I M P R O V E D GAS C H R O M A T O G R A P H I C D E T E R M I N A T I O N

OF ACETIC ACID IN WINES

Bruno Trombella and Anthony Ribeiro
Respectively Analytical Chemist and Senior Technician, E. & J. Gallo Winery, Modesto, Califor-
nia 95353.
The authors gratefully acknowledge the assistance given by M. Ueda, A. Caputi, and
M. Akiyoshi.
Presented at the Annual Meeting of the American Society of Enologists, June 26, 1979, Las
Vegas, Nevada.
Manuscript submitted June 26, 1979.
Revised manuscript May 2, 1980.
Accepted for publication June 4, 1980.

ABSTRACT
An automated gas-liquid chromatographic method accumulation of residues is eliminated by making a
for the analysis of acetic acid in wines is described, dilution of the sample, and using a glass insert in a low
which minimizes the often encountered difficulties of dead-volume injector port. Propionic acid i s u s e d as an
p e a k '¢tailing," b r o a d e n i n g , and ~¢ghosting". The internal standard, and helium carrier gas is saturated
method uses a 6' x 2 mm phosphoric acid-treated glass with formic acid to improve the chromatography. The
column, packed with 60/80 mesh Carbopack C, mod- method is rapid, accurate, a n d precise; giving a stan-
ified with 0.3% Carbowax 20M, and 0.1% H3PO4, dard error of _+ 0.0012 g acetic acid per 100 mL of wine,
which is extremely stable to aqueous injections. The at the 0.005 to 0.054 g per 100-mL concentration level.

To the enologist, the most important volatile acid is
acetic acid. Acetic acid is important in winemaking
because, in addition to being a product of normal fer-
mentation, it can denote bacterial (Acetobacter) spoil-
age of wine. Quality control, as well as state and fed-
eral regulation, makes it imperative to have an accu-
rate and precise analytical method for its determina-
tion.
The method commonly used in the wine industry
for volatile acidity determination includes steam distil-
lation, followed by t i t r a t i o n of the distillate with
sodium hydroxide (3). This method has certain limita-
tions. First, errors can arise due to other volatile com-
ponents such as sorbic acid, lactic acid, SO2, CO2, etc.
(3). Second, the technique used to compensate for these
interferents, e.g., the SO2 titer, can itself introduce er-
rors (6). Third, the recovery in the Cash still is incom-
plete and variable as observed in this laboratory. Fi-
nally, the method does not lend itself to automation.
Wine, being the complex solution that it is, poses
certain unique problems to the direct injection GC
analysis of acetic acid. The nonvolatile components of
wine, e.g., sugars, pigments, proteins, etc., have a det-
rimental effect upon column packing. The alcohol pro-
duces a tailing peak which obscures that of the acetic
acid. In addition, the chromatographic idiosyncracies
associated with fatty acids in general must be dealt
with.
The prospect of a more precise, accurate and readily
automated method by GLC was the motivation for this
study.
MATERIALS AND METHODS
Acetic acid working standards were prepared by
weighing appropriate amounts of reagent grade glacial
acetic acid into 100-mL volumetric flasks, and bringing
to volume with 10% ethanol. The formic acid used to
saturate the helium carrier gas was 90% Baker PCS
Reagent grade. A stock propionic acid solution was
prepared by weighing 1 g of reagent grade propionic
acid into a 100-mL volumetric flask and bringing it to
volume with deionized water. Propionic acid internal
standard solution was prepared by mixing 5 mL of
stock propionic, 2 mL of 70% H3PO4 in a 2-L volumet-
ric flask and bringing to volume with deionized water.
A Hewlett Packard, model 5720A G.C., fitted with
an HP7671A autosampler, F.I.D. detector and low vol-
ume glass i n s e r t injector port, was used for the
analysis. The column used was a 6' x 2 mm glass col-
umn packed with 60/80 mesh Carbopack C, modified
with 0.3% Carbowax 20M, and 0.1% H3PO4 (available
from Supelco, Inc., Bellefonte, Pa.) The following GC
parameters were used: injector temperature, 175°C;
column t e m p e r a t u r e , 90°C; detector t e m p e r a t u r e ,
200°C; helium carrier flow, 32 cc/min; hydrogen flow,
37 cc/min; air flow, 257 cc/min; range, 1; attenuation,
x2; and sample size, 0.5 t~L.
Peak area integration and data storage was done by
an HP3351B Lab Data System, using the following pa-
rameters: method, internal standard; units, g/100 mL;
run time, 7.00 minutes; m i n i m u m area, 500; slope sen-
sitivity, 0.15 mV/min; reference peak retention time

