Purification and Modification of Nanoclay for Adsorption of Vitamin B6 as Nanocarriers

Special Article - Vitamin B Deficiency

Ann Nutr Disord & Ther. 2016; 3(1): 1032.

Purification and Modification of Nanoclay for Adsorption of Vitamin B6 as Nanocarriers

Akbari Alavijeh M, Sarvi MN* and Ramazani Afarani Z

Department of Mining Engineering, Isfahan University of Technology, Iran

*Corresponding author: Mehdi Nasiri Sarvi, Department of Mining Engineering, Isfahan University of Technology, Isfahan 84156-83111, Iran

Received: October 25, 2016; Accepted: November 18, 2016; Published: November 21, 2016


Nanomaterials are widely used for preparation of nanocarriers especially in order to overcome problems associated with deficiency of vitamins. Among them nanoclay has been introduced as a versatile carrier. In this study nanoclay was prepared, modified and applied for adsorption of vitamin B6 as carrier. The nanoclay was prepared from bentonite as a naturally available ore and then modified using a dispersing agent (sodium Hexamethaphosphate (NaHMP)) for improvement of adsorption of vitamin B6 on it. The results showed that the adsorption of vitamin B6 on nanoclay was occurred in early stages of experiments and adsorption was stopped after 5 minutes indicating a high affinity of vitamin B6 to the nanoclay due to cation exchange mechanism. The adsorption kinetics was quit similar in different pH. However, the amount of vitamin B6 adsorbed at pH=6 was higher than pH=3.2. Amount of adsorption was increased when small amount of dispersant was used for modification of nanoclay however it reduced by increasing the amount of dispersant. The results indicated that the nanoclay can be tuned for improvement of adsorption of vitamin B6 in order to develop versatile nanocarriers to fight deficiency of such important species.

Keywords: Nanoclay; Montmorillonite; Vitamin B6; Biomolecule adsorption; Cationic exchange mechanism


Vitamin B6 is one of the water soluble vitamins which occur naturally as pyridoxine, pyridoxal, and pyridoxamine, either free or combined with other substances such as phosphate [1]. It helps red blood cell regeneration and fat and carbohydrate need it as co-factor for neurotransmitters [2,3]. In addition, this vitamin is necessary for proper adsorption of vitamin B12. Maintaining healthy nerve and muscle cells, production of DNA and RNA are responsibility of pyridoxine. Deficiency of vitamin B6 causes Changes in mood, such as irritability, anxiety and depression, confusion, muscle pains, fatigue, irritability, difficulty concentrating and worsening symptoms of anemia. The food sources of vitamin B6 are meat, poultry, fish, egg, potato, starchy vegetables. Banana, nut, whole grains and fortified soy are other sources [3].

Considering, lack of several very important nutrients such as vitamins attempt have been done to prepare food and pharmaceutical supplements to overcome this problem [4-6]. In this regards, carriers of food and pharmaceutical supplements played an important role which among them, nanocarries attracted much attention to compensate deficiency of vitamins and proteins [7-9]. Between these carriers nanoclay is a promising nano-carrier for this purpose due to its superior characteristics such as high surface area, cation exchange capacity, non-toxicity, high biocompatibility, and low price [10].

Among clay minerals montmorillonite has been widely used for preparation of nanoclay as nano-carrier due to its phenomenon cation exchange capacity [11-13]. Montmorillonite is the major component of bentonite that is a natural clay mineral with layered structure. Due to its 2:1 layered structure with exchangeable cations, different molecules could be adsorbed in between the layers.[14]There are many drugs and biomaterials that have been loaded on montmorillonite that proteins (DNA [15], cytochrome c [16], lysozyme and bovine serum albumin [17]), antibiotics (metronidazole [18] and tetracycline [19]) and vitamins (B12 [10], B1 [12], B6 [20], E [11]).

As the vitamin B6 is protonated in the solution it is possible to adsorb it to the montmorillonite through an ionic exchange mechanism. In this regards the accessible sites for adsorption of vitamin to adsorb is not well investigated. In addition, one of the disadvantages of nanoclay in the adsorption of food supplements and drugs is that they tend to accumulate together and form larger cluster. Consequently, clay nanoparticles adhere together and reduce the accessible sites for adsorption. To the best of our knowledge there is not enough information about improvement of adsorption of vitamin B6 onto montmorillonite by increasing the accessible sites for adsorption. Hence, in this study we are planning to apply a dispersing agent for increasing the accessible adsorption sites for vitamin B6 and to improve the adsorption of vitamin B6 onto nanoclay. Adsorption kinetics and Isotherms were tested and calculated by modified montmorillonite with different amount of NaHMP in two different pH for this purpose.

Materials and Methods


Pyridoxine hydrochloride (>98%, Sigma Aldrich) and Sodium hexametaphosphate (>96%, Sigma Aldrich), and Hydrochloric acid, HCl (37% w/w, Merck) was used as received. Deionized water (the water was purified to a resistivity of =18.2 MO.cm) was used in all experiments. The bentonite sample was provided by Salafchegan bentonite mine (Iran).

