Measurement of the 2H/1H Ratios of the Carbon Bound Hydrogen Atoms in Sugars

John Dunbar and H.-L. Schmidt

Lehrstuhl fiir Allgemeine Chemie und Biochemie, Technische Universitfit Mfinchen, D-8050 Freising-Weihenstephan, Federal Republic of Germany

Bestimmung der 2H/1H-Verhiiltnisse kohlenstoffgebundener Wasserstoffatomc in Zuckern

Zusammenfassung. Das 2H/1H-Isotopenverhfiltnis von Zuckern ist yon deren Vorbehandlung abhfingig, da die an Sauerstoff gebundenen Wasserstoffatome rasch und unkon- trollierbar mit Wasser austauschen. Die von solchen Proben erhaltenen 6D-Werte sind deshalb nicht reprfisentativ ffir den D-Gehalt des bei der Photosynthese gebundenen Wasser- stoffs. Mehrere Verfahren f/Jr die Substitution dieser Wasser- stoffatome werden erprobt; als geeignet erweist sich die Herstellung der Salpetersfiureester der Zucker. Das ent- wickelte ,,Nitrierungsverfahren" verl/iuft quantitativ, und bei den am Kohlenstoff gebundenen Wasserstoffatomen treten keine Austauschreaktionen auf. Die Anwendung ffir die direkte Nitrierung des Restzuckers in Wein erweist sich gleichzeitig als M6glichkeit, die Zucker leicht von den anderen Komponenten in einem Lyophilisat abzutrennen. Die Reproduzierbarkeit ffir die D-Bestimmung des an C gebundenen Wasserstoffs ist 1,9%o .

Summary. Problems are encountered with the measurement of 2H/1H ratios in sugars because of the rapid and uncon- trolled rate at which the hydroxyl hydrogen atoms can exchange with water. The values thus obtained may not be representative of the hydrogen bound during photosynthesis. A method for the removal of these hydroxyl hydrogen atoms through replacement with NO 2 groups has thus been de- veloped. This "nitration" process has been shown to be quantitative and no disturbance of the remaining hydrogen atoms through exchange reactions was detected. The direct nitration of the residual sugars in wine is also possible using this method as, when in the nitrated form, the sugars can be easily separated from the other components present. The reproducibility for the measurement of the 2H/all ratios of the carbon bound hydrogen was 1.9 ~ o.

Introduction

Because of the expanding interest in the measurement of natural stable isotope ratios in biological compounds a method for the measurement of 2H/1H ratios of the non- exchangeable hydrogen atoms in sucrose and also in glucose/fructose mixtures isolated from wine is needed.

Offprint requests to." H.-L. Schmidt

The measurement of 2H/1H ratios of sugars has been reported in literature [5 - 7, 15], although always on the total hydrogen present. It is known however that hydroxyl hy- drogen atoms are able to readily exchange with water [3], therefore the values reported are not truely representative of the hydrogen bound during photosynthesis. It is thus neces- sary to replace these hydrogen atoms with non-hydrogen con- taining moieties, for example with - N O 2 or -O(CO)CF3 groups. Various methods for the nitration of sugars have been reported although these have not been in connection with isotopic studies [8, 11, 13, 14].

For the measurement of the non-exchangeable hydrogen atom D/H ratios in cellulose (a glucose polymer), a nitration method is used [4, 9, 10]. Two different nitration reagents have been reported (nitric-phosphoric acid, nitric acid-acetic anhydride) with no significant difference between them having been found [17]. In this paper different methods for the replacement of the exchangeable hydrogen atoms in sugars have been investigated and the reproducibility under different conditions reported.

Methods and Materials

Preparation and Combustion of the Nitro Derivatives

Several of the nitration methods previously mentioned [8, 11, 13] were attempted but the one that led to the most satisfactory results was that using a mixture of nitric and acetic acids with acetic anhydride [13]. This was prepared by first cooling 50 ml of fuming HNO3 (99 % purity) to 0 ~ C, then adding a mixture of 25ml CH3COOH (100%) and 25ml (CHaCO)20 (redistilled) that was also at 0~ As heat was generated during mixing, the system was cooled and the temperature not allowed to rise above + 20~ At higher temperatures an extremely vigorous reaction can occur which may be explosive.

