polarimetry method

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ANALYSIS OF FOOD AND NATURAL PRODUCTS
LABORATORY EXERCISE
Determination of Starch Content
(Ewers’ Polarimetric Method)
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Exercise guarantor: Assoc.Prof. Ing. Marek Doležal, Ph.D.

CONTENT
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Required knowledge ............................................................................................................................ 3
Evaluative criteria ................................................................................................................................ 3
Laboratory exercise content ................................................................................................................. 3
Laboratory exercise schedule ............................................................................................................... 4
Introduction—Method Principle ...................................................................................................... 4
Introduction—Polarimetry ............................................................................................................... 4
Experimental set-up—Preparation of solutions ............................................................................... 6
Experimental set-up—Sample preparation and polarimetric measurement ..................................... 6
Objectives......................................................................................................................................... 6

Required knowledge
1. Starch – chemical structures of amylose and amylopectin.
2. Conditions of starch hydrolysis.
3. Polarimetric methods: principle of measuring the angle of polarisation or optical rotation,
basic notions, related calculations.
4. Solutions – basic calculations.
Evaluative criteria
- proved theoretical knowledge
- quality of practical work execution
- compliance with laboratory work rules (safety, order)
- protocol – integrity of experimental data, results calculation and discussion
Laboratory exercise content
A. Determination of dry matter in cereal products, in accordance with standards for
cereals and cereal products (based on ČSN ISO 712; adapted for teching purposes)
B. Determination of Starch Content after Ewers (based on ČSN 56 0512-16; adapted for
teching purposes)
C.
Specification:
� Each student analyzes own sample (each student performs two determinations)
� Starch content is determined in various cereal products
� Water content is determined in various cereal products

Introduction—Method Principle
Laboratory exercise schedule
TASK DURATION (min) Note
Introduction and testing 30 min
Weighing of samples 30 min
Hydrolysis of samples 30 min
Drying of samples 120 min runs in parallel to hydrolysis
Determination of starch 30 min
Determination of water content 20 min
Cleaning of the working space 20 min
In the polarimetric (or Ewers’) method, the starch is released from the sample by boiling in
dilute hydrochloric acid (HCl). This procedure effectively gelatinises the starch granules and
simultaneously hydrolyses the starch to glucose in a single step. The acid also helps to break
down the endosperm tissue, ensuring complete release of the starch granules from the protein
matrix. Substances, which may interfere with the measurement, are removed by
filtration/clarification and then glucose concentration is determined by measuring the angle of
polarisation or optical rotation. Due to its simplicity this is a relatively inexpensive method.
Introduction—Polarimetry
Molecules that contain an asymmetric carbon atom (chiral compounds) have the ability to rotate
plane-polarised light. A polarimeter is a device that measures the angle that plane-polarised light
is rotated on passing through a solution. A polarimeter consists of a source of monochromatic
light, a polariser, a sample cell of a known length, and an analyser to measure the angle of
rotation (Fig. 1).

Light
source
Normal
light
Figure 1: Polarimeter schematics.
Polariser Plane-polarised
light
Direction of light propagation
Sample tube
containing a chiral compound
Plane-polarised Analyser
light
Detector
The extent of polarisation is related to the concentration of the optically active molecules in
solution by the Eq. 1
t �� � � �c
� � � , (1)
�
where � is the measured angle of rotation, � � t
�
� is the optical activity (which is a constant for each
type of molecule), ℓ is the path length and c is the concentration. The overall angle of rotation
depends on the temperature and wavelength of light used and so these parameters are usually
standardised (e.g. 20°C and 589.3 nm (the D-line for sodium)).

Experimental set-up—Preparation of solutions
� Solution A—1.124% HCl solution: Add approx. 300 mL of distilled water to a 500 mL
volumetric flask; add 13.2 mL of HCl (36%) by a pipette. After mixing, make up the 500 mL
by distilled water. (Note: this procedure must be done carefully in a working digester)
� Solution B—30% ZnSO4 solution (Carrez I): 53.4 g ZnSO4·7 H2O and 46.6 mL of distilled
water
� Solution C—15% K4[Fe(CN)6] solution (Carrez II): 17.2 g K4[Fe(CN)6] · 3 H2O and 82.8 mL
of distilled water
Experimental set-up—Sample preparation and polarimetric measurement
A portion of 5 g of a homogenised sample is weighed in a 100 mL Kohlrausch volumetric flask
and its content is mixed with 25 mL of 1.124% HCl solution (solution A). After addition of another
25 mL of 1.124% HCl solution (solution A), the suspension is heated on a boiling water bath for 15
min (after 3 min the content of a volumetric flask is mixed to avoid coagulation). Once the
hydrolysis is finished, 20 mL of 1.124% HCl solution (solution A) is added. After fast cooling
(using a stream of flowing water), clarification using 5 mL of Carrez I (solution B) and 5 mL of
Carrez II (solution C) solutions is performed (note: for each solution use a separate pipette).
Finally, a volumetric flask is filled up by distilled water, its content is properly mixed, and filtrated
using a filtration funnel. The obtained filtrate is then transferred to a polarisation tube (2 dm) and
measured using a polarimeter.
The obtained value is firstly corrected for a laboratory temperature (t) drift using Eq. 2
corrected
measured
��S� � 0.
0144�t
� � ��S� � � �
20 , (2)
followed by multiplying by a factor of 0.3462.
The amount of starch (X) in the sample is calculated using Eq. 3
X �
4
10
t
�
��
�� � � � � m
, (3)
where � is calculated value of optical rotation, � � t
� � is the optical activity (specific rotation)
depending on the discharge lamp and wavelength of light used and variety of starch, ℓ is the path
length (2 dm), and m is the sample weight (5 g). For a mercury discharge lamp and a wavelength
(�) of 546.1 nm, the � � t
� � values are 214.7, 216.3, 213.3, 213.1, 218.5, 217.0, and 215.5 for
wheat, rye, barley, oaten, rice, maize, and unknown cereal starch, respectively. (Note: the
correction for moisture content is not taken into account in this equation)
Objectives
� Prepare A, B, and C solutions.
� Prepare the sample for the polarimetric measurement.
� Calculate the starch content in the analysed sample.