Appendix I Page 1 of 2

INSTRUMENT STANDARDIZATION

A. CALIBRATION OF ULTRAVIOLET-VISIBLE SPECTROPHOTOMETERS

(International Method)

Reagents (a) Ethyl alcohol. (b) Hydrochloric acid, 3N. (c) Dichromate solution. Weigh 0.500 g potassium

dichromate primary standard, Mallinckrodt1 No. 6772, and dilute to 100 mL with 0.01N H2SO4. Dilute 5.0 mL of this solution to 500 mL with 0.01N H2SO4 for the working solution.

(d) Chromate solution. Weigh 0.400 g potassium chromate, Mallinckrodt1 No. 6870, and dilute to 100 mL with 0.05N KOH. Dilute 5.0 mL of this solution to 500 mL with 0.05N KOH for the working solution. The 0.05N KOH is 3.3 g KOH pellets (85% KOH) per L with water.

(e) Cobalt solutions. Dissolve 1.448 g cobalt ammo-nium sulfate hexahydrate, Matheson-Coleman-Bell1 No. CX 1790, in distilled water containing 1 mL concentrated H2SO4 (1.84 sp gr) and dilute to 100 mL. Prepare a 4-times higher concentration of cobalt ammonium sulfate by dissolving 5.94 g in distilled water containing 1 mL concentrated H2SO4 and diluting to 100 mL with water.

The above calibration reagents should be weighed out on a well-calibrated, recently checked analytical bal-ance. Stability of the calibration solutions has not been established. It is best, therefore, to store them in the dark and use them shortly after preparation.

Apparatus (a) Holmium oxide filter, Beckman Instruments, Inc.,1

catalog No. 96157. (b) Ultraviolet-visible spectrophotometer. (c) Spectrophotometer cells, silica, 1-cm. (d) Volumetric flasks, 100-, 500-, 1,000-mL. (e) Metal-on-quartz filters, available from the Office of

Standard Reference Materials, U.S. Department of Commerce/National Institute of Standards and Tech-nology (NIST), Gaithersburg, MD (www.nist.gov).

Wavelength Accuracy Determination The holmium oxide filter provides sharp absorption

bands at specific wavelengths and should be used to check accuracy of wavelength setting before making spectrophotometer measurements. The filter is designed to fit in the standard 1-cm compartment and includes a

set of instructions on proper use. It may also be used to detect visible stray light and to determine visible resolution.

The filter has discrete absorbance maxima at 279.3, 287.6, and 333.8 nm in the ultraviolet region. In the visible region, the discrete maxima are at 360.8, 385.8, 418.5, 453.4, 536.4, and 637.5 nm. Maximum absor-bance wavelength settings that correspond to these abso-lute wavelengths should be found. This is accomplished by turning the wavelength dial through a small arc encompassing one of the absolute wavelengths and stopping at the point where maximum absorbance deflection occurs. Difference between the observed wavelength settings and the absolute values should be within specifications of the instrument.

If the wavelength error is larger than instrument specifications, instrument service should be requested. If the error is less than specifications, no correction would normally be necessary except for the types of measure-ments where exact wavelength setting is very critical, such as measuring absorbances on shoulders. If a correc-tion is necessary, the wavelength dial should be adjusted in the proper direction away from the desired wave-length to give the true wavelength. For example, if the 279.3-nm holmium oxide band reads 280.3 nm on the dial, then, for a determination that requires 275 nm, the setting would have to be 276 nm to give a true 275 nm. A holmium oxide absorbance maximum should be chosen that is near the wavelength to be corrected.

Cell Selection Cells used for accurate spectrophotometric work

should be from a matched set. The following test may be applied to a pair of cells to determine whether they are well matched. With the standard chromate solution (reagent d), approximately the same absorbance should be obtained when either cell is used as the sample cell with resetting 100% transmission using distilled water after switching the cells. If the difference between readings is larger than the instrumental specifications, then the cell path-lengths are not well matched.

Cells should be clean and unscratched. Silica cells designed for ultraviolet work are specified since they may be used for both ultraviolet and visible spectro-photometry. Cell deterioration may be caused by films deposited from impure solvents or strong wetting agents, by inadequate cleaning, or by etching due to prolonged contact with strong alkali or mineral acids.

