Introduction

The principles of spectroscopic analysis rely on Beer's law. The principle of Beer's law is that passing light of a known wavelength through a sample of known thickness and measuring how much of the light is absorbed at that wavelength will provide the concentration of the unknown, provided that the unknown is in a complex that absorbs light at the chosen wavelength. 

IODP's Agilent Technologies Cary 100 double-beam UV-Vis (ultraviolet–visible) spectrophotometer is ideal for shipboard routine laboratory work. The system measures analytes in interstitial water obtained from sediment cores using standard colorimetric methodology.

The described methods are based on ODP Technical Note 15, Chemical Methods for Interstitial Water Analysis Aboard the JOIDES Resolution, Aug 1991; J.M. Gieskes, T. Gamo, and H. Brumsack.

Methods

Ammonium

Determination of ammonium concentration is of importance because this constituent is an indicator of diagenesis of organic matter in the sediments. The onset of sulfate reduction coincides with initiation of ammonium ion production. Ammonium production increases strongly in the zone of methanogenesis, presumably as a result of associated deammonification reactions. The large potential variation in ammonium concentrations, therefore, suggests that a few preliminary ammonium concentrations should be run in order to set limits to the sample dilution and range of standards to be used. Suggestions for this follow below.

The methodology is based on Solorzano (1969), originally developed to detect very small NH₄⁺ concentrations in seawater. Although background contamination problems in seawater are enormous, the relatively high concentrations of ammonium in pore fluids (as high as 85 mM in ODP Leg 112 samples; Kastner et al., 1990) minimizes this problem when matrix blanks are run along with the samples. In areas of low sedimentation, however, very low ammonium concentrations require careful sample handling to avoid this problem. 

The ammonium method is based on diazotization of phenol and subsequent oxidation of the diazo compound by Chlorox™ to yield a blue color, measured spectrophotometrically at 640 nm.

Reagent Solutions

Standards

Add standard to a 50 mL volumetric flask and bring to volume with nanopure water.

50 mL batches are stable for 1 month. 

Procedure

Concentrations of ammonium may differ quite a bit at different sites. Typically in areas with strong evidence of organic carbon diagenesis (e.g., organic carbon–rich sediments), high concentrations of NH₄⁺ can be expected. In that case, sample aliquots must be made appropriately small or sample dilution may be required. The range can be established by using a sample near the alkalinity maximum. Once the range has been determined, prepare standards that cover this range. In this manner, samples and standards are treated in a similar way.

Note:  Use a smaller aliquot of sample if the result exceeds the linear range of the spectrophotometer, making up the volume with nanopure water. (For example, for a 100 µL aliquot of a sample, add 2.1 mL nanopure water.)

Note:  The order of dilution (below) matters, so do not change this order. Shake samples after EACH addition.

Phosphate

Determination of dissolved phosphate, particularly in rapidly deposited organic carbon–rich sediments, is important in the shipboard analytical program. Phosphate concentrations may vary considerably, and it is therefore advisable to obtain a preliminary idea of the concentration ranges to be expected. This can most easily be accomplished by taking samples in the region of maximum alkalinities. Typically if alkalinities are >30 mM, dissolved phosphate concentrations may be >100 µM; thus, only very small sample aliquots will be needed to establish the concentration range.

This method is, in essence, the colorimetric method from Strickland and Parsons (1968) as modified by Presley (1971) for DSDP pore fluids. Orthophosphate reacts with Mo(VI) and Sb(III) in an acidic solution to form an antimony-phosphomolybdate complex. Ascorbic acid reduces this complex to form a blue color, and absorbance is measured spectrophotometrically at 885 nm.

It is important to note that the concentrations in the final test solution cannot exceed ~10 µM. Thus, for open-ocean (low sedimentation rate, low organic carbon) sediments, one might need to do the determination on 2 mL of sample (expected range 0–10 µM), but in typical continental margin settings, where concentrations can exceed 100–200 µM, a 0.1 or 0.2 mL sample aliquot must be used. The concentration range must be established prior to running samples, and it is highly advisable to make standards that cover the range of concentrations to be expected.

Note: Samples with high silica concentrations may give a false increase in measured concentration of phosphate (http://dx.doi.org/10.1007/BF02071829; S. Noriki, Silicate correction in the colorimetric determination of phosphate in seawater, 1983).

