Apiacta 1, 1972
DETERMINATION OF GLUCIDES BY G.L.C.
AND ITS POSSIBILITIES FOR HONEY QUALIFICATION
M.B. BATTAGLINI, G. BOSSI - ITALY
INTRODUCTION
Before the development of modern techniques of analysis, honey was considered as a saturated
solution of glucose and fructose including small amounts of sucrose.
Starting with 1952, the investigations conducted by German and American scientists showed that
this bee product is much more complex.
The first important investigation in this direction was conducted in the USA, in 1962, by Jonathan
White, who analyzed over 500 American honeys by column chromatography and reached the following
conclusion: glucose and fructose and the most important components (accounting for 80 to 90% of the total
amount of glucides); sucrose is little represented and maltose which was unknown for a long time, is present
in quantities worthy of consideration.
In 1955, Goldschmidt and Burckert isolated new superior sugars in honey: dextrantriose, erlose,
ketose, mellezitose and raffinose.
In 1959, Wite and Hoban identified isomaltose, turanose, maltulose, nigerose; in 1960, Watanabe
and Aso identified kojibiose and leucrose.
The presence of so great a number of sugars resulted in the formulation of different hypotheses as
to their origin.
Honey was belived to include enzymes secreted by the hypopharyngeal glands of worker bees and
of insects producing honeydew and capable to synthetize oligosaccharides. This hypothesis was confirmed
by the discovery of erlose by White and Maher (maltosil-fructoside) as an intermediary element in the
enzymatic reaction of transglucosidation. More recently Siddiqui and Furgala (1967) have discovered new
disaccharides and trisaccharides.
At present, the glucide part of honey is belived to be made up of 2 monosaccharides and at least 11
disaccharides and 12 tri-and-oligosaccharides:
Monosaccharides
Disaccharides
Polysaccharides
1. Fructose
2. Glucose
1. Maltose
2. Kojibiose
3. Turanose
4. Isomaltose
5. Sucrose
6. Maltulose
7. Isomaltulose
8. Nigerose
9. Trehalose
10. Gentibiose
11. Laminaribiose
1. Erlose
2. Panose
3. Maltotriose
4. Ketose
5. Isomaltotriose
6. Mellezitose
7. Isopanose
8. 6 α Glucosilsucrose
9. 3 α Isomaltosilglucose
10. Raffinose
11. Isomaltotetrose
12. Isomaltopentose
The presence of raffinose identified first by Goldschmidt and Burckert and then by Pourtallier is
doubtful in the opinion of Siddiqui and Furgala; the latter maintain that if raffinose existed then the
monosaccharide galactose should also be found; but traces of the latter were never found in honeys
analysed.
All this points out the complexity of the substance in question and also the fact that the classical
methods can no longer provide a systematic research on sugars of a great number of honeys.
This paper attempts to analyse a number of unifloral honey samples to determine whether it is
possible or not to characterize them according to their sugar content. That is why we preferred a rapid and
versatile method, namely the gas chromatography.
Several authors have already tested separation of glucides by using gas chromatography and the
well-known technical method of Sweeley, Bentley, Makita and Wels; we also mention Pourtallier (France),
who worked on honey and Capella and Losi (Italy), who worked on venoms.
Apiacta 1, 1972
MATERIAL AND METHOD
Reagents
- Inositol – 1% solution
- Anhydrous ethyl alcohol
- Anhydrous pyridine (distilled on KOH)
- Hexamethyl disilasane
- Trimethylchorosilane
Apparatus
-Rotative evaporator provided with a thermostatic bath with water
- Calibrated test tubes, 15 ml each, provided with stoppers screwing up on a Teflon gasket capable of
being adapted – through a special sleeve connection to the rotative evaporator
- Agitator
- Centrifuge
- Double columned Carlo Erba gas chromatograph provided with double F.I.D. detector and linear
temperature computer
- Stainless steel column (180m x 3mm)
- O.V. 17 stationary state at 3% on W.A.W. silaned chromosucker
- Column temperature: initial - 180ºC; final 290ºC, increasing by 3ºC per minute
- Temperature of detector and injector 280ºC
- Transport gas N2 (2 kg/cm²)
- W Speedomax recorder (5 mv. At the end of scale)
- Record speed – 10 i/h
- Injected volume – 0.5 µl with an attenuation of 10x64, increased up to 10x8 during the
chromatogram
Preparing samples
A small drop of liquefied honey (15 to 20 mg) is spread by a fine rod on the walls of the test tube
adapted to the rotative evaporator; it is dried and maintained in the appropriate vacuum at 50ºC for 20
minutes.
