the success of endodontic treatment is mainly dependent on thorough cleaning , shaping and disinfection of root canal system . the irrigation protocol thereby plays a key role in the disinfection of the root canal space . a clean root canal system along with a three dimensional seal is the clinician 's road to success . subsequent to biomechanical preparation , an amorphous irregular layer known as the  smear layer  is formed on the root canal walls . it contains inorganic and organic substances that include fragments of odontoblastic process , microorganisms and necrotic debris . its presence increases microflora and the inorganic toxins , decreases the sealing ability and increases the potential for bacterial survival and reproduction . until date , no single irrigant has been capable of demonstrating both tissue dissolving as well as demineralizing properties . current methods to remove the smear layer might involve the use of a chelating agent during irrigation or as a final rinse in combination with other irrigants having tissue dissolving properties . sodium hypochlorite ( naocl ) is the main endodontic irrigant used to dissolve the organic portion of the smear layer . to remove the inorganic portion of the smear layer , a decalcifying agent is used , which can be either a chelator or an acid currently , all the products in the dental market sold to dissolve smear layer are based on ethylenediaminetetraacetic acid ( edta ) or citric acid . therefore , it has been proposed to use peracetic acid ( paa ) instead of these classical decalcifying agents to dissolve the smear layer and also to disinfect the root canal system . apart from edta and paa , a newly introduced qmix solution is also recommended chelator which is used in conjunction with naocl . recent research indicates that qmix ( dentsply tulsa dental , tulsa , ok ) , an experimental irrigant containing a mixture of a bisbiguanide antimicrobial agent , a polyaminocarboxylic acid calcium - chelating agent and a surfactant , might be as effective as edta at removing smear layers when used after an initial rinse with naocl . it is well - known that some chemicals used for endodontic irrigation are capable of causing alterations in the chemical composition of dentin . any change in the ca : p ratio may in turn change the microhardness , permeability and solubility characteristics of dentin and may also adversely affect the sealing ability and adhesion of dental materials . no study until date has been done to compare the effect of paa , qmix and edta on calcium loss and microhardness of root dentin . therefore , the present study has been undertaken to evaluate the effect of different irrigation regimens on calcium loss and its effect on the micro - hardness of the root dentin . ten intact , single rooted human premolars , which were extracted for orthodontic reasons were used for this study . teeth were stored for 1 week in formalin and then in normal saline until use . the teeth were decoronated at the cementoenamel junction using a high speed carbide bur under copious water irrigation . thick transverse sections of 2 mm with a maximum and minimum width of 3 mm and 2 mm respectively were obtained from the coronal third of each root using a low - speed safe sided diamond disc . each section was further divided into 4 quarters , each part constituting a sample specimen from the same tooth for each group . specimens were horizontally embedded in autopolymerizing resin so as to expose the canal part of the dentin . the specimens were ground flat on a circular wet grinding machine with ascending grades of sic abrasive papers ( 320 , 600 , 1000 , 1200 and 1500 grit ) under constant water irrigation using leco grinder polisher . 17% edta was prepared by adding 17 g of disodium salt of edta powder into 100 ml of distilled water and favoring its dissolution by the addition of 5 n sodium hydroxide . 2.25% paa was prepared by dissolving equal volume of hydrogen peroxide ( 26% ) , acetic acid and acetylhydroxyperoxide in equilibrium . nearly 5% naocl solution and qmix ( dentsply , tulsa dental , ok , usa ) were obtained as commercial preparations . the treatment groups were as follows : 
 group 1 ( control ) : 5% naocl for 5 min - distilled water for 5 mingroup 2 : 5% naocl for 5 min-17% edta for 5 mingroup 3 : 5% naocl for 5 min-2.25% paa for 5 mingroup 4 : 5% naocl for 5 min - qmix for 5 min . group 1 ( control ) : 5% naocl for 5 min - distilled water for 5 min group 2 : 5% naocl for 5 min-17% edta for 5 min group 3 : 5% naocl for 5 min-2.25% paa for 5 min group 4 : 5% naocl for 5 min - qmix for 5 min . all the specimens were immersed in a magnetic stirrer bath containing 10 ml of the first test solution for 5 min . they were then immersed into 10 ml of the second test solution of the respective group for another 5 min . each time after irrigation of one specimen per group , the eluates were centrifuged at 4000 g for 10 min . subsequently , 10 ml of the supernatant was transferred to a polypropylene tube with a lid and stored at 20c until further analysis . once all the eluates had been collected , they were thawed and then analyzed for their calcium content using an atomic absorption spectrophotometer ( lab india , india ) with an air acetylene flame . for each specimen after the combined treatment , surface hardness of the root dentin was measured with a vickers hardness tester ( hmv , shimadzu , japan ) . hardness was measured under the load of 300 g with duration of 15 s. in each sample , three indentations were made . the representative hardness value for each sample was obtained as the average of the three indentation values . mean values were compared among different study groups by using one - way anova followed by post hoc tukey test . pearson 's correlation was done to compare the relation between calcium loss and subsequent microhardness of root dentin . 