Sucrose samples were first crushed to a fine powder whereas the wine samples were first lyophilised to remove water and alcohol, then the total residue nitrated (the justification for this will be given in the results section). The reaction mixture was then added to the sugar samples in a 30-fold excess (by weight) and allowed to react for approx- imately 4 h after the sample had dissolved. The temperature during this time was kept at approximately + 5 ~ C. After the reaction was complete the mixture was poured into ice water where the sugar derivatives precipitated. This precipitate was washed with water until all traces of the acid were removed, then dried in vacuum for 12 h. The samples were then stored

Fresenius Z Anal Chem (1984) 317:853-857 �9 Springer-Verlag 1984

bubble counter

! I~

Bunsen burner

cold trap I hydrogen

i . pump ~ gas rubber s e p ~ ~ bottle

vacu~ : z ~ J u"u ~ H g _ 2 n ~

cold trap 2 Fig. 1. Combustion system

in a desiccator with phosphorous pentoxide under vacuum for four days at + 5~ (to prevent decomposition) to ensure complete removal of any water present. In all cases the dried "ni t rosugar" was a clear, colourless or slightly yellow, sticky semisolid mass.

The derivatives could then be combusted in the system shown in Fig. J. In the case of pure sugar (non-nitrated) a CuO filling at 800~ was used in the combustion tube and the sample burnt in a 20 ~ O 2 / 8 0 ~ N2 atmosphere. Fo r the nitrated derivatives, however, a Cu filling at 600~ was used (to remove nitrogen oxides, being principally NO2) and the combustion carried out in a pure N2 atmosphere. In both cases the system was initially flushed with N 2 for 5 min to remove any water vapour that may have entered the com- bustion tube during loading of the sample. The combustion was then carried out with an ethanol bath at approximately - 1 0 0 ~ around trap i in order to collect the H20 (but not the CO2) produced.

A controlled burning of the derivative was not possible even in the absence of oxygen as the sample was relatively unstable (octanitro-sucrose: melting point + 8 1 ~ deto- nation temperature + 170 ~ C, pentanitroglucose: melting point + 54 ~ C, detonation temperature + 250 ~ C). Therefore, during combustion of the nitro derivatives tap 3 was closed to prevent any water being absorbed by the phosphorous pentoxide drying tube at the time of explosion. As soon as the explosion had occurred the tap was reopened and the flow of gas continued. At this stage a small amount of oxygen was mixed into the nitrogen stream and the combustion tempera- ture increased. It was impor tant that as little oxygen as possible was used in order not to reduce the life of the Cu filling. The system was then flushed for a further 5 rain with Nz to ensure that all of the H20 produced was transferred into trap 1.

Sample sizes were chosen in order to produce approx- imately 8 lal H20. The necessary quantities were therefore:

pentanitroglucose: 50 rag, pentanitrofructose : 50 rag, octanitrosucrose : 45 rag, nitrosugar from wine : 50 mg (a mixture of pentanitroglucose and pentanitrofructose); sucrose: 13 mg.

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The water collected in trap 1 was then transferred into trap 2 (N2 carrier gas first pumped away) where it was then allowed to react with uranium turnings (U. AF002000, Soc. Ind. de Combust ible Nucl6aire, Veurey, France) a t 800 ~ C. To assist in this process a small pump was installed to circulate the hydrogen and unreacted water through the uranium oven. This was found to be necessary to achieve reproducible 2H/1H ratios.

