Journal of Analytical Sciences, Methods and Instrumentation, 2012, 2, 74-80
http://dx.doi.org/10.4236/jasmi.2012.22014 Published Online June 2012 (http://www.SciRP.org/journal/jasmi)
Optimal Sample Preservation and Analysis of Cr(VI) in
Drinking Water Samples by High Resolution Ion
Chromatography Followed by Post Column Reaction and
UV/Vis Detection*
Prince Ezebuiro1, Jay Gandhi2, Chunlong Zhang1, Johnson Mathew3, Melvin Ritter3,
Marvelyn Humphrey3
1University of Houston-Clear Lake, Houston, USA; 2Metrohm USA Inc., Riverview, Florida, USA; 3US EPA Region 6 Laboratory,
Houston, USA.
Received November 29th, 2011; revised January 16th, 2012; accepted January 31st, 2012
ABSTRACT
A recent study by the Environmental Working Group reported the detection of hexavalent chromium (Cr(VI)) in tap
water at 31 out of 35 states investigated in the United States. Even though Cr(III) is an essential element for human diet,
Cr(VI) is a potential carcinogen. Previous work has clearly identified a linear trend of increasing risk of lung cancer
mortality with increasing cumulative exposure to water soluble Cr(VI). Regardless, Cr(VI) is still not regulated or
monitored in drinking water in the US. There is an existing method (EPA 218.6) for the analysis of Cr(VI), however,
this analytical method does not addresses detailed sample preservation techniques and optimization process to achieve
lowest detection limit possible. In this study, five buffer solutions with pH of 9 and above were compared to determine
the most suitable buffer to preserve Cr(VI) in drinking water samples for an extended period of time. Results showed
that the five buffers responded very differently to Cr(VI)-fortified drinking water. The best preserving reagent was
found to be Ammonium Hydroxide + Ammonium Sulfate (pH 9.2) and Sodium Carbonate + Sodium Bicarbonate+
Ammonium Sulfate (pH 9.7), whereas a buffer solution with Sodium Hydroxide + Sodium Carbonate (pH 11.5+) re-
sulted in a poor chromatographic resolution. A controlled study with a fortified Cr(III) at 1 ppb was also conducted to
ensure no false positive detection of Cr(VI) due to the potential oxidation of Cr(III) during sample storage. The optimal
preserving reagent identified from this study was compatible with the existing EPA method 218.6 using ion chroma-
tography followed by post column reaction, with a method quantitation limit of 0.020 ppb and matrix spike recovery of
± 10%.
Keywords: Hexavalent Chromium; Ion Chromatography; USEPA Method; Sample Preservation
1. Introduction
Chromium (Cr) exists in oxidation states varying from –2
to +6 [1], but exists predominantly in the environment in
two stable forms, i.e., trivalent {Cr(III)} and hexavalent
{Cr(VI)} chromium. Cr(III) is a known essential element
for both animals and humans, whereas Cr(VI) in either
oxyanionic forms as chromate () or dichromate
() and bichromate (), is a known toxin
and carcinogen. It has been reported that the yellowish
coloration of water is due mainly to the presence of
monomeric specie of Cr(VI) at concentrations greater
than 1.0 mg/L while the orange coloration is due to the
presence of high levels of the dichromate,
2
4
CrO 
4
rO 2
27
Cr OHC
2
27
Cr O

.
Chromium contamination in the environment can occur
through leakages, improper waste disposal or poor stor-
age [2,3].
Conversion between these two major forms of chro-
mium can occur at their respective environmental condi-
tions either in the ambient water or during sample storage
period. Cr(VI) is a strong oxidant, which can be easily
reduced to Cr(III) The equations below show the reduc-
tion of different species of Cr(VI) in the presence of a
reducing agent (an electron donor):
*Disclaimer: Reference herein to any specific commercial products or
nonprofit organization, process, or service by trade name, trademark,
manufacturer, or other-wise, does not necessarily constitute or imply its
endorsement, recommendation, or favoring by the United States Gov-
ernment. The views and opinions of authors expressed herein do not
necessarily state or reflect those of the United States Government and
shall not be used for advertising or product endorsement purposes.

