Journal of Petroleum Science and Engineering, 9 ( 1993 ) 273-279 273

Elsevier Science Publishers B.V., Amsterdam

Xanthan biopolymers: A review of methods for the

determination of concentration and for the measurement of
acetate and pyruvate content

Kevin C. Taylor and Hisham A. Nasr-E1-Din*

Petroleum Recovery lnstitute, -#100, 3512-33rd Street NW,, Calgary, Alta. T2L 2A6, Canada
(Received February 25, 1993;revised version accepted April 8, 1993)

ABSTRACT

Xanthan gum is used extensivelyfor enhanced oil recoveryas a mobilitycontrol agent, in drilling operations to increase
the suspension capacity of the drilling mud, and in gels to improve the volumetric sweep efficiency.Flow properties,
injectivity, and adsorption characteristics depend on acetate and pyruvate content of xanthan. This reviewdiscussesvar-
ious methods and techniques available for measuring the concentration of xanthan and its pyruvate and acetate content
in laboratory and field samples. It includes a description of the principles of each method, advantages, limitations, inter-
ferences, and other information necessaryto understand the strengths and weaknessesof each.

Introduction cation (Sloneker and Jeanes, 1962; Holzwarth

X a n t h a n gum has been used in m a n y oil-
field operations including drilling, polymer-
augmented water flooding, alkaline flooding,
micellar flooding and profile modification
(Chatterji and Borchardt, 1981; Nisbet et al.,
1982; Sutherland and Kierulf, 1987). Its
structure has been reported in detail (Jansson
et al., 1975). Accurate m e a s u r e m e n t o f xan-
than concentration is important both in field
use and in laboratory studies. The pyruvate
and acetate contents o f xanthan have a large
effect on the rheological properties o f the poly-
mer (Sandford et al., 1977; Smith et al., 1981,
1984; Callet et al., 1987a,b; Rochefort and
Middleman, 1987; Kleinitz et al., 1989; Ku-
licke et al., 1990). X a n t h a n can be prepared
with an absence o f either acetate or pyruvate
groups, or with neither, by chemical modifi-
and Ogletree, 1979; R i n a u d o et al. 1983;
Cheetham and Punruckvong, 1985 ) or by ge-
netic engineering (Hassler and Doherty,
1990).
G r a h a m (1977), H o c k w i n (1974) and
Jeanes et al. ( 1976 ) have published limited re-
views of xanthan determination. These re-
views are very limited in their scope, are no
longer current, and do not include methods to
measure acetate and pyruvate content. The
present work reviews in detail methods for the
determination o f xanthan concentration and
the measurement o f acetate and pyruvate con-
tent. Principles and limitations of each method
for use with oilfield samples are discussed.
Discussion
A. Determination of xanthan
Phenol-sulfuric a c i d m e t h o d

*Current address: Laboratories Department, P.O. Box 62, The phenol-sulfuric acid m e t h o d relies on
Saudi Aramco, Dhahran 31311, Saudi Arabia. the formation o f a colored complex from the

0920-4105/93/$06.00 © 1993 Elsevier Science Publishers B.V. All rights reserved.

