Efficient synthesis of superparamagnetic magnetite nanoparticles under air for biomedical applications

Author’s Accepted Manuscript Efficient Synthesis of Superparamagnetic Magnetite Nanoparticles under Air for Biomedical Applications Namita Saxena, Man Singh www.elsevier.com/locate/jmmm

PII: DOI: Reference:

S0304-8853(16)32287-9 http://dx.doi.org/10.1016/j.jmmm.2017.01.031 MAGMA62379

To appear in: Journal of Magnetism and Magnetic Materials Received date: 20 September 2016 Revised date: 20 December 2016 Accepted date: 9 January 2017 Cite this article as: Namita Saxena and Man Singh, Efficient Synthesis of Superparamagnetic Magnetite Nanoparticles under Air for Biomedical Applications, Journal of Magnetism and Magnetic Materials, http://dx.doi.org/10.1016/j.jmmm.2017.01.031 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Efficient Synthesis of Superparamagnetic Magnetite Nanoparticles under Air for Biomedical Applications Namita Saxenaa# and Man Singhb* a)

School of Nano Sciences, Central University of Gujarat, Gandhinagar 382030, India. School of Chemical Sciences, Central University of Gujarat, Gandhinagar 382030, India.

b)

*Corresponding author, Phone: +91-79-23260340, Fax: +91- 79-23260076 Email: [email protected] # Email: [email protected]

Abstract- The facile co-precipitation process of synthesising Superparamagnetic Iron Oxide Nanoparticles (SPIONs) especially magnetite was investigated and simplified, to develop a reproducible and scaled up synthesis process under air, for producing particles with enhanced percentage of magnetite, thus eliminating the crucial and complicated need of using the inert atmosphere. Presence of magnetite was confirmed by XRD, TEM, and Raman spectroscopy. Efficiency of synthesising magnetite was increased up to approx. 58 wt. %, under air with no other phases but maghemite present. Alkali concentration was optimised, and particles with better magnetisation values were synthesised. The approximate weight percentage of magnetite was calculated using the simple and rapid XRD peak deconvolution method. Higher pH values from 13 to14 were investigated in the study while alkali concentration was varied from 0.5 M to 4 M . 1Molar NaOH with a final pH of 13.4 was found to be optimum. Well crystallised particles with approx. 6-12 nm size, narrow size distribution and cubospheroidal shape were synthesised. Particles were Superparamagnetic with high values of saturation magnetisation of up to 68 emu/g and negligible values of remanence and coercivity. A reaction yield of up to 62% was obtained. Hydrophilic coated particles were produced in a single, one step facile process for biomedical applications, using optimised parameters of pH and alkali concentration obtained in the study. Single domain particles with good magnetisation formed stable aqueous dispersions. FTIR, UV-Visible and DLS were used to confirm the coating and dispersion stabilities of the particles. These particles have the requisite properties required for application in different biomedical fields. Key Words: Superparamagnetic iron oxide nanoparticles, Molar solutions, pH, Magnetisation, Co-precipitation.

1

1. IntroductionIron oxide nanoparticles (IONPs) mainly magnetite (Fe3O4) and maghemite (γFe2O3) are an important class of compounds required for wide variety of applications because they have the unique combination of both the magnetic as well as nanoscale properties. As they are nontoxic, bio-compatible and able to form stable dispersions, they are one of the foremost nanoparticles required for different bio-medical applications like drug delivery [1], MRI contrast agents [2, 3], cancer treatment by magnetic hyperthermia [4,5], cancer theranostics [6], immunoassay [7] and various other applications in different other fields like catalysis [8] etc. Critical requirements for bio-medical related fields are good values of magnetisation and ability to form stable aqueous dispersions. Below approximately 30 nm size they start behaving superparamagnetically at room temperature [4]. Larger values of susceptibility and magnetic moment with smaller values of coercivity and remanence make them most desirable for various biomedical applications. After their manoeuvre, when the applied field is removed no magnetisation remains hence no aggregation in vivo is possible. To get the particles suitable for a specific application, synthesis protocol must be modified to get the desired physicochemical properties in the particles. Among the various processes of synthesising IONPs high temperature decomposition methods use toxic chemicals and harsh reaction conditions, with the production of particles with hydrophobic surface ligands that are unsuitable for bio-medical applications. On the other hand co-precipitation process is the method of choice for its scalability, simplicity, non involvement of toxic chemicals and higher yields. Massart [9] reported as early as 1981, synthesis of magnetite under N2 with a ratio of Fe3+/Fe2+ equal to two in the alkaline media. Co-precipitation process has been studied extensively since then [10, 11, 12]. Effects of various synthesis parameters like ratio of iron salts, temperature, pH, stirring speed, mixing rate and the order of addition of the reactants etc. on the properties of the formed magnetic nanoparticles (MNPs) have been variously studied [13,14,15, 16, 17]. Presence of an inert atmosphere during synthesis is necessary in the co-precipitation process to prevent the oxidation of magnetite to maghemite that takes place very easily in the air. Although efforts were made in the past to synthesise magnetite under air by using the altered ratio of iron salts from the preferred Fe

2+ 3+

/

equal to 0.5 [18,19], but the formation

of maghemite is usually reported [18]. Only up to 30-40 vol. % magnetite at pH 13 has been reported to be formed under air, with a surface layer with defective maghemite like structure 2

