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Periodate oxidation analysis of carbohydrates
Arrc~lyrlcu
Chhiccr
Acta,
77 ( 1975) 269-273 Publishing Company.
:<:IElscvicr
Scientific
SHORT
COMMUNICATlON
Periodate
oxidation
analysis
HONDA.
of formic
KAZUAKI
KAKEWI
Pcrctrlr~* 41’ Pl~crrr~lrr~eicri~~trlSciewes. (Rcccivcd
18th August
- Printed
in The Ncthcrlnnds
of carbohydrates
Part II. P.m.r. determination SUSUMU
269 Amsterdam
und
acid liberated KlYOSHl
Osctkcr U~~iwrsify.
by oxidation
TAKIURA
To~~cytrr~~rr. Toyotrctktr.
Osoktr~lir
(Jtrpcrrr )
1974)
In the preceding Part’ a spectrophotometric method was reported for the selective determination of glyoxal in dialdchydc fragments. formed from glycosidcs by pcriodate oxidation. In a continuation of these analytical studies of periodate oxidation fragments. the determination of formic acid was studied. This volatile acid is formed by oxidation ofa-hydroxyaldehydes. vicinal trihydroxy compounds. and the corresponding amino derivatives. Sometimes it is also formed by oxidation which is not of the Malabrade type. The amount of formic acid liberated has been usually determined by titration with dilute alkali and indicators such as methyl red2 and bromocrcsol purple”. byt the use of this method is restricted to the cases where unbuffered systems are employed. and the possibility of the presence of other organic acids is excluded. The selective determination is based on the stoichiometric redox reaction between formic acid and mercury(I1) chloride. The rcsultunt mercury(I) chloride may be determined gravimetrically4, iodimetricallys, or calorimetrically”. Nevertheless, since some neutral fragments also rcducc.mercury( II) chloride. prior isolation of this acid by distillation is necessary to eliminate the interference from these reductants. This paper presents a simple direct method based on proton magnetic resonance spectroscopy, which makes it possible to determine selectively lO-IOO-Itmole quantities of formic acid in oxidation mixtures.
Itt.stntnterltutiort. P.m.r. spectra were obtained at 90 MHz in the frequencysweep mode at 35°C with a Hitachi R-22 spectrometer. which was equipped with a thermostatically controlled permanent magnet and was locked with the external ‘“F signal of trifluoroucctic acid. The sweep speed for signal observation was 1 Hz s-l. Under these conditions the average signal/noise ratio for a lOA2 M DzO solution of sodium 3-(trimetl~ylsilyl)-l-propanesulfonatc(TMS-PS-Na) was 75. Signals of formic acid and TMS-PS-Na( the internal standard) were integrated at a sweep speed of 8 Hz s -1 . Standard mixtwes.
To
procetlwv
a 0.2
M
for
D20
the ckterrnirwtior~
solution
of
qf
sodium
jbrrnic
trcid
iri pehxlntc~
metaperiodate
(0.50
ositlatiim
ml)
con-
SHORT
270
COMMUNICATION
taining TMS-PS-Na (5.0 jcmole), add a carbohydrate sample which is expected to liberate cn. 45 jtmole of formic acid by oxidation. and keep the resultant solution at 25°C on a thermostated wutcr bath. shielding from the light. Observe the p.m.r. spectrum applying a HI lield of 150 ~cgauss. The molar amount of formic acid is obtained as the product of the average value of the aldehydic-methyl proton-signalresponse ratios and the molar amount of the internal standard used. At least five determinations should be done for each sample for averaging.
The aldehydic proton in formic acid resonates at 8.23 p.p.m. as a singlet. In the spectra of period&c oxidation mixtures obtained from carbohydrate samples. this signal is well isolated from those of other fragments. RS seen from a typical example of the oxidation mixture from methyl x-D-glucopyranoside (Fig. I). As indicated in Fig. 2 the signal response of this aldchydic proton increased with increasing H, levels to reach a plateau at 80 jcgauss. but irradiation with HI higher than 250 11 gauss caused a dccrcase in the response, owing to saturation of the nuclei. The curve for methyl protons in TMS-PS-Na (the internal standard) resembled that for the aldchydic proton except for the delay of arrival at plateau to 150 jcgauss. Therefore. the HI level was controlled at 150 /cgauss throughout this work,
p.2OOlI
---I _Ib
10
9
8
7
Fig. 1. OO-MHz p.m.r. spectrum glucopyrunosidc in D20.
