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Stable Isotope Laboratory
Earth and Planetary Sciences
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LC Isolink Liquid Chromatography Operation

LC Startup Overview

  1. Degas all reagents and solvents (HPLC grade water).
  2. Set oxidation oven to 99.9°C.
  3. Start MS pump at 500 µl/min and:
    1. Check waste mobile phase is pumping out liquid.
    2. Check the backpressure (should be > 80 bar, but may be as low as 60 bar).
    3. Inspect lines to ensure there is no leaking
      1. Check for air bubbles (indicative of a leak).
      2. Check CO2 background (high values can = air leak).
  4. Turn MS pump to 50 µl/min.
  5. Turn to purge on Isolink (software).
  6. Purge Acid Pump
    1. Turn on acid pump to 500 µl/min, pause and then to 1000 µl/min.
    2. Check that you have flow to the purge line exiting the back of the Isolink.
    3. Check for leaks in the pulse damper and acid pump.
    4. Then, slowly take the acid pump up to 2950 µl/min flow.
    5. Hold at 3000 µl/min flow for 3 minutes (use timer).
    6. Slowly (over 1-1.5 mins) ramp down the pump at intervals of 1000 µl/min to 50 µl/min
  7. Purge Oxydation pump
    1. Turn on acid pump to 500 µl/min, pause and then to 1000 µl/min.
    2. Check that you have flow to the purge line exiting the back of the Isolink.
    3. Check for leaks in the pulse damper and oxydation pump.
    4. Then, slowly take the acid pump up to 2950 µl/min flow.
    5. Hold at 3000 µl/min flow for 3 minutes (use timer).
    6. Slowly (over 30-45 secs) ramp down the oxydation pump at intervals of 500 µl/min to 1500 µl/min.
  8. Run both the oxidation pump and acid pump together at 1500 µl/min flow for 1 min in purge mode.
  9. Return flow of both pumps at 50 µl/min for normal operation.
  10. Switch from purge mode to Isolink.
  11. Set MS pump to 300 µl/min.
  12. Monitor the mass 44 cup for 1 hour.
    1. Background should be < 2V.
    2. Make sure open split is open (on).
  13. Check backgrounds
    1. Argon < 9V with open split in Cup 3.
      1. High Argon indicates a system air leak.
    2. Water 12V (Mass 18 in Cup 3). If high:
      1. Suggests improperly functioning gas dryer.
      2. Defective membrane within separation unit.
    3. Oxygen <10 V with open split in (select O2 from bottom tab in Isodat, Mass 32 Cup 2).
      1. O2 background reflects the concentration of oxydizer pumped into the LC Isolink. The goal is about 9V as a sufficient amount of oxydizer for the sample.
      2. With low oxidation and acid pump flow rates it takes approximately one hour for the system to reach equilibrium.
  14. "Five Minute Test" – to be done once the above background levels are achieved.
    1. Open Isodat Instrument Control software module.
    2. Select timescan in pull down menu: Options = 300 sec: Start Scan: Monitor Mass 44
      1. Click box (next to Mass 44 on graphic) Note software bug so may be Mass 28.
      2. Select Mass 44 column and calculate
      3. Want standard deviation < 10 mV (< 3 mV for a good system).
      4. Want slope < 0.01 mV/s.

Note: When operating: Total combined flow of MS pump, oxidation pump, and acid pump cannot exceed 700 µl/min. Over-pressuring the line can damage the Isolink separation unit.

Isolink Operational Settings

Reference CO2 0.9 bar
He Purge 1.6 bar
He Carrier 1.6 bar
Reference Purge Off

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LC Shutdown Overview

  1. Flush all pumps with degassed water
    1. In practice do the reverse of the priming steps
      1. Flush oxidation pump at 2950 µl/min for 3 minutes in purge mode.
      2. Flush acid pump at 2950 µl/min for 3 minutes in purge mode.
      3. Then run both the oxidation and acid pumps for 1 minutes while both pumps are set at 1500 µl/min flow, in purge mode.
    2. Set oxidation pump and acid pump to 50 µl/min and run through the Isolink plumbing.
    3. Set the MS pump to 600 µl/min and run for 15 minutes. Check the pH of the effluent to the waste jar. It should be ~ pH=7.
  2. Back flush the Piston Pump
    1. Press "manual" button for Piston flushing the oxidation and acid pumps. Rinse with degassed water for 5-10 seconds once liquid is in the lines.
  3. Turn oxidation oven to 30°C.

