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GCR Agilent Cary Spectrophotometer User Guide

GCR Agilent Cary Spectrophotometer User Guide

Table of Contents


https://www.agilent.com/en/products/uv-vis-uv-vis-nir/uv-vis-uv-vis-nir-systems/cary-100-uv-vis

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

Ammonium Standard (0.10 M)

make once an expedition

Dry ammonium chloride (NH4Cl) overnight in an oven.

Dissolve 5.349 g dried NH4Cl in a 1000 mL volumetric flask. Bring to volume with nanopure water.

  • To make 25 mL - Dissolve 133.728 mg dried NH4Cl in a 25 mL volumetric flask. Bring to volume with nanopure water.

Alkaline

make once an expedition

Dissolve 7.5 g (tri)sodium citrate tribasic dihydrate (Na3C6H5O7·2H2O) and 0.4 g sodium hydroxide (NaOH) in a 500 mL volumetric flask. Bring to volume with nanopure water.

Oxidizing

make fresh daily

Add 1 mL fresh sodium hypochlorite (4% available chlorine) to 50 mL Alkaline solution. This should be adjusted for the amount of samples to be run.

  • Use sodium hypochlorite or regular strength Chlorox™ bleach. DO NOT use Chlorox that contains NaOH, fragrances, or other agents, and do not use another brand of bleach.

  • The Clorox bleach can go bad with time. Absorbance values of the ammonium analysis will be much lower when this happens. Absorbance of the 1000 uM standard falling below 0.2 or the 100 uM standard falling below 0.01 could be indications that a new bottle of bleach is needed.

Phenol

make fresh daily

Add 1 mL liquid Phenol to 100 mL Absolute Ethanol 

Sodium Nitroprusside

make fresh daily

Dissolve 75 mg sodium nitroprusside (Na2[Fe(CN)5NO]) (also known as sodium nitroferricyanide) in 100 mL nanopure water

Standards

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

50 mL batches are stable for 1 month. 

concentration

(µM)

volume of ammonium standard

(mL)

volume of nanopure water

(mL)

concentration

(µM)

volume of ammonium standard

(mL)

volume of nanopure water

(mL)

0

0

50.000

50

0.025

47.975

100

0.050

49.950

150

0.075

49.950

200

0.100

49.900

400

0.200

49.800

600

0.300

49.700

800

0.400

49.600

1000

0.500

49.500

1500

0.750

49.250

2000

1.000

49.000

3000

1.500

48.500

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.

1.

Transfer 200 µL of sample/standard to a vial.

2.

Add 2 mL of nanopure water to each vial. 

3.

Add 1 mL phenol solution to each vial and shake.

4.

Add 1 mL sodium nitroprusside solution to each vial and shake.

5.

Add 2 mL of oxidizing solution to each vial and shake.

6.

Let the color develop (in a dark place) for 6.5 hr and then determine the absorbance at 640 nm wavelength.

(From a series of measurements over 8 h, it was found that results stabilized after 6.5 hr.)

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

Phosphate Standard (0.01 M)

make once an expedition

Dry potassium phosphate monobasic (KH2PO4) in oven at 100°C for two hours; keep in a desiccator while it cools before weighing.

Dissolve 1.361 g dried KH2PO4 in a 1000 mL volumetric flask. Bring to volume with nanopure water.

  • To make 25 mL - Dissolve 34.025 mg dried KH2PO4  in a 25 mL volumetric flask. Bring to volume with nanopure water.

Ammonium Molybdate

stable indefinitely

Dissolve 2 g ammonium molybdate tetrahydrate ([NH4]6Mo7O24·4H2O) in a 1000 mL volumetric flask. Bring to volume with nanopure water.

  • Store in polyethylene @ 4°C

Sulfuric Acid

stable indefinitely

Add 10 mL concentrated sulfuric acid (H2SO4) to ~600 mL nanopure water in a 1000 mL volumetric flask. Bring to volume with nanopure water.

  • Caution: Mixing sulfuric acid and water produces heat. Take appropriate precautions. 

Antimony Potassium Tartrate

make once an expedition

Dissolve 102 mg antimony potassium tartrate trihydrate (KSbC4H4O7·3H2O) in a 1000 mL volumetric flask. Bring to volume with nanopure water.

  • If using antimony potassium tartrate hemihydrate [KSbC4H4O7·½H2O], dissolve 90 mg. 

