Gas Chromatographic Analysis of Volatile Sulfur Compounds

from Heated Milk Using Static Headspace Sampling’

K. R. CHRISTENSEN and G. A. REINECCIUS
Department of Food Science and Nutrition
University of Minnesota
St. Paul 56108

ABSTRACT sonnel trained in dairy judging by sensory

An investigation was conducted to methods (4). Instrumental methods of dairy
test the feasibility of using gas chroma- judging have long been sought, but none has
tography with static headspace sampling met the requirements of processors. To be
as an objective tool to measure milk useful in quality control, an instrumental
flavor quality. Heated milk off-flavor method has to be very fast, reproducible,
was chosen for study. Different strategies highly correlated with sensory scores, and ap-
were tried for increasing the sensitivity plicable to all of the commonly encountered
of a commercially available headspace off-flavors of milk. Off-flavors include heated,
method, including salting out with so- rancid, oxidized, light oxidized, barny, and
dium sulfate, cryofocusing during injec- bitter (4).
tion, and applying backpressure to the In the 1960s, the potential of static head-
sampling loop. With the aid of a sulfur- space sampling coupled with gas chromatogra-
specific detector, the resulting system phy (GC) was recognized (6, 11, 13, 17), but
was sufficiently sensitive to detect the the methods in use at that time were not
sulfur volatiles, H2S and dimethyl sul- sensitive enough. In the last 25 yr, much prog-
fide, at the concentrations found in pas- ress has been made in the field of static head-
teurized skim milk. Milk that was heated space sampling coupled with GC (9, 12). in-
to varying degrees was analyzed, and the cluding some work that was aimed specifically
analytical results were compared with at increasing the sensitivity of the headspace
the intensity of heated flavor as deter- sampler used in this study (18, 19). It seemed
mined by a sensory panel. For skim to us that the methodology had matured suffi-
milk, correlations were moderately ciently to allow a study on heated milk off-
strong: Spearman’s correlation coeffi- flavor using static headspace sampling alone
cients for H2S and dimethyl sulfide were with no other sample preparation.
.75 and .60,respectively. Correlations There appears to be some consensus (16)
were weak for whole milk. that heated milk off-flavor is caused by an
(Key words: heated milk flavor, static increase in the concentration of sulfur com-
headspace, gas chromatography) pounds. These include dimethyl sulfide
Abbreviation key: DMS = dimethyl sulfide, OMS), H2S, dimethyl disulfide, and meth-
FPD = flame photometric detector, GC = gas anethiol (MeSH) (10). Based on threshold
chromatograph, HSS = automatic headspace values from the literature, MeSH, DMS, and
sampler, MeSH = methanethiol. H2S are likely to contribute to cooked milk
flavor (10). Badings and de Jong (2) found
MeSH, DMS, H2S, and dimethyl disulfide in
INTRODUCTION
heated milk; dimethyl disulfide and MeSH
Currently, the task of evaluating the flavor were present in only trace amounts. Of these
quality of fluid milk is entrusted to dairy per- compounds, only DMS appears to be present
above its threshold in milk prior to heating.
Dimethyl sulfide, the fvst compound to be
linked to milk flavor (14), has been shown to
Received May 20, 1991. contribute to the “feed” defect (15).
Accepted January 27, 1992. The log of the concentration of H2S has a
‘Research sponsored, in part, by the Minnesota-South
Dakota Dairy Foods Research Center. Scientific Journal strong linear relationship with the heated fla-
Series Number 18,804, Minnesota Agricultural Experiment vor intensity of milk (3). Thus, there would
Station, St. Paul. seem to be some promise for using sulfur

