Production of L-ascorbic acid

L-ascorbic acid is produced by allowing an acid to act on 2-keto-L-gulonic acid in a mixture solvent of an inert organic solvent and an aliphatic ketone in the presence of water and a surfactant. The method produces L-ascorbic acid in a high yield 90% or more and is an industrially advantageous method.

This invention relates to a method of preparing L-ascorbic acid, in which 
2-keto-L-gulonic acid is employed as the starting material. 
As one of the methods of preparing L-ascorbic acid, there has been known a 
method which comprises employing 2-keto-L-gulonic acid as the starting 
material and allowing an acid to act thereon to prepare L-ascorbic acid in 
one step. 
The known methods include, for example, 1 a method which comprises allowing 
concentrated hydrochloric acid to act on 2-keto-L-gulonic acid using 
acetic acid as the solvent U.S. Pat. No. 2,185,383 Specification (1940)!, 
2 a method which comprises adding ethanol and acetone to sodium salt of 
2-keto-L-gulonic acid, neutralizing with hydrochloric acid, separating 
precipitating sodium chloride by filtration, then maintaining the reaction 
mixture at temperatures ranging from 25.degree. C. to 75.degree. C. to 
thereby obtain L-ascorbic acid Japanese Unexamined Pat. Pub. No. 
58-177986, 3 a method which comprises allowing a mineral acid to act on 
2-keto-L-gulonic acid in an inert solvent in the presence of a surfactant 
(Japanese Examined Pat. Pub. No. 48-15931) and 4 a method which comprises 
causing slurry of substantially anhydrous 2-keto-L-gulonic acid to be 
produced in an inert organic solvent containing a surfactant, then 
allowing a substantially anhydrous acid catalyst to act on this slurry to 
give L-ascorbic acid PCT,WO87/00839 (1987)!. 
On the other hand, fermentative methods of preparing 2-keto-L-gulonic acid 
in a large amount from L-sorbose have been proposed (e.g. U.S. Pat. No. 
4,543,331, EP 132,308), and thus a method of industrial production of 
L-ascorbic acid at one stroke by using this starting material has been 
desired to be established as early as possible. 
However, the known methods mentioned above have still some drawbacks, 
while, some improvement is observed in e.g. yield, including still 
insufficient yield from the viewpoint of industrial production, a large 
content of colored substances in the reaction mixture as impurities, which 
inevitably imposes a burden on the purification process, thus preventing 
them from being employed as methods in an industrial scale. 
The present inventors have conducted study on methods of preparing 
L-ascorbic acid employing 2-keto-L-gulonic acid as the starting material, 
and have established an industrially advantageous method affording a high 
yield of about 90% or more of the desired product with little production 
of impurities More specifically, the present inventors have found that the 
reaction proceeds advantageously by conducting lactonization of 
2-keto-L-gulonic acid in a mixture solvent of an inert organic solvent 
e.g. toluene, benzene, etc. and an aliphatic ketone e.g. acetone, methyl 
ethyl ketone, etc. And, the present inventors have also found that the 
reaction proceeds more advantageously by suitably controlling the amounts 
of water and an acid catalyst. 
Namely, the present invention relates to a method of preparing L-ascorbic 
acid, which comprises allowing an acid to act on 2-keto-L-gulonic acid in 
a mixture solvent of an inert organic solvent and an aliphatic ketone in 
the presence of water and a surfactant. 
The reaction of this invention is conducted in a mixture solvent prepared 
by adding a given amount of an aliphatic ketone to an inert organic 
solvent. 
The inert organic solvent means an organic solvent with which 
2-keto-L-gulonic acid and L-ascorbic acid are not reactive and in which 
2-keto-L-gulonic acid and L-ascorbic acid are insoluble. 
Inert organic solvents usable for the present invention include aromatic 
hydrocarbons which may be substituted with halogen or alkyl, such as 
benzene, toluene, xylene and chlorobenzene; halogenated aliphatic 
hydrocarbons such as chloroform and ethylene chloride; aliphatic 
hydrocarbons such as hexane, heptane and octane; and ethers such as 
tetrahydrofuran, dioxane and isopropyl ether; or a mixture of them. 
