copper is present in a large number of enzymes , which are involved in electron transfer , activation of oxygen , and other small molecules as well as superoxide dismutation [ 1 , 2 ] .
it serves as an essential cofactor for a variety of proteins involved in neurotransmitter synthesis as well as in neuroprotection via the cu / zn superoxide dismutase .
copper in excess is toxic while cu deficiency can lead to serious disease [ 35 ] .
copper ions have also showed antimicrobial activity against a wide range of microorganisms [ 68 ] .
the determination of trace amounts of copper , because of its importance in health , medical , and industrial processes , is of great interest to analytical chemists .
the most common techniques are icp - ms [ 9 , 10 ] , atomic absorption [ 11 , 12 ] , capillary electrophoresis , and uv / vis spectrophotometry [ 1416 ] .
in addition , some methods use organic solvents such as chloroform or acetonitrile which are for the issue of environmental concern or worldwide storage crisis . among these techniques ,
visible absorption spectrophotometry represents the most convenient technique because of the availability of the instrumentation , simplicity , speed , precision , accuracy , and low cost . as it was conducted in our previous studies , 6-naphthyl and anthracenyl substituted , 1,2,4-triazine-3-thione form colored complexes with cu(ii ) in basic media [ 1720 ] . in this investigation
the synthesis of 6-(2-methoxynaphthyl)-2,3-dihydro-1,2,4-triazine-3-thione has been synthesized as a chromogenic reagent for the determination of cu(ii ) and validation of the developed method is reported .
all spectra recordings and absorbance measurements were carried out on a shimadzu , 160a uv / vis spectrophotometer . atomic absorption (
nmr spectra were recorded on a bruker ft-500 spectrometer ( bruker , rheinstetten , germany ) with tetramethyl silane ( tms ) as internal standard .
2-methoxynaphthalene , thiosemicarbazide , amylnitrite , aluminium chloride , and acetyl chloride were used for ligand synthesis and were purchased from fluka ( switzerland ) or merck chemical companies .
solvents ( acetone , acetonitrile , ethyl alcohol , and methanol ) were of hplc grade and prepared from merck ( germany ) . 

copper nitrate solution . a stock standard solution of 1  mg / ml cu(ii )
was prepared by dissolving 0.5  g pure elemental copper in hot concentrated hno3 , cooling , and adjusting the volume to 500  ml by addition of the distilled water .
acetyl chloride ( 0.4  g ) and alcl3 ( 0.67  g ) were mixed in a mortar in dry condition and then 2-methoxynaphthalen ( i ) ( 0.5  g ) was added and mixed in oxygen protected condition .
the product was re - crystallized from ethanol - water to yield 0.454  g ( 72% ) , mp .
c nmr ( 125  mhz , cdcl3 ) was 32.68 ( ch3co ) , 55.36 ( och3 ) , 112.71 , 123.58 , 124.05 , 128.65 , 128.13 , 128.78 , 130.37 , 131.44 , 133.31 , 153.91 , and 205.22 . 

was then refluxed with amyl nitrite in ethanol in presence of sodium ethoxide for 48  h in dry condition .
the product ( iii ) , 2-methoxynaphthylglyoxal aldoxime , was then extracted with diethyl ether , dried , and crystallized from water - ethanol .
the reaction product ( iii ) and thiosemicarbazide were refluxed for 3  h in dilute hydrochloric acid .
the progress of the reaction was monitored by tlc using a mixture of chloroform and methanol as a mobile phase . the reaction mixture
c nmr ( 125  mhz , dmso ) was 57.04 ( och3 ) , 114.25 , 123.56 , 124.45 , 128.03 , 128.56 , 131.21 , 131.26 , 132.48 , 154.85 , 158.53 , 173.76 , and 188.74 .
h nmr ( 500  mhz , dmso ) was 3.87(s , 3h , ch3 ) , 7.387.56 ( m , 3h ) , 7.577.62 ( m , 2h ) , 810 ( s , 1h ) , and 833 ( bs , 1h , nh ) .
mass m / z ( % ) was 269 ( 7 ) , 241 ( 5 ) , 239 ( 20 ) , 165 ( 10 ) , 153 ( 20 ) , 151 ( 100 ) , and 138 ( 4 ) . 

general procedure for determination of cu(ii ) . in a series of 5  ml volumetric flask , 1  ml
( 2.520  g / ml ) of cu(ii ) and 2  ml of mndtt ( 0.002  m ) were taken and the volume was adjusted to 5  ml with methanol .
the absorbance of the solutions was recorded against reagent blank at 475  nm in a 1  cm quartz cell . 