Reagent Solutions

Standards

Add standard to a 50 mL volumetric flask and bring to volume with nanopure water.

Procedure

Note:  Use a smaller aliquot of sample if the result exceeds the linear range of the spectrophotometer, making up the volume with nanopure water. (For example, for a 300 µL aliquot of a sample, add 300 µL nanopure water.)

Silica

Silicon is routinely measured on the ICP, so measurement by spectroscopic analysis can be considered an alternate method.

Dissolved silica determinations are of great importance in interstitial waters. Often they represent the lithology of the sediments, and the concentrations can vary substantially, especially if highly dissolvable phases such as biogenic opal-A, volcanic ash, or smectite are present. Thus, a wide range of concentrations can be expected, typically from 50 to 1200 µM or higher (especially in hydrothermally affected sediments). The method below usually covers the range, although greater dilutions may be appropriate if sediments or sample sizes necessitate this.

This method is based on the production of a yellow silicomolybdate complex. The complex is reduced by ascorbic acid to form molybdenum blue, measured at 812 nm. The blue complex is very stable, which will enable delayed reading of the samples.

Reagent Solutions

Standards

Add standard to a 50 mL volumetric flask and bring to volume with nanopure water.

Procedure

Make sure that all reagents are prepared ahead of time. The method has a time factor built in, and therefore it is of great importance to have all necessary reagents ready to go.

Do not handle more than about thirty samples at a time in order to ensure that the 15 min time limit can be adhered to. Make sure that there are no large fluctuations in room temperature.

Do not use synthetic seawater in dilutions of the primary standard. This could cause the decrease in reactive silica in a few hours as a result of polymerization reactions.

The reason for adding 200 µL of synthetic seawater to the standards is to maintain a reasonably uniform salt content in relation to the samples, this suppressing a potential salt effect on the method.

It is important to wait at least three hours for the blue color to develop; the higher the concentration, the longer the time. The color remains stable for many hours, and reading after 4–5 hours may, in fact, be a good idea. Again, consistency in time limits is advisable.

Sulfide

The sulfide method is based off a method developed by Cline in 1969. This method called for very large volumes of water (50 mL). This method was modified on the BONUS Baltic Gas expedition in 2011 to work with sample volumes in the 1-5mL range.
The method is a bit tricky in that the reagent concentrations change depending on what concentration range your samples fall in:

• High range: 6-80 uM
• Low range: 1-10 uM.

All samples need to be preserved during splitting with a 1% zinc acetate solution. It can require a relatively large amount of sample in order to do this analysis. The high range method uses less sample (~500 µL), but if the sulfide level is below 6 µM, it won't be enough. It may still be worth screening samples with the high-range method in order to conserve interstitial water sample volume. The low-range method requires 4 mL (8x the amount of sample) but is sensitive down to 1 µM. The scientists will have to determine if consumption of 4 mL, possibly 4.5 mL, of sample is worth obtaining the sulfide concentration.

Reagent Solutions

High-Range Method 

Sample Preservation

The IW splits for sulfide should be preserved immediately with the 1% zinc acetate solution to prevent the loss of dissolved sulfide.

Add 40 µL of 1% zinc acetate to 0.5 mL sample aliquots.

Standards

Make dilutions of the 100 µM zinc-sulfide standard for the standards to create 0.5 mL of standard at each level.

Procedure

Low-Range Method

Sample Preservation

The IW splits for sulfide should be preserved immediately with the 1% zinc acetate solution to prevent the loss of dissolved sulfide.

Add 800 µL of 1% zinc acetate to 4 mL sample aliquots.

Standards

Make dilutions of the 100 µM zinc-sulfide standard for the standards to create 4 mL of standard at each level.

Procedure

Analyzing Samples

Preparing the Cary Spectrophotometer

1. Turn on the unit and let it warm up for at least three hours at the wavelength in question.
2. In Varian > Cary WinUV start the Advanced Read application. 
3. Click the Setup button and select the Options tab.
   • Confirm in the Beam Mode area that Double Beam is selected and Normal is also selected.

SPS3 Autosampler

The Agilent Technologies SPS3 autosampler facilitates sample introduction into the Cary Spectrophotometer with minimal operator interaction.  

Figure 1 : Sample inlet tube and flow cell

The SPS3 is connected to the PC via a RS232 cable to a Keyspan USB converter and the SPS3 is connected to the Cary unit via a RSA sample inlet tube (Figure 1).

