TITLE: Measuring dissolved black carbon in water via aqueous, inorganic, high performance liquid chromatography of benzenepolycarboxylic acid (BPCA) molecular markers
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 oxidation products (BPCAs) 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 supplies cleaned according to the above protocol. During sampling, care should be taken to avoid sample contamination (e.g. rinsing sample bottle with sample before collection, collecting water upstream from your position, avoiding the introducing sunscreen/oils from hands to water or inner sample bottle, etc). Samples are typically filtered through pre-cleaned filters with pore sizes ranging from 0.1 to 0.7 μm. Store filtered (un-acidified) water samples in the dark at 4°C until further analysis or up to 48 hours.
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. In order to determine the volume of sample necessary to extract, please see the Estimating Sample Volume tab in the Example PPL-BPCA Calculations spreadsheet. In general, approximately 0.5-mg of extracted DOC is used to quantify DBC in the original sample. As extraction efficiencies and amount of DBC present are sample-dependent, we suggest passing more than the minimum volume through the cartridge and instead aim for a volume that will oxidize to ~20uM of benzenehexacarboxylic acid (B6CA). Please see linked spreadsheet and the rest of the protocol for further discussion and elaboration.   

Once the necessary volume is determined, acidify the filtered water sample to pH 2 using hydrochloric acid.
 


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. Set up SPE apparatus and begin flow of acidified sample through SPE cartridge.

   

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
9.3. 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: 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
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
12. Once extractions are complete, close stopcocks and remove tubing and adapter caps from the sample bottles and SPE cartridges.
SECTION: Solid Phase Extraction: Extraction of DOM
13. Record the mass of the empty sample bottle.
 

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
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 for up to three months before 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. Cartridges should be stored at 4°C.
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: 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. This methanol eluate can be stored for years in the freezer as an archive of SPE-DOC for future BPCA analyses. 
 

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: Sample Oxidation: Transfer of SPE-DOC eluate to ampules
29. Label glass ampules
 

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: 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
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: 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
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
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
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. Evaporate the concentrated nitric acid contained in the ampules.
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: 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: 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: HPLC Analysis of BPCAs: Preparation of HPLC mobile phases and BPCA standard solutions
52. Prepare mobile phase B = 20 millimolar (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
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
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
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: 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: 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
62. Examine the chromatogram for each blank run.
  
 

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
64. Examine the chromatogram for each sample run.
  
 

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).
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.