since the use of chlorine for tap water treatment was adopted in england in the 1880s , water chlorination has been one of the most common disinfectant methods used by waters suppliers .
because chlorine is an efficient agent for inactivating several types of microorganisms , it has been preferred as a disinfecting agent to assure the bacteriological quality of the drinking water .
the water bacterial contamination can also occur in the distribution network ; therefore , to prevent this occurrence , a free chlorine residual in excess of 0.2  mg  l must be maintained throughout the distribution lines [ 3 , 4 ] .
therefore , the availability of sensitive and reliable analytical procedure for chlorine determination can be considered as an essential condition to assure the quality of tap water .
determination of free chloride in tap water has been carried out by employing as detection techniques amperometry , spectrophotometry , ion selective electrode , chemiluminescence , and so forth . in this work ,
we intend to develop a photometric procedure for the determination of residual chlorine in tap water using leuco crystal violet ( lcv ; 4,4,4-methylidynetris ( n , n - dimethylaniline ) , c25h31n3 ) as chromogenic reagent .
lcv is colorless in aqueous solution , but when oxidized at ph 4 , it forms crystal violet dye ( cv ) , which absorbs electromagnetic radiation with a maximum at 592  nm [ 9 , 10 ] .
lcv has been employed in procedures for the determination of hydrogen peroxide , iodine , iodide and hypoiodous acid [ 9 , 12 , 13 ] , iridium , and oxidized manganese .
although , lcv has been widely used in analytical procedures , it has not yet been used for hypochlorite determination , which will be attempted in this investigation .
nowadays , efforts have been made to environmentally friendly develop analytical procedures , and thus accomplishing the green analytical chemistry ( gac ) recommendation [ 1517 ] .
reduction of reagent consumption and waste generation are among the requisites necessary to attain the gac recommendation [ 18 , 19 ] .
multicommuted flow analysis ( mcfa ) [ 2022 ] and multisyringe flow injection analysis ( msfia ) [ 18 , 23 ] are efficient tools for handling low volumes of sample and reagent solutions , thus providing facilities to save them .
solenoid minipumps have been employed to propel solution in flow analysis systems replacing the peristaltic pump , presenting as an advantage small dimension [ 2426 ] , a feature that has been exploited in order to downscale the flow system [ 27 , 28 ] .
this downscaling approach seems to be a powerful tool to develop analytical procedure focused to the gac recommendation . in the research
reported here , these features are exploited to design a miniaturized flow system setup based on the mcfa process for photometric determination of hypochlorite in tap water .
all chemicals were of analytical - grade reagent . purified water with electric conductivity lower than 0.1  s cm was used throughout .   an acetate buffer solution ( 0.2  mol  l )
was prepared by dissolving 1.6408  g of sodium acetate in 20  ml of water .
after dissolution , ph was adjusted to 4.0 using a 2.0  mol  l   hcl solution . afterwards
a 1.0  g  l leuco crystal violet ( lcv ; 4,4,4-methylidynetris ( n , n - dimethylaniline ) , c25h31n3 ) stock solution was prepared by dissolving 0.1  g of solid material ( eastman kodak )   in 1  ml of phosphoric acid ( merck ) 85% ( v / v ) .
a 0.1  g  l lcv working solution was prepared daily by adequate dilution of the stock solution with water .  
a 1000  mg  l hypochlorite ( clo ) stock solution was prepared by adequate dilution of a 10% ( w / v ) reagent solution ( fluka ) in 100 ml of a 0.01  mol  l naoh solution .
hypochlorite working solutions ranging from 0.02 up to 2.0  mg  l were prepared daily by adequate dilution of the clo stock solution using a 10  mol  l naoh solution .  
prior to analysis , samples were alkalized by adding 1.0  ml of a 10  mol  l   naoh solution to a volumetric flask , and the volume was made up to 100  ml with sample .
the apparatus included a microcomputer running a software written in quich basic 4.5 , a digital multimeter with a serial interface ( minipa , et-2231 ) , three solenoid minipumps ( bio - chem valve inc .