40  mg of an amalgam ( 110-plus ) containing ag(i ) ( 45% ) , sn(ii ) ( 30% ) , and cu(ii)(25% ) was taken in a 50  ml beaker and 10  ml hno3  :  hcl ( 1  :  1 , v / v ) solution was added .
the mixture was heated at 120c for 30  min until the dissolution is completed and the resulting solution reaches minimum volume .
the solution cooled and transferred to a 100  ml volumetric flask and diluted to mark with double distilled water .
the reagent 6-(2-methoxynaphthyl)-2,3-dihydro-1,2,4-triazine-3-thione ( mndtt ) was synthesized , due to our recent studies for preparing more sensitive chromogenic reagents , for determination of trace amount of some cations such as cu(ii ) , ni(ii ) , and hg(ii ) . to achieve more sensitivity , complexing moiety , 1,2,4-triazine-3-thione ring ,
different solvent systems ( acetonitrile , water , 0.1  m naoh , chloroform , ethanol , and methanol ) were examined to find out more suitable solubility , better absorption spectra , and greener solvent .
the results showed that the reagent , mndtt , was not soluble in chloroform and water .
both the reagent and cu - mndtt complex were soluble in 0.1  m naoh , acetonitrile , ethanol , and methanol .
acetonitrile not only had lower sensitivity but also was not considered as an environmental and health friendly solvent [ 25 , 26 ] .
eventually , comparison of the absorption value of cu - mndtt complex in methanolic and ethanolic media showed that the most suitable solvent for both reagent and complex was methanol ( table 1 ) .
therefore , methanol which had greater absorbance value and was also considered as one of the greener solvents was selected for subsequent experiments . 

6-(2-methoxynaphthyl)-2,3-dihydro-1,2,4-triazine-3-thion , which is synthesized as a new reagent , reacts with cu(ii ) forming a red - colored complex in methanol .
the absorption spectra of cu - mndtt complex versus the blank and the ligand ( mndtt ) in methanol was recorded in the wavelength region of 200800  nm ( figure 2 ) .
the ligand shows a maximum wavelength at 346  nm while the spectrum of cu - mndtt reveals a maximum at 475  nm which increased as a function of cu / mndtt molar ratio according to the curve reported in figure 3 . 

the influence of ph on the complex formation using britton - robinson buffer ( ph = 512 ) and 1  m  naoh was studied by measuring the real absorbance of the solution containing cu(ii ) ( 20  g / ml ) in the presence of the reagent mndtt against the reagent blank .
the results illustrated in figure 4 reveal that maximum absorbance of the colored complex was obtained at ph = 12 . comparing the results obtained in the absence of buffer ( table 1 ) with the above values
reveals that the absorbance of the produced complex decreased in the presence of aqua buffer solution .
the effect of cationic ( cetrimide ) , anionic ( sodium lauryl sulfate ) , and nonionic ( tween 80 ) surfactants was studied and results showed that surfactants caused turbidity in solution in different surfactant concentrations .
, the absorbance of a 25  g / ml solution of cu(ii ) at the optimum condition was recorded over a period of 3  h with an interval of 30  min , after 24 and 48 hours .
the results showed that the complex was completely stable for at least 3 hours and there were no significant changes ( < 3% ) in the absorbance of the complex after 24 hours . 

