Introduction

Carbon, hydrogen, nitrogen, and sulfur (CHNS) are fundamental elemental components that are analyzed on the ship during IODP expeditions. Fluctuations in the concentration and/or content ratio of carbon, nitrogen, and sulfur define the origin, depositional environment, and diagenetic alteration of source materials. A few options for sample preparation method, instrument settings, and measurement methodology exist. In addition to the pre-generated methods, specific analytical methodology may be required based on the nature of certain sample materials. In this case, new methods will be created by the laboratory technicians working in conjunction with the scientists. Each instrument method is recorded by the USIO and will be associated with the measurements performed under that method.

Carbon Analysis

Most marine sediments and sedimentary rocks contain both carbonate ("inorganic") carbon and organic carbon. The CHNS procedure measures total carbon (inorganic plus organic) when following the standard method. Organic carbon content is then determined by using the inorganic carbon value from coulometric analysis and calculating the difference between total carbon from CHNS analysis and inorganic carbon analyzed by coulometer. Alternative methodologies can be employed to measure organic and inorganic carbon.

Nitrogen Analysis

Nitrogen is one of the important limiting nutrients in the ocean. The global carbon cycle and, consequently, atmospheric CO₂ might be tightly coupled to the nitrogen cycle, and therefore changes in the magnitude of the sinks and sources of fixed nitrogen in the oceans can significantly influence global climate. Biological nitrogen fixation, denitrification, and consumption of nitrate by phytoplankton are the major biological processes of the global nitrogen cycle. Changes in ocean circulation and nutrient supply, which occur in response to changes in environmental conditions, affect the relative importance and spatial extent of the major pathways of the nitrogen cycle.

Carbon-Nitrogen Relationship

C-N signatures indicate diagenesis and changes in productivity in seafloor sediments. Diagenesis may cause a decrease in C/N with decoupled C-N concentration variations, whereas productivity changes tend to produce C-N covariance in concentrations at relatively constant C/N ratios. Without significant superimposed diagenetic effects, linear relationships between C and N compositions can in some cases be interpreted as reflecting sources of organic matter:

• C/N = 6–8: fresh marine organic matter

• C/N = 8–20: degraded marine organic matter

• C/N > 20: continental organic matter

Low C/N values occur in sediment that is poor in organic carbon; these values may be biased by the tendency of clay minerals to absorb ammonium ions generated during the degradation of organic matter. Sediments rich in TOC have higher C/N values than sediments lean in TOC. C/N values that are elevated above algal values are common in organic carbon-rich marine sediments. These values evidently result from the selective loss of nitrogen as organic matter settles from the photic zone because nitrogen-bearing proteins are more labile than other organic matter components such as carbohydrates and lipids. This type of preferential nitrogen depletion and consequent carbon enrichment is recognized in organic carbon-rich sediments. C/N elevations are most pronounced when TOC concentrations are highest, suggesting that a higher rate of organic matter delivery leads to diminished organic matter degradation.

Sulfur Analysis

Cycling of sulfur compounds is a ubiquitous process in marine sediments that supports a range of microbial metabolic strategies. The occurrence of sulfur over a wide range of oxidation states (–2 to +6) allows sulfur species to serve as both electron acceptors and electron donors. In reduced form as sulfide (H₂S = H₂S_(aq) + HS^–), sulfur is also an important sink for reactive iron. The reduction of sulfate to sulfide is by far the most important pathway for sedimentary organic matter oxidation in anoxic marine sediments, and there is evidence that anaerobic oxidation of methane controls microbial sulfate reduction (MSR) in many marine systems.
Much of the sulfide produced during dissimilatory MSR in marine sediments is oxidized back to sulfate by a variety of biological and abiotic pathways, and sulfate produced by oxidation of sulfide may have variable isotope values reflecting the nature and complexity of the abiotic and biological oxidation pathways and relative contributions from different oxidants. These pathways often include the production of intermediate sulfur species such as elemental sulfur and thiosulfate, which can undergo further bacterial disproportionation reactions that may lead to further fractionations of both sulfur and oxygen isotopes in secondary sulfate.
Elemental sulfur is a possible intermediate in pyrite formation and may serve as an indicator for active SO₄ reduction. Elemental sulfur enrichments may form at places where the sulfide concentrations were high, resulting from in situ SO₄^2– reduction. Elemental sulfur forms from partial oxidation of sulfide. In addition, low-molecular-weight organic sulfur compounds are included in elemental sulfur.

