ANALYTICAL BIOCHEMISTRY 24, 470481 (1968)

A New Modification of the Carbazole Analysis:

Application to Heteropolysaccharides

CLARENCE A. KNUTSON AND ALLENE JEANES

Northern Regional ResearchLaboratory: Peoria, Illinois 61604

ReceivedOctober 27, 1967

Dische’s carbazole reaction (1) has been the principal method for
uranic acid determination since 1947. The method as originally described
is limited in applicability by low color intensity of certain uranic acids,
such as n-mannuronic. Among modifications developed to increase sensi-
tivity of the reaction, the most significant are those which use borate
(24). Others have been based on the differential temperature response
of certain uranic acids (5-7).
In this laboratory, the discovery that n-mannuronic acid is a major
constituent of the extracellular polysaccharide from Arthrobacter viscosus
NRRL B-1973 (8, 9) necessitated finding an accurate method for
n-mannuronic acid determination. We adapted the borate-modified car-
bazole method of Bitter and Muir (4) to the analysis of heteropolysac-
charides (lo), and discovered that the color intensity of n-mannuronic
acid increased tenfold over that obtained by the Dische method. In this
initial work we also developed a technique for calculating the relative
proportions of two uranic acids in a synthetic mixture, using data from
carbazole analyses both with and without borate. However, application
of this method to alginates containing known amounts of n-mannuronic
and n-guluronic acids gave unsatisfactory results, apparently because of
interference from traces of n-glucuronic acid. The same difficu1t.y was ex-
perienced by Haug and Larsen (11) using Dische’s carbazole method.
Because the reactivity of n-glucuronic acid is considerably higher than
that of either n-mannuronic or n-guluronic acid both with and without
borate, very small amounts of n-glucuronic acid could affectthese cal-
culations greatly. However, it appeared likely that conditions-could be
found for reducing the reactivity of n-glucuronic acid relative to that of
n-mannuronic acid, since the reactivity of n-glucuronic acid varies much
more with temperature than that of other uranic acids (5). We therefore
‘This is a laboratory of the Northern Utilization Research and Development
Division, Agricultural Research Service, U. S. Department of Agriculture.
470
 MODIFICATION OF CABBAZOLE ANALYSIS 471

have made a systematic study of the combined effects of borate and re-
duced temperature on the stability, sensitivity and specificity of the car-
baeole reaction. Reported here are the standardized procedure established
for a 55” borate-carbazole analysis and comparative data obtained by
this method and other accepted procedures on selected mono-, o&o-, and
polpsaccharides containing uranic acids.

MATERIALS AND METHODS

Reagents
(1) Concentrated H&SO,, sp. gr. 1.84: ACS reagent grade.
(2) Stock borate solution: 24.74 gm H,BO, dissolved in 45 ml 4 M
KOH; solution diluted to 100 ml with distilled water to give a stable
stock solut.ion 4 M in BOa3-.
(3) H,SO,-borate reagent: 25 ml stock borate soIution made to 1 liter
with ACS grade HeSO,. Final solution 0.1 M in H,BO,.
(4) Carbazole reagent: 0.1% solution prepared by dissolving carbazole,
recrystallized from benzene, in absolute ethanol purified by the method
of Stark (12).
Test Materials
All test and standard solutions were freshly prepared before use. Po-
tassium n-glucuronate dihydrate, a-n-galacturonic acid monohydrate,
n-glucose, n-galactose, and n-mannose were high-grade commercia1 sam-
ples. n-Guluronic acid was a gift from Dr. Arne Haug. All other materials,
prepared in this laboratory, were in the form of potassium salts. These
include the a- and P-methylpyranosides and furanosides of n-mannuronic
acid, the extracellular polysaccharide from Arthrobacter viscosus NRRL
B-1973 and the aldobiuronic and aldotriuronic acids isolated from this
polymer, namely 4-0-/?-n-mannopyranosyluronic acid- (l-4)-n-glucose-
and 4-O-P-n-mannopyranosyluronic acid- (l-4) -O-/?-n-glucopyranosyl-
(l-4) -n-galactose (8, 9). All calculations of concentration were based
on the molecular weight of the acid form, rather than the potassium salt
form, so,that alI absorbance values would be comparable. Samples were
of known moisture content; all calculations were on a dry-weight basis.
Standardizatibn’tif Probedtire for 55” Borate Antilysis
Since our primary interest was in developing a method that had high
sensitivity for n-mannuronic acid and low sensitivity for n-glucuronic
acid, experimentation was limited to these two materials during standard-
ization of procedure. Because Radhakrishnamurthy and Berenson (5)
showed exceptionally low color yield for n-glucuronic acid when samples
472 KNUTSON AND JEANEs