1992 J Dairy Sci 75:209&2104 2098

 HEADSPACE ON HEATED MILK
compounds such as DMS and H2S as an index
of cooked flavor when they can be isolated in
amounts large enough to be measured ac-
curately.
The objective of this research was to apply
some of the improvements in technique and
equipment for the analysis of heated milk off-
flavor to determine whether an instrumental
method for quality control was feasible. After
method development, the second step was to
apply the headspace method on milRs that had
been cooked to different degrees to determine
if the correlation with sensory data was statisti-
cally significant.
MATERIALS AND METHODS
Instrumental Method
The chromatograms for this study were gen-
erated with a Hewlett-Packard (Avondale, PA)
model 5880A GC and a Hewlett-Packard
model 19395A automatic headspace sampler
(HSS).The HSS was equipped with a 3-ml
sampling loop. Helium was the carrier gas in
the GC and was also the purge gas in the HSS.
Clear 22-ml borosilicate glass vials were
sealed with 19-mm diameter PTFE (Teflon@,
E. I. duPont de Nemours & Co., Wilmington,
DE)-silicone septa and aluminum ''tear-away"
crimp seals. For each chromatogram, two vials
were filled with 15 ml of milk and allowed to
equilibrate for 1 h at 50°C. Following equili-
bration, the headspace of both vials was sam-
pled at a vial pressurization of 304 kPa (45
psi) and cryofocused together at the head of
the column.
TABLE 1. Events program within the gas chromatograph (GC).
Line
no. Command
_______~
10 wait
20 Valve 12 on
21 wait .01
22 Valve 12 off
30 Valve 11 on
31 wait .01
32 Valve 11 off
40 Start valve 7 on
90 Stop program
_ _ _ _ ~
Automatic headspace sampler.
2099
A Hewlett-Packard flame photometric de-
tector (FPD)was used in the sulfur mode.
Heated zones were set to the following temper-
atures: sample carousel, 50°C; sample loop,
55°C; injection port, 200°C; and FPD,225°C.
The GC oven temperature profile was as fol-
lows. The oven was held at 27 to 30°C until
the end of cryofocusing, then heated ballisti-
cally to 5 0 T , and then ramped up at the rate
of 5Wmin to 180°C. Gas flows to the FPD
were optimized for sensitivity prior to the be-
ginning of the study. The column used for the
study was a 30-m, .32-mm id., DB-5 (J&W
Scientific, Rancho-Cordoba, CA). Identities of
the peaks were determined by comparison with
the retention times of authentic compounds on
two columns of differing polarity. The
columns used for this part were the DB-5 and
a 30-m, .25-mm i.d., DB-Wax from J&W Sci-
entific.
To maximize sensitivity, the injection port
was bypassed completely, so there was no
split. The capillary column was connected di-
rectly to the transfer line of the HSS via a
1.56-mm to 1.56-mm (1/16" to 1/16") Swage-
lok union (Supelco, Bellefonte, PA), and the
column was operated under a constant linear
velocity (equivalent to a flow rate of 5 mY
min), controlled from the HSS.
Making long injections onto a capillary
column necessitated cryofocusing, which was
done manually. A 30-cm loop of the column
was immersed into a Dewar flask (7 cm i.d. x
13 cm deep) filled with liquid N. The GC oven
was held at ambient temperature during
cryofocusing. When the injection was com-
plete, the Dewar flask was removed, the oven
Action taken
____
Program stops until all setpoints are reached.
H S S ~moves carousel one vial over.
HSS begins sampling sequence.
Gc Starts run.
Prom StODS.
Journal of Dairy Science Vol. 75. No. 8. 1992
2100 CHRISTENSEN AND REINECCIUS
TABLE 2. Three-minute, double injection sampling se-
quence stored in the memory of the automatic headspace
sampler.
Action
Insert sampling needle
Pressure on
Pressure off
Vent on
Vent off
Begin injection
End injection
Pressure on
Pressure off
Vent on
Vent off
Begin injection, second vial
End injection, second vial
