TITLE: Measurement of dissolved black carbon in water via benzenepolycarboxylic acid (BPCA) oxidation and quantification using aqueous, inorganic, high-performance liquid chromatography
AUTHORS: Riley  Barton, Sasha  Wagner

[DESCRIPTION]
Dissolved black carbon (DBC) is the condensed aromatic portion of dissolved organic matter produced from the incomplete combustion of biomass and other thermogenic processes. DBC quantification facilitates the examination of the production, accumulation, cycling, transformation, and effects of biologically recalcitrant condensed aromatic carbon in aquatic environments. Due to the heterogeneous nature of DBC molecules, concentrations are difficult to measure directly. Here, the method for DBC quantification consists of oxidizing condensed aromatic carbon to benzenepolycarboxylic acids (BPCAs), which are used as proxies for the assessment of DBC in the original sample. The concentrations of BPCA oxidation products are quantified using high-performance liquid chromatography. DBC concentrations are determined from the concentration of BPCAs using a previously established conversion factor. Details and full descriptions of the preparative and analytical procedures and techniques of the BPCA method are usually omitted for brevity in published method sections and method-specific papers. With this step-by-step protocol, we aim to clarify the steps of DBC analysis, especially for those adopting or conducting the BPCA method for the first time. 

[BEFORE_START]
Prior to sample preparation and DBC analysis, all water samples should be:
Collected and filtered using trace-clean sampling protocols. Samples are typically filtered through pre-cleaned filters with pore sizes ranging from 0.1 to 0.7 μm. Store filtered water samples in the dark at 4°C until further analysis.
Analyzed to obtain DOC concentrations. These DOC concentration data are essential for determining volumes for accurate sample preparation and robust DBC quantification. In addition, DOC concentrations also allow for proper contextualization of DBC data obtained using this protocol.

All glassware and plasticware should be cleaned according to procedures described herein.

[GUIDELINES]
A helpful spreadsheet detailing necessary calculations is included here and utilized multiple times throughout the protocol:   

Expected Protocol Timeline: From start to finish, completion of this protocol for dissolved black carbon (DBC) analysis will take approximately one week. However, there are multiple points in the protocol where the preparative procedures can be paused and resumed at a later time. Before starting each step in the procedure, read ahead to make sure you have allocated sufficient time to complete each step before reaching a designated pause point. To achieve maximum sample preparation efficiency and data consistency, we recommend that samples are prepared in relevant batches, rather than one at a time. For example, solid phase extracted dissolved organic carbon (DOC) can be archived in the freezer until an entire sample set is collected, then the samples can be analyzed for DBC in one batch.

Chemical considerations: Analytical grade chemicals and solvents should be used at all times.

Cleaning glassware*:
 Triple rinse with ultrapure or deionized water
 Soak in 0.01M hydrochloric acid bath (ultrapure water acidified to pH 2) for 6+ hours
 Triple rinse with ultrapure water
 Place in a drying oven set to ~60°C until glassware is completely dry
 Remove glassware from oven and cover any openings with aluminum foil
 Combust foiled glassware in a muffle oven at 500°C  for 5 hours

*Exceptions to this procedure include:
Glass ampules and HPLC vials, which are simply wrapped in foil and combusted (steps 5 and 6 only)
HPLC vial inserts, which are used directly from the package
Volumetric glassware, such as volumetric pipettes or flasks, should be cleaned following the "Cleaning plasticware" procedure below

Cleaning plasticware:
Triple rinse with ultrapure or deionized water
 Soak in 0.01M hydrochloric acid bath (ultrapure water acidified to pH 2) for 6+ hours
 Triple rinse with ultrapure water
 Place in a drying oven set to ~60°C until plasticware is completely dry

[STEPS]
SECTION: Solid Phase Extraction: Prepare and condition cartridges
1. The solid phase extraction (SPE) cartridges used in this protocol (Agilent Bond Elut PPL cartridge, 1g, 6 mL) is packed with a styrene-divinylbenzene polymer resin that recovers high proportions of dissolved organic matter (DOM) on a per-carbon basis. The resin must first be conditioned to enable DOM extraction from filtered water.

To set up for conditioning of SPE cartridges, construct an apparatus that securely holds the cartridges upright above a solvent-compatible container. 

 

 

SECTION: Solid Phase Extraction: Prepare and condition cartridges
2. Condition SPE cartridges with analytical grade methanol (3 headspace volumes).
 

SECTION: Solid Phase Extraction: Prepare and condition cartridges
3. Condition SPE cartridges with ultrapure water (3 headspace volumes).
 

SECTION: Solid Phase Extraction: Prepare and condition cartridges
4. Condition SPE cartridges with pH 2 water (3 headspace volumes).
 

SECTION: Solid Phase Extraction: Prepare and condition cartridges
5. Fill headspace of SPE cartridges with pH 2 water and immediately cap top and bottom.

  
 



SECTION: Solid Phase Extraction: Prepare and condition cartridges
6. Store SPE cartridges in the refrigerator at 4 °C  until DOM extraction.
 


SECTION: Solid Phase Extraction: Extraction of DOM
7. At this point, the water sample should have been filtered and analyzed for dissolved organic carbon (DOC) concentration.

Acidify the filtered water sample to pH 2 using hydrochloric acid.
 


SECTION: Solid Phase Extraction: Extraction of DOM
9. Set up SPE apparatus and begin flow of acidified sample through SPE cartridge.

   

SECTION: Solid Phase Extraction: Extraction of DOM
10. Open each stopcock sequentially, checking to make sure sample is flowing to and effluent is flowing from each cartridge and that flow is exiting the manifold. 


