I=resenius' Journal of
Fresenius J Anal Chem (1993) 345:420-423
Separation of bromide, bromate, iodide, iodate, nitrite, nitrate
and selenite anions by capillary zone electrophoresis
M. B. Amran 1, M. D. Lakkis 1, F. Lagarde 1, M. J. F. Leroy 1, J. F. Lopez-Sanchez 2., and G. Ranret 2
1Laboratoire de Chimie Min6rale et Analytique, URA 405 du CNRS, EHICS, 1 rue Blaise Pascal, F-67008 Strasbourg Cedex, France
2 Departament de Quimica Analitica, Universitat de Barcelona, Av. Diagonal 647, E-08028 Barcelona, Spain
Received December 23, 1991;revised June 11, 1992
Summary. Capillary zone electrophoresis (CZE) has been
used for the separation of bromide, bromate, iodide, iodate,
nitrite, nitrate and selenite anions. The separation was achie-
ved using a fused silica capillary (72 cm long x 50 gm i.d.)
filled with an acidic phosphate buffer (pH = 3, 25 mmol/1),
and with on-column UV detection at 200 nm. The influence
of different experimental parameters such as pH, ionic
strength and voltage, was studied. The nitrate concentration
of a Rhine water sample was then determined under selected
conditions and the results were compared to those obtained
by high performance ion chromatography (HPIC).
Introduction
Capillary zone electrophoresis (CZE) is a very powerful tech-
nique for the separation of ionic substances, and extensive
reviews have been recently published [1-6]. The use of nar-
row diameter capillaries (50-75 gm i.d.) leads to an efficient
dissipation of heat and suppresses convection, whereas the
high voltages applied cause fast separations with high effÉ-
ciency. The separation mechanism in CZE is based on diffe-
rences existing between the electrophoretic mobilities of
charged species in the presence of an electroosmotic flow.
Therefore, separation depends on the structural properties of
the solutes (size, shape and charge), and is also affected by
the physico-chemical properties of the carrier electrolyte,
such as pH, ionic strength, viscosity and dielectric constant.
Generally, CZE is applied to biochemistry (separation of
nucleotides, peptides, proteins and other organic com-
pounds). However, many inorganic ions such as iodate and
periodate [7], metal ions [8, 9], hexacyanoferrate (II) and
(III) ions [10], alkaline, alkaline-earth and ammonium ions
[11] have been separated by CZE. The recent developments
* On leave from the Universitat de Barcelona (postdoctoral fellow-
ship from ECC-BCR).
Correspondence to: M. J. F. Leroy
@ Spnnger-Verlag 1993
in electrochemical detectors [12-15] for CZE open good per-
spectives for the analysis of electrochemical active species. In
this work the separation of bromide, bromate, iodide, iodate,
nitrite, nitrate and selenite anions is presented. The
influence of different experimental parameters such as pH,
ionic strength, voltage, injected sample volume, etc., is stu-
died. Finally the nitrate concentration in a River Rhine water
sample is determined and the results compared with those
obtained by high performance ion chromatography (HPIC).
Experimental
Capillary electrophoresis
A capillary electrophoresis system from Applied Biosystems
(Foster City, CA, USA) model 270 A was used. A 72 cm long
x 50 g m i.d. fused silica capillary, filled with a phosphate
buffer, served as the separation column operating at high
voltages (10-30 kV), using a cathodic injection/anodic detec-
tion scheme. A small section of the polyimide capillary coat-
ing was removed to get an optical window for UV detection
at 50 cm from the injection site. Detection was carried out at
200 nm with a rise time of 0.5 s. The column temperature was
set at 40°C and samples were injected by the vacuum techni-
que. All the electropherograms were recorded using a Spec-
tra-Physics (San Jose, CA, USA) Chromjet integrator.
Ion chromatography
The chromatographic system used in this work consisted of a
Dionex Corporation (Sunnyvalley, CA, USA) model 4500i
ion chromatograph equipped with a pulsed electrochemical
detector (used in conductometric mode), a guard column
(AG4A), an analytical column (AS4A), and a membrane
suppressor, all supplied by Dionex Corporation. The flow-
rate of the mobile phase, composed of 0.0018 mol/L Na2CO3
and 0.0017 mol/L NaHCO3, was 2 mL/min. The chromato-
grams were recorded on a Spectra Physics model SP4270
integrator.
 42;[

:!
Reagents 10

Commercially analytical grade (Prolabo, France) sodium or

potassium salts of bromide, bromate, iodide, iodate, nitrite,
nitrate, selenate and selenite anions were used without fur-
ther purification. Water used for dilutions or for the prepara- 4
tion of buffer solutions was HPLC grade (Fisons). The phos-
phate buffers were prepared by mixing various proportions 2 (A)
of stock solutions of 0.025 mol/L H3PO4, 0.025 mol/L fo
NaH2PO4 and 0.025 mol/L Na2HPO4 filtered through a
0.65 # m cellulose acetate filter (Millipore). J8