Optimal Sample Preservation and Analysis of Cr(VI) in Drinking Water Samples by High Resolution Ion
Chromatography Followed by Post Column Reaction and UV/Vis Detection
75
2+ 3
27 2
Cr O14H6e2Cr7HO

  (1)

2
42 4
CrO4H O3eCrOH4OH


  


2
(2)
3
4
HCrO7H3eCr4H O
 
  (3)
The conditions favorable for the reduction of Cr(VI) to
Cr(III) include acidic pH [4], presence of low dissolved
oxygen concentration, organic matter [5] or humic sub-
stances [6] and the presence of reducing agents such as
Fe(II) [5] and sulfide [7]. The mechanism and chemical
pathway for such redox reactions has been extensively
studied [8]. The conversion of Cr(III) to Cr(VI) has also
been investigated under the presence of high dissolved
oxygen [7], presence of oxidizing agent such as manga-
nese oxide [9] under acidic or slightly alkaline conditions
[0].
It is therefore essential that a method for the reliable
analysis of Cr(VI) in environmental samples should en-
sure the integrity of the Cr species to be preserved and
such a preserving reagent will be compatible with the
existing method. Currently, the US EPA method 218.6 is
the commonly used regulatory method for the analysis of
Cr(VI) in surface and drinking water samples. Unfortu-
nately this method does not specify details regarding the
preservation of Cr(VI) and Cr(III) originally present in
samples, thereby having the potential to report Cr(VI)
either false positive or false negative due respectively to
the oxidation of Cr(III) and reduction of Cr(VI). This
study was initiated in part due to the discovery of Cr(VI)
by the Environmental Working Group (EWG) (December
2010) [4] that 31 out of 35 investigated states in the U.S.
detected the presence of Cr(VI) in the drinking water. In
certain states, detected Cr(VI) averaged about 300%
above the proposed 0.02 ppb limit by the state of Cali-
fornia (June 2011).
Figure 1 shows the basic principles of the ion chro-
matography method used in this study. Cr(VI) after pres-
ervation at pH > 9.5 exists as oxyanion 2
4
CrO

or
2
27
Cr O

. This oxyanion was chromatographically sepa-
rated from common anions in drinking water using high
resolution anion exchange column. After separation,
chromate ion is then reacted with 1,5-diphenyl Carbazide
color reagent in the presence of excess acid to form ma-
genta color complex, which is subsequently subject to the
detection by a UV-VIS spectrophotometer at 530 nm
wavelength.
2. Experimental
2.1. Apparatus and Reagents
Standard solutions of Cr(VI) and Cr(III) were prepared
using DI Water (18 MΩ) and pure reagent grade com-
pounds. A list of reagents used is shown in Table 1.
Figure 1. Principle of the ion chromatography followed by
post column reaction for the analysis of Cr(VI).
Table 1. Chemicals and reagents.
S/No Reagent Chemical Formula Manufacturer CAS Number Percentage Purity (%)
1. Methanol HPLC grade CH3OH Fisher Chemical 67-56-1 99.5
2. 1,5-Diphenylcarbazide C13H14N4O Sigma 140-22-7
3. Conc. Sulfuric Acid ACS grade H2SO4 Mallinckrodt 7664-93-9 98
4 Potassium Dichromate K2Cr2O7 Sigma 7778-50-9 99.9
5 Sodium Hydroxide NaOH ACROS Organic 13-10-73-2 97.7
7 Sodium Carbonate Na2CO3 Sigma Aldrich 497-19-8 99.5
8 Sodium Bicarbonate NaHCO3 Sigma 144-55-8 99.5
11 Ammonium Hydroxide NH4OH Sigma-Aldrich 320145 27-29
12 Ammonium Sulfate (NH4)2SO4 Aldrich 204501 99.9
14 Sodium Tetraborate Decahydrate Na2B4O7·10H2O Sigma-Aldrich 1303-96-4 99.5 - 101.5
15 Chromium(III) Nitrate Cr(NO3)3·9H2O Fisher 7789-02-8 99.9