6
of
liic
-
I
I
I
s
.:
pcriod:ltc
3
oxidation
2
mixture
1
obtained
n
Pl’fJ’
from
mclhyl
z-D-
The abundance of both protons. aldehydic and methyl. was estimated by the signal integration method. When the integration was performed at the maximum sensitivity. the highest reproducibility was obtained for ;I sweep speed of 8 Hz s-l. Under these conditions, the calibration curve was shown to be linear for 10-100 pmoles of formic acid; the relaiive signal response increased from about 0.22 to about 2.22. For this curve. formic acid was added directly to the pcriodate mixture. Table I indicates that this method is accurate and reproducible for various lcvcls of sample amounts. On the basis of the obscrvatlons mentioned above. the standard procedure
SHORT
COMMUNICATION
0
271
200
100
"I
SOD
4 0 (1
,I~clllSR
ICVCl
Fig. 2. Rclotionship bctwccn HI lcvcl and signill rcsponsc. (0) The conccntrution of both kinds of protons was 1.0~ IO-’ ,%I. TABLE
(0)
CHI
in TM%PS-Nil.
I
REPRODUCIBILITY (10 dctcrminntions
IO 50 loo
HCOOH:
OF THE
P.M.R.
DETERMINATION
OF FORMIC
ACID
were done ror each Icvcl.)
9.9 SO 99
-
0.41 I.6 2.3
--
___--.-.~--
devised for the determination of formic acid liberated from carbohydrates by periodate oxidation. Table II summarizes the results obtained from the oxidation of selecte’d carbohydrates. For alditols having different numbers of carbon atoms (erythritol. D-xylitol and D-mannitol), as well as glycosides (methyl cc- and P-Dglucopyranosidcs). the amounts of formic acid as determined by this method w&e in good agreement with those determined by alkalimetry2. It is noticeable that the spectra of the oxidation mixtures obtained from D-arabinose and D-glucose gave a minor singlet at 8.17 p.p.m.. ascribable to the formyl ester proton, along with the major singlet at 8.23 p.p.m. of the aforementioned aldehydic proton in free formic acid., Since the values obtained by alkalimetry lay between those of free and total formic acid as determined by the p.m.r. method, partial hydrolysis of resultant formyl esters should be involved during titration with alkali. Alkalimetry is, therefore, considered to be unsuitable for the determination of formic acid liberated from such aldoses. except when only the total formic acid is to be determined after the formyl esters have been hydrolyzed by e.g. heating. Ketoses, aldonic acids. and uranic acids are groups of compounds the liberation of formic acid from which cannot be determined simply by alkalimetry. was
SHORT
272 TABLE
COMMUNICATION
JJ
LJBERATJON OXIDATION
OF --.
FORMIC
ACJD
FROM
SELECTED
Forr~ric trcici iihcwtteci --.. Pmr. .-
Erythritol D-Xylitol D-Mnnnitol Methyl r-D-pl~~copyrnnosido Methyl /I-D-glucopyrnnosidc DL-Glyccrnltlchydc D-Arabinosc D-Glucose D-Fructose L-Sorbosc D-Gluconic acid 8-lactonc D-Galacturonic ticid D-Glucuronic ncid
BY
PERJODATE
-_
.-
-
CARBOHYDRATES
(lll~JiC?/l,lCJi~~)
~wtiiotl
Aikaiirwtr_v ---.
lh
3 ir
24 /I
J it
3 II
24 ir
2.9 2.9 4.0 0.73 0.73 1.2 2.20 3.1* 2.6” 3.6h I.1 I.2 I.5 4.1 3.9
2.0 3.0 4.0 0.90 0.88 1.5 2.3” 3.3h 3.2” 4.1* I.2 1.4 2.3 4.1 4.0
2.0 2.9 4.0 0.94 0.93 2.0 3.2” 3.gh 4.1” 4.6h 1.x 2.2 2.6 4.2 4.0
1.98 2.97 3.98 0.74 0.73 1.19 2.90
2.01 3.00 4.01 0.88 0.86 I .47 3.03
2.00 2.99 4.00 0.93 0.92 1.90 3.58
3.28
3.79
4.20
I.81 1.91 2.87 5.62 5.39
1.Y6 2.15 3.47 5.64 5.49
2.67 3.10 3.84 5.47 5.40
---
” Free lbrmic acid. I’ Total formic acid.
