Editing Magnetic beads Preparation Methods (section)

== Agarose Preparation methods ==

''Preparation of a Fast-Flow Agarose-Based Chelating Adsorbent with a Novel Dioxime Derivative for Selective Column Preconcentration of Copper Payman HASHEMI,† Zohreh RAHMANI, Ali KAKANEJADIFARD, and Esmaiil NIKNAM''

Preparation of spherical agarose particles Spherical homogeneous agarose particles (beads) of the particle size range (45 – 400 µm) were prepared according to a known emulsification method described by Gustavsson et al.18 A 20 ml solution of 6% (w/v) agarose was prepared by dissolution of the required amount of agarose powder in boiling water. The temperature was then adjusted on 60˚C in a water batch and 40 ml of cyclohexane containing span 85, 4% (v/v), with the same temperature, was added to the solution. The mixture was thoroughly stirred during the addition of cylohexane. After 3 min the mixture was cooled to room temperature by adding ice to the water bath. A cooling rate of about 5˚C/min was used. The spherical particles were then washed with ethanol and water and sized using graded metal sieves. Agarose particles with size ranges of 45 – 100, 100 – 200, and 200 – 400 µm were obtained in this way. Agar particles were prepared by simply grinding and sieving an agar powder (Merck). For increasing the rigidity of the particles, a patented method, described by Lindgren,13 was used with some modifications. Agarose (or agar) was first reacted with allylbromide in an alkaline medium, followed by a treatment with bromic water. In this way agarose was cross-linked in a defined way, and the number of side-reactions was minimized. This process was repeated until the desired rigidity was obtained.



[https://patents.google.com/patent/CN105713212A/en https://patents.google.com/patent/CN105713212A/en]

Embodiment 1

Taking 7.5g agarose (agarose solution concentration is 3%) in 250mL water, stirring at normal temperature 1h, after adding 0.12gKCl continuation stirring 0.5h, heat and seethe with excitement to agarose, solution presents transparence.Prepare 1.5L toluene solution, add 23.49g polyvinyl acetate and 2.349g oleic acid.Organic facies constant temperature at 70 DEG C is stirred to mix homogeneously, the agarose solution of boiling is poured in organic facies, the dispersion and emulsion 1h of the lower agarose of certain stirring.Disposable addition 22.5g epoxychloropropane, the NaOH solution of gradation dropping 20mL50%, dropwise in 1h.Condense again after certain stirring lower solidification 3h, control 2 DEG C/min of rate of cooling and be cooled to less than 10 DEG C to temperature.Prepare, after repeatedly cleaning, the agarose gel microsphere that particle diameter is 100-300 order.

Embodiment 2

Taking 10g agarose (agarose solution concentration is 4%) in 250mL water, stirring at normal temperature 1h, after adding 0.2gKCl continuation stirring 0.5h, heat and seethe with excitement to agarose, solution presents transparence.Prepare 1.75L toluene solution, add 30.45g polyvinyl acetate and 3.045g oleic acid.Organic facies constant temperature at 70 DEG C is stirred to mix homogeneously, the agarose solution of boiling is poured in organic facies, the dispersion and emulsion 1h of the lower agarose of certain stirring.Disposable addition 30g epoxychloropropane, the NaOH solution of gradation dropping 25mL50%, dropwise in 1h.Condense again after certain stirring lower solidification 3h, control 2 DEG C/min of rate of cooling and be cooled to less than 10 DEG C to temperature.Prepare, after repeatedly cleaning, the agarose gel microsphere that particle diameter is 100-300 order.

Embodiment 3

Taking 15g agarose (agarose solution concentration is 6%) in 250mL water, stirring at normal temperature 1h, after adding 0.375gKCl continuation stirring 0.5h, heat and seethe with excitement to agarose, solution presents transparence.Prepare 1.625L toluene solution, add 35.3g polyvinyl acetate and 3.53g oleic acid.Organic facies constant temperature at 70 DEG C is stirred to mix homogeneously, the agarose solution of boiling is poured in organic facies, the dispersion and emulsion 1h of the lower agarose of certain stirring.Disposable addition 45g epoxychloropropane, the NaOH solution of gradation dropping 38mL50%, dropwise in 1h.Condense again after certain stirring lower solidification 3h, control 2 DEG C/min of rate of cooling and be cooled to less than 10 DEG C to temperature.Prepare, after repeatedly cleaning, the agarose gel microsphere that particle diameter is 100-300 order.

