Journal of Chromatography A, 1110 (2006) 235–239

Incorporation of flow injection analysis or capillary electrophoresis withresonance Rayleigh scattering detection for inorganic ion analysis

Li Qi a, Zhi-qiang Han a,b, Yi Chen a,∗a Laboratory of Analytical Chemistry for Life Science, Department of Chemical Biology, Institute of Chemistry,

Chinese Academy of Sciences, P.O. Box 2709, Beijing 100080, PR Chinab Graduate School, Chinese Academy of Science, Beijing 100039, PR China

Received 3 November 2005; received in revised form 27 December 2005; accepted 11 January 2006Available online 7 February 2006

Abstract

Resonance Rayleigh scattering (RRS) has been explored as a detection (RRSD) technique for capillary electrophoresis (CE) or flow injectionanalysis (FIA) of inorganic ions. The detection was achieved through a scattering probe of ion-association complex formed from rhodamine B(Rh B) and iodine. The probe scatters strongly at 630 nm when oxidants such as Cr2O7

2−, MnO4− and ClO− present in a mixed solution of Rh

BpCacsacC©

K

1

iobatobis

E

0d

and iodide. The scattering disappears once iodine is reduced by reductants. Oxidant or reductant species in a sample can thus be detected byositive or negative RRS signal. To verify the RRSD, FIA-RRSD was first constructed and continuous measurement of testing samples containingr2O7

2−, MnO4− and/or ClO− was performed. The detection limits reached a level of decade nM and a linear range was found between peak height

nd concentration at the range of 0.255–2.04 �M for Cr2O72−, 0.158–3.16 �M for MnO4

−, and 1.18–9.43 �M for ClO−, with linear regressionoefficients of all above 0.99. The run-to-run relative standard deviation of peak height was less than 3% (n = 6). CE-RRSD was then set up andtudied, using a capillary of 75 �m i.d. × 33 cm filled with a running buffer of 50 mM citrate and 25 mM Tris (pH 3.32) and worked under −12 kVt room temperature. The CE eluent was at-line conducted into a stream of rhodamine B and iodine flowing inner a wide tube by plugging theapillary outlet into the wide tube. Different mixtures prepared from Cr2O7

2−, MnO4− and ClO− were successfully separated and detected by the

E-RRSD.2006 Elsevier B.V. All rights reserved.

eywords: Resonance Rayleigh scattering detection; Capillary electrophoresis; Flow injection analysis; Iodide; Basic rhodamine B dye; Permanganate; Dichromate

. Introduction

Capillary electrophoresis has been shown to be powerfuln the separation of small ions but its application was oftenbstructed by detection difficulty. Several methods have sinceeen explored, of which indirect UV absorption [1], direct UVbsorption through complexation [2], and conductometric detec-ion (CD) [3–5] were on the top for use. However, they have morer less problems to allow the free selection of running buffer foretter resolution. It remains thus a challenge to detect the smallonic eluent in CE, which has made us thinking about to exploreome other detection techniques if possible.

In theory, light scattering is a candidate for exploration.xcept for Raman spectroscopy [6], Rayleigh scattering (RS)

∗ Corresponding author. Tel.: +86 10 62618240; fax: +86 10 62559373.E-mail address: chenyi@iccas.ac.cn (Y. Chen).

has been tried in this laboratory and resonance Rayleigh scatter-ing (RRS) was found to be worth of further investigation.

Normally, RS is performed at an incident wavelength farfrom the molecular absorption region, and the resulted signalsfrom transparent or homogeneous solutions are too poor to beused for detection [7,8]. However, if the incident wavelengthis selected within the molecular absorption band, the scatter-ing signal will greatly be enhanced due to a mechanism ofstrong adsorption and re-emission. That is the so-called RRS[8–11].

RRS has been explored as a sensitive technique to studythe molecular properties in very dilute solutions [12–17], forinstance, to extract information of size, shape or aggregationnumber from supramolecular assemblies [18–26]. The scat-tering intensity of such species could be enhanced by sev-eral orders of magnitude at or near the absorption band whenchromophore units and a strong electronic coupling existed[27–30].