294
Am. J. Enol. Vitic., Vol. 31, No. 3, 1980
 ACETIC ACID ANALYSIS n 295

window, 0.10 minutes; percent retention time window, employed. When the helium was saturated with formic
5; number of known peaks, 2; reference peak retention acid, as reported by Ackman and Burgher (1), the
time, 2.57 minutes; reference and internal standard feasibility of the procedure became even more evident.
peak, propionic acid; and internal standard peak reten- A gradual optimization of operating parameters re-
tion time, 6.14 minutes. sulted in chromatographic separation of the acetic and
Samples were p r e p a r e d by d i l u t i n g a 0.15-mL propionic acids from the other components (Fig. 1).
aliquot of either standard or sample to 2 mL with prop- Sample injection immediately after the integration of
ionic internal standard solution, using a Fisher dilutor. the propionic acid peak overlapped unimportant peaks
This was the only sample preparation necessary. The and permitted a shortening of the analysis time to
diluted samples, in 2-mL glass vials, were loaded di- seven minutes. This resulted in a substantial savings
rectly into the autosampler carousel. Quantification of
the analysis was with a 0.0500 g/100-mL acetic acid
standard.
RESULTS AND DISCUSSION
Several column packings were examined, including
the column packing suggested by E. Christensen (4).
These were: 3% Carbowax 600, 0.05% glycerol, 0.05%
H3PO4 on 80/100 mesh Chromosorb W-HP; as well as
15% Carbowax 1500, 0.5% polyphosphoric acid on
80/100 mesh Chromosorb W-HP; and 10% FFAP, 1%
HzPO4 on 100/120 mesh acid-washed Chromosorb W.
The major shortcoming was a relatively rapid deterio-
ration of the stationary phases. This deterioration
seemed to be caused by two factors: hydrolysis of some
of the stationary phases due to aqueous injections and
gradual bleeding off of the deactivating phosphoric
acid due to relatively high column temperatures. The STANDARD II WINE
most promising of the column packings tested was the
one suggested by DiCorcia and Samperi (5) for the
analysis of C2-C5 acids in aqueous solutions. This col-
umn packing consists of graphitized carbon black mod-
ified with polyethylene glycol 20M (Carbowax 20M),
and HzPO4.
The dilution of the wines with the propionic acid
internal standard solution reduces the nonvolatile con-
centration in samples, while retaining reasonable peak
areas (1,500 to 30,000 counts per peak) for the acetic
acid within the range of 0.005 g/100 mL to 0.1 g/100
mL. The dilution step also provides a convenient oppor-
tunity to add the propionic acid internal standard.
The use of a low dead-volume injector port fitted
with a glass insert was found to eliminate some of the
"tailing" and ~¢ghosting" caused by the nonvolatiles.
These seemingly are trapped on the surface of the in-
sert, as evidenced by a dark residue on it upon re-
placement. It is convenient to replace the insert after
approximately 70 injections, or two full auto-sampler
trays. This protects against the buildup of nonvolatile
residues on the column packing and minimizes the
generation of "active sites".
An effective way of eliminating "active sites" on the
glass surface of the column and inserts is to soak them
overnight in 1% phosphoric acid solution and to dry
them prior to use.
Helium carrier gas was selected over purified ni-
trogen as suggested by DiCorcia and Samperi (5), be-
cause acetic acid could not be differentiated from the 0 2 4 6 8 1012 14 16 min. 0 2 4 6 8 i0 12 14 l~min.
alcohol within a reasonable analysis time in a six-foot
column. Efforts to automate acetic acid analysis by Fig. 1. Chromatogram of an acetic acid standard and a wine. 1,
GLC became promising only when this carrier gas was solvent; 2, acetic acid; 3, propionic acid.

Am. J. Enol. Vitic., Vol. 31, No. 3, 1980

296 m A C E T I C A C I D A N A L Y S I S

of time over the 15 minutes required by the distillation Table 3. Comparison of distillation and GLC analyses.
method. Wine type Method a
The linearity of the F.I.D. detector response to the Steam GLC Difference
different acetic acid concentrations was determind by distillation
(using HgO)
injecting replicate standards r a n g i n g from 0.005 g/100
Cabernet Sauvignon 0.020 0.024 0.004
mL to 0.054 g/100 mL. The data showed a correlation Flor Sherry 0.036 0.038 0.002
coefficient of 0.997 within this range. Burgundy 0.036 0.040 0.004
Pinot noir 0.045 0.051 0.006
The precision of the method was determined by in- Oak Sherry 0.036 0.038 0.002
jecting, in duplicate, 10 dilutions of a 0.050 g acetic Red Press 0.047 0.052 0.005
acid/100-mL standard and 10 dilutions of a wine. Table Pink Chablis 0.028 0.032 0.004
Chablis 0.047 0.046 0.001
1 shows the standard deviations of _+ 0.0008 g/100 mL Pinot Chardonnay 0.051 0.050 - 0.001
and _ 0.0012 g/100 mL for the standard and the wine, French Colombard 0.075 0.070 - 0.005
Zinfandel 0.031 0.031 0
respectively, are more precise t h a n the _+ 0.005 ob- Riesling 0.021 0.026 0.005
tained by the steam distillation method (2).
a Concentration g acetic acid/100 mL.

