the concentration of ammonium is usually more than micromolar level in mostly continental water and coastal seawater , even up to millimolar level due to environmental pollution [ 24 ] .
however , it is less than micromolar level , even down to nanomolar level in ocean water and some unpolluted freshwater [ 5 , 6 ] .
the accurate measurement of trace ammonium natural water is essential to understand the biogeochemical cycle of nitrogen .
the indophenol blue method ( ipb ) and o - phthalaldehyde ( opa ) fluorometric method were the main methods for the determination of ammonium in natural water [ 715 ] .
the indophenol blue method is based on the berthelot reaction . in the catalysis of nitroprusside
, ammonium reacts with hypochlorite and phenol forming indophenol blue , which has a maximum absorbance of 640  nm .
the ipb method is used as the standard method to determine ammonium in water by the u.s .
the technique has low sensitivity ( lod 0.6  mol / l ) . to meet the determination of trace ammonium in ocean water and some unpolluted freshwater , the sensitivity of the ipb method
was obviously improved using a long - path liquid waveguide capillary cell ( lwcc ) in the later reported work .
however , many accompanying weaknesses such as complicated operations and a high reagent blank appeared .
opa method is based on the fluorometric reaction that ammonium reacts with o - phthaldialdehyde and sulfite producing highly fluorescent isoindole derivatives , which has the maximum excitation wavelength ( ex ) at 361365  nm and the maximum emission wavelength ( em ) at 422425  nm .
the reaction was firstly reported by roth in 1971   and was modified by replacing mercaptoethanol with sulfite to provide an opa method for determination of ammonium in water in 1989 .
afterwards , the opa method was modified to improve the sensitivity and operability by much work [ 1115 ]
. the lower limit of quantitation ( 1.67  nmol / l ) of the opa method was gained by coupling with flow analysis and solid phase extraction , and the method was applied on board to analyze seawater samples collected from 65 stations in the south china sea , and a detailed ammonium profile in the seats station was obtained .
however , uv led ( 360  nm ) had to be used as the excitation light source for conforming to the excitation wavelength of the opa method , and heating device was also needed in the determination system to provide higher reaction temperature ( 75c ) .
uv led source has many defaults , such as lower light intensity , higher price , and complicated technology .
uv led source and heating device are not very suitable for developing a portable fluorescence detection system .
if a new fluorescent reagent is prepared for ammonium determination and it can rapidly react with ammonium at room temperature , the determination of ammonium should become much simpler in field . in this paper , a methoxy group , an electron - donating group ,
was joined to benzene ring of opa molecule , producing a new fluorescent reagent , 4-methoxyphthalaldehyde ( mopa ) .
and then a novel analytical method was developed for trace ammonium in freshwater and seawater using mopa as fluorescent reagent .
all of the other chemicals used for analysis were of guaranteed reagent grade , supplied by aladdin chemical reagent co. , china , unless stated otherwise .
all solutions were prepared in ultrapure water ( resistivity  18.2  mcm at 25c ) . in detail ,
7.8  g / l mopa solution was made by dissolving 1.95  g of mopa in 200  ml of methanol ( hplc grade ) and diluting to 250  ml with ultrapure water ; 1.26 
g / l na2so3 solution was made by dissolving 0.63  g of na2so3 in 500  ml ultrapure water and adding 0.20  ml 37% formaldehyde to prevent the solution from being oxidized ; 2.0  g / l naoh solution was prepared by dissolving 1.0  g naoh in 500  ml of ultrapure water ; 40  g / l naoh solution was prepared by dissolving 20.0  g naoh in 500  ml of ultrapure water ; 300  g / l sodium citrate solution was made by dissolving 30.0  g sodium citrate in 100  ml of ultrapure water ; 15  g / l sodium tetraborate buffer solution ( r4 ) was prepared by dissolving 7.5  g na2b4o710h2o in 500  ml ultrapure water ; 3.4  g / l opa solution was made by dissolving 0.34  g of opa in 20  ml of methanol ( hplc grade ) and diluting to 100  ml with ultrapure water ; ammonium standard stock solution ( 1000  mg
mmol / l ) was purchased from aladdin chemical reagent co. , and the ammonium salt is ( nh4)2so4 in the solution ; ammonium standard substock solution ( 10 
mmol / l ) was prepared monthly by diluting the stock solution with ultrapure water ; the stock and substock solutions were stored at 4c in a refrigerator while not in use ; ammonium working solution ( 0.1  mmol / l ) was prepared daily by diluting 1.0  ml of the substock solution to 100  ml with ultrapure water .
