Chromium Speciation in Waterby HPLC/ICP-MS

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Introduction

Chromium exists primarily in twoforms: trivalent and hexavalent.Trivalent chromium is present inthe cationic form as Cr+3 and is anessential nutrient, but hexavalentchromium is toxic and exists as ananion, either as chromate (CrO4

-2)or dichromate (Cr2O7

-2). Therefore,the analysis of total chromiumdoes not always provide a trueindication of the effects of chromiumpresent in a sample. For example,a high total chromium concentrationmay not be harmful if the majorityis Cr+3. A more accurate assessmentof the true effects of chromiumcan be gained by distinguishingtrivalent and hexavalent species.As a result, chromium regulationshave existed for some time for speciated analysis in water.

A common problem with chromiumspeciation is the well-known interconversion of Cr+3 and Cr+6;as a result, the determinedCr+3/Cr+6 concentrations may nottruly represent what exists in thesample. There has been much

work exploring the interconversionof these species and trying to limitthis process1-9. Although some initial work is presented here,more in-depth discussions aboutchromium species preservationcan be found in the literature. Thefocus of this work is to explorechromatographic and instrumentalconditions and parameters necessaryto distinguish Cr+3 from Cr+6 inwater samples using HPLC/ICP-MS. The work here is an extensionof earlier work.10

Experimental

Instrumental Parameters

Tables 1 and 2 show the chromato-graphic and ICP-MS operatingconditions respectively. The HPLCseparation of chromium species is accomplished isocratically by ion-pair chromatography using tetrabutylammonium hydroxide(TBAH) as the ion-pair reagent.Detection of the chromium species isaccomplished with an ELAN® DRC(dynamic reaction cell) II ICP-MS.

Authors:

Kenneth NeubauerWilhad ReuterPamela Perrone

PerkinElmer Life and Analytical Sciences710 Bridgeport AvenueShelton, CT 06484 USA

Table 1. HPLC Isocratic Method and Operating Parameters.

Parameter Setting

Mobile Phase 1 mM Tetrabutylammonium Hydroxide (TBAH)+0.6 mM EDTA (potassium salt); pH 6.9Then added 2% MeOH

Flow Rate 1.5 mL/min

Run Time 3 min

Column 3x3™ CR C8 (Part No. 0258-0191)

Column Temperature Ambient

Autosampler Flush Solvent 5% Methanol / 95% HPLC-grade Water

Sample Injection Volume 50 µL

Sample Prep Dilute with Mobile Phase

Detection PerkinElmer SCIEX ELAN DRC II ICP-MS

Total Analysis Time 3 min

Mobile Phase Preparation

The mobile phase was prepared bydissolving the appropriate amountsof tetrabutylammonium hydroxide(TBAH, Sigma-Aldrich, 1.0 M solution in water) and the dipotassiumsalt of ethylenediaminetetraaceticacid(EDTA, Sigma-Aldrich) in deionizedwater. The pH was then adjustedwith nitric acid to 6.9, and methanolwas added to make a 2% methanol solution.

Conditioning the HPLC Column

The column used for this applicationis a high-speed, reversed-phase, C8cartridge column. It is 3.3 cm longand packed with 3-µm particles,which permit very rapid analysistimes (typically 2-3 minutes) andreduced solvent consumption. Thecartridge column consists of twopieces: the column holder (part number0715-0028) and the cartridge column(part number 0258-0191, availableonly in a 5-pack). The main advantageof cartridge columns is their reducedcost compared to standard columns.

When the column is first received, itshould be washed for 90 minuteswith 100% methanol at a flow of 1.0mL/min. This procedure ensures thatthe column is clean and does notneed to be repeated.

Prior to using the column for thefirst time each day, mobile phaseshould be allowed to flow throughthe column for 30 minutes. This procedure conditions the columnwith the ion-pair reagent.

After use for the day, the columnshould be washed for 15 minuteswith 5/95 v/v methanol/water toremove all salts from the system andto inhibit bacterial growth. For long-term storage, first rinse thecolumn with 100% water and then40/60 v/v methanol/water. After rinsing the column, cap the columnto prevent it from drying out.

Standards and StandardPreparation

Chromium standards were preparedfrom 1000 mg/L stock solutions of Cr+3 (PerkinElmer, Cr(NO3)3.9 H2O,part number N930-0173) and Cr+6

(Spex Certiprep, (NH4)2Cr2O7).Intermediate 2 mg/L solutions ofeach species were prepared in deionized water. Standards of different chromium concentrationswere made by appropriate dilutionof the intermediate standards withmobile phase.