Preferable ones are aromatic hydrocarbons such as benzene or toluene. 
Aliphatic ketones usable for the present invention include ketones 
containing alkyl having 1 to 6 carbon atoms, and cyclic ketones containing 
cycloalkyl having 5 to 6 carbon atoms. 
The alkyl may be straight-chain or branched one, preferably a one having 1 
to 4 carbon atoms. These two alkyls bonding to carbonyl may be the same as 
or different from each other. Practical examples of such ketones include 
acetone, methyl ethyl ketone, diethyl ketone, methyl isobutyl ketone and 
cyclohexanone, preferably acetone and methyl ethyl ketone. 
Such a ketone as exemplified above is required to be present in a specified 
amount in an inert organic solvent. The amount of a ketone to be mixed is 
about 0.02 to 0.3 volume part, preferably 0.05 to 0.2 volume part, 
relative to 1 volume part of an inert organic solvent. By maintaining the 
ketone concentration within this range throughout the reaction, the 
reaction can be allowed to proceed more smoothly. And, the ketone may be 
supplemented in the course of the reaction, so long as the amount of 
ketone does not deviate the above-mentioned range. 
When the amount of a ketone to be mixed is out of the range specified as 
above, the amount of degradation products increases to cause more strong 
coloration of the reaction mixutre, thus complicating the purification 
procedure, which is far from an industrially advantageous method. When the 
amount of a ketone exceeds the above-mentioned range, the amount of 
unreacted materials tends to become more, thus lowering the yield of the 
object compound. 
In this invention, the lactonization of 2-keto-L-gulonic acid is carried 
out in the above-mentioned mixutre solvent. The concentration of 
2-keto-L-gulonic acid relative to the mixtrue solvent is not limitative, 
but is usually 5 to 40 weight %, preferably 10 to 30 weight % from the 
economical viewpoint. 
In the present invention, the reaction is allowed to proceed by adding an 
acid in the presence of water and a surfactant. In this case, the amounts 
of water and an acid are preferably restricted as follows, respectively. 
The amount of water is 1.5 to 3.5 times as much in molar ratio, preferably 
1.8 to 3 times as much in molar ratio relative to 2-keto-L-gulonic acid. 
Water is allowed to be present within the mentioned range in the solvent 
when the reaction is carried out. When the starting material 
2-keto-L-gulonic acid is, for example, hydrated or contains water, such 
water is taken into account for calculation of the amount of water. And, 
the amount of water contained in an acid catalyst used for promoting the 
reaction or in a mixture solvent then employed is also taken into account 
for calculation of the amount of water. When the amount of water deviates 
from the above-mentioned range, the amount of decomposed materials 
increases to cause lowering of the yield. 
Surfactants that can be used in this invention include nonionic surfactants 
such as polyoxyethylene alkylaryl ether, polyoxyethylene alkyl ether, etc, 
cationic surfactants such as quarternary ammonium salts, pyridinium salts, 
etc., and anionic surfactants such as higher aliphatic alkylaryl 
sulfonates, etc.; each surfactant may be used alone or in combination with 
one or more of them Desirable surfactants for this invention are cationic 
ones, especially quaternary ammonium salts such as trimethyl tetradecyl 
ammonium chloride, trimethyl dodecyl ammonium chloride, trimethylcetyl 
ammonium chloride, trimethyloctyl ammonium chloride, diiethylethylcetyl 
ammonium chloride, trimethylstearyl ammonium chloride, dimethyl butylcetyl 
ammonium bromide and trimethyldodecyl ammonium bromide. The amount of the 
surfactant to be added ranges from 0.01 to 10 w/w %, preferably from 0.05 
to 3.0 w/w % relative to 2-keto-L-gulonic acid. 
