selenium ( se ) has been recognized as an essential nutrient for plant , animal , and human body , but at high concentration it can become toxic . the range between the concentration in which selenium is essential and toxic is very narrow [ 1 , 2 ] .
this element plays an important role in elderly people as well as in the prevention of many age - associated diseases and in maintenance of normal immune function .
se is potent antioxidant involved in cellular defense against free radical reactions , and the risk of deficiency seems to increase in proportion to the age .
evidence is accumulating that most of the degenerative diseases have their origin in deleterious free radical reactions .
these diseases include atherosclerosis , cancer , inflammatory joint , asthma , diabetes , senile dementia , and degenerative eye disease .
but high se concentrations in human can cause loosing hair and nails and irritation of skin and eye .
the required daily amount of se is 20  mcg  day and 4070  mcg day for 46 years old and adult males , respectively .
the essential role of the se is due to its presence in the active site of some enzymes ( i.e. , glutathione peroxidase and iodotironine-5-deiodinase ) and the catalytic effect of selenium compounds on the reaction of intermediate metabolism and inhibition of the toxic effect of heavy metals .
it has been established that diets with deficit in se are associated with some human diseases , but diets with se contents higher than 5  mg / kg are toxic and cause important symptoms in humans and animals .
selenium is present as selenocysteine ( se - cys ) in at least 30 proteins .
all these factors , together with the fact that the concentration levels are extremely low , call for sensitive and accurate method for the determination of this element .  
selenium can be present in 2 , 0 , + 4 , and + 6 oxidation states .
these forms can not simultaneously be evaluated by direct application of certain analytical techniques such as instrumental neutron activation analysis ( inaa ) [ 6 , 7 ] , as well as hydride generation atomic absorption spectroscopy ( hg - aas ) , inductively coupled plasma atomic emission spectrometry ( icp - aes ) , electrothermal atomic absorption spectrometry ( et - aas ) , or inductively coupled plasma mass spectrometry ( icp - ms ) , after digestion of sample [ 810 ] .
the most common methods used for determination of various species of selenium were atomic absorption spectrometry ( hydride generation and electrothermal atomization ) [ 1114 ] , molecular [ 15 , 16 ] and atomic fluorescence spectrometry [ 1719 ] , high - performance liquid chromatography ( hplc ) [ 2022 ] , voltammetry [ 23 , 24 ] , atomic emission spectrometry ( aes ) , such as inductively coupled plasma ( icp )   and inductively coupled plasma - mass spectrometry ( icp - ms ) , and spectrophotometry methods [ 2528 ] . in spite of some of the above methods , spectrophotometric methods are popular because of their simplicity and are based on piazselenol complex formation between the reagents and selenium .  
an interesting alternative to traditional liquid - liquid extraction is the micelle - mediated extraction , firstly developed by watanabe and tanaka .
the cloud point is the temperature above which aqueous solutions of nonionic and zwitterionic surfactant become turbid .
micelles of such well - known nonionic surfactant as triton x-100 or x-114 have a nonpolar core and extended polar layer , where both extractants and extracted complexes can be solubilized . above the cloud point ,
the solution is separated into two phases , a rich phase containing a high surfactant concentration in a small volume and a poor phase with a surfactant concentration close to the critical micelle concentration ( cmc ) .
hydrophobic species ( hydrophobic organic compounds or metal ions after reaction with a suitable hydrophobic ligand ) present in sample are able to interact with the micelles , thus being concentrated in the small volume of the surfactant - rich phase .
the aim of this work was to use a simple , rapid , and sensitive spectrophotometric method ( dithizone as chromogenic reagent ) for determination of trace amounts of selenium ( iv ) ( 5100  ng  ml ) in micellar medium ( triton x-100 ) in cosmetic and pharmaceutical products . to improve
the detection limit of this method , cloud point extraction ( cpe ) has been used .
selenium metal ( purity : 99.9% ) was obtained from the riedel de haen ( germany ) . nitric acid
( > 99.5% ) , sodium hydroxide ( > 97% ) , ascorbic acid , hydrochloric acid ( 36.538% ) , phenol ( > 99.0% ) , methanol ( > 99.8% ) , and sulfuric acid ( 95.097.0% ) were purchased from merck ( germany ) .
