POLARIMETRY

Approximately 25% of all drugs are marketed as either racemates (mixtures of two enantiomers) or mixtures of diastereomers [1]. Whenever a carbon atom has four different groups bonded, two different molecules can be formed which are like mirror images. An example of an enantiomer is the amino acid alanine which occurs as L-alanine (Fig. 1 left) or D-alanine (Fig. 1 right) [2].

Fig. 1: Enantiomers of alanine [2]. If a compound contains two chiral atoms, it may exist in four stereoisomeric forms [3]. Since the configuration at each chiral carbon may be either R or S, there are four stereochemical possibilities: RR, SS, RS, and SR. The RR and SS stereoisomers are enantiomers. The RS and SR stereoisomers are also enantiomers. The RR stereoisomer is a diastereomer of both the RS and the SR stereoisomers. The SS stereoisomer is a diastereomer of both the RS and SR stereoisomers. Confused? Fig. 2 should make things a bit clearer. It shows projections of the four stereoisomers of 2-chloro-3-fluorobutane.

Fig. 2: Stereoisomers of 2-chloro-3-fluorobutane [3].

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The orientation around a chiral center can have a dramatic impact on the pharmacological response of that drug in the human body [1]. A worst case scenario is one which the non desired enantiomer causes serious toxicity. The drug Thalidomide was prescribed to millions of women to suppress morning sickness associated with pregnancy during the late 50s and early 60s. Horrible birth defects, including missing limbs, resulted. The cause of these birth defects was assigned to the S-thalidomide, which did not undergo clinical trials. This assessment is currently being questioned. None the less, this brought about severe tightening in the laws surrounding the introduction of new drugs. Chiral synthesis and purification is a crucial aspect of all successful drug manufacture.

Chiral molecules have an asymmetrical center which responds to light as a lens and rotates the plane of the light. The ability to rotate light is termed as optical activity. Enantiomeric compounds rotate light by exactly the same amount but in the opposite direction. The degree to which a substance rotates light may be used to determine: a) the identity of the substance, b) the enantiomeric purity of the substance or c) the concentration of a known substance in a solution. In order to observe rotation, the light which is passed through the solution must be plane polarized. Ordinary light has (Fig. 3 left) waves which are oriented in all directions. Plane polarized light (Fig. 3 right) is made up of waves which are oriented parallel to a defined plane. The polarized light can be obtained by reflection, refraction or double refraction.

Fig. 3: Ordinary and plane polarized light.

Polarimetry is a sensitive, non-destructive technique for measuring the optical activity exhibited by inorganic and organic compounds [4]. A compound is considered to be optically active if plane polarized light is rotated when passing through it. The amount of optical
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rotation is determined by the molecular structure and concentration of chiral molecules in the substance.

The term specific rotation

has been established in order to be able to compare the

rotation of individual substances. The specific rotation is given by the following equation: (1) where t is the temperature (C), is the measured angle of rotation of the polarized light plane (), w is the concentration of the dissolved compound (the mass fraction of the dissolved substance) (%), is the density of the solution (kg.m-3) and d is the length of the sample tube (m). The specific rotation is a typical property of an optically active substance at a given temperature (t; usually t = 20 C) and a given wavelength () of the used radiation. According to equation (1), the specific rotation equals to the angle of rotation of the polarized light plane which was induced by a 10 cm thick layer that contains 1 g of an optically active compound in 1 cm3 of a solution. In this laboratory exercise you will work with solutions of saccharose. The specific rotation of an aqueous solution of saccharose is .

From equation (1) we can calculate the concentration of the dissolved substance: (2) A polarimeter is a device used for the measurement of optical rotation and its scheme is depicted in Fig. 4.

Fig. 4: Components of a polarimeter [5].

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The main components of the polarimeter are polarizer (Nicol prism), analyzer (Nicol prism) and the sample tube [6]. The polarizer is used to obtain polarized light. The analyzer is used to measure the angle of rotation. The sample tube should be filled with the sample in a way that after the filling a convex surface of the liquid is formed on the tube thread. This surface is then cut off with a lens. This procedure should be performed fast in order to avoid the formation of gas bubbles in the sample tube. After screwing the tube thread the filling of the tube is checked, the tube is wiped and inserted into the polarimeter.