Optimal Sample Preservation and Analysis of Cr(VI) in Drinking Water Samples by High Resolution Ion
Chromatography Followed by Post Column Reaction and UV/Vis Detection
76
2.2. Instrumentation
The ion chromatography system employed in this study
was Metrohm IC system (model # 850 Professional IC
AnCat version) consisting of an auto-sampler (model #
858 Professional AS), with 6 port injection valve (2000
µL injection loop), dual metal-free pumps, a column
oven, post column reactor (PCR Box) and a Metrohm
UV detector (model # 887). Major instrumental condi-
tions are given in Table 2.
A strict QA/QC protocol was maintained for the entire
experiment as per USEPA method guidelines [10]. Each
sequence run contained DI water blank to demonstrate
lack of carryover due to the instrument or any of its
components. A 0.1ppb standard containing the analyte of
interest was measured to show the accuracy of the cur-
rent calibration. The percent recovery was within 10%
(Figure 2).
2.3. Experimental Setup
a) Preparation of Mobile Phase: The IC mobile
phase was prepared by adding 1.3568 g sodium carbonate,
0.336 g sodium bicarbonate, and 0.25 g ammonium sul-
fate in 1 L volumetric flask. Dilution was made by using
18 mega-ohm DI water produced by a series of activated
carbon, cationic/anionic exchange column, and finally
0.22 µm filter. Alternatively, pre-made concentrated
carbonate/bicarbonate can be used.
b) Preparation of Post Column Reagent: The color
reagent was prepared by first dissolving 0.5 g 1,5-diphenyl
Carbazide (DPC) into 50 mL HPLC grade methanol, then
adding this dissolved DPC into approximately 450 mL of
18-mega-ohm DI water in a 1 L volumetric flask. This
was followed by gently adding 28 mL concentrated
H2SO4 and finally diluted with DI water to 1 L. This light
sensitive solution was kept in dark prior to use.
Table 2. Method parameters for the analysis of Cr(VI) using IC with post column reaction.
Method Parameters Analytical Conditions
IC Column Metrosep ASUPP5-150
Oven Temperature (˚C) 45
Mobile Phase 12.8 mmol/L Sodium Carbonate + 4.0 mmol/L Sodium Bicarbonate + 2.5 mM Ammonium Sulfate
Column Flow Rate 0.7 mL/min
Post Column Reagent (PCR) 2 mmol/L 1,5-Diphenyl Carbazide + 1 N H2SO4
PCR Flow rate 0.25 mL/min
UV Wavelength 530 nm
Injection Volume 2000 µL
Figure 2. CCV statistics for 200+ injections.