021-9673/$ – see front matter © 2006 Elsevier B.V. All rights reserved.oi:10.1016/j.chroma.2006.01.053

236 L. Qi et al. / J. Chromatogr. A 1110 (2006) 235–239

It was thus tried to incorporate RRS with CE because RRSwas so sensitive as shown in the literatures that it looked appli-cable to detecting the CE eluents. To produce RRS signal,a suitable enhancing reagent system composed of rhodamineB dye (Rh B) and KI was selected and CE eluent was con-ducted into the reagent stream by at-line hyphenation. Thebackground scattered from Rh B and iodide is commonly veryweak but strong signal will appear once the iodide is oxidizedby some oxidant analytes to give iodine which in turn formscomplexes with Rh B, that is [Rh B+][I3

−]. All ionic and non-ionic (like H2O2, etc.) species able to oxidize iodide or toreduce iodine are in theory detectable by positive or negativesignals.

For demonstration, oxidative inorganic ions such as Cr2O72−,

MnO4− and ClO− were tried and measured first by FIA-RRSD

and then by CE-RRSD (both systems were established in thislaboratory). The results confirmed that FIA-RRSD was able tomeasure �L-to-nL samples at a concentration down to decadenM, which implies that RRS may serve as a type of detec-tor for CE. CE-RRSD of mixed inorganic ions was hencestudied and successful detection of the separated bands hasbeen achieved. This paper presents the demonstration of FIA-and CE-RRSD of either individuals or mixtures of Cr2O7

2−,MnO4

− and ClO−. Some critical conditions will also bediscussed.

2

2

Xdrj

2

((u

2

dm1ca

2

w

2

a

Fig. 1. Set-ups of home-built FIA-RRSD (A) and CE-RRSD (B). 1, pressuredgas; 2, 4, 9, FEP tube; 3, seal vial; 5, injection outlet; 6, injection orifice withrubber septum; 7, sample loop; 8, 4-way value; 10, signal collecting lenses andfilter; 11, laser beam; 12, PMT; 13, laser; 14, slit; 15, detection cell; 16, wastevial; 17, amplified viewing of detection cell; 18, data treating part; 19, T-shapedinterface; 20, fused-silica capillary; 21, buffer vial; 22, negative high voltagepower supply; 23, Platinum electrode (G, grounded).

2.2. Apparatus

Laboratory-built FIA-RRSD and CE-RRSD systems wereschematically shown in Fig. 1A and B, respectively. The RRSDpart was composed of a photoelectron-multiple-tube (PMT, 12),a He–Ne laser (13, Model: 3227 H-PC, U.S.A, operated at632.8 nm) as light source, signal collecting lenses with filter(10), a detection cell (15) and a data acquisition and analysisunit (18). The detector was so designed that its detection flowcell could be varied from decades of microliters to nanoliters.As a consequence, the detector was adaptable to a CE system(Fig. 1B).

The FIA-Unit consisted of only a solution delivering partand a sample injection block. The key part of the injectionblock was actually a four-way valve (home-made, 8), whichconnected a delivering vial (3) to the RRSD via a fluorinatedethylene propylene (FEP co-polymer, Tianjing Plastic Plant,China) or fused silica tube of 100–400 �m i.d. (4 and 9) andbrought injected sample plug into a flowing stream to the

. Experimental

.1. Chemicals and solutions

Sodium hypochlorite solution was purchased from Beijinginfu Chemical Reagent Institute (Beijing, China). Potassiumichromate, potassium permanganate, and other chemicals andeagents were all purchased from Beijing Reagent Work (Bei-ing, China).

.1.1. RRSD background solution (BGS)BGS was prepared just before use by mixing 4 M HCl

0.3 mL, in water), 0.005% (w/w) Rh B (0.9 mL, in water), 10%w/w) KI (0.2 mL, in water) and 6.1 mL distilled water. Watersed was triply distilled.