The polarimeter used in this exercise has two scales. On the left side is the circular scale which is divided into 360 (Fig. 5). One division of the circular scale corresponds to = 1. On the right side is the nonius scale which has a division from 0 to 10. One division of the nonius scale corresponds to 0.05. When reading the measured value look first between which divisions of the circular scale is the zero value of the nonius scale .Read the smaller value of the two values; this will be the integer value of . According to Fig. 5 this value is 2. The tenths and hundredths of will be read from the nonius scale. This should be done by finding a division on the nonius scales which is exactly opposite to a division on the circular scale. According to Fig. 5 this value is 0.85. Finally, add these two values and you have the final value of , following Fig. 5 = 2 + 0.85 = 2.85.

Fig. 5: Scales of a polarimeter [6].

What about measuring with a polarimeter? The first step is to set up the zero value on the nonius scale exactly opposite to the zero value on the circular scale. This should be done by using the polarimeter screw which is under the ocular. The second step is to insert the sample
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tube (filled with the sample) into the polarimeter. The field of vision observed in the ocular is depicted in Fig. 6a. This position is denoted as the first extreme position. Then, turn the polarimeter screw left till you wont observe a change in the field of vision which is depicted in fig. 6c. This position is denoted as the second extreme position. The last step is to find the medium position by which we read the value of . Turn the polarimeter screw slowly backwards till you wont observe the field of vision which is depicted in Fig. 6b.

Fig. 6: Different fields of vision observed during a measurement. Aim of the work: 1. Measuring of angle of rotation () and calculation of concentrations of prepared saccharose solutions. 2. Determination of density using a densimeter.

Equipment: polarimeter, pipettes, densimeter, filter paper

Chemicals: saccharose solutions, distilled water

Experimental procedure: 1. Wash the polarimeter sample tube with a small amount of the measured solution and fill the sample tube with the solution. Close and wipe the sample tube and check that the solution contains no bubbles. 2. Measure five times the angle of rotation of the first solution and write down the values in Tab. 1.

3. 4.

Pour out the solution into the storage bottle and apply the same procedure for the other solutions. When finished measurements determine the angle of rotation without the sample tube inserted (0). The value of 0 should be close to zero. Write down the value of 0 in Tab. 1.

5. 6. 7.

Write down the room temperature. Write down the length of the sample tube d. When finished all measurements disamntle the sample tube, carefully wash all parts of the tube with distilled water, dry and place them.

Processing of the measured data: Calculate the following data and write them down in Tab. 1: The average values of angles of rotation calculated from the measured values.

The corrected values of angles of rotation calculated according to the equation: (3) The values of concentrations of the dissolved substance according to the equation (2). Determine the densities of the solutions by means of a densimeter. The procedure of determination of density using the densimeter is following: 1. Wash the graduated cylinder with distilled water. Fill the graduated cylinder with distilled water. The height of the water level should be 7 10 cm below the rim (Fig. 7).

Fig. 7: Measuring with a densimeter [6].

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2. Immerse slowly and carefully the densimeter into the cylinder. Read the value of density. It is the value on the scale of the densimeter which is equal to the level of the measured liquid. 3. Pour out the distilled water into a beaker and wash the graduated cylinder with a small amount of the first measured solution. Fill the graduated cylinder with the first solution and insert the clean and dry densimeter. Read the value of density. When finished the measurement pour the solution into the storage bottle. 4. Apply the same procedure for the other solutions. Write down the measured values in Tab. 1.

Table 1 t= C d= m =

Solution

1-5 [] [] [] -

[kg.m-3]

w [%]

Distilled water 1 2 3

References

1. http://www.xula.edu/chemistry/documents/orgleclab/StereoPolar.pdf 2. http://www.daviddarling.info/encyclopedia/E/enant.html 3. http://www.usm.maine.edu/~newton/Chy251_253/Lectures/Chirality/Chirality2.html 4. http://www.spectronic.co.uk/polarimeters/polardef.htm 5. http://www.standardbase.hu/tech/SITechPolar.pdf 6. J. Oremusov, K. Sarka, M. Vojtekov, Fyzika Laboratrne cvienia pre farmaceutov, Univerzita Komenskho v Bratislave, 2009, p.42 and 98.

Manual written by Ing. Martin Grf, PhD.