 

SECTION: Solid Phase Extraction: Extraction of DOM
11. Place an appropriately sized container beneath the manifold to capture SPE effluent and wait until extraction is complete (i.e. the entire sample volume has passed through the SPE cartridge by gravity).
 

SECTION: Solid Phase Extraction: Extraction of DOM
14. Rinse SPE cartridges with pH 2 water (3 headspace volumes).
 

SECTION: Solid Phase Extraction: Extraction of DOM
13. Record the mass of the empty sample bottle.
 

SECTION: Solid Phase Extraction: Extraction of DOM
15. At this point, you may continue directly on to the next step or recap DOM-containing SPE cartridges and wrap in aluminum foil and freeze/refrigerate until continuing on to the next step.
SECTION: Solid Phase Extraction: Drying SPE cartridges
16. Set up the drying apparatus and attach DOM-containing SPE cartridges via the following steps:

Suspend and secure manifolds using a clamp and ring stand
Use tubing and connectors to attach manifolds to gas tank*
Attach SPE cartridge to manifold using an adapter cap connector
Lay paper towels or other absorbent material beneath the manifold and cartridges

*Any of the following gases may be used to dry SPE cartridges: high purity nitrogen, argon, or zero air.
    
 


SECTION: Solid Phase Extraction: Drying SPE cartridges
17. Turn on gas flow.
 

SECTION: Solid Phase Extraction: Drying SPE cartridges
18. Allow gas to flow until SPE cartridge resin is completely dry.  
    
 





SECTION: Solid Phase Extraction: Drying SPE cartridges
19. Turn off gas at the tank and remove dry SPE cartridges from manifolds. Repeat drying process as necessary to dry the rest of the SPE cartridges in the sample set.
SECTION: Solid Phase Extraction: Drying SPE cartridges
20. Remember to turn off gas when you are done. If you do not have time to move on to the next step, cap the SPE cartridges and wrap in aluminum foil until you are ready to elute the DOM. 
SECTION: Solid Phase Extraction: Elution of SPE-DOC with methanol
21. Label glass vials with sample identifier or name.
 

SECTION: Solid Phase Extraction: Elution of SPE-DOC with methanol
22. Record the mass of the empty glass vial (with cap on).
 

SECTION: Solid Phase Extraction: Elution of SPE-DOC with methanol
23. Using a test tube rack, or similar apparatus, suspend the SPE cartridges in preparation for DOM elution. Remove caps from vials and place them underneath their corresponding SPE cartridges.
 

   

SECTION: Solid Phase Extraction: Elution of SPE-DOC with methanol
24. In this step, DOM is eluted from the SPE resin with methanol and collected directly in a glass vial. It is important to use analytical grade methanol so as not to contaminate the DOM sample with extraneous material.

Elute DOM from the SPE resin by adding 3 x 3 mL aliquots of methanol to the cartridge.
 



SECTION: Solid Phase Extraction: Elution of SPE-DOC with methanol
25. Once flow from the SPE cartridges has stopped, close each vial with their corresponding cap.
SECTION: Solid Phase Extraction: Elution of SPE-DOC with methanol
26. Record the mass of the eluate-filled glass vial (with cap on).  
 

SECTION: Sample Oxidation: Transfer of SPE-DOC eluate to ampules
28. Using the spreadsheet linked below (PPL tab), calculate the volume of DOM eluate that should be dried down for DBC analysis.
  
 



 

 