[20]. Goethite formation is also reported along with magnetite and maghemite depending on the pH, from 8-10.6 even after using N2 [21]. Medium pH is found to affect the formation of different phases with no effect on particle size, in the study done under N2 at pH from 8-12.5 [22]. Alkali plays a major role in the co-precipitation process, as its nature, concentration, rate and order of addition etc. affect the synthesis outcomes [10, 15, 16, 17]. pH of the solution in the co-precipitation process is a significant factor, as the Fe2+ and Fe3+ hydrolyse at different pH values in the mixed salt solution. In solution Fe3+ ions start hydrolysing at a pH > 3 forming ferric hydroxide, while Fe2+ hydrolyse at around pH 7 but the formation of Fe(OH)2 occurs at around pH 9 [23]. Co-precipitation reaction is usually preferred to be performed between pH 8-14 [24]. Nanoscale magnetite is very sensitive to oxidation so reaction is performed under inert atmosphere. Besides aerial oxidation, different interfacial ion and electron transfer reactions do take place depending on the pH of the medium that can also oxidise magnetite [23]. These electron and/or ion transfer reactions are reversible and formation of magnetite can occur if the medium is sufficiently basic [23]. The ferric hydroxide formed initially from hydrolysis of Fe3+ ions may transform to goethite (α FeOOH) or hematite (α Fe2O3), depending on the conditions of the pH, temperature and conc. of the medium [25]. Presence of water in the medium helps to form well hydrated and loose ferric hydroxide that is very reactive and transforms easily to spinel if Fe2+ ions are also present in the solution [25]. Overlapping of ‘d’ orbitals and easy electron transfer between Fe(II) and Fe(III) helps transform ferric hydroxide into spinel. Excess –OH- ions help in producing the reducing environment in the medium. Degree of hydration, presence of Fe2+ ions and the pH value of the solution, all affect to a great extent the synthesis of magnetite and its resulting crystal structure. Studies done at the lower pH values of 6, 7, 8, and 9 reported the formation of other phases of iron oxide like ε-Fe2O3, goethite and α Fe2O3 along with magnetite [22, 26, 27]. Higher pH values have been reported to be suitable for the formation of pure magnetite. pH values of 9.7-10.6, >11 and 12.5 have been reported to be suitable for synthesising particles with better dispersion and crystallinity [21, 28, 29]. Particle stoichiometry, surface oxidation and purity of magnetite are affected by pH [20]. Going to still higher pH value of 13, formation of particles with only 30-40 vol. % magnetite under air has been reported [20].

3

In the present study we have reinvestigated and optimised the co-precipitation process under air, by using different molar solutions of NaOH, excess alkali and with a higher final pH value between 13 to 14. We varied the alkali concentrations from 0.5 M to 4 M, to see the effects of higher percentage of water in the dilute alkali solutions, on the properties of formed particles. We also relaxed the condition of using an inert atmosphere and were able to achieve good percentage of magnetite with no other significant iron oxide phase present except maghemite. We got well crystallised particles with good dispersions and better magnetisation values compared to other reported works [26, 30, 31]. We also functionalised these particles in a single step with different coating materials, for making stable aqueous dispersions under the optimised pH and alkali concentration, obtained in the study. Well dispersed particles with functional groups for further modification for different applications, are the required criterion of particles for different biomedical applications. It was well achieved in the study. Magnetite and maghemite both are biocompatible, and a simple synthesis method with scaled up properties is needed for production of particles with desired parameters for biomedical applications. The novelty of this work lays in one step synthesis of single domain, well crystallised, cubospheroidal and coated hydrophilic particles, at higher pH values of 1314 under air. Study also involves the investigation into the effects of various concentrations of NaOH, from dilute to more concentrated ones and their effects on the magnetite percentage, bringing out the importance of dilute alkali in synthesising magnetite by coprecipitation process. Significance of pH as well as the concentration of alkali, both are investigated in the study. Not many studies have been done at a higher pH of 14 for synthesising magnetite. Relatively poor crystallinity and wider size distribution are the two main drawbacks of the co-precipitation process which are overcome in the present study. The extreme basic conditions of the medium, at a pH of 13-14 and other reaction parameters prove to be conducive for good crystallinity and lesser defects hence good magnetisation values. The studies reported in literature of synthesising magnetite at different pH values, have reported the formation of different other oxides of iron like goethite and α Fe2O3 also along with magnetite, that was not found in our study [22, 26, 27]. The biomedical applications of these particles depend upon their size, size distribution and magnetic properties besides their ability to form stable aqueous dispersions with suitable coating for further functionalisation, that was well achieved in a single step synthesis. Ordered crystal structure with least number of defects and impurities is required for better magnetic properties in crystalline materials. The used reaction parameters are thus 4

helpful for synthesising well coated, water dispersible MNPs under simplified conditions in a one step process, with good reaction yields. The details of the synthesis process and effects on coating are given in the following sections.

