Patent Application: US-86835507-A

Abstract:
extraction systems comprising acetonitrile , water , and a saccharide selected from the group consisting of a monosaccharide , an oligosaccharide , and mixtures thereof . the systems comprise a first phase and a second phase , and the concentration of the saccharide is at least 0 . 5 weight / volume %.

Description:
the present invention makes use of the discovery that saccharides are capable of causing acn to separate from its water solutions and form two - phase systems . this discovery provides methods for acn dehydration , and extraction methods and systems . a mixture of acn , water and a saccharide forms upper and lower phases ; the upper organic phase comprises mostly acn and small amounts of saccharide . the acn in this “ sugared - out ” upper phase can reach a purity of 95 . 4 %, which cannot be obtained by ordinary distillation . the lower aqueous phase contains most of the saccharide . unequal distributions of several organic species were found between the upper phase and lower phase , with most of the organic species contained in the upper phase . accordingly , salt is no longer required in acn - based solvent extraction systems , since one or more saccharides may be used instead , thereby minimizing equipment corrosion and fouling . also , acn / water extraction can now be performed without metal ions , rendering it amenable for use in systems that are intolerant to metal ions , for example biosystems . without being bound by any particular theory , it may be inferred that the sugaring - out phenomenon is due to the very poor solubility of saccharides in acn . this may account for the repulsion between saccharides and acn , which in turn causes separation of the organic and aqueous phases . preferred saccharides are monosaccharides and oligosaccharides that generate two phases at 0 ° c . from a 1 : 1 acn / water mixture at a concentration of 5 % wt / v . more preferred saccharides are monosaccharides and oligosaccharides that generate two phases at 0 ° c . from a 1 : 1 acn / water mixture at a concentration of 2 . 5 % wt / v . most preferred saccharides are monosaccharides and oligosaccharides that generate two phases at 0 ° c . from a 1 : 1 acn / water mixture at a concentration of 0 . 5 % wt / v . preferred saccharides include arabinose , lyxose , ribose , xylose , ribulose , xylulose , allose , altrose , galactose , glucose , gulose , idose , mannose , talose , fructose , psicose , sorbose , tagatose , mannoheptulose , sedoheptulose , octolose , 2 - keto - 3 - deoxy - manno - octonate , sialose , sucrose , lactose , maltose , trehalose , cellobiose and mixtures thereof . most preferred saccharides include glucose , xylose , arabinose , fructose , sucrose and mixtures thereof . sugaring - out can also be carried out with mixtures of acn , water and optionally one or more additional organic solvents , for instance alkanes such as hexane , halogenated alkanes such as methylene chloride , and cyanated alkanes such as propionitrile . in the presence of an additional organic solvent , the organic phase may be the upper phase or the lower phase , depending on the relative quantities of acn and the additional organic solvent . for example , an acn , methylene chloride and water mixture will yield an organic upper phase if the acn to methylene chloride volume ratio is sufficiently high . there can also be three separate phases coexisting , where the upper phase is sugared - out acn , the middle phase is aqueous and the lower phase is methylene chloride . if the volume of water is too small , there will be only one phase . accordingly , acn can be separated from heterogeneous systems that include water and organic solvents , for example phase transfer catalysis ( ptc ) mixtures . the extraction system of the present invention can be used , for example , in the bioenergy field , to extract inhibitors and by - products , for instance organic acids , plant phenolics , furfural and 5 - hydroxymethylfurfural ( hmt ), from fermentation broths and biomass hydrolysates during the production of alcohols such as ethanol , propanol and butanol , thereby enabling continuous , uninterrupted production under milder conditions than those afforded by salting - out extraction methods . in this regard , it was found that the distribution of solutes in the saccharide / acn / water two phase systems was also influenced by ph . for example , at a ph below 6 , concentrations of plant phenolics such as para - coumaric , ferulic and syringic acids in the organic phase were high . when the ph was adjusted to values higher than 9 , however , no detectable quantities of para - coumaric , ferulic and syringic acids were found in the organic phase , the compounds having re - entered the aqueous phase . thus , at least with certain types of solutes , the addition of a ph - modifying agent such as an acid or base can increase the efficiency of the extraction system . the milder conditions of the sugared - out extraction system are also particularly applicable to the extraction of pharmaceuticals from fermentation broths in the course of manufacturing processes , thereby removing the need for dosing the fermentation broth with acids and / or alkaline compounds and its concurrent yield losses . since acn has a high polarity , dielectric strength and dipole moment that render it an excellent solvent for charged organic compounds [ 7 ], the sugared - out extraction system of the present invention can be used to extract pharmaceuticals and other high - value