Fluorimetric Determination of Free Cyanide by Flow-Injection Analysis

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Fluorimetric Determination of Free Cyanide by Flow-Injection AnalysisH. P. Beck a , Bin Zhang a & Adela Bordeanu aa Institute of Inorganic and Analytical Chemistry and Radiochemistry, Saarland University,Saarbrücken, GermanyVersion of record first published: 02 Feb 2007.

To cite this article: H. P. Beck , Bin Zhang & Adela Bordeanu (2003): Fluorimetric Determination of Free Cyanide by Flow-Injection Analysis, Analytical Letters, 36:10, 2211-2228

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©2003 Marcel Dekker, Inc. All rights reserved. This material may not be used or reproduced in any form without the express written permission of Marcel Dekker, Inc.

MARCEL DEKKER, INC. • 270 MADISON AVENUE • NEW YORK, NY 10016

ANALYTICAL LETTERS

Vol. 36, No. 10, pp. 2211–2228, 2003

OPTICAL METHODS

Fluorimetric Determination of Free Cyanide

by Flow-Injection Analysis

H. P. Beck,* Bin Zhang, and Adela Bordeanu

Institute of Inorganic and Analytical

Chemistry and Radiochemistry,

Saarland University, Saarbrucken, Germany

ABSTRACT

A flow-injection analysis system for the rapid and sensitive

fluorimetric determination of free cyanide is developed. The deter-

mination is based on oxidation of nonfluorescent thiamine to highly

fluorescent thiochrome (�ex/�em¼ 370/440 nm) in alkaline medium by

Cu2þ when CN� is present. After designing an appropriate manifold,

the effects of chemical variables, FIA system parameters and

temperature on the determination of CN� were studied in

detail. Under optimum experimental conditions the working range

was linear from 0.02 to 2.5mg L�1 with a high sampling throughput

of 120 h�1 at room temperature. The precision, in terms of

relative standard deviations for ten replicates of analysis of

1.0mg L�1 CN�, was 1.9%. Interference studies were performed

*Correspondence: H. P. Beck, Institute of Inorganic and Analytical Chemistry

and Radiochemistry, Saarland University, P.O. Box 15 11 50, D-66041

Saarbrucken, Germany; E-mail: [email protected].

2211

DOI: 10.1081/AL-120023712 0003-2719 (Print); 1532-236X (Online)

Copyright & 2003 by Marcel Dekker, Inc. www.dekker.com

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with typical cations and anions in natural waters. CN� spiked in

synthetic water samples was determined by this proposed FI

method with good results. The composition of the reduced product

[Cuþ(CN�)n]1�n was determined using an indirect method. In

addition, results of cyclovoltammetric measurements of thiamine

are reported which throw light on the electrochemical process.

Key Words: Fluorimetric; Cyanide; Flow-injection analysis.

1. INTRODUCTION

Cyanide is an important environmental contaminant that occurs insurface and ground water as a result of the discharge of industrialwaters.[1] Owing to its toxicity, the development or improvement ofmethods for its determination in industrial effluents as well as in naturaland drinking waters is a subject of interest. In order to detect abnormalcyanide levels rapidly, the use of on-line monitoring systems is highlydesirable. Moreover, methods used for cyanide determination shouldbe sufficiently sensitive to detect the low concentration levels allowedby law, e.g., the maximum value for cyanide in drinking waters is0.05 mg L�1. Therefore, a highly sensitive fluorimetric detection withthe help of an on-line monitoring system (FIA) could be advantageous.

The methods reported in literature include spectrophotometricdetections based on Konig’s reaction[2–4] which is a current standardmethod[5] and other electrochemical,[6–7] capillary electrophoresis[8] andFIA[9–11] methods. Problems encountered with these methods are highcost, lengthy analysis time and poor selectivity. Although severalfluorimetric methods[12–14] have been described for batch analysis ofcyanide, little work has been performed with fluorimetric detection inon-line systems.[15–16] Thus we propose a novel fluorimetric method byFIA technique for the rapid determination of free cyanide, which is basedon the oxidation reaction of nonfluorescent thiamine to form fluorescentthiochrome by Cu2þ only in the presence of cyanide.