Withdraw sampling needle
door was closed, and the run proceeded auto-
matically from that point.
In order to have reproducible injections, the
timing of GC and HSS events was controlled
electronically; printed queues came from the
integrator to tell the operator when to remove
the cryotrap and close the oven door. The GC
events were controlled with a keystroke pro-
gram stored in the memory of the GC (Table
1). This program started the GC run, advanced
the sample carousel of the HSS, and initiated
the HSS injection sequence. The HSS injection
sequence was another program stored within
the memory of the HSS, which controlled the
timing of the HSS (Table 2).
Fat Interference
To study the effect of the presence of fat in
this particular system, DMS and dibutylsulfide
were dissolved in distilled water and in whole
milk at a concentration of 100 ng/g. The solu-
tions were analyzed under the conditions just
described.
ADSA Heat Treatments
Pasteurized skim and whole milks, which
were purchased locally, were subjected to the
first three of the four heat treatments
prescribed by the ADSA Committee on
Nomenclature and Standards of Milk Off-
Flavors (16) to produce sulfurous, rich, and
caramelized milks. We did not attempt to pro-
Journal of Dairy Science Vol. 75, No. 8, 1992
duce the scorched off-flavor. The milks were
placed in foil-covered, 946-m1 glass bottles
and subjected to selected heat treatments. The
Time sulfurous and rich milks were heated on a
(min:s) steam bath at 75’C for 2 min at 97’C for 15
0:Ol min, respectively. The caramelized milks were
0:02 autoclaved in a pressure cooker at 95 kPa (14
022 psi) for 15 min. At the end of heat treatment,
0:38 samples were cooled by placing them in an ice
0:40
0:42
water bath.
1 :20 The milks were analyzed by the GC method
1 :22 described with the results reported as peak
1 :42 areas.
158
2:oo
2:02 Sensory and Instrumental Analyses
255
257 To study the relationship between the ana-
lytical results and sensory scores, skim and
whole milks were brought to 75’C and held at
that temperature for different times. A sensory
panel rated the milks according to the amount
of cooked flavor that had developed. The head-
space method was used to follow changes in
the levels of the sulfur volatiles. The sample
procurement, heat treatment, sensory study,
and analytical measurements were completed
in 4 d.
On the 1st d of the study, 18.9 L (5 gal) of
skim and 18.9 L of whole pasteurized milk
were procured, and 15.1 of the 18.9 L were
loaded into 18.9-L “shotgun pails” (round
stainless steel pails having inside radii of about
24 cm) and heated with constant stirring in a
97°C water bath until the temperature of the
milk had reached 75’C. At this point, the pails
were transferred to another bath, which was
held at 75 to 80’C. After intervals of 1, 5, 10,
20, and 30 min, milk was transferred from the
stainless steel pails into foil-covered .98-L (1-
qt) jars and placed on ice. Once cool, they
were stored in the dark at 3 to 5’C until
sensory testing.
Sensory testing took place 16 h after heat
treatment. The sensory panel consisted of 21
volunteers, all of whom were students or fac-
ulty at the University of Minnesota Depart-
ment of Food Science and Nutrition. The
judges’ experience varied considerably, so they
were first presented with a training set consist-
ing of four samples: severely cooked (30 min)
skim milk, severely cooked whole milk, and
freshly pasteurized s k i m and whole milks.
They were asked to taste the reference samples
 HEADSPACE ON HEATED MILK 2101
TABLE 3. Two-factor ANOVA of fat (skim or whole
31 milk) and cook (sulfurous, rich, or caramelized) on H2S
peak area.
Source df SS MS F Test P Value
Fat 1 18,449 18,449 2.508 .1644
Cook 2 134,620 67,310 9.15 .0151
Fat x
cook 2 67,336 33,668 4.577 .0621
Error 6 44,139 7351