Preparing trimethylsilylethers
Anhydrous pyridine (1 ml), hexamethldisilasane (0.25 ml), trimethylchlorsilane (0.25 ml) and some
glass balls are added to the dried sample. The test tube is immediately corked up by the screwing stopper
and kept on the agitator for 1 hour. After centrifugation, the clear supernatant is ready for sampled and
injected.
It should be noted that honey is an aqueous solution including different sugars in which the
allomerous forms α and β, if any, are in a balanced relationship specific to aqueous solutions .Accordingly,
the possible chromatograms of some standard mixtures of sugars should be obtained starting from the
aqueous solution. The same holds good for fructose in which the relationship of the balance concentrations
between the pyranic and furanic forms is influenced by the nature of solvent.
DISCUSSION
We analyzed 100 samples from 12 different honeys. The gas chromatography analysis of the same
samples allowed to separate and value 6 sugars, namely: fructose, glucose (α and β), sucrose, X2, X3 and
mellezitose.
For other disacobarides such as maltose, trialose, isomaltose, gentibiose, X and X1 we did not
succeed in obtaining a good resolution of peaks, hence the valuation is approximate only.
The quantities analysis (relative percentages) was carried out by comparing the peaks of different
maximums with those obtained for an artificial mixture of sugars that copied to a certain extent the glucide
composition of honey including its most important components. We calculated the ratio between the peaks of
maximums and the concentrations referred to one of allomerics of glucose; this made it possible to estimate
the amount of each sugar as against glucose considered as a unit. All these amounts were then referred to
100 without taking into consideration the oligosaccharieds present.
We measured the rotation power for each honey [α] 20D)*)and calculated the fructose to glucose
ratio and the amount of disaccharides and trisaccharides.
____________________
*) C= 10-20 cm polarimetric tube
Apiacta 1, 1972
All data were statistically analyzed
.Honey classification dependent on its floral origin was made by the classical methods of
melissopalinology.
As to Castanea honeydew, this has not yet been found in pure state, since in Italy chestnut tree is
frequently mixed with fire-tree and honey obtained is mixed (Castanea and Abies honey)
One more reason why we took into consideration these honeys is the fact that these are very
common and enabled us to determine by their analysis the proportion of the two components.
The different honeys showed both analogies and differences in their glucide composition.
If we take into consideration at the same time fructose and glucose, which as is known are the most
representative components, we notice that they are present in values ranging from 70% (Abis and Castanea
honey) to 85% (heather honey)
Fructose percentage is rather low in Abies and Castanea honey as well as in Brassica honey, but in
other honey it reaches 42 to 47%
Glucose shows a low percentage in all honeys and also in Robinia honey, but reaches sensibile
values in Brassica ,Erica and Onobrychis honeys.
Sucrose, though present in a small quantity, shows somewhat important difference. Its percentages
range from 0.20 to 1.26%, the highest values being recorded with Robinia honey and Castanea and Abies
honeydew honey.
Maltose is the most important disaccharide in terms of quantity; in Castanea and Abies honey it
reaches 9%.
Trialose is also present in percentages worth of consideration; it reaches its maximum in Abies
honey (5%). The other did and trisaccharides, except mellezitose, are found in minimum percentages; even
though they differ from one honey to another, sometimes we may assume them as a basis for a
characterization. Mellezitose can be of some help to determine the floral source of honey since many honeys
do not contain it, some of them contain traces only and some others contain very high percentages
(Abies,Castanea, Rubinia).
The specific rotation power depends on the amount and quality of sugars present in honey including
oligosaccharides, which were not taken into consideration in the present stage of our investigations. A
comparative examination of data allowed to notice that this physical property is particularly linked to the
fructose to glucose ratio and the percentages of did and trisaccharides.
All honeys, except honeydew ones, are levogyric, the maximum values being found in honey with
the highest fructose to glucose ratio (Robinia,Castanea, etc) or in that with low percentages of did and
trisaccharides (Erica, Onobrychis etc). The same is true even of the honeydew honey coming from Abies and
Castanea, which are very rich in did and trisaccharides (maltose, X2, raffinose and mellezitose) ; but in this
case the positive value should be attributed to superior oligosaccharides.
A comparative examination of unifloral honey points out that:
- Rubinia honey differs from the other floral honeys in that it is richer in sucrose, maltose, trialose
and particularly in mellezitose, which reaches a value of 7.12%, almost equal to that of Abies honeydew
honey. Another feature of this honey consists of its low glucose content and consequently a high fructose to
glucose ratio and one of the highest rotative powers.
- The ratio between fructose and glucose in Castanea honey is equal to that of Rubinia honey, but
the former plainly differs from the latter as well as from the others, because it has a higher maltose and
isomaltose content and mellezitose as well as the peak of X3 are absent.