were heated at 5502 for comparatively short periods, our investigations

were limited to this temperature, and experimentation primarily involved
study of the sequence and duration of heating.
Samples were heated before or after carbazole addition, or both, for
total heating periods of up to 80 min in a 55” constant-temperature water
bath. The stability at room temperature of colors formed was tested.
Certain other experimental details were common to this analytical pro-
cedure and to all the procedures described in the comparison studies below.
All tubes had glass covers to prevent condensation of water vapor inside
the tubes. Absorbance was measured at 530 rn,u in a Beckman3 model B
spectrophotometer. All samples were run in duplicate with both reagent
and sugar blanks. Inclusion of sugar blanks is especially important with
samples containing neutral hexose in order to compensate for color pro-
duced during heating before carbazole addition. Theoretically, 0.2 ml of
alcohol should be added to sugar blanks as a substitute for the 0.2 ml of
carbazole solution. In practice this step was omitted after tests showed
an insignificant difference in absorbance with and without that volume
of ethanol.

Comparison of Modifications of Carbazole Analysis

Relative reactivities of n-glucuronic, n-galacturonic, n-mannuronic,
and L-guluronic acids, as well as D-ghcose, n-galactose, and n-mannose,
were studied under four different reaction conditions. These were: Dische’s
(1) original procedure, samples heated at 100” with no borate; Bitter and
Muir’s (4) borate procedure, samples also heated at 100”; an adaptation
of the procedure used by Radhakrishnamurthy and Berenson (5) at 55”
with no borate; and heating at 55” in the presence of borate. All samples
contained 10-70 pg of uranic acid per tube. For the 100” reactions, a boil-
ing water bath was used which was large enough so that boiling would
resume within 3-4 min after immersion of tubes. Specific details of the
procedure for each reaction condition are given below.
Reaction without Borate (both 55” and 100’). One milliliter of test
solution was pipetted into each tube and equilibrated in an ice bath. Con-
centrated H,SO, (6 ml) was added rapidly to the surface of the sample,
and tubes were again equilibrated in the ice bath. After contents of the
tubes were mixed with a Vortex Jr. mixer, samples were heated 20 min at
the proper temperature and again cooled in the ice bath. After the carba-
zole solution (0.2 ml) was added, samples were mixed and allowed to

’ All temperatures are in degrees Centigrade.

’ Mention of a specific commercial product does not constitute endorsement by the
U. S. Department of Agriculture over similar products.
 MODIFICATION OF CARBAZOLE ANALYSIS 473

stand 3 hr for color development. Color was stable for an additional

l-2 hr.
Reaction with Borate at 100”. Six milliliters of H&SO,-borate reagent
was pipetted into each tube and allowed to equilibrate in the ice bath.
Carefully, 0.7 ml of test solution was layered on top of the cold acid.
Samples were mixed 4 set with a Vortex Jr. mixer and again equilibrated
in the ice bath. After tubes were heated in a boiling water bath for 15 min
and equilibrated in the ice bath, 0.2 ml carbaaole was added. Tubes were
heated for an additional 10 min, cooled l-2 min in the ice bath, and
equilibrated to room temperature in air. Color development was com-
plete at this time and was stable for at least 48 hr.
Reaction with Borate at 65”. Six milliliters of H,SO,-borate reagent
was pipetted into each tube and equilibrated in an ice bath. Carefully,
0.7 ml of test solution was layered on top of the acid and allowed to
equilibrate in an ice bath; the mixture was agitated 4 set with the Vortex
Jr. mixer and equilibrated again in the ice bath. Carbazole solution (0.2
ml) was added and mixed, and the samples were heated in a 55” constant-
temperature bath for 30 min. Color develpoment was then complete, and
color was stable for 2 hr at room temperature.
Application to Oligo- and Polysacchurides
The four isomeric methyl glycosides of n-mannuronic acid, the extra-
cellular poIysaccharide from Arthobacter viscosus NRRL B-1973, and
the aldobiuronic and aldotriuronic acids from this polysaccharide were
analyzed by both the 55” and the 100” borate methods; and results were
compared in order to determine relative accuracy and efficiency of the two
methods for analysis of uranic acids in compounds containing neutral
hexoses.

