MICELLAR SPECTROPHOTOMETRIC DETERMINATION OF COPPER IN THE VARIOUS SAMPLES OF MILK

MICELLAR SPECTROPHOTOMETRIC DETERMINATION OF COPPER IN THE VARIOUS SAMPLES OF MILK

N. Gakkhar ^{* 1} and N. Bhojak ²

GCRC Department of Chemistry ¹, S. S. Jain Subodh P. G. College, Rambagh Circle, Jaipur - 302004, Rajasthan, India.

GCRC Department of Chemistry ², Government Dungar Collge, Bikaner - 334001, Rajasthan, India.

ABSTRACT: Milk is a complex system where analysis of metal ions is a difficult task due to the presence of interfering materials. The formation of a coloured complex between the metal and a coloured or colourless ligand is the basis for the development of spectro-photometric methods. The aim of this study is to analyze and estimate copper present in various samples of milk (i.e., cow, buffalo, goat, camel, soya, and powdered milk) by micellar spectrophotometry method. For this purpose, the ligand N-(2’-Pyridyl)-2-hydroxy-benzamide (NPHB-2) was synthesized by the reaction of 2-aminopyridine with salicylic acid, which is a weak acid and is found to be soluble in alcohol, acetone, and chloroform. It is insoluble in water at a pH less than 7. In addition to the solution of this ligands to copper (II) solution in the presence of micellar medium, a yellowish-green or light green coloured complex is formed immediately. It has been observed that in the presence of a micellar medium, solubility, as well as absorption intensity, has been found to be increased. This phenomenon has been used to develop a novel and selective micellar spectrophotometric method for the determination of metal ions in various samples of milk.

Milk, Spectrophotometry, Copper, Micellar medium

INTRODUCTION: Milk is the most valuable and nutritious product for human consumption because it contains vital nutrients, including proteins, essential fatty acids, lactose, vitamins, and minerals in balanced proportions ^{1, 2}. Milk is also an important source of metallic elements, including trace elements such as alumi-nium, copper, iron, manganese, silicon, zinc etc. for human nutrition ³.

Copper is an important trace element believed to be essential for living beings. However, excess copper catalyzes oxidative changes, which have an adverse effect on flavour of the products, e.g., rancidity is caused in milk by oxidation of ascorbic acid, and excess also impairs the taste of beer ⁴.

For the determination of copper at micro levels there are several analytical techniques have been used including atomic absorption spectrometry ^5-8, atomic emission spectrometry ⁹, electroanalytical techniques ¹⁰, spectrophotometry ^11-16, inductive coupled plasma-emission spectrometry, inductive coupled plasma-mass spectrometry ¹⁷, Voltammetry ¹⁸, flow injection diode array spectro-photometry and X-ray fluorescence spectrometry ^19-22.

Among these, the spectrophotometric methods are preferred as they are cheaper, easier to handle comparable sensitivity, and involve less expensive instrumentation. The aim of this study was to develop a novel and selective micellar spectro-photometric method for the determination of metal ions in various samples of milk because milk is a complex system where an accurate estimation of metal ions is a difficult task due to the presence of interfering materials. The formation of a coloured complex between the metal and a coloured or colourless ligand is the basis for the development of spectrophotometric methods. In order to develop such methods, we have explored the potential use of amide group-containing ligands in the presence of micellar medium, which has been found to increase the solubility as well as absorbance or fluorescence intensity resulting a selective and sensitive micellar spectrophotometric method for the determination of metal ions in various samples of milk knowingly or unknowingly.

EXPERIMENTAL:

Preparation of Solutions: Stock solution of N-(2’-Pyridyl)-2-hydroxybenzamide (NPHB-2) (0.1 M) was prepared by dissolving requisite amount of it in ethanol.

Surfactant Solution: Stock solutions of the following surfactants were prepared, Triton X-100 solution (1.8 × 10^-2 M, 100 CMC), sodium dodecyl sulfate SDS (0.50 M, 75 CMC), hexadecyltrimethyl ammonium bromide HTAB (9.2 × 10^-2 M, 100 CMC).

Buffer Solution: Acetate buffers were prepared by mixing 0.2 M sodium acetate and 0.2 M acetic acid and were used to maintain the pH in acidic medium. Borate buffers were prepared by mixing boric acid and sodium hydroxide and were used to maintain the pH in alkaline medium. A stock solution of copper (II) was prepared by dissolving an appropriate amount of hydrated copper sulfate in double-distilled water and was standardized according to reported procedure ²³.

