Chemistry Lab

ALKALINITY

The use of the METROHM automatic titrator constitutes a great advance in the methodologies of the chemistry laboratory aboard JOIDES Resolution.

The use of this method allows quick determination of the alkalinity very shortly after completion of the pore-water extraction. The titrated sample can be used for many subsequent analyses, provided the dilution caused by the addition of 0.1 M HCl is properly accounted for through the use of a dilution factor. It should, however, be noted that subsequent analysis of potassium may be somewhat suspect because of the use of saturated KCl in the reference electrode.

Theory

In the alkalinity determination we rely on a mathematical evaluation of the second equivalence point of carbonate titration in seawater. This method uses the most stable part of the titration curve, i.e., the part well beyond the equivalence point on the low pH side. The mathematical method is called the GRAN method after its developer, Gran, from Sweden. In essence the method linearizes the titration curve by means of a simple function:

   F = (v + V₀) x 10^E/A,

where:          F = Gran factor,
                v = volume of acid added to the solution in the titration vessel;
                V₀ = original volume of the sample;
                E = EMF (millivolts) at v;
                A = slope of electrode determined on the basis of the electrode calibration.

Usually the slope is about 59 mV at 25^o C, but it can be determined from the electrode calibration.

Of some interest is that the function, F, when plotted as a function of the volume of acid added (v), is linear when obtained sufficiently far removed from the equivalence point. With the combination microelectrodes at hand, this range stretches from 220 mV to 240 mV. For these reasons the computer is directed to use this EMF range in the evaluation of the equivalence point. In the absence of a computer plot of F vs. v, it may be advisable for the operator to take one or two sets of data and plot F vs. v. If anything, this will instill confidence in the method by the analyst in charge of the titration. The value of v at F = 0 is the equivalence point from which the alkalinity can be evaluated.

The slope of the F vs. v plot changes with a variation in the sulfate content of the samples. This is because at lower pH values the reaction

H⁺ + SO₄^2- = HSO₄^-

plays an important role in establishing the pH of the solution through a buffering effect. This change in slope, however, has no effect on the GRAN extrapolation intercept with the y-axis, as demonstrated by Gieskes and Rogers (1973). The slope change, however, is not accurate enough to estimate sulfate concentrations.

The nature of the automatic operation of the titrator is described in the appropriate shipboard manual.

Standardization

Often the alkalinity method is standardized by the titration of IAPSO standard seawater with an alkalinity of about 2.325 mM. It is, however, advisable to make some extra standards at higher alkalinities over a range that will cover the expected alkalinities. For this purpose, standards of NaHCO₃, Na-borate (Borax), and Na₂CO₃ can be prepared:

42 g of sodium bicarbonate (NaHCO₃) in 1000 cm³ of nanopure water (0.5 M),
53 g of sodium carbonate (Na₂CO₃) in 1000 cm³ of nanopure water (0.5 M),
38.1 g of Na₂B₄O₇^.10H₂O (Borax) in 1000 cm³ of nanopure water (0.1 M),
52 g of potassium chloride (KCl) in 1000 cm³ of nanopure water (~0.7 M);

with these solutions, standards can be prepared as indicated below:

10 cm³ of Na₂CO₃ solution made up to 100 cm³ with KCl solution yields an alkalinity of 100 mM;
10 cm³ of NaHCO₃ solution made up to 100 cm³ with KCl solution yields an alkalinity of 50 mM;
10 cm³ of borax solution made up to 100 cm³ with KCl solution yields an alkalinity of 20 mM.

The KCl solution serves as an ionic-strength preserver and does not affect the alkalinity. Preference should be given to the borax standard, followed by the bicarbonate and carbonate standards respectively. Good agreement among all standards instills good confidence.

General Remarks

It should once more be emphasized that under the SI system of units the term "equivalent" has been officially abandoned. In the case of alkalinity one must think in terms of the number of moles of hydrogen ions required to titrate the excess base (alkalinity) in the solution. Thus alkalinity can be expressed straightforwardly in terms of mM without losing continuity with the previous system of units.

Often, especially in continental-margin zones, very high alkalinities may occur. Thus, for instance, in a sample of 100 mM alkalinity it will require about 5 cm³ of acid to titrate 5 cm³ of sample. This is not a major problem, but of course the original concentrations of other dissolved ions have halved in that case. If alkalinities much higher than 100 mM are expected, it would be preferable to titrate only 2 or 3 cm³ of sample, provided the electrodes can still be inserted into the solution.