2. EXPERIMENTAL

2.1. Reagents

All reagents were of analytical grades and solutions were freshlyprepared in ultrapure water (Milli-Q plus system, Millipore, UK). CN�

2212 Beck, Zhang, and Bordeanu

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working solutions were diluted from 1000 mg L�1 standard solution(Merck, Germany). A 0.1 mol L�1 Cu2þ solution was prepared by dis-solving 2.50 g CuSO4�5H2O (Merck, Germany) in water and diluting to100 mL in a volumetric flask. A 0.01 mol L�1 thiamine solution wasprepared by dissolving 3.373 g of thiamine hydrochloride (ACROS,Belgium) in water and diluting to 1000 mL. A 0.06 mol L�1 boraxsolution (to maintain the final mixing solution at pH 9.0) was preparedby dissolving 22.88 g of Na2B4O7�10H2O (Fluka, Switzerland) in waterand diluting to 1000 mL in a volumetric flask.

All solutions used were degased prior to use in a Sonorex bath(Brandelin, Germany).

2.2. Apparatus

Figure 1 illustrates the designed FIA manifold. A 12-channelperistaltic pump (ISMATEC, Model IPC-12, Switzerland) was used togenerate all the flowing streams. A 4-way Teflon rotary valve (Type 50,LATEK, Germany), which was connected to a computer controlledmotor, was used to inject samples automatically. The flow systemconsisted of 1.0 mm i.d. tubing (Bohlender, Germany) throughout,except that 0.38, 0.64 and 0.89 mm i.d. pump tubes (Tygon, England)were used for delivering the aqueous solutions in order to get differentflow rates under the same rotation speed of the pump.

The fluorescence intensity was measured by a Model SFM-23Spectrofluorometer (Kontron, Switzerland) (10 nm of spectral slitwidths are used for excitation and emission monochromators) equippedwith a flow-through cell (inner volume 8 mL), whose output was recorded

D W

R1

S

D

C

R2

R3 P

MC1 (50 cm/1.0 mm i.d.)

MC2 (150 cm/1.0 mm i.d.)

Figure 1. Schematic diagram of the FIA configuration for the fluorimetric

determination of CN�. S—sample injection (100mL), P—peristaltic pump

working at 30 rpm, C—carrier (MQ-H2O), R1—0.0015 molL�1 Cu2þ solution,

R2—0.0025 molL�1 thiamine solution, R3—0.06mol L�1 borax solution,

D—detector of the spectrofluorimeter (�ex/�em¼ 370/440 nm), MC1�2—mixing

coils, W—waste.

Fluorimetric Determination of Free Cyanide 2213

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by a computer via an A/D converter. In addition a manifold using a multi-port valve (Bio-Chem Valve Corp, Germany) injection system was devel-oped so that the measurements of several standard solutions and samplescould be finished automatically under the control of the computer.

The measurement of the electrochemical potential of thiamine wasperformed using cyclic voltammetry with a Model 263A Potentiostat(Princeton Applied Research, USA) with a Model 303A static mercurydrop electrode (SMDE). The three-electrode system consisted of ahanging mercury drop electrode (HMDE), an Ag/AgCl/saturatedaqueous KCl reference electrode and a Pt auxiliary electrode. Thevoltammograms were recorded and analyzed using a Model 270Electrochemical Analysis Software (EG&G PARC, USA).

2.3. Procedure

The FIA system shown in Fig. 1 consisted of a carrier stream (H2O)and three reagent streams R1, R2 and R3, which were each propelled viathe peristaltic pump working at a speed of 30 rpm (revolutions perminute). The CN� sample solution was injected into the carrier streamC, which then merged with R1 (0.0015 mol L�1 Cu2þ solution) through a50 cm long mixing coil (MC1). At the next merge point, the sample-Cu2þ-carrier stream mixed with R2 (0.0025mol L�1 thiamine solution) and R3

(0.06mol L�1 borax solution) through a 150 cm long mixing coil (MC2),in which the redox reaction took place and the fluorescent speciesthiochrome was produced.

The fluorescence signal was monitored as the mixture passed throughthe flow-through cell (�ex/�em¼ 370/440 nm), which is installed in a waterthermostatable holder inside the detector. The transient signal from thedetector was recorded as a peak, the height of which was proportional tothe CN� concentration in the sample. The cycling time was set at 30 sallowing about 120 injections per hour including the time needed forloading and injecting sample solutions. Three replicate injections persample were made in all instances.

