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ATOMIC ENERGY COMMISSION
DETERMINATION OF SMALL AMOUNTS OF BERYLLIUM
BY FLUORESCENCE MEASUREMENT
A. B. Carlson
W. F. Neuman
A. L. Underwood
Published for use within the Atomic Energy Commission. Inquiries for additional copies
and any questions regarding reproduction by recipients of this document may be referred
to the Documents Distribution Subsection, Publication Section, Technical Information Branch,
Atomic Energy Commission, P. O. Box E, Oak Ridge, Tennessee.
Inasmuch as a declassified document may differ materially from the original classified
document by reason of deletions necessary to accomplish declassification, this copy does
not constitute authority for declassification of classified copies of a similar document which
may bear the same title and authors.
Date of Manuscript: February 1947
Document Declassified: May 8, 1947
This document consists of 13 pages.
-- _. .'
The fluorescence of alkaline beryllium solutions with quinizarin has been studied in
detail. pH, dye concentration and many common ions have been shown to influence the
fluorescence of the complex. From these studies a procedure was developed for the de-
termination of beryllium in amounts of 1-10 micrograms. There is good reason to believe
that the method can be extended to determine even smaller amounts of beryllium.
DETERMINATION OF SMALL AMOUNTS OF BERYLLIUM
BY FLUORESCENCE MEASUREMENT
White and Lowe (1) described the fluorescence of alkaline beryllium solutions with 1-
amino-4-hydroxyanthraquinone. Fairhall and his co-workers (2) found that this fluorescence
was proportional to the beryllium concentration in the range of 0.05 to 10 micrograms.
These investigators studied the fluorescence in ultra-violet light by visual comparison. It
is further stated (2) that 1,4-dihydroxyanthraquinone (quinizarin) produced the fluorescence
as well as the amino compound.
Our attempts to reproduce the method of Fairhall (2) led to anomalous results; there-
fore studies of the effects of pH, dye concentration, time of standing, and interfering ions
were undertaken. Quinizarin was employed in these studies. A procedure based on fluores-
cence measurement has been developed for determining beryllium in the range of 1 to 10
micrograms per 20 ml, with a standard error of about 10%.
Instrument-Fluorescence intensities were measured by means of the fluorophotometer
(Fig. 1) designed and built in this laboratory. This instrument employed two phototubes
(and amplifiers), one (the control tube) receiving light directly from the ultra-violet lamp,
the other being activated by fluorescence of the sample. A balanced circuit was thus ob-
tained, although null-point measurement with a slide-wire was abandoned in favor of direct
galvanometer readings. The ultra-violet source was a General Electric Type H4 mercury
discharge lamp. Heat resistant Corning ultra-violet filters (no. 5874) were employed to
reduce visible light produced by the lamp; Corning filters (no. 3486) were interposed be-
tween the sample chamber and the phototube to absorb ultra-violet light and to transmit the
orange-red fluorescent light. The lamp was operated with a constant-voltage transformer;
this, together with the balanced circuit mentioned above and shown in the diagram, rendered
the instrument insensitive to changes in line voltage which are frequently encountered. The
fluorophotometer circuit was operated with four commercial-type, lead storage batteries
arranged in series. Cuvettes made of high-silica glass (Klett Mfg. Co.) were used to hold
the solutions. The instrument was checked for stability and linearity of response by meas-
uring the fluorescence of quinine sulfate solutions of various strengths (Fig. 2), Corning
filters. (no. 4303, 3385, and 3389) being used in place of no. 3496 to pass the bluish fluores-
cence of quinine sulfate.
The adsorption spectrum of quinizarin in alkaline solution was determined, using a
Beckmann photoelectric spectrophotometer. From Fig. 3 it is seen that the solution does
not strongly absorb light of the principle wave-length of the lamp output (3650 Angstroms).
