ANALmcA

CHIMICA
ACTA
ELSEVIER Analytica Chimica Acta 302 ( 1995) 45-52

Indirect conductometric detection of amino acids after liquid

chromatographic separation
Part II. determination of monosodium glutamate in foods
Oi-Wah Lau *, Chuen-Shing Mok
Department of Chemistry, The Chinese University of Hong Kong, Shatin, Hong Kong

Received 31 January 1994; revised manuscript received 9 August 1994

Abstract

A liquid chromatographic (LC) method using indirect conductometric detection is proposed for the determination of mono-
sodium glutamate in foods, where a conductivity detector is used for detection. It involves the use of a 5-pm Econosil CN
column as the stationary phase with a mixture of water, acetonitrile and tetrahydrofuran containing 1 mM perchloric acid or
trichloroacetic acid as the mobile phase. Pre-column or post-column derivatization of the amino acid to increase the sensitivity
of detection is not required. The proposed method employs a short Dowex SOW-X8 (H+ ) column for the sample clean-up prior
to LC determination. Freezedrying is used for pre-concentration and the residue is redissolved in the LC mobile phase before
measurement. The peak due to glutamic acid was well separated from the solvent peak and those from other amino acids or food
components. The calibration graph is linear from 0 to 500 pg/ml of sodium glutamate. The R.S.D. for lo-replicate measurements
of the sodium glutamate content of an oyster flavoured sauce containing 3.00% (m/m) of the compound was 1.8%. The proposed
method has been applied to determine monosodium glutamate, down to the low level of 0.007% (m/m), in a variety of food
samples.

Ke.words: Liquid chromatography; Conductimetry; Foods

1. Introduction the use of an amino acid analyser [ 111. The paper

chromatographic method [8] is not precise. The gas
chromatographic determinations for glutamate (in the
Monosodium glutamate (MSG) is commonly used
form of glutamic acid) require derivatization of glu-
as a flavour enhancer in foods, particularly with salts
tamic acid before measurement as the acid, like other
in meat products, sauces and soups. There are concerns
amino acids, lacks the volatility required for the gas
on the amount of MSG added to foods as large amounts
chromatographic (GC) methods. Liquid chromato-
of MSG may result in certain physiological effects [ l] .
graphic (LC) determinations or determinations using
A number of methods have been proposed for the
an amino acid analyser are common methods, but these
estimation of MSG in foods, which include titrimetric
also require post-column or pre-column derivatization
[ 2,3] and fluorimetric [ 41 methods, gas [ 5-71, paper
of glutamic acid, because the acid lacks functional
[ 81 and liquid [ 9,101 chromatographic methods, and
groups which have strong enough UV absorptivity or
* Corresponding author

0003-2670/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved

SSDIOOO3-2670(94)00423-4
46 O.-W. Lau, C.-S. Mok I Analytica Chimica Acta 302 (1995) 45-52

