ICAR-National Dairy Research Institute

Dairy Chemistry Division

M. Tech. Result Seminar

Studies on the detection of sorbitol addition in milk
using Thin-layer chromatographic approach

Major Advisor
Speaker
Dr. Vivek Sharma
Ranjith kumar N
Principal Scientist
M.Tech. Scholar
Dairy Chemistry Division
OBJECTIVES

1 To standardise a Thin-layer chromatographic method to

separate sorbitol in aqueous system and application of
standardised TLC method in milk system.

2 Effect of formalin on the performance of standardised TLC

method for the detection of sorbitol in milk.
 TLC METHODS

Silica gel 60F (merck) impregnated

with copper Silica gel plates treated with Boric acid
Developing solvnet: n-propanol: Developing solvnet: n-
distilled water (4:1) propanol :distilled water (4:1)

(Hadzija et al., 1994) (Coles et al., 1972)

Detecting reagent: 0.5% potassium permanganate in 0.1M NaOH

 Scheme of optimisation of the TLC process

TLC plates were run by applying reported solvent system i.e. n-

propanol : water (4:1), results were analysed

Optimisation of the solvent system (different combinations of

solvent systems were tried)
Sl. No. Solvent system Ratio
1 n-propanol, ethyl acetate, ammonia and 4:2:2:1
water
2 n-Butanol: acetic acid: ethyl ether: water 9:6:3:1

3 n-propanol: acetonitrile: water (4:2:1)

4 n-propanol: ethyl acetate: water (4:2:1 )

5 n-propanol: ethyl acetate: water (7:1:2)

Cont…

Optimisation of impregnation time of the standardised Cu-

impregnated TLC (0.5 min, 1.5 min, 5 min and 15 min)

Optimisation of conditions in the milk system

 Preparation of Copper impregnated TLC plates

Sample
application

Silica gel 60F Cu- Cu-impregnated TLC

plate impregnation plates (dried in hot hair
solution oven @ 1000 C/ 2 hrs)

TLC plate with spots

 How does Copper impregnated TLC plates work?..

Glu
Sucrose

Lact

Manni

Malti

Sorb

Sorbitol-copper complex (Imamura et al., 1978)

1 2 3 4 5 6 7

Silica gel 60F plate with Cu-impregnation, 1-glucose, 2- lactose, 3-sucrose, 4-sorbitol, 5- mannitol, 6- maltitol,
and 7- mixture of 1, 2, 3, 4, 5 and 6.
 Activity 1

Optimisation of TLC conditions (Cu-impregnated) for sorbitol detection in pure system a) solvent system

Glu Mixture Sorb+malti

Glu Glu Sorb+
Manni malti Sorb+manni
Lact
Lact Lact
Malti

Sorb
Sorb Sorb

TLC chromatogram of sugars and polyols, on Cu-impregnated Silica gel 60F plate with n-propanol: distilled water (4:1)
as the solvent system; (i) plate (1) glucose (2) lactose (3) sorbitol (4) mixture of sorbitol+glucose+lactose+mannitol and
maltitol (5) mannitol (6) maltitol (0.2% each) The (ii) plate (1) Glu+sorb (2) lact +sorb (3) Glu + lact + sorb (4) Sorb +
malti (5) Sorb +manni (6) manni + malti (7) sorb+Manni+malti+glu+lact (8) Sorb
 The Optimized solvent system; n-propanol: ethyl acetate : water (7:1:2)

Glu
Sucrose

Lact

Manni
Malti

Sorb

1 2 3 4 5 6 7

TLC chromatogram of sugars and polyols on Cu-impregnated silica gel 60F plate with n-propanol: ethyl
acetate: water (7:1:2) as the solvent system (1) glucose (2) lactose (3) sucrose (4) sorbitol (5) mannitol
(6) maltitol (7) sorbitol+glucose+lactose+sucrose+mannitol+ maltitol (0.2% each)
 Optimisation of TLC conditions for sorbitol detection in pure system b) The impregnation time

