[Code of Federal Regulations]
[Title 40, Volume 28]
[Revised as of July 1, 2002]
From the U.S. Government Printing Office via GPO Access
[CITE: 40CFR796.1050]
[Page 80-82]
TITLE 40--PROTECTION OF ENVIRONMENT
CHAPTER I--ENVIRONMENTAL PROTECTION AGENCY (CONTINUED)
PART 796--CHEMICAL FATE TESTING GUIDELINES--Table of Contents
Subpart B--Physical and Chemical Properties
Sec. 796.1050 Absorption in aqueous solution: Ultraviolet/visible spectra.
(a) Introductory information--(1) Guidance information. (i)
Molecular formula.
(ii) Structural formula.
(2) Standard documents. The spectrophotometric method is based on
national standards and consensus methods which are applied to measure
the absorption spectra.
(b) Method--(1)(i) Introduction, purpose, scope, relevance,
application and limits of test. (A) The primary environmental purpose in
determining the ultraviolet-visible (UV-VIS) absorption spectrum of a
chemical compound is to have some indication of the wavelengths at which
the compounds may be susceptible to photochemical degradation. Since
photochemical degradation is likely to occur in both the atmosphere and
the aquatic environment, spectra appropriate to these media will be
informative concerning the need for further persistence testing.
(B) Degradation will depend upon the total energy absorbed in
specific wavelength regions. Such energy absorption is characterized by
both molar absorption coefficient (molar extinction coefficient) and
band width. However, the absence of measurable absorption does not
preclude the possibility of photodegradation.
(ii) Definitions and units. The UV-VIS absorption spectrum of a
solution is a function of the concentration, c1, expressed in
mol/L, of all absorbing species present; the path length, d, of the
spectrophotometer cell, expressed in cm; and the molar absorption
(extinction) coefficient,
[]i, of each
species. The absorbance (optical density) A of the solution is then
given by:
[GRAPHIC] [TIFF OMITTED] TC15NO91.045
For a resolvable absorbance peak, the band width
[] is the
wavelength range, expressed in nm=10-9 m, of the peak at half
the absorbance maximum.
(iii) Reference substances. (A) The reference substances need not be
employed in all cases when investigating a new substance. They are
provided primarily so that calibration of the method may be performed
from time to time and to offer the chance to compare the results when
another method is applied.
(B) Reference compounds appropriate for the calibration of the
system are:
(1) Potassium dichromate (in 0.005 mol/L,
H2SO4 solution) from J.A.A. Ketelaar, paragraph
(d)(2) of this section:
log []..............................
[] in nm..........................
(2) Fluoranthene (in methanol) from C.R.C. Atlas of Spectral Data,
paragraph (d)(3) of this section:
log []..............
[ 237 236 288 339 357
<] in nm...................
(3) 4-nitrophenol (in methanol) from C.R.C. Atlas of Spectral Data,
paragraph (d)(3) of this section:
log [].... 3.88 4.04
[] 288 311
in nm..................................................
See also paragraph (d)(1) of this section.
(iv) Principle of the test method. This method utilizes a double-
beam spectrophotometer which records only the absorption differences
between the blank and test solutions to give the spectrum of the
chemical being tested.
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(v) Quality criteria--Reproducibility and sensitivity. (A)
Reproducibility and sensitivity, need not be measured directly. Instead,
the accuracy of the system in measuring the spectra of reference
compounds will be defined so as to assure appropriate reproducibility
and sensitivity. It is preferable to use a recording double-beam
spectrophotometer to obtain the UV-VIS spectrum of the test compound.
Such an instrument should have a photometric accuracy of [plusmn]0.02
units over the absorbance range of 0 to 2 units. It should be capable of
recording absorbances at wavelengths of 200 to 750 nanometers nm with a
wavelength accuracy of [plusmn]0.5 nm. The cells employed with the
instrument must necessarily be transparent over this wavelength range
and must have a path length determined to within 1 percent. To ensure
that the instrument is performing satisfactorily, spectra for test
solutions of K2Cr2O7 (for absorbance
accuracy) and holmium glass (for wavelength accuracy) should be run
periodically.
(B) In the event that a recording double-beam instrument is not
available, it will be necessary to determine the absorbance of the test
solution in a single-beam instrument at 5-nm intervals over the entire
wavelength range and at 1-nm intervals where there are indicated
absorbance maxima. Wavelength and absorbance tests should be done as
with the double-beam instrument.