all chemicals were used without any further purification . deionized water from a milli - q system ( millipore , usa ) was used for preparation of all solutions .
selenium sulphide shampoo ( iran ) , centrum tablet ( usa ) , and selen plus capsule ( germany ) were obtained from market . a shimadzu double - beam uv - vis spectrophotometer ( model 1650 pc , japan ) with 1.0  cm quartz cell
was used for all spectral measurements . in order to investigate the accuracy of method a perkinelmer ( model optima 7000 dv ) inductively coupled plasma optical emission spectrometer ( icp - oes )
separation of surfactant phase and aqueous phase was carried out with a hettich centrifuge ( eba 20 , uk ) .
a microwave system ( microdigest 301 , prolabo , france ) with a maximum irradiation power of 200  w was used .
ph measurements were made with a metrohm ph meter ( model 744 , switzerland ) .
the effect of temperature was investigated by a gfl thermostated bath ( model 1003 , germany ) .
the stock standard solution of selenium ( iv ) ( 1000  g  ml ) was prepared by dissolving 100  mg of selenium metal in hot concentrated nitric acid and diluted to 100  ml with water . from this solution
serial dilutions were made to obtain different concentration levels of 5 , 15 , 30 , 40 , 55 , 70 , 80 , and 100  ng  ml of selenium ( iv ) .
the stock solution of dithizone ( 10  mol  l ) was prepared daily by dissolving 12.8  mg of the reagent in 5  ml of naoh solution ( 0.1  m , containing the 50  mg of ascorbic acid for stabilizing the dithizone ) and diluting to 50  ml with water .
the stock solution was diluted with water to obtain the final dithizone concentration of about 7.5  mol  l. the stock solution of triton x-100 ( 10% ( w / v ) ) was prepared by weighing 10  g of this reagent into 100  ml volumetric flask , dissolving in water by gentle heating , and making up to the mark with water .
the final concentration of triton x-100 ( 0.2% ) was prepared by suitable dilution of stock solution with the diluent ( solution of 0.5% ( w / v ) of phenol ) .
an aliquot of 50  ml of a solution containing selenium ( iv ) ( final concentration 5100  ng  ml ) , triton x-100 ( 0.2% ( w / v ) ) , dithizone ( 7.5  mol  l ) , and hcl ( 0.35  m ) was kept for 15  min to complete the color development . then the mixture was placed in a water bath at 40c for 10  min , when 0.5% ( w / v ) of the phenol was used for inducing the cloud point .
phase separation was accelerated by centrifuging the test tube at 3500  rpm for 15  min .
the bulk aqueous phases were easily decanted by gently inverting the test tubes , and the surfactant - rich phase ( complex rich ) was made up to 5  ml by adding methanol .
the same procedure was applied for preparation of blank solution without the selenium and dithizone .
the absorbance of two methanolic solutions was measured at 424 and 592  nm , against a blank ( prepared in the same way without se and dithizone ) .
the absorption maxima of the complex ( 424  nm ) and dithizone ( 434  nm ) were overlapping ( figure 1 ) .
hence , the corrected absorbance , a 
 corr , was used to overcome the problem : 
 ( 1)acorr = a424(a424a592)la592 , 
 where a 
 424 and a 
 592 are the absorbance of the complex measured at 424 and 592  nm , respectively , and ( a 
 424/a 
 592)l is the absorbance ratio of dithizone at 424 and 592  nm . the complex does not show any absorbance at 592  nm .
under optimum conditions , a calibration graph was used for determination of selenium in cosmetic and pharmaceutical products .
shampoo ( selenium sulphide , iran ) sample dissolution was prepared using the procedure described by afkhami et al . .
approximately 1  g of shampoo sample was weighted into a 100  ml beaker . concentrated sulfuric acid ( 1  ml )
the solution was allowed to cool , and 5  ml of 30% ( w / v ) hydrogen peroxide was added .
the mixture boiled vigorously to eliminate excess hydrogen peroxide and allowed to cool and then the digest transferred to a 100  ml volumetric flask .
finally , 2  ml of this solution was taken for determination of selenium ( iv ) as in the recommended procedure .  
a centrum tablet ( usa ) containing 0.25  mg se ( as sodium selenate ) was placed in digestion vessel
. 5  ml of concentrated nitric acid and 2  ml of hydrogen peroxide were added and the mixture subjected to microwave irradiation ( 25% of power , 5  min ) in order to digest the sample .
then , 10  ml of hydrochloric acid ( 6  m ) was added , and the mixture was irradiated by microwave ( 75% of power , 5  min ) in order to reduce all se to se ( iv ) .