chemicals within relatively mild ph ranges , and with potentially much less degradation and emulsification . fig5 illustrates an example of a sugaring - out two - phase system for the extraction of compound “ i ” from an aqueous phase , such as a fermentation broth . a mixture is formed by mixing acn and optionally one or more additional organic solvents with a fermentation broth or a biomass hydrolysate 902 . optionally , the ph of 902 is adjusted in order to maximize the yield of the extraction . one or more saccharides “ s ” are then added , resulting in the partition of the mixture into aqueous phase 903 and sugared - out organic phase 904 . phase 904 contains most of compound “ i ”, whereas more hydrophilic compounds such as the saccharides “ s ” are retained in the aqueous phase 903 . chemical “ i ” is then isolated from the sugared - out organic phase . for example , 904 can be mixed with an aqueous saccharide solution and subjected to a ph change that induces compound “ i ” to migrate to the aqueous phase 905 . the remaining organic phase 906 is then recycled . when the goal is simply to obtain acn from an aqueous mixture containing water and acn , one or more saccharides may be added to the mixture , causing phase separation . acn ( ch 3 cn , hplc grade ), d - glucose ( c 6 h 12 o 6 , certified acs grade ), sucrose ( c 12 h 22 o 11 , certified acs grade ), d - fructose ( c 6 h 12 o 6 , certified grade ), starch (( c 6 h 10 o 5 ) n , certified acs ) were obtained from fisher scientific company ( pittsburgh , pa ., usa ). l - arabinose ( c 5 h 10 o 5 , hplc grade ), dextran mr ˜ 100000 (“ dextran 100000 ”) and dextran mr ˜ 500000 (( c 6 h 10 o 5 ) n , biochemika grade ) (“ dextran 500000 ”) from leuconostoc spp . bacteria were purchased from sigma - aldrich company ( st . louis , mo ., usa ). sudan i ( c 16 h 12 n 2 o , lot number : a0214906001 ), d - xylose ( c 5 h 10 o 5 , 99 %), maltose monohydrate ( c 12 h 22 o 11 . h 2 o , biochemical reagent ) were supplied by acros ( new jersey , usa ), aldrich chemical company ( milwaukee , wis ., usa ), and thomas kerfoot & amp ; co . ( vale of bardsley , lancashire , england ) respectively . each of the saccharides listed in table 1 was tested for its ability to induce phase separation in acn / water mixtures . distilled water was combined with one of the saccharides to produce a saturated solution . the saturated solution was then mixed with acn in capped test tubes at 1 : 1 v / v ( volume ratio ), stirred vigorously , and left to equilibrate at 25 ° c . for 24 hours . each of the saccharides listed in table 1 was tested for the effects of its concentration on phase separation . water and acn were mixed in capped test tubes at 1 : 1 v / v . the saccharide was separately weighed and added to the water and acn mixtures . the resulting saccharide concentrations were from 5 g / l to 50 g / l at increments of 5 g / l , as illustrated in table 1 . the tubes were vortexed and left to equilibrate at 0 ° c . for 24 hours , and then examined for the formation of two clear phases . the tubes had a minimum graduation of 0 . 1 ml which could lead to a relative deviation of less than 1 %. samples of the upper and lower phases , each 0 . 2 ml to 1 ml of liquid , were taken from the tubes for high - pressure liquid chromatography ( hplc ) and gas chromatography ( gc ) analysis . the phase ratio was defined as r = v upper / v lower , where v upper was the volume of the upper phase and v lower was the volume of the lower phase . sudan i was dissolved in acn at a concentration of 1000 mg / l . mixtures containing 5 ml of the resulting sudan i in acn solution and 5 ml of water were prepared , and different quantities of glucose were added to each mixture . the resulting glucose concentrations were from 5 g / l to 50 g / l with 5 g / l increments . the resulting glucose - added mixtures were stored at 0 ° c . for 24 hours , then samples of the upper phases were collected for later analysis . sugar concentrations in the upper and lower phases were determined by hplc analysis . the hplc system consisted of a waters ( milford , mass ., usa ) 2695 separation module , a waters 410 refractive index detector monitored by an hp chem station computer program ( agilent technologies , germany ), and a prevail carbohydrate es hplc column ( 250 × 4 . 6 mm , 5 μm ; alltech associates , inc ., deerfield , ill ., usa ) equipped with a guide column ( 7 . 5 × 4 . 6 mm , 5 μm ). the column temperature was kept at 30 ° c . the temperature of refractive index detector was also set at 30 ° c . the mobile phase was 75 %: 25 % ( v / v ) hplc grade acn and water ( 0 . 45 μm filtered ) at a flow rate 1 ml / min . standard solutions of glucose , xylose , arabinose , sucrose and fructose , with concentrations from 1 . 2 g / l to 24 g / l were prepared . standard curves were plotted according to calculations and performed on the hp chem station . all samples were filtered through 0 . 4 5 μm filters into autosampler vials . sudan i concentration was determined by absorbance measurements at 478 nm using a hp 8453 uv - visible spectrophotometer ( hewlett - packard , waldbronn , germany ). the correlation of all standard curves was found to be greater than 0 . 