because glycolic or glyoxylic acid is expected to be formed along with formic acid in these cases. Indeed, the p.m.r. determination gave lower values of formic acid than alkalimetry. The differences between these values are considered to give the amounts of such carboxylic acids. From this consideration. the differences for both ketoscs (D-fructose and L-sorbose). corresponding to the, amounts of glycolic acid formed, were 0.7. 0.8. and 0.9 mole per mole of ketose after oxidation for 1 h. 3 h, and 24 h, respectively. However, D-gluconic acid Ci-lactone and two uranic acids (Dglucuronic and D-galacturonic acids) gave differences higher than 1 mole for unknown reasons. The mechanism of the periodate oxidation ofglyoxylic acid, presumed to be formed, should be clarified in future studies.
In the spectra of oxidation mixtures, aldchydic protons in free and esterified formic acid resonate as singlets at 8.23 and 8.17 p.p.m.. respectively, isolated from other signals. From the response of these signals l O-IOO-Itmole amounts of formic acid in both forms can be determined rapidly and simply. REFERENCES I S. Honda
K. Knkchi. I-I. Yuki. and K. Tukiurn. clrrtri. Ciaiw dvfrr. submitted.
SHORT 2 3 4 5 6
COMMUNICATION
E. L. Hirst and J. K. N. Joncs. .I. C’hw. Sot.. Lomht. 73 (1949) 1659. M. Morrison, A. C. Kuypcr and J. M. Ortcn. .I. Amv. C/rem Sot.. 75 (1953) J. R. Dyer. Mcrlrot/s Bloc*/wr~~. Am/.. 3 (1956) I I 1, W. J. Hopton. AII~/. Chirrt. Actu. 8 ( 1953) 429. W. M. Grunt. Arurl. Chcwr., 19 ( 1947) 206.
273 1502.
Chhiccr
Acta,
77 ( 1975) 269-273 Publishing Company.
:<:IElscvicr
Scientific
SHORT
COMMUNICATlON
Periodate
oxidation
analysis
HONDA.
of formic
KAZUAKI
KAKEWI
Pcrctrlr~* 41’ Pl~crrr~lrr~eicri~~trlSciewes. (Rcccivcd
18th August
- Printed
in The Ncthcrlnnds
of carbohydrates
Part II. P.m.r. determination SUSUMU
269 Amsterdam
und
acid liberated KlYOSHl
Osctkcr U~~iwrsify.
by oxidation
TAKIURA
To~~cytrr~~rr. Toyotrctktr.
Osoktr~lir
(Jtrpcrrr )
1974)
In the preceding Part’ a spectrophotometric method was reported for the selective determination of glyoxal in dialdchydc fragments. formed from glycosidcs by pcriodate oxidation. In a continuation of these analytical studies of periodate oxidation fragments. the determination of formic acid was studied. This volatile acid is formed by oxidation ofa-hydroxyaldehydes. vicinal trihydroxy compounds. and the corresponding amino derivatives. Sometimes it is also formed by oxidation which is not of the Malabrade type. The amount of formic acid liberated has been usually determined by titration with dilute alkali and indicators such as methyl red2 and bromocrcsol purple”. byt the use of this method is restricted to the cases where unbuffered systems are employed. and the possibility of the presence of other organic acids is excluded. The selective determination is based on the stoichiometric redox reaction between formic acid and mercury(I1) chloride. The rcsultunt mercury(I) chloride may be determined gravimetrically4, iodimetricallys, or calorimetrically”. Nevertheless, since some neutral fragments also rcducc.mercury( II) chloride. prior isolation of this acid by distillation is necessary to eliminate the interference from these reductants. This paper presents a simple direct method based on proton magnetic resonance spectroscopy, which makes it possible to determine selectively lO-IOO-Itmole quantities of formic acid in oxidation mixtures.