This is important, since penetration of the ultra-violet light is necessary to activate flores-
cence throughout the solution when beryllium is present. It was found, however, that addi-
tion of beryllium did not significantly alter the absorption spectrum of the solution. This
may be disadvantageous, since absorption of the activatingelight is a prerequisite for fluores-
F UORO PHO TOMBE'ER
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Reagents-1,4-dihydroxyanthraquinone (quinizarin), obtained as the technical grade re- 7
agent from Eastman Kodak Co., was sublimed, and recrystallized from ethyl alcohol, yield- ."i
ing an orange-red powder having a melting point of 194-1950C. It is moderately soluble in ,
alcohol, forming an orange solution, and is very soluble in alkalies, giving rise to a deep
purple solution. In the studies reported below, a solution of 0.3 mg per ml 95% ethyl alcohol :I
was used. Solution was effected by gentle warming over a steam-bath.
Standard beryllium solutions were prepared by dissolving 1 g of the metal in dilute HC1,. .
diluting to a liter, and using 1 ml of this solution to prepare a liter containing 1 microgram
beryllium per ml.
Diethylamine (Eastman Kodak Co.) was redistilled (B. P. ca. 550C) and dissolved in water
to give a 1M solution.
Effect of pH- A series of solutions containing 5 micrograms beryllium and 0.2 ml dye .:
solution in a total volume of 20 ml was prepared, using various amounts of NaOH to vary
the pH from 7.9 to 13.5. It is important to add the alkali before the addition of the dye; other-'
wise, a difficultly-soluble precipitate of dye forms. Blanks were prepared for each solu-
tion. The pH of each solution was determined by means of a Beckmann pH meter. It was
found that maximal fluorescence occurs at pH 11.3 11.4 (Table I, Fig. 4).
EFFECT OF pH ON FLUORESCENCE
pH Galvanometer Reading
11.60 48 .
11.80 24 '
Because the pH was shown to be critical, the solutions were buffered in all further stud- "'
ies. It was established by titration studies, that a combination of diethylamine and its hy- .
drochloride was a useful buffer at the pH 11.4. A solution of 2 ml of IM diethylamine plus .
4 ml 0.1N HC1 diluted to 20 ml had a pH of about 11.4 which was unchanged by addition of ...
small amounts of beryllium and dye. By comparison of solutions thus buffered with a soai- : .i
tion adjusted to the same pH with NaOH, it was established hat the buffer does not inter-
fere with the beryllium fluorescence.
... : .I..
~. .. : .
FI G. Y
EFFECT OF PH
If a I1 a
NICV o 3.WO'N ) a.N
9NIV3j Y3L3WONVA1V3 *L3N
Effect of Dye Concentration-Solutions of constant pH and beryllium concentration, with
varying amounts of dye, were prepared. Table H and Fig. 5 show that the dye concentration
is critical. This was repeated for several beryllium levels, and it was found that for each
beryllium concentration there was an optimal amount of dye which gave the maximalfluores-
cence. Fig. 6 shows a plot of beryllium concentration (in micrograms per 20 ml) vs. the
amount of dye in mg. giving maximal fluorescence).
EFFECT OF DYE CONCENTRATION ONFLUORESCENCE
Mg Dye Net Galv.
In preliminary test tube experiments, it was noted that ether extracted dye from the
aqueous layer in alkaline solutions containing no beryllium, while if beryllium was present,
dye remained in the aqueous layer. Therefore an attempt was made to obtain automatically
the optimal dye concentration by adding excess dye to several solutions containing from 1
to 10 micrograms of beryllium and extracting the excess-with ether, with the idea in mind
that the beryllium might hold in the aqueous layer the appropriate amount of dye. This
proved not to be the case. Although excess dye was extracted preferentially, as indicated
by increased fluorescence of solutions low in beryllium, some essential dye was also re-
moved, since solutions of greater beryllium concentration fluoresced less strongly after
Effect of Time Standing-No measurable changes in fluorescence intensity occurred in
solutions standing for as long as an hour-and-a-half.
Interfering Substances- Fairhall (2) mentioned the interference of calcium and magnesium
in determining beryllium in bone samples by the fluorescence method; these precipitated as
phosphates from alkaline solution. Beryllium recoveries from such solutions were cor-
rected by factors obtained with bone solutions having known beryllium content.
