ever, as well as the problem of determination of the actual. GA concentration in fixative mixtures. We, therefore, developed a method for the de- termination of GA.
Taz JOURNAL OF HISTOCHEMISTRY Copyright © 1969 by The Histochemical
A SIMPLE
Society,
METHOD
Inc.
FOR
NORMAN of Biological
Division
Vol.
CYTOCHEMIBTRY
AND
DETERMINATION
A. FRIGERIO
and
Medical
for
MICHAEL
AND
Research,
Received
OF
publication
June
3
GLUTARALDEHYDE’ J. SHAW
National
Argonne
17, No.
in U.S.A.
Printed
Laboratory,
27,
Argonne,
Illinois
1968
A rapid method for determination of glutaraldehyde (GA) is described based on formation of the GA-bisulfite complex followed by iodometric titration of unreacted bisulfite. A 1-mi aliquot of 2-6% GA is added to 20 ml of 0.25 M Na bisulfite. A reaction time of 5 mm is allowed, and the solution is titrated with 0.1 M triiodide. Blanks are run in parallel with 1 ml water or buffer. The method gave precisions of 0.2% or better and was not affected by phosphate, phosphate-sucrose or cacodylate buffers or by glutaric acid. Pure GA was prepared by vacuum distillation, analyzed by combustion and used as a primary standard. Deterioration of GA in water and buffer was studied by ultraviolet spectrometry and bisulfite titration. Ultraviolet extinction coefficients proved to be strong functions of time, temperature and initial concentration so that Beer’s law was not obeyed. Deterioration led to the formation of polymeric materials, first water-soluble and then, with Increasing polymerization, insoluble. Polymerization was most easily followed by observing the disappearance of aldehyde groups titrimetrically, since interpretation of the ultraviolet absorbances at 235:280 mi was subject to a multitude of conditions. Polymerization was independent of light, oxygen or transition metal ions, and nearly independent of temperature between 1 and 25#{176}C. Under the proper conditions a partial, but slow, reversal of polymerization could be obtained. Because of the somewhat erratic deterioration shown by GA, periodic determinations of titer are advised if reproducible fixation is desired. Within has
the
become
solutions excellent
last an
few
years
important
glutaraldehyde
We first
(GA)
component
of fixative
for electron microscopy, fixation for a wide variety
when
in storage has noted
tions
produce
of GA
and shelf life. that different noticeable
quality of tissue fixation. in our laboratories, and GA fixatives, suggested under marked chemical
grounds
deteriorate of oxygen
would
concentration, ucts (acids, might,
GA
(6, 9) one
would
at room temperature, and at neutral or
deterioration
ever,
variations
typical conditions of use and effect on fixation quality.
lead
but also semialdehydes
themselves,
not
affect
‘Work Commission.
supported
by
had a purely
expect
GA
to change
tissue of
the
in the
U.S.
in
Atomic
lots
of 50%
water,
w/w
GA,
precipitated
procedure. Small can be purified
(4). We found as later lots were
termination rapid enough
to
of fixative tion of the
GA
concentration developed
a
lots of GA, by vacuum
it simpler free of this
to discard polymeric
aldehyde at 280 tion, free
of
cedure fixative
Energy 176
in fixative mixtures. a method for the de-
solutions, and applied rates of GA deterioration
by measuring mis.
howof
of GA which was both simple and to be used for the day to day control
fixation conditions. Fahimi and Drochmans
How-
instability
polymer,
the actual GA We, therefore,
of prodwhich
fixation. the
some
distilled
The problem of deterioration remained, ever, as well as the problem of determination
in the presence alkaline pH. Such
only
manufacturing
distillation such lots, material.
obtained (1), using of GA
storage On
to the formation and polymers)
actual measurements have not been reported.
the
containing
Fahimi (4), concentra-
Erratic results those of others that deterioration
that
with
polymeric aldehyde. Furthertests, and correspondence with the supplier, showed that this behavior of GA is eliminated by slight alterations in
providing of plant and
animal tissues (11). Problems of reproducibility do exist, however, especially since GA concentrations vary significantly in commercial lots because of variation for example,
found
diluted
Their
method
(3)
it to determinaunder typical analyzed
its ultraviolet requires
prior
careful temperature control and of deterioration products. Thus, is not directly solutions. Fein
applicable and Harris
glutarabsorption distillaa sample their pro-
to analysis (5) determined
of
SIMPLE the glutaraldehyde iodimetrically, via: 1RCHO
content
in tanning
+ 3C02 I,
+
(1)
+ 2I, + 3Na,CO, RCOONa + Na,S04 + 4NaI
-,
2NaS,O,
Na2S2O3
-
(2)
+ H,O
+ 21
Na,S4O6
-‘
+ 2NaI Their
method, and,
however, requires in our hands, gave
hr/analysis
Anderson droxylamine
(1)
analyzed titration.