Under all circumstances the total volume of acid added to the sample aliquot (record the size of the aliquot also) must be recorded on the storage vial so that future workers on the sample are alerted to the dilution factor to be used in the calculation of original concentrations.

Note that for the HCl, use must be made of ultrapure HCl. Normal (reagent grade) 0.1 M HCl can have a significant amount of iodide (~100-300 mM) in it, which can easily be avoided by using triple distilled ultrapure HCl.

It is of great importance to calibrate the 5 cm³ and 3 cm³ pipettes with distilled water and to note their precise volumes. The volumes should remain fixed for the duration of the cruise. They are important for establishing the dilution factor to be used for further work on the samples. The standardization of the acid, of course, takes account of the volumes.

Note on Alkalinity Calibration (by J. Gieskes)

Until now calibration of the alkalinity has relied on calibration with IAPSO standard seawater. I think it is time to eliminate this and to proceed with a calibration based on a 40 mM (alkalinity) borax solution. The procedure for preparation is described in the "Standardization" section of this chapter, but it is worth repeating here:

1. Make a ~0.7 M KCl solution: 52 g KCl in 1000 cm³ of nanopure water.

2. Make a solution of 38.1 g of Na₂B₄O₇^.10H₂O in 1000 cm³ nanopure water (0.1 M).

3. Take 20 cm³ of borax solution (2) and dilute to a total volume of 100 cm³ with the KCl solution. The KCl serves to make the solution of approximately the same ionic strength as seawater (in this case an ionic strength of about 0.56 M; seawater has an ionic strength of ~0.7 M). The alkalinity of this solution is 40 mM.

With the 5 cm³ sampling pipette, transfer 5 cm³ of this borate standard solution into the titration vessel. Titrate and evaluate the alkalinity with the presently available program (omitting the IAPSO correction-factor step). This titration will yield an alkalinity that is probably not quite equal to 40 mM, for the following reasons:

(a) The volume of the pipette is not quite equal to 5 cm³, and
(b) the acid molarity of the 0.1 M HCl solution is not quite equal to 0.1 M.

Now, from the estimated alkalinity and the actual alkalinity, calculate a calibration factor that goes in the program, much like the previous factor that adjusted for the IAPSO "alkalinity." In a way, that factor also adjusted for the difference in volume, although this fact was not known. Of course this necessitates the creation of different correction factors for a 3 cm³ or a 10 cm³ sample because of differences in true volumes. For these reasons I suggest the following program modifications:

1. Make the program easily accessible to the input of the calibration factor.

2. At the beginning of the cruise, calibrate with three different-sized pipettes, say of 3, 5, and 10 cm³. The volumes of these pipettes should not be changed from then on (tape them up). Work out the calibration factors for each of the pipettes and put them into the program in conjunction with the volume size command now used.

3. Make some extra standards of different alkalinities over the range of interest to serve as a double check. Weighing these standards presented no problems on the ship, and we obtained very good results during Leg 131.

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SULFATE

Determination of dissolved sulfate in interstitial waters recovered from a drill hole is important for assessing diagenetic processes involving the alteration of organic matter and also for analyzing the overall quality of the database. Because of the anionic nature of the sulfate ion, it is not affected directly by ion exchange processes and thus is more independent of artifacts created by the extrusion of pore fluids at other than in situ conditions. Any spurious deviations from an overall trend with depth can be interpreted in terms of potential contamination with surface seawater that is pumped down the hole during the drilling process. Naturally this method works best when sulfate concentrations are zero, in which case any small increase can be directly attributable to contamination.

Determination of sulfate is routinely carried out by means of the DIONEX ion chromatograph, which uses a small amount of sample and provides very reproducible results. However, if a quick semiquantitative estimate of dissolved sulfate is needed an alternate method can be used which involves the precipitation of sulfate in an excess of BaCl₂. A comparison of the degree of cloudiness can be used as a qualitative estimate. Stabilization of the suspension with glycerol can, in principle, be used for a semiquantitative estimate of dissolved sulfate (see below).