2. Materials and Methods 2.1. Materials The chemicals used in this study are Ferric chloride (Sigma-Aldrich, 157740), Ferrous chloride (Aldrich, 372870), Citric Acid, Ascorbic acid and Glutamic acid, all from Sigma Aldrich. NaOH of analytical grade was used. Milli Q water was used in the study. 2.2. Synthesis of Magnetic Nanoparticles (MNPs). The most common and simplest co-precipitation process was used for the synthesis of MNPs that does not require much stringent conditions [24]. For better results and keeping in view the thermodynamics of the reaction, stoichiometric ratio of Fe3+ and Fe2+ in a ratio of 2˸1 was taken and the pH was kept in the range of 13 to 14. The following reaction takes place during the synthesis, 2Fe3+ + Fe2+ + 8OH-

=

Fe3O4 + 4H2O

(1)

If non-oxidising environment of N2 or Ar is not present, oxidation of magnetite to maghemite occurs as follows not only by the O2 of the air but also by the electron or ion transfer reactions depending on the pH of the solution [24], Fe3O4 + 2H+ =

γFe2O3 + Fe2+ + H2O

(2)

Water heated to 70±50C was used in the study to dissolve the iron salts. Reaction was performed at 800 C. In a typical procedure Milli Q water was heated to 70±50 C. In heated water first iron (III) (0.017 moles) and then Fe (II) salt (0.008 moles) was dissolved respectively, in a molar ratio of 2:1 and final volume was made 50 ml. The solution was transferred to a round bottom flask immediately and put in the already heated oil bath at 800C. After keeping for 30minutes at a stirring speed of 1150 rpm, the NaOH solution in the required concentration and volume was added all at once at a rapid rate, with simultaneous vigorous stirring of the solution. The used volumes of NaOH and final pH of the solutions are given in Table. 1. There was formation of the black precipitate immediately after the addition of base in almost all the cases. The solution was allowed to stir at the 1000 rpm and 800C for 30 minutes. After 30 minutes the solution was taken away from the oil bath and allowed to cool. 5

S.No.

Table 1. Synthesis parameters of the as synthesised particles. Sample No. Volume NaOH Initial NaOH (Molarity)

(ml)

Moles

Final pH

1.

S-1 (1M)

225

0.22

13.8

2.

S-2 (2.0M)

75

0.16

13.9

3.

S-3 (4M)

35

0.14

13.9

4.

S-4 (2.0M)

110

0.22

14.0

5.

S-5 (4M)

39

0.16

14.0

6.

S-6 (1.0M)

105

0.11

13.4

7.

S-7 (0.5M)

316

0.16

13.4

8.

S-8 (0.5M)

175

0.09

13.0

9.

S-9 (1M)

100

0.10

13.3

The formed precipitate was separated with the help of the Neodymium magnet by decantation. Solution was washed several times with Milli Q water to remove extra salts and alkali. The washed precipitate was dried in the hot air oven at 800 C for 14-18 hours. In the study four concentrations of alkali were used starting from 0.5 M to 4 M. In all the studies an excess amount of alkali was used. Different volumes of NaOH for reaching the final pH from 13 to 14 were used in the study as shown in Table 1. To see the effect of final solution pH, in helping to coat these particles by different organic compounds, we coated these particles in the post synthesis protocol. After the digestion of precipitated MNPs for 30 minutes, we added the citric acid in a concentration of 25 mol% of the Iron salts, and ascorbic and glutamic acid in a conc. of equal moles to iron salts. The solution was left for one hour at 900 C after the addition of the coating agent. After coating, the formed black precipitate was washed repeatedly 5-6 times and dried. As the glass electrode is not suitable for measuring the pH higher than 11, the pH of the final solution was theoretically calculated from the remaining moles of NaOH after the reaction, using reaction stoichiometry, with known initial moles of interacting iron salts and final volume of the solution. 2.3. Characterisation. 6

Various characterisation techniques like XRD, VSM, TEM, Raman, FTIR, UV-Visible and DLS were used to analyse the particles. The formed nanoparticles were characterised by the XRD at 45kV, 40mA, using Cu Kα radiation source for particle size and wt % of the magnetite. XRD data is obtained from 10º-90º, using a step scan of 0.01º. The data corrected for instrumental broadening and Cu Kα2 contribution was used. The average diffracting crystallite size of the as prepared particles was calculated using Scherrer’s equation, from the most intense peak (311), D = Kλ / β cosθ

(3)

Where D is the average crystallite size, β is the Full Width at Half Maximum (FWHM) in radians; K is the dimensionless shape factor equal to 0.9 for cubic structures, λ is the wavelength of the incident X rays (0.15406 nm). Different efforts to quantify the amounts of magnetite and maghemite by means of various techniques like FTIR, XPS, Raman and Mossbauer have already been reported, but none is found unambiguous as reported in literature [32]. Although Mossbauer is considered as the better technique for determining the stoichiometry and the extent of oxidation of magnetite, but even it suffers from different drawbacks like the difficulty in the interpretation of the data and the ambiguities in fitting parameters for the sextets [33]. The main difficulties are posed by the superparamagnetic nature and the nanometric size of the particles, as well as the surface effects, magnetic spin canting and the different extents of oxidation and formation of solid solutions of the maghemite and magnetite in the partially oxidised particles [33]. In one study [34] the wt. percentage of magnetite and maghemite in the prepared samples is compared by both Mossbauer and XRD, and it was found that both methods give the same results within limits of experimental errors [34]. There is considerable correspondence found in the percentage of magnetite determined by the two methods [34]. Maghemite presence can also be proved by the presence of superstructure peaks at lower angles in XRD, determined by the distribution of vacancies and space group. The maghemite crystal structure can vary from spinel (Fd3m space group) to primitive cubic (space group P4132) or tetragonal (P43212) depending on the ordering of vacancies in the lattice [35]. The peaks found at lower angles of 23.77 (210) and 26.10 (211) are very low in

7

intensity (only about 5%) in XRD for maghemite [32], and in our diffraction pattern these peaks are not discernible from the background clearly. Peak deconvolution method is used in the present study at higher angles. The method depends on the quantification and calculation of magnetite, based on the relation with a high correlation coefficient [32]. Peaks of the (511) and (440) planes were deconvoluted, using the voigt amplitude function, at 2 values of 570, 62.50 for magnetite and 57.30 and 63.030 for maghemite respectively. The individual intensities for both the deconvoluted peaks are obtained and percentage of magnetite is calculated as shown below in equations (4) and (5) for the (511) and (440) planes respectively. The approx. weight percentage of magnetite is calculated by averaging both the values obtained from (511) and (440) peaks.