method
required
so that,
in our
+ I
at erratic
about
2 hr and
by
11 separate
it proved
too
(3)
least results.
glutaraldehyde Unfortunately,
hands,
OF GLUTARALDEHYDE
solutions
-p RCH(OH)SONa
+ NaHSO3
RCH(OH)SO,Na
DETERMINATION
1
hythis steps
cumbersome
and time-consuming for use as a control procedure. We tried, but rejected, methods employing gravimetric determination of Ag following Ag reduction by GA and direct titrimetric oxidation with MnOr (8). These lacked adequate specificity
for
many
common
specific
GA,
for
fixation
showing reductants.
the
aldehyde
depends
on
strong interference from In any case, a method group
was
functional
needed,
since
cross-linking
by
aldehyde. The complexity of Fein’s method suggested that it could be greatly simplified by adding a measured excess of bisulfite to GA, followed by the
titration
standard Ripper
of
I, (10)
adapted
the
unreacted
bisulfite
as had been originally for other aldehydes.
Ripper’s
method
to the
with
suggested by We therefore determination
of
GA. Molarities lems
have
sometimes
Standard
concentrates,
solutions common dicated
been
used
associated
0.1
M
in
Ii,
to avoid
the
with
normalities
intended
to
are
sold
(12).
give
as 0.2
prob-
final
N.
The
use of starch indicator was contrainfor this determination. Its use masked the
otherwise
clear
end point, sensitivity. tions must
warning
of the
approach
of the
and it added little or nothing to final Furthermore, starch indicator solube made afresh every few days or
deterioration
produces
interfering
aldehydes,
stored in a glass-stoppered bottle to prevent excessive loss of SOS. At 25. C, its concentration remained above 0.20 M for at least 5 weeks, which is adequate for this method. During the analysis, the NaHSO was stored in, and delivered from, a loosely stoppered 50-nil burette. Under these conditions the NaHSO3 concentration varied by less than 0.3% over 30 runs during the same day. S. Iodine solution: 0.1 M in Ii. It was prepared (a) by diluting a standard concentrate (Sargent Chemical Company, Chicago, Ill.) or (b) by adding 25.78 g resublimed iodine to 44 g El, dissolving this mixture in about 100 ml distilled water and diluting to 1 liter (2). 4.0.02 M phosphate buffer: pH 6.85. Ten milliliters of 0.1 M Na,HPO4 and 10.0 ml 0.1 M KH,P04 were diluted to 90 ml. When a 6% (w/v) sucrose-phosphate buffer was desired, 6.0 g sucrose were dissolved in the above buffer for each final volume of 100 ml. The pH was adjusted to the desired value with 0.1 M Na,HPO4 or 0.1 M KH,P04 before dilution to 100 ml. 5.0.0275 M cacodylate buffer: pH 7.25. Fifty milliliters of 0.055 M cacodylic acid, (CHs),.ASO2H, and 50 ml of 0.055 M NaOH were mixed well, and the pH was adjusted to the desired value with 0.055 M cacodylic acid or 0.055 M NaOH. 6. Glutaraldehyde: a biologic grade, supplied by Fisher Scientific Company, was used to develop this procedure. Apparatus: 1. 10-ml pipettes. 2. 50-nil burettes. 3. 125-nil glass-stoppered Erlenmeyer flasks. 4. Magnetic stirrer with Teflon-covered magnets. Procedure: If the concentration of GA is greater than 6%, it is quantitatively diluted with water, or buffer, to a value between 0.6 and 6%. One milliliter of this solution is pipetted into a 125-nil glass-stoppered Erlenmeyer flask. One milliliter
of distilled
water
into
a
125-nil
meyer
second Flask.
delivered
to each
same
reaction the
MATERIALS
2. Sodium
mately
0.25
AND
METHODS
The
solution
of the water. (NaHSO3) (26.0 g/liter).
bisulfite
M
solution: approxiThis solution was
flask
ml
from
end
turns
titrating
until yellow of Ii
blank
point,
remains ume
(5-10
Titrate
and
Erlen-
mm)
with
the
at first but,
A carefully
within
turns
3 mm.