Sulfur-Carbon Relationship

In normal marine sediments the relation between sulfur and carbon contents has a slope of 1/2.8 (S_tot/C_org ratio, wt%/wt%) and passes through the origin (assuming that sulfur fractions other than reduced sulfur are relatively negligible). In euxinic marine environments, however, sulfide is omnipresent (independent of local C_org contents) and iron sulfide formation can take place in the water column or at the sediment/water interface.
In addition, even slowly reacting iron compounds may react with sulfide in euxinic environments. Consequently, positive intercepts on the sulfur axis are obtained in sulfur vs. carbon plots for euxinic sediments, and only weak correlations may be observed. Additionally, post-depositional sulfidization of C_org-poor sediments may result in extremely high sulfur/carbon ratios.

Theory of Operation

Dried and powdered samples are combusted in a tin sample crucible with vanadium pentoxide catalyst, purified by a reactor packed with electrolytic copper and copper oxide, separated on a gas chromatographic column, and analyzed using a thermal conductivity detector (TCD). Addition of the V₂O₅ ensures complete conversion of inorganic sulfur in the sample to sulfur dioxide.

When the tin crucible with sample is dropped into the reactor, the oxygen environment triggers a strong exothermic reaction. Temperature rises to 1800°C, causing the sample to combust. The combustion products are conveyed across the reactor, where oxidation is completed. Nitrogen oxides and sulfur trioxide are reduced to elemental nitrogen and sulfur dioxide, and oxygen excess is retained. The gas mixture containing N₂, CO₂, H₂O, and SO₂ flows into the chromatographic column, where separation takes place. Eluted gases are sent to the TCD where electrical signals processed by the Eager 300 software provide percentages of nitrogen, carbon, hydrogen, and sulfur contained in the sample.

Equipement and Reagents

Equipement

Carbon, hydrogen, nitrogen, and sulfur are analyzed on a Thermo Electron Corporation FlashSmart CHNS elemental analyzer (Figure 1) with autosampler (Figure 2).

Instrument Description

C, H, N, and S are analyzed on a Thermo Electron Corporation FlashEA 1112 CHNS elemental analyzer (Figure 1, below) with the following specifications:
–Supplier: Thermo Electron Corporation
–Model: FlashEA 1112
–Detector: Thermal conductivity detector (TCD)
–Instrument Control: Eager 300 software for MS Windows
–Power Supply: 230 V AC; 60 Hz (US); 140 VA
–Dimensions: 500 mm H × 590 mm W × 558 mm D
–Weight: 65 kg
–Measurement Range: 100 ppm to 100% for C, N, H, and S
–Nominal Measurement Time: CNS: 8 min, CHNS: 10 min
–Sample Weight: 0.01–100 mg according to nature of the sample
–Temperature range:15°–35°C Maximum rel. humidity:30%–85%
The basic CHNS instrument configuration contains the following components.

1. Autosampler (highlighted in red in Figure 2 and shown in detail in Figure 3)
2. Gas tubing for helium and oxygen
3. Synoptic panel
4. Furnace (furnace cover plate shown in Figure 4; opened plate in Figure 5)
5. Reactor
6. Oven
7. Water adsorption column (shown in Figure 6)
7. Gas chromatographic column (also in Figure 6)
8. TCD detector

Reagents

Be sure to read the Health, Safety, and Environment section before using any of the chemicals and reagents in this method.

Note that many of these materials are hazardous and before any work is done with them, the user must be familiar with the appropriate Material Safety Data Sheets (MSDS).