Dionex Method

The ion chromatographic method has the advantage that measurements can be made on very small quantities of sample. Typically, the standard representative of seawater concentration and the samples are made of 0.25 cm³ of standard/sample solution diluted with nanopure water to 50 cm³ (representing a final concentration of ~145 mM for IAPSO standard seawater). If less sample is available, smaller amounts can be prepared with the same dilution.

The operation of the DIONEX ion chromatograph is discussed in detail in the operations manual on board ship. In this section only a brief description of the method is presented.

Principle of the Method

This method is based on an ion exchange process between a mobile phase and exchange groups covalently bound to a stationary matrix. Upon introduction into the system, the sample is carried by a mobile phase that holds the counter ions to different degrees, dependant on the bonding strength between the cation/anion and the exchange group of the matrix. Because the method of cation exchange (for Ca²⁺, Mg²⁺, K⁺, and Li⁺) chromatography is somewhat cumbersome and time consuming in nature and also because faster, equally acceptable methods are available on board JOIDES Resolution, the only ion exchange chromatographic method routinely used is the anionic exchange method, which allows the determination of SO₄^2- and potentially also of Br^-.

Although each individual user of the DIONEX system should consult the operations manual in detail, it may be useful to reiterate the principle of operation. Details of the theory can be found in Weiss (1986). Figure 1 (from Weiss, 1986) shows the following flow diagram:

1. The mobile phase is pumped through the system by means of a double reciprocating pump that allows for a pulse-free flow.

2. The sample is introduced through a loop injector and swept to the column by the mobile phase (the eluant) in which the ion exchange reactions separate the various anions (Cl^-, Br^-, SO₄^2-) into separate waves, which then arrive at the detector as the sodium salts (the eluant consists of Na₂CO₃/NaHCO₃).

3. The first part of the detector system (Fig. 2) consists of the suppressor, which consists of a semipermeable membrane. The solution from the separator column runs through the interior of the membrane fiber, whereas a dilute acid (H₂SO₄) runs countercurrently in contact with the exterior of the membrane. This allows H⁺ ions to be exchanged across the semipermeable membrane in exchange for Na⁺ ions. This, in turn, converts the greater conducting HCO₃^- and CO₃^2- ions into less conducting H₂CO₃, and the less conducting sodium salts of Cl^-, Br^-, and SO₄^2- are converted into their greater conducting acids.

4. The final solution then enters the conductivity cell, which records the conductivity of the "wave." This conductivity is proportional to the concentration of the ion at low concentration levels.

Reagents

ELUANT: 0.0022 M Na₂CO₃ ( 0.933 g in 4000 cm³); 0.0028 M NaHCO₃ (add 0.941 g to above solution); pH = 10.23.
REGENERANT: 0.025 M H₂SO₄ (1.4 cm³ concentrated H₂SO₄ in 2000 cm³ nanopure water).

Standards

Although the use of properly prepared artificial standards as described in the "standards" section is encouraged, in many cases the use of standard seawater will suffice. For this purpose one can use the dilution of 0.5 cm³ of IAPSO Standard Sea Water (SO₄^2- = 28.9 mM) to 100 cm³ with nanopure water as a reference. This allows the preparation of a set of sub-standards. For example, 0.1 cm³ IAPSO diluted to 100 cm³ yields a standard representative of 5.78 mM SO4, and 0.6 cm³ of IAPSO diluted to 100 cm³ yields 34.68 mM SO₄. It is suggested that at least five standards are made between 0 and 28.9 mM SO₄, but, when very low values of sulfate occur, it will be advisable to make a few extra low SO₄ standards. Figure 3 is an example of an acceptable calibration curve.

The seawater dilution standards will also serve as standards for potassium and sodium analysis by atomic emission spectroscopy, as will be discussed in the sections on these cations.

Procedure

Instrumental parameters and an example of a chromatogram recorded with the integrator (HP 3392A) are presented in Figure 4.

Though for the actual methodology we refer to the special DIONEX manual on board ship, it is important to stress some aspects of sample methodology here.

Under circumstances of normal interstitial-water recovery, we suggest using a dilution of 0.25 cm³ of sample to 50 cm³ with nanopure water.

