Spectrophotometric Determination of Manganese by Biologically Active 2-Hydroxy-4-Methoxy Acetophenone Oxime

Introduction

A number of reagents are known for spectrophotometric and complexometric determination of Mn(II) and other transition metal ions1-5. The present communication deals with the spectrophotometric determination of Mn(II) with HMAO & compare their biological behaviour with standard drug.

Experimental

Standard stock solution (0.1M) of Mn(II) has been prepared by dissolving appropriate amount of MnCl2 (AR) in double distilled water. The amount of Mn(II) in stock solution was determined by standard method.6

Preparation of 2-Hydroxy-4-methoxy acetophenone oxime ( HMAO)

2,4-Dihydroxy acetophenone(resacetophenone) and 2-hydroxy-4-methoxy acetophenone were prepared by standard methods 7,8 Oxime of HMA has been prepared by  refluxing HMA with hydroxyl amine hydrochloride in the presence of sodium acetate in ethanol medium for 2 hrs. The product separated on concentration and subsequent dilution and recrystallised from ethanol in to colourless needle like crystals (mp101◦С)with m.w. 181.24 gm ( calcd for C9H11NO3 ).The structure of the compound was confirm by elemental analysis. IR spectra, NMR spectra and Mass spectra.

Preparation of Mn(II) –HMAO complex and selection of solvent

When metal ion solution was treated with an ethanolic solution of HMAO and the mixture was stirred for about an hour at room temperature, dark brown precipitates of complex were obtained in the pH range 8.0-11.0. The complex was found to be insoluble

in polar solvents like water, methanol or ethanol but soluble in non polar solvents like chloroform, benzene, CCl4 etc. This complex was more  soluble in chloroform so it was elected as a solvent for extractive spectrophotometric determination of Mn(II).

Apparatus

Systronics UV/VIS spectrophotometer (model-118) was used for absorbance measurement & systronic pH meter (model 335) for pH measurement.               .

Results and Discussion

Selection of optimum wavelength & pH

The formation of Mn(II) complex with HMAO & their stability are depend on the pH of solution. While absorbance is depend upon the wavelength.

The method of Vosburg & cooper showed the formation of only one complex having λmax at 410 nm.The absorbance of complex formation was measured  at room temperature (300 K) at regular intervals of time up to two week & also different temperature varies from 300K  to 325K. The results show that complex is stable for one week & upto 318K without change of absorbance.

The Mn(II) complex exhibit maximum & almost constant absorbance in the pH range 8.0 to 11.0.( Table-2). The subsequent studies were, therefore, carried out at pH 10.0. It was also found that a 16 fold excess of the reagent was necessary to attain the maximum colour intensity.

Table 1: VOSBURG AND COOPER’S METHOD FOR  Mn(II) AND HMAO

Concentration of Mn(II)    =     HMAO      =   1.0  x  10 -2 M

Total Volume                                             =    16.0  ml

Wave length                              Ratio of Metal to ligand
(nm)                  1:1  OD              1:2   OD                    1:3 OD                1:4  OD
410                       0.580                   0.785                        0.785                   0.330
420                       0.540                   0.750                       0.780                   0.290
430                       0.450                   0.670                       0.750                   0.230
440                       0.365                   0.535                       0.690                   0.185
450                       0.315                   0.410                      0.600                   0.150
460                        0.280                  0.340                      0.500                   0.120
480                        0.215                  0.270                       0.390                   0.100
500                        0.150                  0.220                       0.295                    .080
520                        0.110                   0.190                      0.240                    .060
540                        0.100                   0.150                      0.210                    .040
560                        .090                    0.140                       0.200                    .030
580                        .085                    0.135                       0.200                    .030
Metal  (ml)              6.0                      4.0                            3.0                       8.0
Ligand  (ml)             6.0                     8.0                            9.0                       4.0
Fig. 4.33                     A                       B                              C                          D

 

Table 2: EFFECT OF pH ON THE ABSORBANCE OF Mn(II)-HMAO AT 410  nm

Concentration of Mn(II)=HMAO   =  1.0 x 10-3 M

Volume of Mn(II) Solution            =   2.0  ml

Volume of HMAO Solution           =   4.0   ml

Total Volume                                 =    8.0  ml

pH	O.D	pH	O.D
7.0	0.080	9.5	0.250
8.0	0.120	10.0	0.280
8.5	0.180	10.5	0.280
9.0	0.210	11.0	0.280

 

Reproducibility

Absorbance measurements of a set of six solution prepared in a similar way & have the same concentration of all the reagents show that the reproducibility of measurements are quite good with standard deviation ± 0.432 i.e. 0.26 %.

Stoichiometry and stability constant of the complex

Results of yoe and jones mole ratio method9, slope ratio method10  and job’s method of continuous variation11 establish the formation of 1:2 ( metal: ligand) complex.(Fig 2,3)

The stability constant of the complex was calculated from the following relationship using mole ratio method. (Table-3)

Came to be 5.468×107. The value of the standard free energy of the formation of complex from the expression ∆ G = -2.303 RT log K , came to be 1.06 Kcal/ mol at 30◦C.