Optimal Sample Preservation and Analysis of Cr(VI) in Drinking Water Samples by High Resolution Ion
Chromatography Followed by Post Column Reaction and UV/Vis Detection
77
c) Preparation of sample preservation buffer: Five
buffer solutions were tested for their potential as the pre-
serving reagent for Cr(VI). Buffer A was selected be-
cause it is suggested by USEPA method 3060A and 1636,
whereas buffer C was used in USEPA method 218.6 [11,
12]. As noted in Table 3, all buffers have pH of higher
than 9.0.
3. Results and Discussion
3.1. Initial Calibration and Quality Control for
Entire Experiment
Multi-point calibration was established for analytical
range as per Table 4.
3.2. Sample Chromatography and Calibration
Curve (Figures 3 and 4)
Table 3. Preparation of buffer solutions for the preservation
of Cr(VI).
Buffer Chemical Composition Initial
measured pH
A 2 mM Na2CO3 + 10 mM NaOH
in 1000 mL 12.0
B 1.25 mM Na2CO3 + 1.25 mM NaHCO3
in 1000 mL 10.2
C 0.4 g (NH4)2SO4 + 0.65 mL NH4OH
in 1000 mL 10.0
D 1.25 mM Na2CO3 + 1.25 mM NaHCO3
+ 0.15 g/L (NH4)2SO4 in 1000 mL 9.25
E 5 mM NaHCO3 + 10 mM Na
Tetra Borate in 1000 mL 9.10
Table 4. Calibration standards.
Calibration Level Chromium(VI),
parts per billion (ppb)
Level 1 0.025
Level 2 0.050
Level 3 0.100
Level 4 0.250
Level 5 0.500
Level 6 1.000
Level 7 2.000
Level 8 5.000
0.0
2.0
4.0
6.0
mAU
0.01.02.0 3.0 4.0 5.0 6.0min
Chrom-6
Figure 3. Cr(VI) at 0.025 ppb.
0.0
1.0
2.0
3.0
4.0
5.0
6.0
(mAU) x min
0.01.02.0 3.0 4.0 5.0
pp
b
Figure 4. Calibration curve.
3.3. Method Detection Limit (MDL) Study [10]
A method detection limit (MDL) is the minimum con-
centration of a specified analyte that can be detected and
quantified with a 99% confidence level. The MDL was
determined by injecting 7 replicates of known concentra-
tion near the expected limit of detection. The standard
deviation is determined from the results and multiplied
by the t value. The t value for 7 replicates is 3.14 at a
99% confidence level. An MDL study was performed for
Chromium(VI) over three days. Table 5 demonstrates
the data for the study.
a) Minimum Reporting Limit (MRL) study (new QC
parameter for USEPA Chromium (VI) method Analyze
seven replicate at or below the proposed MRL concentra-
tion. Calculate the mean (Mean) and standard deviation
for these replicates. Determine the Half Range for the
Prediction Interval of Results (HRPIR) using the equation
HRPIR = 3.963S

Optimal Sample Preservation and Analysis of Cr(VI) in Drinking Water Samples by High Resolution Ion
Chromatography Followed by Post Column Reaction and UV/Vis Detection
78
where S is the standard deviation and 3.963 is a constant
value for seven replicates.
Confirm that the Upper and Lower limits for the Pre-
diction Interval of Results (PIR = Mean ± HRPIR) meet
the upper and lower recovery limits as shown below.
The Upper PIR Limit must be 150 percent recovery.
PIR
Mean HR100 150%
Fortified Concentration

The Lower PIR Limit must be 50 percent recovery.
PIR
Mean HR100 50%
Fortified Concentration

Table 6 demonstrates analyzed data for MRL study.
b) Continuing Calibration Verification (CCV) for the
entire experiment.
CCV of lower range of calibration (0.1 parts per bil-
lion) was analyzed every 10 samples. Overall average
recovery is 103%.
Table 5. Method detection limit (MDL) study.
Chromium(VI), (ppb)
MDL-1 (Day 1) 0.064
MDL-2 (Day 1) 0.074
MDL-3 (Day 2) 0.073
MDL-4 (Day 2) 0.058
MDL-5 (Day 2) 0.063
MDL-6 (Day 3) 0.062
MDL-7 (Day 3) 0.061
Average 0.065
Standard Dev 0.006
Calculated MDL 0.019
Table 6. MRL study data.
Chromium(VI), ppb
analysis-1 0.018
analysis-2 0.018
analysis-3 0.019
analysis-4 0.02
analysis-5 0.018
analysis-6 0.022
analysis-7 0.018
Mean 0.0190
std.Dev 0.0015
HPPIR 0.0061
True Concentration 0.020
Upper HPPIR 125.2679
Lower HPPIR 64.73208
4. Sample Preservation Buffers Data
Discussion
1 Liter of each buffer solutions was prepared in two
separate 1 Liter plastic containers. One bottle was forti-
fied with 1 part per billion Chromium(VI) and other bot-
tle was fortified with 1 part per billion each of Chro-
mium(III) and Chromium(VI). Every day for consecutive
21 days this solution was analyzed for Chromium (VI)
stability. This solution was preserved at 4 deg.C in re-
frigerator.
4.1. Buffer—A (2 mM Na2CO3 + 10 mM NaOH)
Buffer A (Figure 5) data demonstrated that even at Day1
there is almost 52% oxidation of Chromium(III) into
Chromium(VI) due to high pH of buffer (pH = 12). Pro-
gressively, oxidation is increased and on Day 6 it is
100% conversion. Also, please make a note that data
from Day 7-21 indicates more than 100% conversion to
Chromium(VI). This is due to original Total Chromium
(Cr) contamination in Sodium Hydroxide pellets. This
total Cr converted to Cr(VI) with favorable pH in the
solution.
4.2. Buffer—B (1.25 mM Na2CO3 + 1.25 mM
NaHCO3)
Buffer B (Figure 6) data demonstrated that even at Day1
there is almost 59% oxidation of Chromium(III) into
Chromium(VI) due to high pH of buffer (pH = 10.5).
Progressively, oxidation is increased and on Day 8 it is
92% conversion. Sodium Carbonate/Bicarbonate buffer
preserves Chromium(VI) at a stable rate but if native
Cr(III) is present in drinking water then it will also con-
vert it to Cr(VI) very quickly.
Figure 5. Buffer A data for Cr(III)--> Cr(VI) conversion.