. 1  ml of treated sample solution was dropwise passed through an eichrom cation exchange resin ( sulfonic acid groups , mesh ranges : 50100  m , 8  cm ) , for removal of interferences , and selenium was determined with the recommended procedure .  
a selen plus capsule ( germany ) was dissolved in 10  ml hcl ( 6  m ) , and the mixture was irradiated by microwave ( 75% of power , 5  min ) in order to reduce all se to se ( iv ) .
the mixture was then diluted to 50  ml with water , and then 1  ml of this mixture was taken for determination of selenium ( iv ) as in the recommended procedure .
from the plot obtained by job 's method of continuous variation , the se- ( iv ) to- dithizone ( h2dz ) ratio forming the complex was found to be 1 
:  4 at ph  <  1 . the complex can , therefore , be represented as se(hdz)4 which is in agreement with the reported composition in the organic phase .
the conditional stability constant of the 1  :  4 ( se  :  h2dz ) complex was calculated , assuming the following equilibrium in the system :  
 ( 2)h2seo3 + 4  h2dzse(hdz)4 + 3h2o . 
 using the plot obtained by job 's method ,
xmax  ( chdz / ctotal ) was found to be 0.8 , and therefore the ratio of se  :  hdz was calculated 1  :  4 ( figure 1 ) .
selenium ( iv ) reacts with dithizone ( hdz ) in micellar medium and forms a hydrophobic complex , se(hdz)4 , which is subsequently trapped in the surfactant micelles and separated from the aqueous phase .  
figure 2 shows the absorption spectra for the dithizone and selenium - dithizonate complex in surfactant - rich phase against a reagent blank .
the absorption maximum of the complex was 424  nm , and the absorption maxima of the dithizone were 434 and 592  nm .
solution ph plays a unique role on metal - chelate function and subsequent extraction .  
figure 3 shows the influence of hcl concentration on the corrected absorbance of the selenium dithizonate complex at 424  nm .
as can be seen , the complex extraction was increased with the increased hcl concentration and then showed a decrease .
the reason for this behaviour might be because of decreasing the dithizone stability at higher phs . hence , an hcl concentration of 0.35  m was chosen for further studies . the influence of the dithizone ( hdz ) concentration on analytical response is shown in figure 4 .
this figure shows that the absorbance increased up to a known concentration of dithizone ( 7.5  mol  l ) and then reached a plateau that can be attributed to complete extraction .
therefore , the concentration of 7.5  mol  l of dithizone was selected as the optimum .  
the nonionic surfactant triton x-100 was chosen because of its commercial availability of high purified homogeneous form , low toxicological properties , and cost .
the cloud point temperature of triton x-100 is 63.7c . to decrease the cloud point temperature of triton x-100 to room temperature ( desirable temperature in cloud point procedure ) ,
this solution was prepared by the diluent ( solution of 0.5% ( w / v ) of phenol ) .
the variation of absorbance at  
 max of complex as a function of the concentration of triton x-100 is shown in figure 5 . at lower concentrations of triton x-100
, the low extraction efficiency of the complex may be due to the inadequacy of the assemblies to entrap the hydrophobic complex quantitatively .
the concentration of 0.2% ( w / v ) triton x-100 was selected for further studies .
optimal incubation time and equilibration temperature are necessary to complete the reaction , easy phase separation , and preconcentration as efficient as possible . providing that the complexation reaction has been completed under certain conditions , corrected absorbance data
show that , for equilibration times of 10 to 30  min , extraction efficiency is almost constant .
on the other hand , it appears that the phase volume ratio of all nonionic surfactants decreases as the equilibration temperature increases . as can be seen in figure 7 , the corrected absorbance increases very gently up to 40c and starts to decrease afterwards due to decrease in the stability of the dithizone and consequently the extraction efficiency .