************************************************************************

Maximum Flow Rate (Pumps)

  1. When purging the interface (oxidation and acid) pumps: ² 3000 µl/min.
  2. For interface (oxy, acid) pumps to interface: ² 200 µl/min.
  3. Total flow (HPLC + interface pumps): ² 700 µl/min.

 

************************************************************************

Detailed LC Instructions

 

  1. Preparing reagents and degassing the solvent (water).
    1. Degas HPLC grade or MiliQ water, and reagents (oxidative and acid/catalyst) in an ultrasonic bath whist under house vacuum for 2 hours (or until bubbles stop forming in the water when swirled).

                                         i.    Slight Vacuum: Apply a slight vacuum.

                                        ii.    Ultrasonic Bath: Treat the reagents in an ultrasonic bath at this slight vacuum and 30 ¼C-40 ¼C. CO2 dissolved in the liquid will thus be volatilized. As both oxidation reagent and acid/catalyst mixture are acidic media, nearly no CO2 will be re-dissolved.

                                        iii.    Helium Stripping: As a protective measure, perform a slight helium stripping using e.g. a capillary that is directly introduced and continuously fed with helium. The helium atmosphere prevents re-gassing, and dissolved CO2 will thus be stripped out of the liquid. The same procedure is valid for the water at the HPLC pump, which even is already equipped with a degasser: in addition to this degasser, it is necessary to degas the water before use (first via slight vacuum followed by ultrasonic bath and concluded by helium stripping). Leave the water under a helium atmosphere by providing a constant helium stream.

    1. Note. Change the water at least every three days. Additionally, slightly acidify it to eliminate possible organic material via protonation.
  1. Preparing & maintaining the equipment
    1. Purging HPLC Pump and HPLC Pulse Damper

                                          i.    Backpiston Flushing of HPLC Pump

                                         ii.    Bacteria Note: If the CO2 background signal nevertheless is still considerably high after purging with water, eliminate remaining bacteria by oxidation: therefore, purge the HPLC unit including the HPLC pulse damper with oxidation reagent daily before measurement. A lower concentration of M2S2O8 (M+ = Na+, K+, NH4+) than that used for measurements is sufficient, e.g. 50-100 g/l. Finally purge several times using pure water.

    1. Purging the HPLC Pump lines (If air is in lines after prolonged operation pauses).

                                          i.    Fill the solvent bottle(s) with degassed water and/or water inorganic solvent mix.

                                         ii.    Insert the tip of a 10 mL syringe into the tubing that is connected to the wingnut of the LDA (liquid displacement assembly).

                                        iii.    Open the wingnut by turning it counter-clockwise.

                                        iv.    Turn-on the pump power.

                                         v.    Open direct control software in Isodat.

1.     Enter percentages of solvent lines (100% Line A if just water).

2.     Enter flow rate to 2000 µL/min.

3.     Click Start Run (blue triangle) to start pump flow.

 You may need to stop the pump to empty the syringe periodically.

4.     After purging is complete, stop the pump and close the wingnut.

    1. Priming the Pulse Damper (only done after flushing the HPLC pump lines and pusle damper with weak oxydizing solution to eradicate bacteria)

                                          i.    Fill the solvent reservoir with degassed water, or weak inorganic oxidizing solution.

                                         ii.    Make sure a flow restrictor or HPLC column is attached to the pulse dampener outlet

                                        iii.    Insert the tip of a 10 ml syringe to the manual bleed valve of the pulse dampener, and then open the wingnut by turning it counter-clockwise.

                                        iv.    Set the pump flow to 1000 µl/min.

                                         v.    Fill the loop completely to expel any air bubbles that may be in the line, or continue flushing to free the lines of bacteria followed by a degassed water (solvent) flush.

                                        vi.    Set the pumprate to the appropriate for the application.

                                       vii.    After the Pulse Dampener has been refilled with degassed wager, close the manual feeder valve (wingnut clockwise).

    1. Backpiston flushing of the hplc pump  (Maintenance done ???)

                                          i.    Cut the 1/8Ó OD x 1/16Ó ID PTFE tubing of the outer loop of the piston.

