleep(3); takeshot s1 1.0 30 3.0".
  • When run multiple samples from Sample Rack page, data collection command will be generated from Sample Rack page, not here.

    • Syringe/Valve Position

  • Check syringe position, the info of syringe and valve position will be display in the white box.
  • Change valve position: Select position L1-L6 first, then click Change.

    • Tweaks

  • Tweak+ is drawing liquid towards the syringe through a certain valve
  • Tweak- is pushing liquid away from the syringe.
  • Combining Tweak and Change Valve, one can manually do various operations on the liquid samples.

    • Commands: & Execute

  • for software developers's use, needs knoweldge on pump python library.
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    3. Autosampler Needle and Rack Position Calibration

    Only for staff and experienced users's operation

    Figure 8. Rack and Needle configuration

    Sample Rack Motor Operation

  • srx, sry, srz are the X-, Y- and Z-linear slide motors.
  • Pos: current position.
  • Move to: type in the desired position and enter to move motor to the desired position.
  • Tweak step: tweak motor position.
  • Reset To: reset current motor position to ZERO.
  • A (0,0,0) position is marked on the autosampler. combine above motions to calibrate motor/rack position.

  • srz_low#1: needle Z-position to pick up samples.
  • srz_low#2: needle Z-position to return samples.
  • srz_high: needle Z-position to freely move around without clashing with tubes, etc.li>
  • go to srz_XXX: function bottun to move to the position.

    • Cleaning Station Location

  • Define the positions of Waste Well and two clean rinse water, and function buttons.
  • Clean rinse water helps remove droplet and clean needle tip.
  • Bottom and surface position in blue color just reference. need to recalibrate at the beginning of experiment.

    • Plate Configuration

  • Define the poistion (X,Y) of first sample on 32-tube plate or 96-well plates or 60-vial plate(12-ID-C).
  • Those position values are relative to the (0,0,0) mark.
  • To change the poistion values, type in and click "Save Config" to change the confirmed values.
  • Only the confirmed values will be taken by the program and used in the rack position configure.
    •

    4. Autosampler Sample Operation

    Fig 9. Sample Operations.


    Load correct plate (1-3)

  • 1. Choose correct plate, 32-tube plate or 96 well plate. 96 well palte #1 is the default.
  • 2. Click "Load Plate Config" and poistion parameters will be loaded and displayed in area 3. Plate Position Config.
  • And corresponding sample parameters (e.g., xPos & yPos etc) will be loaded in 4-11.

    Sample Parameters (4-11)

  • 4. Sample index, and row and col position.
  • 5. Sample name, most speical characters like whithspace, and "/\&*@#$~^*()!%=+?<>{}[]|,:;" are not allowed in file name. "_" is ok.
  • 6. Exposure time in seconds.
  • 7. Period time in seconds.
  • 8. Number of images to collect.
  • 9. xPos yPos: X,Y position relative to the first sample (in mm).
  • 10. wash after? Whether needs to wash the flow cell after this current sample. Choice "y" and "n".
  • 11. Collect? Whether need to run this current sample. will be skipped if "n".

    • Utility Functions on Sample Parameters(12-14)

  • 12. "Save Sample File" : save sample parameters in above table into a file for later use.
  • 13. "Load Sample File": load sample parameter file to the above table.
  • 14. "from WebDB": Only for 96 well plate. Load sample parameter file from the website database.
  • 15. "Change Expt": Change exposure time for all samples.
  • 16. "Period": Change period time for all samples.
  • 17. "Imag#": Change number of image to collect for all samples.
  • 18. "Verify Table Params": validate xPos & xPos values. Invalid position value will be in red.

    • Convienient Function Buttons (19-20)

  • 19. Function buttons for three needle positions.
  • 20. Function buttons for Waste well and rinse waters.
  • Note: If use semi-manual washing, remember rinse the needle before running samples.

    • Sample Operations(24-30)

  • 24. Sample number sequence to run. Can be single, multipe, or a combined number sequence seperated by "," and/or "-".
  • For examples: (a) 2 (#2 only); (b) 2,3,6(three samples to run); (c) 2-10 (from #2 to #10); (d) 2,5,8, 10-22,2,25,27-32 (multiple).
  • 25. "Run sample #": click to run the sample sequence in listed 24.
  • 26. "Go to Sample": Needle moves to the position (X-Y only) of the sample (ONE number) specified in 24.
  • 27. "Run All/Selected Samples #": Not fully function yet, Don't use now!.
  • Here how samples be run: (1) running by the number squenece; (2) for every sample, needle comes to the specified position (xPos, yPos, srz_low_xxx); (3) draw air & sample, shake/flow sample and collect data at the same time; (4) after data collection, samples will be returned; (5) if "wash after"="y", needle will move to Cleaning station for flow cell washing and needle rinse twice; (6) move to the next sample; (7) repeat 2-6 until all specified samples done.

  • 28. "Pause": pause the autosampler after finishing the current line.
  • 29. "Resume": resume the operation.
  • 30. "!!Stop!!": Stop the sample operation after the current line and cancel the rest samples.
  • Note: When "!!Stop!!" operation, wait for enough time to let all running processes finish. Otherwise could damamge the needle!!

    • Message on Autosampler Status (31-32)

  • 31. Syringe and valve position.
  • 32. Message on autosampler processes. More printing-out info can be found in the linux terminal.

    • Other Useful Functions (21-23)

  • 21. If checked, last bit ( amount of "Manual Return") of volume will be dumped into the Waste well.
  • 22. Wait time (in seconds) before running. Can be set as equlibrium time in temperature study.
  • 23. Wait time (in seconds) between samples.
    •

    Additional instruction videos available:


    Fig 4. Start Autosamper.


    Fig 7. Syringe pump operations (duplicate)


    Fig 8. Syringe pump operations (duplicate)


    Fig 9. Sample operations

    1. Start AutoSampler

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    • 2. Update Parameters

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    • 3. Semi-manual Flow Cell Washing

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    • 4. Run Samples

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