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Raffinose
UV-method
for the determination of raffinose in foodstuffs and other
materials
BOEHRINGER MANNHEIM / R-BIOPHARM
Enzymatic BioAnalysis / Food Analysis
For use in in vitro only Store at 2-8°C
Cat. No. 0 428 167
Test-Combination for approx. 30 determinations
Principle (Ref. 1)
Raffinose is hydrolyzed at pH 4.5 to D-galactose and sucrose in the presence
of the enzyme -galactosidase (1).
D-Galactose is oxidized by nicotinamide-adenine dinucleotide (NAD) to Dgalactonic
acid in the presence of the enzyme galactose dehydrogenase
(Gal-DH) (2).
The amount of NADH formed in the above reaction is stoichiometric to the
amount of raffinose. The increase in NADH is determined by means of its
light absorption at 334, 340 or 365 nm.
This principle allows the determination of raffinose besides sucrose and Dglucose.
The Test-Combination contains
1. Bottle 1 with approx. 320 mg lyophilizate, consisting of:
citrate buffer, pH approx. 4.5; NAD, approx. 28 mg
2. Bottle 2 with approx. 1.6 ml suspension -galactosidase, approx. 36 U
3. Bottle 3 with approx. 34 ml solution, consisting of:
potassium diphosphate buffer, pH approx. 8.6
4. Bottle 4 with approx. 1.6 ml suspension -galactose dehydrogenase,
approx. 30 U
Preparation of solutions
1. Dissolve contents of bottle 1 with 8.0 ml redist. water.
2. Use suspension of bottle 2 undiluted.
3. Use solution of bottle 3 undiluted.
4. Use suspension of bottle 4 undiluted.
Stability of reagents
The contents of bottle 1 are stable at 2-8°C (see pack label).
Solution 1 is stable for 3 months at 2-8°C, for 5 months at 15 to 25°C.
Bring solution 1 to 20-25°C before use.
The contents of bottles 2, 3 and 4 are stable at 2-8°C (see pack label).
Bring solution 3 to 20-25°C before use.
Procedure
Wavelength1: 340 nm, Hg 365 nm or Hg 334 nm
Glass cuvette2: 1.00 cm light path
Temperature: 20-25°C
Final volume: 3.400 ml
Read against air (without a cuvette in the light path) or against water
Sample solution: 3-250 μg raffinose/assay3 (in 0.100-0.500 ml sample
volume)
If the sample solution contains free D-galactose, this D-galactose is to be
determined in a separate assay without suspension 2 (D-galactose
sample).
1 The absorption maximum of NADH is at 340 nm. On spectrophotometers, measurements
are taken at the absorption maximum; if spectralline photometers equipped with a mercury
vapor lamp are used, measurements are taken at a wavelength of 365 nm or 334 nm.
2 If desired, disposable cuvettes may be used instead of glass cuvettes.
3 See instructions for performance of assay
(1) Raffinose + H2O
-galactosidase
D-galactose + sucrose
(2) D-Galactose + NAD+ Gal-DH
D-galactonic acid + NADH + H+
* Pipette solution 1, suspension 2, redist. water and the sample solution, each, onto the bottom
of the cuvette and mix by gentle swirling. When using a plastic spatula, remove it from
the cuvette only directly before measuring absorbance A1.
** Rinse the enzyme pipette or the pipette tip of the piston pipette with sample solution before
dispensing the sample solution.
*** For example, with a plastic spatula or by gentle swirling after closing the cuvette with Parafilm
(trademark of the American Can Company, Greenwich, Ct., USA)
Determine the absorbance differences (A2-A1) for blanks and samples. Subtract
the absorbance differences of the blanks from the absorbance differrences
of the corresponding samples.
A = (A2-A1)sample - (A2-A1)blank
It follows AD-galactose (from “D-galactose sample”) and
Araffinose + D-galactose (from “raffinose sample”).
The difference of these values stands for Araffinose.
The measured absorbance differences should, as a rule, be at least 0.100
absorbance units to achieve sufficiently precise results (see "Instructions of
performance of assay" and “Sensitivity and detection limit”, pt. 3).