RESULTS

Standardization of 55” Bomte Analysis

For both n-mannuronic acid and n-glucuronic acid, considerable vari-
ation in absorbance values occurred with changes in the sequence and
duration of heating. The absorbance of n-glucuronic acid continued to in-
crease not only with continued heating, but also at room temperature
after heating had been stopped. Because of this behavior D-glucuronic
acid could not be used to determine optimum conditions for the standard
procedure. Therefore, the standard conditions were based on the behavior
of n-mannuronic acid.
Of particular interest was the effect of the sequence of heating on both
maximum absorbance and color stability of n-mannuronic acid. If the
474 KNUTSON AND JEANES

total heating time was kept constant, but if the proportion of the heating
done before and after carbazole addition was varied, considerable vari-
ation in absorbance occurred. As shown in Figure 1, absorbance is highest
if all heating is done after addition of carbazole, even when samples are
kept at room temperature for 3 hr, although the differences are not so
great after this length of time. Thus the procedure of heating only after
addition of carbazole was adopted as standard for all subsequent tests.

Time at
Room Temp.

I I I I
0’ 10 20 30 40
Heating Time After Carbazole Addition, min

FIG. 1. Effect of heating sequence on sensitiyity of borate-carbazole analysis of

n-mannuronic acid at 55”. Total heating time 40 min. Abscissa shows amount of heat-
ing done after carbazole addition. Absorbance of samples was measured at 0 hr (-),
1 hr (--), and 3 hr (-•-) after co,mpletion of heating.
With heating only after carbazole addition, the effect of heating time
on absorbance of n-mannuronic and n-glucuronic acid was determined
(Fig. 2). Absorbance. of n-mannuronic acid (50 pg) reached a maximum
in 50 min, then decreased slightly, in contrast to that of n-glucuronic acid
(50 fig); which -continued to increase as long as heating was continued
(80 min). This behavior of n-glucuronic acid is quite similar to that ob-
served by Radhakrishnamurthy and Berenson (5) at 55” without bora’te,
/
the only difference being one of magnitude.. .
Color stability of n-mannuronic acid samples heated 20, 30, and 40 min
was determined by plotting absorbance vs. time at room temperature
after completion of heating (Fig. 3). The sample heated 30 min showed
greatest stability and that heated 20 min apparently had not reacted com-
pletely, whereas the sample heated 40 min had begun to degrade. When
 MODIFICATION OF CABBAZOLE ANALYSIS 475

Heating Time. min

FIG. 2. Variation of absorbance with heating time in borate-carbazole analysis

at 55”.

tested in the same manner, samples of n-glucuronic acid continued to in-

crease in absorbance on standing at room temperature.
On the basis of color stability and high absorbance of D-mannuronic
acid and of relatively low absorbance of D-glucuronic acid, 30 min was
chosen as the heating period for the standardized 55” borate procedure.