When very low sulfates occur, a lesser dilution can be used, but it will be advisable to make a set of low sulfate standards using a similar dilution procedure. One more important aspect is that in order to assure continuous monitoring of the potential drift in the measurements, standards bracketing the range of expected sulfate concentrations (low SO₄, low standard; high SO₄, high standard) are measured during the sulfate run at a rate of one standard after each four samples. With the automatic sample changer, this is not a serious problem.

Diluted samples should be stored carefully (all samples stored in plastic polyethylene bottles) for future use in the analysis of sodium and potassium (see also above).

Nephelometry

(A Sulfate method to be used as crosscheck or in case of Dionex failure).

The nephelometric method for the determination of dissolved sulfate has been used for a number of years in freshwater research. It is based on the precipitation of barium sulfate by the addition of an excess of BaCl₂ solution to a sample aliquot. In freshwater research the precipitation was effected by means of the addition of well-defined grain-sized barium chloride crystals, but the addition of a 0.25 M solution of BaCl₂ to the test solution seems to suffice if a precision of ~1 mM is desired. In the method described below a few drops of glycerol are added in order to enhance the viscosity of the solution, which in turn keeps the suspension of BaSO₄ in a better condition. We emphasize that this method is semiquantitative in nature, but it can be used for the following purposes:

1. The determination of dissolved sulfate in pore-water samples in case of failure of the DIONEX analyzer.

2. The determination of dissolved sulfate on an ad-hoc basis to determine sulfate concentrations on selected samples from pore waters in order to establish the concentration range of samples.

3. Any routine checks on water quality of drilling fluids, potable water, etc.

Reagents

BaCl₂ SOLUTION: Make a roughly 0.25 M solution of BaCl₂ (~10 g in 200 cm³ nanopure water) and filter the solution through a Whatman no. 1 paper.
GLYCEROL: Put glycerol in an eye-drop bottle.

Procedure

The method requires about 0.05 to 0.3 cm³ of sample, depending on the sulfate concentration. It appears prudent to start with small quantities, which can be augmented when greater accuracy is required.

One (1) cm³ of nanopure water is added to a 10 cm³ glass vial with a snap cap. Add 0.1 cm³ (or any other appropriate amount) of sample or standard to the vial, followed by three drops of glycerol. Then add 3 cm³ of the BaCl₂ solution. Set the spectrophotometer at 400 nm and read the absorbance. Plot the absorbance vs. standard curve.

Figure 5 below indicates that there is no exact linear fit and that the accuracy suffers somewhat at higher concentration levels. Nevertheless, reproducibility is quite good. The low-concentration-range samples still show a reasonable precision.

Below we document an even better nephelometric method for sulfate determination. The method uses a better stabilization of the barium sulfate suspension. The details were provided through the courtesy of Dr. Rick Jahnke.

Reagents

STOCK SULFATE SOLUTION: Dissolve 1.8141 g of K₂SO₄ in nanopure water and dilute to 1 dm³. 1 M HCl: Add 85 cm³ concentrated HCl to 300 cm³ nanopure water and dilute to 1 dm³.
STOCK GELATIN SOLUTION: Dissolve 1.5 g of Difco-Bacto Gelatin (Detroit, MI) in 500 cm³ of hot (~70 C) nanopure water; cool, and store at 4 C for at least 16 hours before use (bring water to 70 C on hot plate/stirrer, move flask to cold magnetic stirrer, add gelatin, stir 15 minutes, let cool to room temperature and transfer to refrigerator). This solution will be stable for about 28 days if kept in refrigerator.
BARIUM/GELATIN REAGENT: Bring 150 cm³ of stock gelatin solution to room temperature (by leaving it on the bench; do not heat on hot plate). Add 1.5 g of BaCl₂^.2H₂O and stir for 1 hour. Let stand 1 additional hour at room temperature and then transfer to refrigerator. Stable for 7 days if kept cold.

Procedure

1. Add 10 cm³ of nanopure water to a large test tube as precisely as you can.

2. Add 500 L of 1M HCl.

3. Add 40 L of sample or standard.

4. At precise 1 or 1.5 minute intervals, add 500 L of Ba/Gelatin reagent; mix gently.

5. After 30 minutes plus/minus 10-15 seconds, read turbidity in spectrophotometer at 420 nm.

Note: If reagent is added at 1.5 minute intervals, samples can be read at the same intervals 30 minutes later. It is an easier time limit to adhere to. Modify method as convenient. For lower sulfates, of course, one can use larger aliquots with matching standards. Also, use IAPSO standard seawater and dilution thereof as extra standards.

