Cary Spectrophotometer: User Guide

Table of Contents

Manual Information

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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 significantly at the chosen wavelength.
Beer's law, expressed for liquids, can be represented as an equation, where A = absorbance, k = an experimentally determined constant, b = path length, and c = concentration:
A = kbc.
Thus, concentration can be determined.
IODP's Agilent Cary 100 double-beam UV-Vis (ultraviolet–visible) spectrophotometer is ideal for shipboard routine and research laboratory work. The system measures analytes in interstitial water obtained from sediment cores using standard colorimetric methodology.

Hardware and Materials

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 1). More details can be found below.

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Figure 1. Schematic of Cary Spectrophotometer.

Electrical

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An external computer workstation provides control, communication, and data analysis.

Software

Agilent provides a full suite of applications for various user requirements; for our needs we use the Simple Reads application. Simple Reads is used to perform simple absorbance readings of single samples. There is, however, a built-in programming language, Applications Development Language (ADL), which allows complete customization of Cary WinUV to specific applications.

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In a 1000 mL volumetric flask, dissolve 0.102 g of antimony potassium tartrate trihydrate (KSbC₄H₄O₇·3H- 3H₂O) in ~600 mL of nanopure water. (If using antimony potassium tartrate hemihydrate [KSbC₄H₄O₇·½H- ½H₂O], dissolve 0.09 g.) Dilute to 1000 mL with reagent water.

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In a 1000 mL volumetric flask, dissolve 2 g of ammonium molybdate tetrahydrate ([NH₄]₆Mo₇O₂₄·4H- 4H₂O) in ~600 mL of nanopure water. Dilute to 1000 mL with reagent water.

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In a 500 mL volumetric flask, shake 50 g of analytical-grade oxalic acid dihydrate [(C₂H₄O₂)·2H- 2H₂O] in 500 mL of nanopure water and allow to stand overnight. Let stand overnight. Decant saturated solution of oxalic acid from crystals before use.

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From the Exp. 366 Methods Chapter
Hydrogen sulfide (expected to be HS^- at the pH of most IW samples) concentrations were analyzed following the method of Cline (1969) with modifications as adapted for small volumes of pore fluids by Ferdelman et al., 2011. Initially, 500 uL sample was fixed with 40 ¿L of a 1% zinc acetate solution, and reagents used were for a range of 6 to 80 ¿M. However, because most samples were below detection for the first set of samples, 4 ml sample was fixed with 800 ¿L zinc acetate solution, and the analyses were conducted following the lowest range (1 to 3 ¿M) outlined in Cline (1969), which had a linear range for the calibration curve at least up to 10 ¿M. The zinc acetate fixed sample was vigorously shaken and 320 ¿L of a diamine solution consisting of 0.5 g N,N-dimethyl-p-phenylenediamine sulfate and 0.75 g ferric chloride (FeCl3 * 6H2O) per 500 ml DI water, was added. The solution was shaken, and left for 30 minutes in the dark, then measured by spectrophotometry at 670 nm. If the blue color of the sample was visually darker than that of the highest calibration standard (10 ¿M), the sample was diluted with DI water until a lighter color was observed.

Analyzing Samples

Preparing the Instrument

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Figure 4. Peristaltic Pump.

Running Samples

Preparing Samples

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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 and rinse with tap water.

QAQC

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

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Archive

• Cary_UG_374_draft.pdf