Itt.stntnterltutiort. P.m.r. spectra were obtained at 90 MHz in the frequencysweep mode at 35°C with a Hitachi R-22 spectrometer. which was equipped with a thermostatically controlled permanent magnet and was locked with the external ‘“F signal of trifluoroucctic acid. The sweep speed for signal observation was 1 Hz s-l. Under these conditions the average signal/noise ratio for a lOA2 M DzO solution of sodium 3-(trimetl~ylsilyl)-l-propanesulfonatc(TMS-PS-Na) was 75. Signals of formic acid and TMS-PS-Na( the internal standard) were integrated at a sweep speed of 8 Hz s -1 . Standard mixtwes.
To
procetlwv
a 0.2
M
for
D20
the ckterrnirwtior~
solution
of
qf
sodium
jbrrnic
trcid
iri pehxlntc~
metaperiodate
(0.50
ositlatiim
ml)
con-
SHORT
270
COMMUNICATION
taining TMS-PS-Na (5.0 jcmole), add a carbohydrate sample which is expected to liberate cn. 45 jtmole of formic acid by oxidation. and keep the resultant solution at 25°C on a thermostated wutcr bath. shielding from the light. Observe the p.m.r. spectrum applying a HI lield of 150 ~cgauss. The molar amount of formic acid is obtained as the product of the average value of the aldehydic-methyl proton-signalresponse ratios and the molar amount of the internal standard used. At least five determinations should be done for each sample for averaging.
The aldehydic proton in formic acid resonates at 8.23 p.p.m. as a singlet. In the spectra of period&c oxidation mixtures obtained from carbohydrate samples. this signal is well isolated from those of other fragments. RS seen from a typical example of the oxidation mixture from methyl x-D-glucopyranoside (Fig. I). As indicated in Fig. 2 the signal response of this aldchydic proton increased with increasing H, levels to reach a plateau at 80 jcgauss. but irradiation with HI higher than 250 11 gauss caused a dccrcase in the response, owing to saturation of the nuclei. The curve for methyl protons in TMS-PS-Na (the internal standard) resembled that for the aldchydic proton except for the delay of arrival at plateau to 150 jcgauss. Therefore. the HI level was controlled at 150 /cgauss throughout this work,
p.2OOlI
---I _Ib
10
9
8
7
Fig. 1. OO-MHz p.m.r. spectrum glucopyrunosidc in D20.
6
of
liic
-
I
I
I
s
.:
pcriod:ltc
3
oxidation
2
mixture
1
obtained
n
Pl’fJ’
from
mclhyl
z-D-
The abundance of both protons. aldehydic and methyl. was estimated by the signal integration method. When the integration was performed at the maximum sensitivity. the highest reproducibility was obtained for ;I sweep speed of 8 Hz s-l. Under these conditions, the calibration curve was shown to be linear for 10-100 pmoles of formic acid; the relaiive signal response increased from about 0.22 to about 2.22. For this curve. formic acid was added directly to the pcriodate mixture. Table I indicates that this method is accurate and reproducible for various lcvcls of sample amounts. On the basis of the obscrvatlons mentioned above. the standard procedure
SHORT
COMMUNICATION
0
271
200
100
"I
SOD
4 0 (1
,I~clllSR
ICVCl
Fig. 2. Rclotionship bctwccn HI lcvcl and signill rcsponsc. (0) The conccntrution of both kinds of protons was 1.0~ IO-’ ,%I. TABLE
(0)
CHI
in TM%PS-Nil.
I
REPRODUCIBILITY (10 dctcrminntions
IO 50 loo
HCOOH:
OF THE
P.M.R.
DETERMINATION
OF FORMIC
ACID
were done ror each Icvcl.)