Gases

• Helium (carrier gas): chromatography grade

• Oxygen (sample oxidation gas): 99.995% minimum purity

Other Consumables and Tools

• Pre-packed reactor: Thermo Electron PN 468 020 15; also CE Elantech 061110

• Cleaning devices: Thermo Electron PN 205 006 25 and 276 060 10

• HPAR reactor extractor

• Crucible extractor

Liquid Sample Consumables

An expedition-specific science strategy or sample materials may require additional apparatus to handle liquid sample analysis.

Automatic sampling system with injection assembly
Water container and vials for solvent
Syringe (0.5 µL): PN 36504045
Syringe (10 µL): PN 36500525
Syringe (250 µL): PN 013680 (36504042)

Sample Preparation

Samples are freeze-dried, crushed, and homogenized using a mortar and pestle or an electric mill and weighed into a tin sample cup (crucible). If sulfur is being analyzed, vanadium pentoxide is also added, acting as a catalyst. The crucibles are then closed (referred to as "wrapping" the sample) for instrumental analysis.

The following amounts are currently used:

• Standards: 1, 3, 5, 10, 15, 20, 25 mg

• Unknown samples: 12–15 mg

• Vanadium pentoxide (if used): ½ small spatula for blanks, standards, and unknowns

Drying and Homogenizing the Sample

1. For faster free-drying, samples can be taken out of sample tubes and broken into pieces.

2. Freeze-dry samples for at least 12 hrs until samples are completely dry (may require additional freeze-drying time, up to 3 hrs). The sample is dry when there is no more condensate in the cold part of the tube. Also when a sample bag is felt immediately after releasing the vacuum, if the sample still feels cold, there is residual moisture in it. Freeze-dried samples should not feel cold coming out of the freeze-dryer. Note: do not overload the freeze-dryer; it will not dry properly.

3. Wipe an agate mortar and pestle with ethanol and allow to dry completely before grinding each sample. Be sure the mortar and pestle are completely dry; ethanol residue can cause false C and H results.

4. Grind and homogenize sediment using a cleaned and dry agate mortar and pestle.

5. Fill a sample bottle with powdered homogenized sediment.

Sample Volume

Because the balance system is of unknown lower precision aboard ship than in a shore-based laboratory, we recommend 6–9 calibration points to obtain a high CHNS measurement precision. The usual quantities of sample materials and standards used are as follows:

• Vanadium pentoxide: ½ of small spatula for blank, standard, and unknown samples

• Standard (sulfanilamide): 1, 3, and 5 mg

• Standard (Buffalo River Sediment): 5, 10, 15, 20 mg

• Standard sediment or sulfanilamide (for QA/QC evaluation; see Quality Assurance/Quality Control): 11 mg, minimum 10 pieces may be required for one sequence of the measurement.

• Unknown samples: The amount used depends on the nature of the sample material. If material is unusual, it may be necessary to weigh out several different quantities of sample to correctly analyze the material. Typical masses of some materials are:

  • Hemipelagic sediments: 11–12 mg

  • Pelagic sediments (low productivity): 11–15 mg

  • Upwelling area (high productivity area): 10–12 mg

Clean Preparation Equipment

Wipe spatula, spring tweezers, balance, and other stainless equipment with ethanol between each sample. Be sure these are completely dry before using to avoid contaminating samples.

Weighing and Wrapping Samples

1. Double-click the Cahn Balance icon.

2. Log into the system by entering LIMS user name and password.

3. Enter the Text ID and the Container number of the sample or standard.

4. Enter 100, 200 or 300 as the Measurement Count depending on sea state.

5. Place an empty crucible (tin container) on the REFERENCE balance pan (right pan).

6. Place a new crucible into the small hole of the metal plate.

7. Flare the upper half of the tin cup and place a crucible on the UNKNOWN balance pan (left pan).

8. Tare the balance using the software. (Do not press the Tare button on the Cahn balance.)

9. Click Start to start weighing.

10. Once the mass volume is acceptable, click Get Mass.

11. Remove the crucible from the UNKNOWN balance pan, place the crucible on the metal plate, and place the appropriate amount of sample material (see Sample Volume) into the crucible.