Heatrng
mm

0.2 -

I I I L
0 I 2 3 4
Time at Room Temperature. hr

FIG. 3. Stability of n-mannuronic acid color at room temperature in borate-

carbazole analysis at 55”.
 LIE/*-
*.-*
v
04

U2

u
,$
/
---,--
2"
_/

----
/
*.--

-i---------
40 60J

FIG. 4. Standard curves for modifications of the carbazole analysis: (A) n-galac-
turonic acid; (B) n-mannuronic acid; (C) L-guluronic acid; and (D) n-glucuronic acid.
476
-.
55".
_______--

--air---
no 08lale
..----

IO
 MODIFICATION OF CABBAZOLE ANALYSIS 477

Comparison of Modifications of the Carbazole Analysis

Standard curves for each of the four uranic acids studied under each
of the reaction conditions used are shown in Figure 4. The 55” borate re-
action is the most sensitive for all the acids studied, except n-glucuronic.
The other three acids show strikingly similar patterns: absorbance is
much higher with borate than without, and 55” samples give slightly
higher absorbance than those at 100”. This pattern is not strictly obeyed
by n-mannuronic acid without borate, but absorbance of this acid is negli-
gibly low at any temperature if borate is not present. The primary dif-
ferences in the reactions of n-galacturonic, n-mannuronic, and n-guluronic*
acids are in the magnitude of absorbances and in the magnitude of
changes arising from the different reaction conditions. Whereas the be-
havior of n-galacturonic acid is changed little by variation in reaction
conditions, that of n-mannuronic acid is grossly affected by the presence
or absence of borate. n-Guluronic acid reacts similarly to n-galacturonic
acid, but with lower absorbance values for corresponding reaction
conditions?
TABLE I
Carbasole Reaction: 50 rg Absorbance Values under Different React’ion Conditions
Absorbance at 530 mp
No borate Borate
Sample 100” 55” 100” 5<5”

D-Glucuronic acid 0.66 0.087 I. IX 0.47

n-Galacturonic acid 0.76 0.90 1 .n4 1.11
n-Mannumnic acid 0.062 0.058 n.67 O.)il
n-Guluronic acid 0.17 0.23 0.41 0.54
n-Glucose 0.025 0.015 0 091 0.050
D-Galactose 0.027 0.009 o.o63 0.043
o-Mannose 0.018 0.007 n ,043 0.035

The absorbance values of these four acids, as well as of n-glucose,

n-gala&se, and n-mannose (50 pg in all cases), have been tabulated for
more direct comparison (Table 1). From this table the best method for
analysis of each uranic acid can be determined both from the point of
view of maximum absorbance as well as from minimal interference from
neutral sugars or traces of other uranic acids.

*The reactions of n-glucuronic acid appear to be quite anomalous when compared

to reactions of the other three acids. However, Radhakrishnamurthy and Berenson
(5) reported that L-iduronic acid also showed a high degree of temperature depend-
ence in reactions without borate. It would therefore be expected that the reaction of
r.-iduronic at 5P with borate would be similar to that of n-glucuronic acid.
478 KNUTSON AND JEANES

Application to Oligo- and Polysaccharides

From the outset, our primary concern with this analysis has been to
develop a satisfactory method for determination of n-mannuronic acid in
heteropolysaccharides. Data shown in Table 2 compare the observed ab-
sorbances of our di-, tri-, and polysaccharides containing n-mannuronic
acid with theoretical values calculated from standard uranic acid and
neutral sugar analyses by both the 55” and the 100” reaction. By both
methods, actual absorbance of the saccharides agrees closely with theo-
retical values. In any oligo- or polysaccharide in which the identity and
TABLE 2
Borate-Carbazole Analysis, 100” and 55”: Observed and Theoretical Absorbance
of Heterosaccharides Containing n-Mannuronic Acid
100” 55”
Sample Pz Absorbance % of theory Absorbance % of theory

o-Mannuronic acid 50” 0.690 - 0.812 -

~-Ghcose 46” 0.074 0.042 -
n-Gala&Me 46” 0.050 - 0.029 -
Sum of individual values 142 0.874 - 0.883
Aldotriuronic acid from 158b 0.844 103.7 0.875 99.2
polysaccharide B-1973
Aldobiuronic acid from 112b 0.775 191.4 0.847 99.3
polysaccharide B-1973
Polysaccharide B-1973 204b 0.848 104.1 0.875 99.2

