EXPERIMENTAL PROCEDURE
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1 EXPERIMENTAL PROCEDURE The present experimentation is carried out on biosorption of chromium and lead from aqueous solutions by biosorbents Ageratum conyzoides leaf and Anacardium occidentale testa powder. These experiments are carried out in batch operations. The experimental procedure consists of the following steps: 4.1 Preparation of the biosorbents 4.2 Characterization of the biosorbent 4.3 Preparation of stock solutions 4.4 Biosorption studies. 4.1 Preparation of the biosorbents: Preparation of Ageratum conyzoides leaf powder: Ageratum conyzoides leaves were collected from ICFAI campus, Dehradun. Ageratum conyzoides leaves were washed thoroughly with water, distilled water and completely dried in sunlight. The dry mass was grinded and the resulting powder was separated into different sizes (53, 75, 105, 125 and 152 µm) using BSS sieves. These size fractions were stored in air tight packing for further use as biosorbent Preparation of Anacardium occidentale testa powder: Anacardium occidentale testa was collected from Vetapalem, Prakasam, Andhra Pradesh. The testa were cleanly washed with water and then with distilled water, dried in sun until it became colorless and crispy. They were grinded to powder, separated into desired sizes using BSS sieves and stored in air tight plastic covers. 209
2 4.2 Characterization of biosorbents Scanning electron Microscope (SEM) studies: The pretreated biosorbent samples were examined in Scanning Electron Microscope and electron probe micro analyzer. The samples were coated with ultra thin film of gold by an ion sputter JFC-1100 and exposed under SEM. The working height was 15 mm with a voltage ranging from 10 to 25 kv. The compositional image analyses of samples were also taken using same equipment at 20 kv and na beam current. 4.3 Preparation of stock solutions Preparation of chromium stock solution: The standard stock solution of chromium (1000 mg/l) was prepared by dissolving g of 99.9 % analytical grade K 2 Cr 2 O 7 in 1000 ml of distilled water. All the required solutions are prepared with analytical reagents and double-distilled water. Synthetic samples of different concentrations of chromium are prepared from this stock solution by appropriate dilutions. 20 mg/l chromium stock solution is prepared by diluting 20 ml of 1000 ppm chromium stock solution with distilled water in 1000 ml volumetric flask up to the mark. Similarly solutions with different metal concentrations such as (50, 100, 150 and 200 mg/l) are prepared. The ph of aqueous solution is adjusted to the desired value by addition of 0.1 N HNO 3 or 0.1N NaOH solution. 210
3 (Molecular wt. of K 2 Cr 2 O 7 X 100) Chromium equivalent to 1gm = (Atomic wt. of Chromium X purity) Preparation of lead stock solution: Pb(NO 3 ) 2 is used as the source of lead stock solution. All the required solutions are prepared with analytical reagents and double-distilled water g of 99% Pb(NO 3 ) 2 is dissolved in distilled water in 1 L volumetric flask up to the mark to obtain 1000 ppm (mg/l) of lead stock solution. Synthetic samples of different concentrations of lead are prepared from this stock solution by appropriate dilutions. 20 mg/l lead stock solution is prepared by diluting 20 ml of 1000 ppm lead stock solution with distilled water in 1000 ml volumetric flask up to the mark. Similarly solutions with different metal concentrations such as (50, 100, 150 and 200 mg/l) are prepared. The ph of aqueous solution is adjusted to the desired value by addition of 0.1 N HNO 3 or 0.1N NaOH solution. (Molecular wt. of lead nitrate X 100) Lead equivalent to 1gm = (Atomic wt. of lead X purity) 4.4 Biosorption studies The initial concentrations of chromium and lead in the aqueous solutions were analyzed in an Atomic Absorption Spectrophotometer (Perkin Elmer A Analyst 200 model) and found to be 50 mg/l and 50 mg/l respectively. Wave length is nm for chromium and nm for lead. The sensitivity check is 4.0 mg/l for chromium and 10 mg/l for lead. The procedures adopted for the biosorption of chromium and lead are given below. 211
4 4.4.1 Biosorption of chromium Effect of agitation time: 50 ml of aqueous solution (initial concentration of chromium was 20 gm/l) was taken in a 250 ml conical flask and 10 g/l of 53 μm size biosorbent was added. This sample was shaken on an orbital shaker at 180 rpm at 30 o C for 1 min. Similarly 14 more samples were prepared in conical flasks adding 10 g/l of biosorbent and exposed to varying agitation times (3, 5, 10, 15, 20, 25, 30, 40, 50, 60, 90, 120, 150 and 180 min). These samples were filtered separately with Whatman filter papers and the filtrates were analyzed in AAS to obtain final concentrations of chromium. The percentage biosorption of chromium is calculated as (C o -C e ) x 100/C o, where C o is initial concentration of chromium in metal solution and C e is equilibrium concentration of chromium in metal solution. The equilibrium agitation time was calculated from the data Effect of biosorbent size: 50 ml of aqueous solution (containing 20 mg/l of Cr) was taken in each of five 250 ml conical flasks. 10 g/l of biosorbent of different sizes (53, 75, 105, 125 and 152 µm) were added and the contents were agitated in an orbital shaker for equilibrium agitation time. The mixtures thus obtained were filtered and filtrates were analyzed in AAS for residual Cr concentrations in the solutions. The optimum biosorbent size was determined from the data. 212