Glu Glu
Sucrose
Manni
Lact Malti
Lact
Sorb Manni
Glu
Malti

Lact
Sorb
ManniMalti
Sorb

1 2 3 4 5 1 2 3 4 5 6 7

0.5 min 1.5 min 15 min

TLC chromatograms of 0.5 min Cu-impregnated plates (1) sorb (2) glu (3) lac (4) manni (5) malti; 1.5 min Cu-
impregnated plates (1) glu (2) lac (3) suc (4) sorb (5) manni (6) malti (7) sorb+glu+lact+sucrose+manni+ malti
(0.2% each) ; 15 min Cu-impregnated plates (1) glu (2) lac (3) sorb (4) manni (5) malti (6)
sorb+glu+lact+sucrose+manni+ malti (0.2% each)
 Effect of concentration of polyols on the standardised TLC method

mannitol
Sorbitol
0.2% 0.5%
2% 0.1% maltitol
0.1% 0.5%
0.2%
1%
0.5%
0.1%
0.2%

6 7 8 9 10 11
1 2 3 4 5

Silica gel 60F plate with Cu-impregnation, (1) 0.1%, (2) 0.2%, (3) 0.5%, (4) 1%, (5) 2%
sorbitol. (6) 0.1%, (7) 0.2%, (8) 0.5% mannitol (9) 0.1%, (10) 0.2% (11) 0.5% maltitol
 Standardised Copper impregnated TLC conditions

Plate: Silica gel 60F

Impregnation time: 1.5 minutes
Solvent System: n-propanol: ethyl acetate and water (7:1:2)
Sample size: 2µl
Run time: 2 hours
Drying temperature after Run: 70 0C/ 10 min
Detecting Reagent: Potassium permanganate 0.5% in 0.1M NaOH/
30sec
Final drying temperature: 650C/ 10 min
Standardisation of Boric acid impregnated TLC for the detection of sorbitol in aqueous and milk system

Glu
Lact Manni
sorb Malti

1 2 3 4 5 6
1 2 3 4 5 6
n-propanol: water (4:1) n-propanol: ethyl acetate: water (7:1:2)

TLC chromatograms of sugars and polyols on Boric acid impregnated plates (1) glucose, (2) lactose, (3)
sorbitol (4) mannitol (5) maltitol (6) sucrose
Cont…

1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8

n-Butanol: acetic acid: ethyl ether: water (9:6:3:1) 2-propanol, ethyl acetate, water, ammonia (4:2:2:1)

TLC chromatograms of sugars and polyols on Boric acid impregnated plates (1) glu+lac+sorb (2) sorb (3)
glu+lac+sorb+manni+malti (4) sorb+manni (5) sorb+malti (6) manni+malti (7) malti (8) manni
Activity 2
Effect of other carbohydrates and carbohydrate mixture on the separation behavior of the standardized TLC
method

Preparation of mixture of glucose, lactose, sucrose,

maltitol, mannitol and sorbitol in 1:1:1:1 ratio

Apply the sample of 2µl of each sample on the

activated Cu-TLC plate as stated in activity 1-’a’

Check the suitability of the standardised conditions of

TLC in the mixture
Detection of sorbitol in aqueous system (in the mixture of individual sugars and polyols) by the developed TLC method

Glu Lane No. Sugars concentration

Sucrose 1 Sorbitol + Glucose 0.5% each
Manni
2 Sorbitol + Sucrose 0.5% each
Lact 3 Sorbitol + Lactose 0.5% each
Malti 4 Sorbitol + Mannitol 0.5% each
sorb 5 Sorbitol + maltitol 0.5% each
6 Mixture of glucose, 0.2% each
lactose, sucrose, sorbitol,
mannitol and maltitol

1 2 3 4 5 6
Activity 3

a) Separation and identification of sorbitol extracted from sorbitol spiked milk

Procedure for the analysis of milk for sorbitol adulteration by

Standardised TLC method
 Hot hair oven@
700 C/ 10 min
Milk sample 2 ml Acetone 4 ml
Alkaline KmNO4/30 sec
Dried again
@ 650C/
10-12 min
TLC chamber/2hrs

Milk+ acetone The filtrate obtained

was diluted to, 1:2, 1:3
and 1:4

Vortex mixing/ 5 min Centrifugation @ 1500 RPM / 5 min Sample application-2µl Test results
 Lactose

sorb

1 2 3 4 5

TLC chromatograms of sorbitol in milk without dilution 1) sorbitol standard (0.2%), 2) sorbitol in milk
(0.5%), 3) sorbitol in milk (1%), 4) sorbitol standard (1%), 5) sorbitol in milk (1%)
 Detection of Sorbitol in the milk system ( individual sugars)

i ii

sorb

1 2 3 4 5 6 1 2 3 4 5 6

TLC chromatograms of (i) cow milk (1:3 dilution ) (ii) buffalo milk spiked (1:3 dilution ) with (1) glucose (2)
sucrose (3) blank (4) sorbitol (5) mannitol (6) mannitol (0.5% each)
Activity 3

b) Effect of other sugars and sugar alcohol on the separation of sorbitol in milk filtrate
obtained from the spiked milk sample