Q Relative weights of these three monomers in a 1: 1: 1 ratio, as in the aldotriu~oriir:

acid or the repeat unit of the polysaccharide.
b These sample weights of the potassium salts of oligomers and polymer contain
50 ag of n-mannuronic acid.
an estimation of the neutral sugars are known, accurate determination of
the uranic acid content can be made. For preliminary determinations in
polysaccharides where neutral sugars are not known, only an estimation
of uranic acid content is possible. In such cases the 55” borate method
will give more accurate results owing to the lower interference from the
neutral sugars, except when the uranic acid is n-glucuronic. Data in Table
2 show the relative accuracy of the two methods under such circumstances.

DISCUSSION
Although there have been numerous publications on carbaeole method-
ology, comparatively little is known about the chemistry of the reactions
involved. 5-Formylfuroic acid has been identified as the chromogen re-
sponsible for the carbazole reaction color (13), and it has been shown to
be the preferential product formed from -uranic acids in concentrated
 MODIFICATION OF CARBAZOLE ANALYSIS 479

sulfuric acid (14). It is not known why the formation of this chromogen
should vary so much from one uranic acid to another, or why such factors
as addition of borate or variation of heating temperature should cause
large and specific changes in amount of reaction. The second reaction in-
volved in the analysis, the formation of the chromophore from carbazole
and 5-formylfuroic acid, has been described only empirically. Gauthiei
and Kenyon (6) have reported that the sensitivity of Dische’s original
method can be increased if samples are heated after carbazole addition,
and Galambos (7) reported that 95% of the color lost upon standing at
room temperature can be restored by further heating at 190”. Possibly the
increased absorbance observed with most of the acids studied by our
method resulted from both reactions proceeding simultaneously. Carhazolc
present in the reaction mixture might react immediately with nascent
5-formylfuroic acid and thus reduce immediate degradation of the chro-
mogen and permit a greater proportion of the chromogen t.o form
chromophore.
TABLE 3
Ratios of Reactivity of Uranic Acids under Various Reaction Conditions
to Reactivity of D-Ghxuronic Acid at 100” without Borate 15 w
Uranic acid
Reaction conditions D-Gluouronic wGalacturonic r+Mannuronic L-Guluronin

loo”, no borate 1.00 1.15 0.09 0.26

55”, no borate 0.13 1.36 0.09 0.35
loo”, borate 1.79 1.58 1.01 0.62
55”, borate 0.71 1.68 1.23 0.82

Our results demonstrate the flexibility of the basic reactions and the
high sensitivity and specificity that can be obtained. Shown in Table 3
is the relation of the absorbance of all four acids studied under all four
reaction conditions to the absorptivity of n-glucuronic acid under the
standard Dische condition. Under any reaction condition, the reaction of
each acid is quite specific. A similar comparison is shown in Table 4, ex-
cept that the absorptivity of each acid under each condition is compared
to its own absorptivity at 100” without borate. This comparison reveals
not only the specificity of each condition for each acid but the much
greater specificity of the absorbance pattern of each acid when all con-
ditions are considered together. Such data are useful in identifying un-
known uranic acids or in determining whether a compound or mixture
rontains more than one uranic acid. This technique has been especially
valuable in this laboratory for confirming identification of the uranic acid
in a heteropolysaccharide.
480 KNUTSON AND JEANJCS

Analyses of LY- and j?-methylpyranosides and (Y- and p-methylfurano-

sides of n-mannuronic acid gave standard curves which were identical
within experimental error. This similarity confirms that conformation and
linkage have no effect on the extent of reaction of the uranic acid.
Reactions of aldobi- and aldotriuronic acids from B-1973 polysac-
charide, as well as of the polysaccharide itself, agree closely with theo-
retical values for the constituent monosaccharides in the 55” borate re-
action (Table 2). The 100” borate values are less accurate, probably
because of the greater color obtained from the neutral hexoses at the
higher temperature.