3. RESULTS AND DISCUSSION

Cu2þ is a weak oxidant, which can be reduced usually by formingsuch reaction products as insoluble cuprous compounds (e.g., Cu2O, CuI)or stable cupro complexes. However, in the presence of CN�, Cu2þ

acquires a powerful oxidizing characteristic and can react with many


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reductants, due to the formation of extremely stable cuprocyanide com-plexes.[17] The corresponding half reactions and their electrode potentials(vs. NHE, normal hydrogen electrode) are as follows[18]:

Cu2þþ 2e��!Cu E0

¼ 0:337 V ð1Þ

Cu2þþ e��!Cuþ E0

¼ 0:152 V ð2Þ

Cu2þþ 2CN�

þ e��!½CuþðCN�

Þ2� E 0

¼ 1:12 V ð3Þ

A more detailed discussion of the measurement of the electrochemi-cal potential of thiamine is given in Appendix A. Because of the highredox potential of Cu2þ/[Cuþ(CN�)2]

�, Cu2þ can be reduced by somereductants in the presence of cyanide. One example of its application is aAAS/FIA method,[19] in which an on-line column containing cupricsulfide (CuS) was used to form a soluble species [Cuþ(CN�)4]

3�. Thenthrough measuring the amount of Cuþ by AAS the concentration ofcyanide was determined.

The application of the organic reagent thiamine in analyticalchemistry is based on the fact that it can be oxidized by many substancesto form thiochrome, which is a fluorescent compound and also a chemi-luminescent emitter.[20] Many related analytical methods for the determi-nation of thiamine or oxidizing agents have been established throughmeasuring the fluorescence intensity of thiochrome. The oxidation reac-tion of thiamine to form thiochrome takes place in alkaline solution andthe strongest fluorescence signal can be observed between pH 8–10.

In this proposed FIA fluorimetric method, the nonfluorescent thia-mine can be oxidized by Cu2þ after injecting CN� into the flow system toproduce the fluorescent thiochrome at pH 9.0, which can be continuouslydetected when the stream flows through the detector (�ex/�em¼

370/440 nm). The maximum peak height of the FI response curve(relative fluorescence intensity) is proportional to the concentration ofCN�. The related reaction is as follows:

Cu2þþ nCN�

þ thiamine �!pH 9:0

½CuþðCN�

Þn1�n

þ thiochrome ð4Þ

(see also Appendix B).This redox reaction takes place only in the presence of CN�. No

fluorescence of thiochrome has been observed apart from the signal ofthe baseline of solutions containing only Cu2þ, thiamine and buffer. Bymeasuring the fluorescence intensity of the thiochrome formed in the flowsystem after injecting a CN� containing solution, the concentration of


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CN� can be determined. This is the basic mechanism of the FI fluorim-etric method for the quantitative determination of CN�.

3.1. Optimization of the Chemical Variables

The effects of reagent concentrations on the sensitivity (peak height)when measuring 1.0 mg L�1 CN� were studied with fixed manifoldparameters at room temperature: flow rates for the stream of C, R1, R2

and R3 were 1.15, 0.6, 0.6 and 0.2 mL min�1 respectively under the pumprotation speed of 30 rpm, 0.06 mol L�1 borax was used as buffer solution(to keep the final mixing solution in MC2 at pH 9.0), sample injectionvolume was 100 mL, the lengths of mixing coils MC1 and MC2 were 50and 150 cm.

The peak height increased by increasing the Cu2þ concentration from0.0002 up to 0.0015 mol L�1. A greater amount of Cu2þ results in adecreased sensitivity, as shown in Fig. 2A. Thus 0.0015 mol L�1 Cu2þ

was selected for all further optimization experiments.The effect of varying the thiamine concentration on the peak height

was tested from 0.0002 to 0.004 mol L�1. Figure 2B shows that the peakheight reaches a maximum and remains quite constant at a concentrationabove 0.0025 mol L�1, which was thus selected for all further optimi-zation experiments.

The effect of pH on the sensitivity of the redox reaction in the flowsystem is not discussed in this paper, because the oxidation reaction ofthiamine by different oxidants to form fluorescent thiochrome is wellknown to proceed in alkaline medium and best results could be obtainedbetween pH 8.0–10.0. Therefore 0.06 mol L�1 borax was used as buffersolution to maintain the final mixing solution in MC2 at pH 9.0.