Also built into the reduction system was a rubber septum for the injection of water samples where the VSMOW standard (for the definition of VSMOW see later section) could be introduced directly into the oven. A mercury manometer monitored the H 2 pressure present in the system (typically 100 ~) and could be used to determine the reaction endpoint. After approximately 8min the pressure in the system was constant, however it was found necessary to wait another 5 rain until a stable isotopic equilibrium was reached. At this point tap 10 was closed and the H 2 allowed to expand into the evacuated sample bottle (volume 100 ml). Because of the sample sizes used (8 gl), it was not necessary to use a Toepler pump to obtain an increase in pressure in the sample bottle. The 2H/1H ratio could then be measured with the mass spectrometer.

Exchange of the Hydroxyl Hydrogen Atoms with Standard Water

2gin of sucrose was dissolved in 50ml of s tandard water which was then equilibrated at 25~ for 2 h. The solution was subsequently quickly frozen in a methanol cold bath at - 60 ~ C, then the water removed by lyophilisation. This process was repeated twice to ensure complete removal of the original sugar hydroxyl hydrogen atoms, then the samples were stored in a desiccator under vacuum with phosphorus pentoxide. Combust ion was carried out as previously described.

Preparation and Combustion of Trifluoroacetic Acid Derivatives

The fluoro derivative was prepared by mixing 60 mg of sucrose with 1 ml of trifluoroacetic acid anhydride at 40 ~ C in a pressure resistant test tube equipped with a screw cap. Depending on the crystal size of the sample, the reaction was complete after between 2 and 5 h at this temperature (yield determinations showed more than 99 ~ substitution). The volatile reaction products (CF3COOH/unreac ted tr if luoroacetic acid an- hydride) were removed under vacuum in a rotary evaporator at approximately 90 ~ C.

Combust ion of the derivative was carried out in a multi- component/mult i - temperature combustion tube. This con- tained CeO2 and CuO at 800 ~ C followed by PbCrO4, Cu and Ag wool at 600 ~ C. The CeO2 was used in order to remove H F produced during the combustion which was carried out with a mixture of 80 ~ He and 20 ~ 02. The tube was flushed after the combustion with pure He.

The collection of the water produced and its subsequent reduction to H 2 gas was carried out as previously described.

Inversion of Sucrose

25 ml of a J2 ~ sucrose solution was heated with 2 ml of conc. HC1 for 10 min in a water bath at 6 7 - 70 ~ C. The solution was then quickly cooled and neutralised. Using this method only glucose and fructose, i.e. no side products, are formed [1].

Enzymatic Measurement of Acetate Concentrations

These measurements were made following the method of Bergmeyer [2]. The relevant equations are: Sample

CH3COOH + ATP ~ce~ ki,~%~ CHaCOOPOaH 2 + A D P

CH3COOPO3H 2 + HzNOH--+ C H 3 C O N H O H + H3PO 4. 1

The enol form of the acetohydroxamic acid forms a 2 red/red-violet complex with Fe(III) salts in acid conditions 3 which can be measured between 436 nm and 546 nm. 4

Initially, the bound acetyl groups were freed from the 5 derivative by treatment with K O H in methanol. The pH was 6 adjusted to 8 with HC1 and the amount of acetate thus formed 7 determined as outlined above.

Measurement of Isotopic" Ratios

All isotope ratios were measured on hydrogen gas with a VG- Micromass 902 isotope rat io mass spectrometer. A gas s tandard was produced by direct reduction of 8gl of the international water s tandard V S M O W (Vienna Standard Mean Ocean Water). To ensure accuracy, a further two aliquots of the water s tandard were reduced and used for comparison against the first.

Analyses are reported in the 6-notation, where:

= 1 000%0 (2H/1H)vsMo w

All samples were measured in triplicate, i.e. the com- bustion, reduction and isotopic determination steps were repeated three times.