SECTION: Solid Phase Extraction
None. Transfer to ampules:  Materials:
SECTION: Solid Phase Extraction
None. ·         Micropipette and glass Pasteur pipette (precombusted and connected to pipette via tubing), methanol, waste beaker, DOM eluate vials from Step 3, 2-mL ampules  Method:
SECTION: Solid Phase Extraction
None. ·         Triple-rinse glass pipette with methanol (always do this between samples as well to prevent carryover)
SECTION: Solid Phase Extraction
None. ·         Using the volume of methanol eluate to dry value from calculations (V MeOH to dry from BPCA prep example spreadsheet), transfer eluate to ampule with the correct Sample ID
SECTION: Solid Phase Extraction
None. o   When transferring the methanol extract, insert tip of Pasteur pipette into methanol extract and gently pump up and down 3-5 times until headspace in the Pasteur pipette is equilibrated and bubbles no longer form during pumping in the sample. After a few pumps (and no bubbles forming), draw in the target amount of methanol extract and transfer to the ampule.
SECTION: Solid Phase Extraction
None. o   Put pipette tip all the way down the neck of the ampule and try to make sure the solution goes directly to the bottom of the ampule (not the sides and especially not the neck)
SECTION: Solid Phase Extraction
None. ·         Triple rinse pipette with methanol; dispose each washing in waste beaker
SECTION: Solid Phase Extraction
None. ·         Repeat for each sample   
SECTION: Solid Phase Extraction
None. Dry ampules  Materials:
SECTION: Solid Phase Extraction
None. ·         Drying oven, ampules with DOM eluate, Kimwipe, container to hold ampules  Method:
SECTION: Solid Phase Extraction
None. ·         Place ampules in drying oven (lightly place a Kimwipe over them to keep dust, liquid, etc. from dropping in the ampule and contaminating the sample)
SECTION: Solid Phase Extraction
None. ·         Make sure to dry to completion (at least overnight and maybe a second night to be safe if you are unsure if it is fully dry) as the reaction between nitric acid and undried methanol is explosive … and you will lose sample.  Notes:
SECTION: Solid Phase Extraction
None. o   See if any has creeped up the sides during evaporation
SECTION: Solid Phase Extraction
None. o   Note any vials that have eluate in the neck so you can pay special interest in them later   
SECTION: Solid Phase Extraction
None. Add nitric acid to ampules (do not start unless you can do Step 8-9 right after!)  Materials:
SECTION: Solid Phase Extraction
None. ·         Concentrated nitric acid, micropipette with glass or plastic tip, dried DOM eluate in ampules  Method:
SECTION: Solid Phase Extraction
None. ·         Once the methanol is evaporated from the ampules, in a fume hood, add 500uL of nitric acid to each ampule
SECTION: Solid Phase Extraction
None. o   Check notes and visually examine the ampules to see if there is any DOM in the neck.  Notes:
SECTION: Solid Phase Extraction
None. o   If there is any visible DOC in the neck, attempt to rinse it down with the nitric. If this is not done the DOC can char during the sealing of the ampule, potentially producing a BPCA artifact during subsequent analyses
SECTION: Solid Phase Extraction
None. o   Do not start this step if you do not have the time to immediately seal them, wait until you can because the reaction starts once you add the nitric acid   
SECTION: Solid Phase Extraction
None. Seal the ampules  Materials:
SECTION: Solid Phase Extraction
None. ·             Blow torch, long tweezers, safety goggles, beaker of water     Method (see image above):
SECTION: Solid Phase Extraction
None. ·         Turn on torch
SECTION: Solid Phase Extraction
None. ·         Hold ampule at a 45 degree angle. Use your hand for the base and long, metal tweezers for the top
SECTION: Solid Phase Extraction
None. ·         Roll the upper neck of the ampule in the flame until the glass is malleable
SECTION: Solid Phase Extraction
None. ·         Gently stretch the glass by pulling the tweezers farther from the flame until it is separated from the rest of the ampule
SECTION: Solid Phase Extraction
None. ·         Drop the glass top from the tweezers into a beaker of water
SECTION: Solid Phase Extraction
None. ·         Examine the ampule:
SECTION: Solid Phase Extraction
None. o   There should be no holes and the end should resemble a candle
SECTION: Solid Phase Extraction
None. ·         Repeat with all samples  Notes:
SECTION: Solid Phase Extraction
None. o   If you are new to sealing columns, practice with empty ampules. Wait until you are confident with the technique before working with real samples. This will prevent sample loss due to incorrect sealing.
SECTION: Solid Phase Extraction
None. o   You don’t want to twist the glass too much as the seal won’t be as good. Spinning back and forth with the fingers holding the bottom of the ampule and gently clicking the tweezers to allow the top of the ampule to also spin but keep it from flopping when the glass gets malleable.
SECTION: Solid Phase Extraction
None. o   Leave the tip of the sealed ampule in the flame as you place the top end in the beaker of water. This ensures a rounder top and better seal
SECTION: Solid Phase Extraction
None. o   If there is a hole, reseal using the same method described above. It is easier to reseal if your original seal is farther from the neck
SECTION: Solid Phase Extraction
None. o   Do not pick up the ampule by the top right after sealing (I know it’s obvious that it is hot, but you will definitely end up doing it and it will hurt)   
SECTION: Solid Phase Extraction
None. Oxidize  Materials:
SECTION: Solid Phase Extraction
None. ·         Sealed ampules, tin foil, glass beaker (to hold ampules), programmable oven (we typically use an old GC oven)  Method:
SECTION: Solid Phase Extraction