3.2. Optimization of the FIA System

The optimum of the FIA system variables was obtained as a com-promise between sensitivity and sampling throughput with the optimumreagent concentrations. In order to get a high sensitivity, the flow rate ofcarrier C should be kept higher than that of the reagents. After testing,0.89, 0.64, 0.64 and 0.38 mm i.d. pump tubes were selected to deliver C(H2O), R1 (Cu2þ), R2 (thiamine), R3 (buffer solution) respectively.

High sensitivity could be obtained when a low flow rate was selected.But too low flow rates yield a poor sampling throughput. Figure 3A clearly


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reflects the effect of flow rate on the sensitivity by only changing the pumprotation speed for the selected pump tubes, which will influence the reac-tion time in the mixing coil greatly but has little effect on the concentrationof each reagent. As a compromise, 30 rpm was selected for the furthermeasurements, under which flow rates of the four streams C, R1, R2 andR3 were 1.15, 0.6, 0.6, 0.2 mL min�1 and sampling throughput was 120 h�1

if the cycling time was set at 30 s including the sample injection time.The length of the mixing coil MC1 had a certain influence on the

sensitivity, hence 50 cm long 1.0 mm i.d. tubing was used to mixthe injected sample and Cu2þ solutions. Figure 3B shows the effect ofthe length of MC2 on the peak height by measuring 1.0 mg L�1 CN�. Onehundred and fifty centimeters of an 1.0 mm i.d. tubing three-dimension-ally braided was selected for the further measurements.

Conc. o f Cu2+(m ol L-1)

Con c. o f th iam in e (m ol L-1)

A

B

0,000 0,001 0,002 0,003 0,004

0

5

10

15

20

25

pea

k he

ight

0,000 0,001 0,002 0,003 0,004 0,005

0

5

10

15

20

25

pea

k he

ight

Figure 2. Effect of Cu2þ and thiamine concentrations on the sensitivity (peak

height) of measuring 1.0mg L�1 CN�. All the other conditions are the same as

indicated in Fig. 1.


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It is known that the dispersion coefficient (D) is an important param-eter in the design of a FIA system, which in fact reflects the degree ofdilution of the injected sample from the injection point until it arrives inthe detector. Some standard dyes are usually used to determine the valueof D (within their linear ranges). For the FIA system shown in Fig. 1,0.1 mg L�1 of standard fluorescein solution (�ex/�em¼ 470/525 nm) wasused for such a measurement.

According to the definition of the dispersion coefficient, the value ofD for this FIA system is calculated as follows:

D ¼ C0=Cmax¼ H0=hmax

¼ 3:9 ð5Þ

where C0 being the original sample concentration to be injected, Cmax

the concentration of the fluid element at the peak maximum of thedispersion zone; H0 and Hmax are signals (peak height) corresponding toC0 and Cmax.

3.3. Effect of Temperature on the Sensitivity

The effect of reaction temperature on the sensitivity (peak height) ofmeasuring 1.0 mg L�1 CN� was studied by putting MC2 into a thermo-static bath. Increasing the temperature from 20 to 70C for the redoxreactions between Cu2þ and thiamine in the presence of CN� (in MC2)had a significant effect on the peak height, as shown in Fig. 4. Thesensitivity of determination can be increased considerably at highertemperatures. This acceleration of kinetics has also been observed for

10 20 30 40 50 600

5

10

15

20

25

30

35

peak

hei

ght

0 50 100 150 200 2500

5

10

15

20

25

30

peak

hei

ght

Pump rotation speed (rpm) Length of mixing coil (MC2, cm)

A B

Figure 3. Effects of flow rate (pump rotation speed, rpm) and mixing coil length

(MC2) on the sensitivity of measuring 1.0mg L�1 CN�. All the other conditions

are the same as indicated in Fig. 1.


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reactions between thiamine and other oxidants. For all the measurementsthe whole FI system was kept at room temperature and the flow-throughcell was installed in a water thermostatable holder inside the detector inorder to avoid the temperature increase of the cell resulting from the longtime irradiation or the instrument itself.