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BORON

The boron method is modified from a colorimetric technique described by Grinstead and Snider (1967) and by Presley (1971). In this method the boron concentration is measured from the formation of a boron-curcumin complex in acidic conditions, which shows an orange-red color.

Reagents

CURCUMIN: Dissolve 0.125 g of curcumin (C₂1O₂0H₆) in 100 cm³ of glacial acetic acid.
ACID REAGENT: Mix 50 mL of 96% sulfuric acid and 50 mL of glacial acetic acid.
AMMONIUM ACETATE-ACETIC ACID: Dissolve 250 g of ammonium acetate plus 300 cm³ of glacial acetic acid in sufficient nanopure water to make 1 dm³.

Standards

Always choose standards that bracket the concentration range of the samples. This curcumin method can be used to measure the concentration in pore-water samples which typically contain 0-3 mM boron. It is therefore advisable to make standards of 0, 0.5, 1.0, 1.5, ......, 3.0 mM boric acid (in an artificial seawater matrix (0.6 M NaCl, 0.05 M MgCl₂). In addition, IAPSO standard seawater should be run (B = 0.45 mM). Standard solutions (and samples) should be diluted in such a manner that the highest concentration does not exceed 0.5 mM.

A good stock solution will be 6.183 g of boric acid in 1 dm³ of nanopure water (=100 mM).

Boron Procedure

1. Pipette a sample aliquot (25 mL or 50 mL) into a plastic bottle.

2. Add 0.1 cm³ curcumin reagent.

3. Add 0.4 cm³ acid reagent (with repipet), mix on vortex mixer, and wait for at least 30 minutes.

4. Add 2.0 cm³ ammonium acetate-acetic acid buffer and mix.

5. Measure absorbance at 555 nm in a 1 cm cell after 1.5 hours.

6. Standard solutions should be run at the same time as the samples.

7. Make a standard curve. If linear, read concentrations directly. If curved, use lower concentration range of final solution.

CAUTION: If a crystalline precipitate forms after addition of the buffer (Step 4) it may be necessary to use slightly less buffer, e.g., 1.9 cm³ (by trial and error).

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IODIDE

In this method we really measure I^- and IO₃^-, but in our DSDP/ODP samples we usually work with samples from reducing environments in which the predominant species will be iodide or an organic complex with iodide. The method was adapted from Pedersen (1979).

Reagents

NaCl: 0.7 M (20.5 g NaCl/500 cm³ H₂O).
ACETATE BUFFER: 30 g NaAc in 1 dm³ flask, add 7 cm³ glacial acetic acid, make up to 1 dm³ with distilled water.
BROMINE WATER: saturated (fresh daily).
FORMIC ACID
KI: 10 g KI in 100 cm³ distilled water (make this fresh before use).
SULFAMIC ACID: 1 g sulfamic acid (NH₂SO₃H) in 100 cm³ distilled water.

Standards

Always choose standards that bracket the range. Typically in Legs 67 and 111 samples with high I (high NH₄) we use: 0, 200, 400, 600, 800, 1000 mM (use 0.7 M NaCl solution as matrix) and then use 25 mL samples. If samples have much lower (e.g., typical anoxic box cores would have 0-50 M I) use 0, 10, 20, 30, 40, 50 mM I standards and use 500 mL samples. Make sure to bracket samples with the appropriate range of standards. A good stock solution is 0.415 g KI in 250 cm³ distilled water (10 mM in I^-). The procedure is outlined on the following page.

Iodide Procedure

1. Pipette a sample aliquot (25 mL or 500 mL^--see above) into a glass vial.

2. Add 1 cm³ acetate buffer.

3. Add 0.04 cm³ bromine water (fresh), mix on vortex mixer, and gently heat on hot plate for ~5 minutes (do not boil).

4. Allow to cool and add 0.1 cm³ of a 10% Na-azide solution (optional--if you fear NO₂ is present, this will kill the NO₂).

5. After cooling to room temperature, add 0.04 cm³ formic acid and mix on vortex mixer.

6. Add 0.03 cm³ of 10% KI and 0.5 cm³ sulfamic acid solution and mix on vortex mixer.

7. Measure absorbance at 355 nm in 1 cm cell 3 minutes (±5 seconds) after KI is added.

8. Standards should be run at the same time as samples, using same aliquots.

9. Make a standard curve; if linear, you can read the I concentrations directly. If curved, use lower concentration range of final solution.

