Effect of Toa Concentration, pH and Temperature on Nicotinic Acids

Nicotinic acid (NA) is a water soluble B complex vitamin; possess antipallegra, antidiarrhoea and antidepression activity. It is a novel active substance against the deadly disease cancer and diabetes. It is an important compound used significantly in food, pharmaceutical and biochemical industries. The recovery study of NA is investigated using tri-n-octylamine (TOA) in dodecane and dodecanol mixture in 1:1 (w/w). n-dodecane (log Pa = 6.6) toxicity study reveals that it is the most suitable diluent for the simultaneous extraction of products during fermentation. However, 1-dodecanol (log Pa = 5.0) can also be used which provides a very low phase level toxicity with very high salvation power for extraction and blending a non toxic diluent (dodecane) with less toxic diluent (decanol) in 1:1(w/w) yield a biocompatible diluent with good extraction efficiency. Equilibrium study of NA (0.014-0.083 mol.kg-1) is performed by varying different parameters like effect of TOA concentrations (0.148 – 1.201 mol.kg-1), effect of initial pH of aqueous solution (2.5- 6) and effect of temperature (298- 328 K). Using the equilibrium data, the distribution coefficient (KD), extraction efficiency (% E) and loading ratio (z) are calculated. A maximum KD is obtained as 10.8 using optimum TOA (0.706 mol.kg-1) and acid concentration (0.055 mol.kg-1) with 91.53% recovery of NA. Maximum extraction is observed near pH 3.3. It has negligible influence on NA-TOA 1:1 complex based on model values and FT-IR spectra. Thermodynamic parameters, enthalpy (ΔH0) and entropy (ΔS0) are determined in the temperature range of 298 to 318K. The number of theoretical stages (NTS) for counter current extraction at optimum condition is found to be 4. In addition, the back-extraction rate was found to be reached 99.0 % using 0.05 mol kg−1 NaOH as stripping agent.

Keywords: Nicotinic acid, reactive extraction, tri-n-octylamine, equilibrium, pH, temperature, column design.

Introduction

Niacin or nicotinic acid (pyridine-3-carboxylic acid) is a water soluble B-complex vitamin, which participates in the formation of NAD and NADP coenzymes. These coenzymes are involved in the catabolism of carbohydrates, fats and proteins with energy production, along with fatty acids and cholesterol synthesis 1-3. Nicotinic acid is widely used in food, pharmaceutical and biochemical industries. The acid plays a key role in DNA repair and helps in the formation of steroid hormones in the adrenal gland. Deficiency of nicotinic acid may leads to pellagra, red tongue, diarrhoea, apathy, depression, disorientation and even memory loss 4, 5. Nicotinic acid also plays key role in the causes and treatment of other diseases, such as cancer 6, 7, diabetes 8, 9 and cardiovascular diseases associated with high level of cholesterol 10, 11. As human body is inept to produce nicotinic acid by its own, hence to maintain its ideal concentration in body, it should be taken in the form of food such as eggs, fish and green leafy vegetables or nutritional supplements. However, the overall production of nicotinic acid, worldwide have reached to about 22, 000 tonnes/y 12, 13, and still more emphasis is going on to increase its production rate.

Nowadays the complicated chemical synthesis methods become unalluring, due to worldwide awareness towards growing ecological problems. Hence, the production of nicotinic acid is intensified by biosynthetic route or enzymatic conversion of 3-cyanopyridine. The successful production of nicotinic acid (Lonza, Switzerland, China) on commercial scale using nitrilase enzyme has proved its industrial application 14, 15-17. In nature, it is present extensively in bacteria, filamentous fungi and plants 18, 19. However, the downstream processing of bioproducts costs about 60% of total production cost 20. Hence, for large scale production, it is not preferred over the chemical synthesis route. Thus, an alternate efficient method is required for its recovery from dilute fermentation broth via biosynthetic route 21. Reactive extraction is one of the efficient alternative to remove valuable organic acids/ biocompounds from an aqueous medium continously, using a suitable organic extractant/diluents system 22- 24. The toxicity of diluents in the bioreactor is the major concern when the in situ technique is used in separation. Molecular toxicity due to dissolution of solvent usually causes less damage to the cell than does phase toxicity because the former is limited by solvent solubility in the aqueous phase. The best possible way to reduce the toxicity of extracting medium is by blending a non toxic diluents (log Pa ≥6) with a less toxic diluents (6 ≥ log Pa ≥4) 25,26.