None. ·         Place three ampules in tinfoil pouches (particularly useful if you are analyzing replicates; if one ampule explodes you won’t mess up the labels on the rest of them)
SECTION: Solid Phase Extraction
None. ·         Place pouches together in a beaker (to keep them from rolling around) and place in the oven
SECTION: Solid Phase Extraction
None. ·         Set oven to ramp up from 25 degrees C to 160 degrees C with a ramp of 5 degrees per minute
SECTION: Solid Phase Extraction
None. ·         The oven should be set to stay at 160 degrees for 360 minutes
SECTION: Solid Phase Extraction
None. ·         Allow samples to cool to room temperature before storing them in the freezer or for continuing with the next steps     Notes:
SECTION: Solid Phase Extraction
None. o   You must be around when it finishes to put in the freezer. Plan accordingly because there’s 6-7 hours from when you put them in the oven to when you need to put them in the freezer   
SECTION: Solid Phase Extraction
None. Evaporating the nitric acid      Materials:
SECTION: Solid Phase Extraction
None. ·         Sandbath, hot plate, needles, tubing, ring stands  Method:
SECTION: Solid Phase Extraction
None. ·         Set up drying apparatus (see image above)
SECTION: Solid Phase Extraction
None. o   Turn on hot plates and allow sand to heat up (1 hr); hot plates should be set such that the sand bath reaches a temperature of ~60*C – The exact knob settings will need to be optimized for your specific hotplate
SECTION: Solid Phase Extraction
None. o       Only prepare samples that you can dry that day (in photos you can do 10 at a time and it takes approx. 1 hr for the samples to dry)
SECTION: Solid Phase Extraction
None. o   Will also need a glass and plastic waste container
SECTION: Solid Phase Extraction
None. o   Crack off top of the ampules (SAVE THE TOP, see image below)
SECTION: Solid Phase Extraction
None. §  Hold bottom upright in one hand and, with your other hand,  place your thumb under the bulb and your pointer finger on the opposite side up the neck
SECTION: Solid Phase Extraction
None. §  Crack open away from you – If you are having trouble opening the ampules, ampule opening devices are available for purchase
SECTION: Solid Phase Extraction
None. ·         Rinse the ampule tops
SECTION: Solid Phase Extraction
None. o   Pipette 40uL of nitric acid into the tops of each ampule
SECTION: Solid Phase Extraction
None. o   Roll & swirl top of ampule to rinse the entire inner surface area to dissolve any BPCAs that may be stuck in the top of the ampule
SECTION: Solid Phase Extraction
None. o   Using a fresh pipette tip (change after each sample), transfer the nitric from the top of the ampule to the corresponding bottom (where the rest of the sample is)
SECTION: Solid Phase Extraction
None. ·             Evaporate the nitric acid in the ampule bottoms
SECTION: Solid Phase Extraction
None. o   Push ampule into sand, just below the shoulder (5 per sand bath, it is easier to start with the centermost position)
SECTION: Solid Phase Extraction
None. o   Remove needle covers and clean each needle with methanol on a Kimwipe
SECTION: Solid Phase Extraction
None. o   Insert needle into the headspace of the ampule (as low as you can without submerging)
SECTION: Solid Phase Extraction
None. o   Open N2 or Ar tank
SECTION: Solid Phase Extraction
None. §  Slowly turn up the N­2 or Ar flow
SECTION: Solid Phase Extraction
None. §  Should make the liquid shimmer (not bubble up)
SECTION: Solid Phase Extraction
None. §  You should see fumes comping from the ampules
SECTION: Solid Phase Extraction
None. §  Feel free to adjust flow or needle height periodically as the liquid level decreases to optimize evaporation; this step requires a watchful eye and some degree of “babysitting” and adjusting to ensure efficient evaporation
SECTION: Solid Phase Extraction
None. ·         Allow to dry completely (~1hr if monitored/adjusted as described above)
SECTION: Solid Phase Extraction
None. o   There should be absolutely know condensation on the ampule or needle
SECTION: Solid Phase Extraction
None. o   Turn off flow of N2 or Ar
SECTION: Solid Phase Extraction
None. o   Carefully remove needles from the ampules
SECTION: Solid Phase Extraction
None. o   Remove ampules from the sand bath – If at this point, it is obvious that not all acid has dried, the ampules must go back in the sandbath for further drying
SECTION: Solid Phase Extraction
None. o   Wipe needles with methanol and a Kimwipe
SECTION: Solid Phase Extraction
None. §  Either replace the needle covers or repeat drying procedure with more samples  Notes:
SECTION: Solid Phase Extraction
None. o   Be careful when inserting and removing needles: you could poke yourself or accidentally get sand on your samples!
SECTION: Solid Phase Extraction
None. o   If sand does get into the ampules, don’t panic – Take notes on which samples were affected and check chromatograms later for possible contamination.   
SECTION: Solid Phase Extraction
None. Transferring from ampule to HPLC vial  Materials:
SECTION: Solid Phase Extraction
None. ·         Pipette, glass waste container, Buffer A, 2-mL HPLC vials, 300-uL glass inserts for HPLC vials  Method:
SECTION: Solid Phase Extraction
None. ·         Fill labeled HPLC vials with 300uL inserts – Inserts may not be necessary if large amounts of OC were oxidized initially (>>> 0.5 mg-OC), but this is uncommon for field-collected DOC samples
SECTION: Solid Phase Extraction
None. ·         Pipette target volume of Buffer A (Wagner et al. 2017) into dried ampule
SECTION: Solid Phase Extraction
None. o   Normally adding 200uL for 0.5mg of C but could go down to 150uL or up to 250uL depending on the expected BPCA concentration of your samples – These adjustments will become more obvious as you run more samples and understand environmental BPCA trends and expected ratios of BC:OC.