3.4. Interferences

In order to assess the possible analytical application of this method,the effect of some concomitant species on the determination of free CN�

which could exist in natural waters was studied (Table 1). It was foundthat most of the selected ions except S2� had no or weak interferences onthe determination of 1.0 mg L�1 CN� when their concentration was up to20 mg L�1. In the presence of 20 mg L�1 S2�, 1.0 mg L�1 CN� could notbe detected. This may result from the formation of the insoluble CuS inMC1, which consumed large amounts of Cu2þ before it mixed withthiamine in MC2 for the redox reaction. Therefore almost no detectablethiochrome formed. Twenty milligrams per liter EDTA had no effect onthe determination inspite of its chelate reaction with Cu2þ (logK¼ 18.8),because the formation of the cyanocuprous complex has a much greaterstability constant. Since EDTA is a representative for complexing agentsthis suggests that other ones, such as humic or tannic acids etc., doprobably not interfere with the determination of cyanide.

20 30 40 50 60 700

10

20

30

40

50

60

70

80

peak

hei

ght

Temperature (°C)

Figure 4. Effect of reaction temperature on the sensitivity of measuring

1.0mg L�1 CN�. All the conditions are the same as indicated in Fig. 1.


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3.5. Analytical Performance Characteristics

Under the selected optimum experimental conditions, the peakheight of the FI response curve was a linear function of the CN� concen-tration in the range of 0.1–2.5mg L�1 using the FIA manifold as shownin Fig. 1 at room temperature, with a sampling throughput of 120 h�1.The precision of this FI fluorimetric method, evaluated as the relativestandard deviation (RSD) of ten replicates of 1.0 mg L�1 of CN� was1.9%. Figure 5A gives the FI response curves of CN� standard solutionswith different concentrations.

One of the merits of FIA is that its performance and working rangescan be varied by changing some parameters of the FIA configurationsystem, i.e., the length of mixing coils, the size of pump tubes and thevolume of sample injection etc., which can affect the sensitivity of themeasurement by affecting either the reaction time or the degree ofdilution of the sample in the flow system. The optimum result can beobtained usually as a compromise between sensitivity and samplingthroughput. For example, in the FIA manifold shown in Fig. 1, theresponse signal was linear with the CN� concentration up to2.5 mg L�1, when the size of the pump tube for carrier stream C wasselected to be 0.89 mm i.d. When substituting it for smaller ones, 0.64or 0.38 mm i.d. pump tubes (diluting the injected samples to a greater

Table 1. Determination of 1.0mgL�1 CN� in the presence

of 20 fold (w/w) interfering ions.a

Interfering

ions

Relative peak

height

Interfering

ions

Relative peak

height

NHþ4 110 F� 98

Ca2þ 100 Cl� 96

Mg2þ 92 NO�2 89

Al3þ 92 NO�3 89

Fe3þ 83 CO2�3 57

Cd2þ 82 SiO2�3 75

Cr6þ 96 PO3�4 92

Ni2þ 78 SCN� 64

Pb2þ 87 S2� 0

EDTA 97 I� 108

aThe relative peak height of the FI response curve for

1.0 mgL�1 CN� standard solution without interfering ions

was considered as 100.


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degree), the linear range of measurement can become wider. Figure 5Bshows calibration curves for 1.0–5.0 mg L�1 CN� with different sizes ofpump tubes for carrier stream C.

By increasing the volume of sample injection to 200 mL and using a1.02 mm i.d. pump tube for the carrier stream C, the measurement limitcan be reduced to 0.02 mg L�1 with a sampling throughput of 90 h�1 if thecycling time was set to 40 s. Figure 5C gives the FI response curves ofCN� standard solutions from 0.02–0.1 mg L�1.

It is possible to determine even lower concentrations of CN� if themeasurement is performed at higher temperature.

Time (min)

A

0 3 6 9 12 15

20

40

60

80

2.5

2.0

1.5

1.0

0.5

0.250.1re

lativ

e fl

uore

sce

nce

inte

nsity

Conc. o f CN- (m g L- 1)

B

0 1 2 3 4 50

10

20

30

40

50

0.89 mm i.d.

0.64 mm i.d.

0.38 mm i.d.

peak

hei

ght

Time (s)

C

0 20 40 60 80 10050

60

70

80

90

100

0

0.02

0.04

0.06

0.08

0.1

Re

lativ

e fl

uore

sce

nce

inte

nsity

Figure 5. Analytical performance characteristics of the FI fluorimetric deter-

mination of CN�. All the conditions are the same as indicated in Fig. 1 except

the selected parameters described in B and C. A: FI response curves of CN�

standard solutions from 0.1 to 2.5 mgL�1 using the manifold as shown in Fig.

