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Trace Determination of Rhodamine B and

Rhodamine 6G Dyes in Aqueous Samples by
Solid-phase Extraction and High-performance
Liquid Chromatography Coupled with
Fluorescence Detection

Article · April 2012

DOI: 10.1002/jccs.201100318

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Detection of Rhodamine Dyes by HPLC-FLD J. Chin. Chem. Soc., Vol. 59, No. 1, 2011 1

Trace Determination of Rhodamine B and Rhodamine 6G Dyes in Aqueous

Samples by Solid-phase Extraction and High-performance Liquid
Chromatography Coupled with Fluorescence Detection

Tsung-Ling Chiang, Yu-Chen Wang and Wang-Hsien Ding*

Department of Chemistry, National Central University, Chung-Li 320, Taiwan, R.O.C.

Received May 30, 2011; Accepted October 17, 2011; Published Online October 25, 2011

A simple and reliable method was developed to detect two basic synthetic dyes, rhodamine B (RB)
and rhodamine 6G (R6G), in wastewater and surface water samples by high performance liquid chroma-
tography with fluorescence detection (HPLC-FLD). These dyes have been reported to be both mutagenic
and carcinogenic in various organisms. The contents of these two dyes in water samples were extracted by
Oasis HLB solid-phase extraction (HLB-SPE), and were then determined by an isocratic HPLC using an
AtlantisÒ T3-C18 column. Water samples at various pH conditions and the compositions of eluents for
SPE were evaluated. The results indicate that the proposed method is precise and sensitive in analyzing
these two basic synthetic dyes, and the limits of quantitation were 1.5 ng/L for RB and 0.3 ng/L for R6G in
100 mL of water samples. The recovery of analytes in spiked surface water and municipal wastewater
treatment plant (WWTP) effluent samples ranged from 61 to 90% with the precision (RSD) ranging from
2 to 12%. The concentrations of analytes were detected in various water samples ranging from 0.7 to 81
ng/L.

Keywords: Water analysis; Solid-phase extraction; Rhodamine B.

INTRODUCTION thetic dyes at trace-level for the first time in the environ-
Rhodamine B (RB) and rhodamine 6G (R6G) with mental water samples by HPLC coupled with fluorescence
the chemical structures shown in Fig. 1, are among the old- detector. The precision and recovery of the SPE methods in
est and most commonly used synthetic dyes. They are various conditions were evaluated, and the effectiveness of
widely used as color additive in cosmetic, food, pharma- the method in determining the concentrations of RB and
ceutical, and also used as colorant in textiles and plastic in- R6B in various environmental water samples was demon-
dustries.1-3 However, they may cause irritation to the skin, strated.
eyes and respiratory tract, moreover, the carcinogenicity,
reproductive and developmental toxicity toward humans
and animals have also been experimentally proven.4,5 Due
to their potential toxicity and the aesthetically unpleasant
color they leave behind in the water bodies, many investi-
gations have focused on the removal of these synthetic
dyes from wastewater treatment or from highly colored
wastewater containing hazardous industrial chemical efflu-
ent.6-13 Few examinations concerned about the concentra-
tion of these toxic synthetic dyes discharged directly into
the aquatic environment.
In this study, we developed a simple, sensitive and re-
liable method to routinely determine these two basic syn- Fig. 1. Structures of Rhodamine B and Rhodamine 6G.