Results

Three methods for the removal/replacement of the hydroxyl hydrogen atoms in various sugar samples have been exam- ined. Fo r the initial evaluation of these methods a series of identical sucrose samples were prepared and the results obtained compared.

a) Exchange with standard water: It was hoped that the 2H/1H ratio of the carbon bound hydrogen atoms in sugar could be calculated from the 2H/1H ratio of the total hydrogen present if the hydroxyl atoms could be replaced with hydrogen atoms of a known 2H/1H ratio, i.e. after exchange with s tandard water. While the 6D values of the corresponding nitric acid esters and even those of the non- equil ibrated sugars were within relatively narrow limits (Table 1), this was not the case after equilibration with the water s tandard, where a rather large spread in the 6 values was observed. As the exchange between the hydroxyl atoms and the hydrogen atoms of water is in the order of milliseconds [16], it would be impossible to freeze the sugar/s tandard water solution quickly enough prior to lyophilisation in order to obtain a 25~ equilibrated hydroxyl hydrogen a tom in the sucrose molecule. In addition, uncontrolled deuterium en- richments could take place during lyophilisation in the remaining water. The deuterium content of the exchangeable hydrogen seems to be more a function of the lyophilisation procedure rather than that of the s tandard water; thus the development of this technique was taken no further.

b) Trifluoroacetic acid derivatives: SbF 5 and other fluorine compounds are used for the measurement of oxygen

Table 1. Comparison of the 2H/1H ratios of sucrose after pretreat- ment of the hydroxyl hydrogen atoms

Pure H20 Nitrated c~D ~ o exchanged a ~D ~ 0

6D %o

-79.4 -48.8 - 125.4 -85.3 - 12.4 - 121.2 - 73.2 - 37.5 - 123.8 -75.1 -16.1 -120.1 -82.3 -40.7 -130.4 -64.3 -21.4 - 124.7 - 71.0 - 30.3 - 126.4

2 = -75.8 2 = -29.4 2 = -124.6 or= 7.2 a = 13.5 a = 3.4

a After exchange with water with a 6D value of -65.6%0

isotope ratios in inorganic oxides [12], however previous work (unpublished results) has shown that from organic com- pounds COF2, H F and other fluorine containing compounds are produced. The most stable hydrogen containing com- pound at high temperature in a system containing H, C, O and F must be H F however this compound was not considered to be suitable for direct mass spectrometry. Reaction with CeOz should convert H F to an equivalent amount of water. Experimentally the format ion of water was found to occur (after combustion via the procedure described under "Methods and Materials"), however no acceptable reprodu- cibility of the 2H/1H ratios could be obtained. Deviations in ratio between measurements of an identical sample were as much as 50%0 , indicating that for this work the method was not suitable. These deviations were probably due to the formation of hydrogen containing side products during the combustion which resulted in a loss of hydrogen from the system with a subsequent isotopic fractionation.

c) Nitric acid derivatives: All the values of the nitrated sugars are within a narrow range and could be reliably reproduced. As the nitrat ion method has already been reported in literature for the measurement of 2H/1H ratios in cellulose [9, 10], and as it is technically relatively simple to apply, it was used for both the sucrose and the sugar samples from wine (glucose + fructose) that were studied.

The following experiments were carried out in order to determine the optimal parameters as well as the limits of accuracy of the method.

I) Completeness of Reaction

This experiment had a double function in that it was a test for hydroxyl groups remaining in the sugar derivative as well as a test for residual water. As water contains a large mole fraction of hydrogen its presence represents a large hydrogen contamination, hence possible error in the 2H/1tt ratios of the derivatives.

Addit ionally, tests were carried out to determine if acetyl groups (or alternatively free acetic acid) were present in the derivative. Because of the extreme conditions under which the nitration was carried out, it is possible that a small amount of acetylation could occur resulting in the introduction of foreign hydrogen into the derivative. The presence of free acetic acid would indicate incomplete washing of the de- rivative after reaction in the nitrating mixture.