Embodiment 4

Taking 20g agarose (agarose solution concentration is 8%) in 250mL water, stirring at normal temperature 1h, after adding 0.36gKCl continuation stirring 0.5h, heat and seethe with excitement to agarose, solution presents transparence.Prepare 1.7L toluene solution, add 32.5g polyvinyl acetate and 3.25g oleic acid.Organic facies constant temperature at 70 DEG C is stirred to mix homogeneously, the agarose solution of boiling is poured in organic facies, the dispersion and emulsion 1h of the lower agarose of certain stirring.Disposable addition 60g epoxychloropropane, the NaOH solution of gradation dropping 50mL50%, dropwise in 1h.Condense again after certain stirring lower solidification 3h, control 2 DEG C/min of rate of cooling and be cooled to less than 10 DEG C to temperature.Prepare, after repeatedly cleaning, the agarose gel microsphere that particle diameter is 100-300 order.





[https://patents.google.com/patent/US3860573A/en https://patents.google.com/patent/US3860573A/en]

EXAMPLE 1

5 g of agarose or agar are suspended in benzene; n X 0.18 g triethylamine (n 1 are added. The reaction mixture is cooled close to the freezing point of benzene (about +6C) and n X 0.1 g oxalic acid chloride are added under powerful agitation. The temperature is allowed to rise up to +C (not higher) and the agitation is continued for another 6 hrs. The crosslinked agarose thus obtained is filtered and carefully washed with benzene, water and acetone and allowed to dry at room temperature.

EXAMPLE 2

5 g of agarose are suspended in ether; n X 0.14 triethylamine (n l 10) are added. The suspension is cooled to about +6C and thereafter n X 0.1 g maleic acid dichloride are added. Continuation as in Example 1.

EXAMPLE 3

5 g of agarose are suspended in petrol ether; n X 0.12 g N,N-dimethylaniline (n 1 10) are added. To the cooled (+6C) suspension n X 0.1 g cyclohexane l,2- dicarboxylic acid chloride are added. Continuation as in Example 1.

5 of agarose. are suspended in dioxane. In the mixture, n X 0.11 g (n'=1 10) of crystal water-free, recently dried sodium acetate are dissolved, and at about +10C, under agitation, n X 0.1 g succinic acid dichloride are added Continuation as in Example 1.

EXAMPLE 6 5 g of agarose are suspended in ether; n X 0.075 g triethylamine (n, 1 5) and n X 0.1 g benzene-1,3-.

disulphonic acid chloride are added. Reaction at +10 30C for 2-4 hrs is allowed. Continuation as in Example 1.

EXAMPLE 7 g of agarose are suspended in toluene; n X 0.1 g (n l-. 20) p-phenylene-diisothiocyanate are added. Under powerful agitation, the mixture is heated under a reflux cooler at the boiling point of toluene (+1 10C) for 8 hrs. Upon filtration, the cross-linked agarose thus obtained is washed with toluene and allowed to dry at room temperature.

EXAMPLE 8 5 g of agarose are suspended in dioxane; n X 0.1 g n l butyl-l,4-diisothiocyanate are added. Heating at the boiling point of dioxane (+l0lC) as in Example 7 is followed by washing with dioxane and acetone and drying at room temperature.



