Succinic acid tests

The isolation of an aliphatic acid from its aqueous solution, particularly in the presence of metallic salts, is a tedious operation (cf. p. 56), although a few such acids, e.g., succinic acid, can be extracted with ether. Since, however, a solution of an acid or one of Its salts is admirably suited for most of the tests in this series, the isolation of the free acid is rarely necessary except as a nieans of distinguishing (as in (i)) between aliphatic and aromatic members. 

I. Fluorescein test. Fuse together carefully in a dry test-tube for about 1 minute a few crystals of resorcinol and an equal quantity of succinic acid or a succinate, moistened with 2 drops of cone. H2SO4. Cool, dissolve in water and add NaOH solution in excess. A red solution is produced which exhibits an intense green fluorescence.-f-... 

Both succinic and phthalic anhydride respond to the hydroxamic acid test (see 5 above). 

The fluorescein test for succinic acid (p. 349) and the phthalein and fluorescein tests for phthalic acid (p. 351) are obviously given also by succinic anhydride and phthalic anhydride, as these tests depend upon the initial formation of the anhy dride in each case. 

With some acids (e.g., succinic acid and sulplianilic acid) more satisfactory results are obtained by reversing the order of mixing, i.e., by adding the solution of the so um salt of the acid to the reagent. It should be pointed out that the melting points of the derivatives as determined on the electric hot plate (Fig. II, 11, 1) may differ by 2-3° from those obtained by the capillary tube method. In view of the proximity of the melting points of the derivatives of many acids, the mixed m.p. test (Section 1,17) should be applied. 

The discrete protonation states methods have been tested in pKa calculations for several small molecules and peptides, including succinic acid [4, 25], acetic acid [93], a heptapeptide derived from ovomucoid third domain [27], and decalysine [61], However, these methods have sofar been tested on only one protein, the hen egg lysozyme [16, 61, 71], While the method using explicit solvent for both MD and MC sampling did not give quantitative agreement with experiment due to convergence difficulty [16], the results using a GB model [71] and the mixed PB/explicit... 

Dlugosz M, Antosiewicz JM (2004) Constant-pH molecular dynamics simulations A test case of succinic acid. Chem Phys 302 161-170. 

The applicability of Eq. (2.63) was tested for some 180 individual solutes (with up to 10 carbon atoms) and 25 dry solvents by stepwise multivariable Unear regression and for 28 solutes and 9 dry solvents by target factor analysis essentially the same conclusions and universal coefficients were obtained by both methods. As an example of the application of Eq. (2.63), the distribution of succinic acid between water and chloroform (a dry solvent with = 0.0048) and tri-n-butyl phosphate (a wet solvent with Xw = 0.497) may be cited [13]. 

Many industrial organic acids can be produced by fermentation, such as acetic, citric, and lactic acids. Succinic acid is a dicarboxylic acid of potential industrial interest as a platform chemical (1-3). Separation and purification of succinic acid by adsorption was tested to replace current precipitation methods and their associated waste disposal problems. Succinic acid is a valuable intermediate value chemical with a moderate market. For succinic acid to have an economic and energy impact, it will need to become a commodity chemical intermediate with a much lower price. This target price hasbeen estimated to be between 0.22 and 0.30 / lb ( 0.48- 0.66/kg) and is potentially achievable with advanced technology (1). At this price, succinic acid can be catalytically upgraded into other higher valued chemicals suchastetrahydrofuran, 1,4-butanediol, y-butyrolactone, 2-pyrrolidinone, and N-methylpyrrolidinone. 

Adsorption has shown good potential and some data have been gathered for the distribution properties of other carboxylic acids, including acetic, lactic, and citric acids. These sorbents were tested with succinic acid. The key desired properties for an ideal sorbent are high capacity, complete low-cost stable regenerability, and specificity for the product. The economics of the adsorption process have not been evaluated in detail however, as a stand-alone primary separation, an ultimate goal of a final concentration of >100 g of succinic acid/L was chosen as likely to be economically feasible if achieved. 

It may also be economical to remove the inhibitory product directly from the ongoing fermentation by extraction, membranes, or sorption. The use of sorption with simultaneous fermentation and separation for succinic acid has not been investigated. Separation has been used to enhance other organic acid fermentations through in situ separation or separation from a recycled side stream. Solid sorbents have been added directly to batch fermentations (18,19). Seevarantnam et al. (20) tested a sorbent in the solvent phase to enhance recovery of lactic acid from free cell batch culture. A sorption column was also used to remove lactate from a recycled side stream in a free-cell continuously stirred tank reactor (21). Continuous sorption for in situ separation in a biparticle fermentor was successful in enhancing the production of lactic acid (16,22). Recovery in this system was tested with hot water (16). 

The extraction of the desired product from the sorbent and the stable regeneration of the resin for further use are essential. The regeneration of some of these sorbents by methanol has also been tested (23). Hot water regeneration has been proposed as well (24). Back extraction with trimethy-lamine has been tested for succinic acid with consideration of formation of a succinate ester (13,25). 