Earlier, several investigations on equilibrium and kinetic studies of nicotinic acid are done by different researchers. However, no recent literature on column design of nicotinic acid using biocompatible diluents mixture has been performed. Earlier, Kumar and Babu 27 review the different manufacturing processes of nicotinic acid. It includes both chemical and enzymatic method. They found separation of nicotinic acid through reactive extraction via enzymatic route is a promising technique in terms of enhancing nicotinic acid production. Sushil and co-workers 28 reported equilibrium study of nicotinic acid using organophosphorous extractant, TOPO and TBP dissolve in different inert and active diluents like n-heptane, n-decane, kerosene, 1-octanol, 1-decanol, MIBK etc. Maximum extraction yield is obtained by dissolving TOPO in MIBK. They found the mechanism of reaction is controlled by extractant type and solvent polarity. However, pure solvents without extractant are not capable of efficient extraction of nicotinic acid. Depaloy et al. study the equilibrium and kinetic study of nicotinic acid using TOA in MIBK 29. The equilibrium results show the formation of both 1:1 and 2:1 acid-amine complex. Kinetic study show that speed of stirring has no effect, however rate of extraction increases with increasing volume ratio of the phases. This group has also done equilibrium study of benzoic acid and nicotinic acid using TOA and TBP dissolved in different binary mixture 30. Results suggest that extraction strongly depend on polarity and ionizing strength of acid. Deliang et al. study the reactive extraction of nicotinic acid with TOA in n-octanol 31. It is found that the proton– donating n-octanol is efficient diluents when trialkylamine (N235) is used for extracting nicotinic acid. The favourable operating conditions are equilibrium aqueous pH 4.2 to 5.5.

The present article reports the determination of suitable column and it’s designing for optimization of industrial scale recovery of nicotinic acid from dilute aqueous/fermentation broth. This is based on experimental determination of equilibrium data by varying different parameters. Nicotinic acid is recovered by reactive extraction method using Tri-n-octylamine (TOA) as extractant dissolve in lauryl alcohol (log Pa = 5.032) + dodecane (log Pa = 6.633) in 1:1(w/w) ratio throughout the experiments. However, the combined use of diluent with polar modifier also affect the extraction yield positively. Nevertheless, with increase in number of carbon chain, toxicity of alcohol and alkane decreases. Hence, the combination of dodecane and lauryl alcohol as diluent system will create a non toxic, biocompatible environment friendly organic phase with good extraction efficiency. Finally, solvent regeneration is performed using different concentration of NaOH (stripping agent) solution.

Reactive Extraction Chemodel

The solvent dodecane +lauryl alcohol and extractant (tri-n-octyl amine) used in this study are poorly soluble in water. The solubility of alkane and alcohol decreases with increase in the number of carbon chain. Hence, no change in volume in aqueous and extract phase is accounted and all the calculations are done by assuming negligible solubility of solvents in the aqueous phase. Also, the solvent or organic phase co-extract water in negligible amount 34, 35.

Case I: n =1Separation of nicotinic acid from fermentation broth with TOA dissolved in dodecane and lauryl alcohol mixture (1:1 w/w) have been shown in eq. (1), assuming that m molecule of acid reacts with one TOA (T) molecule, with corresponding equilibrium constant(KE).

Experimental

Materials. Nicotinic acid (SRL Pvt. Ltd, Mumbai, India), tri-n-octylamine (Spectrochem, Mumbai, India), lauryl alcohol (Alfa Aesar), dodecane (Spectrochem, Mumbai, India) were used without any pre-treatment. All the aqueous phases were prepared by using Millipore water (Milli -Q Advance A 10 TOC, Flix, Bangalore). pH of aqueous solution is maintained using reagent grade NaOH and H2SO4 solution. The physiological properties of chemicals used in the experimental study are given in table 1.

Table 1. Physical characterization of reagents used in the experimental study.

Reagents IUPAC name supplier Purity (% w) Mol. wt.