1. Calculation result: �I¼ 0.05þ 23.91C (R¼ 0.999, N¼ 7). B: Calibration curves

of 1.0–5.0 mgL�1 CN� standard solutions with different sizes of pump tubes for

carrier stream C. C: FI response curves (three replicates) of CN� standard

solutions from 0.02 to 0.1mgL�1 with 200 mL sample injection volume and

1.02mm i.d. of pump tube for the carrier stream C giving a calibration curve

with �I¼�1.63þ 265.5C (R¼ 0.996, N¼ 5).


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3.6. Determination of CN2 in Synthetic Samples

As another procedure to evaluate the application of this method, astandard addition method was used for the determination of CN� spikedin river water (Nied) and nonindustrial waste water, because in the caseof industrial accidents, these natural waters can possibly be contaminatedwith cyanide. Prior to analysis these samples were filtered using the No. 1filter paper (Whatman, England) without any other treatment proce-dures. The results are summarized in Table 2 and demonstrate the generalreliability of this method.

4. CONCLUSION

This FIA system provides a novel simple, rapid and sensitivefluorimetric method for the determination of free CN�. Different linearranges can be obtained by selecting various parameters of the FIAsystem. With the manifold shown in Fig. 1 the peak height of FI responsecurves was a linear function of the CN� concentration in the range from0.1 to 2.5 mg L�1 with a high sampling throughput of 120 h�1. With otherparameters of the FIA configuration system the measurement limit (orlimit of quantification, LOQ) could reach as low as 0.02 mg L�1 which isunder the maximum value allowed in drinking waters (0.05mg L�1).Compared with other reported FIA determination methods, thismethod excels by low cost, good selectivity and high samplingthroughput. The result of CN� determination in synthetic andnatural water samples demonstrates that this method has great potentialfor the sensitive and rapid determination of free cyanide in real watersamples.

Table 2. Determination of cyanide in synthetic samples.

Sample

CN� spiked

(mgL�1)

CN� found

(mgL�1)

CN� found after

3 daysa (mgL�1)

River water 0.10 0.10 0.103

0.50 0.51 0.56

Waste water 0.10 0.12 0.089

0.50 0.55 0.56

aSamples were stored at 4C in the refrigerator.


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APPENDIX A

Measurement of the Electrochemical Potential of Thiamine

Values for the electrochemical potential of thiamine have beenreported in the literature (see Table 3). The values show considerablescatter indicating that the specific conditions such as pH value, concen-tration and temperature play a decisive role. In order to get a value for theconditions used in our analytical procedures we have used cyclic voltam-metry. The measurement was done at pH¼ 9.0 using a 0.1 mol L�1

NH3/NH4Cl buffer solution. Figure 6A gives a typical result of such anexperiment. Its interpretation could lead to the conclusion that the oxida-tion process takes place at about �336 mV (vs. Ag/AgCl) and that it is notreversible. (The large peaks near 0mV are due to the fact that at about�150 mV the reduction of calomel (Hg2Cl2) takes place which is formedwhen the potential at the working electrode is made positive enough tocause the oxidation of the mercury which is maximum at 0.4 V.) However amore elaborate study shows that the detected features depend on the para-meters of the scanning procedure. Weak additional peaks indicating areversible reaction appear during the second scan cycle in the region�700 to �500 mV (Fig. 6B) and they become stronger when beginningto scan at more positive potentials (Fig. 6C). The amplitude of thesepeaks as well as that of the one at�336 mV strongly depend on the startingconditions of the cycle indicating that differently charged species areabsorbed at the HMDE depending on its potential. The proposed mech-anisms for the formation of thiochrome[20,22] show different pathways withdifferent intermediates for the oxidation reaction which indeed showdifferent charges or polarization states and will therefore adhere tothe electrode surface differently depending on the change of its potentialwith time. The scatter of potential values given in Table 3 may also

Table 3. Half wave potential of thiamine in literature.

Solution pH E1/2 (V, vs. SCEa) Reference

Phosphate 7.2 �1.26 (catalytical wave) [21]

Sorensen 11.0 �0.38 (anodic wave) [21]

0.1N LiOH — �0.46 [21]

NH3/NH4Cl 9.0 �0.336 (vs. Ag/AgCl) [this work, see

Appendix A]

aSCE, standard calomel electrode.


