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Universiti Brunei Darussalam

BI2209 Cell biology and biochemistry

Identification of milk sugar by TLC and estimation of lactose

Hj. Mohammad Iqzan Bin Hj. Jamil 07b0879 B.Sc.Edu

The identification of milk sugars in this practical was done by using the TLC method (It is the abbreviation for Thin-Layer Chromatography). It involves the use of a plastic plate coated with a solid absorbent. Drops of each sample are placed on the plate. The plate is dipped into a running solvent. After a period of time, the plate will undergo certain procedures before the data can be collected.

Using TLC, we can identify what sugar is present in the milk sample. The identification of sugar will be done by comparing the milk sample Rf value with known sugar sample Rf values that will be done side-by-side on the TLC plate.

In the second part of the practical, we will then estimate the lactose that is present in the milk sample by titrating it with benedicts reagent. Then, we need to repeat the same procedure to a sample of lactose.

Methods and materials

As stated in the practical handout (no deviation from the suggested method).


Figure 1



1 2 3 4 5 6 7

Milk Glucose Galactose Fructose Sucrose Lactose Maltose

Distance travelled by the sample on the plate (cm) 2.70 5.30 4.55 5.35 4.55 2.60 3.65
Table 1

Distance travelled by the solvent on the plate (cm)

Rf value 0.273 0.535 0.460 0.540 0.535 0.263 0.369


Volume of boiled milk used when titrated with Boiled Milk 5 benedicts reagent ( ) 1st 2nd 3rd Initial 1.00 4.70 8.00 Final 4.70 8.00 11.30 Total 3.70 3.30 3.30 Table 2 Volume of standard lactose used when titrated with 5 benedicts reagent ( ) 1st 2nd 3rd 1.10 4.10 7.20 4.10 7.20 10.30 3.00 3.10 3.10
Table 3

Standard Lactose

Initial Final Total

Discussions By measuring the distance travelled by each sample and dividing with the distance travelled by the running solvent from Figure 1, we obtained the sets of Rf values for the samples of Milk, Glucose, Galactose, Fructose, Sucrose, Lactose, and Maltose. The Rf values for each sample were recorded into Table 1. Observing the Rf values for each sample of known sugar and comparing with the Rf value of the milk sample, we can see that only lactose sugar has the nearest Rf value to that of the milk sample. Whereas, the Rf values of other sugar samples are very far from the milk samples Rf value. Therefore, we can safely assume that the sugar that is present in the milk sample is lactose. In the second part of the practical, we recorded the volume of milk sample that was titrated with benedicts reagent until the blue colour disappears, given in Table 2. Then, the test was repeated with standard lactose, recorded in Table 3. From Table 3, 3.10 of standard lactose sample reacted with 5 of benedicts reagent. It was stated that the standard lactose has lactose concentration of 50 . 3.10 50 = 155 = 0.155 5 . 0.031 1 .

In Table 2, 3.30 of reacted with 5 of benedicts reagent. We knew that 0.155 g of lactose reacts with 5 of benedicts reagent. In 100 0.155 3.30 0.0470 = 4.7 100 100 0.155 3.30 4.697 %

In %

= In

47 = 12(12) 1(22) 16(11) 0.137

Sugar that is present in the milk sample is Lactose. Concentration of lactose sugar in milk is . , 4. %

and . .