  I (511) maghemite    1.0136  wmaghemite  0.2371  I (511) maghemite  I (511) magnetite 

Magnetite Wt. Percentage



I ( 440) Magnetite I ( 440) Magnetite  I ( 440) Maghemite

 100

(4)

(5)

TEM studies (Philips, CM200, 200kV) were done by dispersing the powdered samples in water by ultrasonication and putting a drop of the dispersion on 300 mesh carbon coated Cu grid. The size distribution of the particles was investigated using the Image-J software and counting up to 200 particles. Raman spectroscopy was done on these particles to further prove the formation of magnetite in the synthesised samples using 532 nm solid state laser. The values of saturation magnetisation were obtained from the applied field Vs magnetic moment graphs, from the magnetisation studies done on a VSM at room temperature at an applied field from (-20kOe to +20kOe) for uncoated particles. For the coated particles magnetisation studies were done from -10 to +10 Tesla by a Quantum Design (USA) physical properties measurement system (PPMS) having VSM, at two temperatures of 5 K and 300 K. M/T plots for ZFC and FC curves were obtained at an applied field of 100 Oe. To further prove the presence of coating on the synthesised particles FTIR, UV-Visible and DLS studies were done. DLS (Microtrac Zetatrac, NPA 152-31A-0000-000-90M) studies were done in Milli Q water (pH 5.7) to determine the hydrodynamic size and Zeta potential of the coated particles and to find the dispersion stability of the as prepared coated particles.

8

FTIR studies (Perkin Elmer, SP-65) were done by grounding the sample with KBr and forming the pallet, and taking the spectra in the 400-4000 nm range. FTIR studies provided the proof of coating of different organic compounds on these particles. Further UV- Visible spectra (Analytical Instruments, 2060+) of the coated and uncoated particles in water, were recorded in the range of 200-800 nm, with a quartz cell of 1 cm path length, to study the coating of different materials on these particles. The plots were corrected for background for FTIR, DLS and UV-Visible spectroscopy.

3. Results and Discussion. The synthesised particles are studied by various characterisation techniques as detailed below.

3.1. Size and the presence of magnetite. Size and the presence of magnetite in the prepared samples was determined by analysing the XRD, TEM and Raman spectra.

3.1.1 XRD Analysis.

Fig. 1. XRD pattern of the synthesized MNPs at different pH values. As shown in Fig. 1 the XRD pattern confirms the presence of crystalline cubic spinel structure of MNPs, with the peaks corresponding to the planes for (220), (311), (400), (422), (511), and (440) present. XRD patterns of the samples proved that no other phase of iron oxide like goethite was present in the synthesized samples. The XRD patterns of four samples 9

prepared at different values of pH are shown in Fig.1. As shown in the diffraction pattern all the samples show nearly similar peak positions with significant broadening signifying the presence of nanosized crystalline Fe3O4. The average sizes of the crystallites calculated by Sherrer’s equation are given in Table 2.The average crystallite size is found to be nearly 8-13 nm. The sizes of the MNPs synthesized in our experiment conform to the size regimen of the Superparamagnetic particles.

Table 2. Different observed parameters of the MNPs from XRD (S.D. – Standard Deviation)

S.No.

Sample No.

Final

(Molarity)

pH

Lattice

Size

Parameter

TEM(nm)

(Å)

±(S.D)

Size(nm) XRD

Approx. Wt. Percentage Fe3O4

1.

S-1

( 1M)

13.8

8.370

12.9

47.7

2.

S-2

(2.0M)

13.9

8.363

8.4

41.0

3.

S-3

(4M)

13.9

8.358

8.6

32.0

4.

S-4

(1.0M)

14.0

8.360

9.2

39.5

5.

S-5

(4.0M)

14.0

8.357

8.7

37.5

6.

S-6

(1.0M)

13.4

8.366

9.6

58.1

7.

S-7

(0.5M)

13.4

8.368

11.4

52.1

8.

S-8

(0.5M)

13.0

8.355

10.5

35.0

9.

S-9

(1M)

13.3

8.375

9.2

45.6

9.06 ±2.8

9.30±3.12

The values of Lattice parameters as calculated from the XRD data are also shown in Table 2. The Lattice parameter of magnetite is 8.396 Aº (JCPDS File 19-629) and maghemite is 8.346 Aº (JCPDS File 39-1346), as also reported in the literature [36]. Maghemite lattice parameter depends on the ordering of vacancies in the crystal [35]. The observed values are midway between the values for magnetite and maghemite. The approx. wt. percentage of the magnetite is calculated as described above from the deconvoluted peak intensity ratio, for (511) and (440) peaks and the average percentages obtained are given in the Table 2. Deconvoluted peaks for one of the sample are shown in Figs. 2 and 3. Highest wt. percentage 10

of approx. 58% of magnetite is obtained with 1M NaOH with a pH of 13.4. Next highest percentages are obtained with the 0.5 M NaOH at the same pH of 13.4 (52%), and 1 M NaOH with a value of 47% at pH 13.8. Due to their large surface area nanocrystalline particles are more prone to oxidation, hence resulting in the formation of maghemite but higher percentage of magnetite is obtained with lower alkali concentrations.