Results

Figure 1 shows a chromatogram of amixture of Cr+3 and Cr+6 (10 µg/L(ppb) each) acquired while monitoring52Cr+. The data was acquired in lessthan 3 minutes under the conditionsstated above. Shorter analysis timescan be accomplished using somewhathigher methanol concentrations. Forthis study, a 1mM TBAH concentrationappeared to be optimal. Though2mM TBAH has been used success-fully with other 3x3 C8 columns, significantly increasing or decreasingthe TBAH molarity may reduce peak resolution and may result inpeak broadening.

The separation is accomplished byinteraction of the chromium specieswith the different components of themobile phase. The Cr+3 forms a complex with the EDTA; it is thiscomplex that is retained on the col-umn. Cr+6 exists in solution asdichromate. The net negative chargeof the chromium-EDTA complex andthe negative charge of the dichromateinteract with the positive charge ofthe tetrabutylammonium group. Inturn, the hydrocarbons of the tetra-butyl group interact with the C8 ofthe stationary phase, thus permittingthe separation of a charged specieson a reversed-phase column. Thepurpose of the methanol is to assistin the elution of these chromiumspecies from the column.

Table 2. DRC ICP-MS Operating Conditions and Parameters.

Parameter Setting/Type

Nebulizer Meinhard® Type A quartz (Part No. WE02-4371)

Spray Chamber Quartz Cyclonic (Part No. WE02-5221)

RF Power 1100 w

Plasma Ar Flow 15 L/min

Nebulizer Ar Flow 0.96 L/min

Aux. Ar Flow 1.2 L/min

Injector 2.0 mm i.d. Quartz (Part No. WE02-3915)

Monitored Ion m/z 52Cr+

Dwell Time 1000 ms

Total Acquisition Time 180 sec

CeO+/Ce+ <2%

Reaction Gas NH3

NH3 Flow 0.50 mL/min

RPq 0.70

Figure 1. Optimal separation.

Time (minutes)

Cr+6

Cr+3/EDTA Complex

Column Washing

The effect of washing the columnwith methanol is shown in Figure 2.Figure 2a displays the chromatogramof Cr+3 and Cr+6 (10 µg/L each) on anew, unwashed C8 column. Clearly,there is no baseline resolutionbetween the peaks. Figure 2b showsthe same sample run on the samecolumn after it has been washed for90 minutes with 100% methanol at 1 mL/min. After the column wash,the individual peaks, resulting fromthe two different chromium species,are clearly distinguishable. The initialpeak is an unidentified componentthat was not investigated further; it wasonly observed when polypropylenevials were used.

The reason that washing the columnis important is due to the mechanismof ion pairing. With ion pairing, theorganic side of the ion-pair reagent(TBAH in this work) interacts withthe organic C8 stationary phase ofthe column, and the ionic side of theion-pair reagent interacts with theanalyte (Cr+3). With a new column,the original condition of the stationaryphase prevents the TBAH from establishing a stable interaction equilibrium with the C8; therefore, atfirst, the chromium species are notwell separated (Figure 2a). When thecolumn is washed with methanol,the stationary phase is properlyconditioned. Thus, indirectly, thisallows the chromium species to bebetter retained and separated on thecolumn (Figure 2b).

The use of a C18 column was alsoexplored for this work. As with theC8 column, a before-and-after column wash experiment was performed on this column. As theresults show (Figures 2c, 2d), therewas no adequate peak separationachieved using the C18 column.

Mobile Phase Concentrations

There are numerous parameters thatmay be optimized in a reversed-phase ion-pair separation. Theseparameters include:

• Type of ion-pair reagent

• Alkyl chain length of the ion-pair reagent

Figure 2. Effect of washing new columns with MeOH. Washing scheme: 90 minutes, 100%methanol at 1mL/min. X-Axis: mV (millivolts), Y-Axis: Time (min).

Figure 3. Effect of TBAH concentration on chromium separation.

• Concentration of the ion-pair reagent

• Percent organic modifier

• pH

In this work we investigated three ofthese parameters - the type of ion-pair reagent, the concentration of theion-pair reagent and the percentorganic modifier.