Glucose, sucrose, mannitol, maltitol was spiked in conjunction with sorbitol in milk

Sample number Adulterant(0.5 and 1%) ratio

1 Sorbitol +glucose 1:1

2 Sorbitol +sucrose 1:1

3 Sorbitol +maltitol 1:1

4 Sorbitol +Mannitol 1:1

5 Sorbitol +glucose+ maltitol+ Mannitol +sucrose 1:1: 1:1:1

 Detection of sorbitol in the milk system (in the mixture of individual sugars and polyols)

Lane. NO. Sugars Concentration

Glu
1 B -
Sucrose
2&3 Sorbitol and glucose 0.5% and 1% each
B
Lactose 4&5 Sorbitol and sucrose 0.5% and 1% each
Manni Malti
6&7 Sorbitol and 0.5% and 1% each
Sorb Sorb mannitol
8&9 Sorbitol and maltitol 0.5% and 1% each

1 2 3 4 5 6 7 8 9 10 11
10 &11 All the sugars 0.5% and 1% each
Buffalo milk
Cont…

1 2 3 4 5 6 7 8 9 10 11

Cow milk
Objective 2
Effect Of Formalin On The Performance Of Standardised TLC Method For The Detection Of
Sorbitol In Milk

Cow and buffalo milk samples were spiked with

varied concentration of sorbitol i.e. 0.5 and 1%
and mixture of sorbitol, mannitol and maltitol

Each of these spiked milk samples were further

spiked with 0.4% formalin.

Samples were stored in both room and

refrigerated temperature (40C)

Samples were analysed by the standardised TLC

method on 1st day as well as 30th day of
storage.
Effect of formalin on the performance of the standardised TLC method ( 1 st day of storage)

1 2 3 4 2 3 4 1 2 3 4 2 3 4

Ref Room Ref Room

Cow milk Buffalo milk

TLC chromatograms of milk samples added with 0.4% formalin, (1) blank, (2) 0.5%, (3) 1%
sorbitol and (4) mix-sorbitol, mannitol and maltitol (0.5% each)
Effect of formalin on the performance of the standardised TLC method ( 30 th day of storage)

1 2 3 4 2 3 4 1 2 3 4 2 3 4
Ref Room Ref Room
Cow milk Buffalo milk

TLC chromatograms of milk samples added with 0.4% formalin, (1) blank, (2) 0.5%, (3) 1%
sorbitol and (4)mix-sorbitol, mannitol and maltitol (0.5% each)
To check the effect of urea on the developed TLC method and comparison with the Colour based method.

Both Cow and Buffalo milk samples Finally, the results obtained were
were spiked with 0.2, 0.5 and 1 % compared with the colour based
urea. method, developed by Madhavi,
2021.

Impact of urea adulteration on

Each of those urea spiked milk
analysing of sorbitol was
samples were further spiked with
determined by using standardised
0.2-1% of sorbitol
TLC method
Effect of Urea on the performance of the standardized TLC method and comparison with the Colour- based method

1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6

0.5% Urea 1% Urea

0.2% Urea

TLC chromatogram of urea spiked milk , (1) blank, (2) 0.1% (3) 0.2% (4) 0.5% sorbitol (5)1% sorb (6)
mixture of sorbitol, mannitol and maltitol
 Effect of Urea (0.5%) on colour based method to detect sorbitol in milk

1 2 3 4 5

Colour based method to detect sorbitol in milk (Madhavi, 2021) 1) blank (2) 0.2% and (3) 0.5 % (4) 1%
(5) Mixture of sorbitol, mannitol and maltitol (0.5% each)
To determine the effect of acidity of milk and neutralisation on the standardised TLC method and its comparison with
colour-based method.

Cow and buffalo milk samples were kept in room temperature for 6-7 hours to reach
the acidic pH of (5.9-6.04), pH and acidity of milk were noted.

These acidic milk samples were then spiked with 0.2, 0.5, and 1% of sorbitol,
mixture of polyols and analysed by using standardised TLC method

The same acidic milk samples were neutralised to natural pH of milk i.e. pH 6.7 and
analysed using standardised TLC method.