998 . when necessary , samples were diluted to fit in the concentration ranges of the standard curves before analysis . using acetone as an internal standard , the acn concentration in the upper phase was determined with a gas chromatographic ( gc ) system . c acn was defined as the concentration of acn in the upper phases , and calculated according to equation ( 1 ): ( 1 ) c acn =( weight of acetone in std × rf * peak area of acetonitrile × dilution factor )/ peak area of acetone where the rf ( response factor ) was calculated according to equation ( 2 ): ( 2 ) rf =( internal std peak area / acn peak area )/( acetone weight in std / acn weight in std ) the distribution coefficient d was defined as d = c upper / c lower , where c upper is the concentration of the solute in the upper organic phase and c lower is the concentration of solute in the lower aqueous phase . the extraction rate was defined as e = 100 ×( c upper × v upper )/( c initial × v initial ), where c upper and v upper were sudan i concentration in the upper phase and the upper phase volume , c initial and v initial were the solute concentration in the initial solution and the initial solution volume . all experiments above were duplicated . at 25 ° c ., all saturated solutions of the tested monosaccharides and disaccharides , specifically glucose , xylose , arabinose , fructose , sucrose ( a disaccharide of glucose and fructose ) and maltose ( a disaccharide of two units of glucose ) can sugar out acn from its water solution . two clear layers , one an upper organic phase , the other an aqueous lower phase , were formed . starch , dextran 100000 and dextran 500000 are complex polysaccharides composed of many glucose units . neither of these polysaccharides induced the sugaring - out of acn ; instead , starch deposited from the acn / water mixture , whereas dextran 100000 and dextran 500000 gelatinized the mixture . 5 weight / volume % of any of the tested saccharides could not sugar out the 1 : 1 acn / water mixture at 25 ° c . however , all saccharides tested , except for starch and dextran , generated two phases at 0 ° c . at a concentration no higher than 3 weight / volume %. a lowering of the temperature therefore appears to favor the phase separation of the acn / water systems . saccharide concentration appears to be an important factor to the sugaring - out effect . with each saccharide , the higher the concentration , the larger the upper phase obtained ( table 1 ). for all tested saccharides , the concentration in the upper phase was much lower than in the lower phase . higher concentrations yielded lower distribution coefficients between the upper phase and lower phase ( fig2 ). glucose , xylose , arabinose , fructose , sucrose exhibited different sugaring - out capabilities . mixed with identical volumes of acn at 0 ° c ., xylose required the least weight of saccharide ( 15 g / l ) to sugar out acn , while sucrose required the largest , i . e . 30 g / l to attain the sugaring - out effect . under the same conditions , xylose was first in terms of relative saccharide distribution in the upper phase , glucose and fructose were next and sucrose was last ( fig2 ). xylose ( fig1 ( a )) and arabinose are pentoses , glucose ( fig1 ( b )) and fructose are hexoses , while sucrose ( fig1 ( c )) is composed of glucose and fructose . without being bound to any particular theory , it appears that the smaller the molecular size , the easier it is for the saccharide to enter the organic phase . when a glucose and xylose mixture ( 1 : 1 wt / wt ) was tested as a sugaring - out agent , possibly because of synergistic effects , the sugaring - out occurred at concentrations even lower than those required with xylose . the volume of the sugared - out upper phase was intermediate between those obtained by equal concentrations of xylose and glucose alone ( table 1 ). in such “ glucose + xylose ” mixture systems , as the total concentration of glucose and xylose increased , the upper phase concentration of xylose increased at a relatively faster rate than the glucose concentration . without being bound to any particular theory , the incompatibility between acn and saccharide molecules might be the principal factor occasioning the creation of two phases and the unequal saccharide distribution . the smaller , 5 - carbon xylose might have relatively better “ compatibility ” than the larger , 6 - carbon glucose in the upper organic phase . accordingly , because of this difference in compatibility , more acn could enter the lower xylose solution , so the upper , acn phase sugared - out by xylose was smaller than that sugared - out by glucose . as illustrated in table 2 , acn concentration in the upper phase is also proportional to the saccharide concentration ; higher saccharide concentrations yielded higher concentrations of acn in the upper phase . also , glucose appeared to yield higher acn concentrations in the upper phase . when glucose was added at 50 g / l , the acn concentration reached 95 . 4 %, a purity yet unattained by ordinary distillation methods . moreover , all the tested saccharides could sugar out acn with a purity of more than 90 %. the extraction capabilities of sugaring - out systems was confirmed by the unequal distribution of sudan i between the upper phase and lower phase , as illustrated in fig3 . in fig3 . 1 , sudan i distributed evenly between tube a ( left ) and b ( right ). after dissolving 2 . 5 % ( wt / v ) glucose in tube a and 5 % ( wt / v ) glucose in tube b , the system turned opaque , an emulsion - like layer having formed in the solution ( fig3 . 2 ). the emulsion - like layer then decreased , while a separate upper phase and lower phase formed . the upper phase turned darker and the lower became transparent , but the interface was not yet visible ( fig3 . 3 ; 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