Determination of Copper in Samples of Milk: Pipetted out 25 ml of milk slowly at the rate of approximately one drop per second into a slowly heated silica crucible; when all the moisture was removed the temperature was raised to approxi-mately 450-500 °C avoiding loss of sample by foaming and swelling. At this temperature, ignition was continued until grey ash was obtained. The crucible was allowed to cool.

The ash was dissolved in a minimum amount of concentrated nitric acid and evaporated to dryness and ignited again at 450-500 °C for 1h. Dissolved the resulting white ash in a minimum amount of dilute nitric acid, and the amount of copper was determined by following the recommended pro-cedure.

Recommended Procedure: Into 25 ml standard flask, add 2.5 ml of buffer solution of pH 6.5, a suitable aliquot of copper, 2.5 ml of 1.8 × 10^-2 M Triton-X-100 solution, and 0.5 ml of 5 × 10^-3 M NPHB-2 solution. Dilute the solution to volume with double distilled water and then record the absorbance at l_max 388 nm. For finding the precision and accuracy, experiments were repeated five times each.

RESULTS AND DISCUSSION: N-(2’-Pyridyl)-2-hydroxybenzamide (NPHB-2) has been used for the estimation of copper metal. Various effects i.e., pH, micellar medium, ligand concentrations, copper concentration on absorbance, the stability of complexes, and effect of diverse ions etc., have been observed at λ_max 388 nm and pH at 6.5.

Effect of pH on Absorbance: Absorption spectra of a solution containing copper- NPHB-2 complex in the micellar medium was recorded against reagent blanks by adjusting the pH in the range of 2.5 to 10.0 at l_max 388 nm.

FIG. 1:  EFFECT OF pH ON CU-NPHB-2 COMPLEX

The effect of pH was investigated on copper- NPHB-2 complex.  The absorbance has been found to increase with the increase in pH up to 6.5 and remained constant up to pH 7.2, thereafter it started decreasing. For further, studies acetate buffer of pH 6.5 was selected with NPHB-2. Absorption spectra of solution containing copper- NPHB-2 complex in micellar medium is represented in Fig. 1.

Effect of Micellar Medium: The absorption spectra of a solution containing copper- NPHB-2 complex have been recorded in the presence of different surfactants, nonionic Triton-X-100, cationic - HTAB, and anionic-SDS for comparison purpose at pH 6.5 and l_max 388 nm.  In Triton-X-100 the absorbance was found to be maximum. The absorption spectra are given in Fig. 2.

FIG. 2: EFFECT OF MICELLAR MEDIUM AND SOLVENTS ON CU-NPHB-2 COMPLEX

Effect of Ligand Concentration: For maximum complexation, 5 times molar excess of NPHB-2is required. However, in subsequent studies, 10 times molar excess of NPHB-2 has been maintained. A plot of absorbance versus mole ratio of NPHB-2/ NPHB-3/ NPHB-4 is represented in Fig. 3.

FIG. 3: EFFECT OF NPHB-2 ON CU-NPHB-2 COMPLEX

Effect of Copper Concentration on the Absor-bance: The effect of copper concentration was investigated on Cu-NPHB-2 Complex. A linear curve was obtained in the range 0.11 ton 0.95 ppm of Cu (II) ions, and the optimum range for the accurate determination of Cu (II) is recommended as 0.11 to 0.85 ppm.

A plot of Cu (II) concentration (in ppm) versus absorption intensities at l_max 388 nm has been represented in Fig. 4.

FIG. 4: EFFECT OF CONCENTRATION OF COPPER ON CU-NPHB-2 COMPLEX (BEER’S LAW)

Stability of the Complexes: The stability of copper-NPHB-2/NPHB-3/NPHB-4 complexes has been carried out in the presence of Triton-X-100. The complex is found to be reasonably stable over a period of approximately 90 min during which the analysis can easily be completed. The complex solution, however, is unstable when stored for prolonged periods of time. The complex solution should be prepared fresh before analysis. The present investigations have revealed that NPHB-2, like a spectrophotometric reagent for copper, compares well with other well-known methods. The method is highly sensitive, simple, and rapid in Triton-X-100 micellar medium. Although nickel and cobalt ions interfere and make it less selective, it ranks amongst the most sensitive reagents known so for the purpose. A plot of absorption intensity versus time has been represented for copper- NPHB-2 complex in the presence of Triton-X-10 in Fig. 5.