  • Store in amber glass @ 4°C

Ascorbic Acid

make fresh weekly

Dissolve 3.5g ascorbic acid (C6H8O6) in a 1000 mL volumetric flask. Bring to volume with nanopure water.

  • If the reagent is discolored upon creation, the dry ascorbic acid is probably oxidized and must be replaced.

Mixed Reagent

make fresh every 6 hours

Mix the following solutions. Mix well after each addition. Can adjust the volumes to be suitable for the number of samples as long as the proportions of each reagent are maintained.

  • 50 mL Ammonium Molybdate

  • 125 mL Sulfuric Acid

  • 25 mL Antimony Potassium Tartrate

  • 50 mL Ascorbic Acid

Standards

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

concentration

(µM)

volume of phosphate standard

(mL)

volume of nanopure water

(mL)

concentration

(µM)

volume of phosphate standard

(mL)

volume of nanopure water

(mL)

0

0

50

5

0.025

49.975

10

0.050

49.950

15

0.075

49.925

20

0.100

49.900

40

0.200

49.800

60

0.300

49.700

80

0.400

49.600

100

0.500

49.500

150

0.750

49.250

200

1.000

49.000

300

1.500

48.500

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.)

1.

Transfer 600 µL sample/standard to a vial.

2.

Add 2 mL nanopure water to each vial.

3.

Add 4 mL mixed reagent to each vial and shake.

4.

After a few minutes a blue color develops, which remains stable for a few hours.

It is best to make the readings at 885 nm ~ 30 min after addition of the mixed reagent.

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

Silica Standard (3000 µM)

make once an expedition

Dry sodium silicofluoride in a vacuum desiccator overnight to remove excess water.

Do not heat.

Sulfuric Acid

make fresh monthly

Slowly add 250 mL concentrated sulfuric acid to ~200 mL nanopure water in a 500 mL volumetric flask. Allow to cool to room temperature, then bring flask to volume with nanopure water. 

  • Caution: Mixing sulfuric acid and water produces heat. Take appropriate precautions. 

Synthetic Seawater

make fresh monthly

Dissolve 25 g sodium Chloride in ~800 mL nanopure water in a 1000 mL volumetric flask. Add and disolve 8 g magnesium sulfate heptahydrate (MgSO4·7H2O). Bring flask to volume with nanopure water

Ammonium Molybdate

make fresh monthly

Dissolve 4 g ammonium molybdate tetrahydrate ((NH4)6MO7O24·4H2O) in ~300 mL nanopure water in a 500 mL volumetric flask. Add 12 mL concentrated hydrochloric acid (HCl). Bring flask to volume with nanopure water. 

Metol Sulfite

make fresh monthly

Dissolve 6.0 g anhydrous sodium sulfite (Na2SO3) in a 500 mL volumetric flask. Add 10 g Metol (p-methylaminophenol sulfate [(C7H10NO)2SO4]). Bring flask to volume with nanopure water. When the Metol has dissolved, filter the solution through a Whatman No. 1 filter paper. 

  • Note: This solution may deteriorate quite rapidly.

Oxalic Acid

make fresh monthly

Add 50g oxalic acid dihydrate [(C2H4O2)·2H2O] to ~300 mL nanopure water in a 500 mL volumetric flask. Shake well, and bring flask to volume with nanopure water. Let stand overnight. Decant saturated solution of oxalic acid from crystals before use.

Reducing

make fresh daily

  • mix 50 mL Metol Sulfite solution with 30 mL Oxalic Acid solution.

  • add slowly, with mixing, 30 mL Sulfuric Acid solution

  • add 40 mL nanopure water to bring total volume to 150 mL

Standards

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

concentration

(µM)

volume of primary standard

(mL)

volume of nanopure water

(mL)

concentration

(µM)

volume of primary standard

(mL)

volume of nanopure water

(mL)

0

0

50

30

0.5

49.5

60

1

49.0

120

2

48.0

240

4

46.0

360

6

44.0

480

8

42.0

600

10

40.0

900

15

35.0

1200

20

30.0

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.

 

1.

add 4 mL of nanopure water to each vial (3.8 mL for standards).

2.

for standards, pipette 200 µL of synthetic seawater to each vial.

3.

add 200 µL sample/standard to each vial.

4.

Record time.

5.

add 2 mL ammonium molybdate solution.

6.

a yellow color will develop; allow to mature for exactly 15 minutes (± 15 s).