Figure 2 : SPS3 Reservoir peristaltic pump

The reservoir peristaltic pump (Figure 2) allows nanopure water to be pumped through the system between samples to flush lines. Pump speed is controlled by a dial above the pump. This pump fills the reservoir but does not change the flow into the Cary's flow cell; that is controlled by the spectrometer's peristaltic pump.  

Figure 3 : SPS3 Sample trays

Sample trays allow up to 180 samples to be run (Figure 3). First vial is a "zero" (nanopure water).

Software setup

Advanced Reads setup

Figure 4 : Advanced Reads main screen

In the Advanced Reads software, click the Setup button (figure 4).

Figure 5: Cary (Enter wavelength)

On the Cary tab (figure 5), the wavelength can be changed to the necessary wavelength for the analysis type. 

Ave Time (sec) is the length of time over which readings will be taken and averaged.  The default setting is 0.1 seconds; increasing this time is beneficial for improving precision and reducing the effect of noise on the resultant measurement.

1 second is a good minimum setting for Ave Time. There are diminishing returns for precision as time increases. Setting it higher than 5 seconds provides almost no more benefit at the expense of taking more time.

Figure 6 : Setup (Entering sample information)

On the samples tab (figure 6), the samples to be run can be entered.

Set the number of samples to the total combined amount of samples and standards. Enter sample names, including the standards.

An alternative is that a text file containing sample names can be imported by clicking import names. A text file with textID for sample names can be filled quickly with a barcode scanner (one sample per line). 

Figure 7 : Setup (Sipper settings [Cary peristaltic pump])

On the Acessories2 tab (figure 7), make sure sipper on is checked and internal RSA is selected. Set fill/return to 32 seconds and delay to 8 seconds.

Figure 8 : Setup (Rinse/Sample trays)

On the samplers tab (figure 8) make sure use sampler is checked along with rinse. Verify that SPS3 Autosampler is selected and displayed. Click configure for the next window.

Figure 9 : Setup (Rinse/Rack type)

Click on a tray area and then select the 5x12 positions rack type from the sample rack type drop down menu.

To position your samples on the autosampler. Click on zero, then click on the first tube position (top right). Then click on sample and click on the second position. This will then populate the sample rack with the samples that were entered on the sample tab.

Verify that the display matches the loading of samples on the sample rack.

Figure 10 : Setup (Comm Setup/RS232 settings)

Com port settings (figure 10) can be reached by clicking comm setup (figure 9). This is just a reference for the proper com port setup in case the setup is lost or forgotten by the software.

Running samples

1. • Prepare samples according to the protocol outlined in the above sections for the appropriate analyte.  
   • Engage the tubing on the peristaltic pump for both the Spectrophotometer and the Autosampler. Make sure that the waste lines go into a receptacle.
   • Click on START in the main Advanced Reads window (Figure 4).
   • A window will pop-up showing the samples entered in the previous steps (Figure 12). Click OK.
   • Save the data to a file for later viewing (Figure 13).

Figure 12 : Samples ready for analyses

Figure 13 : Assigning a filename for the results

• • Observe where the samples need to go (Figure 14). Click OK.

Figure 14 : How to load the vials in the sample racks

• • View the results in real time (Figure 15).

Figure 15 : Viewing results in real-time

• • Save the results to a .csv file.
    • Select File > Export report (*.csv)
    • Enter the location/file name.
  • Copy/paste into a calibration spreadsheet for manipulation and subsequent upload via Spreadsheet Uploader.
    • Using the results from the reads of the standards, create a calibration curve from the plot of the concentration vs. absorbance values. Use this equation to extrapolate the sample concentrations from their corresponding absorbance value. This can be done in the same spreadsheet as created above in the Advanced Reads application. This sheet can be loaded into the LIMS Spreadsheet Loader application as outlined in the LIMS Integration section.

Shutting down the Instrument

Aspirate approximately eight cycles of nanopure water, release the tubing on the peristaltic pump, turn off power to the unit and exit from the software. Clean any spills that may have occurred.
Empty the waste container into a gallon size ziploc bag of corn cob absorbent in a fume hood. Label the bag "CHEM LAB WASTE - DO NOT OPEN". Let evaporate for a week and place in burnables bag with other burnables. 