5 Effect biosorbent dosage: The experiments were repeated for nine more dosages (0.25, 0.75, 1.0, 1.25, 1.5, 1.75, 2.0, 2.5 and 3.0 g) of 53 µm size biosorbent for equilibrium agitation time. From these data, optimum biosorbent dosage was identified Effect of ph of the aqueous solution: To study the influence of ph on chromium biosorption, 50 ml of aqueous solution was taken in each of seven conical flasks. The ph values of the solutions were adjusted to 2, 3, 4, 4, 5, 6, 7 and 8 in separate flasks by adding required amounts of 0.1 N H 2 SO 4 or 0.1N NaOH. 10 g/l of 53 μm size biosorbent was added separately to these flasks. The samples were shaken on an orbital shaker at 30 o C for equilibrium agitation time. Then samples were allowed to settle and are filtered. The chromium concentrations of the filtrates were determined by using AAS Effect of initial concentration of the chromium in aqueous solution: 50 ml of aqueous solution was taken in a 250 ml conical flask and 10 g/l of 53 μm size biosorbent was added. The sample was kept in continuous contact for equilibrium agitation time by shaking on an orbital shaker at 30 o C. The sample was allowed to settle and then filtered. The final chromium concentration of the filtrate is determined in an AAS. The same procedure was repeated for other initial concentrations of chromium in aqueous solutions (20, 50, 80, 120 and 150 mg/l). 213
6 Kinetic studies: In order to determine the order of the biosorption rate, 50 ml of aqueous solution containing 20 mg/l of chromium was taken in each of twelve conical flasks. 10 g/l of 53 μm size biosorbent was added in each flask. The conical flasks were shaken on an orbital shaker for different time intervals at 30 0 C. They were settled, filtered and the filtrates were analyzed to find the final concentrations of chromium Thermodynamics studies: The following procedure is followed to determine the effect of temperature on biosorption rate and to evaluate the enthalpy of adsorption (ΔH), entropy of adsorption (ΔS) and Gibbs free energy (ΔG). 50 ml of aqueous solution containing 20 mg/l of chromium was taken in each of five conical flasks. 10 g/l of 53 μm size adsorbent was added in each of these flasks. The above five conical flasks were shaken in an orbital shaker for equilibrium agitation time for five different temperatures (283, 293, 303, 313 and 323K). These samples were settled and filtered. The chromium concentrations of the filtrates were determined by using AAS. The values of parameters investigated are compiled in table 4.1 to table
7 Table-4.1 Experimental conditions investigated for biosorption of chromium Biosorbent: Ageratum conyzoides leaf powder S.No. Parameter Values investigated 1 Agitation time, t, min 1, 3, 5, 10, 15, 20, 25, 30, 40, 50, 60, 90, 120, 150 & Biosorbent size, D p, µm 53, 75, 105, 125 & Biosorbent dosage, w, g 0.25, 0.5, 0.75, 1.0, 1.5, 1.75, 2.0, 2.5 & Initial chromium concentration, C 0, mg/l 20, 50, 100, 150 & ph of aqueous solution 2, 3, 4, 5, 6, 7 & 8 6 Temperature, K 283, 293, 303, 313 & 323 Table-4.2 Experimental conditions investigated for biosorption of chromium Biosorbent: Anacardium occidentale testa powder S.No. Parameter Values investigated 1 Agitation time, t, min 1, 3, 5, 10, 15, 20, 25, 30, 40, 50, 60, 90, 120, 150 & Biosorbent size, D p, µm 53, 75, 105, 125 & Biosorbent dosage, w, g 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 1.75, 2.0 & Initial chromium concentration, C 0, mg/l 20, 50, 100, 150 & ph of aqueous solution 2, 3, 4, 5, 6, 7 & 8 6 Temperature, K 283, 293, 303, 313 &