SECTION: Solid Phase Extraction
None. o   Be sure to record the volume of Buffer A you use to transfer as this is important for calculating the concentrations of BPCAs (see V mobile in attached BPCA prep example spreadsheet)
SECTION: Solid Phase Extraction
None. ·         Shake/swirl/run along every inner surface area of the ampule
SECTION: Solid Phase Extraction
None. o   However you do this, be consistent between samples
SECTION: Solid Phase Extraction
None. o   Example: shake/shimmy the ampule for a while, then tilt the ampule bottom up and down to get the sides (rotating after going up-down side to dissolve any BPCAs on a different portion of the side of the ampule). Repeat these steps multiple times.
SECTION: Solid Phase Extraction
None. ·         Using fresh glass or plastic pipette tips for each sample, transfer the liquid from the ampule to the HPLC vial with insert
SECTION: Solid Phase Extraction
None. o   Make sure to put the tip of the pipette all the way down into the insert to keep an air bubble from forming. If this bubble forms capping the vial and gently shaking should dislodge it
SECTION: Solid Phase Extraction
None. ·         Cap the vials
SECTION: Solid Phase Extraction
None. o   Now the sample is ready to analyze using HPLC or ready to be frozen until then!           Running BPCAs on HPLC  Materials:
SECTION: Solid Phase Extraction
None. ·         HPLC vials, samples in labeled HPLC vials, HPLC w/Chromeleon software, o-phosphoric acid, NaH2PO4 monohydrate, Na2HPO4 heptahydrate, two 1L volumetric flasks, BPCA standards (benzenehexacarboxylic acid (B6CA), benzenepentacarboxylic acid (B5CA), 1,2,4,5-benzenetetracarboxylic acid (B4CA), 1,2,3-benzenetricarboxylic acid, 1,2,4-benzenetricarboxylic acid 1,3,5-benzenetricarboxylic acid (B3CAs))
SECTION: Solid Phase Extraction
None. Prepare HPLC Buffers:
SECTION: Solid Phase Extraction
None. ·         Make sure there are fresh buffer solutions (if not make some)
SECTION: Solid Phase Extraction
None. o   Buffer A: 40 mL of o-phosphoric acid in 1L of MilliQ water (use volumetric flask)
SECTION: Solid Phase Extraction
None. o   Buffer B: 2.42 g of NaH2PO4 monohydrate & 0.64 g of Na2HPO4 heptahydrate in 1L of MilliQ water
SECTION: Solid Phase Extraction
None. ·         Put ~1.5 mL of the Buffer A solution into a HPLC vial labeled “Blank” and cap
SECTION: Solid Phase Extraction
None. Prepare BPCA standards and MilliQ:
SECTION: Solid Phase Extraction
None. ·         Make a 5000 uM-C BPCA standard stock solution in a 100 mL volumetric flask with each BPCA standard
SECTION: Solid Phase Extraction
None. o   Weigh out BPCA Standards:
SECTION: Solid Phase Extraction
None. §  Make sure to record actual mass of each BPCA weighed out as this actual value is what will be used for calculations (see calibration tab in BPCA example spreadsheet).
SECTION: Solid Phase Extraction
None. §  Example Table:
SECTION: Solid Phase Extraction
None. Standard
SECTION: Solid Phase Extraction
None. Mass (mg)
SECTION: Solid Phase Extraction
None. Actual Mass (mg)
SECTION: Solid Phase Extraction
None. 1,2,3-B3CA
SECTION: Solid Phase Extraction
None. 11.680
SECTION: Solid Phase Extraction
None. 11.661
SECTION: Solid Phase Extraction
None. 1,2,4-B3CA
SECTION: Solid Phase Extraction
None. 11.680
SECTION: Solid Phase Extraction
None. 11.638
SECTION: Solid Phase Extraction
None. 1,3,5-B3CA
SECTION: Solid Phase Extraction
None. 11.680
SECTION: Solid Phase Extraction
None. 11.655
SECTION: Solid Phase Extraction
None. 1,2,4,5-B4CA
SECTION: Solid Phase Extraction
None. 12.700
SECTION: Solid Phase Extraction
None. 12.749
SECTION: Solid Phase Extraction
None. B5CA
SECTION: Solid Phase Extraction
None. 13.560
SECTION: Solid Phase Extraction
None. 13.618
SECTION: Solid Phase Extraction
None. B6CA
SECTION: Solid Phase Extraction
None. 14.260
SECTION: Solid Phase Extraction
None. 14.145
SECTION: Solid Phase Extraction
None. o   Put all of the standards into a 100 mL volumetric flask
SECTION: Solid Phase Extraction
None. o   Dissolve standards in Buffer A
SECTION: Solid Phase Extraction
None. §  If necessary, submerge flask in warm sonicator to help facilitate dissolution
SECTION: Solid Phase Extraction
None. o   These standards are made based on concentration of Carbon. The concentrations of each BPCA will be calculated later on (see Calibration Tab of BPCA example spreadsheet).
SECTION: Solid Phase Extraction
None. Notes:
SECTION: Solid Phase Extraction
None. o   All BPCAs are included in the standard solution but not all of them are measured for dissolved black carbon concentrations. Please see Wagner et al. (2017) and Stubbins et al. (2015) supporting information for further details and justification
SECTION: Solid Phase Extraction
None. o   Weighing tips:
SECTION: Solid Phase Extraction
None. §  Cut weigh paper into a small square and fold in half diagonally (this makes it easier to pour into the volumetric flask later)
SECTION: Solid Phase Extraction
None. §  Use a microbalance
SECTION: Solid Phase Extraction
None. ·         Dilute Stock solution to make 2000, 1000, 500, 200, 100, 50, 25 uM standards
SECTION: Solid Phase Extraction
None. o   We usually make dilutions using 10mL volumetric flasks and dilute with mobile phase A each time
SECTION: Solid Phase Extraction
None. o   Consider transferring standard dilutions from 10mL volumetric flasks into glass vials for longer term storage and easy access when running them during analysis
SECTION: Solid Phase Extraction
None. ·         Put ~1 mL of each standard solution (including the stock 5000uM-C) into a labeled HPLC vial and cap
SECTION: Solid Phase Extraction
None. ·         Put ~1.5 mL of MilliQ water into a HPLC vial labeled “MilliQ” and cap
SECTION: Sample Oxidation: Transfer of SPE-DOC eluate to ampules
29. Label glass ampules
 