Acids employable as the catalyst are exemplified by mineral acids 
including, for example, hydrochloric acid, phosphoric acid, etc., and 
hdyrochloric acid is espacially preferable The amount of an acid to be 
added for allowing the reaction to proceed advantageously, in the presence 
of water of an amount within the above-mentioned range, is 0.5 to 2 times 
as much in molar ratio, preferably 0.5 to 1.5 times as much in molar ratio 
relative to 2-keto-L-gulonic acid. As water is required for the reaction, 
it is preferable to employ hydrochloric acid of 20 to 45% concentration, 
usually a one having 35% concentration. The ketone solution containing 
hydrogen chloride is employable for adjusting the amount of an acid. 
Addition of an acid may be conducted in two installments within the 
above-mentioned range. 
Under the above-mentioned conditions, the starting material 
2-keto-L-gulonic acid is lactonized by the action of an acid to give 
L-ascorbic acid. The reaction proceeds in a heterogenous system. As the 
reaction proceeds, 2-keto-L-gulonic acid becomes gruel-like to oily 
(usually in 30 minutes to one hour after initiating the reaction). At this 
point of time, it is preferable to adjust the amounts of water and acid to 
the given ones. It is also possible to adjust the volume of water 
remaining in the reaction mixture by eliminating water in the reaction 
mixture by means of a conventional dehydrating procedure such as 
azeotropic distillation. This dehydration process is preferable to 
complete within a period as short as possible. When an aliphatic ketone or 
an acid catalyst is distilled off together with water, for example, by 
azeotropic dehydration, they may be suitably supplemented. Reaction 
temperatures range from about 40.degree. C. to about 80.degree. C., 
preferably from 50.degree. C. to 70.degree. C. The recation completes 
usually in 3 to 8 hours. 
For separating the object compound from the reaction mixture, a per se 
conventional process, for example, filtration, concentration, extraction, 
etc is employable. Further, if necessary, the object compound may be led 
to a highly purified one through, for example, recrystallization. 
According to the method of this invention, L-ascorbic acid can be prepared 
in a high yield of 90% or more. In addition, due to a little amount of 
impurities (e.g decomposition products) which may cause coloring occurs, 
no troublesome purification process is required, which is of a very 
industrial advantage. The following Examples and Comparative Examples will 
explain the present invention in more detail.

EXAMPLE 1 
To a mixture solvent of toluene (570 ml) and acetone solution containing 
23.5 weight % of hydrogen chloride (65 ml as acetone) was added 
2-keto-L-gulonic acid (content 91.2%, water content 8.4%)(100 g). To this 
solution were further added water (7.7 ml) and trimethylcetylammonium 
chloride (110 mg)(water content was 1.90 times as much in molar ratio 
relative to 2-keto-L-gulonic acid). The reaction mixture was stirred for 
six hours while heating at 60.degree. C., which was then cooled to 
20.degree. C. The resultant was poured into water (1 l), and the mixture 
was stirred, which was then left standing, followed by separating the 
aqueous layer. To the organic layer was added water (400 ml) for 
re-extraction. The aqueous layers were combined and subjected to 
quantitative determination by means of a high performance liquid 
chromatography to reveal the existence of L-ascorbic acid78.2 g (yield 
94.5%)!, while unreacted 2-keto-L-gulonic acid remaining in an amount of 
2.7%. The absorbance (E.sub.430.sbsb.nm.sup.1%) of this aqueous layer was 
0.160. 
The high performance liquid chromatography was conducted under the 
following conditions (the same applies to all the subsequent Examples and 
Comparative Examples). 
Column: Aminex HPX-87H manufactured by Bio Rad Co. 
Eluent: 0.1M ammonium sulfate 
Column temperature: room temperature 
Detection e UV.sub.210 nm and refractive index 
EXAMPLE 2 
To a mixture solvent of toluene (576 ml) and acetone (30 ml) was added 
2-keto-L-gulonic acid (content 89.9%, water content 8.4%) (101.4 g), to 
which was added trimethyltetradecylammonium chloride (110 mg), followed by 
further addition of 35% hydrochloric acid (10 ml)(d=1.17, 4.1 g as HCl, 
7.7 g as water). The water content in the reaction mixture was calculated 
to be 16.2 g (1.19 times as much in molar ratio relative to 
2-keto-L-gulonic acid). 