                                         ii.    Connect a syringe filled with degassed HPLC grade water to the bottom piece of the tubing and gently flush water through the system.

                                        iii.    Reattach a new piece of tubing (DO I NEED TO FINISH BY PUSHING AIR THROUGH THE SYSTEM WHEN DONE FLUSHING????).

  1. LC Isolink Procedures
    1. Note. To prevent damage from the membrane, do not exposure it to overall flows above the critical value of 700 μl/min (that is, 500 μl/min at the HPLC unit and 200 μl/min overall at both interface pumps together)!
    2. Prepare your reagent containers as follows:

                                         i.    Rinse the containers using LC-grade solvent to remove any dust.

                                        ii.    Fill the containers with appropriate LC-grade solvent.

                                      iii.    The bottle caps are pre-assembled to include an inlet line and filter. Ensure that each filter is tightly assembled to its fitting, and that the filter fitting is firmly attached to the inlet line. Mace the solvent filter inlet line into each bottle, making sure that the inlet filter rests on the bottom of the bottle. Cap the bottle.

                                      iv.    Attach the appropriate label to each solvent bottle cap to identify it.

                                        v.    Run vent lines from each bottle to an appropriate exhaust apparatus.

    1. Example for Reagent Concentrations: values below should be taken as an example:

                                         i.    oxidation reagent

1.     200 g/l M2S2O8 (M+ = Na+, K+, NH4+); this will yield a strongly oxidative solution.

2.     For UCSC test: 40 gm Na2S2O8 was dissolved in 200 ml H2O. However, what ultimately worked best was dissolving 15 g Na2S2O8 in 500 ml H2O.

3.     If no AgNO3 catalyst is used, increase the concentration to 50 gm Na2S2O8 in 200 ml H2O.

4.     Measure water using a graduated cylinder.

                                        ii.    acid

1.     1.5 molar solution of H3PO4 in water

                                      iii.    catalyst

1.     Catalytic amounts of solid AgNO3 are used, e.g. three tips of spatula in 200 ml water. Compared to an aqueous solution of AgNO3, solid AgNO3 is advantageous, as:

a.      no impurities due to additional water will enter the system and

b.     no additional water needs to be degassed.

                                        iv.    The acid/catalyst mixture is composed of:

1.     aqueous 85% ortho-phosphoric acid, H3PO4

2.     silver nitrate, AgNO3. Silver ions (Ag+) are catalytically active as they temporarily fix oxygen and transfer it to the organic compounds. These, in turn, will be oxidized to CO2.

3.      Warning. In case of sulfide- or halogenide-containing samples, e.g. chloride in seawater, do not use silver nitrate, AgNO3, as an oxidative catalyst! As precipitates, sparingly soluble silver sulfide, Ag2S or silver halogenides, AgX would be formed causing damage to the separation unit.

4.     These are the recommended acid/catalyst concentrations given by Dieter (of Thermo-Bremen), as relayed to UCSC by installation engineer Burt Wolff.

 

H3PO4 (ml, d = 1.88 g/ml)

H2O (ml)

AgNO3 (spatula dollops)

15

140

3

20

187

4

48

448

8

50

467

8

 

                                         v.    Note. It is unreasonable to recommend any generally accepted concentration values. Rather, the optimum concentrations of all reagent components must always be determined empirically.

                                        vi.    Principle: On the one hand, oxidation reagent concentration must be sufficiently high to ensure complete oxidation (check via oxygen background; see Oxygen Background (m/z 32) on page 7-6).

                                     vii.    If its concentration is too high, however, the solutions show increased viscosity. Therefore, they are more difficult to be pumped, and lifetime of the system might be reduced due to corrosion.

  1. Troubleshooting Problems
    1. Incomplete oxidation is indicated by some peak shape characteristics:

                                          i.    double peaks (a small peak is followed by a big one)

                                         ii.    peak broadening

                                        iii.    extraordinary long tailing (additionally, δ 13C values may diverge).

 

 

 

 

 

 

 

  1. Things to Order
    1. 1 in-line filter, ss, i.d. 0.25 mm, frit ss 5 μm 116 9390  (should have this as part of our startup package).
    2. The volume of the sample loop depends on the particular analysis. Various loops are available for different sample volumes; see www.rheodyne.com. Fisher is a distributor for rheodyne.