, 090sp115 - 8 ) , a homemade interface to drive the solenoid pumps , which was coupled to the microcomputer through the parallel output port , a homemade regulated power supply ( 12  v , + 12  v ) to feed the photometer , an orange high - bright   led   (  = 590  nm ) , and a photodiode opt301 ( burr brawn ) .
a homemade flow cell machined in acrylic , with an optical path length of 20  mm and an inner diameter of 1.0  mm , and a homemade bubble - removing microdevice machined in acrylic , with an inner volume of 10  l , was similar to that   used elsewhere .
the system control and data acquisition were performed by the microcomputer , running software written in quick basic 4.5 .
the network constituted by a transistor ( te ) , and resistors were used to control the intensity of the light beam emitted by the led . the glass cylinders ( g )
conducted the light beams  i1 and i2 from the led up to the flow cell channel , and from there up to the photodetector ( det ) , respectively .
when the software was run , the solenoid minipumps p1 , p2 , and p3 were switched in an alternating on / off sequence , as shown in table 1 , so as to fill each flow line with the respective solution . afterwards , mini - pump p2 was switched on / off 40 times to wash the flow cell with carrier solution .
the calibration step included the reading of both the dark signal ( dk ) and the reference signal ( ss ) .
in the first case , the reading was done with led switched off while , in the second one , the led emission was enabled by turning forward the variable resistor ( 10  k ) wired to the base of the transistor ( tr ) .
the emission intensity was adjusted to obtain an electric potential difference ( ss ) of 2000  mv generated by the photodetector ( det ) .
the measurements dk and ss were converted to digital signal by the multimeter and sent to the microcomputer through the serial interface .
a micropumping on / off switching event is named as a sampling cycle , thus the volumes of sample and reagents solutions necessary to carry out the analysis were found by varying the number of sampling cycles . as shown in table 1 ( step d ) , micro - pumps p1 and p2 were switched simultaneously , so that the coil ( b1 ) was loaded with a mixture of reagent and buffer solutions . afterwards ( step e ) ,
micro - pumps p2 and p3 were switched on / off also simultaneously ,   so that slugs of the sample and a mixture of buffer and reagent solutions merged into the coil ( b2 ) .
the reaction for forming the compound to be monitored proceeded while the sample zone was displaced towards the photodetector ( det ) .
while steps ( d ) and ( f ) were performed , the microcomputer carried out data acquisition and saved this data as an ascii file , to allow further processing .
the absorbance was calculated using the following equation : absorb = log  [ ( s0  dk)/(ss  dk ) ] , where s0 was the sample signal , with ss and dk defined as in the previous paragraph .  
while sample processing was occurring , a plot of the analytical signal was displayed on the screen , in order to allow its visualization in real time .
after the last event ( step f ) , the software returned to step ( d ) , so as to start another analytical run .   to find the adequate volume of reagent solution ,
the assays were carried out using 20 sampling cycles for sample and varying from 1 up to 5 the on / off switching event applied to minipumps p1 and p2 ( step e ) .
the assays described above were carried out using a chromogenic reagent solution with a concentration of 0.1  g  l lcv and a 1.0  mg  l clo standard solution . to find the appropriate reagent concentration ,
experiments were performed applying 12 and 8 sampling cycles for sample and reagent solution , respectively . maintaining the hypochlorite concentration at 1.0  mg 
l , the lcv concentration was varied from 0.02 up to 0.1  g  l.   aiming to prove the effectiveness of the proposed setup , a set of tap water samples was analyzed . to allow accuracy assessment ,
hypochlorite in a ph 6.3 medium reacts with the chromogenic reagent n , n-diethyl - p - phenylenediamine ( dpd ) , which is oxidized to a red - colored semiquinonoid cationic radical ( dpd * ) that was monitored at 520  nm .
according to hatch   and lambert et al . , the stoichiometric relationship of lcv oxidation by hypoiodous acid or iodine in acid medium results in the generation of two electrons for each oxidized molecule , as indicated by the equations shown below :  
 ( 1)hio+lcvi+cv++h2o , 
 ( 2)i2+lcv2i+cv++h+ , 
							 where lcv is leuco crystal violet and cv is the colored crystal violet specie .  
since lcv could be oxidized by hypochlorite , following a reaction mechanism similar to that described for hio , one would expect that a relationship according to the equation shown below occurs :  
 ( 3)hclo  + lcvcl+cv++h2o . 
 
preliminary assays carried out using hypochlorite working solutions showed that absorbance at 592 nm was directly proportional to hypochlorite concentration , thus this feature was employed to develop the analytical procedure .  
reduction of reagent consumption and waste generation are among the requisites necessary to attain the gac recommendation [ 18 , 19 ] , and the volumes of solutions delivered by the solenoid micropumps per stroke have been used to calculate reagent consumption and waste generation .
nominally , the solenoid micropumps used in this work should deliver a solution volume of 8  l per stroke , nevertheless , in an earlier work , it was verified that volume delivered per stroke was lower than the expected value .
thus , considering this fact , the volume of solution delivered by stroke under working condition was determined , and was done by applying for each micropump 10 on / off pulses .
the water volume was collected into a vial and weighted to determine the actual volume , which was found to be 6.0  l per stroke .