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-1000 -800 -600 -400 -200 0 200 400 600-2,0

-1,5

-1,0

-0,5

0,0

0,5

1,0

1,5

2,0

A (µ

A) -1500 -1000 -500 0 500

-100

-50

0

50

100

150

E vs. Ag/AgCl (mV)

C

Figure 6. Determination of electrochemical potential of thiamine with

cyclic voltammetry. Conditions: 0.0025mol L�1 thiamine, 0.1 molL�1 NH3/

NH4Cl, pH¼ 9.0, Scan speed: 200mV s�1. A. Scan: 0!�1500mV, single

cycle; B. Scan: 0!�1500 mV, two cycles; C. Scan: 400!�1500mV, two cycles.

-800 -700 -600 -500 -400 -300 -200 -100 0 100 200 300-5

-4

-3

-2

-1

0

1

2

3

4

A (µ

A)

-1600 -1200 -800 -400 0-20

0

20

40

60

80

E vs. Ag/AgCl (mV)

B

-1600 -1400 -1200 -1000 -800 -600 -400 -200 0-3

-2

-1

0

1

2

3

0.1 M NH3/NH

4Cl

0.1 M NH3/NH

4Cl + 0.0025 M thiamine

A (

µA)

E vs. Ag/AgCl (mV)

A


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be due to the fact that different steps of these pathways have beenmonitored in the experiments. A detailed study of this system is under way.

APPENDIX B

Determination of the Composition of the

Coordination Complex [CuQ(CN2)n]1�n

Under the optimum experimental conditions with the designed FIAmanifold as shown in Fig. 1, the peak height of the FI response curve wasa linear function of CN� concentrations (the amounts of Cu2þ andthiamine were always in excess). Therefore the value of n should be con-stant in the reaction product although the concentration of CN� variedin a great range. In order to measure n an indirect determination proce-dure using batch experiments according to the mole ratio method (satu-rated method) was designed by measuring the fluorescence intensity ofthiochrome which was produced after mixing all the reagents, i.e., Cu2þ,CN�, thiamine and buffer solutions. It is assumed that an unstableintermediate [Cu2þ(CN�)n]

2�n forms at first, which can then react withthiamine and be reduced to [Cuþ(CN�)n]

1�n. The result shows that thevalue of n was approximately 2 (The crossing point of the two linear fitsin Fig. 7 was 2.3).

Mole ratio of [CN-]/ [ Cu2+ ]0 1 2 3 4 5

10

20

30

40

50

60

peak

hei

ght

Y1 = -2.85 + 18.88 * X

1

Y2 = 36.3 + 1.1 * X

2

Figure 7. Indirect determination of the composition of the coordination com-

plex [Cuþ(CN�)n]1�n using the mole ratio method (saturated method). Conc.:

10�5 mol L�1 Cu2þ, 10�4 mol L�1 thiamine, pH¼ 9.0. The peak height at

�em¼ 440 nm in the fluorescence emission spectrum of thiochrome (�ex¼

370 nm) was measured after mixing all the reagents and waiting for 10 min.


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Thus the overall reaction 3 can be described as:

Cu2þþ 2CN�

þ thiamine �!pH 9:0

½CuþðCN�

Þ2�þ thiochrome ð6Þ

with the intermediate steps:

Cu2þþ 2CN�

�!½Cu2þðCN�

Þ2 ð7Þ

½Cu2þðCN�

Þ2 þ thiamine �!pH 9:0

½CuþðCN�

Þ2�þ thiochrome ð8Þ

The offset of the mole ratio of [CN�]/[Cu2þ] from 2.0 could be due to tworeasons. The first one might be the effect of fluorescence inner filteringwhich would generally reduce the fluorescence values and cause a shift ofboth lines. However, the experimental result with an 8 mL flow throughcell having shorter pathways of light showed that this is not the reason.Because of the low CN� concentration at the beginning too little thio-throme is formed and the fluorescence is below the detection limit (seeFig. 2B). This causes a shift of the first segment of the plot away from theorigin by about 0.3.

ACKNOWLEDGMENT

The authors gratefully thank the DAAD (DEUTSCHERAKADEMISCHER AUSTAUSCHDIENST) for financial support ofthis work.

REFERENCES

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Received December 11, 2002Accepted March 4, 2003


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Documents

Fluorimetric Determination of Free Cyanide by Flow-Injection Analysis