The reaction mixture was heated at 60.degree. C. and stirred for one hour 
to cause the crystals of 2-keto-L-glulonic acid to become oily, to which 
was added an acetone solution containing hydrogen chloride (15.8 weight %) 
(51. 8 g) (8.18 g as HCl, 55 ml as acetone). The whole mixture was stirred 
for 5 hours at the same temperature, which was then cooled to 20.degree. 
C.(the total amount of acid relative to 2-keto-L-gulonic acid was 0.72 
times as much in molar ratio). 
The reaction mixture was poured into water (1 l), which was stirred, then 
was left standing. The aqueous layer was separated. To the organic layer 
was further added water (600 ml) for re-extraction. The aqueous layers 
were combined. 
Quantitative determinaiton of the resultant L-ascorbic acid by means of a 
high performance liquid chromatography revealed that 77.7 g (yield: 94.0%) 
of the compound was produced, while 2-keto-L-gulonic acid remained 
unreacted in an amount of 2.3% ,The absorbance (E.sub.430.sbsb.nm.sup.1%) 
of this reaction mixture was 0.17. 
EXAMPLE 3 
To a mixture solvent of toluene (576 ml) and methyl ethyl ketone (24 ml) 
was added 2-keto-L-gulonic acid (content 89,9%, water content 8.4%) (101.4 
g), to which was added trimethylcetyl ammonium chloride (110 mg), followed 
by further addition of 35% hydrochloric acid (13.2 ml) (d=1.17, 5.4 g as 
HCl, 10.0 g as water). The water content in the reaction mixture was 
calculated to be 18.5 g (2.14 times as much in molar ratio relative to 
2-keto-L-gulonic acid). 
The reaction mixture was stirred for one hour at 60.degree. C., then 
crystals of the 2-keto-L-gulonic acid became oily, to which was added a 
methyl ethyl ketone solution containing 18 weight % hydrogen chloride (45 
g) (8.1 g as HCl, 46 ml as methyl ethyl ketone). The whole mixture was 
stirred for 5 hours at the same temperature, followed by cooling to 
20.degree. C. (the total amount of acid relative to 2-keto-L-gulonic acid 
was 0.79 times as muchlin molar ratio). 
The reaction mixture was poured in water (1 l), which was stirred, then 
left standing, followed by separating the aqueous layer. To the organic 
layer was added water (600 ml) for re-extraction, and the aqueous layers 
were combined. 
Quantitative determination of the resultant L-ascorbic acid by means of a 
high. performance liquid chromatography revealed that the 76.9 g (yield: 
93.0%) of the compound was produced, while 2-keto-L-gulonic acid remained 
unreacted in an amount of 2.0%. The absorbance (E.sub.430nm.sup.1%) of the 
reaction mixture was 0.155. 
EXAMPLE 4.about.6 
Completely the same procedure as that in Example 2 was conducted, excepting 
using, in place of methyl ethyl ketone, methyl isobutyl ketone, diethyl 
ketone or cyclohexanone, respectively, to obtain the following results. 
______________________________________ 
Yield of L- Color Absorbance 
Example No. ascorbic acid (%) 
(E.sub.430 nm.sup.1%) 
______________________________________ 
4(isobutyl ketone) 
91.6 0.160 
5(diethyl ketone) 
92.7 0.162 
6(cyclohexanone) 
92.4 0.170 
______________________________________ 
EXAMPLE 7 
To a mixture solvent of benzene (180 ml) and acetone (20 ml) was added 
2-keto-L-gulonic acid (content 91.2%, water content 8.4%)(32 g), to which 
was added trimethyloetylammonium chloride (140 mg), followed by further 
addition of 35% hydrochloric acid (10 ml)(d=1.17, 4.09 g as HCl). The 
water content in the reaction mixture was calculated to be 10.3 ml (3.8 
times as much in molar ratio relative to 2-keto-gulonic acid). The 
reaction mixture was stirred for one hour at 60.degree. C., then crystals 
of 2-keto-L-gulonic acid became oily, followed by subjecting the reaction 
mixture to distillation by means of a Soxhlet extractor to distill off 
about 8 ml as distillate (aqueous layer) under reduced pressure. It was 
revealed that the aqueous layer was composed of water (3.8 g), acetone 
(3.0 g) and HCl (0.8 g). 