In the present study, various sorbents were tested for the critical properties of capacity for succinic acid, regenerability of the sorbent, and coadsorption of substrates. These criteria were evaluated in order to choose a suitable sorbent for use with either a primary purification step or an in situ separation and fermentation. Other factors that were considered were the pH, temperature, and potential ability to concentrate the product. 

Some resins were tested for in-process removal of succinic acid. Industrial-scale sorption is performed in columns. A packed column (1-cm id,... 

Approximately half of the better resins were then tested for successive loading of succinic acid in simulated medium and regeneration using hot water. The final pH was near pH 5.0 This cycle was repeated for multiple cycles. Some of the hot water regeneration results are excerpted in Table 2. The best resins (Reillex 425, XUS 40285, and XUS 40091) have a stable capacity of about 0.06 g of succinic acid/g of resin. MWA-1 was not included in these tests because of its lower capacity however, it was reexamined in later... 

In a similar manner, the sorbents were tested for their capacity for the substrate of the planned fermentation, glucose. Glucose was found to adsorb with all tested resins (data not shown). Maximum glucose capacities ranged from 0.01 to 0.04 g/g. However, the resinpreferred succinic acid at a ratio near 4 1 for Reillex 425,4 1 for XUS 40091, and 8 1 for XUS 40285. Sorbents that preferentially sorbed glucose over succinic acid were eliminated from further testing. 

Two resins were tested for the removal of succinic acid from simulated medium on a packed column of sorbent to simulate an actual process on a small scale. It is important to test the sorption with medium, because salts and other nutrients can interfere with the sorption. Table 4 presents the results for XUS 40285 MWA-1 was comparable. This indicates that either sorbent can remove succinic acid efficiently from the fermentation broth without direct loss of product. Both columns were then stripped or regenerated with hot water. Stripping with hot water recovered 70-80% of the succinic acid from the XUS 40285 resin whereas less (50-60%) was recovered from the MWA-1. The XUS 40285 column was stripped with 2 column volumes of hot water with eluent concentrations up to 49 g / L. Succinic acid was concentrated on average to 40 g/L in the XUS resin by this operation and to 30 g/L by the MWA-1. The 10-fold concentration factor bodes well for the use of sorbents to purify the fermentation broth. 

More traditional acid and base regeneration steps were tested for this high-capacity resin. The column of sorbent XUS 40285 was loaded with succinic acid at 35 g/L and extracted with 1M HC1. Eighty-one percent of the succinic acid was recovered however, there was no increased concentration of the succinic acid in the final product stream. The recovered product stream must be at least as concentrated as the fermentation broth and should be significantly concentrated. 

Tests were performed with both simulated broth containing succinic acid at various concentrations and actual broth provided by MBI. Seven resins were tested for regenerability and stability with acid XUS 40285, Dowex 1x2, XUS 40283, XUS 440323, XFS-40422, IRA-35, and IRA-93. Previous results had shown a decrease in capacity with repeated hot water regeneration. It is essential for economical operation that the organic acid recovery be >90% and that the sorbents be stable for at least 20 cycles (based on industrial comments). Several resins were tested for stability with a single-step dilute-acid regeneration. The resins were either low capacity after five cycles or had incomplete recovery of the succinic acid (data not shown). Therefore, we modified the procedure to extract the succinic acid first with dilute base, then hot water. 

Several sorbents were tested more exhaustively for uptake of succinic acid and for successive loading and regeneration using hot water. One resin, XUS 40285, has a good stable isotherm capacity, prefers succinate over glucose, and has good capacities at both acidic and neutral pH. 

Succinic acid was removed from medium on a packed column of sorbent. The resin XUS 40285 was tested in a packed column with simulated medium containing salts, succinic acid, acetic acid, and sugar. The packed column completely separated the fermentationbyproduct, acetate, from succinate. A simple hot water regeneration successfully concentrated succinate from 10 g/L (inlet) to 40-110 g/L in the effluent with a pH of about 5.0. The end pH indicates that succinate salt was sorbed from the "medium" and released in a partially acidic form by the hot water regeneration. If successful, this would lower separation costs by reducing the need for chemicals for the initial purification step. 

Despite promising initial results of good capacity (0.06 g of succinic acid/ g of sorbent), 70% recovery using hot water, and a recovered concentration of >100 g/L, this regeneration was not stable over 10 cycles. Alternative regeneration schemes using acid and base were examined and more sorbents screened. Tests were performed with simulated broth containing succinic acid at various concentrations. 

Seven of the most promising resins were tested for regenerability and stability using a modified extraction procedure combining acid and hot water washes. Two (XUS 40285 and XFS-40422) showed both good stable capacities for succinic acid over 10 cycles and >95% recovery in a batch operation. 

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