International Federation

Sugars, detection I.FJ.U.-Analyses

of Fruit Juice Froducers (Characterization by thin-layer chromatography on cellulose) No. 31

Principie
T h e m a i n s u g a r s , glucóse, fructose and s u c r o s e p r e s e n t i n fruits and derived

layer chromatography i n one unidimensional r u n on cellulose p l a t e s . T h e y a r e

m a d e visible by m e a n s of two s p r a y r e a g e n t s that give c h a r a c t e r i s t i c c o l o r a t i o n s .

WORKING INSTRUCTIONS

1. A p p a r a t u s a n d reagents
*)
T L - S t a n d a r d equipment of t h e f i r m CAMAG ^, consisting of
1 coater for p l a t e s up to 20 c m width
1 gauge for t h e adjustment of the l a y e r t h i c k n e s s (feeler gauge)
glass plates 20 x 20 c m o r 10 x 20 c m
1 drying r a c k
1 spotting témplate
1 development t a n k
1 automatic m i c r o - p i p e t t e (self-füling), 5 ¡ÍL CAMAG*^ o r OLIAG**^
1 hot air d r i e r
1 hot cabinet o r p r e f e r a b l y hot píate, adjustable to 150°C
1 spraying a p p i r a t e , consisting of a p r e s s u r e bottle with attachable plástic
s p r a y - h e a d (Supplier: e . g . Polycolor 6052 H e r g i s w i l NW, Switzerland)
1 desiccator o r drying c a s e with s i l i c a - g e l drying agent (to t a k e plates)
1 electric mixer
benzine o r chloroform for d e - g r e a s i n g p l a t e s
1) R e a d y - m a d e cellulose plates and films now available c o m m e r c i a l l y can
r e p l a c e tiie T L - p l a t e s p r e p a r e d a s d e s c r i b e d l a t e r . See further under
n u m b e r 3f.
*) e . g . CAMAG, H o m b u r g e r s t r a s s e 24, 4132 Muttenz B L (Switzerland)
**) e . g . OLIAG AG, K a p p e l i s t r a s s e , 8703 E r l e n b a c h ZH (Switzerland)
No. 3 1 , page 1
1965
 International Federation Sugars, detection I.FJ.U.-Analyses
of Fruit Juice Froducers (continuation) No. 31

cellulose powder CAMAG DS 8 or a m i x t u r e of 75 % MN 300 G and 25 %

MN 300
s o d l u m acétate, J^ía.C^Il^O^ • 3H2O, 1 %
Solvent s y s t e m (prepared f r e s h daily) consisting of a . m i x t u r e of
8 v o l u m e s n-butanol
3 v o l u m e s glacial acetic acid
3 volumes water
T h e individual components a r e m e a s u r e d singly in dry m e a s u r i n g cylinders.'
Spray reagent:
Solution I 10 g t r i c h l o r a c e t i c acid
5 g phthalic acid
1.2 g p - a m i n o h i p p u r i c acid, dis solved in
200 m i ethyl alcohol 9 6 %
(keeps in d a r k bottle in r e f r i g e r a t o r for ca. 1 week)
Solution H 15 g u r e a , dis solved in
45 m i 2-n hydrochloric acid
(made up f r e s h every other day)

2. D e t e c t i o n procedure

a) Preparatíon of T L - p l a t e s
25 g CAMAG-cellulose DS 8 a r e homogenized for 1 minute with 125 m i
1 % sodium acétate in an electric m i x e r . T h e plates a r e washed with
dilute soda and HCl solutions, r i n s e d with dist. water and dried: they
a r e then coated in the CAMAG-Coater according to the m a k e r ' s instructions.
It i s r e c o m m e n d e d that the surfaces of the plates be freed f r o m the l a s t
t r a c e s of g r e a s y m a t e r i a l by wiping over with a pad of cotton wool
m o i s t e n e d with benzine or chloroform just before they a r e s p r e a d . The
l a y e r t h i c k n e s s should be 0.3 m m ; with adjustable s p r e a d e r s , the depth
of the cellulose can b e fixed at 0.3 m m by m e a n s of a milled s c r e w and

No. 3 1 , page 2
1965
 International Federation Sugars, detection I.FJ.U.-Analyses
of Fruit Juice Froducers (continuation) No. 31

a feeler gauge. L a t e r models a r e obtainable with fixed adjustments a t

0.3 and 0.5 m m . (With other spreading devices the proportions of
cellulose and w a t e r m u s t be found by previous t r i a l . ) P l a t e s that leave
the spreading apparatus with a non-uniform coating a r e pushed b a c k once
or twice; poorly coated p l a t e s can thus b e r e d u c e d to a mínimum.
After spreading, the plates a r e left in the air for a p e r i o d of 2 h o u r s ,
then placed in a píate-rack and activated for one hour in t h e hot cabinet
at 105°C. The p l a t e s , now r e a d y for u s e , a r e kept i n a d e s i c c a t o r .