-The ratio between fructose and glucose in Hedysarum honey exceeds very little the unit, its rotative
power is low, the did and trisaccharide content is also rather low, and mellezitose and the X3 peak are
absent.
- Brassica honey is easy to identify because the ratio between fructose and glucose is the lowest of
all those examined, glucose is present in high quantity and fructose is relatively low;
- Citrus honey is characterized by a high percentage of maltose; it shows other features too, which
allow to differentiate it from the other honeys.
- Onobrychis and Medicago honeys are similar; the only features that differentiate them are the
rotative power and the ratio between fructose and glucose. Eucaliptus and Erica honeys are also like but the
percentage of did and trisaccharides is higher in the former (it is richer in maltose and poorer in glucose).
As to honeydew honey, there are several elements to distinguish it.
Quercus honeydew honey shows a negative rotative power but it is considerably lower than that of
floral honey. It contains little glucose, but it is rich in maltose and trialose.
Abies honeydew honey shows a high positive rotative power, contains a low percentage of glucose
and fructose, but high percentages of maltose, trialose and mellezitose.
Castanea and Abies honey has, just like the previous one, a positive rotative power, but its value is
flatly inferior. It is also poor in glucose and fructose and rich in did and trisaccharides. The features of this
honeydew honey does not allow to establish the proportions of the two components (Castanea and Abies)
Apiacta 1, 1972
A comparative examination of the percentages of sucrose, maltose and mellezitose of the floral
honey coming from Castanea and of that coming from Abies allow to exclude the presence of chestnut-tree
nectar.
The correlation between the specific rotative power and percentages of different sugars gave
different and sometimes even contradictory results. The correlation between the specific rotative power and
the percentage of fructose was in principle positive, whereas the correlation between glucose and maltose
and that between glucose and mellezitose were negative with all honeys. This makes us presume that the
formation of the two sugars mentioned above is due to glucose.
CONCLUSIONS
The analysis by gas chromatography of 100 unifloral honeys coming from 12 different species
allowed to perfectly separate and estimate 6 sugars, namely; fructose, glucose, sucroset X2, X3, and
mellezitose.
As to the trisaccharide X3 we think, in accordance with S i d d i q u i and F u r g a l a, that there is no
question of raffinose, though, it has the same retention time, since in honey as well as in its hydrolysis
products we found neither galactose nor melibiose, that is the monosaccharide and the disaccharide that
compose it.
For some disaccharides we could not obtain a good solution of peaks, nor their valuation, hence this
is approximate only (maltose, trialose, isomaltose, gentibiose X and X1)
The results of analyses supplied sufficiently valuable parameters to distinguish honeydew honey
from floral honey and to determine the floral source of honey of each group.
The correlation between the specific rotative power of honey and the components of its glucide
fraction turned out to be somewhat helpful.
REFERENCES
GOLDSCHMIDT, S & BURKERT, H. (1955) – Uber das Vorkommen im Bienenhonig bisher unbekannter Zucker. Hoppe-Seyler’s Z.
physiol. Chem. 300:188-200
POURTALLIER, J. (1964) – Détermination par chromatographie en couche mince des sucres du miel. Bull, apic. Inf. Docum. Scient.
Tech. 7 (2): 197-211.
POURTALLIER, J. (1968) – Uber die Benutzung der Gaschromatographie für die Bestimmung der Zucker im Honig. Zeit. Bienenfor. 2:
217-221
SIDDIQUI, I. R. & FURGALA, B. (1967) – Isolation and characterization of oligosaccharides from honey. Part. I. Disaccharides. Journal
of Apicultural Research 6 (3): 139-145.
SIDDIQUI, I. R. & FURGALA, B. (1968) – Isolation and characterization of oligosaccharides from honey. Part II. Trisaccharides. Journal
of Apicultural Research (7) 1: 51-59.
SWEELEY, BENTLEY, MAKITA and WELLS (1963) – The method for the trimethylsilylation of sugars and related substances. J. A. C.
S. 83 2597.
WATANABE, T. & ASO, K. (1960) – Studies on honey. II. Isolation of kojibiose, nigerose, maltose and isomaltose from honey. Tohoku
J. agric. Res. 11 (1): 109-115.
WHITE, J. W. Jr. & MAHER, J. (1953) – Transglucosidation by honey invertase. Archs Biochere Biophys, 42 (2): 360-367.
WHITE, J. W. & HOBAN, N. (1959) – Composition of honey. IV. Identification of the disaccharides. Archs. Biochem. Biophys. 80: 368392.
WHITE, J. W. Jr., MARY, L. RIETHOF, MARY, H. SUBERS, and KUSHNIR, I. (1962) – Composition of American honeys. Technical
Bulletin No. 1261 – Washington, D. C.