SECTION: Sample Oxidation: Evaporation of methanol from SPE-DOC eluate
31. In this step methanol is evaporated completely from the eluate to obtain the dry SPE-DOC residue for oxidation.

Place ampules in a properly vented drying oven set to 60 °C . Loosely place a Kimwipe over the ampule openings to prevent contamination from extraneous material during drying. Depending upon the eluate volume, methanol evaporation can take anywhere from 12 to 36 hours. 
 




SECTION: Sample Oxidation: Nitric acid addition and sealing ampules
32. Do not begin this step if you do not have the time to flame-seal ampules and perform sample oxidation immediately thereafter.

Using a volumetric, mechanical pipette, add 0.5 mL of analytical grade nitric acid (15.8N) to each ampule.
 

SECTION: Sample Oxidation: Nitric acid addition and sealing ampules
33. Flame seal the glass ampules.
 

SECTION: Sample Oxidation: Nitric acid addition and sealing ampules
34. Examine the ampule for a proper seal and repeat the previous step for each ampule in the sample set.

 

SECTION: Sample Oxidation: Oxidation of SPE-DOC with nitric acid
35. Wrap ampules in aluminum foil pouches, usually 3 to 5 ampules per pouch.
 

SECTION: Sample Oxidation: Oxidation of SPE-DOC with nitric acid
36. Place pouches together in a glass beaker or other glass container and place in a temperature controlled oven.
 

SECTION: Sample Oxidation: Oxidation of SPE-DOC with nitric acid
37. Oxidize samples at 160 °C  for 6 hours. 
 

 


SECTION: Sample Oxidation: Oxidation of SPE-DOC with nitric acid
38. Note that you must be present when the oxidation finishes to transfer ampules from the oven. This is especially important if you are using a non-programmable oven that does not automatically cool after the oxidation period. 

After oxidation is complete, you may immediately move on to the next step or place fully cooled ampules into a freezer up to one week before continuing to the next step.
SECTION: HPLC Analysis of BPCAs: Evaporation of nitric acid from ampules
39. At this point, the sealed glass ampules contain sample benzenepolycarboxylic acids (BPCAs) and other SPE-DOC oxidation products dissolved in the concentrated nitric acid. In this step, the concentrated nitric acid is removed through evaporation under a stream of inert gas so the BPCAs can be prepared for separation and quantification by high performance liquid chromatography (HPLC).

   
Set up the drying apparatus* and sand bath in a chemical hood via the following steps:

Suspend and secure drying apparatus using a clamp and ring stand
Use tubing and connectors to attach drying apparatus to gas tank†
Place sand bath on a hot plate beneath the drying apparatus

*Note that this drying apparatus is different from the one used to dry SPE cartridges in step 16.

†Either of the following gases may be used to dry nitric acid in ampules: high purity nitrogen or argon.
 

SECTION: HPLC Analysis of BPCAs: Evaporation of nitric acid from ampules
40. Turn on hot plate and allow sand bath to heat up to approximately 60 °C . 

It usually takes approximately 1 hour for the bath to reach and stabilize at this temperature. You will need to determine the exact setting for your hot plate to reach this desired temperature.
SECTION: HPLC Analysis of BPCAs: Evaporation of nitric acid from ampules
42. Carefully break off the top of the ampules. Keep the ampule tops organized in a way that it is clear that each top corresponds to the bottom. At this point, the ampule tops still contain trace amounts of BPCA products and we will quantitatively transfer this material to the rest of the ampule in the next step.
 

 

SECTION: HPLC Analysis of BPCAs: Evaporation of nitric acid from ampules
43. Quantitatively transfer BPCA oxidation products from ampule top to the rest of the solution in the ampule bottom.
SECTION: HPLC Analysis of BPCAs: Evaporation of nitric acid from ampules
44. Evaporate the concentrated nitric acid contained in the ampules.
SECTION: HPLC Analysis of BPCAs: Evaporation of nitric acid from ampules
45. Repeat previous step until all samples are dried down. Wipe down needles with a Kimwipe soaked in methanol between each batch of samples added to sand bath to prevent cross contamination.
SECTION: HPLC Analysis of BPCAs: Transfer of BPCAs from ampules to HPLC vials
46. In this section, the BPCA-containing residue in the ampule is re-dissolved in mobile phase and transferred to the HPLC vial for analysis.

Label HPLC vials with sample identifier or name, then add inserts to reduce interior volume. 
 

SECTION: HPLC Analysis of BPCAs: Transfer of BPCAs from ampules to HPLC vials
47. Transfer the target volume of mobile phase A to the ampule containing the dry BPCA-containing residue using a volumetric pipette. Record the volume of mobile phase A added to the ampule.
 

SECTION: HPLC Analysis of BPCAs: Transfer of BPCAs from ampules to HPLC vials
48. Rinse the entire interior surface of the ampule with the volume of mobile phase A added.
 

SECTION: HPLC Analysis of BPCAs: Transfer of BPCAs from ampules to HPLC vials
49. Using a clean glass or plastic pipette, transfer the mobile phase in the ampule, which now contains re-dissolved BPCAs, from the ampule to the insert in the corresponding HPLC vial.
 

SECTION: HPLC Analysis of BPCAs: Transfer of BPCAs from ampules to HPLC vials
50. Cap the HPLC vials. At this point, samples can be analyzed immediately, or stored in the freezer for years without compromising sample integrity.

SECTION: HPLC Analysis of BPCAs: Preparation of HPLC mobile phases and BPCA standard solutions
51. Details regarding the HPLC analysis of BPCAs are previously described by Wagner et al. (2017).

Prepare mobile phase A = 0.6 Molarity (M) phosphoric acid (pH 1).
 


 

SECTION: Solid Phase Extraction: Extraction of DOM
8. Record the mass of sample bottle containing the acidified sample.
 

SECTION: Solid Phase Extraction: Extraction of DOM
9.1. Insert open end of Teflon tube into sample bottle until it touches the bottom (stopcock should be closed at this point). Tape the Teflon tube to the outside of the bottle to keep it in place during the extraction process.
 

SECTION: Solid Phase Extraction: Extraction of DOM
9.2. Cover sample bottle opening (e.g. with aluminum foil). Place sample bottle on a shelf, or other high and stable area, that will be located above the SPE cartridge.
SECTION: Solid Phase Extraction: Extraction of DOM
9.4. Connect Teflon tubing from sample bottle to SPE cartridge via the following steps: 

Insert stopcock into a plastic adapter cap
Label body of SPE cartridge with sample identifier or name
Remove SPE cartridge caps and place into manifold (all stopcocks should be closed to prevent flow)
Open stopcock and let sample drip into SPE cartridge until headspace is nearly full
Close stopcock and snap adapter cap to the top of the SPE cartridge

Repeat steps above for each sample until the manifold is full.