b) The s a m p l e to be examined i s applied by m e a n s of a self-filling m i c r o -

pipette of 5 |al capacity using the CAMAG-Applicator. (The spotting points
a r e m a r k e d with a needle, the slide being drawn b a c k and u s e d a s a hand
r e s t . ) It i s r e c o m m e n d e d that the liquid m e a s u r e d out by the pipette be
put on in 5 - 6 approximately equal portions (each d r i e d with t h e blower).
With a fine spatula a s e p a r a t i n g line 2 - 3 m m wide i s m a d e 3 c m f r o m
the upper edge; this m a r k will c o r r e s p o n d to t h e position of t h e solvent
front when t h e píate has been developed. It i s also r e c o m m e n d e d that
the two l a t e r a l b o r d e r s and that p a r a l l e l to the starting line b e s c r a p e d
off with the thumb to a width of ca. 2 - 3 m m .

c) Preparatíon of the s a m p l e s for spotting

The b e v e r a g e s should be diluted with water according to their s u g a r
contents, so that the application of 5 ^ of t h e diluted solution puts on

1
ca. 12. 5 |Lig of the p a r t i c u l a r s u g a r . As a r u l e , fruit juices a r e diluted
in the proportion 1 : 10.
If the sugar content i s v e r y low, i t i s r e c o m m e n d e d to put on 2 o r
3 X 5 pl of the undiluted b e v e r a g e .
It i s also r e c o m m e n d e d to r u n c o m p a r i s o n solutions of the c o r r e s p o n d i n g ^
s u g a r s on each c h r o m a t o g r a m .

No. 3 1 , page 3
1965
International Federation Sugars, detection I.FJ.U.-Analyses
of Fruit Juice Froducers (continuation) No. 31

Preparatíon of standard sugar solutions

A9l]?ÍL9.^s _of_ single _s_ugajs_
0.125 g of glucose, fructose o r síicrose, and 0.25 g of lactose or rhatnnose
a r e each dissolved in 50 m i of 20 vol. % alcohol. The solutions a r e stable
for s e v e r a l m o n t h s .

Standard solution of a sugar m i x t u r e

0.125 g each of glucose, fructosa and s u c r o s e with 0.25 g each of lactose
and r h a m n o s e a r e taken up a s above i n 50 m i of 20 vol. % alcohol.

Amounts for_ spotting

5 |Lil a r e put on for the individual sugars. or for the sugar m i x t u r e .
If t h e sugar content i s not exactly known, i t i s suggested that dilutions
b e p r e p a r e d a s d e s c r i b e d above and that s e v e r a l concentrations b e put on
t h e píate ( e . g . 2 . 5 ; 5 o r 10 |LI1). Strongly pigmented s a m p l e s should b e
boíled up with a knife-point of active carbón and fíltered, u n l e s s the colour
intensity can b e r e d u c e d by using a g r e a t e r dilutíon. P l a t e s can be labelled
by m e a n s of a pointed i n s t r u m e n t , e.g» with a needle.

d) Development of the plates

After the s u b s t a n c e s have been put on, 1 - 2 plates a r e placed in a
development tanlc containing 1 c m depth of the solvent n-butanol-acetic
a c i d - w a t e r . T h e glass container i s covered by a sheet of ground glass
and p r o t e c t e d f r o m draughts by a c a r d b o a r d box. - T i m e of development: C2
3 h o u r s ; Rimning distance: ca. 1 4 - 1 5 c m .

e) D r y i n g t h e plates
After development t h e plates a r e dried for ca. 40 minutes i n a cabinet
(with exhaust I ) ; the drying t i m e can be reduced to 20 m i n . by using an
auxilíary fan.

No, 3 1 , page 4
1965
 Sugars, detection I.FJ.U.-Analyses
International Federation
of Fruit Juice Froducers (continuation) No. 31

f) Spraying t h e plates and r e v e a l i n g the spots

Using the s p r a y assenably (Spray-Bombe) ^ t h e plates a r e uniíormly s p r a y e d
from. a distance of ca. 50 c m with Solution I for 30 - 35 s e c . ; they a r e
o* **)
then put for 10 - 15 min. i n a drying oven at 135 C o r on a hot píate '
í (150°C). When t h e colour of the sugar spots, which a r e mainly brown,
shows no further i n c r e a s e in intensity, the heating i s stopped (also check
the grouping of t h e tints after t h e first spray). The c h r o m a t o g r a m i s
immediately inspected for possible r e d or r e d - b r o w n spots of galacturonic
acid and l a c t o s e , for after the second spraying t h e s e spots will b e c o m e
p a l e r . The píate, while still hot, i s now s p r a y e d as before with Spray
Reagent H but only for 15 s e c . : i t i s then heated as d e s c r i b e d above. After
a few minutes the s u g a r s will appear as well-defined and isolated spots
with c h a r a c t e r i s t i c c o l o u r s . T h e background i s lightened by Spray Reagent
U. T h e spot colours p e r s i s t for s o m e days if p r o t e c t e d f r o m the light,
but in t h e c o u r s e of t i m e they all t a k e on b r o w n t o n e s . The accompanying
table l i s t s t h e R^, valúes and colours of t h e spots.
Table 1

