CHEM2410

Determination of Lactose Content of Milk by Polarimetry

Before you come to lab

Review the operation of each type of polarimeter. Practice reading the vernier scale on the practice readings. Look
up the safety data for mercuric iodide and complete the other parts of the pre-lab.

Introduction
One of the easiest methods of determining the lactose concentration in milk is through polarimetry. For the method
to be successful, one must remove all optically active compounds except for the lactose (for instance the proteins).
The solution must be transparent enough for the polarized light to pass through readily. Setting the polarimeter to
the correct angle for the optical rotation value is tricky and must be done carefully to avoid erroneous values.

Procedure
Sample Preparation
Use a volumetric pipette to transfer 50.0 mL of milk to a 125 mL Erlenmeyer flask. Add 7.5 mL of concentrated
sulfuric acid to the milk (CAUSTIC!). To this mixture add 7.5 mL of a mercury iodide/potassium iodide solution
(TOXIC!). Note the precipitation of milk proteins. Filter the solution to remove this precipitate. Rinse the
Erlenmeyer and wash the protein solids with water to ensure that the dissolved lactose is transferred completely.
Transfer the filtrate to a 100 mL volumetric flask and carefully dilute the solution to the mark. You may discard the
solidified proteins.

Fill a clean polarimeter tube with the diluted milk solution. Use a pipette as necessary to fill as completely as
possible to avoid any air bubbles. Place the glass window (please don’t break it) over the top (once again
minimizing air bubbles) followed by the gasket. Screw the top on snugly but do not overtighten (too tight will stress
the glass and stressed glass interferes with polarized light).

Measuring the optical rotation

With the sample compartment empty, record the “zero” value. Place your filled sample tube in the polarimeter and
adjust the angle according to the directions on the attached sheet. Record the value of αobserved. A value less that 10°
is reasonable. Measure the length of the tube as accurately as possible (inside of window to inside of window).

Calculations
An aqueous solution of lactose at equilibrium (it is subject to mutarotation) has a specific rotation or [α] of +52.3°
(Merck Index, 11th edition, entry 5221).

(1) Use the following expression and solve for c to calculate the concentration of the diluted milk:
αobserved
[α] = --------------- where c is concentration in grams per mL, l is the path length in dm, [α] is the specific
c x l rotation, and αobserved is your measured value

If your “zero value” was not zero, then you must adjust you’re αobserved accordingly before using it in the above
equation.

(2) Use the concentration from calculation (1) to calculate the number of grams of lactose present in the diluted
milk (which is the same as grams in the undiluted milk).

(3) Use the grams of lactose from calculation (2) to calculate the percent concentration of lactose (g/mL) in the
original (undiluted) sample of milk.
Discussion
Consider your measurements and calculations- do you feel they are accurate? What part of the procedure actually
led to the greatest uncertainty/error in your results (be specific)? List a few assumptions required for this method to
be accurate (one of them is given above in the introduction!).
Answer the following questions:
1. If you (unknowingly) used less than 50 mL of milk, will your calculated value (of percent lactose in milk) be
greater than, less than, or same as the actual value based on this error? Explain your reasoning.
2. If you measured your tube as being shorter than it actually is, then will your calculated value (of percent lactose
in milk) be greater than, less than, or same as the actual value based on this error? Explain your reasoning.
3. If you (unknowingly) diluted your milk to more than 100 mL, then will your calculated value (of percent lactose
in milk) be greater than, less than, or same as the actual value based on this error? Explain your reasoning.
4. If the previous class had been using your polarimeter tube for an aqueous fructose sugar solution ([α] –92°) and
the tube was not thorough cleaned out, what effect will this likely have on your results?
(TA note: HgI2/KI soln prep: dissolve 40 g KI in 200 mL H2O, add 55 g HgI2, dilute to 500 mL, filter if necessary)

Operating the polarimeter

(The two types of polarimeters at ASU work similarly.)

1. Make sure the light source is on and adjusted to give the brightest image through the eyepiece. The Linos
polarimeter has a built-in light source. The Cenco-kern is a bit more difficult and will require you to adjust a
mirror to direct the light into the polarimeter properly.
2. Place your sample in the tube.
3. Focus the eyepiece so that the image is as sharp as possible.
4. Rotate the front ring until there is even illumination across the circle that you view through the eyepiece. On
the Linos polarimeter, the circle is split into two halves, whereby one half or the other will be darker until it is
properly set (see example below). In the Cenco-kern polarimeter, the circle is split by a “stripe” down the
middle whereby the stripe will either be lighter or darker (just like the other polarimeter) than the outside
portions until properly set (see example below)
5. Once set, read the vernier as carefully as possible. The Cenco-kern polarimeter has better resolution than the
Linos, but both give adequate results. You can follow the example below for reading the vernier and then
practice on the other readings.

Cenco-kern polarimeter view Linos polarimeter view- view split into

correct halves instead of thirds
adjustment

incorrect
adjustment

incorrect
adjustment