TABLE 4
Ratios of Reactivity of Hexuronic Acids under Three Reaction Conditions Compared
with Standard Carbazole Procedure at 100” without Borate
Reactivity ratio
Borate
No borate,
55” 100” 55”

n-Glucuronic acid 0.13 1.80 0.72

D-Galacturonic acid 1.18 1.36 1.45
D-Mannuronic acid 0.94 10.8 13.1
I,-Guluronic acid 1.36 2.39 3.36

The modifications discussed here are by no means all the possibilities

nor necessarily the best for determining specificity and sensitivity of
uranic acids. For our purpose we believe conditions for a 55” borate anal-
ysis give optimum sensitivity and specificity. The other basic reactions,
however, could probably be improved further, particularly if all reagents
were added before heating. In addition, other temperatures should be in-
vestigated to determine whether greater specificity or sensitivity, or both,
might be achieved. Further utilization of such data is described in the
paper following (15), wherein the specificity of the reactions is used to
determine relative proportions of uranic acids in mixtures and compounds,
with extreme accuracy and minimal interference from extraneous
materials.
In any of the modifications of the carbazole analysis, reproducible re-
sults can be obtained only with careful attention to procedural detail.
Items which can cause particular trouble are slight impurities in the sul-
furic acid, carbazole, or absolute ethanol and also uncontrolled heating of
samples during sulfuric acid addition. If pure reagents are used and if
uncontrolled heating is kept to a minimum by use of an ice bath and the
layering technique followed by mixing with a Vortex mixer, duplication
and reproducibility will be good.
 MODIFICATION OF CAIlBAZOLE ANALYSIS 481

SUMMARY
The carbazole analysis when run at 55” in the presence of borate gives
increased sensitivity for n-galacturonic, n-mannuronic, and L-guluronic
acids, while greatly decreasing that of n-glucuronic acid. Comparison of
values from these reaction conditions with values from other modifications
of the carbazole analysis reveals a high degree of specificity of the re-
action for each acid under any defined set of conditions. This specificity
is of value for identification of unknown uranic acids. The new modifi-
cation is especially useful for determination of n-mannuronic acid in
heteropolysaccharides.
ACKNOWLEDGMENTS
We wish to thank Dr. Ame Haug of the Institute of Seaweed Research, Trondheim,
Norway, for his gift of L-guluronic acid and Dr. J. H. Sloneker of thie laboratory for
samples of the methylglycoeides of n-mannuronic acid and for the aldobiuronic and
aldotriuronic acids from the extracellular polysaccharide from Arthrobacter viscose
NRRL B-1973.

REFERXNCES
1. DI~CHE, Z., J. B&d. Chem. 191,189(1947).
2. GREQCJRY, J. D., Arch. Biochem. Biophys. 89, 167 (1960).
3. BITTER, T., AND EWINS, R., Biochem. J. 81,43P (1961).
4. BITIXR, T., AND Mom, H. M., Anal. Biochem. 4,330 (1962).
5. RADHAERIGHNAMURTHY, B., AND BEBENSON, G. S., And. Chem. 35, 1316 (1963).
6. GAUTHIER, P. B., AND KENYON, A. J., Biochim. Biophy. Acta 130, 551 (1966).
7. GALAMBOS, J. T., Anal. B&hem. 19, 119 (1967).
8. JEANES, A., KNUTSON, C. A., PITTSLEY, J. E., AND WATSON, P. R., J. Appl. Polymer
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9. SLONEKER, J. H., ORENTAS, D. G., KNUTSON, C. A., WATSON, P. R., AND JEANES,
A., Can. J. Chem., in press.
10. KNUTSON, C. A., AND JEANES, A., Abstracts of Papers, 148th Meeting, American
Chemical Society (1964), p. 16D.
11. RAna, A., AND LARSEN, B., Acta Chem. Scud. IS,1908 (1962).
12. STARK, S. M., JR., Anal. Chem. 22, 1158 (1950).
13. BOWNESS, J. M., Biochem. J. 70, 107 (1958).
14. ANDERSON, D. M. W., AND GARBUTP, S., J. Chem. Sot. 3294 (1963).
15. KNUTSON, C. A., AND JEANES, A., Anal. Biochem. 24,482 (1X%3).