  
SECTION: Solid Phase Extraction: Extraction of DOM
12. Once extractions are complete, close stopcocks and remove tubing and adapter caps from the sample bottles and SPE cartridges.
SECTION: Sample Oxidation: Nitric acid addition and sealing ampules
33.1. The flame sealing of ampules requires a beaker of water, long metal forceps, safety glasses, and a blow torch that is sufficiently hot enough to melt borosilicate glass.
SECTION: Sample Oxidation: Nitric acid addition and sealing ampules
33.2. Turn on the torch. Hold ampule at a 45 degree angle. Use your hand to stabilize the base of the ampule and the forceps to stabilize the neck of the ampule.
SECTION: Sample Oxidation: Nitric acid addition and sealing ampules
33.3. Hold the upper neck area of the ampule in the flame and slowly rotate until the glass begins to soften. Once the glass is sufficiently malleable, gently rotate and pull apart the top and bottom portions of the ampule by pulling the forceps further away from the flame until the top portion is removed and the neck of the ampule is pinched closed. After removing the top part of the ampule, hold the sealed portion in the flame for an extra second or so to ensure a good seal.
 

 

SECTION: Sample Oxidation: Nitric acid addition and sealing ampules
33.4. Place the top piece of the ampule (pulled off using forceps) into the beaker of water to cool. Carefully place sealed ampule into a tray using the forceps.
 

SECTION: HPLC Analysis of BPCAs: Evaporation of nitric acid from ampules
41. Remove ampules from freezer and place in a rack. Gently flick the top of each ampule to dislodge any nitric acid solution that may be trapped near the sealed ampule tip and let the solution drip down to the bottom of the ampule. 
SECTION: HPLC Analysis of BPCAs: Evaporation of nitric acid from ampules
43.1. Pipette 40 μL of concentrated nitric acid into the tops of each ampule.
SECTION: HPLC Analysis of BPCAs: Evaporation of nitric acid from ampules
43.2. Roll and swirl the ampule top to rinse the entire inner surface area and to collect any BPCA-residue in the top of the ampule.
SECTION: HPLC Analysis of BPCAs: Evaporation of nitric acid from ampules
43.3. Using a fresh pipette tip, transfer the nitric acid rinsing from the ampule top to the corresponding bottom. Repeat this step for all samples, changing pipette tips between each sample to prevent cross contamination.
SECTION: HPLC Analysis of BPCAs: Evaporation of nitric acid from ampules
44.1. Gently push opened ampule into the sand to just below the shoulder of the ampule.

   
 

SECTION: HPLC Analysis of BPCAs: Evaporation of nitric acid from ampules
44.2. Remove steel needle covers and clean each needle with a Kimwipe soaked in methanol.
SECTION: HPLC Analysis of BPCAs: Evaporation of nitric acid from ampules
44.3. Insert needle into the headspace of the ampule so the tip of the needle is hovering just above the surface of the nitric acid in the ampule.
 

SECTION: HPLC Analysis of BPCAs: Evaporation of nitric acid from ampules
44.4. SLOWLY increase gas flow. The nitric acid solution in each ampule should shimmer slightly, not sputter aggressively. You may observe dark orange-to-light colored fumes emitting from the ampules.
 

SECTION: Solid Phase Extraction: Elution of SPE-DOC with methanol
27. At this point, you may continue directly to the next step or store eluate in the freezer until you are ready to continue. 
 

SECTION: HPLC Analysis of BPCAs: Preparation of HPLC mobile phases and BPCA standard solutions
52. Prepare mobile phase B = 20 Milimolar (mM) sodium phosphate buffer (pH 6).
 


SECTION: HPLC Analysis of BPCAs: Preparation of HPLC mobile phases and BPCA standard solutions
53. Prepare stock solution of BPCA standards = 5000 Micromolar (µM) of BPCA-carbon (BPCA-C). 

Stock solution concentrations used here are consistent with those described by Wagner et al. (2017). Note that concentrations are μM of BPCA-C (i.e. carbon concentrations), not BPCA molecular concentrations - this is taken into account during quantification of DBC via the conversion factor described in step 66.
 

SECTION: HPLC Analysis of BPCAs: Preparation of HPLC mobile phases and BPCA standard solutions
54. Conduct a series of simple dilutions to produce solutions of 2000, 1000, 500, 200, 100, 50, and 25 uM BPCA-C standards. These solutions will be used for external calibration of BPCAs in unknown samples.
 

SECTION: HPLC Analysis of BPCAs: Preparation of HPLC mobile phases and BPCA standard solutions
56. Transfer ~1 mL of each BPCA standard solution directly into a clean, separate HPLC vials. Label each vial with the BPCA standard concentration and cap and set aside.
SECTION: HPLC Analysis of BPCAs: Preparation of HPLC mobile phases and BPCA standard solutions
53.1. Using a microbalance, weigh out each BPCA standard to the target mass listed in the table below. Transfer all measured BPCAs to the same 100 mL volumetric flask. 

Since it is difficult to weigh out accurately to the μg, record the actual mass of each BPCA standard that was weighed out. Actual masses will be used to calculate actual BPCA stock solution concentrations.

 

 

SECTION: HPLC Analysis of BPCAs: Preparation of HPLC mobile phases and BPCA standard solutions
53.2. After all BPCA standards have been weighed out and transferred to the volumetric flask, fill with mobile phase A and mix to dissolve BPCAs.