(2) Description of the test procedure--(i) Preparation--(A)
Preparation of test solutions. (1) Solutions should be prepared by
accurately weighing an appropriate amount of the purest form of the test
substance available. This should be made up in a concentration which
will result in at least one absorbance maximum in the range 0.5 to 1.5
units.
(2) The absorption of a compound is due to its particular chemical
form. It is often the case that different forms are present, depending
on whether the medium is acidic, basic, or neutral. Consequently,
spectra under all three conditions are required where solubility and
concentration allow. Where it is not possible to obtain sufficient
concentrations in any of the aqueous media, a suitable organic solvent
should be used (methanol preferred).
(3) The acid medium should have a pH of less than 2, and the basic
medium should be at least pH 10. The solvent for the neutral solution,
and for preparing the acidic and basic ones, should be distilled water,
transparent to ultraviolet radiation down to 200 nm. If methanol must be
used, acidic and basic solutions can be prepared by adding 10 percent by
volume of HCl or NaOH in aqueous solution ([HCl], [NaOH]=1 mol/L).
(4) In theory, all chemical species other than that being tested are
present in both beams and would therefore not appear in the recorded
spectrum of a double-beam instrument. In practice, because the solvent
is usually present in great excess, there is a threshold value of
wavelength below which it is not possible to record the spectrum of the
test chemical. Such a wavelength will be a property of the solvent or of
the test medium. In general, distilled water is useful from 200 nm
(dissolved ions will often increase this), methanol from 210 nm, hexane
from 210 nm, acetonitrile from 215 nm and dichloromethane from 235 nm.
(B) Blank solutions. A blank must be prepared which contains the
solvent and all chemical species other than the test chemical. The
absorption spectrum of this solution should be recorded in a manner
identical to that of the test solution and preferably on the same chart.
This ``baseline'' spectrum should never record an absorbance reading
varying more than [plusmn]0.05 from the nominal zero value.
(C) Cells. Cell pathlengths are usually between 0.1 cm and 10 cm.
Cell lengths should be selected to permit recording of at least one
maximum in the absorbance range of 0.5 to 1.5 units. Which set of cells
should be used will be governed by the concentration and the absorbance
of the test solution as indicated by the Beer-Lambert Law. The cells
should be transparent over the range of the spectrum being recorded, and
the path-lengths should be known to an accuracy of at least 1 per cent.
Cells should be thoroughly cleaned in an appropriate manner (chromic
acid is useful for quartz cells) and rinsed several times with the test
or blank solutions.
(ii) Performance of the test. Both cells to be employed should be
rinsed with
[[Page 82]]
the blank solution and then filled with same. The instrument should be
set to scan at a rate appropriate for the required wavelength resolution
and the spectrum of the blank recorded. The sample cell should then be
rinsed and filled with the test solution and the scanning repeated,
preferably on the same spectrum chart, to display the baseline. The test
should be carried out at 25 [deg]C.
(c) Data and reporting--(1) Treatment of results. (i) The molar
absorption coefficient []
should be calculated for all absorbance maxima of the test substance.
The formula for this calculation is:
[GRAPHIC] [TIFF OMITTED] TC15NO91.046
where the quantities are as defined above (see Definitions and units).
(ii) For each peak which is capable of being resolved, either as
recorded or by extrapolated symmetrical peaks, the bandwidth should be
recorded.
(2) Test report. (i) The report should contain a copy of each of the
three spectra (3 pH conditions). If neither water nor methanol solutions
are feasible, there will be only one spectrum. Spectra should include a
readable wave-length scale. Each spectrum should be clearly marked with
the test conditions.
(ii) For each maximum in each spectrum, the
[] value and bandwidth
(when applicable) should be calculated and reported, along with the
wavelength of the maximum. This should be presented in tabular form.
(iii) The various test conditions should be included, such as scan
speed, the name and model of the spectrophotom-eter, the slit width
(where available), cell type and path length, the concentrations of the
test substance, and the nature and acidity of the solvent medium. A
recent test spectrum on appropriate reference materials for photometric
and wavelength accuracy should also be submitted (see Reproducibility
and sensitivity).
(d) Literature references. For additional background information on
this test guideline, the following references should be consulted:
(1) Milazzo, G., Caroli, S., Palumbo-Doretti, M., Violante, N.,
Analytical Chemistry, 49: 711 (1977).
(2) Katelaar, J.A.A., Photoelectric Spectrometry Group Bulletin, 8,
(Cambridge, 1955).
(3) Chemical Rubber Company, Atlas of Spectral Data, (Cliffland,
Ohio).
[50 FR 39472, Sept. 27, 1985]