leather tanning is one of the polluting industrial sources in terms of both conventional and toxic parameters , which include cod , bod , tannic acid , protein , grease , sulfide , chloride , chromium , suspended substances , and so on .
it is one of the main persistent organic pollutants in leather wastewater , causing environmental pollution [ 1 , 2 ] . tannic acid may interact with metal to form toxic tannic acid - metal compound .
tannic acid may also interact with toxicants in aquatic ecosystems and may change their toxicity .
therefore , it is very important to determine tannic acid in wastewaters in order to evaluate water pollution from leather wastewaters .
several methods have been used for the determination of tannic acid such as spectrophotometry [ 4 , 5 ] , chemiluminescence [ 6 , 7 ] , fluorescence [ 8 , 9 ] , voltammetry [ 10 , 11 ] , liquid chromatography [ 12 , 13 ] , thin - layer chromatography , titration , protein precipitation , flow injection analysis , and so forth . however , some are troublesome , time - consuming , and expensive .
modern environmental monitoring calls for automatic , simple , rapid , and sensitive methods for online determination .           
a rapid , simple and automatic method was proposed for online determination of tannic acid in colored tannery wastewater by automatic reference flow injection analysis .
the proposed method is based on the fact that tannic acid can reduce phosphotungstic acid to form blue compound in alkaline solutions . in the folin - denis method
, saturation sodium carbonate reacts with phosphoric acid to form carbon dioxide which will affect the determination of tannic acid .
thus , in the proposed method , the alkaline solution of sodium tetraborate - sodium hydroxide buffersolution is substitutedfor saturation sodium carbonate solution to avoid the formation of carbon dioxide . compared with manual methods , the proposed method not only consumes small sample , reagent and little time but
a model zj - la automatic analyzer developed and produced successfully by professor xinshen zhang ( sichuan university , chengdu , china ) was used .
the analyzer has the functions of flow injection analysis ( fia ) , ion chromatography , and automatic sample injection .
two analytical pumps ( shanghai huxi analytical instrument plant , p.r . china ) of flow injection were used to deliver all solutions .
one was used to deliver reagent solution while the other delivered sample solution and reference reagent solution .
polytetrafluoroethylene ( ptfe ) tubing of 0.5  mm in internal diameter was used as the channels for all solutions to circulate .  
ultraviolet and visible spectrometer was obtained by using a spectrumlab $ 54 ( lengguang technology co. , shanghai , china ) .
data acquisition and processing were performed with hw-2000 chromatography software ( qianpu software co. , ltd , shanghai , china ) running under windows xp .
all reagents used including sodium tungstate dihydrate , phosphomolybdicacid , tannic acid ( tianjin ruijinte chemical reagent plant , tianjin , china ) , sodium tetraborate , sodium hydroxide , and phosphoric acid ( chengdu fang zhou chemical reagent plant , chengdu , china ) were of analytical grade and all solutions were prepared with deionized water .
tannic acid stock standard solution : tannic acid stock standard solution at a concentration of 1000  mg  l was prepared by dissolving 1.000  g of tannic acid and diluting to 1000  ml . working solution was prepared by suitable dilution of the stock solution .  
phosphotungstic acid stock solution : phosphotungstic acid stock solution was prepared by dissolving 50  g sodium tungstate dihydrate , 10  g phosphomolybdicacid , and 25  ml of 85% phosphoric acid in 350  ml deionized water , distilling for 2 hours and then diluting to 500  ml .  
reagent r1 : 10  ml phosphotungstic acid stock solution was diluted to 500  ml .
reference reagent r2 : 400  ml of 0.2  mol  l sodium tetraborate and 600  ml of 0.1  mol  l sodium hydroxide were mixed and the mixture diluted to 1000  ml .
a smooth baseline was obtained when reference reagent r2 was injected into the six - way injection valve , mixed with sample in the three - way mixer , and pushed into the detector . when the instrument was set into  analyzing  position ,
reagent r1 was injected , mixed with reference reagent r2 , and sample in the three - way mixer and reacted to form blue compound in the reaction coil .
the absorbance intensity of the blue compound was determined at 760  nm by detector and transformed to a signal of the peak which was recorded by hw-2000 software on a pc .
in ph 12.38 of sodium tetraborate and sodium hydroxide media , tannic acid reacts with reagent r1 and reference reagent r2 to form a blue compound .
the absorbance of the blue compound was scanned in the range of 400800  nm by ultraviolet - visible spectrometer .
the result showed that the maximum absorbance wavelength of the blue compound is 760  nm . in the fia
, the flow rate could affect the sensitivity of the proposed method . in the study ,
, the effect of the flow rate of reagent r1 , reference reagent r2 , and sample on the peak height was studied from 0.10  ml  min to 1.00  ml  min , respectively .
the test results showed that the most peak height was obtained when the flow rates of reagent r1 , reference reagent r2 , and sample were 0.22  ml  min , 0.40  ml  min , and 0.72  ml  min , respectively .
the effect of amount of phosphotungstic acid on the peak height was tested from 0.2  ml to 4.0  ml ( diluting to 100  ml ) in figure 2 .