Calculation
According to the general equation for calculating the concentration:
V = final volume [ml]
v = sample volume [ml]
MW = molecular weight of the substance to be assayed [g/mol]
d = light path [cm]
ε = extinction coefficient of NADH at:
340 nm = 6.3 [l × mmol-1 × cm-1]
Hg 365 nm = 3.4 [l × mmol-1 × cm-1]
Hg 334 nm = 6.18 [l × mmol-1 × cm-1]
It follows for raffinose:
for D-galactose:
If the sample has been diluted during preparation, the result must be
multiplied by the dilution factor F.
Pipette into
cuvettes
Blank
raffinose
sample
Raffinose
sample
Blank
D-galactosesample
D-Galactosesample
solution 1*
sample solution**
suspension 2
redist. water
0.200 ml
-
0.050 ml
0.100 ml
0.200 ml
0.100 ml
0.050 ml
-
0.200 ml
--
0.150 ml
0.200 ml
0.100 ml
-
0.050 ml
Mix*, and incubate for 15 min at 20-25°C. Add:
solution 3
redist. water
1.000 ml
2.000 ml
1.000 ml
2.000 ml
1.000 ml
2.000 ml
1.000 ml
2.000 ml
Mix***, read absorbances of the solutions after approx. 2 min (A1). Start reaction
by addition of:
suspension 4 0.050 ml 0.050 ml 0.050 ml 0.050 ml
Mix***, wait unil the reaction has stopped (approx. 20 min) and read absorbances
of the solutions (A2).
c =
V × MW
× A [g/l]
ε × d × v × 1000
c =
3.400 × 504.5
× Araffinose =
17.15
× Araffinose [g raffinose/l
ε × 1.00 × 0.100 × 1000 ε sample solution]
c =
3.400 × 180.16
× AD-galactose=
6.125
× AD-galactose [g D-galacε
× 1.00 × 0.100 × 1000 ε tose/l sample solution]
0101.1. 462 632 rb bz
2
When analyzing solid and semi-solid samples which are weighed out for
sample preparation, the result is calculated from the amount weighed:
:
1. Instructions for performance of assay
The amout of raffinose present in the assay has to be between 5 g and
250 g (measurement at 365 nm) or 3 g and 130 g (measurement at 340,
334 nm), respectively. In order to get a sufficient absorbance difference, the
sample solution is diluted to yield a raffinose concentration between 0.5 and
2.5 g/l or 0.25 and 1.3 g/l, respectively.
Dilution table
If the measured absorbance difference (A) is too low (e.g. 0.100), the
sample solution should be prepared again (weigh out more sample or dilute
less strongly) or the sample volume to be pipetted into the cuvette can be
increased up to 0.500 ml. Adjust sample solution to pH 4.2-4.8, if necessary.
The volume of water added after incubation must then be reduced so as to
obtain the same final volume in the assays for sample and blank. The new
sample volume v must be taken into account in the calculation.
2. Specificity
Besides raffinose, -galactosidase also hydrolyzes other -galactosides, e.g.
galactinol, melibiose, and stachyose. Substances with -galactoside
such as lactose are not hydrolyzed and do not interfere with the assay,
even when present in high concentrations.
Apart from D-galactose, Gal-DH also oxidizes L-arabinose. Small amounts of
L-arabinose only occur in foodstuffs, if it has been released from its natural
glycosidic
In the analysis of commercial raffinose, results of approx. 99 % have to be
expected.
3. Sensitivity and detection limit (Ref. 1.2)
The smallest differentiating absorbance for the procedure is 0.005 absorbance
units. This corresponds to a maximum sample volume v = 0.500 ml and
measurement at 340 nm of a raffinose concentration of approx. 3 mg/l, resp.
1 mg D-galactose/l sample solution (if v = 0.100 ml, this corresponds to 14
mg raffinose/l, resp. 5 mg D-galactose/l sample solution).
The detection limit of 5 mg raffinose/l, resp. 2 mg D-galactose/l is derived
from the absorbance difference of 0.010 (as measured at 340 nm) and a
maximum sample volume v = 0.500 ml.
4. Linearity
Linearity of the determination exists from approx. 3 g raffinose +
D-galactose/assay (5 mg raffinose + D-galactose/l sample solution; sample
volume v = 0.500 ml) to 250 g raffinose + D-galactose/assay (2.5 g
raffinose + D-galactose/l sample solution; sample volume v = 0.100 ml).