274
reaction of sugars with a phenol compound or
thioglycolic acid in concentrated sulfuric acid.
The method measures the total carbohydrate
content of a sample. Phenol, anthrone, orci-
nol, 3-phenyl phenol, and thioglycolic acid
have been used in this method (Auerbach,
pers. commun., 1991"*; Dubois et al., 1956;
Jeanes et al., 1976; Philips et al., 1985; Que-
mener, 1986; Ranjbar-Hamghawandi, 1988,
1990, 1992). The method is accurate within
_+2%, with a range of 2.5-35 mg/1 sugar (Du-
bois et al., 1956) and has been used for bio-
polymer determination in high salinity brine
(Ranjbar-Hamghawandi, 1990). The concen-
tration of glucose or other sugars must be
known, because they will result in a positive
interference (Jeanes et al., 1976). Color gen-
eration is affected by high salinity or the pres-
ence of Fe 3+ (Philips et al., 1985). Littmann
et al. (1992) used the method to determine
xanthan concentration in brine produced from
a polymer flood field test. Samples were di-
alyzed prior to analysis to remove salts and
potential interferences.
The phenol-sulfuric acid method provides
good sensitivity and reproducibility. How-
ever, the method is relatively slow, and the ef-
fect of interferences common in oilfield brines
(especially formaldehyde) is largely unknown.
Size exclusion chromatography
This method uses a column to separate the
polymer from low-molecular-weight impuri-
ties. This allows the use of ultraviolet (UV) or
refractive index detectors that would other-
wise be unsuitable with unpurified samples.
The UV detector is more sensitive, with detec-
tion limits as low as 0.6 mg/1 (Hunt et al.,
1988). Beazley (1985) examined samples
from a viscous emulsion produced by a par-
tially hydrolysed polyacrylamide/sulfonate
**M.H. Auerbach, pers. commun., 1991. Pfizer Proce-
dure: "Total Carbohydrate Assay by Phenol-Sulfuric Acid
Method". Pfizer Central Research, Easter Point Road,
Groton, Conn., USA.
K.C. TAYLOR AND H.A. NASR-EL-DIN
surfactant flooding project and concluded that
no interference with polymer determination
occurred. Similar results are expected for
emulsions containing xanthan.
Size exclusion chromatography is ideally
suited for highly contaminated samples. Sam-
ple throughput rates of three to five samples
per hour are typical.
Radioactive labelling
Carbon-14, a fl-ray emitter, was incorpo-
rated into xanthan by adding carbon-14-1a-
belled glucose to the fermentation broth late in
the growth cycle (Sorbie et al., 1987). They
produced a carbon-14-labelled xanthan with a
specific activity of 1.8 MBq/cm 3 ( 48.6 X 10- 6
Ci/cm 3) at a xanthan concentration of 3200
mg/l. Quantitation is generally performed with
a commercial liquid scintillation counter.
Goodyear et al. (1992) used carbon-14-la-
belled xanthan in the measurement of polymer
retention levels. Crushed rock with adsorbed
polymer was heated to decompose the poly-
mer into carbon dioxide and water. Radioac-
tively labelled carbon dioxide was absorbed in
a scintillation cocktail, and radioactivity was
measured.
The most significant drawback of radioac-
tive labelling as a method for xanthan deter-
mination is that it can not be used for com-
mercial samples. Labelled samples must be
grown in culture, and their properties may not
exactly match a commercial sample of interest.
Organic carbon content
Measurement of total organic carbon in
aqueous samples has been used by Lecourtier
et al. ( 1987 ) and Muller et al. ( 1986 ) for xan-
than determination. Solutions containing high
amounts of salt may interfere with the com-
plete combustion of carbon compounds, be-
XANTHAN BIOPOLYMERS
cause sodium carbonate may be formed pref-
erentially. Any carbon containing species will
interfere with the method, making it unsuita-
ble for the determination of polymer concen-
tration in the presence of surfactants, biocides
such as formaldehyde, or emulsions.
Metachromatic method
The metachromatic acid method by Mo-
raine and Rogovin ( 1971 ) uses the dye tolui-
dine blue O which forms an insoluble complex
with aqueous xanthan. When the mixture is
extracted with a petroleum ether, the complex
adsorbs at the interface. The method measures
the decrease in absorbance of the aqueous
phase as dye is lost due to the formation of the
insoluble complex. It is reported to be of use
at xanthan concentrations as low as 20 mg/l.
The effect of interferences is not known.
Precipitation
Xanthan can be purified and isolated by first
dissolving the polymer in water and then pre-
cipitating it in a water-miscible nonsolvent
such as isopropanol (Hassler and Doherty,