9.9 SO 99
-
0.41 I.6 2.3
--
___--.-.~--
devised for the determination of formic acid liberated from carbohydrates by periodate oxidation. Table II summarizes the results obtained from the oxidation of selecte’d carbohydrates. For alditols having different numbers of carbon atoms (erythritol. D-xylitol and D-mannitol), as well as glycosides (methyl cc- and P-Dglucopyranosidcs). the amounts of formic acid as determined by this method w&e in good agreement with those determined by alkalimetry2. It is noticeable that the spectra of the oxidation mixtures obtained from D-arabinose and D-glucose gave a minor singlet at 8.17 p.p.m.. ascribable to the formyl ester proton, along with the major singlet at 8.23 p.p.m. of the aforementioned aldehydic proton in free formic acid., Since the values obtained by alkalimetry lay between those of free and total formic acid as determined by the p.m.r. method, partial hydrolysis of resultant formyl esters should be involved during titration with alkali. Alkalimetry is, therefore, considered to be unsuitable for the determination of formic acid liberated from such aldoses. except when only the total formic acid is to be determined after the formyl esters have been hydrolyzed by e.g. heating. Ketoses, aldonic acids. and uranic acids are groups of compounds the liberation of formic acid from which cannot be determined simply by alkalimetry. was
SHORT
272 TABLE
COMMUNICATION
JJ
LJBERATJON OXIDATION
OF --.
FORMIC
ACJD
FROM
SELECTED
Forr~ric trcici iihcwtteci --.. Pmr. .-
Erythritol D-Xylitol D-Mnnnitol Methyl r-D-pl~~copyrnnosido Methyl /I-D-glucopyrnnosidc DL-Glyccrnltlchydc D-Arabinosc D-Glucose D-Fructose L-Sorbosc D-Gluconic acid 8-lactonc D-Galacturonic ticid D-Glucuronic ncid
BY
PERJODATE
-_
.-
-
CARBOHYDRATES
(lll~JiC?/l,lCJi~~)
~wtiiotl
Aikaiirwtr_v ---.
lh
3 ir
24 /I
J it
3 II
24 ir
2.9 2.9 4.0 0.73 0.73 1.2 2.20 3.1* 2.6” 3.6h I.1 I.2 I.5 4.1 3.9
2.0 3.0 4.0 0.90 0.88 1.5 2.3” 3.3h 3.2” 4.1* I.2 1.4 2.3 4.1 4.0
2.0 2.9 4.0 0.94 0.93 2.0 3.2” 3.gh 4.1” 4.6h 1.x 2.2 2.6 4.2 4.0
1.98 2.97 3.98 0.74 0.73 1.19 2.90
2.01 3.00 4.01 0.88 0.86 I .47 3.03
2.00 2.99 4.00 0.93 0.92 1.90 3.58
3.28
3.79
4.20
I.81 1.91 2.87 5.62 5.39
1.Y6 2.15 3.47 5.64 5.49
2.67 3.10 3.84 5.47 5.40
---
” Free lbrmic acid. I’ Total formic acid.
because glycolic or glyoxylic acid is expected to be formed along with formic acid in these cases. Indeed, the p.m.r. determination gave lower values of formic acid than alkalimetry. The differences between these values are considered to give the amounts of such carboxylic acids. From this consideration. the differences for both ketoscs (D-fructose and L-sorbose). corresponding to the, amounts of glycolic acid formed, were 0.7. 0.8. and 0.9 mole per mole of ketose after oxidation for 1 h. 3 h, and 24 h, respectively. However, D-gluconic acid Ci-lactone and two uranic acids (Dglucuronic and D-galacturonic acids) gave differences higher than 1 mole for unknown reasons. The mechanism of the periodate oxidation ofglyoxylic acid, presumed to be formed, should be clarified in future studies.
In the spectra of oxidation mixtures, aldchydic protons in free and esterified formic acid resonate as singlets at 8.23 and 8.17 p.p.m.. respectively, isolated from other signals. From the response of these signals l O-IOO-Itmole amounts of formic acid in both forms can be determined rapidly and simply. REFERENCES I S. Honda
K. Knkchi. I-I. Yuki. and K. Tukiurn. clrrtri. Ciaiw dvfrr. submitted.
SHORT 2 3 4 5 6
COMMUNICATION
E. L. Hirst and J. K. N. Joncs. .I. C’hw. Sot.. Lomht. 73 (1949) 1659. M. Morrison, A. C. Kuypcr and J. M. Ortcn. .I. Amv. C/rem Sot.. 75 (1953) J. R. Dyer. Mcrlrot/s Bloc*/wr~~. Am/.. 3 (1956) I I 1, W. J. Hopton. AII~/. Chirrt. Actu. 8 ( 1953) 429. W. M. Grunt. Arurl. Chcwr., 19 ( 1947) 206.
273 1502.