5. Precision
In a double determination of raffinose using one sample solution, a
difference of 0.010 to 0.015 absorbance units may occur in the presence of
D-galactose in the sample. With a sample volume of v = 0.100 ml and
measurement at 340 nm, this corresponds to a raffinose concentration of
approx. 25-40 mg/l. (If the sample is diluted during sample preparation, the
result has to be multiplied by the dilution factor F. If the sample is weighed in
for sample preparation, e.g. using 1 g sample/100 ml = 10 g/l, a difference of
0.25-0.4 g/100 g can be expected.)
In a double determination of D-galactose using one sample solution, a
difference of 0.005 to 0.010 absorbance units may occur. With a sample
volume of v = 0.100 ml and measurement at 340 nm, this corresponds to a
D-galactose concentration of approx. 5-10 mg/l. (If the sample is diluted
during sample preparation, the result has to be multiplied by the dilution
Contentraffinose =
craffinose [g/l sample solution]
× 100 [g/100 g]
weightsample in g/l sample solution
ContentD-galactose =
cD-galactose [g/l sample solution]
× 100 [g/100 g]
weightsample in g/l sample solution
Estimated amount of
raffinose per liter
measurement at
Dilution
with water
Dilution
factor F
340 or 334 nm 365 nm
1.3 g
1.3-13 g
13-130 g
2.5 g
2.5-25.0 g
25.0-250 g
-
1 + 9
1 + 99
1
10
100
factor F. If the sample is weighed in for sample preparation, e.g. using 1 g
sample/100 ml = 10 g/l, a difference of 0.05-0.1 g/100 g can be expected.)
The following data for the determination of raffinose and D-galactose have
been published in the literature:
x = 39.8 mg/l CV = 2.6 % (Ref. 1.1)
x = 130 g/assay raffinose solution CV = 0.26 %
x = 220 g/assay raffinose solution CV = 0.43 % (Ref. 1.2)
x = 25 g/assay D-galactose solution CV = 0.95 %
x = 50 g/assay D-galactose solution CV = 0.64 % (Ref. 1.4)
Milk and milk products:
D-galactose: r = 0.10 × (contentD-galactose in g/100 g) g/100 g
R = 0.12 × (contentD-galactose in g/100 g) g/100 g (Ref. 2.1)
6. Recognizing interference during the assay procedure
6.1 If the conversion of D-galactose has been completed according to the
time given under “Procedure“, it can be concluded in general that no
interference has occurred.
6.2 On completion of the reaction, the determination can be restarted by
adding D-galactose (qualitative or quantitative): if the absorbance is
altered subsequent to the addition of the standard material, this is also
an indication that no interference has occurred.
The reaction cannot be restarted with raffinose as, subsequent to altering
the reaction conditions from pH 4.5 to pH 8.6 (“change of the buffer“),
raffinose is no longer cleaved.
6.3 Operator error or interference of the determination through the presence
of substances contained in the sample can be recognized by carrying
out a double determination using two different sample volumes (e.g.
0.100 ml and 0.200 ml): the measured differences in absorbance should
be proportional to the sample volumes used.
When analyzing solid samples, it is recommended that different
quantities (e.g. 1 g and 2 g) be weighed into 100 ml volumetric flasks.
The absorbance differences measured and the weights of sample used
should be proportional for identical sample volumes.
The use of “single“ and “double“ sample volumes in double determinations
is the simplest method of carrying out an assay control in the determination
of raffinose.
6.4 Possible interference caused by substances contained in the sample can
be recognized by using an internal standard as a control: in addition to
the sample, blank and standard determinations, a further determination
should be carried out with sample and assay control solution in the
same assay. The recovery can then be calculated from the absorbance
differences measured.
6.5 Possible losses during the determination can be recognized by carrying
out recovery tests: the sample should be prepared and analyzed with
and without added standard material. The additive should be recovered
quantitatively within the error range of the method.
7. Reagent hazard
The reagents used in the determination of raffinose are not hazardous
materials in the sense of the Hazardous Substances Regulations, the
Chemicals Law or EC Regulation 67/548/EEC and subsequent alteration,
supplementation and adaptation guidelines. However, the general safety
measures that apply to all chemical substances should be adhered to.