the effect concerning the lcv concentration was investigated using a 1.0  mg  l clo   standard solution and applying 12 and 8 sampling cycles for sample and reagent solution , respectively .
the lcv concentration varied from 0.02 up to 0.10  g  l. the absorbance increased up to a concentration of 0.08 
thus , the 0.10  g  l solution was selected , so as to assure an excess of reagent throughout the sample zone .
the assays commented on in previous sections were carried out by inserting aliquots of both lcv and buffer solution , each one with a volume of 48  l and an aliquot of sample solution with a volume of 72  l .
as is shown in table 1 ( step e ) , micropumps p2 and p3     were also switched on / off 12 times , therefore , the volume of the sample zone was 144  l . aiming to minimize the reagent consumption , a set of experiments
was carried out by varying the volume of the solution aliquot from 6 up to 30  l , which was done by varying the number of sampling cycles ( table 1 , step d ) from 1 to 5 .
the curve in figure 2 shows that for volumes higher than 18  l , the variation of the analytical signal was not significant .
thus , the volume of 24  l was selected as a compromise , in order to save reagent solution while , on the other hand , assuring a sufficient amount for allowing the reaction development to occur . with the objective of
verify the effect caused by the sample volume on the analytical signal , assays were carried out by varying the volume of sample aliquot from 6 up to 102  l , yielding the results shown in figure 3 . analyzing this curve
, we observe that for volumes higher than 66  l , the signal increment decreased step by step .
when sample volume varied within the range of 18 to 60  l , a linear relationship ( r = 0.998 ) was achieved .
this behavior is quite different from that observed in the usual fia system employing injection by loop . in order to achieve a compromise between sensitivity and throughput , a sample volume of 72 l was selected .
once the operational conditions of the system were established , a set of assays was carried out , in order to verify the overall performance of the system .
the results achieved are compared with other procedures and are summarized in table 2 . as we can see , the proposed procedure presented useful features :   a wide linear response range , a low limit of detection , which was estimated as three times standard deviation of blank divided by the slope of the analytical curve , high throughput , low volume of waste generated , and low reagent consumption . comparing these data with those presented in the referenced papers [ 3234 ] , we observe that , excepting throughput ( see ) , the results are highly favorable for the proposed system .
an additional assay was carried out in order to verify whether lcv also reacted with combined chlorine .
the first one containing 1.0  mg  l clo plus 1.0  mg  l nh4 and 2.0  mg  l clo plus 2.0  mg  l nh4 ; the second one , prepared to contain equal clo concentration without ammonium . in the first case ,
absorbances generated processing both solutions were 97% lower than those observed when standard solutions were prepared without ammonium .
therefore , we could consider these results as an indication that the lcv does not reacted with combined chloride .
a set of tap water samples was analyzed , employing the operational conditions shown in table 1 , yielding the results shown in table 3 .   in order to make accurate assessment ,
student 's paired t - test calculated for the values obtained by both procedures for the 95% confidence level was found to be 1.360 .
since the theoretical value is 2.447 , there is no significant difference at the 95% confidence level .
the performance of the proposed setup proved that multicommuted flow injection analysis ( mcfa ) , implemented employing solenoid minipumps to propel solutions , afforded facilities for downscaling the flow setup .
its reduced dimension allowed a drastic reduction of reagent consumption and waste generation , without any sacrificing of precision , accuracy , and throughput .  
although the lcv had been employed for many analytical purposes , it had not yet been used for hypochlorite determination . in this work
, we proved that lcv can be used as a chromogenic reagent for the photometric determination of clo in tap water .
 a photometric procedure for the determination of clo in tap water employing a miniaturized multicommuted flow analysis setup and an led - based photometer is described . 
 the analytical procedure was implemented using leucocrystal violet ( lcv ;   4,4,4-methylidynetris ( n , n - dimethylaniline ) , c25h31n3 ) as a chromogenic reagent . 
 solenoid micropumps employed for solutions propelling were assembled together with the photometer in order to compose a compact unit of small dimensions . 
 after control variables optimization , the system was applied for the determination of clo in samples of tap water , and aiming accuracy assessment samples were also analyzed using an independent method . applying the paired t - test between results obtained using both methods , no significant difference at the 95% confidence level was observed . 
 other useful features include   low reagent consumption , 2.4  g of lcv per determination , a linear response ranging from 0.02 up to 2.0  mg  l1  clo , a relative standard deviation of 1.0% ( n = 11 ) for samples containing 0.2  mg  
 l1  clo , a detection limit of 6.0  g  l1  clo , a sampling throughput of 84 determinations per hour , and a waste generation of 432  l per determination . 
 
1. Introduction
2. Experimental
3. Results and Discussion
4. Conclusions