Cross-linked and epoxy activated agarose (Novarose) was a gift from Inovata AB (Stockholm, Sweden) with 40–60 �m particle size. Before use, Novarose was thoroughly washed with a 20ml portion of water, hydrochloric acid (1 mol l−1) and acetate buffer (0.1 mol l−1, pH 5.5), respectively

About 0.6 g of the ligand was dissolved in 40 ml 0.5 mol l−1 Na2CO3 and the solution’s pH was adjusted by 0.1 mol l−1 sodium hydroxide on 12.2. The solution was then added to a magnetically stirred suspension of 2ml of epoxy activated Novarose particles in 10 ml water and agitated for 24 h. The mixture was then filtered and the adsorbent was washed by 20 ml portions of 0.5 mol l−1 HCl, water, ethanol and water again, respectively. Deactivation of the remaining active sites was made by suspension of the adsorbent in 25 ml of a 1 mol l−1 sodium hydroxide solution and agitation for an extra 24 h. Finally, the adsorbent was washed by water and ethanol and stored at 4 ◦C in 20% ethanol.

'''Synthesis of Agarose'''



High-melting agarose D5 (Mr = 120 KDa, gelation temperature 46°-52° C, depending on concentration, composition and setting conditions, ρ(dry) = 1.64 g mL-1 ) (supplied by Millipore, UK) was used as raw material for the manufacturing of the adsorbent. White mineral oil Marcol N82 (ν(90° C) = 3.5 mm2s-1, ρ(90° C) = 812.3 kg m-3) (ESSO, UK) was used as the continuous phase. Sodium chloride (Sigma Aldrich, UK) was used as a co-solute for controlling the pore size of the adsorbent. Oil-soluble surfactants Span 80 (sorbitan monoleate, HLB 4.3, ρ(20° C) = 986 kg m-3, Mr = 446.62 g mol-1) and Span 85 (sorbitan trioleate, HLB 1.8, ρ(20°C) = 956 kg m-3, Mr = 975.51 g mol-1) (Sigma Aldrich, UK) were used for controlling particle size.

Agarose solution preparation Agarose powder at a concentration of 4&nbsp;% (w/w) was dissolved in saline solutions of distilled water containing a certain amount of NaCl in a glass beaker. Distilled water was also added to account for water loss during subsequent heating. The mixture was then placed in a microwave oven for a total of 3 mins. To ensure complete dissolution of the agarose, the mixture was stirred by hand every 0.5 mins and for about 10 s. After obtaining a transparent solution the beaker was sealed and placed in a submersion water bath at 90° C where it was held for at least 30 mins to allow air bubbles to escape. Due to water evaporation a thick crust of dried agarose was formed on the surface of the solution. For this reason it was necessary to load the pre-heated syringe for the bottom of the beaker were the bulk concentration of the agarose solution remained constant. This was done by penetrating the crust of dried agarose with the syringe and immersing it into the beaker until it touched the bottom. Considering the viscous nature of agarose solutions, later observation of agarose beads with the SEM confirmed the mixing procedure followed resulted in homogenous mixture as the internal structure of the particles appeared fairly uniform. Emulsification A volume of 800 mL (80&nbsp;% (v/v), continuous phase) of mineral oil was loaded to the emulsification vessel. Oil soluble surfactant of desired type and concentration was added to the vessel from the top port using a syringe. The mixture was allowed to equilibrate to 90° C while stirring at 1000 rpm for about 30 mins. Then, 4 L of mineral oil were placed in a plastic bottle and stored in deep freezer (-70°C) for approximately 45 mins thus cooling the oil to -10°C. After cooling the bulk volume of oil, 3.8 L were loaded to the quenching vessel and allowed to equilibrate for 30 mins to the desired quenching temperature while stirring at 300 rpm. As soon as the two vessels were equilibrated to the desired temperatures and air bubbles had escaped from the agarose solution, the high shear mixer rotor was fixed at the desired speed. Then, using a preheated syringe, 200 mL (20&nbsp;% (v/v), dispersed phase) of the agarose solution were injected in the high shear mixer from the top port. After 5 mins of shearing the emulsion was discharged in the cooling vessel using compressed air (0.5 bars). To ensure complete gelation of the agarose particles, the mixture was allowed to equilibrate to the final cooling temperature (20° C) for at least 15 mins while stirring at 300 rpm. This relatively low speed was chosen in order to avoid altering the resulting particle size distribution coming out of the high shear mixer. The above concentrations and conditions were kept constant during the following experiments. To study the effect of ionic strength on pore size / size distribution and particle size / size distribution, NaCl was dissolved in the agarose solution at the following concentrations: 0, 0.01, 0.025, 0.05 and 0.1 molal. In this set of experiments the emulsion contained 0.1&nbsp;% (v/v) Span 85, was emulsified at 30.7 W kg-1 (3500 rpm) and was quenched at 0° C. To study the effect of quenching temperature on pore size / size distribution and particle size / size distribution the emulsion was quenched at three different temperatures: 0°, 10° and 20° C, and three concentrations of salt were used at each quenching temperature (0, 0.01, 0.05 m NaCl), 0.1&nbsp;% (v/v) Span 80 was added as a surface active agent. In all three experiments, formulations were emulsified at 30.7 W kg-1 (3500 rpm). To study the effect of surfactant concentration the resulting particle size / size distribution Span 85 was added to the continuous phase prior to emulsification at the following concentrations: 0, 0.1, 0.5, 1.0 and 1.35&nbsp;% (v/v). The salt-free dispersion was emulsified at 2.87 W kg-1 (2000 rpm) and quenched at 0° C. The effect of surfactant type on particle size was studied by preparing two separate emulsions. In the first, Span 80 at a concentration of 0.1&nbsp;% (v/v) was added to the continuous phase. In the second emulsion, Span 85 was added at the same concentration (0.1&nbsp;% (v/v)). No salt was used in this experiment while the dispersions were emulsified at 30.7 W kg-1 (3500 rpm) and quenched at 0° C. Finally, to study the effect of rotor speed on the resulting particle size / size distribution two salt-free agarose solutions, each containing 0.1&nbsp;% (v/v) Span 85, were emulsified at 2.87 (2000 rpm) and 30.7 W Kg-1 (3500 rpm) respectively , and quenched at 0° C.