1:
E
o

Results and discussion

x
Effect of pH on the separation
(B)
I
The pH value is an important parameter for a CZE system --t3--BROMIDE
because it governs the ionization degree of ionic compounds IODIDE
and the value of the electroosmotic flow, by changing the NITRATE
-~- NITRITE
charge of the capillary surface. So the apparent mobility is 6~ -~- 8ROMATE
highly dependent on the pH. It can be calculated by the ~-- IODATE
expression ~tapp = Ld" Lt/V" tm where Ld is the total length of ~ L E N I T E I
the capillary (in cm), L t is the capillary length from the inlet 2~
to the detection window (in cm), V is the applied voltage (in (c)
Volt) and tm the observed migration time (in seconds) [2, 16]. oL
2 4 6 8 pH
Three different experiments were carried out in order to
determine the effect of the pH on the apparent mobilities: Fig. 1. Effect ofpH in apparent mobility (#app)-(A): washing step with
a) variation of the pH by a preliminary washing step with 0.1 mol/L HC1, (B): no washing step, (C): washing step with 0.1 tool/
0.1 mol/L HCI, b) variation of the pH without a preliminary L NaOH. Working condition: capillary 72 cm long x 50 #m i.d.;
25 mmol/L phosphate buffers; T = 40°C; 20 kV applied voltage; and
washing step, and c) variation of the pH with a preliminary injected volume of 15 nL
washing step with 0.1 mol/L NaOH. In the experiments pre-
ceding the washing step the capillary was flushed with the
3
reagent solution for 5 rain, then flushed with the appropriate
buffer solution for 10 min, and the sample was then injected. 1
The procedure was carried out two times for each pH value.

5l
S
In the experiments without the washing step the capillary
was equilibrated overnight with the appropriate buffer solu-
tion. Then the sample was injected until reproducible results
were obtained. Between the injections the capillary was
flushed with the appropriate buffer solution for 10 rain. r ] r - - m q r i
' ; ; '~0 1, 1
3 5 3 5 4 6
The results obtained for these experiments are presented t (rain) t (rain) (min) t (mini t (mini
in Fig. 1. Generally, we can observe that apparent mobilities pH=5.99 pH=7.00
pH=2.98 pH=4.02 pH=5.05
decrease when the pH of solution increases. This fact can be
explained taking into account that the electroosmotic flow is Fig. 2. Effect ofpH on separation. Solutes are 10 mg/L of each bro-
opposite to the electrophoretic mobility (for an anion) and mide (1), iodide (2), nitrate (3), nitrite (5) and 50 mg/L ofselenate (4).
increases with increasing pH values. For anions of low elec- Working conditions as in Fig. 1
trophoretic mobility (as selenite) we found that they migrate
to the cathode at pH values greater than 5. For nitrite and
selenite anions a maximum value of gapp is observed, the time of analysis increases, efficiency decreases and only
because their respective PKa values [PKa (nitrite) = 3.37; PKa] nitrate, nitirite, bromide and iodide anions migrate in times
(selenite) = 2.46] lies in the pH range under study, and the lower than 20 min. Finally, we decided to use a washing step
variation of the pH modifies both the electrophoretic with NaOH, because we observed a lower background to
mobility (by changing the ionization degree) and the signal ratio in comparison to other pretreatments.
electroosmotic flow.
On the other hand the effect of capillary preconditioning Effect of phosphate buffer concentration
is also observed; the apparent mobilities observed for each Apparent mobilities for four buffer concentration (10, 15, 20
anion are greater by using acid than basic washing solutions, and 25 retool/L) were determined. No significant effect was
and an intermediate response is obtained when only a buffer observed in the concentration range studied.
solution is used. This can be explained by the pH hysteresis
effect previously described by Lambert and Middleton [17]. Effect of applied voltage
For further experiments pH = 3 was selected, because the
separation is good and is not affected by preconditioning We have observed that the increase of voltage from 10 to
steps. However bromide and iodide separation is only achie- 30 kV does not modify significantly the apparent mobilities
ved at high pH values (Fig. 2), under these conditions and the resolution. On the other hand, the time of analysis
422

decreases whereas the b a c k g r o u n d signal increases, there-
fore we decided intermediate voltage o f 20kV for further
experiments.
Effect of injected sample volume
Table 1. Short and long term reproducibility of migration time for five
nonconsecutive days. Working conditions: capillary 72 cm long x 50
gm i.d.; 25 mmol/L phosphate buffer, pH = 3.0; T = 40°C; 20 kV
applied voltage; injected volume of 15 nL, and sample composition:
10 mg/L of each bromide, iodide, nitrate, nitrite and iodate, 25 mg/L
of both bromate and selenite