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Table 2. Determination of the completeness of the reaction

Water/hydroxyl groups Atom ~ ~ HzO/OH groups ~ H contamination e

1) Octanitrosucrosr 6D a = - 117.6 ~ o 0.013774 0.17 0.95 6D b = + 981.7~ 0.030867

2) Pentanitroglucose/fructose 6D ~ = -77 .7%o 0.014365 0.56 3.6 6D d = + 1722.2~ 0.042401

Acetic acid/acetyl groups Calculated as ~o Contamination ~ H contamination

Octanitrosucrose (after KOH treatment) i) free acetic acid 0.49 1.6 ii) acetyl groups 0.48 1.2

" Before treatment with 10~ D20 b After treatment with 10 ~ D20

Before treatment with 5 ~ D20

d After treatment with 5 ~ D20 Hydrogen contamination calculated as HzO

The test for hydroxyl groups/water was carried out with a sucrose derivative as well as a glucose/fructose mixed derivative from wine, whereas the test for acetyl groups/free acetic acid was performed only with a sucrose derivative.

The presence of hydroxyl groups or free water can be detected by measuring the 2H/1H ratio of the derivative before and after 2h contact with deuterated water. If hydroxyl groups remain in the derivative or if water is present a large increase in the 2H/1H ratio should be observed. The results of both tests are shown in Table 2.

The first section of Table 2 indicates that fewer hydroxyl groups/residual water are present in the octanitroglucose than in the pentanitroglucose/pentanitrofructose mixture. In the last column (right hand side) the percentage of hydrogen contaminat ion (calculated as if present in the form of water) has been shown. This figure has been obtained from a calculation of hydrogen mole ratios in the water and in the derivative. In the case of the glucose/fructose derivative this was 3.6 ~ , which is acceptable when the natural biological variat ion that can occur between samples is considered.

The percentage of acetyl groups/acetic acid present, as well as the corresponding hydrogen contamination, are given in the second section of the table and are also considered to be sufficiently small enough to be neglected.

II) Test for Hydrogen Exchange During Derivatisation

Because of the extreme reaction conditions present during nitration, it is possible that exchange between the carbon bound hydrogen atoms and the medium could take place. As a test for this, sucrose, fructose, glucose and the sugar extracted from wine were nitrated normally and then in the presence of a small amount of deuterated water. I f exchange takes place, an increase in the 2H/1H ratio of the derivative should be observed. DEO (99 ~ purity) was added to the nitrating mixture at the rate of 250 lal per 100 ml. It was not possible to use more than this because through hydrolysis of acetic acid anhydride the addi t ion of water causes a reduction in the yield of derivative obtained. The results of this experiment are shown in Table 3.

In the case of the fructose, glucose and the sugar from wine no enrichment of the derivative after nitrat ion in the presence of deuterated water has occurred within the limits of experimental error (for a discussion of the experimental

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Table 3. Test for hydrogen exchange during derivatisation

Sample Standard Nitration Difference nitration with D20 (normal-DzO) 6D %0 6D %o %0

Sucrose - 130.4 - 125.2 + 5.2 Fructose - 110.1 - 109.4 + 0.7 Glucose - 30.1 - 31.3 - 1,2 Wine sugar - 78.5 -75.9 +2.6

Table 4. Determination of the length of time necessary for complete derivatisation

Time Sucrose Fructose (h) 6D ~ 0 6D ~ o

1 - 133.0 - 109.0 4,5 -131.3 -109.7

28 -131.3 -110.7

errors see later section). In the case of sucrose a very small enrichment was observed, that can probably be at tr ibuted to incomplete washing of the derivative which was difficult because of its sticky semisolid form.

The results thus indicate that for the compounds studied, no exchange of the carbon bound hydrogen atoms (via HeO) occurs during the derivatisation process.

III) Determination of the Length of Time Necessary for Complete Derivatisation

An experiment was carried out to determine the time necessary for complete derivatisation to occur. Sucrose and fructose samples were left in contact with the nitrating mixture for either 1, 4, 5, or 28 h after the sample had completely dissolved. The subsequent purification and com- bustion steps were as previously outlined. The results of these measurements are shown in Table 4. I t is clear that derivati- sation occurs quickly and that within the limits of experimen- tal error there is no difference in the result obtained after 1 h and that obtained after 28 h reaction time. To be sure of complete reaction however, 4 h were used.