exhibit fluorescence. Most procedures requiring deri- tetrahydrofuran (77:20:3) containing 1 mM trichlo-
vatization are tedious. roacetic acid. Instrumental settings: flow rate, 1 ml/
The current AOAC titrimetric method [ 21 is based min; column temperature, 30°C; detector zero suppres-
on the method proposed by Fernandez-Flores et al. [ 31 sion, 2; detector range, 1 or 10; and chart speed, 0.5
The glutamic acid content is estimated by titration with cm/min.
sodium hydroxide in the presence of formaldehyde The peaks were detected as negative changes in con-
after extraction and clean-up with an ion-exchange col- ductance, and the detector-integrator connections were
umn. However, the titrimetric method cannot deter- reversed in polarity to give positive display of peaks
mine trace amounts of MSG in foods and cannot on the integrator.
indicate cross-contamination from other amino acids. Freezedryer (Model Freezemobile 6 from Virtis)
Subsequent to our findings on the determination of was used for preconcentration.
amino acids with LC using indirect conductometric
detection as detailed in Part I [ 121, an application of 2.2. Reagents
the method is now proposed for the determination of
monosodium glutamate in foods. Unlike most other LC
LC grade solvents were used to prepare the mobile
methods for amino acids, pre-column or post-column
phase. All other chemicals were of analytical reagent
derivatization of the amino acid to increase the sensi-
grade. The water used was distilled, and deionized by
tivity of detection is not required for the method and
passing through a Millipore Milli-Q 50 Ultrapure water
the analysis time is significantly reduced. The method
system.
employs a short Dowex 5OW-X8 (H + ) column for the
sample clean-up as proposed by Coppola et al. [ 41 prior
to LC determination. The method uses freezedrying for 2.3. Standard solutions
pre-concentration, and the residue is redissolved in the
LC mobile phase before measurement. Monosodium glutamate standard solutions were pre-
The proposed method has been applied to determine pared by dissolving the compound (BDH, minimum
monosodium glutamate, down to the low level of assay 99%) in water, and diluted with the mobile phase
0.007% (m/m), in a variety of prepared foods. The to the required concentrations.
results obtained using this method were compared with
those obtained by LC determination after derivatization
2.4. Sample preparation
with dansyl chloride [ 91.

The sample preparation procedure was based on the

2. Experimental method described by Fernandez-Flores et al. [ 31.

2.1. Apparatus Non-starchy food samples

Liquid samples were homogenized by shaking,
The LC system used consisted of a Beckman 1lOB while solid or semi-solid samples were homogenized
solvent delivery pump with an Alltech Free-Flow pulse in a blender. Approximately 20 g of each sample were
dampener, a Wescan conductivity detector (Model weighed accurately into a 250-ml beaker and 100 ml
21511001) with a temperature controller (Model of water were added. The mixture was shaken in an
24020001) and column compartment (Model ultrasonic bath for 15 minutes, and then about 6 g of
26650051), a Rheodyne injection valve with a 20-~1 activated charcoal were added. The mixture was let
sample loop, and a Beckman 427 signal integrator. An stand for half an hour with occasional agitation, and
analytical column from Alltech Associates, 5 pm Econ- was then filtered through a 12.5-cm Whatman No. 542
osil CN (250 mm X 4.6 mm i.d.) cartridge, was used. filter paper into a 200-ml volumetric flask. The residue
The following mobile phases were used: (i) Water- was washed with several portions of water. The filtrate
acetonitrile-tetrahydrofuran (77:20:3) containing 1 and washings were combined and diluted to volume
mM perchloric acid; and (ii) Water-acetonitrile- with water.
 O.-W. Lau, C.-S. Mok IAnalytica Chimica .4cta 302 (1995) 45-52

Starchy food samples

Approximately 20 g of the starchy food sample,
homogenized as described above, was weighed accu-
rately into a 250-ml beaker containing 100 ml of ace-
tone-water ( 1: 1) . The solution was shaken for 15 min
in an ultrasonic bath, and then about 6 g of activated
charcoal were added. The mixture was let stand for half
an hour with occasional agitation, and was then filtered
through a 12.5 cm Whatman No. 542 filter paper into
GIU
a 500-ml beaker. The residue was washed with six
portions of 25 ml of the acetone-water mixture. The 1
filtrate and washings were combined followed by the
addition of two drops of 4 M hydrochloric acid. The
mixture was evaporated to about 40 ml and was then
transferred to a 200-ml volumetric flask and made up
to volume with water.