FIG. 5: EFFECT OF TIME ON ABSORBANCE OF Cu-NPHB-2 COMPLEX

Effect of Diverse Ions: The effect of various ions on the absorption intensity of the Cu- NPHB-2 complex has been investigated following the recommended procedure. The absorbance was measured at l_max 388 nm.

An ion is considered to be interfering if its presence produces a change greater than ± 5% in absorption intensity. The investigated ions and the maximum concentration at which they do not interfere are given in Table 1.

TABLE 1: CONCENTRATION RATIO OF FOREIGN IONS THAT DO NOT PRODUCE A CHANGE GREATER THAN ± 5% IN THE ABSORPTION INTENSITY OF CU- NPHB-2 COMPLEX. [Cu²⁺] = 0.7 ppm [Triton X - 100] = 1.8 x 10^-3M (NPHB^-2] = 1.0 x 10^-4M pH = 6.5 λ_max = 388 nm Absorbance (in the absence of foreign ion) = 0.697.

Analytical Applications of the Method: A novel and selective method for the determination of copper in milk samples has been developed, which meets the criteria of Green Chemistry. This method was compared with the existing methods. The results for the determination of copper in various samples of milk are presented in Table 2.

TABLE 2: DETERMINATION OF COPPER IN MILK SAMPLES

*Mean of five determinations

CONCLUSION: The present investigations have revealed that NPHB-2, as spectrophotometric reagent for copper, compares well with other well-known methods. The method is highly sensitive, simple, and rapid in Triton-X-100 micellar medium. Although nickel and cobalt ions interfere and make it less selective but it ranks amongst the most sensitive reagents known so for the purpose.

ACKNOWLEDGEMENT: The authors are thankful to Green Chemistry Research Centre, P. G. Department of Chemistry, Government Dungar College (A-Grade), the University of Bikaner for providing lab facility.

CONFLICTS OF INTEREST: None Declared

REFERENCES:

1. Licata P, Trombetta D, Cristani M, Giofre F, Martino D, Calo M and Naccari F:  Levels of ‘‘toxic’’ and ‘‘essential’’ metals in samples of bovine milk from various dairy farms in Calabria, Italy. Environment International 2004; 30: 1-6.
2. Gakkhar N, Bhatia A and Bhojak N: Comparative study on physiochemical properties of various milk samples. International J of Recent Sci Res 2015; 6(6): 4436-39.
3. Khan S, Cao Q, Zheng YM, Huang YZ and Zhu YG: Health risks of heavy metals in contaminated soils and food crops irrigated with wastewater in Beijing, China. Environmental Pollution 2008; 152: 686-92.
4. Alexander FW, Clayton BE and Delves HT: Mineral and trace-metal balances in children receiving normal and synthetic diets. QJ Med 1974; 43: 89-111.
5. Ghanemi K, Nikpour Y, Omidvar O and Maryamabadi A: Sulfur-nanoparticle-based method for separation and preconcentration of some heavy metals in marine samples prior to flame atomic absorption spectrometry determination. Talanta 2011; 85: 763-69.
6. Mahmoud ME, Kenawy IMM, Hafez MAH and Lashein RR: Removal, pre-concentration and determination of trace heavy metal ions in water samples by AAS via chemically modified silica gel N-(1-carboxy-6-hydroxy) benzyl-idenepropylamine ion exchange. Desalination 2010; 250: 62-70.
7. Baghban, N, Yilmaz E and Soylak M: Nanodiamond/MoS2 nanorod composite as a novel sorbent for fast and effective vortex-assisted micro solid phase extraction of lead (II) and copper (II) for their flame atomic absorption spectrometric detection. J Mol Liq 2017; 234: 260-67.
8. AL Saidi HS: Biosorption using chitosan thiourea polymer as an extraction and preconcentration technique for copper prior to its determination in environmental and food samples by flame atomic absorption spectrometry: Synthesis, characterization and analytical applications. Int J Biol Macromol 2016; 93: 390-401.
9. Simmons WLiP, Shrader D, Herrman TJ and Dai SY: Anal Sci Microwave plasma-atomic emission spectroscopy as a tool for the determination of copper, iron, manganese and zinc in animal feed and fertilizer.Talanta 2013; 112: 43-48.
10. Yang W, Chow E, Willett GD, Hibbert DB and Gooding JJ: Exploring the use of the tripeptide Gly-Gly-His as a selective recognition element for the abrication of electrochemical copper sensors. Analys 2003; 128: 712-18.
11. Uddin MN, Shah NM, Hossain MA and Islam MM: Copper and Mercury in Food, Biological and Pharmaceutical Samples: Spectrophotometric Estimation as Cu (DDTC) 2 . Am J Anal Chem 2014; 5: 838-50.
12. Madan PU, Barhate VD: Extractive spectrophotometric determination of copper (II) using 2-(5-bromo-2- oxoindolin-3-ylidene) hydrazine carbothioamide as an analytical reagent. Eu J Biom Pharma Sci 2016; 3: 392-96.
13. Babayeva K, Demir S and Andac MA: Novel spectrophotometric method for the determination of copper ion by using a salophen ligand, N, N -disalicylidene-2, 3-diaminopyridine. J Taibah Univ Sci 2017; 11: 808-14.
14. Subudhi KS, Sreevani D and Rao KA: Spectrophotometric determination of trace copper in hair, milk, banana, rice and pharm products with 5-{α´–methyl-3-hydroxy-benzylidene} rhodamine [5M, 3H-Br] reagent. Int J Res Anal Rev 2019; 6: 809-17.
15. Barache UB, Shaikh AB, Lokhande TN, Kamble GS, Anuse MA and Gaikwad  SH: An efficient, cost effective, sensing behavior liquid-liquid extraction and spectrophotometric determination of copper(II) incorporated with 4-(4’-chlorobenzylideneimino)-3-methyl-5-mercapto-1, 2, 4-triazole: Analysis of food samples, leafy vegetables, fertilizers and environmental samples. Spectrochim. Acta A Mol Biomol Spectrosc 2018; 189: 443-53.
16. Alharthi SS and Al-Saidi HM: Spectrophotometric Determination of Trace Concentrations of Copper in Waters Using the Chromogenic Reagent 4-Amino-3-Mercapto-6- [2-(2-Thienyl) Vinyl]-1, 2, 4-Triazin-5(4H)-One: Synthesis, Characterization and Analytical Applications Appl Sci 2020; 10(11): 1-17.
17. Martin-Camean A, Jos A, Puerto M, Calleja A, Iglesias-Linares A, Solano E and Camean AM: In-vivo determination of aluminum, cobalt, chromium, copper, nickel, titanium and vanadium in oral mucosa cells from orthodontic patients with mini-implants by Inductively coupled plasma-mass spectrometry (ICP-MS). J Trace Elem Med Bio 2015; 32: 13-20.
18. El-Raheem HA, Hassan RYA, Khaled R, Farghali A and El-Sherbiny IM: Polyurethane-doped platinum nano-particles modified carbon paste electrode for the sensitive and selective voltammetric determination of free copper ions in biological samples. Microchem J 2020; 155: 104765.
19. Gouda AA and Amin AS: Cloud-point extraction, preconcentration and spectrophotometric determination of trace quantities of copper in food, water and biological samples. Spectrochim Acta A 2014; 120: 88-96.
20. Nalawade RA, Nalawade AM, Kamble GS and Anuse MA: Rapid, synergistic extractive spectrophotometric determination of copper (II) by using sensitive chromogenic reagent N′′, N′′′-bis[(E)-(4-fluorophenyl) methylidene] thiocarbonohydrazide. Spectrochim Acta A 2015; 146: 297-306.
21. Tarighat MA: Orthogonal projection approach and continuous wavelet transform-feed forward neural networks for simultaneous spectrophotometric determination of some heavy metals in diet samples. Food Chem 2016; 192: 548-56.
22. Yadav N and Bhojak N: Microwave assisted synthesis, spectral and antibacterial investigation on Co (II) complexes with amide ligands. The Int J Eng Sci 2013; 2(2): 166-68.
23. Barua S, Verma YS, Garg BS, Singh RP and Singh I: Spectrophotometric determination of copper in blood serum with 4-(2-quinolylazo) phenol. Analyst 1981; 106: 799-802.
    

    ---

    How to cite this article:

    Gakkhar N and Bhojak N: Micellar spectrophotometric determination of copper in the various samples of milk. Int J Pharm Sci & Res 2021; 12(4): 2344-48. doi: 10.13040/IJPSR.0975-8232.12(4).2344-48.

    ---

    All © 2013 are reserved by the International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.

    ---