. the chemical structure of cu - mndtt complex was determined by limiting logarithmic method . as it is shown in figure 5 the logarithm of absorbance intensities of cu - mndtt complex versus log [ cu ] 1.57    104.68    10  m ) at fixed concentration of mndtt ( 5.58  10  m ) and log [ mndtt ] ( 5.75    101.72    10  m ) at fixed concentration of cu(ii ) ( 1.57  10  m )
the values of the slopes of these lines were 0.8463 and 1.586 , confirming the 1  :  2 ratios for the complex formation reaction .
the opposed method was calibrated using 6 series in the range of 2.520  g / ml . the analytical parameters of the proposed method are given in table 2 . the detection limit
( 3 sd / k ) and quantification limit ( 10 sd / k ) ( where sd is the standard deviation of the y - intercept and k represents the slope of the straight line ) , as defined by iupac , were found to be 0.33 and 1.10  g / ml , respectively . 

the accuracy and precision of the proposed method were determined at three different concentrations within the same day ( n = 3 ) and over three different days ( n = 9 ) .
percentage relative standard deviation ( rsd% ) as precision and percentage relative error ( er% ) , which shows accuracy of the suggested method , was less than 3.68% and 2.40% , respectively .
the effect of diverse ions was determined using a standard solution containing 20  g / ml of cu(ii ) and 20  g / ml of the studied ions .
the method was completed according to the general procedure and the absorbance value was obtained against the reagent blank at 475  nm . similar to the results obtained for the naphthyl derivative of 1,2,4-triasine-3-thione ( 6-(2-naphthyl)-2,3-dihydro-1,2,4-triasine-3-thione ) obtained in previous study , the new reagent had no interference with fe(ii ) , ba(ii ) , mg(ii ) , ca(ii ) , cd(ii ) , co(ii ) , mn(ii ) , and sr(ii ) .
ni(ii ) , hg(ii ) , and pd(ii ) ions form complexes with mndtt which have maximum absorbance between 400 and 500  nm .
as there was no suitable masking agent for these ions , derivative spectrophotometric method has been recommended for analyzing cu(ii ) in the presence of ni , hg , or pd ions . 

application of the method to real sample . in order to evaluate the analytical applicability of the proposed method , it was applied for the determination of cu(ii ) in a dental amalgam 110-plus .
the results and recoveries presented in table 4 indicate the percentage of recovery 97.5 and 96.5 for spectrophotometry and atomic absorption method , respectively . using the two - tailed t - test and f - test methods
, it was revealed that there was no significant difference between the results obtained from these two methods ( p value > 0.05 ) . 
 relative recovery .
the relative recovery was determined using the standard addition method ( n = 3 ) .
the percent relative recovery of 101.66  1.24 indicates that no interference with other components in amalgam has been observed .
a simple , rapid , sensitive , and accurate method for determination of cu(ii ) using the newly synthesized reagent , 6-(2-methoxynaphthyl)-2,3-dihydro-1,2,4-triazine-3-thion , was developed .
comparison of characteristic features of some spectrophotometric methods reported earlier for the determination of copper reveals the suitability of the present work in terms of molar absorptivity , linear range , interferences , lod , and so forth ( table 5 ) .
the use of mndtt as a complexing reagent was utilized for nonextractive determination of cu(ii ) in dental amalgam .
additionally , the method was much safer , since only a small amount of methanol was used which is considered as  green chemistry  for determination process . therefore , the proposed method can be recommended for pharmaceutical and industrial samples .
 a simple , sensitive , accurate , and green spectrophotometric method for the determination of cu(ii ) using newly synthesized reagent , 6-(2-methoxynaphthyl)-2,3-dihydro-1,2,4-triazine-3-thione ( mndtt ) , has been developed . 
 mndtt was synthesized based on the acylation of methoxy naphthalene and reaction of the product with amyl nitrite , which upon reaction with thiosemicarbazide yielded 6-(2-meyhoxynaphthyl)-2,3-dihydro-1,2,4-triazine-3-thione . 
 mndtt produces a dark red complex with copper in methanol according to the 1  :  2 stoichiometry . 
 beer 's law was obeyed over the concentration range of 2.520  g / ml with r 
 2 = 0.992 . 
 the limit of detection and limit of quantification were 0.33 and 1.10  g / ml , respectively . 
 within - day and between - day precision values were less than 3.68% . 
 finally , the method has been applied to a dental alloy ( 110-plus ) successfully and the results were compared with atomic absorption method . 
 the results showed that there was no significant difference between the two methods ( p > 0.05 ) . 