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

doi:10.4236/jasmi.2012.22014

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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.

Email: [email protected]

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,

CrO 

rO 2

Cr OHC

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

2+ 3

27 2

Cr O14H6e2Cr7HO



  (1)



42 4

CrO4H O3eCrOH4OH







 





(2)

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

CrO



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

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

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

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

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

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

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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