1990), methanol (Moraine and Rogovin,
1966) or ethanol (Jeanes et al., 1961; Milas
and Rinaudo, 1984). For careful analytical
work, several reprecipitations may be neces-
sary. This method can be a useful technique
for polymer concentrations greater than 10
g/l.
Viscosity
This method requires that viscosity varies
proportionally with polymer concentration,
and assumes that the viscosity of xanthan so-
lutions is independent of salinity. By measur-
ing viscosity at a fixed shear rate for a range of
polymer concentrations, Philips et al. (1985)
obtained a different nonlinear calibration
curve for each commercial polymer used. Any
change in temperature, pH, shear rate, or mo-
275
lecular weight distribution will affect the solu-
tion viscosity, reducing the accuracy of
measurement.
Refractive index
This method requires polymer concentra-
tion to be proportional to the refractive index
of the solution and has been used by Queme-
n e r ( 1 9 8 6 ) . The specific refractive index in-
crement of xanthan was found to be 0.144
cm3/g at 633 nm (Hunt et al., 1988 ) and 0.155
cm3/g at 546 nm (Milas and Rinaudo, 1984).
Lindstr~Sm and Srremark (1976) used a
Knauer refractometer thermostatted to
_+0.005°C, and claimed an accuracy of _+1
mg/1 for acrylamide copolymers in wood pulp
suspensions in distilled water. The refractive
index increment of this polymer is similar to
that ofxanthan (Hunt et al., 1988).
The use of refractive index for the determi-
nation of polymer concentration can produce
acceptable results only when solutions of low,
constant ionic strength are used. Temperature
must be accurately controlled to reduce
changes in refractive index. Ideally, only solu-
tions of xanthan in distilled water or dialyzed
samples should be considered for this method.
Hydrolysis with glucose determination
Hassler and Doherty (1990) quantitated
xanthan by first hydrolysing the polymer to its
constituent sugars, then determining glucose
concentration with an enzyme test kit (Sigma
Procedure No. 16-UV) using hexokinase and
glucose-6-phosphate dehydrogenase. This
method is also useful for determining glucose
impurities in xanthan. The efficiency and re-
producibility of the hydrolysis step has not
been established, nor has the method been
evaluated for interferences common in oilfield
brines.
276
Carbazole method
Glucuronic acid ( 18.4 wt% ofxanthan) can
be determined colorimetrically by the carba-
zole method (Wernau, 1981 ), which is similar
to the phenol-sulfuric acid method. The origi-
nal carbazole method of Dische (1947) was
modified by Knutson and Jeanes (1968) for
use with polysaccharides. The range is from 10
to 70 mg/l glucuronic acid. Bitter and Muir
( 1962 ) reported that glucose, formaldehyde,
and salts interfere with the method.
Gas chromatography
Gas chromatography has been used to deter-
mine xanthan concentration after hydrolysis
and derivatization (Lawrence and Iyengar,
1985). Xanthan is hydrolysed and the result-
ing sugars converted into volatile aldonitrile
acetates. Xanthan concentration is deter-
mined from the concentration of the constitu-
ent sugars. The presence of other carbohy-
drates will result in interference with the
method.
B. Measurement of acetate and pyruvate
content
1. Methods for measurement of both acetate
and pyruvate
High performance liquid chromatography
(HPLC)
The HPLC method for the determination of
acetyl and pyruvyl groups in xanthan uses a
strong acid cation exchange resin as the col-
umn packing material. After hydrolysis of a
small sample ( 10 to 20/tl of a 5 g/1 polymer
solution), acetate and pyruvate groups are de-
termined (Ash et al, 1983; Cheetham and
Punruckvong, 1985; Hassler and Doherty,
1990).
K.C. TAYLOR AND H.A. NASR-EL-DIN
Nuclear magnetic resonance (NMR)
spectroscopy
Several groups (Morris et al., 1977; Smith
et al., 1981 ) have used NMR to determine only
the pyruvate to acetate ratio. They used an in-
dependent method to determine pyruvate
content and then calculated the acetate con-
tent. Other groups have used an internal stan-
dard so that acetate and pyruvate can be mea-
sured directly (Paradossi and Brant, 1982;
Lambert and Rinaudo, 1985; Milas and Ri-
naudo, 1986; Callet et al., 1987a; Milas et al.,
1988). Rinaudo et al. (1983) and Kulicke et
al. (1990) measured pyruvate and acetate
content directly by relating the peak area of
these groups to the only equatorial anomeric
proton of the xanthan molecule. Kulicke et al.
found good agreement between their NMR
measurements and enzymatic methods.
High measurement temperatures (90 °C)