In the paper presented by Chang and Jiang10 the ion-pair reagent usedwas tetrabutylammonium phosphate(TBAP). TBAP has two disadvantages.First, it is an expensive reagent.Second, TBAP is very hydroscopic,and hence, difficult to work with.Therefore, an alternative to TBAPwas investigated.

Tetrabutylammonim hydroxide(TBAH), a very commonly used ion-pair reagent, was found to be anacceptable replacement.

Figure 3 shows the effect of TBAHconcentration. If the TBAH concen-tration is increased, the retentiontimes become longer and the peaksbroaden. The reason for these phenomena is that, at higher concentrations, more TBAH caninteract with the stationary phase,thus providing more ion pairing sites for the chromium species to be retained. Too much retention ultimately results in broader peak shapes.

C8- Before a)

C18- Before c)

Cr+3/EDTA Complex1 mM TBAH

2 mM TBAH

Time (minutes)

Cr2O7-2

C18- After d)

C8- After b)

The purpose of the methanol in themobile phase is to reduce the inter-action between the stationary phaseand the ion-pair reagent. As a result,it is expected that higher methanolconcentrations would result in shorterretention times and narrower peaks.The results shown in Figure 4 support this theory.

For the rest of the work presentedhere, a combination of 1 mM TBAHand 2% methanol was found to giveadequate separation in a desiredamount of time.

Vial Material and Stability

An important aspect of an HPLCmethod is its reproducibility. To testthis, consecutive injections of a 10 µg/L (ppb) mixed standard weremade from a polypropylene HPLCautosampler vial. The chromatogramsare displayed in Figure 5 and showthat, over time, the Cr+6 peak decreaseswhile the Cr+3 peak increases. Theseresults suggest that something iscausing the conversion of Cr+6 to Cr+3.

Another test was performed whereconsecutive injections of a 10 µg/LCr+3 from a polypropylene vial weremade over 1 hour. Figure 6 displaysan overlay of six chromatogramstaken at equal intervals throughoutthe hour. These chromatograms overlap very well, thus demonstratingthe inherent stability of the method.This also suggests that the problemsobserved in Figure 5 resulted from aconversion of Cr+6 to Cr+3, but notvice versa.

The above test was then repeated,except a glass autosampler vial wasused instead of a polypropylene vial;the results are displayed in Figure 7.From this figure, it can be seen thatin consecutive chromatograms, theintensities of both peaks remainedquite constant. These results suggestthat by using glass vials, as opposedto polypropylene vials, the unwantedconversion of Cr+6 to Cr+3 is signifi-cantly minimized. All further analyseswere performed using glass HPLCautosampler vials.

Figure 4. Effect of mobile phase MeOH on chromium separation.

Figure 5. Cr+3 / Cr+6 reproducibility test using polypropylene vials.

Figure 6. Cr+3 reproducibility test using polypropylene vials: 10 µg/L Cr3+ over 1 hour; 6 replicates.

2% MeOHCr+3

Cr+6

4% MeOH

6% MeOH

Cr+3/EDTA ComplexCr2O7

-2Time = 0

Time = + 4.5 min

Time = + 9 min

Time = + 13.5 min

Figure 7. Cr+3 / Cr+6 reproducibility test using polypropylene vials.

Figure 8. Comparison of DRC to Standard Mode (ELAN data): 1 ppb Cr+3 and Cr+6.

Cr+3/EDTA Complex

Standard Mode

DRC Mode

Cr2O7-2

Time = 0

Time = + 4.3 min

Time = + 8.6 min

Interference Reduction

A potential interference for chromiumdetermination by ICP-MS is ArC+

(argon carbide), which forms at m/z52, the same m/z as the majorchromium isotope. Using 2% methanolin the mobile phase amplifies thisinterference, which would limit theability to look at low levels ofchromium. To solve this problem,ammonia was used as a reaction gasin the ELAN DRC II. Ammonia reactswith and eliminates ArC+, thus leaving52Cr+ interference-free. This effectcan be seen in Figure 8, which displays two chromatograms of 1 µg/L Cr+3 and 1 µg/L Cr+6, oneacquired without a reaction gas (standard mode) and the otheracquired with ammonia as a reactiongas (DRC mode).

Time = + 12.9 min

Time = + 17.2 min

Time

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The top chromatogram was obtainedin the standard mode. The baselineis noisy and has an intensity ofabout 18,000 cps, the result of ArC+.The amplitude of the baseline noiseis about 1000 cps, and the peaks areabout 3,000–4,000 cps above thebaseline, thus leading to a signal tonoise (S/N) of about 3–4. In addition,a broad, unidentified peak is seen inthe top chromatogram.