It appears that the injected sample v o l u m e (according to the

Day Br- I- NO3- NO2- B r O 3 - I O 3 - SeO32-
v a c u u m technique, and calculated from the Poiseuille equa-
tion) can be increased up to 45 nL without great loss o f effici-
ency and resolution. Analysis time slightly increases where 1 xa 3.58 3.63 3.98 9.26 5.24 7.38 11.10
the sensitivity is improved, allowing determinations in the %RSD b 1.13 1.02 1.02 2.46 0.99 0.93 1.59
m g / L range. Figure 3 shows the electropherogram o b t a i n e d 2 xa 3.61 3.66 4.01 8.68 5.28 7.46 10.47
when a standard sample is injected u n d e r the selected condi- %RSD b 1.26 1.24 1.10 1.97 0.99 0.57 0.86
tions.
3 xa 3.61 3.66 4.02 8.94 5.29 7.49 10.97
%RSD b 1.21 1.11 1.04 3.46 0.88 0.90 2.26
Reproducibility (Precision)
4 xa 3.51 3.55 3.94 9.83 5.17 7.33 11.50
The precision was calculated using a standard solution con- %RSD b 0.56 0.60 0.50 1.26 0.41 0.27 0.46
taining 5 m g / L nitrate, 10 m g / L bromide, iodide, iodate and
5 xa 3.57 3.62 4.00 9.80 5.25 7.43 11.53
nitrite, and 25 m g / L b r o m a t e and selenite. F o r short t e r m %RSD b 0.67 0.69 0.56 1.48 0.46 0.25 0.42
precision this solution was injected six times and the per-
centage relative standard deviation (% RSD) was calculated xc 3.57 3.62 3.99 9.30 5.24 7.42 11.17
from the migration time and the ratio o f peak areas analyte to %RSD d 1.44 1.43 1.07 5.42 1.09 0.98 2.95
bromate. F o r long term precision the same p r o c e d u r e was
carried out on five non-consecutive days (27 days period), a Day mean (six values); b Day relative standard deviation; c Between
and the overall % R S D was calculated from the thirty values day mean (thirty values); a Between day standard deviation
obtained. In Tables 1 and 2 the m e a n and calculated % R S D
values are shown. T h e % RSD values obtained for nitrite and Table 2. Short and long term reproducibility for the analyte peak area
selenite are significantly higher. Both anions have their re- to bromate peak area ratio for five nonconsecutive days. Working
spective pKa close to the working pH. Therefore, slight varia- conditions as in Table 1
tions o f p H can change their ionization state and conse-
quently their electrophoretic mobilities. Day Br- I- NO3- NO2= B r O 3 - I O 3- SeO32-

Determination of nitrate anions in a real sample 1 xa 2,57 1.09 1.47 1.98 0.97 1.36
°/0RSD b 0,98 4.27 1.17 1.77 2.66 1.50
The nitrate concentration in a Rhine water sample collected
at Strasbourg was d e t e r m i n e d by C Z E and by HPIC. T h e 2 xa 2.55 1.09 1.48 1.90 0.95 1.29
%RSD b 0.84 2.22 2.31 3.73 1.30 0.76
3 xa 2.59 1.08 1.44 1.92 0.94 1.30
3 %RSD b 1.53 1.99 1.04 2.62 1.35 2.26
4 xa 2.56 1.14 1.45 2.10 0.93 1.40
%RSD b 2.72 2.54 1.79 1.95 1.43 2.74
5 xa 2.63 1.08 1.47 2.08 0.94 1.40
°/0RSD b 1.48 2.64 0.83 1.22 1.11 0.42
xc 2.58 1.10 1.46 1.99 0.95 1.35

l
0.005 AU
'
t (mira)
°
°/0RSD d 1.99 1.43 1.07 4.78 1.85 3.85

a Day mean (six values); b Day relative standard deviation; c Between

day mean (thirty values); a Between day standard deviation

sample was filtered through a 0.45 g m acetate cellulose filter

0 3 6 9 12 15
time (rain)
Fig. 3. Electropherogram of a standard sample composed by 10 mg/L
of each bromide (1), iodide (2), nitrate (3), nitrite (5) and iodate (7), 25
mg/L of both bromate (6) and selenite (8) and 50 mg/L ofselenate (4).
Reprocessed electropherogram at higher chart speed is placed in the
inset. Working condition: capillary72 cm long x 50 gm i.d.; 25 mmol/
L phosphate buffers; pH = 2.9; T = 40~C; 20 kV applied voltage; and
injected volume of 15 nL
(Millipore) to eliminate s u s p e n d e d matter. T h e electro-
p h e r o g r a m s obtained by C Z E and the c h r o m a t o g r a m s
obtained by H P I C for a standard sample, the Rhine water
sample, and the spiked sample with standard are shown in
Figs. 4 and 5, respectively. In the electropherogram o f the
water sample with a d d e d standard a very large peak migrates
before nitrate peak. This is caused by the chloride ions
present in the sample as it is shown in Fig. 6. T h e chloride
concentration increases the peak width for b r o m i d e and
iodide and this can interfere with the nitrate peak for high
chloride contents.
 423