all vessels used in the experiments were firstly soaked with 1  moll hcl for more than 12 hours , cleaned with reverse osmosis water ( resistivity 0.5  mcm at 25c ) , and then soaked with 1  moll naoh at least for 12 hours and cleaned thoroughly with ultrapure water before use . synthetic route of mopa is showed in figure 1 . 
 compound 
 1 .
a flask was charged with 15.2  g 3-methoxybenzoic acid and 50  ml dioxane .
the mixture was stirred at room temperature and a solution of 50  ml 37% aqueous formalin solution and 50  ml 37% aqueous hcl was added .
the reaction was continued at 60c for 3 days , and then it was cooled at room temperature .
the contents of the flask were washed with ch2cl2 ( dichloromethane / cas number 75 - 09 - 2 ) .
the combined organic layer was dried with anhydrous mgso4 , filtered , and concentrated under reduced pressure to give crude white solid .
this solid was recrystallized from 95% ethyl alcohol to afford 6-methoxyphtalide ( compound 1 ) as white needle ( 11.42  g , 70.1% yield )
a flask was charged with 11.4  g 6-methoxyphtalide and 80  ml dry tetrahydrofuran ( thf ) , and the mixture was stirred at room temperature .
a mixture of 3.4  g lialh4 and 50  ml dry thf was added slowly when a clear solution was obtained .
the reaction was continued at room temperature for 30 minutes , and then it was heated at 80c for 8 hours .
subsequently , 4  ml h2o and 2  ml 15% aqueous naoh were added to the reaction mixture , respectively .
the filtrate was dried with anhydrous mgso4 , filtered , and concentrated under reduced pressure to give crude yellow oil ( compound 2 , 8.46  g , 72.2% yield ) . 
 compound 
 3 . a 250  ml round - bottomed flask equipped with an addition funnel
was charged with 6  ml ( cocl)2 ( oxalyl chloride / cas number 79 - 37 - 8 ) and ch2cl2 ( 60  ml ) and the mixture was stirred at 78c .
after the addition of 13.6  ml dmso ( dimethyl sulfoxide / cas number 67 - 68 - 5 ) and 20  ml ch2cl2 , the mixture was stirred for 35 minutes at 78c .
a solution of compound 2 ( 0.06  mol , 10.0  g ) and 20  ml ch2cl2 and dmso ( v  : v = 10  :  1 ) was added to the mixture .
after the addition of 80  ml et3n , the mixture was stirred for another 10 minutes at 78c and then the mixture was allowed to warm to room temperature .
the combined organic layer was dried with anhydrous mgso4 , filtered , and concentrated in vacuo .
the residue was purified by silica gel flash column chromatography to give the 4-methoxyphthalaldehyde as yellow needle ( 5.9  g , 60.1% yield ) , mp 7678c .
h nmr ( 500  mhz , cdcl3 )  10.61 ( s , 1h ) , 10.29 ( s , 1h ) , 7.90 ( d , j = 8.5  hz , 1h ) , 7.41 ( d , j = 2.6  hz , 1h ) , 7.19 ( dd , j = 8.5 , 2.6  hz , 1h ) , 3.92 ( s , 3h ) ; c nmr ( 125  mhz , cdcl3 )  192.0 , 191.0 , 163.9 , 138.6 , 134.7 , 129.5 , 118.8 , 114.8 , 56.0 .
ms(esi ) , m / z : 165 ( [ m+h ] ) . 10  ml of standard ammonium solution or sample solution with a concentration range of 0.0250.300 
appropriate amounts of sodium citrate solution , mopa solution , na2so3 solution , and naoh solution were added into the bottle .
the concentrations of sodium citrate , opa , and sodium sulfite in the final solution were 16.8  g / l , 0.12 
after all the reagents were added , the mixed solution was tightly sealed and allowed to react for a certain time at room temperature .
the fluorescence intensity was measured on a fluorescence spectrophotometer ( rf-5301pc , shimadzu co. , ltd . ,
japan ) with excitation wavelength set at 370  nm and emission wavelength at 454  nm . both the excitation and emission slits of the instrument were set as 5  nm , unless stated otherwise .