Fig. 2. Deconvoluted (511) XRD peak of the as synthesized MNPs at 2 values of 57 º and 57.30 for magnetite and maghemite respectively.

Fig. 3. Deconvoluted (440) peak of the synthesized MNPs at 2 values of 62.530 and 63.03o, corresponding to magnetite and maghemite at 1M NaOH, pH 13.4. 11

3.1.2 TEM Micrographs. The TEM micrographs of two of the samples are shown in Figs. 4 and 5 to further confirm the crystallite size and the presence of magnetite. The electron diffraction patterns are used to determine the lattice spacings ‘d’ of the crystallites as shown in Table 3. As shown in Fig. 4 the particles are well crystalline. The size distribution curve in inset shows that the maximum particles are of about 6-10nm in size with a narrow size distribution. Most of the particles are cubo-spheroidal shaped.

Table 3. Measured Lattice spacings ‘d’(nm) from TEM diffraction rings of MNPs (0.5 M citric and 1 M Ascorbic acid coated). Lattice NaOH Molarity 1M

Measured lattice spacing ‘d’ (nm) from diffraction rings (TEM) 0.1213

(Coated ) (444)

0.1514

0.1991

0.2403

0.2914

0.498

(440)

(400)

(311)

(220)

(111)

4M

0.1242

0.1506

0.2114

0.2544

0.3039

0.1736

(Blank)

(444)

(440)

(400)

(311)

(220)

(422)

0.5M

0.1557

0.2049

0.2487

0.2889

0.1639

(Coated)

(440)

(400)

(311)

(220)

(511)

The HR-TEM micrograph as shown in Fig. 4, clearly indicates the presence of single crystals as is also confirmed by the visible lattice planes. The almost same particle size obtained from the XRD and TEM also confirms the formation of single crystals. The crystal planes in Fig.4 confirm the presence of (111) plane of magnetite with a ‘d’ spacing of 4.98 Å. The uncoated particles as shown in Fig. 5 are well dispersed and less agglomerated. Most of the particles are 6-10 nm in size on average and cubo-spheroidal in shape with narrow size distribution. Few particles are although 20-25 nm, deviating from this trend in size and shape.

12

Fig. 4. TEM image of particles, top inset ‘d’ spacing for (111) plane and bottom inset size distribution of particles at 1.0M NaOH, pH 13.4.

Fig. 5. TEM image of MNPs synthesised with 4.0M NaOH (pH 13.9) with particle size distribution in insets.

13

3.1.3 Raman Spectra. The dominating presence of the Fe3O4 phase was further confirmed by Raman Spectroscopy. The Raman spectra of the nanoparticles is shown in Fig. 6. The band at 667 cm

-1

is the major band for magnetite. It confirms the presence of the A1g mode of

magnetite. The band at 700 cm-1 is visible only after the deconvolution of the peak. Band at 700 cm-1 is important as this is the major phonon band of maghemite. Further the presence of bands at 350 cm-1(T2g) and 500 cm-1(Eg) also confirm the presence of maghemite [37, 38]. Thus the presence of maghemite with magnetite is proved as previously shown by the XRD results also. Deconvoluted Raman peaks are further used to calculate the ratio of the peak intensity of magnetite and maghemite using the Lorentzian fit and thus to further prove the presence of 60% magnetite in synthesised particles. The values of peak width with the other related parameters are given in Table 4. The ratio of peak intensity of magnetite/maghemite is equal to 1.74.

Table 4. Deconvoluted Lorentzian fit of the Raman data. Sample

A1g ( Fe3O4 )

A1g(γFe2O3)

Intensity Ratio

T2g(350)

Eg (500)

M (pH)

X1/ W

X1/ W

I667/I700

W/(γFe2O3)

W/(γFe2O3)

S-6,1M

667/ 72.58

700/ 50.60

1.74

64.25

30.78

(13.4) X1 peak position, W- Width

14

Fig. 6. Lorentzian fit of the deconvoluted Raman peaks of the synthesized MNPs at 1.0M NaOH, pH 13.4 (S-6).

3.2 Effects of excess alkali and high pH The effects of the excess alkali and high pH during the precipitation were studied using the strong base NaOH of different concentrations. From the beginning with the start of the precipitation, it provided large no. of –OH- ions immediately after the addition, thus increasing the reaction pH and producing a highly reducing environment. Higher values of pH also helped in the simultaneous precipitation of the Fe2+ and Fe3+ ions. Initial dissolution of the iron salts in hot water lead to an increased activity of the ions, and homogeneous and uniform nucleation conditions were ensured by vigorous shaking. An excess of water was present in the dilute alkali solutions (0.5 M and 1.0 M) compared to the concentrated ones (2.0 M and 4.0 M). The optimum temperature, stirring speed and stoichiometric ratio of iron salts in the presence of alkali, helped in the initial ordering of spinel in the formed precipitate. The very rapid addition rate of alkali, with simultaneous vigorous stirring helped to form uniformly basic solution for rapid and maximum nucleation. Initially solutions of 1 M, 2 M and 4 M conc. of NaOH at higher pH value of reacted solution of 13.8-13.9, were used to study its effects on the formed percentage of magnetite. The effects of different molar solutions with different final pH values are shown in Fig.7. There was observed a decreasing trend in the wt% of formed magnetite with increase in molar 15