1,2
3 O.OlOAU
1 1
0.010 AU

1
6 T 5 8
6;7,"
1
A B
,-...-
i , / i i I . i , i

1A
0 4 8 12 I
0 4 8 12 8 12 I
t (rain) t (min) t (min)

Fig. 4. Electropherograms of standard mixture (A), Rhine water sam-

ple (B), spiked sample with standard mixture (C). All working conditi- r r - -
ons as in Fig. 3 3 4 ; 4 ; ;-; 4 ; 4 3 3 4
t (min) t (rain) t (rain) t (min) l (rain) t (min) t (mln)

Fig. 6. Effect of chloride concentration in separation. A: no CI-;

B: 10 mg/L C1-; C: 25 mg/L C1-; D: 50 mg/L C1-; E: 100 mg/L C1-;
T
2.5 #S F: 500 mg/L C1-; G: solution containing only 500 mg/L C1-. Peaks are
bromide (1), iodide (2) and nitrate (3)
1

ab 1 and the efficiency of the separation o f different anions has

c been improved by optimizing the different experimental
parameters. Finally, under optimum conditions the determi-
nation o f nitrate in river water sample with CZE at ppm level
L I yields comparable results to those obtained with HPIC.
13
o ~ 6' r ~ 6' i
0 3
time (rain) time (rain) time (min)
Acknowledgments. Dr. J. F. Lopez-Sanchez thanks the Commission of
Fig. 5. Chromatograms of standard mixture (A), Rhine water sample the European Communities for the grant received for his stay in
(B) and spiked sample with standard (C). Peaks are fluoride (a), chlo- Strasbourg during the year 1991.
ride (b), nitrate (c), phosphate (at), sulfate (e) and bromate (/)

For both techniques the nitrate concentration was deter-
mined by the calibration plot m e t h o d and the standard addi-
tion method. The use of an internal standard has been
r e c o m m e n d e d for CZE quantitative measurements when
vacuum technique is used for injection [18], in order to
remove all systematic variations.
Thus, for CZE measurements, we have used bromate as
internal standard. The nitrate concentration obtained by
CZE and H P I C is 6.4 m g / L and 6.8 m g / L for the calibration
plot method, 5.7 m g / L and 6.3 m g / L for the standard addi-
tion m e t h o d (each value is the mean of the result for the
three injections), respectively. The nitrate concentration
level found is comparable to data presented for nitrate levels
in different French rivers [19].
Conclusions
CZE is a powerful and flexible separation technique for the
separation o f inorganic anions, and provides complementary
information to HPIC determinations because the separation
mechanisms o f both techniques are different. The selectivity
References
1. Ewing AG, Wallingford AR, Olefirowicz TM (1989) Anal Chem
61:292A-303A
2. Gareil P (1990) Analusis 18:221-241
3. Gareil P (1990) Analusis 18:447-468
4. Schomburg C (1990) Chromatographia 30:500-508
5. Kuhr WG (1990) Anal Chem 62:403R-414R
6. Yeung ES, Kuhr WG (1991) Anal Chem 63:275A-282A
7. Honda S, Suzuki K, Kakechi K (1989) Anal Biochm 177:62-66
8. Tsuda T, Nomura K, Nakagawa G (1983) J Chrotogr 264:385-392
9. Swaile DF, Sepaniak MJ (1991) Anal Chem 63:179-184
10. Aguilar M, Huang X, Zare RN (1989) J Chromatogr 480:427-431
11. Yeung ES, Gross L (1990) Anal Chem 62:427
12. Wallingford AR, Ewing AG (1987) Anal Chem 59:1762-1766
13. Huang X, Pang TKJ, Gordon MJ, Zare RN (1987) Anal Chem
59:2747-2749
14. Huang X, Zare RN, Sloss S, Ewing AG (1991) Anal Chem
63:189-192
15. Huang X, Zare RN (1991) Anal Chem 63:2193-2196
16. Jorgenson JW, Luckas KD (1983) Science 222:266-272
17. Lambert, JW, Middleton, DL (1990) Anal Chem 62:1585-1587
18. Dose EV, Guiochon, GA (1991) Anal Chem 63:1154-1158
19. Fustec E, Schenck C, Cloots-Hirsch AR, Soulie M, Bouton D,
Ackerer P, Pinay G, Tournoud MG (1991) Les nitrates dans les
vall6es fluviales. Report of CNRS, Paris