The solution was stirred for 5 hours at the same temperature, which was 
then cooled to 20.degree. C. The reaction mixture was poured into water 
(300 ml), stirred and left standing, followed by separating the aqueous 
layer. To the organic layer was added water (200 ml) for re-extraction. 
The aqueous layers were combined and subjected to a high performance 
liquid chromatography to quantitatively determine the L-ascorbic acid 
produced to reveal that the compound was produced in an amount of 24.9 g 
(yield: 94.1%) and the 2-keto-L-gulonic acid remained unreacted in an 
amount of 4.6%. The absorbance (E.sub.430nm.sup.1%) of the aqueous layer 
was 0.170. 
EXAMPLE 8 
To a mixture solvent of benzene (90 ml) and acetone (8 ml) was added 
2-keto-L-gulonic acid (15 g)(content: 99.4%, water content: 0.5%), to 
which were added trimethylcetylammonium chloride (100 mg) and 35% 
hydrochloric acid (6 m,)(d=1.17, 2.46 g as HCl). The water content in the 
reaction mixture was calculated to be 4.6 g (3.3 times as much in molar 
ratio relative to 2-keto-gulonic acid). The reaction mixture was stirred 
for about 75 minutes at 60 to 65.degree. C. to give an oily 
2-keto-L-gulonic acid, which was then subjected to distillation under 
reduced pressure by means of a Soxhlet extractor to eliminate about 2.5 ml 
(weight: 2.4 g) as distillate (aqueous layer). It was revealed that the 
aqueous layer was opposed of water (1.4 g), acetone (0.8 g) and HCl (0.1 
g). The solution was stirred for 3 hours at the same temperature, which 
was cooled to 30.degree. C., followed by collecting the resulting 
precipitates by filtration. The precipitates were washed with a small 
volume of benzene, then dried under reduced pressure to obtain a grayish 
crude L-asoorbic acid (13.4 g) (purity: 94.2%, yield: 93.4%) This 
crystalline crude product contained 2-keto-L-gulonic acid (0.16 
g)(remaining ratio: 1%). This crude crystalline product was dissolved in 
water, and the absorbance (E.sub.430nm.sup.1%) of the aqueous solution was 
0.145. The mother liquor was subjected to extraction twice with 50 ml each 
portion of water. The aqueous layer was subjected to a high performance 
liquid chromatography to quantitatively determine the L-ascorbic acid to 
reveal that the yield was 0.23 g (1.7%), while the 2-keto-L-gulonic acid 
was detected in an amount of 0.36 g (remaining ratio 2.4%). 
EXAMPLE 9 
To a mixture solvent of toluene (120 ml) and acetone (10 ml) in which was 
dissolved hydrogen chloride (3 g) was added 2-keto-L-gulonic acid (purity: 
91.2%, water content: 8.4%)(30 g) to which was added 
trimethylcetylammonium chloride (300 mg), then the whole mixture was 
heated to 65.degree. C. To the reaction mixture was added 35% HCl (3 
ml)(d=117, 1.23 g as HCl). The water content in the reaction mixture was 
calculated to be 4.8 ml (1.89 times as much in molar ratio relative to 
2-keto-L-gulonic acid). The reaction mixture was stirred for about one 
hour, then about 1.5 ml (weight: 1.3 g) of water layer was distilled off 
under reduced pressure. The water layer was composed of water (0.3 g), 
acetone (0.9 g) and HCl (0.1 g). The reaction mixture was stirred for 3 
hours at the same temperature, which was then cooled to 30.degree. C. or 
below, followed by extraction twice with 300 ml each portion of water. The 
aqueous layers were combined and subjected to a high performance liquid 
chromatography to find that L-ascorbic acid was produced in an amount of 
23.5 g (yield: 94.9%), while the 2-keto-L-gulonic acid remained unreacted 
in an amount of 0.4 g (remaining ratio: 1.5%). The absorbance 
(E.sub.430nm.sup.1%) of the extract solution was 0.19. 