17% edta was prepared by adding 17 g of disodium salt of edta powder into 100 ml of distilled water and favoring its dissolution by the addition of 5 n sodium hydroxide . 2.25% paa was prepared by dissolving equal volume of hydrogen peroxide ( 26% ) , acetic acid and acetylhydroxyperoxide in equilibrium . nearly 5% naocl solution and qmix ( dentsply , tulsa dental , ok , usa ) were obtained as commercial preparations . the treatment groups were as follows : 
 group 1 ( control ) : 5% naocl for 5 min - distilled water for 5 mingroup 2 : 5% naocl for 5 min-17% edta for 5 mingroup 3 : 5% naocl for 5 min-2.25% paa for 5 mingroup 4 : 5% naocl for 5 min - qmix for 5 min . group 1 ( control ) : 5% naocl for 5 min - distilled water for 5 min group 2 : 5% naocl for 5 min-17% edta for 5 min group 3 : 5% naocl for 5 min-2.25% paa for 5 min group 4 : 5% naocl for 5 min - qmix for 5 min . all the specimens were immersed in a magnetic stirrer bath containing 10 ml of the first test solution for 5 min . they were then immersed into 10 ml of the second test solution of the respective group for another 5 min . each time after irrigation of one specimen per group , the eluates were centrifuged at 4000 g for 10 min . subsequently , 10 ml of the supernatant was transferred to a polypropylene tube with a lid and stored at 20c until further analysis . once all the eluates had been collected , they were thawed and then analyzed for their calcium content using an atomic absorption spectrophotometer ( lab india , india ) with an air acetylene flame . for each specimen after the combined treatment , surface hardness of the root dentin was measured with a vickers hardness tester ( hmv , shimadzu , japan ) . hardness was measured under the load of 300 g with duration of 15 s. in each sample , three indentations were made . the representative hardness value for each sample was obtained as the average of the three indentation values . mean values were compared among different study groups by using one - way anova followed by post hoc tukey test . pearson 's correlation was done to compare the relation between calcium loss and subsequent microhardness of root dentin . the mean calcium loss and dentin microhardness and its standard deviation along with intergroup comparison are given in table 1 . intergroup comparison for calcium loss and microhardness mean calcium loss in naocl + distilled water group ( 2.79  0.97 ppm ) and naocl + qmix group ( 5.72  0.91 ppm ) are least followed by naocl + edta group ( 6.38  1.76 ) and maximum in naocl + paa group ( 9.00  0.55 ppm ) . there was a statistically significant difference between all groups except between naocl + qmix group and naocl + edta group ( f = 50.215 , df-3 , 36 ; p < 0.001 ) . the mean dentin microhardness in naocl + distilled water group ( 77.39  2.16 vickers hardness number [ vhn ] ) and naocl + qmix group ( 70.68  4.97 vhn ) were maximum followed by naocl + edta group ( 69.70  4.14 ) and least in naocl + paa group ( 62.98  8.17 vkn ) . there was statistical significant difference between all the groups except between naocl + qmix group and naocl + edta group ( f = 12.26 , df-3 , 36 ; p < 0.001 ) . a negative correlation existed between the calcium loss and reduction in the microhardness of root dentin . dentin is composed of inorganic components of hard dental tissues , in which calcium and phosphorus are distributed in the form of hydroxyapatite crystals . the ca / p ratio in hydroxyapatite is approximately 1.67 and it depends on many factors such as level of mineralization , type of crystals , age of tissue and anatomical site . during biomechanical preparation , removal of the smear layer requires the use of irrigants that can dissolve both the organic and inorganic components . since chelating agents act only on the inorganic component of the smear layer , therefore they were used in combination with naocl which acts on its organic component altering the mechanical and chemical properties of root dentin . however , reports have indicated that the use of edta and naocl may lead to dentinal erosion of the root canal walls . further , it has been reported that surface treatment of dentin with different agents may cause alterations in the chemical and structural composition of dentin , which in turn may change its permeability and solubility characteristics and subsequently a loss of ca / p ratio of root dentin resulting in an impact on the microhardness . in the design of this study , the issue of biological variability among different teeth was addressed by comparing the effects of different solutions on the dentin sections from the same patient . this allowed the comparison of the demineralizing capacity of different irrigating solutions on identical sections having similar degree of calcification geometry and surface area . also , coronal parts of the roots were used to prepare the dentin specimens because of the