0.20  mol / l standard ammonium solution was allowed to react with mopa and sodium sulfite according to section 2.3 .
the product had the maximum excitation wavelength ( ex ) at 370  nm and the maximum emission wavelength ( em ) at 454  nm .
compared with the product of opa reacting with ammonium , the maximum excitation and emission wavelength appeared as red shift phenomenon ( see figures 3 and 4 ) , and the maximum excitation and emission wavelength increased 9  nm and 32  nm , respectively . though the maximum excitation wavelength was located in the nearly uv region , it should be noticed that the fluorescence intensity was still sizable in the wavelength range of 380410  nm .
this means that visible led at which wavelength ranged from 380 to 410  nm could be chosen as excitation light source to make a portable fluorescence detection system if mopa is used as fluorescent reagent .
six solutions were separately prepared and stood for the same time , marked ( a ) , ( b ) , ( c ) , ( d ) , ( e ) , and ( f ) , and their components are listed in the cutline of figure 5 .
30 minutes was set as the standing time but is not necessary , more or less time is also feasible for the experiment in this section .
the excitation wavelength was set at 370  nm , and the fluorescence emission spectra of these six solutions were determined , shown in figure 5 .
corresponding to the six solutions , curves in figure 5 are separately marked ( a ) , ( b ) , ( c ) , ( d ) , ( e ) , and ( f ) from the bottom up .
curve ( a ) which has a background peak at 425  nm is the emission spectrum of ultrapure water , and curve ( b ) almost overlaps curve ( a ) , illuminating that the mixed solution of ammonium , sodium hydroxide , sodium sulfite , and sodium citrate had no fluorescent properties
. curve ( c ) is the fluorescence emission spectrum of mopa , showing that mopa had weak fluorescence .
curve ( d ) is the fluorescence emission spectrum of the mixed solution of ammonium , mopa , and sodium sulfite .
compared with curve ( c ) , the intensity of curve ( d ) had obviously increased , displaying that a fluorescent compound had been produced in solution ( d ) .
the difference of solution ( d ) and solution ( e ) was ph , and the ph of solution ( e ) is higher because of addition of sodium hydroxide .
the maximum emission wavelength of curve ( e ) was the same as curve ( d ) , but the fluorescence intensity of curve ( e ) was much higher than that of curve ( d ) , illuminating that ph was obviously affecting the fluorescence intensity of the solution
. curve ( f ) almost overlaps curve ( e ) , and the difference of solution ( e ) and solution ( f ) lies in the existence of sodium citrate , explaining that sodium citrate had no obvious effect on the fluorescent reaction of the proposed method .
the maximum emission peaks of both curve ( e ) and curve ( f ) were located in 454  nm , which appeared as red shift of 29  nm in comparison with curve ( a ) , illuminating that the interference of ultrapure water could be avoided using mopa as fluorescent reagent .
all of above results had confirmed that ammonium reacting with mopa could produce strong fluorescent compound in the existence of sodium sulfite and sodium hydroxide . in this section ,
effects of parameters such as concentration of mopa , addition of na2so3 solution , ph in reaction , and concentration of sodium citrate solution on the fluorescence intensity ( fi ) of blank and 0.200  mol / l ammonium working solution were investigated based on univariate experimental design .
the concentration of other reagents and experimental conditions were controlled as the described in section 2.3 .
the relationship between the fluorescence intensity and the concentration of mopa in the reaction solution was investigated , shown in figure 6 .
the fluorescence intensity of both blank solution and 0.200  mol / l ammonium working solution increased with an increase in the concentration between 0 and 0.12 
g / l and was closed to constant when the mopa concentration was more than 0.12 
the results above illuminated that mopa was an essential fluorescent reagent in the proposed method , and 0.12  g / l of mopa was enough for the fluorescence reaction . consequently , the concentration of mopa in the solution was controlled at 0.12  g / l in the consequent experiment .
effects of the solution ph on the fluorescence intensity of blank and 0.200  mol / l ammonium working solution were investigated in the range of 9.212.2 .
the results in figure 7 illuminated that the fluorescence intensity of both solutions rapidly increased when the ph ranged from 9.2 to 11.2 , then closed to a constant in the ph range of 11.212.0 , and decreased in the final when ph is more than 12.0 .
the concentration of other reagents and experimental conditions were set as described in section 2.3 .
the effect of na2so3 concentration on the fluorescence intensity of blank and 0.200  mol / l of ammonium working solution was investigated .