concentration of alkali as 1 M >2 M >4 M. At 1 M NaOH the percentage of formed magnetite was 47.7% while with 2 M solution it was 41.0% and with 4 M only 32% magnetite was formed. We investigated higher concentrations of 2 M and 4 M NaOH solutions further with a still higher pH of 14, but no major changes could be observed for the wt% of magnetite. The wt % was 39.5% for 2 M and 37.5% for 4 M at a pH of 14, as compared to 41% and 32% for the pH of 13.9. So the higher pH with high NaOH concentration was not showing good outcomes with respect to the magnetite percentage. The higher alkali concentrations were not proving useful as not more than 40% of the magnetite was obtained. In comparison to that, lower concentration of NaOH at 1 M is showing better results in terms of the formed wt% of magnetite. So we shifted our focus towards lower pH values with lower molar solutions of alkali. We studied further the lower pH value of 13.4 with 1 M NaOH. This time the wt% of magnetite was found to be 58.1%, much higher than previous ones. We now decided to compare the still lower conc. of 0.5 M with 1 M NaOH keeping the pH value the same at 13.4. But we found a downward trend in the wt% of magnetite formed with a value of 52.1%, as shown in Fig. 7.

Fig. 7. Variation of formed percentage of Fe3O4 with different molar conc. of NaOH, pH values are given with percentage in the figure. This value was lower than obtained at 1 M but higher when compared to 2 M and 4 M NaOH. While going still down to a pH of 13 with 0.5 M alkali we only got a value of 35% of 16

magnetite. There is a great decrease in the wt% of formed magnetite with only slight change of pH. It showed clearly that the lower concentrations of alkali are definitely better compared to the higher ones of 2 M and 4 M but the optimum value is obtained at a pH of 13.4 for 1 M NaOH. The next better value is obtained with 0.5 M NaOH at pH 13.4 under the set reaction parameters. We also prepared a solution of 1 M NaOH with a pH of 13.3, but dried the sample at 530 C instead of 800 C, to see if this affects the percentage of formed magnetite. This time although the pH was almost the same, there was decrease in the formed percentage of magnetite. The wt % of magnetite was only 45.6%.Thus drying temperature also affects the magnetite percentage besides the pH of the medium. It needs to be further explored. Although this value is lower compared to the value at pH 13.4, but it is still better than higher molar concentrations of 2 M and 4 M of NaOH. The variation of pH with magnetite wt. percentage is given in Fig. 8.

Fig. 8. Variation of percentage of Fe3O4 with different pH values.

Thus it is proved that lower concentrations of alkali are giving good results in terms of the wt% of formed magnetite compared to the higher ones. The optimum pH is obtained at 13.4, with 1 M NaOH (Fig.8). Although the particle size reduces at higher alkali conc. but in our study not much variation was found.

17

3.3 Magnetic Characteristics. For studying the magnetic properties of the nanoparticles, VSM studies were done at room temperature (≈300K) from -20kOe to +20kOe for uncoated particles and for ±100kOe for coated particles. The size, crystallinity, surface structure, crystal defects etc. of the nanoparticles affect the magnetic behaviour to a great extent. The magnetic properties of the similar sized and size distributed particles depend to a great extent on the crystallinity and the surface properties of the particles [4, 33]. Better magnetic properties obtained in our study support the presence of better crystallinity and lesser defects, as in case of nanoparticles these effects play a major role. The observed magnetic properties of the particles are shown in Table 5.The magnetization versus magnetic field graphs are shown in Figs. 9 and 10, and it can be seen that all the samples show non-linear reversible graphs with good saturation magnetisation values. Coercivity and remanence values are negligible thus particles are conforming to the Superparamagnetic (SPM) behaviour with higher values of saturation magnetisation as compared to other reported work [26, 30,31].

Table 5. Magnetic properties of the synthesised particles. S.No.

pH

Sample No.

Saturation

Coercivity (Hc)

Remanence (Mr)

(Molarity)

magnetisation(Ms)

(Oe)

(emu/g)

(emu/g) 1.

13.4

S-6(1M)

67.02

26.82

1.88

2.

13.8

S-1(1M)

68.46

11.38

0.88

3.

13.4

S-7(0.5M)

61.78

4.01

0.25

4.

13.0

S-8(0.5M)

68.62

20.18

1.46

5.

13.9

S-2(2M)

63.38

31.14

2.44

6.

14.0

S-4(2M)

63.99

17.83

1.24

7.

13.9

S-3(4M)

64.00

20.74

1.49

8.

14.0

S-5(4M)

61.97

27.18

1.85

18

Fig. 9. Magnetization curves for MNPs at different pH values measured at room temperature.