 

SECTION: HPLC Analysis of BPCAs: Preparation of HPLC mobile phases and BPCA standard solutions
55. Transfer ~1 mL of mobile phase A directly into a clean HPLC vial. Label as "blank" and cap and set aside.
SECTION: HPLC Analysis of BPCAs: Chromatographic separation of individual BPCAs
57. The HPLC separation method described here was first presented by Wagner et al. (2017) for online compound-specific stable carbon isotopic analysis. In this protocol, we use the same chromatographic parameters, but with UV detection, for the efficient quantification of DBC.

HPLC system and user-related assumptions - For the purposes of this protocol, we assume that the user will have access to an HPLC system that is equipped with an autosampler, binary or quaternary pump (for gradient separation), a UV or photo diode array detector, and software to assemble methods and sequences and for instrumental operation. We also assume that the user has basic knowledge of how to operate an HPLC system and/or has access to someone who can assist them with basic operation of an HPLC system. For example, we do not go into detail about how to flush solvent lines or properly hook up and equilibrate an HPLC column. 

HPLC instrument settings for the chromatographic separation of BPCAs are as follows: 

Column: Agilent Poroshell 120 phenyl-hexyl column (4.6 x 150 mm, 2.7 μm) with corresponding guard column (4.6 x 5 mm, 2.7 μm)
Mobile phase A: Phosphoric acid (0.6 M; pH 1)
Mobile phase B: Sodium phosphate buffer (20 mM; pH 6)
Flow rate: 500 μL min-1
Column temperature: 25°C (if available)
Autosampler temperature: 25°C (if available)
Injection volume: 25 μL
Peak detection: 240 nm

Set up the HPLC according to the parameters listed above.
SECTION: HPLC Analysis of BPCAs: Chromatographic separation of individual BPCAs
58. Using the HPLC software, create a method file for gradient separation of BPCAs.  

 

 

SECTION: HPLC Analysis of BPCAs: Chromatographic separation of individual BPCAs
59. Order your HPLC sequence by first starting with a blank injection, then BPCA standards in order of decreasing concentration, then another blank, and then the unknown samples. Perform a blank injection and re-run one of the mid-concentration BPCA standards every ten or so samples - Doing so enables you to assess the effects of potential carryover (uncommon) and instrumental/column drift (more common).

 

SECTION: HPLC Analysis of BPCAs: Chromatographic separation of individual BPCAs
60. Place blank, BPCA standard, and sample vials into the appropriate autosampler positions.
 

SECTION: HPLC Analysis of BPCAs: Chromatographic separation of individual BPCAs
61. At this point, the HPLC system should be set up for BPCA analysis and the column is properly equilibrated with mobile phase (70% A and 30% B).

Run the HPLC sequence.
 

SECTION: HPLC Analysis of BPCAs: Chromatographic separation of individual BPCAs
63. Examine the chromatogram for each BPCA standard run.
  
 

SECTION: HPLC Analysis of BPCAs: Chromatographic separation of individual BPCAs
62. Examine the chromatogram for each blank run.
  
 

SECTION: Calculation of DBC Concentrations
66. Using an external calibration approach, establish calibration equations for B6CA and B5CA.

Using the HPLC software, integrate the area under B6CA and B5CA peaks for each BPCA standard run. Enter peak retention times and peak areas for B6CA and B5CA into the spreadsheet below (DBC Calculations tab). Also enter the mass of B6CA and B5CA weighed out for the 5000 μM-C BPCA stock solution. If these steps are followed, the standard concentrations will be automatically converted from μM-C to μM-BPCA. 

The slope and intercept of the B6CA and B5CA will be calculated automatically and carried through to the DBC calculations spreadsheet.

  
 



SECTION: Calculation of DBC Concentrations
67. Using the HPLC software, integrate the area under B6CA and B5CA peaks for each sample run and BPCA standard runs that are interspersed within the sample queue. 

Enter peak retention times and peak areas for B6CA and B5CA into the calculations spreadsheet.
SECTION: Calculation of DBC Concentrations
68. The data entered into the spreadsheet up to this point is used to automatically calculate DBC concentrations in the original sample. We strongly encourage users to try these calculations by hand to fully understand how the final DBC concentrations are obtained.

The equations used in the spreadsheet are summarized below. 

 
 
 

 

SECTION: HPLC Analysis of BPCAs: Chromatographic separation of individual BPCAs
64. Examine the chromatogram for each sample run.
  
 

SECTION: Solid Phase Extraction: Extraction of DOM
9.3. Open stopcock to allow sample water to flow through it by gravity. Allow 5 to 10 mL of sample to flow out to waste, then close the stopcock.
 

SECTION: Sample Oxidation: Transfer of SPE-DOC eluate to ampules
30. Using a mechanical pipette, fitted with a glass Pasteur pipette tip, quantitatively transfer the calculated volume of methanol eluate ("V MeOH to dry" in spreadsheet) to the corresponding ampule. 

Triple-rinse the glass pipette tip with fresh methanol before use and between each sample to prevent cross contamination. Dispose of each rinsing to the appropriate solvent waste container.
 

SECTION: HPLC Analysis of BPCAs: Evaporation of nitric acid from ampules
44.5. After nitric acid has completely evaporated, turn off gas flow and remove needles from ampules and remove ampules from the sand bath. Gently wipe down the exterior of each ampule to remove excess sand.
 

SECTION: HPLC Analysis of BPCAs: Chromatographic separation of individual BPCAs
65. To our current knowledge, sample-containing HPLC vials can be stored in the freezer indefinitely. 

BPCAs stored in mobile phase A are stable and can be reanalyzed at a future time if desired, or used for online compound-specific stable carbon isotopic analysis of BPCAs following Wagner et al. (2017).