EXAMPLE 10 
To a mixture solution of toluene (580 ml) and acetone (30 ml) dissolving 
therein hydrogen chloride (11.1 g) was added 2-keto-L-gulonic acid 
(content: 89.9%, water content: 8.4%), (100 g), to which were further 
added 35% hydrochloric acid (11.8 g)(d=1.17, 4.1 g as HCl) and 
trimethylcetyl ammonium chloride (0.12 g). The water content in the 
reaction micture was calculated to be 16.1 ml (1.93 times as much in molar 
ratio relative to 2-keto-L-gulonic acid). The reaction mixture was stirred 
for six hours at 60.degree. C., which was then cooled to 20.degree. C. and 
poured into water (1 l). The whole mixture was stirred, which was then 
left standing, followed by separating the aqueous layer. To the organic 
layer was added water (400 ml) for re-extraction. The aqueous layers were 
combined and subjected to a high performance liquid chromatography for 
quantitative determination to find that L-ascorbic acid was produced in an 
amount of 76.6 g (yield: 93.9%), while the 2-keto-L-gulonic acid was 
remianed unreacted in an amount of 1.4%. The absorbance 
(E.sub.430nm.sup.1%) of this aqueous extract was 0.168. 
EXAMPLE 11 
To a mixture solvent of toluene (580 ml) and acetone (110 ml) dissolving 
therein hydrogen chloride (11.1 g) was added 2-keto-L-gulonic acid 
(content: 89.9%, water content: 8.4%) (100 g), to which were added 35% 
hydrochloric acid (11.8 g) (d=1.17, 4.1 g as HCl) and 
trimethylcetylammonium chloride (0.12 g). The water content in the 
reaciton mixture was calculated to be 16.1 ml (1.93 times as much in molar 
ratio relative to 2-keto-L-gulonic acid). The reaction mixture was heated 
to 60.degree. C. and stirred for six hours, which was cooled to 20.degree. 
C. and poured into water (1 l). The reaction mixture was stirred and left 
standing, followed by separation of the aqueous layer. To the organic 
layer was added water (400 ml) for re-extraction. The aqueous layers were 
combined. The L-ascorbic acid produced was quantitatively determined by 
means of a high performance liquid chromatography to find that the 
compound was produced in an amount of 74.6 g (yield: 91.5%), while 
2-keto-L-gulonic acid remained unreacted in an amount of 2.5%. The 
absorbance (E.sub.430nm.sup.1%) of this aqueous extract was 0.135. 
COMATIVE EXAMPLE 1 
In benzene (180 ml) was suspended 2-keto-L-gulonic acid (content: 91.2%, 
water content: 8.4%) (32 g), to which was added trimethylcetylammonium 
chloride (140 mg) and then was added 35% hydrochloric acid (10 ml)(d=1.17, 
4.09 g as HCl). The water content in the reaction mixture was calculated 
to be 10.3 ml (3.8 times as much in molar ratio relative to 
2-keto-L-gulonic acid). 
The reaction mixture was heated to 60.degree. C., which was then stirred 
for about one hourlthen crystals of 2-keto-L-gulonic acid became oily, 
followed by distilling off water layer (ca. 8 ml) by means of a Soxhlet 
extractor under reduced pressure. The residue was stirred for further 5 
hours at the same temperature, which was then cooled to 30.degree. C. The 
reaction mixture was poured into water (300 ml) which was left standing, 
followed by separating the aqueous layer. To the organic layer was added 
water (200 ml) for re-extraction. 
The aqueous layers were combined and subjected to a high performance liquid 
chromatography for quantitative determination of L-ascorbic acid to reveal 
that the compound (22.1 g) (yield: 83.5%) was present, while the 
2-keto-L-gulonic acid remained unreacted (1.3%). The absorbance 
(E.sub.430nm.sup.1%) of this reaction mixture was 0.45. 