the results were shown in figure 8 . when the na2so3 concentration in the reaction solution was between 0.041 and 0.081 
g / l , the fluorescence intensity of both solutions was closed to maximum constant , illuminating that the amount of na2so3 in the range of 0.0410.081 
g / l in the following experiment . according to section 3.2.2 , an optimum fluorescence intensity of the reaction solution
could be obtained in the ph range of 11.212.0 . however , precipitation easily occurred in this ph range due to the existence of metal ions in natural water samples . to avoid precipitation of the metal ions , appropriate amount of sodium citrate
experiments testified that 19  g / l sodium citrate was an optimal choice for most of natural water samples such as seawater , groundwater , and mountain spring water . to investigate the effect of 19  g / l sodium citrate on the fluorescent reaction , different concentration ammonium working solutions in existence and in the absence of sodium citrate were allowed to react with mopa according to section 2.3 , and the fluorescence intensity signals of these solutions
the relationships between fluorescence intensity and concentration of ammonium ( cnh4 ) in existence and in the absence of sodium citrate were fi = 989.3cnh4 + 132.46 ( r = 0.9974 ) and fi = 1025cnh4 + 133.06 ( r = 0.9975 ) , respectively .
the results above illustrated that 19  g / l sodium citrate had no obvious effect on the fluorescent reaction .
0.200  mol / l ammonium working solution was allowed to react with mopa in the conditions described in section 2.3 .
the fi was detected in different reaction time . to control the fi signal in the range of instrument ,
the excitation and emission slit were set as 5  nm and 3  nm , respectively .
it was obvious that the fi rapidly increased as the time ranging from 0 to 100 minutes and closed to constant in the range of 100250 minutes , illuminating that the equilibration time of the reaction was 100 minutes . according to
, the reaction equilibration time of opa and ammonium was 180 minutes at room temperature .
consequently , the fluorescent reaction of mopa and ammonium was much more rapid than that of opa and ammonium . a typical calibration curve of the proposed method was determined according to section 2.3 .
the regression equation of the linear curve is fi = 833.4cnh4 + 109.2 ( r = 0.9946 , n = 6 ) .
according to , a typical calibration curve of the opa method was determined at the same reaction time and same temperature as the proposed method .
the curve regression equation of the opa method is fi = 127.2cnh4 + 16.5 ( n = 5 , r = 0.9989 ) , and linearity range of the curve was ranged from 0.25 to 1.2 
the slope of the proposed method was 6.56 times that of the opa method , illuminating that the proposed method was much more sensitive than the opa method .
the reproducibility of the proposed method was evaluated with 4 repetitive determinations of a 0.100  mol / l ammonium working solution .
four blanks solutions were determined at the same time as the calibration curve , the average fi was 99.581 , and the standard deviation was 1.621 .
the method detection limit , estimated as three times the standard deviations of the blank , was 0.0058 
much lower determination limit should be gained by prolonging the reaction time . a surface seawater sample collected from the south china sea
mountain spring water and a groundwater were collected at yaoshan scenic area in guilin at the day of the experiment .
these three samples were separately used as matrix to investigate the recovery of the proposed method .
the recoveries were in the range of 93.6%108.1% , showing that both the seawater matrix and freshwater matrix had no interference in ammonium determination .
mopa was successfully synthesized and could be used as a novel fluorescent reagent for determination of ammonium .
ammonium could rapidly react with mopa at room temperature producing a strong fluorescent compound , at which maximum excitation and emission wavelength were 370  nm and 454  nm , respectively .
compared with the fluorescent product of ammonium reacting with opa , the strong fluorescent compound of this proposed reaction appeared as red shift phenomenon , and the excitation emission wavelength was closed to the visible light zone . based on this , a novel analytical method was proposed for trace ammonium in natural water using mopa as fluorescent reagent .
 a novel fluorescent reagent for determination of ammonium , 4-methoxyphthalaldehyde ( mopa ) , was successfully synthesized in this study . under alkaline conditions , 
 mopa could reacted with ammonium rapidly at room temperature , producing fluorescent substance which had maximum excitation at 370  nm and emission wavelength at 454  nm . 
 based on this , a novel fluorescence analysis method was established for the determination of trace ammonium in natural water . 
 experimental parameters including reagent concentration , ph , reaction equilibrium time , and metal ions masking agent were optimized . 
 the results showed that the optimized mopa concentration was 0.12  g / l , ph was in the range of 11.212.0 , and sulfite concentration was 0.051  g / l , respectively . 
 metal ions masking agent had no obvious effect on the fluorescence signal . with the reaction time of 15 minutes , linear range of 
 this method was between 0.025 and 0.300  mol / l , and the method detecting limit was 0.0058  
 mol / l . 
 the matrix recovery of the proposed method was in the range of 93.6108.1% . compared with the opa method , 
 this method was much more sensitive and rapid without the interference of background peak and would be more suitable for developing a portable fluorescence detection system . 