QAQC

QA/QC for analysis consists of calibration verification using check standards, blanks and replicate samples.

QA/QC Types

Check Standard

A check standard for each set of analytes is run ~ every fifteen analyses depending on batch size. Check standards consist of a standard from approximately the midpoint of the calibration curve.
The check standard result is evaluated against the threshold for % variance limits for calibration verification standard against true value:

• • • Within ±10%: calibration is verified and sample analysis can continue.
    • Outside of ±10%: check reagent solutions and rerun all samples in the corresponding analytical batch.

Blank

A blank is run with every batch to determine if high background levels are interfering with accurate sample results.

Replicate Samples

During each batch, a single sample should be run in duplicate and the variation of the results compared.

Data Available in LORE

Interstitial Waters Standard Report 

• Exp: expedition number
• Site: site number
• Hole: hole number
• Core: core number
• Type: type indicates the coring tool used to recover the core (typical types are F, H, R, X).
• Sect: section number
• A/W: archive (A) or working (W) section half.
• Top offset on section (cm): position of the upper edge of the sample, measured relative to the top of the section.
• Bottom offset on section (cm): position of the lower edge of the sample, measured relative to the top of the section.
• Top depth CSF-A (m): position of observation expressed relative to the top of the hole.
• Top depth [other] (m): position of observation expressed relative to the top of the hole. The location is presented in a scale selected by the science party or the report user.
• Sampling tool: tool used to collect sample
• Data columns: header lists parameter measured and concentration units, followed by wavelength (for ICP-AES) and then analysis method.

Expanded IC Report

• Exp: expedition number
• Site: site number
• Hole: hole number
• Core: core number
• Type: type indicates the coring tool used to recover the core (typical types are F, H, R, X).
• Sect: section number
• A/W: archive (A) or working (W) section half.
• text_id: automatically generated unique database identifier for a sample, visible on printed labels
• sample_number: sample number of sample. text ID with sample type prefix removed.
• label_id: id combining exp, site, hole, core, type, sect, A/W, parent sample name (if any), sample name
• sample_name: name of sample
• x_sample_state:
• x_project: expedition project the sample is uploaded under. typically the same as Exp.
• x_capt_loc: 
• location: location sample was taken
• x_sampling_tool: tool used to collect sample
• changed_by: name of person who uploaded sample
• changed_on: date and time sample was uploaded
• sample_type: type of sample. typically LIQ, for liquid.
• x_offset: top offset of parent sample where sample was taken in m
• x_offset_cm: top offset of parent sample where sample was taken in cm
• x_bottom_offset_cm: bottom offset of parent sample where sample was taken in cm
• x_diameter: 
• x_idmp: 
• x_orig_len: 
• x_length: length of sample in m
• x_lengeth_cm:  length of sample in cm
• status:
• old_status:
• original_sample: 
• parent_sample:
• standard:
• login_by: name of person logged into LIMS application used for this test
• sampled_date: 
• legacy:
• test changed_on: date of last edit of analysis
• test date_started: date analysis was started
• test group_name: 
• test status:
• test old_status:
• test test_number: unique number associated with the instrument measurement steps that produced these data
• test date_received: date analysis was uploaded to LIMS
• test instrument: instrument used to perform analysis
• test analysis: analysis type
• test x_project: project test was assigned to
• test version: 
• test order_number:
• test replicate_test:
• test replicate_count:
• rest sample_number: sample number for sample the analysis was performed on

• Top depth CSF-A (m): position of observation expressed relative to the top of the hole.

• Bottom depth CSF-A (m): position of observation expressed relative to the top of the hole.

• Top depth CSF-B (m):

• Bottom depth CSF-B (m):

• analyte: the analyte measured for this test
• concentration (uM):  concentration of analyte in uM
• ssup_assman_id:  link to download the batch of data uploaded with spreadsheet uploader
• ssup_filename: filename of the batch of data uploaded with spreadsheet uploader

• sample description: observations recorded about the sample itself

• test test_comment: observations about a measurement or the measurement process; some measurement observations may be under Result comments

• result comments: observations about a measurement or the measurement process; some measurement observations may be under Test Comments

Data collected is transferred to the LIMS database using IODP's Spreadsheet Loader application.

This is best done by entering the results into an Excel spreadsheet in a format similar to the pattern below, keeping the appropriate number of columns blank, and omitting the column headers.