the dependence of corrected absorbance upon centrifugation time was studied within the range of 530  min .
it was found that a time of 15  min is adequate for separation of two phases .
calibration graph was obtained by preconcentrating 50  ml of the sample containing 5 , 15 , 30 , 40 , 55 , 70 , 80 , and 100  ng  ml of selenium ( iv ) under the optimal experimental conditions .
analytical parameters for the determination of the selenium - dithizonate complex are presented in table 1 .
the relative standard deviation was 2.18% for six replicate measurements of standard selenium solution ( 50  ng  ml ) .  
the effect of various cations and anions on the determination of 0.05  g  ml of se(iv ) was studied , and the results are summarized in table 2 .
the tolerance limit was taken as the concentration of added ion causing less than 5% relative error .
cations such as ag(i ) , cu(ii ) , hg(ii ) , and fe(iii ) were interfered with the determination of se(iv ) ion .
these cations can be removed using a cation exchange resin ( sulfonic acid groups , mesh ranges : 50100 , 8  cm ) . for this purpose , 25  ml solution containing 2.5  g of se(iv ) and various amounts of desired cations
was passed through 5  g cation exchange resin ( 8  cm wet resin ) dropwise and collected in a 50  ml volumetric flask containing optimum amount of dithizone ( 7.5  mol  l ) , hcl ( 0.35  m ) , and triton x-100 ( 0.2% w / v ) .
after washing the column with water ( three times , with 15  ml ) , 0.5% ( w / v ) phenol was added and the solution was diluted to the mark with water and the amount of se ( iv ) was determined using recommended procedure . to evaluate the analytical applicability ,
the method was applied to the determination of selenium in two multivitamin tablets ( centrum and selen plus ) and a selenium sulphide shampoo ( for treatment of dandruff ) .
the results for selenium sulphide shampoo , centrum , and selen plus multivitamins are given in tables 3 , 4 , and 5 , respectively .   in order to evaluate the accuracy of procedure
, selenium was determined in centrum tablet ( containing 0.25  mg se as sodium selenate ) by icp - oes as reference method . in both methods the results agreed well ( table 6 ) .  
the limit of detection and relative standard deviation of the proposed method were compared with methods in the literature ( table 7 ) .
as observed from the results limit of detection by the proposed method was lower than other methods .
the simplicity , rapidity , and inexpensiveness of the proposed method in combination with the use of dithizone as a chromogenic reagent were utilized for selenium determination in cosmetic and pharmaceutical samples .
precision and recovery data clearly indicated the reproducibility and accuracy of the method . in this investigation ,
spectrophotometric determination of selenium was carried out in micellar medium without the need for an extraction step .
because of the overlapping of absorption maximum of complex at 424  nm with absorption maximum of dithizone at 434  nm , a 
 corr was applied .
 a simple , rapid , and sensitive spectrophotometric method for the determination of trace amounts of selenium ( iv ) was described . in this method 
 , all selenium spices reduced to selenium ( iv ) using 6  m  hcl . 
 cloud point extraction was applied as a preconcentration method for spectrophotometric determination of selenium ( iv ) in aqueous solution . 
 the proposed method is based on the complexation of selenium ( iv ) with dithizone at ph  <  1 in micellar medium ( triton x-100 ) . 
 after complexation with dithizone , the analyte was quantitatively extracted to the surfactant - rich phase by centrifugation and diluted to 5 ml with methanol . since the absorption maxima of the complex ( 424  nm ) and dithizone ( 434  nm ) overlap , hence , the corrected absorbance , acorr , was used to overcome the problem . 
 with regard to the preconcentration , the tested parameters were the ph of the extraction , the concentration of the surfactant , the concentration of dithizone , and equilibration temperature and time . 
 the detection limit is 4.4  ng ml1 ; the relative standard deviation for six replicate measurements is 2.18% for 50  ng ml1 of selenium . 
 the procedure was applied successfully to the determination of selenium in two kinds of pharmaceutical samples . 
1. Introduction
2. Experimental
3. Results and Discussion
4. Conclusion