Record
cleaned
in
0.1 M I.
colorless. again
sample
NaHSO3
standardized
solution
The
to allow Allow the
for both unreacted
are
burette.
loosely enough the solution.
for at least
used.
pipetted
M NaHSO,
a 50-nil
the
yellow
0.25
it becomes
the
is then
glass-stoppered
20.0
time
blank.
the sample
Reagents: 1. Distilled
Next,
or buffer
burette should be corked the drain of bisulfite into and
etc.
177
0.2 ml
Continue yellow the burette
and volis
178
FRIGERIO
necessary this solution
to
deliver tends
the iodine accurately, since to adhere to the walls. (GA) concentration may be the expression
Glutaradehyde
calculated
from
AND
SHAW
of Giukirakiehyde Buffer as a Function
Volume
(I) %GA(w/v)
(V2
-
V1) (100.12)
(100)
25
=
where I is the concentration of the standard I expressed in moles per milliliter, V, is the miiiliters of iodine used in the blank titration, V1 is the milliliters in the sample titration, 100.12 is the molecular weight of GA and S is the sample volume. If sample weight is used for 5, the expression gives % w/w.
II
TABLE Analysis
of
6.24% GA in PhosphateSucrose Buffer Added
Volume
ml
ml
0.0 0.0 1.0 1.0 5.0 5.0
Concentration
of
Water AAdSd
1.0 1.0 1.0 1.0 1.0 1.0
in Phosphate-Sucrose of Concentration
tion of Glutaraldehyde
Found
%
of GA
Normalized
to
diluted
%
(is/i.)
6.234
6.234
6.236 3.121 3.120 1.040 1.041
6.236 6.241
Un-
Value
(is/i.)
6.240
6.237 6.246
RESULTS
A typical experiment, which demonstrates the linearity of the method between 0.6 and 6.0% GA, is shown in Table I. A 6% solution of GA was prepared by diluting 6.4 g of a 48.5% (w/w) solution (Fisher Scientific Company, lot 760579) with distilled water to 50 ml in a volumetric flask. One milliliter of this solution was diluted with varying amounts of water to give effective concentrations between 0.6 and 6.0% GA. Results are shown in Table I. Similar experiments are shown in Tables II and III. For these, GA was dissolved in 0.02 M phosphate with 6% sucrose, pH 6.85, or 0.0275 M cacodylate buffer, pH 7.25. To obtain increased precision, the analyses were done by using the solution weight method (7).
Volumetric
Analysis as a Function
Volume of 69’ Glu taradeh;de AddeJ
Volume Water Added
ml
ml
1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
0.0 0.0 1.0 1.0 3.0 3.0 5.0 5.0 7.0 7.0 9.0 9.0
of
TABLE I of Aqueous Glutaraldehyde of Concentration Concentration of Glu taraldeh;de Found
% (w/v) 6.004 6.119 2.989
8.008 1.511 1.504
1.028 0.984
0.754 0.749 0.587 0.603
% (tv/v)
6.004 6.119 5.978 6.016 6.045 6.014 6.169 5.902 6.034 5.989 5.869 6.033
Mean
± standard
deviation.
±
Volume of 6.24% GA in
0.78k
±
0.0039
III
TABLE
Analysis Buffer
of Glutaraldehyde in Cacodylate as a Function of Concentration Volume
Cacodylate Buffer
of
Concentration
of Glu. Found
ml
ml
1.0 1.0 1.0 1.0 1.0 1.0
0.0 0.0 1.0 1.0 5.0 5.0
%
Concentration Normalized Undiluted
%
(is/is)
6.239 6.247 3.137 3.140 1.046 1.042
of GA to Value
(is/is)
6.239 6.247 6.274 6.281 6.274 6.254 6.261
Concentration of GA Normalized to Undiluted Value
6.014
6.239
±
0.0156
Thus 1.0-mi aliquots of GA solution were weighed into tared, 50-mi, glass-stoppered Erlenmeyer flasks on an analytical balance. Thereupon, 10 ml 0.25 M NaHSO3 were delivered, and the flasks were weighed again. A reaction time of 5 min was used. The solutions were titrated with standard I, and weighed again. Blanks were run in parallel. Table IV shows results obtained for two different GA solutions, prepared at 2.5 and 2.0%. Comparison of Table I with Table IV shows the increased precision available with the weight method, and completely supports the proposed stoichiometry of the reactions. The volumetric method is more than adequate, however, for control of GA concentration in fixatives. Varying the reaction time between 5 and 30 mm had no significant effect (i.e.,