04 ’ 1 . 1 ‘ 1 . 1 ’ 3 . ’ hour of operation. Nevertheless, it was decided

0 10 20 30 40 50
Concentration of added dimethyl
sulfide (nglg)
Figure 1 . Standard curve for dimethyl sulfide in whole
milk: Y = .683 + 3.39 x X. GC = Gas chromato-
graph.
until they knew what defect they were evaluat-
ing and what range to expect. The judges were
then presented with 12 samples for evaluation,
6 skim and 6 whole milk samples that had
been cooked for various times, and asked to
rate the milks on a continuous 160-mm scale
ranging from “not cooked” to “very cooked”.
The samples were randomly presented and
identified by a three-digit code.
As soon as sensory analysis was complete,
instrumental analysis began. Samples were an-
alyzed for DMS and H2S by the GC method
described. The DMS was quantitated by the
external standard method. Figures 1 and 2
show the standard curves for DMS in pasteur-
ized whole and skim milks, respectively.
RESULTS AND DISCUSSION
Effectiveness of the Method
60
to proceed with the study to see whether fur-
ther development of equipment was merited.
Of the sulfur compounds noted in the litera-
ture, only DMS and H 2 S were detectable by
this method. Figures 1 and 2 show that the
sensitivity of the method for DMS is more
than adequate for the purpose at hand but that
the variance of the data appears to increase in
the lower concentrations. Based on the analysis
of variance of the data from the ADSA heat
treatments study (Tables 3 and 4), the standard
deviation of DMS areas is estimated to be
about .6 area units. For H2S, the standard
deviation is about 86 area units.
Difficulties arose during the sensory and
instrumental analyses that made quantitative
standards for H 2 S impossible. For this reason,
the level of H 2 S is reported as peak area only.
m
f

The headspace method used in this research

proved to be quite tedious and slower than
would be predicted from a run time of 15 min.
The occasional breaking through of H 2 S from
the cryogenic zone of the column made it
0 10 20 30 40 50 60
necessary to include a blank run between each
run, and the nonlinear response of the FPD Concentration of added dimethyl
made it necessary to have multilevel calibra- sulfide (nglg)
tion that was repeated daily. So many extra Figure 2. Standard curve for dimethyl sulfide in skim
runs had to be made that only 1 to 1.5 data- milk: Y = .449 + 4.29 x X. GC = Gas chromato-
generating runs could be made in a typical graph.

Journal of Dairy Science Vol. 75, No. 8, 1992

2102 CHRISTENSEN AND REINECCIUS
400 TABLE 4. Two-factor ANOVA of fat (skim or whole) and
mas cook (sulfurous, rich, or caramelized) on dimethyl sulfide
0 DMS. rep. 2
peak area.
Source df SS MS F Test P Value
Fat 1 171 171 476 .OOO1
Cook 2 1806 903 2512 .OOO1
Fat x
cook 2 213 137 380 .OOO1
Error 6 2.156 .359

Water Whole milk

significantly, lowering the sensitivity of head-
Matrix space analysis (5).
Figure 3. Effect of sample matrix on the response of
the method for dimethyl sulfide OMS) and dibutyl sulfide ADSA Heat Treatments
(DBS). rep. = Replicate.
Measurement of H2S and DMS for the three
ADSA heat treatments produced the data for
Figures 4 and 5 . Both sulfur volatiles increased
Fat Interference and then decreased with more severe heat
A study on fat interference was conducted treatment. The analytical precision was poorer
for the whole milk data than for the skim milk
because skim and whole milks seemed to give
data. The analysis of variance performed on
very different area responses for the same the data (Tables 3 and 4) bore this out. The
amount of added DMS. Dibutyl sulfide was statistical difference was significant between
chosen for study because it is more nonpolar the different heat treatments (P< .01) for both
than DMS and, thus, is more fat soluble. Rela- sulfur volatiles in skim milk and in whole
tive to water, the vapor pressure of DMS is milk.
depressed in milk, and the dibutyl sulfide is no The variability is higher for H2S than for
longer detectable at all (Figure 3). Therefore, DMS. The most likely explanation for this is
the approximately 4% fat in the milk has likely that some of the H2S escapes cryofocusing.
had this effect because as little as 1% fat can Thus, the analytical precision of the H2S data
lower the vapor pressure of some compounds depends, in part, on the skill with which the
operator terminates cryofocusing and initiates
the GC run.
Others have observed that the concentration
of sulfur volatiles decreased on continued heat
m treatment. In one study, H2S concentrations
s?
m 40 - increased from .5 to 50 pg/kg for pasteurized
Y
m and UHT milks, respectively, but no H2S was
a 30- detected in milk sterilized in the bottle (2).
m
0
r:
One possible explanation for the decline is that
UJ
20 - the sulfur compounds, themselves the products
-% of early heat-induced changes, are participating
5 10 -
E in further reactions with browning products
6 producing nonvolatile end products (1, 8).
n, Other mechanisms are also possible. For exam-
Sulfurous Healed Carmelized
ple, H2S oxidizes readily in milk to produce a
Cook Level variety of odorless products. Also, pressure
drops rapidly inside the pressure cooker on
Figure 4. Effect of different heat treatments on the
relative amount of dimethyl sulfide detected in milk. The
cooling, which could cause flash vaporization
cook levels were established by the ADSA (16). rep. = of the sulfur volatiles as it does in some UHT
Replicate. processes.
Journal of Dairy Science Vol. 75, No. 8, 1992
 HEADSPACE ON HEATED MILK 2103