After use, the reagents can be disposed of with laboratory waste, but local
regulations must always be observed. Packaging material can be disposed
of in waste destined for recycling.
8. General information on sample preparation
In carrying out the assay:
Use clear, colorless and practically neutral liquid samples directly, or
after dilution according to the dilution table, and of a volume up to 0.500 ml;
Filter turbid solutions (use membrane filter if necessary);
Degas samples containing carbon dioxide (e.g. by filtration);
Crush or homogenize solid or semi-solid samples, extract with water or
dissolve in water and filter if necessary; resp. remove turbidities or dyestuffs
by Carrez clarification;
Deproteinize samples containing protein with Carrez reagents;
Extract samples containing fat with hot water (extraction temperature
should be above the melting point of the fat involved). Cool to allow the fat
to separate, make up to the mark, place the volumetric flask in an ice bath
for 15 min and filter; alternatively clarify with Carrez-solutions after the
extraction with hot water;
Break emulsions with Carrez-solutions.
Carrez clarification:
Pipette the liquid sample into a 100 ml volumetric flask whick contains
approx. 60 ml redist. water, or weigh sufficient quantity of the sample into a
100 ml volumetric flask and add approx. 60 ml redist. water. Subsequently,
carefully add 5 ml Carrez-I-solution (potassium hexacyanoferrate(II) (ferrocyanide),
85 mM = 3.60 g K4[Fe(CN)6] × 3 H2O/100 ml) and 5 ml Carrez-IIsolution
(zinc sulfate, 250 mM = 7.20 g ZnSO4 × 7 H2O/100 ml). Adjust to pH
7.5-8.5 with sodium hydroxide (0.1 M; e.g. 10 ml). Mix after each addition. Fill
the volumetric flask to the mark, mix and filter.
9. Application examples
Determination of raffinose in molasses, syrup and other products of
sugar manufacture
Before starting the assay, precipitate quantitatively any galactinol which may
be present in the sample by addition of basic lead acetate. For performance
of the sample preparation see Ref. 2.3. In the analysis of e.g. molasses, a part
of raffinose is adsorbed to the precipitate (see Ref. 2.4).
Determination of raffinose in soybean flour and flour of cereals
Accurately weigh approx. 2-5 g of the ground and homogenized sample into
a 100 ml volumetric flask and heat with approx. 50 ml water for 30 min in a
water-bath at 60°C. Stir occasionally (alternatively a magnetic stirrer can be
used). For clarification add 5 ml Carrez-I-solution (3.60 g potassium hexacyanoferrate(
II), K4[Fe(CN)6] × 3 H2O/100 ml), 5 ml Carrez-II-solution (7.20 g
zinc sulfate, ZnSO4 × 7 H2O/100 ml) and 10 ml sodium hydroxide (0.1 M).
After each addition mix or stir, respectively, cool to 20-25°C, fill up to the
mark with water, and filter. Use the clear, possibly slightly turbid solution
diluted, if necessary, for the assay.
Soybean flour contains up to 10 % raffinose; flours of cereals contain up to
0.3 % raffinose.
10. Further applications
The method may also be used when analyzing biological samples.
Determination of raffinose in fermentation samples and cell culture
media
Place the sample (after centrifugation, if necessary) in a water-bath at 80°C
for 15 min to stop enzymatic reactions. Centrifuge and use the supernatant
(diluted according to the dilution table, if necessary) for the assay.
Alternatively, deproteinization can be carried out with Carrez-solutions. See
the above-mentioned examples.
Homogenize gelatinous agar media with water and treat further as
described.
References
1.1 Bergmeyer, H.U., Bernt, E. & Gutmann, I. (1974) in Methoden der enzymatischen
Analyse (Bergmeyer, H.U., Hrsg.) 3. Aufl., Bd. 2, S. 1217-1220; Verlag Chemie, Weinheim
and (1974) in Methods of Enzymatic Analysis (Bergmeyer, H.U., ed.) 2nd ed., vol 3, pp.