Neutralised milk samples were further neutralised to pH 7.0 by 0.1 N NaOH, and
analysed for sorbitol using developed TLC method and results were compared with
colour based method.
 Effect of Acidity on the standardised TLC method for detection of Sorbitol

1 2 3 4 5 6
1 2 3 4 5 6

Cow milk (pH 5.9) Buffalo milk (pH 6.04)

TLC chromatogram of acidic milk , (1) blank, (2) 0.1% (3) 0.2% (4) 0.5% sorbitol (5)1% sorb (6)
mixture of sorbitol, mannitol and maltitol
Cont…

1 2 3 4 5

Buffalo milk- pH 6.04

Colour based method to detect sorbitol in milk (Madhavi , 2021) 1) blank (2) 0.2% and (3) 0.5 % (4) 1%
(5) Mixture of sorbitol, mannitol and maltitol (0.5% each)
Effect of neutralization on the standardised TLC method for detection of sorbitol and comparison with the colour based
method

1 2 3 4 5 6
1 2 3 4 5 6 1 2 3 4 5 6

Cow milk (pH 6.72) buffalo milk (pH 6.74) cow milk (pH 7)

TLC chromatogram of neutralised milk , (1) blank, (2) 0.1% (3) 0.2% (4) 0.5% sorbitol (5)1% sorb (6)
mixture of sorbitol, mannitol and maltitol
Cont…

1 2 3 4 5

Buffalo milk- pH 6.74

1 2 3 4 5

Cow milk- pH 6.72

Colour based method to detect sorbitol in milk (Madhavi, 2021) 1) blank (2) 0.2% and (3) 0.5 % (4) 1%
(5) Mixture of sorbitol, mannitol and maltitol (0.5% each)
Effect of sorbitol addition to milk on the Lactometer reading/ SNF content of the milk

Lactometer reading of fresh cow and buffalo milk was recorded.

Both the milk samples were diluted with 11, 25, and 43% of water and their
lactometer readings were recorded.

Both the milk samples were diluted with 11, 25, and 43% with 10% sorbitol solution
instead of water as in above step and their lactometer readings were recorded.

The amount of sorbitol that should be added to compensate the Lactometer reading
was calculated

Milk samples diluted with sorbitol solution were anlysed by the standardised TLC
method and results were compared with the colour based method
 Effect of sorbitol concentration on the Lactometer reading

Milk quantity (in ml) Dilution rate Sorbitol content LR @ 27

250 0 0 30
225 25 ml (11%) 0 22
200 50 ml (25%) 0 20
175 75 ml (43 %) 0 -

Milk quantity (in ml) Dilution by 10% sorbitol Sorbitol content LR @ 27

250 0 0 30
225 25 ml (11%) 2.5 g 27
200 50 ml (25%) 5g 28.5
175 75 ml (43 %) 7.5g 32

Cow milk
Cont…

Milk quantity (in ml) Dilution rate Sorbitol content LR @ 27

250 0 0 33
225 25 ml (11%) 0 23.5
200 50 ml (25%) 0 20
175 75 ml (43 %) 0 -

Milk quantity (in ml) Dilution by 10% sorbitol Sorbitol content LR @ 27

(% dilution)
250 0 0 30
225 25 ml (11%) 2.5 g 27
200 50 ml (25%) 5g 28.5
175 75 ml (43%) 7.5g 32

Buffalo milk
 Effect of sorbitol concentration on the Lactometer reading

1 2 3 4 5 6 1 2 3 4 5 6
Cow milk Buffalo milk

TLC chromatograms of milk diluted with 10% sorbitol solution (1) blank (2) sorbitol 1.1% (3) 2.5%
and (4) 4.3 % (5) Sorbitol standard (0.5%) (6) lactose standard (0.5%)
Cont…

1 2 3 4

Cow milk

1 2 3 4

Buffalo milk

Colour based method to detect sorbitol in milk (Madhavi, 2021) 1) blank (2) sorbitol 1.1% (3) 2.5% and (4) 4.3 %
 Analysis of Market Milk samples of both renowned brands and unbranded for sorbitol adulteration

1 2 3 4 1 2 3 4 5 6

Brand- A and B Brand- C , D and Brand- E

TLC chromatogram of market milk samples (1) and (2) Brand A (3) and (4) Brand B (5) and (6) Brand C (7)
and (8) Brand D (9) and (10) Brand E
Cont…

1 2 3 4
1 2 3 4 5 6
Fresh Cow milk, Buffalo milk and
Unorganised 1 and 2
mixture of cow and Buffalo milk

TLC chromatogram of market milk samples (1) and (2) Fresh cow milk (3) and (4) Fresh buffalo milk (5) and
(6) mixture of cow and buffalo milk (7) and (8) unorganised-1 (9) and (10) unorganised-2
Analysis of Market Milk samples of both renowned brands and unbranded for sorbitol adulteration by colour-based
method (Madhavi, 2021)