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BROMIDE

In the bromide method we make use of an adaptation of the iodometric method proposed by Kremling (1983). In this method, use is made of sodium hypochlorite to oxidize both iodide and bromide to iodate and bromate under alkaline conditions. Then this is reacted with fresh KI solution in acidic medium to create iodine. This iodine is then measured with a spectrophotometer (similar to the measurement of iodide). We found that both "reagent-grade" and chlorox-grade hypochlorite has sufficient iodide/bromide in it to cause a rather large but reproducible blank. We combat this blank by adding sufficient sodium thiosulfate to titrate the excess iodine, thus making a good standard curve possible within a reasonable absorbance range (0-1.5). The necessary blank must be determined by the individual investigator. The method has also some time-dependent steps built in. A careful investigator, however, can obtain accuracy better than 3% on 100 mL samples.

Reagents

PHOSPHATE BUFFER: Dissolve 5 g sodium dihydrogen phosphate (NaH₂PO₄^.H₂O) in 100 cm³ volumetric flask and make to volume with double deionized water.
SODIUM HYPOCHLORITE SOLUTION: Use reagent-grade sodium hypochlorite solution.
SODIUM FORMATE SOLUTION: (HCOONa) 50% (w/v). Dissolve 15 g sodium hydroxide (NaOH) in ~50 cm³ water. Cool down to room temperature and add 16 cm³ of 90% formic acid (HCOOH) while stirring. Then dilute to 100 cm³.
AMMONIUM MOLYBDATE SOLUTION: ((NH₄)6Mo₇O₂₄^.4H₂O) 3% (w/v).
SULFURIC ACID: (H₂SO₄) 3 M. Slowly add 16 cm³ concentrated sulfuric acid into 80 cm³ deionized water while stirring. Cool down to room temperature.
POTASSIUM IODIDE: Dissolve 10 g KI in 100 cm³ double deionized water; make it fresh.
SODIUM THIOSULPHATE SOLUTION: (Na₂S₂O₃^.5H₂O) 0.6 mM. Pipette 3.5 cm³ of 0.05 M sodium thiosulphate solution (dissolve 3 g of Na₂S₂O₃^.5H₂O in 250 cm³ double deionized water) into a 250 cm³ volumetric flask; make to volume with deionized water.

Standards

STANDARD SOLUTION: Use NaBr solutions as standard series. Use IAPSO for comparison. If all data fall on one standard curve, then of course there is no serious salt effect. For highly saline solutions this should be double checked.

STANDARDS: Use 0, 500, 1000, 1500, 2000 mM NaBr solutions and IAPSO (860 mM) as standards.

Procedure

1. Add 1.5 cm³ of phosphate buffer into a 5 cm³ glass vial.

2. Add 1.0 cm³ of distilled water.

3. Pipette a sample aliquot 25 to 100 mL (wash the tip of pipette with (1.+2.) solution several times).

4. Add 0.25 cm³ sodium hypochlorite solution.

5. Heat the solution on a hot plate and boil for about 5 seconds.

6. Remove from the hot plate and add 0.5 cm³ sodium formate solution and cool down to room temperature.

7. Add 0.03 cm³ of molybdate solution.

8. Add 0.05 cm³ of 10% KI solution (fresh daily).

9. Add 0.3 cm³ of sodium thiosulphate solution and 0.75 cm³ of sulfuric acid.

10. Measure absorbance at 355 nm in 1 cm cell 10 minutes after sulfuric acid is added (wait about 1 minute after sucking the solution into the cell, then write down the reading of absorbance).

11. Standard solutions should be run at same time as samples using same aliquots.

12. Make a standard curve; if linear, you can read the Br concentrations directly. If curved, use lower concentration range of final solution or adjust the added amount of sodium thiosulphate solution. Note: Please do not forget to correct for the iodide content; this method determines total I + Br.