Optimal Sample Preservation and Analysis of Cr(VI) in Drinking Water Samples by High Resolution Ion
Chromatography Followed by Post Column Reaction and UV/Vis Detection
79
4.3. Buffer—C (0.4 g (NH4)2SO4 + 0.65 ml
NH4OH)
Buffer C (Figure 7) data demonstrates that this buffer
preserves Cr(VI) very well for at least 21 days.
Buffer—D (1.25 mM Na2CO3 + 1.25 mM NaHCO3
+ 0.15g/L (NH4)2SO4)
Buffer D (Figure 8) data demonstrated that this buffer
preserves Cr(VI) very well for at least 21 days It is be-
lieved that Ammonium Salt in the buffer system forms
Chloramine products due to free chlorine in drinking
water samples and hence prevents Cr(III) oxidation to
Cr(VI).
Figure 6. Buffer B data for Cr(III)--> Cr(VI) conversion.
Figure 7. Buffer C data for Cr(III)--> Cr(VI) conversion.
4.4. Buffer—E (5 mM NaHCO3 + 10 mM Na
Tetra Borate)
Borate Buffer originally recommended by CADPH (May
2010) [13]. Buffer E (Figure 9) data demonstrated that
this buffer preserves Cr(VI) very well for at least 4 days
Up to 20% Cr(III) oxidizes to Cr(VI). This may be due to
native total Chromium present in Tetraborate salt. Ana-
lyst had to validate each batch of pure buffer chemicals
purchased.
5. Conclusion
This study concludes that there are several choices and
flexibility in the selection buffers for the preservation of
Cr(VI) in water matrix. We also conclude that buffers
containing ammonium salt is favorable for drinking
Figure 8. Buffer D data for Cr(III)--> Cr(VI) conversion.
Figure 9. Buffer E data for Cr(III)--> Cr(VI) conversion.

Optimal Sample Preservation and Analysis of Cr(VI) in Drinking Water Samples by High Resolution Ion
Chromatography Followed by Post Column Reaction and UV/Vis Detection
80
water samples as it contains free chlorine which can
serve as a strong oxidizer. Buffer D containg 1.25 mM
Na2CO3, 1.25 mM NaHCO3, and 0.15 g/L (NH4)2SO4
added in the solid form of field samples is recommended.
This preserving chemical is also compatible with the
subsequent analytical method (USEPA method EPA
218.6) [11,14] using IC with post column reaction. Addi-
tional results for the evaluating the effects of various
oxidizers, reducers, organic material potentially impor-
tant in drinking water, and pH effect on sample preserva-
tion will be reported in a forthcoming paper.
6. Acknowledgements
Authors would like to acknowledge the following indi-
viduals for their valuable contribution to this study, in-
cluding Dr. Steve Wendelken (USEPA-ODW), Mr.
David Neliegh (USEPA R6), Dr. Katinka Ruth (Metrohm
AG) [15] and Dr. Hari Narayanan (Metrohm USA) [16].
Dr. Carl Zhang acknowledges the Welch Foundation for
partial sponsorship of this work.
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