(kg.kmol-1) M.P

(K ) Density

(kg.m-3) Viscosity

(mPa.s) Dipole moment

(D) Dielectric constant

Nicotinic acid Pyridine-3-carboxylic acid

SRL Pvt. Ltd. 99 123.11 509.6 1470 - 0.219 -

Tri-n-octylamine N,N, dioctyl octan-1-amine Spectrochem, Mumbai, (India)

95 353.67 134 809 8.32

(296 K) - -

Lauryl alcohol 1-Dodecanol Alfa Aesar 98 186.33 299 830 18.8

(293 K)

1.60 6.50

n-

dodecane dodecane Spectrochem, Mumbai, (India) 99 170.34 263 750 1.40

(293 K) 0 2

Equilibrium. The stock solution of NA is prepared by adding weighed nicotinic acid in Millipore water. Different concentrations, 0.0136, 0.0341, 0.0548 and 0.0825 mol.kg-1 of nicotinic acid are prepared by diluting the stock solution. The organic phase is prepared by dissolving different concentrations of tri-n-octylamine (TOA) in mixture (1:1 w/w) of n-dodecanol plus dodecane. The extraction equilibrium experiments are carried out on a constant temperature water bath shaker (Daihan Labtech co. Ltd) for 6 h at 120 ± 5 rpm in 100 ml Erlenmeyer flasks with screw cap by taking equal volumes (20ml) of aqueous and organic phases at 298 ± 1 K. After reaching equilibrium, the mixture was allowed to settle for 120 min in a 60 ml separating funnel in an incubator ( REMI CIS-24 plus ), maintain at that temperature to achieve complete separation. The concentration of acid in aqueous phase was determined on a UV-Vis spectrophotometer, (Shimadzu UV-3600 plus) at 262 nm. Further, experiments were performed at different temperatures, 288, 298, 308 and 318 ±1 K. The extract phase chemicals used in this study have negligible solubility in aqueous solution.

Results And Discussion

Physical Extraction: It is previously known that low molecular weight diluents are miscible with water while high molecular weight diluents are not. However, mixing of non toxic diluents with polar modifier also enhances the extraction efficiency and reduces toxicity. The degree of toxicity of a solvent is defined by a parameter logPa which is logarithm of distribution of solvent between 1-octanol and water. For toxic solvents, the logPa value is less than 4 and for non toxic solvent logPa value are greater than 6. In this study, we have used dodecane and dodecanol in 1:1 (w/w) throughout the experiments. LogPa value of dodecane and dodecanol are 6.6 and 5.13 respectively. However, the physical extraction of the extract phase is also studied, as shown in Table 2. Maximum extraction efficiency of only 18.76% with 0.034 mol.kg-1 of NA is obtained. Therefore, to increase its extraction efficiency an extractant, TOA is also investigated for its further recovery.

Table 2. Physical Extraction of nicotinic acid in dodecane plus lauryl alcohol in 1:1

(w/w) at temperature 298 ± 1K and pressure 101.325 ±1 kPa.

Cin

(mol.kg-1) CHNc

(mol.kg-1)

(mol.kg-1) KD

- %E

-

0.014 0.0120 0.0020 0.166 14.26

0.034 0.0276 0.0064 0.231 18.76

0.055 0.0454 0.0096 0.213 17.54

0.082 0.0688 0.0132 0.191 16.05

aRelative standard uncertainities in molalities, ur (mHNc) = 0.1 ; standard uncertainities in temperature, u(t)= 1 K; standard uncertainities in pH, u(pH)= 0.01 ; standard uncertainities in pressure, u(p)= 1mPas

Effect of TOA concentration: Equilibrium study of NA was performed by varying different concentrations of acid (0.0136, 0.0341, 0.0548 & 0.0825 mol.kg-1) and TOA (0.148, 0.313, 0.499, 0.706, 0.943 and 1.201 mol.kg-1) in dodecane and lauryl alcohol mixture (in 1:1 w/w) at 298 ±1K and pressure 101.325±1kPa. The effects of both these parameters are observed simultaneously. However, the concentrations of NA studied are based on its concentration in the fermentation broth [ref].

The effect of TOA concentration is observed at six different points from a lowering value of 0.148 mol.kg-1 to a higher concentration at 1.2011 mol.kg-1 as shown in figure 1. Maximum extraction efficiency of NA is observed at 0.943 mol.kg-1 and on moving further slight plunge in degree of extraction is observed at 1.2011 mol.kg-1. The decrease may be due to increase in physical parameters like density, viscisity etc. However, very slight increase in degree of extraction is achieved when TOA concentration is raised from 0.706 to 0.943 mol.kg-1. Hence, 0.706 mol.kg-1 of TOA concentration is found to be the optimum concentration for cost efficient recovery process.

Nevertheless, the effect of acid concentrations is also investigated in this study and it is found that on moving from lower to higher TOA concentration convergence in degree of extraction is observed or the effect is more predominant in lower TOA concentration rather than at higher TOA concentration as shown in figure 1. However, on further investigating all the NA concentration range, it is found that maximum extraction is achieved at 0.055 mol.kg-1 of NA for all the TOA concentration range.