COMATIVE EXAMPLE 2 
In toluene (80 ml) was suspended 2-keto-L-gulonic acid (content: 92.1%, 
water content: 8.3%)(20 g), to which was added hexadecyl trimethylammonium 
chloride (200 mg), and the mixture was heated to 65.degree. C. To the 
mixture was further added 35% hydrochloric acid (1.2 ml), followed by 
introducing hydrogen chloride for 150 minutes at a rate of 80 ml/min. The 
solvent was then distilled off under reduced pressure at temperatures not 
exceeding 30.degree. C. To the residue was added toluene (100 ml), and the 
mixture was sufficiently stirred, followed by distilling off the solvent 
under reduced pressure at temperatures not exceeding 30.degree. C. To the 
residue was again added toluene (100 ml), and the mixture was sufficiently 
stirred, followed by cooling to temperatures not exceeding 20.degree. C. 
Resulting precipitates were collected by filtration, washed with a small 
volume of toluene, and then dried under reduced pressure. 
The product was dissolved in water (300 ml), which was subjected to a high 
performance liquid chromatography for quantitative determination of the 
L-ascorbic acid produced to reveal that the compound was present in an 
amount of 14.1 g (yield: 84.4%), while no 2-keto-L-gulonic acid was 
detected The absorption (E.sub.430nm.sup.1%) of ths aqueous solution was 
0.47. 
The mother liquor was concentrated to dryness under reduced pressure, and 
the residue was dissolved in water (300 ml). Qauntitative determination of 
ascorbic acid by means of a high performance liquid chromatogrpahy 
revealed the existence of the compound (0.18 g)(yield: 1.1%). 
COMATIVE EXAMPLE 3 
To toluene (552 ml) was added 2-keto-L-gulotic acid (content: 91.2%, water 
content: 8.4%)(92.4 g). To the mixture was added trimethylcetyl ammonium 
chloride (100 mg), to which was further added 35% concentrated 
hydrochloric acid (9.4 ml)(d=1.17, 3.85 g as HCl, 7.1 g as water). The 
volume of water contained in the reaction mixture was calculated to be 
14.9 g (1.90 times as much in molar ratio relative to 2-keto-L-gulonic 
acid). Into the reaction mixture was blown, while heating at 60.degree. 
C., hydrogen chloride (10.4 g) taking six hours, followed by cooling to 
20.degree. C. (the total acid amount relative to 2-keto-L-gulonic acid was 
0.9 times as much in molar ratio) The amount of L-ascorbic acid produced 
by processing the reaction mixture in a manner similar to Example 1 was 
found to be 30.6 g (yield: 40%) by a high performance liquid 
chromatography for quantitative determination The amount of 
2-keto-L-gulonic acid remaining unreacted was 46.3 g (remaining ratio 
55%). The absorbance (E.sub.430nm.sup.1%) of this extract solution was 
0.12. 
COMATIVE EXAMPLE 4 
To a mixture solvent of toluene (550 ml) and acetone (40 ml) was added 
2-keto-L-gulonic acid (content: 91.2%, water content: 8.4%)(92.4 g), to 
which was then added trimethylcetyl ammonium chloride (100 mg), followed 
by further addition of 35% conc. hydrochloric acid (11 ml)(d=1.17, 4.55 g 
as HCl, 8.45 g as water). The reaction mixture was heated to 60.degree. C. 
and stirred for one hour, followed by addition of an acetone solution (200 
g)(containing 9.55 g of hydrogen chloride). The whole mixture was stirred 
for 5 hours at the same temperature, then cooled to 20.degree. C. (the 
total acid amount relative to 2-keto-L-gulonic acid was 0.9 times as much 
in molar ratio) 
The reaction mixture was processed in a manner similar to that of Example 1 
to produce L-ascorbic acid, which was subjected to quantitative 
determination by means of a high performance liquid chromatography to find 
that the amount of the compound was 64.9 g (yield: 84.9%). The amount of 
2-keto-L-gulonic acid remaining unreacted was 8.4 g (remaining ratio: 
10.0%). The absorbance (E.sub.430nm.sup.1%) of this extract solution was 
1.5.