Table 1. S u m m a r y of analytical precision.

Standard a Wine b
Mean concentration
(g/100 mL) 0.0494 0.0464
Standard deviation
(g/100 mL) 0.0008 0.0012
o
Coefficient of variation 1.62 2.64
/
Range 0.003 0.004 /
/
a n = 20. 0.06
b n = 20.

The recovery rate of acetic acid, following addition ~ 00,

to a Cabernet Sauvignon and a Riesling wine, was r u n ,.,'~
in triplicate and the averages are shown in Table 2.
The table also shows the concentrations (corrected for 0.04

volume change) and compares these values with the

GLC results. The calculated and experimentally de-
0.03
termined values are plotted in Fig. 2. Extrapolation to
zero-addition resulted in concentrations which were
identical to those of the original wines. 0.02

Table 2. Known additions to wine.

Acetic acid Theoretical Average Difference
added vol. corrected GLC (g/100 mL)
(g/100 mL) concentration results
(g/100 mE) (g/100 mL)
0.01 0.02 0.03 0.04 0.05 0.06 0.07
Cabernet Sauvignon
ACETIC AClD ADDED
0 -- 0.022 -- ( g1100-mL )
0.010 0.032 0.032 0
0.020 0.041 0.041 0 Fig. 2. Known acetic acid addition to wines. © Oabernet Sauvignon"
0.040 0.060 0.059 - 0.001 I-I Riesling.
0 ~ Riesling 0.020
0.015 0.035 0.035 0
0.030 0.050 0.046 - 0.004 CONCLUSION
0.045 0.065 0.070 + 0.005
The automated GLC method described above, with
minimal sample preparation, provides an accurate and
A comparison of the results obtained by steam dis- precise determination of acetic acid in wines. The use
tillation using mercuric oxide as an SO2 complexing of helium carrier gas saturated with formic acid re-
agent (6) with those obtained by GLC was made on 12 sulted in an approximate 50% savings of analysis time
different wine types (Table 3). Although the results in compared to the steam distillation method. The 0.3%
the table show close a g r e e m e n t , the GLC r e s u l t s Carbowax 20M, 0.1% H3PO4 on 60/80 mesh Carbopack
tended to be higher t h a n those found by the steam dis- C packing performed approximately 16,000 determina-
tillation method. An investigation of the recovery of tions before any sign of chromatographic detrioration
the acetic acid by steam distillation method compared was observed.
to the GLC method showed t h a t only 85% was re-
covered by distillation in the Cash still. This indicates L I T E R A T U R E CITED
t h a t a significant portion of the acetic acid is not de- 1. Ackman, R. G., and R. D. Burgher. Quantitative gas liquid
termined. chromatographic estimation volatile fatty acids in aqueous media.

Am. J. Enol. Vitic., Vol. 31, No. 3, 1980


Anal. Chem. 35:647-52 (1963).
2. Amerine, M. A. Laboratory Procedures for Enologists. p.
80. Associated Students Store, Univ. Calif., Davis, Ca. (1965).
3. Amerine, M. A., and C. S. Ough. Wine and Must Analysis.
p. 17-20. John Wiley and Sons, Inc., New York, N.Y. (1974).
4. Christensen, E. N. Determination of acetic acid in wine.
Am. J. Enol. Vitic., Vol. 31, No. 3, 1980
ACETIC ACID A N A L Y S I S - 297
Ann. Meeting, Am. Soc. Enol., San Francisco, Ca., June 27, 1975.
5. DiCorcia, A., and R. Samperi. D e t e r m i n a t i o n of trace
amounts of C2-C5 acids in aqueous solutions by gas chromatog-
raphy. Anal. Chem. 46:140-3 (1974).
6. Pilone, G. S., and B. C. Rankine, and C. J. Hatcher. Evalu-
ation of an improved method for measuring volatile acid in wine.
Aust. Wine Brew. Spirit Rev. 91:62, 64, 66 (1972).