higher possibility of sclerotic dentin in apical root . the exposure time of all the irrigants was kept 5 min and was standardized for all the groups . atomic absorption spectroscopy is the technique used in this study to evaluate the demineralization effect of the chelators used and to determine the concentration of calcium in each sample . it is a single element technique which is less cost - effective than newer multi - element , techniques such as inductively coupled plasma atomic emission spectrometry . in the present study , the vickers microhardness test was done to evaluate the surface changes of dental hard tissue specimens , treated with the chemical agents . further microhardness determination can provide indirect evidence of mineral loss or gain in the dental hard tissues . a significant negative correlation between loss of calcium from root dentin and microhardness panighi and gsell found a simple linear correlation between hardness of dentin and calcium ion concentration and between hardness of dentin and wettability . in this study , irrigation with 5% naocl followed by distilled water as a final rinse eluted minimum amount of calcium from the dentin ( 2.7 ppm mean ) and when compared with other groups , the difference was statistically significant . ( 2009 ) where naocl and distilled water hardly eluted any ca . in the control group , no chelating agent was used but still some calcium loss was seen as a result of its mechanical flushing action on smear layer formed on root dentin . since chelating agents cause demineralization of dentin , resulting in its softening , we find that more ca loss was seen in groups having chelating agents . this might be the reason why all experimental groups showed significantly lower microhardness when compared with the control group . when the mean ca loss of group 3 ( 5% naocl - paa ) was compared with group 2 ( 5% naocl + edta ) and group 4 ( 5% naocl - qmix ) , the result showed that group 3 extracted significantly more ca ions . more ca loss shown by paa can be explained by the fact that because of its acidity , the calcium stays in solution and does not reprecipitate . despite the weak chelating power of this agent , more amounts of ca ions were eluted from the root canal as compared to edta , which is a much stronger chelator but can only be in solution at a slightly alkaline ph . however , in our study the ph used was neutral and thus edta could not remain precipitated in the solution . ( 2009 ) also found that edta removed slightly more smear layer than paa ( p < 0.05 ) . more ca loss shown by paa could have resulted in a significant reduction in microhardness when compared with edta and qmix group . an insignificant difference in ca loss and microhardness was seen between 5% naocl - edta and 5% naocl - qmix groups . as qmix contains edta in its composition along with chlorhexidine and a detergent , the effect of qmix on root dentin could have been almost similar to edta . a study carried out by dai et al . ( 2011 ) showed that the smear layer removing ability of qmix was comparable to that of 17% edta . from the present study , it can be concluded that : 
 irrigation with 5% naocl + 2.25% paa caused the maximum calcium loss from root dentin and minimum microhardness.irrigation with 5% naocl + distilled water caused minimum calcium loss from root dentin and maximum microhardness.a reduction in the microhardness of root dentin was observed with increase in calcium loss from root dentin . irrigation with 5% naocl + 2.25% paa caused the maximum calcium loss from root dentin and minimum microhardness . irrigation with 5% naocl + distilled water caused minimum calcium loss from root dentin and maximum microhardness . a reduction in the microhardness of root dentin was observed with increase in calcium loss from root dentin .
objectives : the objective of this in vitro study was to assess the effect of different chelating agents on the calcium loss and its subsequent effect on the microhardness of the root dentin.materials and methods : ten single rooted lower premolars were selected . 
 the teeth were decoronated and thick transverse sections of 2 mm were obtained from the coronal third of the root . each section was then divided into four quarters , each part constituting a sample specimen from the same tooth for each group . 
 the treatment groups were : group 1 ( control ) : 5% sodium hypochlorite ( naocl ) for 5 min + distilled water for 5 min ; group 2 : 5% naocl for 5 min + 17% ethylenediaminetetraacetic acid ( edta ) for 5 min ; group 3 : 5% naocl for 5 min + 2.25% peracetic acid ( paa ) for 5 min and group 4 : 5% naocl for 5 min + qmix for 5 min respectively . 
 the calcium loss of the samples was evaluated using the atomic absorption spectrophotometer followed by determination of their microhardness using vickers hardness tester . 
 data was analyzed using one - way anova , post hoc tukey test and pearson correlation.results:the maximum calcium loss and minimum microhardness was observed in group 3 followed by group 2 , group 4 and group 1 . 
 there was a statistically significant difference between all the groups except between groups 2 and 4.conclusions:irrigation with naocl + 2.25% paa caused the maximum calcium loss from root dentin and reduced microhardness . 
 a negative correlation existed between the calcium loss and reduction in the microhardness of root dentin .