Drawing and Filtering Chlorophylls & HPLCs

Sample Drawing:

1. Chl samples are drawn on all rosette bottles tripped at 200m and less so sampling on a standard 20-bottle cast usually starts at #7 (sample vol is ~140mls).  For shallow stations, all the bottles may be sampled.  Noontime prodo casts may have extra bottles to sample. Duplicate bottles are usually skipped.  Refer to the sample log sheet to verify sampling or ask the CTD operator or watchleader.

2.  Drawing from the middle valve, add ~20mls, cap loosely, shake then dump; three rinses.  Double-check the sample bottle number matches the rosette bottle number.

3.  Chl samples are volumetric so after rinsing, fill it completely, cap loosely, tap the bottle gently against the rosette to dislodge any bubbles then top-off, cap tightly, invert the bottle – if you see a bubble, top-off and check again. Squeezing the sides of the bottle can change the sample volume and create a persistent bubble; cup the bottle in your palm during the final fill to minimize this problem.

4. Once all the chlorophylls have been drawn, draw the HPLCs – two volumetric samples drawn into dark bottles from the second depth (usually – check the sample log).  The sample volume will vary with chlorophyll concentration so check the sample log or with the watchleader for bottle size.

5. Again, three rinses then fill completely.

6. Once both chl & HPLC samples are drawn, initial the sample log.

7. Take the chlorophyll sample form from the clipboard (under the sample log) along with the chlorophyll samples and HPLCs (use a milk-crate to carry it all) to the chl van to filter ASAP.  The prodo experiment filtrations in the evening may delay the filtrations so ask to be notified when the chl van is available.

 
Filtrations:

1. The chl van filter manifold should have filters installed but double-check.  Turn on the vacuum pump and carefully pour each sample into the filter funnel.  These are volumetric samples so it is important not to lose any by spilling or ill -fitted funnels.  You may want to start the HPLCs first since they can take a long time and can be filtering while the chl samples are processed.

2. If you spill any sample, discard the remainder, replace the filter, redraw the sample from the rosette.  You can wait to do this after all the other samples are done filtering but do it before removing the filters so the vial/sample sequence remain intact.  If you wait too long the rosette bottle may be drained or dumped.  Be sure to pour the redrawn sample into the correct funnel.

3. Monitor the sample filtration and close the valve immediately after the sample is filtered.

4. Once all the samples are filtered, turn off the vacuum, and remove an empty tube rack from the fridge. Open the sample tube storage bin and fill the rack with the next set of tubes; the tubes are consecutively numbered and should continue the sequence from last station  – verify this by looking at the chl sample log (clipboard) from the previous station. Also check the tube volume contains 8 mls (level should be above label area).

5. Remove the filter funnel and the tube cap and carefully tweeze the filter into the tube - rolling it on the filter base then sliding it carefully into the tube, 1cm below the surface; if the filter tears, be sure all the pieces are retrieved and submerged.  If the filter drops onto the counter or floor you will have to redo the sample (you can protect the counter with a fresh paper towel or bench protector sheet). Repeat for all the samples.  Be sure all the tubes are capped tightly. Keep track of the tube, sample & funnel order

6. Fill out the chlorophyll sample log with filtration time, your initials, the vial numbers.

7. Return the vials to the fridge; bungie the door closed.  Samples are light-sensitive so be sure they are in the dark (in the fridge, box or foil-covered) until they are analyzed.

8. Turn on the vacuum and open the valves slightly. Tweeze new GFF filters onto each filter base, centering them carefully on the frit then reattach the funnel, being sure the filter stays centered (vacuum helps) and funnel seats properly by wriggling it slightly.  (If this step is not done correctly then next sample will spill out the sides and require a sample redraw).

9. Check the HPLCs and if they are not done filtering you will have to come back every 15-20mins to check them.

10.  Return the empty chl sample bottles to the wet lab and check-in with the watchleader (unless we have already left station).

11.  Once the HPLCs are filtered, label two cryovials with the station info (follow the example) and
record the time, volume filtered and initial the HPLC log.

12.   Tweeze each filter into a cryovial and cap tightly.  Take these vials and the empty sample bottles to the wet lab.

13.  The filters are frozen in the liquid nitrogen (~196°C) dewar, use eye protection and gloves; clip the vials into a labeled cane and load into the labeled canister to freeze.

14.  Return the two dark bottles to the HPLC milk-crate.

Common mistakes: spillage - sample loss from pouring or squeezing the sides of the bottle when uncapping; filter bypass - filter was not centered on the frit; funnel was loose or skewed; filter falls onto counter or floor when funnel is removed or during transfer to vial; vial skipping and/or disorder; pour a sample into a funnel that already has a sample; new filters are not installed for next station.