i3
Y
I 1120
m
a
Q

a,
e
=3
m
c
Q
0
e
R
r
0 10 20 30
Sulfurous Heated Carmelized
Heating time at 75°C (rnin)
Cook Level
Figure 6. Comparison of the cooked flavor score (A)
Figure 5. Effect of different heat treatments on the to the hydrogen sulfide peak area (m) for skim milk. Error
relative amount of H2S detected in milk.rep. = Replicate. bars correspond to 1 SE.

Whatever the cause, the implications for were moderate, and the correlations for whole
instrumental measures of cooked flavor are milk were weak.
clear: the level of sulfur volatiles is a useful The Spearman procedure indicates that a
predictor of cooked flavor only for mild heat relationship exists between analytical and sen-
treatments. It is possible that a more useful sory results for skim milk, but it does not
relationship might have been found if a non- reveal what the relationship is. Figures 6 and 7
selective detector had been used because the reveal that the flavor scores increase on heat
caramelized flavor in milk is thought to come treatment and then remain fairly constant. The
from the products of browning reactions, same pattern occurs for the H2S areas, suggest-
which may contain little sulfur (16). ing that H2S could be used for predicting
sensory scores. In the case of DMS, there is no
such relationship between sensory scores and
Statistical Analyses
peak areas. There appears to be more DMS in
Spearman's rank correlation coefficients the untreated pasteurized milk than in any
were generated to see whether there was a
statistically significant correlation between
sensory scores and adjusted peak areas (Table
5 ) . The Spearman procedure was chosen be-
cause it is robust against nonlinear relation-
ships and outliers (7). The prescribed ranges
for weak, moderate, and strong correlations are
0 to f.5,f . 5 to .8, f.8 to 1, respectively. By
these criteria, the correlations for skim milk

TABLE 5. Spearman's correlation coefficients for average

sensory scores on adjusted peak areas for sulfur volatiles.
Skim Whole 10 20 30
milk milk
Heating time at 75°C (rnin)
.75 .25
.60 .03 Figure 7. Comparison of the cooked flavor score (A)
to the dimethyl sulfide concentration).( for skim milk.
lDimethyl sulfide. Error bars correspond to 1 SE.

Journal of Dairy Science Vol. 75, No. 8, 1992

2104 CHRISTENSEN AND REINECCIUS
other sample. Except for an increase at approx-
imately 5 min, which may or may not be
significant, the overall trend is of decreasing
amounts of DMS on further heating.
CONCLUSIONS
In conclusion, the headspace method used
in this research, which requires no sample
preparation, had adequate sensitivity for this
study, evidenced by the ability to detect H 2 S
and DMS in pasteurized milks that did not
have a noticeable cooked flavor. Also, there
was a moderately strong correlation between
the level of H 2 S and the intensity of the
cooked flavor in skim milk. Sulfur volatiles
were maximal after moderate heat treatments
and then decreased or remained the same on
more severe heat treatment. This might limit
their usefulness in a quality control setting
because a mild heated flavor is not a severe
defect.
There was no correlation between GC and
sensory data for milk that contains fat. The
milk fat could have been acting as a competing
solvent for the flavor volatiles, lowering their
vapor pressure below the detectable limits of
the analytical system.
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