2.5. Sample clean-up with an ion-exchange column

A short column (6.5 cm X 0.9 cm i.d.) of Dowex

5OW-X8 (H+ ) ( 100-200 mesh) used for the sample
clean-up was first cleaned by passing 100 ml of 4 M
hydrochloric acid followed by 100 ml or more of water
until the eluent gave a negative test with silver nitrate.
Ten ml of sample solution were transferred quantita-
tively and slowly to the column and with the eluent
I I I
flow rate adjusted to 0.5 ml/min. The column was 0 5 10
washed with 5 ml of water, followed by 10 ml of 0.8
Time (min.)
M hydrochloric acid (with the flow rate maintained at
Fig. I. Chrumatogram of 1.2 pg of monosodium glutamate with
0.5 ml/min) which, as reported by Fernandez-Flores
indirect conductometric detection. Mobile phase, water-acetoni-
et al. 131, would elute serine, threonine and aspartic
trile-tetrahydrofuran (77:20:3). 1 mM HCIO,. MSG peak at 7.X
acid. Then 18 ml of 1 M hydrochloric acid were added min.
( with flow rate adjusted to 1.5 ml/min) to elute mon-
osodium glutamate, in the form of glutamic acid, from solution was diluted to 100 ml with water. The rest of
the column, and 5 ml of water were added to wash the the procedure as described in Ref. [ 9 1 was followed.
column. The eluate was collected into a lOO-ml volu-
metric flask and diluted to volume with water. Ten ml 2.6. Quantitative determination of monosodium
of the solution were freezedried in a polypropylene glutamate in food samples
bottle and then redissolved in 10 ml of the mobile phase
for LC determination. Additional dilution with the The amount of monosodium glutamate in each sam-
mobile phase was necessary for solutions outside the ple was determined in triplicate by injecting 20 ~1 of
calibration range. For counter checking with LC using the sample solution prepared as described above into
dansylation, a separate portion of the solution obtained the chromatograph.
from sample preparation was passed through the ion- The calibration graph was obtained by plotting the
exchange column and eluted in the same way as peak area (arbitrary units) against the concentration of
described above except that the final eluate was neu- the respective standard. The concentration of mono-
tralized with sodium hydroxide to pH 7.0 before the sodium glutamate in the sample solution was deduced
48 O.-W. Lau, C.-S. MokiAnalytica ChimicaActa 302 (1995} 45-52
Glu
I
Time
Fig. 2. Chromatogram of monosodium glutamate from a soup base sample. Conditions as for Fig. 1.
from the regression equation of the calibration graph,
and its content in the sample was then calculated.
3. Results and discussion
Since the theoretical aspect of the proposed method
has previousIy been discussed in detail [ 121, it is not
elaborated here. The present work puts into practice
the concept of indirect conductometric detection in the
determination of monosodium glutamate in foods and
the technical aspect is discussed below. It is noted that
monosodium glutamate was converted into glutamic
acid after passing through the ion-exchange column in
the clean-up step.
(min.)
3.1. Ionic strength of solutions for LC measurement
The ionic strength of the sample solutions for the
proposed method needs to be kept low otherwise the
conductivity detector would be saturated. Thus the
hydrochloric acid present in the eluent must be removed
before LC measurement. Freezedrying of the sample
prior to LC determination has been found in the present
study to be effective in removing hydrochloric acid
from the sample solution. Further, monosodium glu-
tamate was very soluble in the mobile phase and the
recovery of the compound was found to be 95-100%.
This means that the residue obtained after freezedrying
can be reconstituted with the mobile phase in any suit-
able volume, and hence, freezedrying can significantly
 O.-W. Lau, C.-S. Mok J Analytica Chimica Acta 302 (1995) 45-52
I 1
0 10
Fig. 3. Chromatogram of monosodium glutamate
lower the concentrations of monosodium glutamate in
foods to be determined by the method.
3.2. Interferences from other amino acids
The glutamic acid peak was well separated from the
solvent peak (Figs. l-3) and there was baseline sepa-
ration of glutamic acid from the neighbouring peaks
due to other amino acids, namely, L-threonine, glycine
and L-glutamine [ 121. The retention times of these
amino acids differ from that of glutamic acid by more
than 0.5 min when measured using the proposed chro-
matographic conditions. Small amounts of other amino
acids, namely aspartic acid, serine, glycine, alanine and
valine, when coeluted with glutamic acid can be easily
I I I
20 30 40
Time (min.)
(approximately 0.12 pg) from a baby food sample. Conditions as for Fig. 1.
identified by their respective retention times. However,
even with around 0.1 pg of monosodium glutamate
(Fig. 3)) the glutamic acid peak was well resolved from
the nearest peak due to the coeluted amino acids.
A large peak started to appear around 26-28 min for
all the food samples determined, however, it was well
separated from the glutamic acid peak (Figs. 2 and 3)
and hence did not interfere with the determination. Fur-
ther, the peak was not observed for the standards, and
the intensity of this peak was found to vary from sample
to sample. Presumably it was caused by some food
components coeluted from the clean-up column with
glutamic acid. Apart from this, no other peaks were
observed close to the glutamic acid peak. Hence, for
the food samples examined, there were negligible inter-
50 0. W. Lay C. -S. Mok 1Analytica Chimica Acca 302 (I 995) 45-52