8 Biosorption of lead Effect of agitation time, biosorbent size and dosage: 50 ml of aqueous solution is taken in a 250 ml conical flask and 0.5 g of biosorbent having a size of 53 μm is added. This sample is shaken on an orbital shaker at 180 rpm at room temperature (30 o C) for 1 min. Similarly 14 more samples are prepared in conical flasks adding 0.5 g of biosorbent and exposed to varying agitation times (1, 3, 5, 10, 15, 20, 25, 30, 40, 50, 60, 90, 120, 150 and 180 min). These samples are filtered separately with what man filter papers and the filtrates are analyzed in AAS to obtain final concentrations of lead. The percentage removal of lead is calculated as (C 0 -C t ) x100/c 0. Graphs are plotted between the agitation time and % biosorption of lead to identify the equilibrium agitation time Effect of ph of the solution: To study the influence of ph on lead biosorption, 50 ml of aqueous solution is taken in each of seven conical flasks. The ph values of the solutions are adjusted to 2, 3, 4, 5, 6, 7 & 8 in separate flasks by adding required amounts of 0.1 N HNO 3 or 0.1N NaOH. 10 g/l of 53 μm size biosorbent is added separately to these flasks. The samples are shaken on an orbital shaker at room temperature for equilibrium agitation time. Then samples are allowed to settle and are filtered. The lead concentrations of the filtrates are determined by using AAS Effect of initial concentration of the lead in aqueous solution: 50 ml of aqueous solution containing 50 mg/l lead is taken in a 250 ml conical flask and 10 g/l of 53 μm size biosorbent is added. The sample is kept in continuous contact for equilibrium agitation time by shaking on an orbital shaker 216
9 at room temperature. The sample is allowed to settle and then filtered. The final lead concentration of the filtrate is determined in an AAS. The same procedure is repeated for other initial concentrations of lead in aqueous solution (20, 100, 150 & 200 mg/l) Studies on biosorption kinetics: In order to examine the adsorption or chemisorptions process and to determine the order of the rate of adsorption, 50 ml of aqueous solution containing 20 mg/l of Pb (II) was taken in each of the fifteen 250 ml conical flasks. 10 g/l of powder of 53 µm size was added in each of the flasks. The conical flasks were shaken on an orbital shaker and the samples were collected for 1, 3, 5, 10, 15, 20, 25, 30, 40, 50, 60, 90, 120, 150 and 180 min. The samples thus obtained were filtered and analyzed using AAS to get the residual concentration of Pb (II) in aqueous solution Studies on Thermodynamics: The procedure followed to determine the effect of temperature on the rate of adsorption and to evaluate the enthalpy of biosorption ( H), entropy of biosorption ( S) and Gibbs free energy ( G) is explained below. 50 ml of the lead solution of 20 mg/l concentration was taken in each of five 250 ml conical flasks. Then 10 g/l of powder of 53 µm size was added to each of the conical flasks. The flasks were then agitated on an orbital shaker for five different temperatures i.e. 283, 293, 303, 313 & 323 K for an optimum agitation time. The samples thus obtained were filtered and the filtrates were analyzed by using AAS Experimental design for biosorption studies of chromium and lead In order to obtain the optimum condition for % removal of chromium / lead, four independent parameters were selected for the study. The range of study for ph 217
10 (X 1 ), Co (X 2 ), w (X 3 ) and T (X 4 ) are chosen based on preliminary experiments. The relationship between the parameters and response were determined using Central Composite Deign (CCD) under RSM of STATISTICA 6.0 (Stat-Ease Inc., Tulsa, OK, USA) a statistical software package. The CCD design is chosen in this study as it is efficient, flexible and robust. Thirty experiments are conducted according to CCD. The percentage of chromium / lead removal was taken as a response (Y) of the experimental design. Samples taken after the desired incubation period are analyzed with an AAS. The regression analyses, graphical analyses and analyses of variance (ANOVA) were done using the STATISTICA software (version6.0), Stat Ease Inc, USA. The statistical significance of the coefficient was determined by Student s t-test and p-values. The proportion of variance obtained by the model was explained by the multiple coefficient of determination, R 2. Table-4.3 Experimental conditions investigated for biosorption of lead Biosorbent: Ageratum conyzoides leaf powder S.No. Parameter Values investigated 1 Agitation time, t, min 1, 3, 5, 10, 15, 20, 25, 30, 40, 50, 60, 90, 120, 150 & Size of biosorbent, D p, µm 53, 75, 105, 125 & Biosorbent dosage, w, g 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 1.75, 2.0 & Initial lead concentration, C 0, mg/l 20, 50, 100, 150 & ph of aqueous solution 2, 3, 4, 5, 6, 7 & 8 6 Temperature, K 283, 293, 303, 313 &
11 Table-4.4 Experimental conditions investigated for biosorption of lead Biosorbent: Anacardium occidentale testa powder S. No. Parameter Values investigated 1 Agitation time, t, min 1, 3, 5, 10, 15, 20, 25, 30, 40, 50, 60, 90, 120, 150 & Size of biosorbent, D p, µm 53, 75, 105, 125 & Biosorbent dosage, w, g 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 1.75, 2.0 & Initial lead concentration, C 0, mg/l 20, 50, 100, 150 & ph of aqueous solution 2, 3, 4, 5, 6, 7 & 8 6 Temperature, K 283, 293, 303, 313 &
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