Centrifugation

Following solid bead formation, the oil-particle mixture was transferred from the cooling vessel to the centrifuge (model Jouan C4 22, Thermo Fisher Scientific, UK). Centrifugation at 3000 rpm (1830 g) for 10 mins successfully separated the particles from the bulk volume of the oil as the liquid removed from the centrifuge bottles was transparent. A 25&nbsp;% (v/v) aqueous ethanol solution was used to wash out the particles from the centrifuge bottles.

Washing

Prior to any characterization measurements particles where washed using the following procedure: A small volume of particles ( ̴30 ml) was placed in a sintered glass funnel connected to a vacuum pump. Particles where then washed with 3 volumes of 25&nbsp;% (v/v) aqueous ethanol, 3 volumes of 50&nbsp;% (v/v) ethanol, 3 volumes of 75&nbsp;% (v/v) ethanol, 30 volumes of ethanol, 3 volumes of 75&nbsp;% (v/v) ethanol, 3 volumes of 50&nbsp;% (v/v) ethanol and finally 3 volumes of 25&nbsp;% (v/v) ethanol. The successive washing with increasing following decreasing concentration of ethanol aqueous solutions prevents particles from possible structural damage caused by the rapid dehydration when washing directly with pure ethanol

(Amsterdam et al. 1975). After washing, particles were kept in 25&nbsp;% (v/v) aqueous ethanol solution to prevent bacterial contamination.

Particle sizing

Particle size distributions, mean diameters (D(0,1), D(0,5), D(0,9) and D(3,2)), and yield (obtained from undersize curve) were determined with the Mastersizer 2000 (Malvern, UK). Refractive index values of 1.58 and 1.33 (Malvern manual) were used for agarose particles and distilled water respectively. The 25&nbsp;% (v/v) aqueous ethanol solution was exchanged with distilled water prior to sizing. A 10&nbsp;% (v/v) dispersion of particles was slowly added to the dispersion unit using a pipette until laser obscuration reached 10&nbsp;% of its total value. A stirrer speed of 2000 rpm was used in the measuring cell (loop) to ensure complete dispersion of particles. Three measurements were performed per sample and the results presented in Chapter 3 are the average of said measurements.