1 2 3 4 5

6 7 8 9 10

Colour based method to detect sorbitol in milk (Madhavi, 2021) 1) fresh cow milk (2) fresh
buffalo milk (3) cow + buffalo milk and (4) brand A (5) Brand B (6) Brand C (7) Brand D (8)
Brand E (9) unorganised-1 (10) unorganised -2
 Summary and Conclusion

• The optimised Cu-impregnated TLC conditions were, impregnation of silica gel 60F with
Cupper for1.5 min use of n-propanol: ethyl acetate: water (7:1:2) as developing solvent.
Sample size was 2 µl, TLC run time was 2 hours and detecting reagent used was 0.5 %
potassium permanganate in 0.1 M NaOH
• Due to Undesirable results the boric acid treated TLC was discontinued from the study
• Standardised TLC method is simple and multiple samples can be applied simultaneously
• Sorbitol can be detected in the presence of glucose, sucrose, lactose and polyols such as
mannitol and maltitol in the milk
• This method can also be used to detect commonly adulterated sugars such as glucose and
sucrose, along with sorbitol
• The pH of the sample has no effect on the standardised TLC method
• The preservative, Formalin and Urea has no effect on the standardised TLC method
• The test can detect minimum of 0.2% of sorbitol in milk
• None of the market milk samples tested were found to be adulterated with the sorbitol
 TLC plate without Cu-impregnation

Sorbitol

1 2 3 4 5

Silica gel 60F plate without Cu-impregnation, 1- Glucose, 2-lactose, 3-sorbitol, 4- mannitol, 5- maltitol
 Effect of mannitol and maltitol (polyols) on the separation of sorbitol

Lane No. Sugars Concentration

1 Sorbitol and maltitol 0.2% each

2 Sorbitol and maltitol 0.5% each

3 Sorbitol and maltitol 1% each

4 Sorbitol and mannitol 0.2% each

5 Sorbitol and mannitol 0.5% each

6 Sorbitol and mannitol 1% each

1 2 3 4 5 6
 To detect sorbitol concentration in milk by using Colour based method

1 ml of sorbitol spiked milk and 2 ml of acetone is taken in a centrifuge tube

Vortex the above mixute for 5 minutes and centrifuge it at 1500 RPM / 5 minutes

1 ml of each filtered supernatant and boric acid was taken in a test tube and mixed
properly

60l of mixed dye prepared from methyl red and methylene blue is added

Formation of green colour indicates absence of sorbitol, brownish to purple colour

indicates the positive test for sorbitol.
Activity 2
a) Separation of sorbitol extracted from sorbitol spiked milk

Cow and buffalo milk were spiked individually with

sorbitol and mixture of sorbitol and other sugars such
as glucose, sucrose, mannitol and maltitol

2ml of sugars spiked milk and 4 ml of acetone were

taken in 15 ml centrifuge tubes, vortexed for 5
minutes and centrifuged at 1500 RPM/ 5 min.

Supernatant obtained was filtered through Whatman

no.42 filter paper.

Filterate was diluted in 2 dilutions, 1:3 and 1:4 i.e. 1

ml of filterate was diluted with 3 ml of water.
To determine sorbitol adulteration in Market milk, unbranded milk by using standardised TLC method and comparing
with the Colour based method

Market milk samples ( toned) from brands such as AMUL, Modern Dairy, Mother
Dairy, Verka, Vita and unbranded (2 samples) were collected

2 ml of each of these milk samples were mixed with 4 ml of acetone in 15 ml

centrifugal tubes and vortexed for 5 minutes , and then centrifuged 1500 RPM for 5
minutes

Supernatent obtained was filtered and diluted to 1:3 and 1:4 ratio with distilled water.

Diluted samples were applied on standardised TLC plate and results were analysed
and compared with the colour based method develped by Karra, 2021.
 Contents

Introduction

Results and discussion

Conclusion
Sorbitol is used as an artificial sweetening agent in milk.
Apart from this, sorbitol is also used, in combination or
alone, to increase the SNF content in the milk. Easy
availability and cheap cost of sorbitol are other factors
which render it to be used as an additive in milk.(Sen et
al.,2016)
Sorbitol is added to milk to increase the specific gravity
of milk and is one of the emerging adulterants affecting
the dairy sector (Nuzhat et al.,2018)

(Nizamani,2014)
Cont…

Sugars Concentration
Lane.
No.
1 Sorbitol and maltitol 0.5% and 0.2%, respectively

2 Sorbitol and mannitol 0.5% and 0.2%, respectively

3 Sorbitol, mannitol and maltitol 0.5%, 0.2% and 0.2%,

respectively

4 Sorbitol, mannitol and maltitol 0.5% each

5 sorbitol 0.5%

6 Maltitol 0.5%