In the present investigation, various common ions were added in ratios to beryllium of
10:1, 100:1, and 1000:1. Na* PO0, F HCO~, C1; NO3, and SOf were found not to inter-
fere with the fluorescence in the above ratios. Ca++, Mg+ Fel* Cu*, and Mn+definitely
interfered in ratios of 10:1, fluorescence measurements being low by as much as 30-50%.
-10- MDDC 941
OPTIMAL DYr IN MG
-11- MDDC 941
AMOUNr BERYLLIUM JNM s
In larger amounts, the hydroxides of these metals precipitated, making fluorescence meas-
Procedure for Determining Beryllium in Pure Solution-The total volume selected as
convenient for fluorimeter employed was 20 ml. Unknown saturations containing from 1 to
10 micrograms of beryllium were treated as follows: 2 ml 1M diethylamine were added,
followed by 0.3 ml dye solution and 4 ml O.1N HCI; the solutions were then diluted to 20 ml
and the fluorescence measured. Beryllium content was read from a standard curve obtained
by treating known beryllium solutions in the same manner. Larger amounts of beryllium
could be determined by dilutions to give samples within the concentration range required.
A typical standard curve is shown in Figure 7.
Results-Two groups of "unknowns", one with 24 samples, the other with 20 samples,
were analyzed, using the procedure outlined above. Comparisons of the analytical recovery
with the true values are shown in Table II. The standard deviation (sigma) was computed
according to the formula: a in % = _(d_) where d is the percentage deviation for each
sample and N is the total number of samples.
Group 1 Group 2
Analyst's Report Actual Be Content Analyst's Report Actual Be Content
micrograms micrograms micrograms micrograms
3.2 3.4 5.6 5.0
3.5 4.0 3.4 3.0
7.9 6.9 10.0 10.0
4.3 5.1 1.0 1.0
4.3 4.6 8.3 6.2
5.3 6.0 2.4 2.0
9.0 9.0 4.5 4.1
10.0 7.0 3.2 3.0
1.9 2.0 7.8 7.0
8.6 8.0 6.0 5.0
3.6 4.0 4.3 4.4
5.2 5.4 10.0 8.0
4.8 5.5 3.7 3.5
1.3 1.0 10.0 9.0
3.4 2.5 5.4 5.0
4.3 4.5 7.4 6.0
2.9 3.0 10.0 7.0
6.8 8.5 5.7 5.5
3.5 3.5 4.0 4.0
5.5 6.0 6.2 6.0
a in % = 17 a in % = 14
The accuracy to be expected by application of the method to pure beryllium samples can
be seen from Table m above. The errors are larger than might be desired, but the method
may be useful, for the determination of small amounts of beryllium in the absence of more
accurate methods. Interferences of common cations as described above indicate that isola-
tion of beryllium will be necessary for any general application of the method, as, for ex-
ample, in analysis of biological samples, minerals, and metals.
In the work described here, the sensitivity of the instrument was reduced to obtain greater
stability fcr performing fundamental studies of the method. The fact that the blank does
not appreciably fluoresce suggests the possibility of applying the method to smaller amounts
of beryllium (0.1 microgram and possibly 0.05 microgram per 20 ml.)
It will be seen from Fig. 7 that the standard curve is not linear thorughout the range 1
to 10 micrograms. This was to be expected from the studies of the effect of dye concentra-
tion on the fluorescence. If a constant amount of dye, optimum for the center of the range,
be added to solutions containing from 1 to 10 micrograms, solutions in the upper part of the
range will not contain sufficient dye to activate maximal fluorescence, while in the lower
samples, excess dye, which perhaps absorbs part of the fluorescent light, causes lower
readings than would otherwise be expected. Thus, workers wishing to apply the method to
amounts of beryllium not considered in this report should use care in selecting the amount
(1) White and Lowe: "Fluorescent Tests for Beryllium and Thorium"
Ind. Eng. Chem., Anal. Ed., 13, 809 (1941)
(2) Fairhall et al: "The Toxicology of Beryllium"
Nat'l Inst. of Health, Bull. No. 181, p. 10,
U.S. Public Health Service
uNIVERSITY OF FLORIDA
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