The extent of cell deterioration, using a silica cell, may be checked as follows: The instrument is zeroed with the cell compartment empty of cells. The cell to be checked is then filled with distilled water and should

1 Mallinckrodt Chemical Works (website: www.mallinckrodt.com);Beckman Instruments, Inc., 2500 Harbor Blvd., Fullerton, CA 92634.

doi: 10.1094/ASBCMOA-Appendix-1

Appendix I Page 2 of 2

give at least 70% transmission at 220, 240, and 270 nm as compared to air in the reference cell space. The most reliable single beam spectrophotometric results are obtained by using the same cell for zeroing the instru-ment and for making measurements.

Cell Cleaning Procedure Cells are to be cleaned immediately after use, prefer-

ably using the pure solvent that was employed in the determination. Use a mixture of 3N HCl (reagent b) and ethyl alcohol (reagent a) (1 + 1) for hard-to-remove deposits. Use a final rinse of spectro-grade methanol or ethanol to avoid leaving a film, allowing the cells to air-dry rather than using forced air. Use an ultrasonic cleaner only with one-piece cells. Do not use a cleaning implement, such as a brush, which may scratch the cells. Never use strong alkali, abrasives, etching materials, or hot concentrated acid for cleaning.

Absorbance Calibration The NIST 2031 metal-on-quartz filters (apparatus e;

Ref. 5) are recommended for use as a standard to verify accuracy of spectrophotometers from 200 to 800 nm. Follow instructions received with the filters when calibrating instruments. If metal-on-quartz filters are unavailable, one or a combination of the solutions in the paragraphs below can be used for spectrophotometer absorbance calibration.

The following absorbance calibration measurements are to be made only after checking the spectrophotometer and correcting wavelength inaccuracy if necessary. Measurements should be made as close to 25°C as possible. Zero and 100% transmission adjustments are made with distilled water, rechecking before each reading. Absorbance readings are made against distilled water.

With dichromate solution (reagent c), ultraviolet absorbances are noted at 235, 257, 313, and 350 nm.

The EBC absorbances (Ref. 3) with a 1-cm cell at these wavelengths are 0.626, 0.728, 0.244, and 0.535, respectively.

An alternate for ultraviolet absorbance checks is the chromate solution (reagent d). This solution can also be used for low-wavelength visible checks. Ultraviolet absorbances are noted at 250, 260, 280, 290, 340, and 350 nm. National Bureau of Standards (NBS) absor-bances (Ref. 6), with a 1-cm cell, are 0.496, 0.633, 0.712, 0.428, 0.316, and 0.559, respectively. Visible absorbances are noted at 360, 380, 410, and 430 nm with respective NBS absorbances of 0.830, 0.932, 0.199, and 0.084.

Using the two cobalt solutions (reagent e), note the absorbances at 430, 435, 520, 530, and 540 nm in the visible range. NBS absorbances (1-cm cell) for the lower-concentration cobalt solution are 0.034, 0.043, 0.169, 0.145, and 0.111, respectively. The 1972 ASBC Instrumentation Subcommittee absorbance averages for the higher concentration cobalt solution were 0.147, 0.183, 0.674, 0.587, and 0.458, respectively (Ref. 1).

References 1. American Society of Brewing Chemists. Report of Subcommit-

tee on Instrumentation. Proc. 1968, p. 257; Proc. 1969, p. 249; Proc. 1970, p. 257; Proc. 1971, p. 363; Proc. 1972, p. 140.

2. American Society of Brewing Chemists. Report of Subcommit-tee on Revised Methods of Hops Analysis. Journal 47:122, 1989.

3. European Brewery Convention. Analytica-EBC, 2nd ed., p. 53. Elsevier, Amsterdam, London, 1963.

4. Hudson, J. R. J. Inst. Brew. 77:348, 1971. 5. Mavrodineanu, R., and Baldwin, J. R. Standard Reference

Materials: Metal-On-Quartz Filters as a Standard Reference Material for Spectrophotometry—SRM 2031. NBS Spec. Publ. 260-268. U.S. Dep. Commerce/National Bureau of Standards, Washington, DC, 1980.

6. U.S. Department of Commerce. Letter Circ. LC-1017. National Bureau of Standards, Washington, DC, Jan. 1967.

1972, rev. 1983, corrected 1987, rev. 1990