* Corresponding author. E-mail: wanghsiending@gmail.com

2 J. Chin. Chem. Soc., Vol. 59, No. 1, 2011
EXPERIMENTAL
Chemicals and reagents
Unless noted otherwise, all chemicals and solvents
were obtained at high purity and used without further puri-
fication. Rhodamine B (RB), rhodamine 6G (R6G), formic
acid and phosphoric acid were purchased from Sigma-
Aldrich (Milwaukee, WI). Acetonitrile and methanol of
HPLC-grade were purchased from Tedia (Fairfield, OH).
Stock solutions of each analyte (1.0 mg/mL) and the mix-
tures of the analytes for working standard were prepared in
methanol. All stock solutions and mixtures were stored in
the dark at -4 °C. Deionized water was further purified us-
ing a Millipore water purification device (Billerica, MA).
Sample collection
Three sets of water samples were collected and used
to evaluate the method. Two municipal wastewater treat-
ment plant (WWTP) effluents (average specific conduc-
tance: 1200 mS/cm, pH 6.5) were collected from the An--
Ping community in the city of Tainan (Taiwan). This WWTP
performs mechanical clarification and flocculation filtra-
tion; population equivalent 380000. Two river water sam-
ples (average specific conductance: 440 mS/cm, pH 6.6)
were collected from a river located 500 m downstream
from the industrial effluents outlet of the detergent manu-
facturers located in Taichung County (Taiwan). Two ground-
water samples were collected directly from the wells in Na-
tional Central University, Taiwan (specific conductance:
220 mS/cm, pH 6.8). All samples were collected in dupli-
cates (500-mL for each) and shipped to the laboratory in
ice-packed containers. Upon arrival, the samples were im-
mediately passed through a 0.45-mm membrane filter
(Advantec MFS, CA), adjusted to pH 3.0 by adding con-
centrated HCl to depress microbial degradation, stored at 4
ºC, and the samples were analyzed within a week.
Sample preparation
Three commercially available Oasis SPE cartridges
were tested: HLB, mixed-mode cationic exchange (MCX)
and weak mixed-mode cationic exchange (WCX) SPE car-
tridges (3 mL, 60 mg, from Waters, Milford, MA). Before
extraction, each cartridge was sequentially pre-conditioned
with 2 mL of eluting solution, 3 mL of methanol, and then
rinsed by 3 mL of deionized water on an SPE manifold
(VacMaster, IT Sorbent Technology, Cambridge, UK). For
optimal procedure (see the Optimization of SPE section),
water sample 100-mL was passed through the HLB-car-
tridge at a flow rate of about 3-5 mL/min. When the extrac-
tion was completed, the cartridge was washed with 2 mL
Chiang et al.
5% methanolic solution, and subsequently air-dried under
vacuum for at least 20 min. The analytes were then eluted
from the cartridge by 3 mL of methanol mixed with 4%
(w/v) of formic acid. The extracts were then completely
evaporated to dryness by a stream of nitrogen. The residue
was then redissolved in 100 mL of deionized water (con-
taining 0.1% (w/v) phosphoric acid), and made ready for
HPLC analysis. To prevent HPLC column blockage, all
extracts were filtered through a glass-fiber filter prior in-
jection.
HPLC Analysis
Analyses were performed on a HP-1100 high-perfor-
mance liquid chromatograph system coupled with a time-
programming wavelength fluorescence detector (Agilent,
Palo Alto, CA). The excitation and emission wavelengths
were set at 535 nm and 580 nm for RB, respectively, and
515 nm and 554 nm for R6G. An AtlantisÒ T3-C18 column
(15 cm ´ 2.1 mm i.d., 3 mm packing, Waters, Milford, MA)
was used at a flow rate of 0.2 mL/min at ambient tempera-
ture (around 20 °C), and the injection volume was 10 mL.
Isocratic elution was performed by a 45% of acetonitrile
with 55% of 0.1% (w/v) of phosphate buffer (optimized,
see the HPLC separation section 3.1) for 16 min.
RESULTS AND DISCUSSION
HPLC separation
Since the properties of the mobile phase primarily
govern the separation of the analytes, optimizing the mo-
bile phase composition to obtain a chromatogram with
good resolution in a reasonable time is important in HPLC
analysis. Preliminary experiment was carried out on a com-
monly used C-18 column (Zorbax XDB-C18; 15 cm ´ 2.1
mm i.d., 3.5 mm packing, Agilent) with 50% of 0.1% (w/v)