.1.2. Sample IFIA samples at different concentrations were prepared by

issolving target solutes in BGS and stirred completely. Theixture was allowed to react at room temperature for 10 s to

0 min depending on the concentrations of the analytes. Theompletely reacted solution was stable at room temperature forbout 1 h.

.1.3. Sample IICr2O7

2−, MnO4− and ClO− were separately dissolved in

ater and then synthetically mixed up whenever required.

.1.4. CE running bufferIt consisted of 25 mmol/L Tris and 50 mmol/L citrate,

djusted to pH 3.32 by hydrochloric acid.

L. Qi et al. / J. Chromatogr. A 1110 (2006) 235–239 237

detector. The sample was injected into a loop (7) through theinjection port (6) using a syringe. By turning the valve (8)clockwise, the injected sample was carried into the main streamand a second injection could be started again after restoringthe valve. Scattering light was detected downstream at 20 cm(15) apart from the injection port and the signal was acquiredat 1 Hz. The carrier liquid was driven by a peristaltic pump(Yinghu, SL-2800, China) through FEP tube (2) and vial 3.

The CE-RRSD (Fig. 1B) was modified from FIA-RRSD(Fig. 1A). The injection block was replaced by a T-shaped inter-face (19) and the outlet of CE capillary (20) was fitted intothe interface to let CE eluents transport continuously into theBGS stream for RRS detection. The inlet of the CE capillarywas plugged in a seal buffer vial with a platinum wire electrode(21) and connected to a negative high voltage power supply (22,Beijing Institute of New Technology Application, China). TheT-interface, the delivering vial and waste vial were all grounded(G) through platinum wire electrodes. The distance from the CEcapillary outlet to the detection cell was also 20 cm.

In addition to the home-made systems, a spectrophotometerof Techcomp UV-8500 (Shanghai, China) and a spectrofluo-rimeter of Shimadzu RF5301PC (Japan) were used at roomtemperature to measure absorption spectra or scattering lightintensity.

2.3. FIA-RRS procedure

Htatswi

2

icd(irwta

3

3

ato

Fig. 2. Absorption spectra of BGS before and after the addition of solutes.Solutes: 11.3 �M MnO4

−, 53.8 �M ClO− and 9.72 �M Cr2O72−; BGS: 0.005%

(w/w) rhodamine B, 10% (w/w) potassium iodide, 4 M hydrochloric acid andwater at a volume ratio of 4.5:1:1.5:30.5.

630 nm when solutes were added (Fig. 2). As a consequence,weak background RRS signal obtained at the selected wave-length of 632.8 nm even in the presence of an excess amount ofI−, but strong resonance scattering signal was measured afterthe addition of solutes (Fig. 3) due to the production of I2.In this case, ion-association complex of [Rh B+][I3

−] forms inthe solution and the resonance scattering cross section enlargestremendously.

To form stable ion-association complex, the solution shouldbe acidified. Hydrochloric acid, phosphoric acid and sulfuricacid were hence tested separately, of which hydrochloric acidwas found to be the best. Further studies revealed that the detec-tion sensitivity was a peak-shaped function of HCl, with themaximum signal at 0.48 mol/L HCl (Fig. 4). In this study, solu-tions of 0.4 mol/L HCl were adopted to simplify pH adjustment(not required to verify the HCl concentration very carefully) andalso to improve the operation reproducibility somewhat since theslop is not very sharp here.

The concentration of KI was checked in between 0.01and 0.05 mol/L, and the optimum signal was measured at

FSF

Before measurement, all the channels were rinsed with 0.1 MCl, 0.1 M NaOH and distilled water for 5 min each. Water was

hen pumped into the system at a flow rate of 0.30–3.0 mL/minnd 40 �L Sample I was injected to generate peak serials. Allhe peaks were detected by PMT working at −800 V while theignals were acquired at 1 Hz and treated via a chromatographicorking station of SC 1100 (Institute of Dalian Physical Chem-

stry, CAS, Dalian, China).

