a simple method for determination of glutaraldehyde - SAGE Journals

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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.,