phosphate buffer in acetonitrile as the mobile phase by
isocratic elution.1,2 Under this condition, co-elution (Reso-
lution < 0.8) of RB and R6G was observed. Moreover, re-
placing phosphate buffer by formate or trifluoroacetate
buffer did not afford satisfactory separation for these two
analytes (data not shown). However, newly commercially
available AtlantisÒ T3-C18 column showed an improve-
ment in selectivity for these two analytes. This column pro-
vides a di-functionally bonded silica-based C18 stationary
phase which exhibits better retention and less tailing for
protonated basic analytes via reversed-phase HPLC analy-
sis. Table 1 lists the chromatographic retention times and
better resolutions of these two analytes obtained using the
Detection of Rhodamine Dyes by HPLC-FLD
AtlantisÒ T3-C18 column with various mobile phases. RB
and R6G eluted in the same order for each of the mobile
phases, and longer retention times but better resolution oc-
curred when formate buffer was replaced by phosphate
buffer. Table 1 also indicates that the RB and R6G were
baseline separation from each other when higher percent-
age of phosphate buffer was employed. Therefore, an iso-
cratic elution using 45% of acetonitrile with 55% of 0.1%
(w/v) of phosphate buffer was adopted as the mobile phase
in all of the following experiments.
Optimization of SPE
The efficiency of SPE depends on the types of sor-
bent, the pH values of the water samples, and the content of
Fig. 2. SPE extraction recoveries of analytes from a
spiked water sample in (a) various SPE sorbent,
(b) various pH values of water sample, and (c)
various percentage of HCOOH mixed with
methanol as SPE eluents. Extractions were per-
formed on three replicates, standard deviation
is reported as an error bar.
J. Chin. Chem. Soc., Vol. 59, No. 1, 2011 3
Table 1. Comparison of the retention times and resolutions with
various HPLC mobile phase compositions
Retention time Resolution
Mobile phase composition
(min) (R)
(a) 50% of 0.1% HCOOH in CH3CN RB: 4.1 1.3
R6G: 4.6
(b) 50% of 0.1% TFA in CH3CN RB: 8.7 1.2
R6G: 9.2
(c) 50% of 0.1% H3PO4 in CH3CN RB: 6.3 1.6
R6G: 7.2
(d) 55% of 0.1% H3PO4 in CH3CN RB: 10.8 2.3
R6G: 12.8
organic modifier in the elution solvent. Three Oasis SPE
cartridges (HLB, MCX and WCX) were investigated to de-
termine the optimal sample extraction procedures. The ef-
fectiveness of SPE cartridges were evaluated initially to
extract RB and R6G from the spiked deionized water sam-
ples (pH 3.0 and spiked final concentration was 20 ng/L).
Figure 2(a) shows that comparison with mixed-mode
cationic exchange cartridges (MCX and WCX), the HLB
SPE cartridge yielded the best results: above 92% mean re-
covery for both analytes. Possibly, the HLB sorbent pro-
vides better hydrophilic and lipophilic balance characteris-
tics, and also more excellent wetting properties of the hy-
drophilic N-vinylpyrrolidine monomer. 14,15 Various pH
values of water samples were then evaluated to assess the
optimal conditions. Figure 2(b) shows the adjusted pH of
the spiked water samples from 3 to 11, and the maximized
recoveries (up to 92%) obtained by adjusting the pH of the
water sample to 3.0. Furthermore, various compositions of
SPE eluents were also evaluated to determine the best ex-
traction efficiency. Figure 2(c) displays that methanol
mixed with 4% (w/v) of formic acid was found to be an ef-
fective elution solvent for RB and R6G. A breakthrough for
the extraction of 100 mL of a spiked deionized water sam-
ple was made using tandem cartridges, and no significant
amounts of analytes (<1%) were detected in the eluting sol-
vent from the second cartridge. These results indicate that
the best conditions for extracting of RB and R6G from wa-
ter samples using HLB-SPE cartridges were achieved by
adjusting the pH of the water sample to 3.0, and then
eluting the analytes from the cartridge by 3 mL of methanol
mixed with 4% (w/v) of formic acid. To determine the effi-
ciency and precision of the method, the recovery from
HLB-SPE was further evaluated using three replicate
analyses with actual environmental water samples from
4 J. Chin. Chem. Soc., Vol. 59, No. 1, 2011 Chiang et al.

