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and Brown.3 At the peak (660 mp), xylose, ribose and arabinose (not shown in Fig. 2)
give the same absorption, viz., about 15 times that shown by fructose and 30 times that
of glucose or galactose. Hence this reaction alone allows for estimation of pentose when
an excess of hexose is not present. Brown3 allowed for hexose by measuring the absorption
a t two wavelengths, at the pentose peak of 660 mp, and also at 520 m p where the difference
between pentose and hexose was less marked. Although in principle his method could be
used for determining both pentose and hexose present together, it has its disadvantages.
The reagent blank at 520 m p is considerable and variable. The large blank absorption
a t the blue end of the spectrum also limits the usefulness of measurements at what would
otherwise be the most suitable wavelength, vix., 425mp, when the absorption for both
yentose and hexose is a t a maximum and of the same order of magnitude. In the method
recommended here the mixture of sugars is heated with orcinol and acid, alone or with
ferric chloride, and the absorption is read a t the most suitable wavelengths, vix., 425mp
and 660mp, respectively. The concentrations of both pentose and hexose can then be
read from a nomogram.
TABLEI
COLORIMETERREADINGS WITH VARIOUS SUGARS
Readings with acid
Readings with acid orcinol- ferric
orcinol reagent and chloride reagent and
Sugar 621 (blue) filter 608 (red) filter
Glucose . . .. .. .. 19 2
Galactose .. .. .. 20 5
Fructose . . .. .. .. 64 2
Sucrose . . .. 13 2
Maltose (C,,H,,d,;.H,Oj * .. 17 2
Xylose . . .. .. .. 26 78
Ribose . . .. .. .. 26 78
METHOD
PREPARATION OF MATERIAL FOR ASSAY-
When only monoses or oligosaccharides are present, no preliminary preparation is required
except for deproteinisation ( e g . , with trichloro-acetic acid) when necessary. Polysaccharides
and combined sugars are hydrolysed with N hydrochloric acid at 100" C for 4 to 6 hours in a
sealed tube.
REAGENT-
Orcinol-Traces of impurities are liable to yield derivatives similar to, but more deeply
coloured than, those of orcinol itself. The blank does not provide full correction, and if the
colour value much exceeds the normal range the linear relationship between carbohydrate
present and colour produced no longer holds. The orcinol must therefore be freshly re-
crystallised from hot water with a little charcoal. This yields the monohydrate, which
should be converted to the anhydrous material by drying in a vacuum-desiccator. If orcinol
containing water is used for making the orcinol- sulphuric acid reagent a high blank may
result.
ORCINOL - SULPHURIC ACID REACTION^-
+
Dissolve dry orcinol (0.2 g) in 100 ml of diluted sulphuric acid (2 1) without heating.
The reagent is stable for 2 days at 0" C, but must be discarded if it shows any discoloration.
This is particularly likely to happen if freshly-recrystallised orcinol is not used. Add 10 ml
of the reagent to 1 ml of the test sample (containing 5 to 25 pg of carbohydrate) in a
6 x 9-inch test tube. After mixing, cover the mouth of the tube with a glass bulb, heat
the tube for 15 minutes a t 100"C and cool rapidly to room temperature. Read the colour
after 30 minutes, either in an "EEL" colorimeter with a 621 (dark blue) gelatin filter* or in a
* Transmission maximum at 450 mp.
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Fig. 3. Nomogram for calculating quantities of pentose and hexose from colori-
meter readings. A, total carbohydrate (pentose plus hexose), p g per ml; B, colori-
meter readings, orcinol- sulphuric acid method ; C, colorimeter readings, orcinol-
ferric chloride method; D, pentose, pg per ml
reagent to 2 ml of the sample (containing 5 to 25 pg of carbohydrate per ml) in a 6 x #-inch
test tube. After mixing, cover the mouth of the tube with a glass bulb, heat the tube a t
100” C for 20 minutes, and then cool rapidly to room temperature. Read the colour after
30 minutes either in an “EEL” colorimeter with a 608 (red) filter* or in a spectrophotometer
at 660 mp. The colour is stable for 2 hours. Make a blank determination and correct
the zero setting of the colorimeter.
PRECAUTIONS-
Careful attention must be paid to the following points-
(1) The test solutions must not be diluted until shortly before the determination, or
they may suffer appreciable decomposition.
(2) A large water-bath should be used, as time and temperature of heating are critical.
(3) The test tubes must be covered with loose glass bulbs or stoppers during heating
so that the sulphuric acid is not diluted by absorption of steam or the hydrochloric
acid by loss of hydrogen chloride.
CALCULATION OF RESULTS-
The amounts of hexose and pentose present can be calculated from the colorimeter
readings by means of a nomogram (Fig. 3). The scales B and C correspond to the colorimeter
readings for the acid-orcinol and the acid-orcinol - ferric chloride reactions, respectively. A
line, e.g., bc, is drawn connecting the two readings for the test sample. This line is extended
* Absorbs all wavelengths shorter than about 640 mp.
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