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AMMONIUM

The determination of ammonium concentrations is of importance because this constituent is an indicator of the diagenesis of organic matter in the sediments. The onset of sulfate reduction coincides with the start of ammonium ion production. The production of ammonium, however, appears to increase 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 the method of Solorzano (1969), originally developed to detect very small NH₄⁺ concentrations in seawater. Although blank problems in seawater are enormous, the relatively high concentrations in pore fluids (up to 85 mM in Leg 112 samples; Kastner et al., 1990) reduce this blank problem. In areas of slow sedimentation, however, very low ammonium concentrations require great caution to avoid this problem. The method is based on the diazotization of phenol and the subsequent oxidation of the diazo compound by chlorox to yield a blue color.

Method

Reagents

PHENOL-ALCOHOL SOLUTION: Dissolve 0.8 g reagent-grade crystalline phenol in 100 cm³ 95% ethyl alcohol. Make fresh each day of use. Instead of the crystalline phenol, 1 cm³ of liquified phenol can be used.
SODIUM NITROPRUSSIDE SOLUTION: Dissolve 0.15 g sodium nitroprusside (sodium nitroferricyanide) in 200 cm³ of nanopure water (make fresh each day).
ALKALINE SOLUTION: Dissolve 7.5 g of trisodium citrate and 0.4 g sodium hydroxide in 500 cm³ nanopure water. This is a fairly stable solution.
OXIDIZING SOLUTION: Add 1 cm³ fresh sodium hypochlorite (4% available chlorine)- chlorox will do equally well-to 50 cm³ alkaline solution and use the same day.
AMMONIUM STANDARD: Dry ammonium chloride in oven overnight. Dissolve 5.345 g ammonium chloride in 1 dm³ of nanopure water. One can also use non-dried NH₄Cl and determine the chloride content of the standard solution by means of a chloride titration. For reasonable accuracy, use a 0.5 cm³ aliquot (Cl = 0.1M) to obtain almost the same concentration of Cl as in IAPSO reference standard seawater.

Procedure

For each specific area, different concentrations of ammonium may occur. Typically in areas with a strong evidence of organic-carbon diagenesis (e.g., organic-carbon-rich sediments), very high concentrations of NH₄ can be expected. In that case, sample aliquots must be made appropriately small, and indeed sample dilution may be required. The range can be established easily by using a sample near the alkalinity maximum. Once the range has been determined, standards must be prepared that cover this range. In this manner samples and standards are treated in a similar way. Below we describe the procedure for relatively low ammonium concentrations.

Care should be taken to use clean glass vials, preferably not used for Si and PO₄ determinations, in which ammonium molybdate is used as a reagent.

Use a 100 lambda (100 microliters) Eppendorf pipette to transfer 0.1 cm³ of sample to a 5 cm³ glass vial. Add 1 cm³ nanopure water to each, then 0.5 cm³ phenol-alcohol solution, 0.5 cm³ sodium nitroprusside, and finally 1 cm³ of oxidizing solution. Adding these solutions with Eppendorf pipettes is fast and convenient and ensures proper mixing during addition. Shake samples after each addition. Standards should range from 0 to 1000 mM, or any other appropriate range as discussed above. Let the color develop for at least 1 hour (longer would be better, up to 3 hours) and then determine the absorbance at 640 nm wavelength.

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SILICA

Dissolved silica determinations are of great importance in interstitial waters. Often they represent the lithology of the sediments, and the concentrations can vary widely, 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 mM or higher (especially in hydrothermally affected sediments). Thus the method below usually covers the range, although greater dilutions may become appropriate if sediments or sample sizes necessitate this.

The method is based on the production of a yellow silicomolybdate complex and the subsequent reduction of this complex to yield a blue color. The blue complex is very stable, which will enable delayed reading of the samples.