Table 1
Description of the food samples

Sample Description Label ingredients

1 Soup base (Brand 1) Chicken, salt, sugar, hydrolysed plant protein, spices, modified starch, preservatives.
2 Soup base (Brand 2) Ingredients were not declared
3 Soup base (Brand 3) Salt, monosodium glutamate, hydrolysed vegetable protein, spices, prawn flavouring.
4 Baby food Water, chicken, carrots, rice, potatoes, modified cornflower, dried skimmed milk,
vegetable oil, gelatin, tomato puree, yeast extract, iron sulphate, herbs, vitamin B1
5 Oyster extractives Oyster extractives.
6 Oyster flavored sauce (Brand 1) Water, sugar, salt, oyster extractives, corn starch, hydrolysed vegetable protein, caramel.
7 Oyster favored sauce (Brand 2) Oyster extractives, salt, sugar, starch, caramel.
8 Spice sauce Water, soy, monosodium glutamate, spice, salt, caramel, wine, sugar, sorbic acid.
9 Pistachios Pistachios, salt.

ferences from other amino acids and substances co-
eluted from the column.
3.3. Calibration graphs
Calibration graphs were plotted employing respec-
tively the two mobile phases described in the Experi-
mental section. The mobile phases differed only in the
acid which they contained, the acids being perchloric
acid and trichloroacetic acid, respectively. The linear
concentration range of sodium glutamate was found to
be from 0 to 500 pg/ml, equivalent to 0 to 10 ,ug of
monosodium glutamate in 20 ~1 of the sample solution.
The correlation coefficients for the calibration graphs
were found to be larger than 0.9995, indicating a linear
relation between peak area and concentration.
Table 2
Comparison of the results obtained using the proposed method with
mobile phase (i) (method (i) ) and mobile phase (ii) (method (ii) )
with those using the LC method with dansylation (LC method)
3.4. Precision and recovery tests
The precision of the method was studied by meas-
uring the content of monosodium glutamate of an oys-
ter flavoured sauce ten times by the proposed method.
The average content was found to be 3.00% (m/m)
with a relative standard deviation (R.S.D.) of 1.8%,
which indicated the high degree of precision of the
proposed method.
The R.S.D. for the determination of monosodium
glutamate near the limit of detection using the proposed
method was around 14%.
The reliability of the method was studied by per-
forming recovery tests on a standard monosodium glu-
tamate solution containing 108 pg/ml of the salt, where
the peak intensity of the standard solution was meas-
ured after running through the whole test procedure and
compared with that obtained by injecting the standard
solution directly into the chromatograph. The mean
recovery of four analyses for added glutamate was
96.1%.