.4. CE-RRS procedure

CE separation was performed in a capillary of 75 �m.d. × 33 cm (Yongnian, Hebei, China). Prior to each run, theapillary was rinsed sequentially with 0.1 M NaOH (10 min),istilled water (5 min) and running buffer (5 min). Sample IImixed Cr2O7

2−, MnO4− and ClO− or individual) was then

njected by siphoning at the height of 15 cm for 10 s and sepa-ated at −12.0 kV and room temperature. The separated bandsere eluted into the BGS to react with an excess amount of I− in

he flowing stream (0.30–3.0 mL/min). The peaks were detectednd treated as in FIA-RRSD.

. Results and discussion

.1. Selection of RRS probe system

Rh B and KI were chosen to form a RRS probe systemccording to the literature [31]. Experimental data revealed thathe probe system had only weak absorption at a wavelengthf longer than 600 nm but strong absorption between 610 and

ig. 3. RRS spectra of BGS before and after the addition of oxidative solutes.olutes: 0.753 �M MnO4

−, 15.1 �M ClO− and 0.162 �M Cr2O72−; BGS as in

ig. 2.

238 L. Qi et al. / J. Chromatogr. A 1110 (2006) 235–239

Fig. 4. Influence of HCl concentration on the intensity of RRS, measured from amixture of 0.005% rhodamine B and 10% KI, with the relative standard deviation(RSD) of 0.80–1.5%.

0.016 mol/L. With these conditions, ion-association complex of[Rh B+][I3

−] could be formed within 10 s to 10 min dependingon the concentration of analytes: the higher was the concen-tration of analytes, the earlier was measured the RRS signal.For instance, it took about 6 min to obtain enough RRS sig-nal for Cr2O7

2− at a concentration of 0.14 �M, but the timewas reduced to about 10 s at 3.00 �M Cr2O7

2−. In general, thereaction time was kept at 10 min for very diluted analytes (�Mor lower). Oppositely, to obtain RRS signals in <1 s, concen-trated analytes (∼mM) should be used. This is critical to performCE-RRSD.

3.2. Quantitation of Ions by FIA-RRSD

To check the performance of RRSD, FIA-RRSD of ClO−was studied by injecting a series of Sample I prepared fromNaClO at various concentrations. Fig. 5 shows an example.In order to improve the detection sensitivity, sample volumeand the flow rate of carrier were investigated from 0.30 to3.0 mL/min. A “steady state” or the most stable measurementcondition was found at the flow rate of 3.0 mL/min and aninjection volume of 40 �L. The repeatability was measured interms of relative standard deviation (RSD) of peak height andall the data were better than 3% (n = 6) as shown in Table 1.The limit of detection (LOD) was in the range from decadenbt>

Fig. 5. Peak series of ClO− produced by FIA-RRSD at an increasing concen-tration sequence. Peak identity: 1:background or [Rh B+][I−]; 2: 1.18 �M; 3:2.36 �M; 4: 4.71 �M; 5: 9.42 �M; 6: 18.9 �M; 7: 37.7 �M; 8: 377 �M.

3.3. CE-RRSD

CE of the tested ions were tried and detected by at-columnRRSD. In order to well control the CE separation, different fac-tors had been studied including running buffer, its pH, ionicstrength, voltage, capillary, injection, detection volume, andtemperature, etc. Among the studied factors, the buffer and itspH were shown to be the key to modulate the separation anddetection sensitivity.

The results obtained in this work revealed that buffer pH hadvery complicated influence on detection, depending on the prod-uct of ion-complexes and the current. Generally, acidic buffersat pH 1.0–2.0 gave better resolution than others but baseline driftwas observed. A bit higher pH (pH 3.2) was thus adapted. As inFIA-RRSD, HCl was used to adjust the pH of running buffer sothat the CE eluent did not disturb too much to BGS.