Method

Reagents

MOLYBDATE REAGENT: Dissolve 4 g of ammonium paramolybdate, (NH₄)₆Mo₇O₂₄^.4H₂O (preferably fine crystalline), in ~300 cm³ nanopure water using a 500 cm³ volumetric flask. Add 12 cm³ concentrated hydrochloric acid (12N), mix, and make up the volume to 500 cm³ with nanopure water. This reagent is stable for many months if stored in a dark bottle. The reagent should be discarded immediately when any white precipitate forms. If unable to store properly, or if time permits, make fresh for each run.
METOL SULFITE SOLUTION: Dissolve 6.0 g anhydrous sodium sulfite, Na₂SO₃, in a 500 cm³ volumetric flask. Add 10 g Metol (p-methylaminophenol sulfate) and then nanopure water to make the volume to 500 cm³. When the metol has dissolved, filter the solution through a Whatman no.1 filter paper and store in a clean glass bottle, preferably dark glass, which is tightly stoppered in the refrigerator. This solution may deteriorate quite rapidly and erratically so it should be prepared fresh every month.
OXALIC ACID SOLUTION: Prepare a saturated oxalic acid solution by shaking 50 g of analytical-grade oxalic acid dihydrate, (COOH)₂^.2H₂O, with 500 cm³ of nanopure water. Let stand overnight. Decant solution from crystals before use. This solution is stable and can be stored in a glass bottle.
SULFURIC ACID SOLUTION: 50% v/v. Using a 500 cm³ volumetric flask, pour 250 cm³ concentrated analytical-grade sulfuric acid into ~200 cm³ nanopure water. Cool to room temperature and bring volume to 500 cm³ with a little extra water. Store in a polyethylene bottle.
REDUCING SOLUTION: Mix 50 cm³ Metol sulfite solution with 30 cm³ oxalic acid solution. Add slowly, with mixing, 30 cm³ 50% sulfuric acid solution and bring volume to 150 cm³ with nanopure water. This solution should be made daily, just before using it.
SYNTHETIC SEAWATER: Dissolve 25 g sodium chloride (NaCl) and 8 g of magnesium sulfate heptahydrate (MgSO₄^.7H₂O) in 1 dm³ of nanopure water and store in a polyethylene bottle. The silica content of the solution should not exceed 1-2 M.

Standards

SILICATE STANDARD: Use is made of sodiumsiliocofluoride (Na₂SiF₆) for this purpose. When dissolved in water, this substance hydrolyzes to form reactive dissolved silica. The Na₂SiF₆ is placed in an open plastic vial and placed in a vacuum desiccator overnight to remove excess water. Do not heat or fuse.

PRIMARY STANDARD: Dissolve 0.5642 g of Na₂SiF₆ in a 1 dm³ polyethylene volumetric flask. Dissolution is slow, so allow at least 3 minutes. This cannot be rushed. Use nanopure water for this purpose. The concentration of the standard is 3000 mM. Store in a 500 cm³ polyethylene bottle. The standard is stable indefinitely.

DILUTIONS FROM PRIMARY STANDARD: When making dilutions, use distilled water and store in polyethylene containers. Using a 50 cm³ volumetric flask, add the following amounts of primary standard and then bring to a total of 50 cm³:

                30    µM Si        0.5 cm³ primary standard
                60                 1.0
                120                2.0
                240                4.0
                360                6.0
                480                8.0
                600                10.0
                900                15
                1200               20                      et cetera

Method

1. First, 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.

2. Label and set up 3-dram plastic vials and caps (well washed with nanopure water).

3. Measure into the vials 4.0 cm³ of silica free nanopure water (3.8 cm³ for standards and blank).

4. For standards and blank only, pipette in 0.2 cm³ of synthetic seawater.

5. Pipette 0.2 cm³ of sample or standard or blank (nanopure water) into the vial with an Eppendorf pipette.

6. Record time.

7. Add 2.0 cm³ of molybdate solution to the vials. A yellow color will develop, and this is allowed to mature for exactly 15 minutes (±15 seconds). Then add 3.0 cm³ of the reducing solution. Cap the vials and let stand for at least 3 hours.

8. Read absorbances on the spectrophotometer at 812 nm. Please consult procedural notes of the following page.

Notes:

Do not handle more than about 30 samples at a time in order to ensure that the 15- minute time limit can be adhered to. Make sure that there are no wild fluctuations in room temperature, which is not normal in an air-conditioned room.

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 0.2 cm³ of synthetic seawater to the standards is to maintain a reasonably uniform salt content in relation to the samples, thus suppressing a potential salt effect on the method.

Although it is suggested that strict adherence to the 15-minute time limit is advisable, tests have shown that there is some leeway, i.e., the yellow molybdate complex is stable from 10 to 20 minutes. However, consistency in the time limit will eliminate any potential error.

It is important to wait at least 3 hours for the blue color to develop; the higher the concentration, the longer the time. The color remains stable for many more hours, and reading after 4 or 5 hours may, in fact, be a good idea. Again, consistency in time limits is advisable.

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