Table 2. Linear range, linearity, limits of detection and quantitation, precision and accuracy data
Intra-day (n = 5) Inter-day (n = 20)
linear range IDL LOD LOQ
Analyte equation r2 %RSD Recovery Recovery
ng mL-1 pg ng L-1 ng L-1
(%RSD) (%RSD)
RB 2 - 50 y = 0.9516x + 0.2442 0.9950 10 5 0.5 1.5 83a (3)b 85 (4)
50 - 1000 y = 2.9673x + 23.19 0.9999 8
R6G 0.5 - 20 y = 0.9308x + 0.3618 0.9940 8 1 0.1 0.3 89 (3) 90 (3)
20 - 400 y = 3.5392x + 33.15 0.9994 6
a
Mean spiked recovery (n = 5 or n = 20).
b
The relative standard deviation (%RSD) are given in parentheses (n = 5 or n = 20).
Final spiked concentration was 20 ng/L for each analyte.

various sources, as described in the Applications section. mean recovery, ranged from 83 to 90%. These results re-
veal that the HLB-SPE method coupled with isocratic
Method validation HPLC and fluorescence detection ensures good reprodu-
The analytical characteristics of the method, such as cibility with excellent linearity and sensitivity for the
linear response range, reproducibility, and quantitation quantitation of these two basic synthetic dyes in water
limit, were investigated to evaluate the efficiency of the samples.
method and the possibility of the method application to ac-
tual water samples. Table 2 lists the results. The linearity of Applications
the analytes was calculated from five-level calibration The versatility of this method is demonstrated in Ta-
curve over the range from 2 to 50 ng/mL (for lower range) ble 3, which lists the recovery of the spiked water samples
and 50 to 1000 (for higher range) for RB, and 0.5 to 20 (spiked final concentrations was 20 ng/L) and the concen-
ng/mL and 20 to 500 ng/mL for R6G, respectively. The trations of RB and R6G detected in various environmental
precisions of the curves, as indicated in terms of the rela- water samples. Fluorescence detection of these two dyes is
tive standard deviations (RSDs) of the calibration factors highly sensitive and selective due to the high fluorescence
(CF = peak area/amount), were less than 10%, and the coef-
ficient of estimation (r2) exceeded 0.9940. The curve cov- Table 3. Concentrations of RB and R6G detected in various
ered a range equivalent to the concentrations of the ana- water samples and their spiked recoveries
lytes in 100 mL water samples after the extract was concen- Analyte
Sample
trated to 100 mL. The limit of detection (LOD), defined as Rhodamine B Rhodamine 6G
the concentration that yielded an S/N ratio of higher than or Tap water conc. (ng/L) n.d. n.d.
equal to 3, and the limit of quantitation (LOQ), defined as Spiked recovery (%) 82a(10)b 83 (8)
the concentration that yielded an S/N ratio of higher than or Groundwater conc. (ng/L) n.d. 0.7
Spiked recovery (%) 85 (3) 90 (3)
equal to 10, were determined by the SPE extraction of the WWTP influent conc. (ng/L) 81 1.4
spiked deionized water samples. LOD values were 0.5 and Spiked recovery (%) 87 (5) 64 (8)
0.1 ng/L, and LOQ values were 1.5 and 0.3 ng/L, for RB WWTP effluent-I conc. (ng/L) 62 0.7
and R6G, respectively (Table 2). The intra- and inter-day Spiked recovery (%) 90 (12) 77 (11)
WWTP effluent-II conc. (ng/L) 37 n.d.
precisions, as well as the accuracies, were evaluated. The Spiked recovery (%) 68 (10) 61 (10)
intra-day precision was determined by analyzing five spiked River water-I conc. (ng/L) 4.8 1.3
River water-II on the same day (n = 5). The inter-day preci- Spiked recovery (%) 78 (7) 86 (6)
sion was evaluated by determining five replicates on four River water-II conc. (ng/L) n.d. n.d.
Spiked recovery (%) 70 (11) 75 (10)
consecutive days (n = 20). The accuracy was evaluated by
a
comparing the mean recovery from five or 20 analyses to Mean spiked recovery (%, n = 3) at final concentration of 20
ng/L for each analyte.
the nominal concentration value. Table 2 also indicates that b
Relative standard deviation (%RSD) of recovery is given in
the intra- and inter-day precisions (RSDs) for the analytes parentheses (n = 3).