 

Table 3:COMPOSITION OF THE Mn(II)-  HMAO COMPLEX BY MOLE RATIO METHOD                                                        AT410 nm AND pH 10.0

Concentration of  Mn(II) Solution  = HMAO   = 0.5 x 10-3 M

Constant Volume of Mn(II)                                = 2.0  ml

Total Volume                                                     = 16.0 ml

Ionic strength                                                    =   0.1 M NaClO4

Volume of HMAO		Volume of HMAO
Solution (ml)	OD	Solution (ml)	OD
1.0	0.175	6.0	0.278
2.0	0.225	7.0	0.298
3.0	0.245	8.0	0.298
4.0	0.254	9.0	0.298
5.0	0.257

Validity of Beer’s law and optimum concentration range

It was observed that the Beer’s law is obeyed in the concentration range 1-12 ppm of Mn(II). The optimum concentration range for determination of Mn(II) in solution as deduced from Ringbom plot 12. From the slope ratio curve, the molecular extinction coefficient of the complex is 2.40×102 while the value of photometric sensitivity as per sendell’s scale is found to be 0.228 μg-Mn/cm2

Effect of foreign ions

The effect of foreign ion on the spectrophotometric determination of manganese was studied by adding different ions quantitatively in a stock solution of Mn(II) ion. Mn(II) was extracted as Mn(II)-HMAO complex in the usual manner after adjusting the pH of solution at 10.0. It was observed that at 9 ppm of Mn(II),1500 ppm of Cl-,SO42- &CH3COO-,1200 ppm of NH4+, K+,Na+, NO3-,Br- and I-: 800 ppm of SO32- & NO2-,  500 ppm of  Ca2+, Ba2+,Sr2+, tartarate & citrate; 150 ppm of Zn2+,Cd2+ and Be2+ could be tolerated. However Cu2+, Pd2+, Co2+, Fe3+ & UO22+ interfered seriously.

The precision  & accuracy of spectrophotometric method were tested by analysing sample containing the known amount of Mn(II). It was found that the standard  deviation of the method does not exceed 2 % & the accuracy of the procedure in limits accepted for spectrophotometric determinations.

Determination of Manganese from different samples

The usefulness of the reagent in estimation of manganese was determined from various samples of Mn(II) concentration having different concentration. The sample mixture containing manganese metal were taken for spectrophotometric analysis at different wavelength. The result are given in table (IV)

Table-4: Analysis of Manganese in various sample

Sample	Mn taken	Mn found	Absorbance	Relative error
	μg	μg
Synthetic Mixture No.1	150	0.420 0.410 0.180 Avg.	164.5 159.3 167.4 163.7	0.13
Synthetic Mixture No.2	120	0.310 0.326 0.334 Avg.	112.6 120.1 123.4 118.7	0.91
Synthetic Mixture No.3	206	0.549 0.547 0.552 Avg.	202.3 209.6 211.6 207.8	0.87

Analysis

The extracted complex was concentrate & crystallized in desiccator. The elemental analysis of the complex shows metal:ligand  (M:L) ratio to be 1:2

Founed          C 53.21           H   4.93             H    7.26        Mn    12.75

Calculated      C 53                H  4.82             H     7.48        Mn    12.99

 

The IR spectra of HMAO and complex revealed that the –OH ( Stretch) band at 3280

cm-1 for the HMAO disappears during complexation .i.e. complex formation takes place through the N of oximino group and oxygen of the hydroxyl group.

The general composition of the complex is [  C18H20N2O6Mn ]

Antimicrobial activity

HMAO & Mn-HMAO were screened for their antibacterial activity against E.Coli & antifungal activity against Aspergillus niger using agar diffusion 13 method & dry weight increased method14. The results indicate that HMAO & its Mn(II) complex exhibit more antimicrobial activity with respect to standard drug.

Mode of action

Antimicrobes exert their action in the different ways

Inhibition of cell wall synthesis.

Inhibition of cell membrane

Inhibition of Biosynthesis ( i.e. production of purines,pyrimidine,Amino acid, Vitamins, Protien,  RNA, DNA)

Inhibitors of energy production ( inhibit the respiration or by uncoupling of oxidative phosphorylation ( Disruption the metabolic activities.)

The studies demonstrated that chelation can increase antimicrobial activities than ligand. It has been suggested that metal chelation reduced polarity of metal ion 15 mainly because of the partial shairing of its positive charge with the donor group and possibility of  the d-electron  delocalization occurring with in the whole chelate ring system formed during coordination. This process of chelation thus increase the lipophilic nature of the central metal atom which in turn favours its permeation through the lipoid layer of the membrane.16

 

 

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