LC method LC method Proposed

3.5. Determination of monosodium glutamate in
vs. proposed vs. proposed method (i)
method (i) method (ii) vs. proposed foods
method (ii)
The proposed method was applied to determine mon-
Mean difference, d 0.2 0.2 0.01 osodium glutamate in various food samples. A total of
S.D. of the 0.406 0.430 0.129
nine food samples were analysed. The ingredients of
differences, s,,
Calculated t value 1.48 1.39 0.23 the food samples as declared by the manufacturers are
(r=&hls,) described in Table 1. The results of analysis using the
Critical 1 value for 2.31 2.31 2.31 proposed method and an established LC method after
8 degrees of derivatization with dansyl chloride using spectropho-
freedom and
tometric detection [ 93 are shown in Table 3. The paired
P = 0.05
t-test [ 131 has been applied to compare the proposed
 O.-W. Lau, C.-S. Mok IAnalytica Chimica Acta 302 (1995) 4%S2 Sl

Table 3
Determination of monosodium glutamate in food products with mobile phases: ( i) water-acetonitriie-tctrahydrofuran C77:X):3). ! mM in
HCIO, and (ii) water-acetonitrile-tetrahydrofuran (77:20:3), 1 mM in CCl,COOH

Sample Description Proposed Method (%, m/m) 1-C method with dansylation (“/r )

Mobile phase (i) Mobile phase (ii)

Found Mean ” Found Mean L’

Soup base (Brand 1) 0.71 0.71 0.73 0.74 0 74

0.72 (0.8) 0.73 ( 1.h)
0.71 0.75
Soup base ( Brand 2) 16.3 16.3 16.1 16.5 173
16.3 (0.4) 16.8 ( 2.3)
16.4 1h.7
Soup base (Brand 3) 17.5 17.2 16.‘) 18.0
16.9 (1.8) 17.1
17.1 16.X
Baby food 0.007 0.007 O.OOh 0.007 0.007
0.007 (7.9) 0.007 (14.3)
0.008 0.008
Oyster extractives 0.41 0.41 0.41 0.42 0.40
0.40 (1.4) 0.1 I 12.X)
0.4 I 0.43
Oyster flavored sauce (Brand 1) 0.32 0.3 I 0.37 0.33 0.33
0.31) (3.2) 0.34 (3.5)
0.31 0.32
Oyster favored sauce (Brand 2) 3.0 I 3.03 2.YS 3.00 ?.XY
3.0x (1.4) 2.03 (3.3)
3.00 3.11
Spice sauce 1.77 1.76 1.75 1.71 I .x‘-l
1.74 (0.9) 1.74 (3.h)
1.76 I.64
Pistachios 0.068 O.OhY O.Oh’J O.Oh7 iLlIf>&
0.071 (3.0) O.Ohi (3.1)
0.067 O.Ohfl

d Mean of triplicate measurements in percentage by mass and relative standard deviation of the results ( 94)in parenthcsc\.

method with the established LC method [9] and the
calculated t values are shown in Table 2, where it can
be seen that they are both less than the critical t values
for the significance level P= 0.05, indicating that the
proposed method and the established LC method do
not give significantly different values for the mean
sodium glutamate content. In general, the mean results
obtained using the two methods agree to within 5%.
The precision of these determinations was found to be
very satisfactory as the R.S.D. values of triplicate anal-
ysis of the food samples are in general within 4%, with
the exception of a baby food sample, which contained
0.007% (m/m) of monosodium glutamate and the
R.S.D. was found to be 7.9% and 14.3% using mobile
phases (i I and (ii) respectively, which are high com-
pared with those obtained for the other samples.
The paired t-test has also been applied to compare
the proposed method using the two different mobile
phases. The calculated t value ( also included in Table
3) has a value of 0.23 and is much smaller than the
critical t value, indicating that no significantly different
results are obtained using the two mobile phases con-
taining different conducting species, and hence the
choice of conducting species for background conduc-
tance is rather flexible. The only requirement is that the
conducting species should be able to maintain a stable
background conductance.
52 0. - W.Lau, C.-S. Mok I Analytica Chimica Acta 302 (1995) 45-52

4. Conclusion References

A simple and accurate method has been developed

[l I H. Egan, R.S. Kirk and R. Sawyer, Pearson’s Chemical
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The method does not require derivatization of glutamic 1981, p. 426.
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vatization process such as completeness of the deriva-
[31 E. Femandez-Flores, A.R. Johnson and V.H. Blomquist, J.
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