the results showed that the peak height rose when the amount of phosphotungstic acid changed from 0.2  ml to 1.5  ml ( diluting to 100  ml ) and the peak height declined when amount of phosphotungstic acid changed from 1.5  ml to 4.0  ml ( diluting to 100  ml ) . therefore ,
1.5  ml ( diluting to 100  ml ) of phosphotungstic acid were selected for further work .
according to the chemical reaction kinetics , the reaction rate will inevitably increase when concentrations of the reagent increase .
however , the reaction rate will not increase when concentrations of the reagent reach a certain value .
therefore , when phosphotungstic acid consumption reaches a certain value , the concentration of phosphotungstic acid which reacts with tannic acid should be certain because tannic acid concentration is fixed in the sample .
thereby , the absorbency will not increase when phosphotungstic acid concentration increases . on the contrary , the background color ( baseline noises ) will increase because the free state phosphotungstic acid in the system will increase . in the flow injection analysis ,
the effect of ph on the blue compound was tested in the ph range 413 .
the test results showed that the maximum peak height could be obtained when ph was 12 or so . in the folin - denis method
, saturation sodium carbonate reacts with phosphoric acid to form carbon dioxide which will affect the determination of tannic acid .
therefore , the ph 12.38 alkaline solution of sodium tetraborate - sodium hydroxide solution ( reference reagent r2 ) was selected to substitute for saturation sodium carbonate solution to avoid the formation of carbon dioxide in the proposed method . in the fia , the reaction time is adjusted by the length of reaction coil .
if reaction coil was too short , the reagents would react incompletely . if reaction coil was too long , it would be time - consuming . therefore
the results in figure 3 showed that 3  m of the reaction coil length was the optimum and any further increase of reaction coil length did not increase peak height .
thus , 3  m of the reaction coil length was chosen as optimal parameter .
therefore , different sample volumes ( 100 , 200 , 250 , 300 , 350 , 400  l ) were tested .
the results in figure 4 showed that the peak height rose when sample volume changed from 100  l to 300  l and peak height did not increase with further increase of sample volume .
the interference of some possibly coexisting foreign ions was examined to make sure the proposed method can be applied to determine tannic acid in tannery wastewaters .
the tolerance limit was defined as the concentration of added solutions causing less than 5% relative error during the determination of 500  g l tannic acid .
therefore , the proposed method had good selectivity . to assess the reproducibility and accuracy of the proposed method , under the selected conditions above , tests of linearity , precision , and detection limit were conducted .
the linearity was tested by a series of working standard solutions of tannic acid ranging from 100  g  l to 100  mg  l. test showed that the peak height versus tannic acid concentration was linear within the range of 200  g  l to 20  mg  l and 20  mg 
l to 80  mg  l. the curves were h = 5.2147c + 1.3624 and h = 2.3485c + 21.8542 ,   respectively , [ h : peak height ; c : concentration of tannic acid ( mg 
the detection limit ( 3 multiples of baseline noises ) was 0.58  g  l. the test of precision was conducted by ten injections of 500  g  l and 40  mg 
the results showed that the relative standard deviation ( rsd ) was calculated as 3.08% and 2.43% , respectively .
the proposed method was applied to determine tannic acid in tannery wastewaters samples using the zj - la automatic analyzer .
the results of samples were shown in table 1 . to prove the accuracy of the method , a test of recovery was conducted by accurately adding known concentration of tannic acid into each sample whose original tannic acid concentration had been determined .
the test results in table 1 showed that the recovery was between 96.4% and 106.2% and analytical results of the proposed method were satisfactory .
therefore , the proposed method was suitable for determination of tannic acid in tannery wastewaters .
the proposed method was significant with regard to the development of a rapid , simple , sensitive , and automatic analytical   method for online determination of tannic acid .
high sensitivity , broad determination range ( 200  g  l to 80  mg 
l of tannic acid ) , rapid testing , and fully automated analysis were just some of the advantages of the proposed method .
especially , compared with conventional methods , the proposed method not only consumed small sample and reagent but also eliminated the interference of the colority without sample pretreatment .
the model zj - la automatic analyzer employed in this study is inexpensive , small and easily portable .
the proposed method had been successfully applied to determine tannic acid in colored tannery wastewaters .
the proposed method is suitable for determination of tannic acid in both colored and colourless tannery wastewaters for its good selectivity and the above mentioned advantages .
 a simple , rapid and sensitive method was proposed for online determination of tannic acid in colored tannery wastewater by automatic reference flow injection analysis . based on the tannic acid reduction phosphotungstic acid to form blue compound in ph 12.38 alkaline solutions , the shade of blue compound is in a linear relation to the content of tannic acid at the point of the maximum absorption peak of 760  nm . 
 the optimal experimental conditions had been obtained . 
 the linear range of the proposed method was between 200  g l1 to 80  mg l1 and the detection limit was 0.58  g l1 . 
 the relative standard deviation was 3.08% and 2.43% for 500  g l1 and 40  mg l1 of tannic acid standard solution , respectively , ( n = 10 ) . 
 the method had been successfully applied to determination of tannic acid in colored tannery wastewaters and the analytical results were satisfactory . 
1. Introduction
2. Experiment
3. Results and Discussion
4. Conclusion