NOTE: DO NOT use the holes on the metal plate if any standard material is spilled in or near it because even a small amount of standard may contaminate the measurement.

1. Place crucible with sample material back on the UNKNOWN balance pan and close door.

2. Click Weigh and Start.

3. Click Get Mass if the mass value is acceptable.

4. Enter any part of a sample ID or Label ID and click Search.

5. Select the sample that was just weighed from the list and click Assign.

6. Enter a container number (sample holder number, not an autosampler number) in the Container# field.

7. Click Save.

Instrument Setup

The shipboard technician will set up and maintain the reactor and gas chromatographic column. The technician will also instruct the scientist on the operation of the instrument.
Instrument startup and conditioning are conducted by the shipboard technician, who also loads the analytical configuration (method). If a specific analytical configuration is required because of the nature of the samples, scientists should collaborate with the technician to develop the analytical method and run parameters. When the instrument is ready, the Eager 300 CHNS screen displays. To confirm the status of the CHNS analyzer instrument, select View > View Elemental Analyzer Status (see Figure 4, below).(Figure 3). Analytical determination takes approximately 40, 100, 150, and 640 seconds for nitrogen, carbon, hydrogen and sulfur, respectively, for a 2 m long separation column. The integration time should be set to approximately 15 min (900 sec) for runs in which sulfur is measured due to the breadth of the signal peak. Either purchase pre-filled reactor columns or prepare them according to the manufacturer's instructions. The adsorption filter should be filled with fresh magnesium persulfate regularly. The JRSO uses pre-packed reactor columns from CE Elantech and Costech; these have always proved to be of high quality, without gaps or faults in the reagents that could lead to a poorly-reacted gas stream.

Starting the Instrument

Perform any required maintenance before starting the system (see Instrument Maintenance section).

Powering on the System

Proceed according to the following operating sequence. Early start up (6 hr before measurement) is recommended for this instrument.

1. Open gas line/bottles.

2. Power on the FlashSmart.

3. Power on the PC and monitor.

Open Instrument Program

Run the program Eager Xperience. Select Analyzer #1.

Open

Setting Instrument Parameters

Verify gas pressure by checking pressure regulators by opening the EA door with the control panel on it.

• Helium pressure: 260 kPa

• Oxygen pressure: 300 kPa

Parameters can be edited by going to Edit > Edit Elemental Analyzer Parameters

• Temperature

  • Left furnace: 950°C

  • Oven: 65°C

  • Set instrument to standby: Unchecked for running samples, checked when idle to conserve gas

• Flow / Timing

  • Carrier: 140 ml/min

  • Oxygen: 250 ml/min

  • Reference: 100 ml/min

  • Cycle: 600 sec

  • Sampling Delay: 12 sec

  • Oxygen Injection end: 5 sec

• Detector

  • Filament: On (can turn off when idle)

  • Time Event: not checked

Select Send to send the paramenters to the CHNS.

Current status can be viewed and confirmed by going to View > View Elemental Analyzer Status

At the above settings, the TCD should settle at ~1000 uV. There's an option to auto-level to 1000 within the View Elemental Analyzer Status window.

Note: Peak retention times are confirmed based on the chromatogram of the Bypass sample obtained using standard materials, which is then used to validate the calibration curve.

Shut Down

• Edit > Edit Elemental Analyzer Parameters

• Uncheck the furnace, oven, filament.