Then run the Spreadsheet Loader application. Go to File > Load and it should import something like below (Figure 16).

Before uploading, click Edit > Validate Sheet to validate the data.  

To upload into the database, go to  Lims > Upload and status messages will appear in the blank window.

Figure 16 : Uploading results with Spreadsheet Uploader

Maintenance and Troubleshooting

Cleaning

Any spills in the sample compartment should be immediately wiped up and any deposits on the sample compartment windows should also be removed. The exterior surfaces should be cleaned with a soft cloth and, if necessary, this cloth can be dampened with water or a mild detergent.

Source Lamps

Instructions for how to change and align both the visible and UV lamps are included in the Help provided with the software. Care must be taken when removing lamps. Touching the glass envelope will reduce its efficiency. NEVER touch the glass surface of a new lamp. Always handle a lamp by its base, using a soft cloth.

Fuses

To replace a fuse, disconnect the unit from the power supply, and replace the blown fuse with one of the type and rating as outlined in the hardware specifications section.

1. Disconnect the instrument from the power supply.
2. Undo the fuse cap by pressing the cap and turning it counter-clockwise.
   
3. Carefully pull out the cap. The fuse should be held in the fuse cap.
   
4. Check that the fuse is the correct type and is not damaged. If necessary, replace it.
   
5. Place the fuse into the cap, push the cap in and then turn the cap clockwise.
   
6. Reconnect the instrument to the power supply.
   

Cary Win UV Help

Varian provides extensive help resources, available from the software CD. After installing the help utilities, go to START > All Programs > Varian > Cary WinUV > Cary Help.
Cary Help offers troubleshooting information, maintenance information like how to replace lamps, aligning lamps and mirrors, and replacing fuses and cleaning the flowcells.

Contact Information

Varian Instruments

1 800 926 3000

customer.service@varianinc.com

References

Gieskes, J.M., Gamo, T., and Brumsack, H., 1991. Chemical methods for interstitial water analysis aboard JOIDES Resolution, ODP Tech. Note, 15. doi:10.2973/odp.tn.15.1991.

Kastner, M., Elderfield, H., Martin, J.B., Suess, E., Kvenvolden, K.A., and Garrison, R.E., 1990. Diagenesis and interstitial water chemistry at the Peruvian margin—major constituents and strontium isotopes. In Suess, E., von Huene, R., et al., Proc. ODP, Sci. Results, 112: College Station, TX (Ocean Drilling Program), 413–440. doi:10.2973/odp.proc.sr.112.144.1990

Noriki, S. 1983. Silicate correction in the colorimetric determination of phosphate in seawater. J. Oceanograph. Soc. Japan, 39(6):324–326. doi:10.1007/BF02071829

Presley, B.J., 1971. Techniques for analyzing interstitial water samples: Appendix Part 1: determination of selected minor and major inorganic constituents. In Winterer, E.L., et al., Init. Repts. DSDP, 7(2): Washington, DC (U.S. Govt. Printing Office), 1749–1755. doi:10.2973/dsdp.proc.7.app1.1971

Solorzano, L., 1969. Determination of ammonia in natural waters by phenol-hypochlorite method. Limno. Oceanogr., 14:799–801.

Strickland, J.D.H., and Parsons, T.R., 1968. A manual for sea water analysis. Bull. Fish. Res. Board Can., 167.

Appendix

Hardware

The Varian Cary 100 is a double-beam, dual-chopper, monochromator UV-Vis spectrophotometer, centrally controlled by a PC. It has a high-performance R928 photomultiplier tube, tungsten halogen visible source with quartz window, and deuterium arc ultraviolet source. 

Figure 17 : Schematic of Cary Spectrophotometer

Electrical

Replacement Parts

Pumps

A double-action peristaltic pump services the feed and waste. 

(1) plastic sleeve. (2) metal hook tubing. (3) waste outlet.

Credits

This document originated from Word documents Cary_UG_374_draft and CarySPS3_QSG_374_draft that were written by E. Moortgat (20111212). Credits for subsequent changes to this document are given in the page history.

LIMS Component Table

Archived Versions

• LMUG-CarySpectrophotometerUserGuide-230220-1918-164.pdf
• Cary_UG_374_draft_20181126.pdf
• Cary_UG_v1.1_20181130.pdf
• Cary_QSG_378P_20181130
• Spec User Guide: 29th September 2022