were all less than 4%. The accuracies, determined as the n.d. Not detected at the LOQ, as listed in Table 2.
Detection of Rhodamine Dyes by HPLC-FLD
quantum yields and the relatively high wavelength of the
maximum fluorescence, which sets these analytes apart
from the most naturally-occurring interferences. Combined
with the baseline separation of each analyte, the target
peaks were identified using comparison with the retention
times of the standard solutions (within one standard devia-
tions of the retention time window), and could be also veri-
fied by the standard addition of target compounds. The
quantities were calculated using calibration factors. The
spiked recovery values of the analytes ranged from 61 to
90% with the RSD ranging from 2 to 12%. These two dyes
were detected in almost all the WWTP effluents and one
river water sample ranging from 0.7 to 81 ng/L. Fig. 3
shows the typical isocratic HPLC chromatograms obtained
from (a) the standard and (b) the River water-I sample. This
developed method appears to be an appropriate technique
for analyzing these two basic synthetic dyes at trace-level
in various aqueous samples.
CONCLUSIONS
The analytical procedure developed herein demon-
strates that a HLB-solid-phase extraction and isocratic
HPLC method is a reliable and sensitive process. It is a con-
venient analytical technique for determining RB and R6G
in various environmental water samples. The sorbent of
SPE, pH of the water sample, and the composition of SPE
eluent are the three parameters that most influence the effi-
ciency of the extraction of these two basic synthetic dyes
Fig. 3. Typical isocratic HPLC-FLD chromatograms
obtained from (a) the standard and (b) the River
water-I sample.
J. Chin. Chem. Soc., Vol. 59, No. 1, 2011 5
by SPE. The preliminary results revealed that RB and R6G
are ubiquitous in WWTP effluents and surface water in Tai-
wan. Although the toxicological information available on
RB and R6G is limited, this study may provide further in-
sights to promote environmental protection and conserva-
tion. This may also be able to support pollution control
policies and sustain development in Taiwan.
ACKNOWLEDGEMENTS
This research was supported by a grant from the Na-
tional Science Council of Taiwan under contract No. NSC
97-2113-M-008-001-005-MY3. We thank the Instrumental
Center of National Central University for instrumental sup-
port. We also thank Erica Ding for her kind assistance with
the English language of this article.
REFERENCES
1. Gagliardi, L.; Orsi, D. D.; Gavazzutti, G.; Multari, G.;
Tonelli, D. Chromatographia 1996, 43, 76.
2. Scalia, S.; Simeoni, S. Chromatographia 2001, 53, 490.
3. Valianou, L.; Karapanagiotis, I.; Chryssoulakis, Y. Anal.
Bioanal. Chem. 2009, 395, 2175.
4. Jain, R.; Mathur, M.; Sikarwar, S.; Mittal, A. J. Environ.
Manage. 2007, 85, 956.
5. Mull, D. S.; Liebermann, T. D.; Smoot, J. L.; Woosley, L. H.
EPA 904/6-88-001; US Environmental Protection Agency:
Atlanta, Georgia, 1988; pp 90-103.
6. Tünay, O.; Kabdasli, I.; Eremektar, G.; Orhon, D. Water Sci.
Technol. 1996, 34, 9.
7. Cassano, A.; Molinari, R.; Romano, M.; Drioli, E. J. Membr.
Sci. 2001, 181, 111.
8. Lourenco, N. D.; Novais, J. M.; Pinheiro, H. M. J. Bio-
technol. 2001, 89, 163.
9. Pokhrel, D.; Viraraghavan, T. Sci. Total Environ. 2004, 333,
37.
10. Forgacs, E.; Cserháti, T.; Oros, G. Environ. Int. 2004, 30,
953.
11. Crini, G. Bioresour. Technol. 2006, 97, 1061.
12. Lee, C. K.; Liu, S. S.; Juang, L. C.; Wang, C. C.; Lin, K. S.;
Lyu, M. D. J. Hazard. Mater. 2007, 147, 997.
13. Fernández, C.; Larrechi, M. S.; Callao, M. P. Trends Anal.
Chem. 2010, 29, 1202.
14. Thurman, E. M.; Mills, S. M. Solid Phase Extraction: Prin-
ciples and Practice, Chemical Analysis, Vol. 147; Wiley:
New York, 1998.
15. Waters Oasis HLBâ Sample Extraction: Environmental and
Agrochemical Applications Notebook, Waters, Milford,
MA, USA, 2001.