Preparation of nanoclay

In order to prepare nanoclay from bentonite montmorillonite particles smaller than 2.5μm were collected using centrifuge force. In addition, the dispersant agent was used in different amount in order to prepare nanoclay with dispersed particles of montmorillonite. Typically, 12 g of dry raw montmorillonite and defined amount of NaHMP (0.1, 0.5, 1.5 and 5 g) dispersed in 400mL of deionized water and the mixture was mixed for 16hours. Centrifuge force is used for physical purification and separating <2.5μm particles of montmorillonite [21]. Veiskarami et al. showed that according to type of centrifuge, nanoclay and falcon’s diameter montmorillonite particles smaller than 2.5μm was achieved by centrifuging at 1000 rpm for 260s [22]. The time of centrifugation was calculated based on stock’s law as followed [21]:

t=[ . log 10 ( R S ) ]/[ 3.81. N 2 . r 2 .ΔS ] MathType@MTEF@5@5@+=feaaguart1ev2aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLnhiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr4rNCHbGeaGqiVu0Je9sqqrpepC0xbbL8F4rqqrFfpeea0xe9Lq=Jc9vqaqpepm0xbba9pwe9Q8fs0=yqaqpepae9pg0FirpepeKkFr0xfr=xfr=xb9adbaqaaeGaciGaaiaabeqaamaabaabaaGcbaaeaaaaaaaaa8qacaWG0bGaeyypa0ZaamWaa8aabaWdbiabl2==Ujaac6caciGGSbGaai4BaiaacEgapaWaaSbaaSqaa8qacaaIXaGaaGimaaWdaeqaaOWdbmaabmaapaqaa8qadaWcaaWdaeaapeGaamOuaaWdaeaapeGaam4uaaaaaiaawIcacaGLPaaaaiaawUfacaGLDbaacaGGVaWaamWaa8aabaWdbiaaiodacaGGUaGaaGioaiaaigdacaGGUaGaamOta8aadaahaaWcbeqaa8qacaaIYaaaaOGaaiOlaiaadkhapaWaaWbaaSqabeaapeGaaGOmaaaakiaac6cacqWI9=VBcaWGtbaacaGLBbGaayzxaaaaaa@55B4@

In this equation parameters are defined as follows: t: time of centrifuge (s), R: distance from the sediment surface to the axis of the centrifuge rotor (13.1cm), S: distance from the liquid to the axis of the centrifuge rotor (4.5cm), N: rotation speed, r: maximum radius of the desired particles and the liquid dispersion (0.00528gcm-1), ?: viscosity of the fluid (0.00748P for deionized water).

Hence in order to achieve <2.5μm particles of montmorillonite the mixture was centrifuged at 1000rpm for 260s and 25°C. The supernatant as a final product was collected and used for adsorption experiments.

Adsorption vitamin B6 on montmorillonite

Adsorption kinetics was carried out at initial vitamin B6 concentration of 0.8mg/mL in deionized water solution at 25°C and two different pH (3.2 and 6). 5mL of abovementioned solution in desired pH was added to 50mg of montmorillonite. In order to plot adsorption kinetics mixing times was set for different durations of 5,10,20,30,45, 60,90,120,180,270, and 360 minutes. Then the mixture was centrifuged at 5000rpm, 25°C for 5min and supernatant concentration was measured by UV-visible spectroscopy at the detection wavelength of 291nm. The amount of vitamin B6 adsorbed on nanoclay was calculated by the difference between initial and final concentration. For adsorption isotherm 5mL of water with different concentration of vitamin B6 (0.1,0.3,0.8,1.5,2.5 and 4.5 mg/mL) was added to 20mg of nanoclay and mixed for 6 hours (equilibrium time). After that the mixture was centrifuged and amount of vitamin remained in the solution was measured by UV-visible spectroscopy.


Normal XRD analysis in the range of 2? from 3 to 80 degree for determination of impurities in nanoclay samples was done using Philips PW1800. Vitamin B6 concentration was measured using Unico UV-2100 spectrophotometer and calculated using the standard curve.

Results and Discussion

Table 1 shows the amount of NaHMP that is used to prepare each type of nanoclay samples. Figure 1 shows the results of XRD analysis of nanoclay samples along with raw montmorillonite. The results represent that physical purification increased purity of montmorillonite. According to the peak of montmorillonite in the range of 7 to 8 degree the peak became broader by increasing the concentrations of NaHMP as a dispersant and this can be a reason for decreasing particle size of montmorillonite. There are three peaks at 9,12 and 17-19 degree that related to muscovite, illite, and gypsum respectively. These peaks were removed when NaHMP was used and purity of montmorillonite. However, indication of cristobalite and calcite can be seen in the XRD patterns of all samples and physical purification and modification with NaHMP couldn’t remove this impurity completely while quartz was removed.