are required, and the polymer must be puri-
fied and dry. A measurement time of about 45
min is required after the molecular weight of
the polymer has been reduced by ultrasonifi-
cation. The method of Kulicke et al. (1990) is
recommended.
2. Methods for measurement of acetate only
Hydrolys& and titration
Xanthan is hydrolyzed under basic condi-
tions. Acetate content is determined by titrat-
ing the hydrolyzed product with acid (Slo-
neker and Jeanes, 1962; Rinaudo et al., 1983 )
or by acidification, distillation, and titration
of resulting acetic acid (Schultz, 1965). Any
acidic materials present which can react with
base may interfere with the method. Pyruvate
may decompose under acidic conditions to
produce acetic acid, resulting in a positive
interference.
XANTHAN BIOPOLYMERS
Hydroxamic acid method
The acetate ester in xanthan reacts with hy-
droxamic acid to produce acetohydroxamic
acid. This acid forms a water-soluble complex
with iron that absorbs light at 520 nm. The
method is applicable for the determination of
2 to 10 micromoles of acetate groups per mL
(200 #1 sample ), with an accuracy within + 2%
(Hestrin, 1949; McComb and McCready,
1957; Sutherland and Wilkinson, 1968).
This method does not require hydrolysis of
the acetate groups present in xanthan prior to
the determination. Interferences, however,
from species present in oilfield brines are
unknown.
3. Methods for measurement of pyruvate only
Lactate dehydrogenase method
The lactate dehydrogenase method for the
determination of pyruvate in polysaccharides
is discussed in detail by Duckworth and Yaphe
(1970). Application of the method to xanthan
is discussed by Jeanes et al. (1976). Both
methods are based on the work of Hadjivassi-
liou and Reider (1968 ). The pyruvate group
in xanthan is first hydrolyzed using oxalic acid
or dilute HC1.
This is the method of choice for the deter-
mination ofpyruvate content in xanthan. It is
specific, requires only 3 to 5 mg of polymer,
and is readily carried out with a minimum of
equipment.
2, 4-dinitrophenylhydrazine method (2, 4-DNP)
Pyruvic acid reacts with 2,4-DNP to pro-
duce a 2,4-dinitrophenylhydrazone derivative
which is measured colorimetrically. The
method has been used extensively for the de-
termination of pyruvate in xanthan (Koepsell
and Sharpe, 1952; Sloneker and Jeanes, 1962;
Sloneker and Orentas, 1962; Cheetham and
Punruckvong 1985; Shatwell et al., 1990). It
277
requires 2 ml of a sample containing approxi-
mately 2 g/1 of acid-hydrolyzed xanthan, and
is reproducible to within 2%. The most serious
disadvantage of the method is the relatively
large number of steps involved and interfer-
ence from formaldehyde (Hirase, 1957),
which is usually added to xanthan as a biocide.
Oxidation and determination of carbon
dioxide
Hirase ( 1957 ) has determined the pyrnvate
content of agar by oxidation with sodium me-
taperiodate and gravimetric determination of
the liberated carbon dioxide. The same method
can be used to determine pyruvate content in
xanthan.
Conclusions
The determination of concentration is im-
portant to the study of xanthan in enhanced
oil recovery, in drilling operations and in gels.
The measurement of acetate and pyruvate
content is important because these two param-
eters have a large effect on the rheological be-
haviour, adsorption, and injectivity of xan-
than solutions.
Size exclusion chromatography with ultra-
violet detection was found to be the most suit-
able method for xanthan determination in oil-
field samples. The method is accurate and is
not affected by impurities common in oilfield
brines. The phenol-sulfuric acid method is ac-
ceptable if interferences from other carbohy-
drates, iron, and salinity are accounted for.
Proton NMR is the method of choice for the
simultaneous measurement of acetate and py-
ruvate content in xanthan. Alternately, pyru-
vate content can be measured using the lactate
dehydrogenase method, or both acetate and
pyruvate can be measured using HPLC. The
effect of salts, divalent ions, and formalde-
hyde, common interferences in oilfield brines,
is not generally known for methods that mea-
sure acetate and pyrnvate content in xanthan.
278
Acknowledgements
The authors would like to thank Janet Noy
for conducting a literature search which iden-
tified many of the papers used in this report;
Lynne Story and Wendy Faid, I.N. Mckinnon
memorial library, for their help in obtaining
many of the papers reviewed; and Prof. G.V.
Chilingarian for useful discussions.
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