Colours after the Colours of spots after

Sugars e t c . Rj,-valúes f i r s t spraying t h e second spra5ring

Lactose 0.13 red-brown violet

Galacturonic acid 0.16 red grey-blue
Sucrose 0.22 brownish grey-black
Glucose 0.28 red-brown brown-red
Fructose 0.35 brownish grey-blue
Arabinose 0.36 red reddish
Rhanmose 0.52 y e l l o w - g r e e n (weak) brown-red
HMB' 0.88 brown blue

*) Supplier: e . g . I>OLYCOLOR, 6052 H e r g i s w i l NW (Switzerland)

**) Supplier: e . g . KONTRON AG, 8005 Z u r i c h (Switzerland) No. 3 1 , page 5
1965
International Federation Sugars, detection I.FJ.U.-Analyses
of Fruit Juice Froducers (continuation) No. 31

3. R e m a r k s
a) T h e method d e s c r i b e d above p e r m i t s , in a single r u n , the clean separation
and Identification of t h e m a i n types of s u g a r p r e s e n t in b e v e r a g e s , v i z .
f r u c t o s e , glucose and s u c r o s e , together with t h e l a c t o s e p r e s e n t in w h e y -
d r i n k s . T h e s i z e s of the spots give a semi-ctuantitative estímate of the
concentrations of s u g a r s p r e s e n t .

b) If t h e sugar concentration of the fruit juice u n d e r t e s t i s not exactly known,

it i s advisable to u s e different dilutions and to put on 5j 10 or 15 m i c r o -
l i t r e s of liquid.. T h e optimal conditions -vary f r o m 10 - 25 m i c r o g r a m s
a c c o r d i n g t o t h e type of s u g a r .

c) T h e limit of detection for the different substances i s about 0 . 2 g / l for

glucose, f r u c t o s e , s u c r o s e , arabinose and H M F , and for l a c t o s e ,
galacturonic acid and r h a m n o s e about 0 . 4 g / l .

d) If t h e p r o p o r t i o n of w a t e r in t h e s l u r r y used for coating the p l a t e s i s

íncreased f r o m 125 m i to 155 m i ( s p r e a d e r 0.3 m m ) , t h e running t i m e i s
extended t o 7 h o u r s . Such p l a t e s give an even b e t t e r and s h a r p e r spot
s e p a r a t i o n and m a y b e u s e d if the t i m e factor is not i m p o r t a n t . T h e amount
spotted on should then be r e d u c e d to a half.

e) Galacturonic acid, a b r e a k - d o w n product f r o m the enzymatic degradation

of pectin, i s also d e t e r m i n e d by the method d e s c r i b e d . It i s r e c o g n i s e d
by t h e a p p e a r a n c e of a r e d - b r o w n spot below t h e s u c r o s e spot (after
s p r a y i n g with Solution I). Since l a c t o s e , with an R ^ - v a l u e corresponding
t o galacturonic acid, does not occur in t h e juices of p o m e fruits, g r a p e s
and b e r r i e s , (hydroxymethylfurfurol), r h a m n o s e , f r u c t o s e , glucose, s u c r o s e
and galacturonic acid can be detected in one operation.

f) R e a d y - m a d e coated T L - c e l l u l o s e plates of 1 m m thickness on glass have

recenüy b e c o m e c o m m e r c i a l l y available, as also coated plástic f i l m s .

N o . 3 1 , page 6
1965
International Federation Sugars, detection I.FJ.U.-Analyses
of Fruit Juice Froducers (end) No. 31

F o r technical r e a s o n s i t h a s not y e t b e e n possible to p r e p a r e these p l a t e s

with gypsum as a binder, so that the c h r o m a t o g r a m s obtained a r e of
lower quality than those given by s e l f - p r e p a r e d p l a t e s . P l a t e s and films
a r e either kept in a d e s i c c a t o r or activated for n s e by heating for 30 m i -
ñutes at 105°C. The thinner s u p p o r t s (disposable g l a s s or film) r e q u i r e ,
f\irthermore, that lower drying t e m p e r a t u r e s (120°C) be u s e d for s p r a y e d
p l a t e s or f ñ m s (normal g l a s s p l a t e s a r e , of c o u r s e , d r i e d at 150°C).
R e a d y - m a d e g l a s s plates a r e offered by the f i r m M e r c k , D a r m s t a d t ,
whüe suitable ceUnlose films designated MN-POLYGRAM C E L 300 a r e
obtainable f r o m the f i r m M a c h e r e y and Nagel, D u r e n (BRD). The R -valúes
can differ slightly f r o m those found with s e l f - p r e p a r e d p l a t e s . Usually the
concentrations of s u g a r put on can b e r e d u c e d by a half.

4.. R e f e r e n c e
H. T a n n e r : Schweiz. Z e i t s c h r . f. O b s t - und Weinbau, 102, 261-267 (1966)

No. 3 1 , page 7
1965