• Check 'Set instrument to standby'

• To fully power down, wait for temperatures to fall below 100°C, then flip the power switch on the back of the CHNS

Checking for Leaks

1. Select Edit > Edit Elemental Analyzer parameter.
2. Select the Flow / Timing tab (Figure 8).
3. Select (check) the Carrier, Oxygen, and Reference parameters.
4. Click Send to transfer the parameters.
5. Click OK to close window.
6. Click NO to exit without saving the method.
7. Select View > View Elemental Analyzer Status.
8. Select the Special Functions tab (Figure 9).
9. Click Leak Test to open the Leak Check screen.
10. Click Start to begin the operation.
11. Click Yes for the Eager 300 to automatically test for leaks.
12. After a few seconds, the reference gas flow must be 0.
Note: After ~560 sec, carrier flow will decrease. Carrier flow must be between 0 and 3 mL/min. Higher values indicate the system is not leak free. Leaks in the system are generally due to incorrect closure of the reactor and filter locking nut. Rarely, leaks may be due to the autosampler.

Combustion Column (a.k.a. reactor tube)

• A combustion column will last approximately 100–120 samples. The life of a combustion column can be extended by using a quartz insert. 

• To condition a new column, run a standard 2 times as an Unk (unknown). Put a scoop of standard in a tin cup without weighing it. Wrap the sample as normal. Enter mass 1. Verify that you see a peak for nitrogen and carbon if using Buffalo River standard. Below is a sample chromatogram from a Buffalo River check standard run. With conditioning we are only verifying that we get a "normal" looking chromatogram with peaks. The height of the peaks is unimportant at this point. 

Open

Open

Typical Buffalo River check standard chromatogram 

Sulfur and Hydrogen

• If analyzing for sulfur, add vanadium pentoxide to the sample cups.

Method Setup

Creating Data Folders and Copying Method File

The sequence of data files must be saved in the folder with the method file. Create new folders in the following directory: Program data\CHNS\Expedition No.

Create subfolders for each column used, or by Site and Hole.

Select File > Save Method and save the method (rename as the expedition number) in the created folder for the expedition and subfolder. There should now be a mth, eam, and cc file in the folder.

Editing the Component Table

1. On the CHNS screen, select Edit > Sample table (Figure 12, below). Clear the sample table and create two new rows.

2. Fill in the sample table as follows:

–Row 1:

• Sample name = Blank test

• Filename = Blank_test_ddmmyy

• Type = blank

–Row 2: Insert one blank line to stop measurement automatically after the blank.
–Row 3:

• Sample name = Bypass test

• Filename = Bypass_test_ddmmyy

• Type = Bypass

1. Prepare Blank (tin cup with V2O5) and Bypass using a standard material (~0.6–0.7 mg of sulphanilamide or BBOT with V2O5 and tin cup). Place the Blank in the sample tray of the MAS 200 R autosampler for the FlashEA 1112.

2. Note: Acquire a chromatogram that contains all the components to be identified.

3. Click OK to close the sample table.

4. Select File > Save method to save the sample table.

5. On the CHNS screen, click the green arrow toolbar icon to start running the samples.

6. Select View > View sample being acquired to confirm run status.

Note: Confirm chromatogram from the Blank to check the condition of the elemental analyzer. If the chromatogram has unexpected peaks and/or an unacceptable (disturbed) baseline, identify the cause of problem before running the Bypass sample. If the Blank is fine, continue measurement of Bypass.

1. After the Bypass sample is done, select Edit > Component table to identify the analysis peaks (Figure 13, below). Instructions for editing the component table are given in the Eager 300 Software Operating Manual, p. 101–115.

2. Select File > Load Chromatogram, and select bypass test-ddmmyy (last sample run).

10. Click Open to view the chromatogram.
11. Select Time of any component on the table.
12. Move the cursor near the red-gray arrow on the screen and right-click.
13. Adjust the red line on the screen to intersect the top of the peak.
14. Repeat Steps 11–13 for each peak on the chromatogram, and click OK to save changes.
Warning: If peak retention times have changed a lot, measure an additional Bypass sample to confirm the new peak positions. Possible causes of peak retention time shifts include (1) gas leaks (include outside of the instrument), (2) gas flow changes, (3) atmospheric pressure changes, and (4) others.

Editing the Integration Parameters

1. After finishing peak identification in the previous section, select Edit > Edit Method on the CHNS screen. Method editing instructions are shown in the Eager 300 Software Operating Manual, p. 74–76.