1172-1175; Verlag Chemie, Weinheim/Academic Press, Inc. New York and London
1.2 Beutler, H.-O. (1984) in Methods of Enzymatic Analysis (Bergmeyer, H.U., ed.) 3rd ed.,
vol. VI, pp. 90-96, Verlag Chemie, Weinheim, Deerfield Beach/Florida, Basel
1.3 Kurz, G. & Wallenfels, K. (1974) in Methoden der enzymatischen Analyse, (Bergmeyer,
H.U., Hrsg.) 3. Aufl., Bd. 2, S. 1324-1327, Verlag Chemie, Weinheim, and (1974) in Methods
of Enzymatic Analysis (Bergmeyer, H.U., ed.) 2nd ed., vol. 3, pp. 1180-1184 and
pp. 1279-1282, Verlag Chemie, Weinheim/Academic Press, Inc. New York and London
1.4 Beutler, H.O. (1984) in Methods of Enzymatic Analysis (Bergmeyer, H.U., ed.) 3rd ed.,
vol. VI, pp. 104-112, Verlag Chemie, Weinheim, Deerfield Beach/Florida, Basel
2.1 Amtliche Sammlung von Untersuchungsverfahren nach §35 LMBG; Untersuchung von
Lebensmitteln: Bestimmung des Lactose- und Galactosegehalts von Milchprodukten
(02.00-9/November 1983)
2.2 Schiweck, H. & Büsching, L. (1969) Die enzymatische Bestimmung des Raffinose- und
Galactinolgehaltes in Zuckerfabriksprodukten ber die bei der Spaltung entstehende
Galactose mit Hilfe von Galactose-Dehydrogenase, Zucker 22, 377-384
2.3 Schiweck, H. & Büsching, L. (1975) Die enzymatische Bestimmung des Raffinosegehaltes
in Zuckerfabriksprodukten über die bei der Spaltung entstehende Galactose
mit Hilfe von Galactose-Dehydrogenase, Zucker 28, 242-243
2.4 Hollaus, F., Wieninger, L. & Braunsteiner, W. (1977) Erfahrungen mit der enzymatischen
Raffinose-Bestimmung mittels Galactose-Dehydrogenase in Rüben und Zuckerfabriksprodukten,
Zucker 30, 653-658
3. Internal standard:
The assay control solution can be used as an internal standard in order to
check the determination for correct performance (gross errors) and to see
whether the sample solution is free from interfering substances:
The recovery of the standard is calculated according to the following
formula:
Pipette into
cuvettes
Blank
raffinose
sample
Raffinose
sample
Raffinose
standard
Raffinose
sample +
raffinose
standard
solution 1
suspension 2
sample solution
assay control sln.
redist. water
0.200 ml
0.050 ml
--
0.100 ml
0.200 ml
0.050 ml
0.100 ml
--
0.200 ml
0.050 ml
-
0.100 ml
-
0.200 ml
0.050 ml
0.050 ml
0.050 ml
-
Mix. Continue as described in the pipetting scheme under “Procedure”.
Follow the instructions given under “Instructions for performance of assay”
and the footnotes.
recovery =
2 × Asample + standard - Asample × 100 [%]
Astandard
Raffinose assay control solution
The assay control solution serves as a control for the enzymatic determination
of raffinose in foodstuffs and other materials.
Reagents
Raffinose penta-hydrate (M = 594.52 g/mol; contains 84.9% raffinose), AR
grade
Preparation of the assay control solution
Accurately weigh approx. 150 mg raffinose penta-hydrate to the nearest
0.1 mg into a 100 ml volumetric flask, fill up to the mark with redist. water,
and mix thoroughly (corresponds to approx. 1.3 g raffinose/l).
Prepare assay control solution freshly before use. The assay control solution
may be frozen in portions.
Application:
1. Addition of raffinose assay control solution to the assay mixture:
Instead of sample solution the assay control solution is used for the assay.
(For the calculation of results use the molecular weight of the assay control
material weighed in for analysis.)
The measurement of the assay control solution is not necessary for calculating
the results.
2. Restart of the reaction, quantitatively:
A restart of the reaction with assay control solution after completion of the
reaction cannot be performed as under the assay conditions (potassium
diphosphate buffer, pH 8.6) raffinose is not cleaved. If necessary, restart with
0.050 ml of a D-galactose solution (0.5 g/l).
R-BIOPHARM GmbH
Dolivostraße 10
64293 Darmstadt/Germany
Telefon + 49 61 51 / 81 02-0
Fax + 49 61 51 / 81 02-20
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