IV) Inversion of Sucrose

As a further check that derivatisation occurs without any form of hydrogen exchange with either sucrose, fructose or glucose samples, and as proof that the glycosidic hydroxyl group is not involved in the nitration, the following experi- ment was carried out.

A sucrose sample was inverted to glucose and fructose as outlined in the "Methods and Materials" section. Nitro derivatives were prepared from samples taken before and after the inversion and the 2H/tH ratios measured. The su- crose had a 2H/1H ratio of - 134.1%o, whereas the resultant glucose and fructose mixture had a ratio of -133.4~ . Within the limits of experimental error there is no difference between the two values indicating that the method of derivatisation is suitable for all three sugars.

V) Isolation of Glucose and Fructose Nitric Acid Esters from Wine

The main components in wine are water, ethanol, sugars (fructose and glucose, although not necessarily in the ratio of 1:1), glycerine, organic acids (malic and tartaric) and the tannins (only at significant levels in red wines). By lyophilising a wine, the water, ethanol and the volatile aroma substances are easily removed leaving, in a white wine, a mixture of predominantly fructose, glucose, malic and tartaric acid and glycerine. As all of these compounds contain hydroxyl groups it is possible that they will all react during the nitration step, hence some method for their separation is necessary. It is fortunate that the solubilities of the nitrated forms of these compounds are different which therefore enables this to be carried out.

Trinitroglycerine and 2-nitromalic acid are soluble in both the nitrating reaction mixture and in water (trinitro- glycerine is soluble in water over + 5 ~ C), therefore can be separated from the pentanitroglucose/fructose during the water wash step (the glucose/fructose derivatives are water insoluble). It is difficult to determine if dinitrotartaric acid is formed because solid tartaric acid added to the nitrating mixture does not appear to dissolve. However, if this solid is removed and heated, it breaks down with the release of N O 2. Fleury et al. [11] reported that the nitrated form of tartaric acid is easily prepared, however that at room temperature it is unstable. In any case this solid, whether it be dinitrotartaric acid, tartaric acid or a mixture of the two, is soluble in water, hence can also be separated during the water washing step.

As a test of the completeness of the purification step, a mixture of fructose/tartaric acid, fructose/malic acid (in the ratio 30 % organic acid, 70 % fructose) and fructose alone were nitrated and purified as outlined in the "Methods and Materials" section and the 2H/1H ratios measured.

In all three cases the 6D values were the same. If the nitrated/partially nitrated organic acids remained as an impurity in the nitro fructose, a change in the 2H/IH ratio of the derivative would be expected. As this did not occur, it can be assumed that the acids have been completely removed.

VI) Reproducibility

The reproducibility of the experimental method for the measurement of 2H/1H ratios in water, sucrose and nitrated

sucrose has been determined with the respective values being: water 0 .8~ (four determinations), sucrose 0.7%o (seven determinations), nitrated sucrose 1.9 %o (six determinations).

The reason for the poor reproducibility of the derivative 6-value probably lies in the combustion step. As the burning of the sample is very rapid and to a large extent uncontroll- able, it is possible that some of the water produced is lost with the sudden flow of gas through the cold trap. If this water is isotopically fractionated, an error will be introduced into the measurement, hence a poor reproducibility will be obtained. For some samples a better standard deviation (under 1%o) was observed although the reason for this is at the moment unknown.

Discussion

The results presented indicate that through formation of the nitric acid ester the hydroxyl hydrogen atoms of certain sugars can be removed in order that the 2H/1H ratios of the carbon bound hydrogen can be measured. This "nitration" process has been shown to be quantitative and no disturbance of the remaining hydrogen atoms through exchange reactions was detected indicating that this method would be suitable for general application in the measurement of 2H/1H ratios of sugars.

Isotopic studies of the residual sugars in wine can be carried out because of the relative ease at which the pure "nitrated" form can be separated from the other components present. This could thus possibly lead to an isotopic method for the determination of the origin of this sugar in wine.

Acknowledgements. John Dunbar thanks the Deutscher Aka- demischer Austauschdienst for financal support throughout the course of this work.

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Received July 9, 1983

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