The separation selectivity for Cr2O72−, MnO4

− and ClO−was influenced by the composition of the electrolyte. Severalchemicals that might maintain the pH of the selected runningbuffer have been investigated including citrate, acetate, phos-phate and so forth. Citrate was turned out to be the best in relevantto peak shape.

Mixed ions of Cr2O72−, MnO4

− and ClO− were then sep-arated and detected with success in a running buffer composedof 50 mM citrate and 25 mM Tris at pH 3.32 (Fig. 6). The LODsof Cr O 2−, MnO − and ClO− were 37.76 mM, 34.17 mMatT0b(

TQ

S

CMC

was

M to �M, depending on analytes. Linear curves were obtainedetween peak height and analyte concentration over one towo orders of magnitude, with linear regression coefficients of0.99.

able 1uantitation features of FIA-RRSD from six measurements

ample LODa (nM) Linear range (�M)

r2O72− 63.7 0.255–2.04

nO4− 79.1 0.158–3.16

lO− 1178 1.18–9.43

a LOD was measured at S/N = 3 by continuous dilution of Sample I; �(Rel I)

2 7 4nd 94.25 mM, respectively. This is about one order of magni-ude worse than FIA-RRSD but comparable to UV absorption.he linear range in CE-RRSD of Cr2O7

2− and MnO4− was

.170–1.70 mM and 0.316–3.16 mM, respectively. The repeata-ility of migration time was 3.17% (n = 6) for MnO4

− and 1.36%n = 6) for Cr2O7

2−.

Linear regression function r RSDa (%)

�(Rel I)a = 1.00 + 3.07C 0.9999 2.79�(Rel I) = 1.00 + 0.390C 0.9998 2.90�(Rel I) = 1.00 + 0.0988C 0.9946 2.19

defined as relative intensity of RRS; RSD was related to peak height (n = 6).

L. Qi et al. / J. Chromatogr. A 1110 (2006) 235–239 239

Fig. 6. Electropherogram of oxidative ions obtained by CE-RRSD. Runningbuffer: 25 mM Tris and 50 mM citrate (pH 3.32); voltage: −12.0 kV; capil-lary: 75 �m i.d. × 33 cm. The concentration of Cr2O7

2−, MnO4− and ClO−

were 1.70 mM, 3.16 mM and 125 mM, respectively. Peak identity:1: MnO4−; 2:

Cr2O72− ; 3: ClO−.

It should be noted that these data were measured at the BGSflow rate of 3.0 mL/L, which allowed only less than 1s’ reactionand largely diluted the sample zone. The detection sensitivitywas thus severely reduced. It is in theory possible to lower theLOD of CE-RRSD by decreasing the flow rate of BGS or prolongthe distance between CE outlet and detection window. Both casesincrease reaction time and are able to produce more I3

− andhence more [Rh B+][I3

−]. Unfortunately, the experimental datashowed that the CE peaks were dramatically distorted, causingdecrease in resolution.

Another theoretical way to improve the detection sensitivityis to replace the tube of 400 �m ID by a narrower one to conductBGS. This concept has been tried by using a fused silica capillaryof 100 �m ID as the conducting tube, but the electropherogramswere not as acceptable as those from the wide tube. Furtherinvestigations are needed to improve the performance of CE-RRSD and will be discussed elsewhere because it is out off thescope of this paper.

4. Concluding remarks

Coupling of RRSD to FIA or CE has been demonstrated. TheFIA-RRSD produced better detection sensitivity than CE-RRSDbut mixed solutes should be analyzed by the latter method.The LOD of FIA-RRSD reached decade nM while CE-RRSDreached �M, which is comparable to UV. The main reason for

sensitivity loss in CE-RRSD was resulted from the very shortmixing and traveling distance between the CE outlet and detec-tion cell.

Acknowledgement

We gratefully acknowledge the financial supports from NSFC(No. 20375042 and No. 20435030), Chinese Academy of Sci-ences (No. KJCX2-SW-H06), and Ministry of Science and Tech-nology of China (No. 2002CB713803).

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