In contrast, the bottom chromatogramwas acquired in the DRC mode. Thebaseline is much less noisy andshowed an intensity of only ~700cps. The peaks are about 4,000–5,000cps above the baseline, which fluctuates about 50 cps, yielding aS/N of about 80-100. Therefore, thequantitative sensitivity in DRC mode

is clearly superior to that in standardmode. In addition, in DRC mode, the unidentified peak disappeared,suggesting that this interference is acarbon species.

Detection Limits

To determine how low a Cr level canbe detected in DRC mode, successive50 µL injections were made of varyingchromium concentrations in mobilephase. Figure 9 shows chromatogramsof 25, 50, and 100 ng/L chromiumspikes (baselines artificially offset forclarity). From this figure, 50 ng/L (ppt)of each chromium species can be seenat about S/N - 3, thus demonstratingdetection limits of about 50 ng/L foreach chromium species.

Figure 9. Cr+3 / Cr+6 detection limits.

Figure 10. Chromium calibration curves.

Cr+3

Cr(III)

Hei

ght

Hei

ght

Adjusted Amt Adjusted Amt

Cr(VI)

Cr+6

100 ng/L50 ng/L25 ng/L

Time (minutes)

Quantification

The ability to quantitatively determinethe amount of each species presentwas tested on a sample of tap waterfrom our lab. External calibrationswere made from 50 µL injections of0.1, 0.5, 1, 5, and 10 µg/L of eachchromium species in the mobile phase,and peak heights were measured.The resulting curves are shown inFigure 10. The regression for eachspecies is 0.999, thus demonstratingthe linearity and reproducibility ofthe technique.

The tap water sample was then analyzed, but the resulting chro-matogram did not yield any peaks,thereby indicating the absence of orextremely low levels of chromium.(The same sample was later analyzedfor total chromium, and the results

show < 75 ng/L of chromium present.)The tap water was then spiked with0.5 µg/L of each chromium speciesand run against the calibrationcurves. The results of this analysisshow that Cr+3=0.62 µg/L andCr+6=0.36 µg/L; the chromatogramsof the spiked sample and 0.5 µg/Lcalibration standard appear in Figure11. Subsequent analyses and samplepreparations yielded the sameresults; therefore, it appears that theCr+6 is converted to Cr+3 after it isspiked into the sample.

To attempt to minimize this conver-sion, the water sample was dilutedfive times with mobile phase prior tospiking the chromium species. Figure12 shows the chromatograms of the diluted spiked sample and the0.5 µg/L calibration standard. These

chromatograms show that the Cr+6

conversion in the spiked water sampleis minimized since the peaks closelymatch those of the calibration stan-dard. Quantitative analysis of thespiked sample shows that Cr+3=0.57µg/L and Cr+6=0.47 µg/L, thus confirming that conversion of Cr+6 toCr+3 is minimized. These results suggest that something present in thewater sample catalyzes the chromiumconversion; the addition of mobilephase to the sample either dilutes thecatalyzing species or preferentiallylocks the chromium in its originalstate. Much work has been publishedabout the interconversion of chromiumspecies and ways to preserve theoriginal oxidation state, but thisremains a difficult and tricky part ofthe analysis.

Figure 11. Chromatograms of 0.5 µg/L chromium spike in tap water (black) and 0.5 µg/L chromium calibrationstandard (green).

Figure 12. Chromatograms of 0.5 µg/L chromium spike in tap water diluted 5x (blue) and 0.5 µg/L chromium calibrationstandard (black).

Cr+6

Cr+3

Conclusions

This work has shown that trivalentand hexavalent chromium can beseparated using ion pair HPLC anddetected with ICP-MS in less than 3minutes. It was seen that many factorsmust be considered and optimized inorder to achieve successful separationand reproducibility. These includecolumn cleaning and pretreatment,ion-pair reagent concentration,organic modifier concentration

(methanol), vial material, and detectionmode. Once these parameters aredeveloped, low concentrations canbe measured, with an estimateddetection limit of 50 ng/L (ppt) foreach species in DRC mode. Theadvantages of using a DRC ICP-MSare clearly evident in reducing interferences and minimizing base-line noise, making the method morerugged for the variety of matricesand concentrations that may be encountered.

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