The value of saturation magnetization and magnetic parameters of particles depend on many factors including the synthesis protocol, crystal structure, surface properties etc. [39]. As the size increases the value of saturation magnetization increases [39, 40]. In the case of superparamagnetic particles, surface and spin effects also play a major role in determining the magnetic properties [40]. Size of the particles determines the single domain character and the surface spin disorder effects [40]. The value of Ms in our particles is less than the bulk value of the magnetite ( 92emu/g) because of the surface and size effects [40] but we have got better results compared to the other reported work of the particles of comparable size [26, 30]. The good values of saturation magnetisation also indicate the absence of any goethite in the samples as goethite is antiferromagnetic and reduces the magnetisation value, as also proved by the XRD results. The lower concentration of NaOH of 0.5 M and 1.0 M are giving better parameters for magnetic properties compared to higher concentrations, Table 5. In the case of coated particles, citric and ascorbic acid coated particles are having good values of saturation magnetisation at 300 K of 57.1 and 56.6 emu/g respectively, as is evident from the M/H graphs in Fig. 10. These values are comparable to that reported in the literature [41, 42]. The values are lower compared to that for the uncoated particles because of the presence of the nonmagnetic coating layer on the surface, proving successful coating of 19

the particles. The value of saturation magnetisation Ms is 63.3 emu/g and 63.2 emu/g at 5K for citric and ascorbic acid coated particles respectively. The magnetisation value Ms is higher at 5K compared to that at 300K, most probably due to the thermal fluctuations of moments at higher temperature. The nature of the coating agent and bonding by functional groups of coating agents with surface atoms of particles, both affect the value of saturation magnetisation. These factors lead to surface spin disorders that are the reasons for the decreased magnetisation values in coated particles [40]. There is almost no hysteresis at 300K proving again the superparamagnetic nature of the prepared particles. Presence of a blocked state is evident by a distinct hysteresis present in the M/H graphs at 5K. Particles show ferromagnetic behaviour at 5K, and their remanence values are 15.8 emu/g and 15.6 emu/g and hysteresis values are 280 Oe and 320 Oe for citric and ascorbic acid coated particles respectively as shown in Table 6. The values of reduced remanence or squareness (SQ) Mr / Ms is only 0.24 and 0.25 in citric and ascorbic acid coated particles respectively at 5K, considerably smaller than the theoretical value of 0.5 proving that the particles possess uniaxial anisotropy and are single domain [40]. In Fig. 11, M/T graphs show the temperature dependence of the magnetisation by the ZFC and FC curves. ZFC curves are unmistakably proving the single domain nature of the prepared particles by the presence of maxima in the ZFC curve for blocking temperature (TB). At the blocking temperature thermal energy becomes comparable to the anisotropic energy. Below the blocking temp. the susceptibility decreases due to the frozen spin states along the easy direction and their random orientations.

Table 6. Magnetic parameters of the Citric and Ascorbic acid coated particles. Coating Material

Sauration Magnetisation Ms (emu/g) 300K 5K

Size (nm) ±S.D.

Squareness(SQ) Mr / M s

Mr(emu/g )

Coercivity Blocking Hc (Oe) Temp.

300

300

Remanence

5K

K Citric

57.1

63.3

0.24

8.5

acid Ascorbic 56.6

5K

(K)

K

1.3

15.8

7.8

280

200

1.7

15.6

13

320

180

±1.3 63.2

0.25

8.6

acid

±1.5

20

Fig. 10. M/H graphs of the citric (a) and ascorbic acid (b) coated MNPs at two different temperatures. Above the blocking temperature the thermal energy is able to overcome the aniosotropy energy barrier and thus spin is able to switch in the magnetic field direction. The presence of the blocking temperature (TB) well below the room temperature at around 200 K and 180 K in both the citric and ascorbic acid coated particles respectively is an evidence for their small sizes as also proved by the TEM and XRD results. These particles thus have magnetic properties suitable for biomedical applications.

(a)

(b)

Fig. 11. ZFC/FC curves of Citric (a) and Ascorbic acid (b) coated particles.

21

3.4 Coating and dispersion of particles. The particles synthesized under the optimum conditions of low molar concentration of NaOH, and pH were coated effectively and successfully with citric, ascorbic and glutamic acid (some data is shown for glutamic acid in the present article). The high pH values of 1314 provided a highly basic medium that brings about the changes in the coating molecules by deprotonation. These deprotonated functional groups, -CO- , -COO- and NH2 help in the better interaction with the surface ions. These coated particles may find applications in different biomedical related fields because of their stable dispersion and hydrophilicity. The presence of stretching bands of Fe-O bonds of Iron Oxides at lower wave number of ≤ 700 cm-1 in the FTIR spectra of the uncoated particles confirm the presence of magnetite with maghemite [18], as shown in the following Fig.12 and 13. Bands at 562 cm-1 and 443 cm-1 are due to the stretching vibrations of Fe-O bonds, confirming magnetite [18], while the

623 cm-1

shoulder confirms the presence of maghemite [43]. Absence of any band at 400 and 800 cm-1 confirm the absence of goethite. In the FTIR spectra multiple bands between 400-800 nm typical of maghemite are also not seen in our samples [33, 44].

Fig. 12. FT-IR spectra of citric acid coated particles.

22

In the case of citric acid coated particles (0.5 M NaOH) the presence of the bands near 1403 cm-1 and ~1610 cm-1 show the presence of the asymmetric and symmetric C-O stretching bands of the acid respectively [11, 41]. Strong band of C=O group in acid near 1700 cm-1 is shifted to lower wave number due to it’s partial single bond character as a result of the bonding to the surface of the MNPs. Citric acid may bind to the surface of the MNPs by one or two –COOH groups depending on the steric necessity and the curvature of the surface [24]. Particles were also coated with ascorbic acid to observe the coating efficiency under the optimum pH and lower conc. of alkali. As shown in the FTIR spectra Fig.13. particles are well coated with ascorbic acid with 1 M NaOH, pH 13.4. In the FTIR spectra the pure ascorbic acid spectra is little bit shifted for clarity. The bands in the pure ascorbic acid spectra between 3000-3520 cm-1 correspond to the vibrations of the different hydroxyl groups [45].

Fig.13. FT-IR spectra of Ascorbic acid coated MNPs. The band at 1755 cm-1 that is due to the vibration of the C=O group of the five membered lactone ring, disappears after coating. The C=C stretching band at 1673 cm-1 shifts to 1651 cm-1 after coating and there is a shift of ≈22 cm-1, showing the binding of the ascorbic acid on 23

to the nanoarticles surface [45].The band at 1396 cm-1 is due to the –CH2 wagging [46]. The band at 796 cm-1 is due to the shifting of the 821 cm-1 band of C-C ring stretching [46]. The presence of coating by citric and ascorbic acid is further confirmed by the UV-Vis spectroscopy. The graphs of the spectra are shown in Fig.14 for the uncoated and coated particles. The results of UV-Vis spectra are also in good agreement of the coating of citric and ascorbic acid on the particles . The absorption band present in the wavelength range of 330450 nm is from the absorption and scattering of UV radiation from the nano sized magnetic nanoparticles in Fig. 14. [47]. Significant absorption is not observed after 230 nm in pure citric acid [48].A broad band in the region of 340 nm for citric acid coated particles is because of the changes in the band gap due to the quantum size and surface effects of nanostructures. Peak at 255 nm for the pure Ascorbic acid is due to the C=C Π-Π* transition. In the spectra of coated particles there is a broad band and the sharp peak is absent, signifying the presence of coating on the particle surface [45].

(b)

(a)

Fig.14. UV-Visible absorption spectra of the uncoated, citric (a) and ascorbic acid (b) coated MNPs. For knowing the stability of the dispersions in aqueous solution, DLS studies were done in water of pH .7. The zeta potential values for the coated particles are given in Table 7. The zeta potential values are well represented with the values of -24.06mV, 43.37 mV and 40.45 mV for the citric, ascorbic and glutamic acid coated particles respectively, proving the well stabilised dispersions. The –OH and/or –NH2 groups present on the particle surface adsorb H+ ions. H+ ions may give rise to positive surface charge. The sign of the zeta potential is same as the surface 24

charge on the particles. This might be the probable reason for the positive zeta potential for ascorbic and glutamic acid coated particles. For citric acid coated particles the value is -24.06 mV. Citric acid gets attached by one/two –COOH groups out of three on to the nanoparticle surface [24].The volumeweighted size distribution for the ascorbic acid coated particles is also shown in Fig. 15, proving the narrow size distribution of the particles.

Table 7. DLS parameters of the coated particles. Coating Material

Hydrodynamic

Zeta Potential

PDI

(NaOH Molarity)

Size

(mV)

Citric acid (0.5M)

97.7 nm

-24.06

0.58

Ascorbic acid (1M)

28.16 nm

43.37

0.53

Glutamic acid (1M)

42.0 nm

40.45

1.00

Fig. 15. Volume weighted size distribution of ascorbic acid coated MNPs by DLS.

The TEM micrographs shown in Figs. 16 and 17 are also confirming the successful coating of the particles at the lower NaOH concentrations and other reaction parameters. The

25

particles are well dispersed as is visible from the micrographs with narrow size distribution, and most of the particles are between the size range 7-9 nm.

Fig. 16. TEM micrograph of the citric acid coated particles with 0.5M NaOH (size 8.5±1.3 nm).

Fig. 17. TEM micrograph of glutamic acid coated particles with 1.0 M NaOH, pH 13.4 (size 8.6±1.5 nm)

26

4. Conclusion. Present study was done to simplify the facile co-precipitation process and produce

hydrophilic MNPs that may find suitable applications in biomedical fields,

optimising the alkali concentration. Formation of magnetite under air in the presence of excess alkali and the higher pH values of 13-14 was studied. Different concentrations of alkali from 0.5 M - 4 M were used in the study. After analysis it was proved that the pH value of 13.4 with 1 M concentration of NaOH was optimum for the formation of maximum percentage of magnetite, up to approx. 58 wt%. Only maghemite with magnetite was present in the samples, and as both are biocompatible so particles are useful for biomedical fields. The presence of magnetite was confirmed by the XRD, TEM and Raman spectroscopy. The particles were small, in the size range of 6-13 nm and superparamagnetic in nature. They were synthesised with good crystallinity, cubo-spheroidal shape and monocrystalline nature.There good magnetisation values, single domain and uniaxial nature as confirmed by the ZFC and M/H curves further proved their future applicability. We needed hydrophilic particles so optimum synthesis parameters of pH and alkali concentration as obtained from the study, were used and found to be successful in obtaining the well coated particles as proved by the FTIR, UV-Visible and TEM results. Stability of the aqueous dispersions was successfully proved by the DLS results. To conclude we were successful in obtaing SPIONs applicable for biomedical fields by simplifying and optimising the facile co-precipitation process.

Note- The authors declare no conflicts of interest.

Acknowledgements. We would like to thank Dr Ravi Prakash Singh, IISER, Bhopal for the XRD. We acknowledge SAIF, IITB for the TEM images. We also acknowledge the use of infrastructural and instrumentation facilities of the Central University of Gujarat, Gandhinagar. We are very thankful to the UGC-DAE Consortium for Scientific Research, Mumbai and Dr. P.D.Babu from CSR, Mumbai for the VSM of the coated samples.We are thankful to Dr. V.R. Reddy UGC-DAE-CSR, Indore for the fruitful discussions.

27

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