CHEM 334L Organic Chemistry Laboratory Revision 1.0 Isolation of Lactose from Milk In this experiment we will isolate the carbohydrate Lactose [β-d-galactopyranosyl-(1,4)- D-Glucose] from non-fat powdered Milk. We will then determine if Lactose is a reducing or non-reducing Sugar. Finally, we will construct models of Lactose and its constituent monosaccharides to better understand its chemistry. Lactose Milk and honey are two of the few substances with the sole purpose of being a food. Milk is probably the most nutritionally complete food that can be found in nature. Whole milk contains vitamins, minerals, proteins, carbohydrates and lipids. The only important elements in which milk is seriously deficient are Iron and Vitamin C. Whole milk is an oil-water type of emulsion, containing about 4% fat dispersed as very small (5-10 micron diameter) globules. The globules are so small that a drop of milk contains about a million of them. Because the fat in milk is so finely dispersed, it is digested more easily than fat from any other source. The fat emulsion is stabilized to some extent by complex phospholipids and proteins that are absorbed on the surface of the globules. The fat globules, which are lighter than water, coalesce on standing and eventually rise to the surface of the milk, forming a layer of cream. Since Vitamin A and Vitamin D are fat soluble vitamins, they are carried to the surface with the cream. Commercially, the cream is often removed by centrifugation and skimming and the milk that remains is called Skimmed Milk. Skimmed milk, except for lacking the fats and Vitamins A and D, has approximately the same composition as whole milk.
P a g e 2 There are three kinds of globular proteins in milk; caseins, lactalbumins, and lactoglobulins. Casein is a phosphoprotein, meaning that phosphate groups are attached to some of its amino acid side-chains. These are attached mainly to the hydroxyl groups of the serine and threonine moieties forming a type of alkyl phosphate. Casein exists in milk as the calcium salt, calcium caseinate. The salt has a complex structure which forms soluble micelles. Calcium caseinate has its isoelectric (neutrality) point at ph 4.6. Therefore, it is insoluble in solutions of ph less than 4.6. The ph of milk is about 6.6; thus, casein has a negative charge at this ph and is solubilized as a salt. If acid is added to milk, the negative charges on the outer surface of the micelles are neutralized and the neutral protein precipitates. Albumins are globular proteins that are soluble in water and in dilute salt solutions. They are, however, denatured and coagulated by heat. The second most abundant protein types in milk are the lactalbumins. Once the caseins have been removed, and the solution has been made acidic, the lactalbumins can be isolated by heating the mixture to precipitate them. A third type of protein in milk is the lactoglobulins. These are present in smaller amounts than the albumins and generally denature and precipitate under the same conditions as the albumins. The lactoglobulins carry the immunological properties of milk. They protect the young mammal until its own immune system has developed. When the fats and proteins have been removed from milk, the carbohydrates remain, as they are soluble in an aqueous solution. The main carbohydrate in milk is Lactose. Lactose, also called Milk Sugar, is not found in plants, and is one of the less sweet sugars. Lactose, being only about 1/6 as sweet as Sucrose, is the reason why milk has a rather bland taste. This sugar is only formed in the mammary glands of lactating mammals and is the only carbohydrate that mammals can synthesize. Sugars, also known as Saccharides (derived from the Greek σακχαρον), are a simpler form of carbohydrate that are frequently used as a food, or energy source. Because their chemical formulas often occur as C n (H 2 O) n, they were once thought of as Hydrated Carbon; (e.g., the sugar Glucose has a chemical formula of C 6 H 12 O 6 = C 6 (H 2 O) 6. Lactose is a disaccharide, meaning it can be hydrolyzed into the two simpler sugars D-Glucose and D-Galactose. Within Lactose these monosaccharides are linked via the number one Carbon of Galactose, in its β configuration, and the number four Carbon of Glucose. This linkage is referred to as a β1,4 glycosidic linkage and is really nothing more than the reaction product of a hemiacetal and an alcohol; an acetal. This acetal linkage is relatively stable in an aqueous environment. Nutritionally, the enzyme Lactase is requires to break-down Lactose and it is produced in the small
P a g e 3 intestine. Lactase will break Lactose into its Glucose and Galactose components. Lactose can be removed from whey (milk without fats and proteins) by adding Ethanol. Lactose is insoluble in Ethanol, and when the Ethanol is mixed with the aqueous solution, the Lactose is forced to crystallize. Finally, it should be noted that as represented above, the number one Carbon of both Glucose and Galactose exist as hemiacetal functionalities. This functionality is labile and can exist in equilibrium with the aldehyde and alcohol that form it. Thus, a molecule such as Glucose can exist either as a free aldehyde or a hemiacetal: (Open Chain Form of Glucose) (Pyranose Form of Glucose) It should also be noted that when the hemiacetal forms, it can form in two different stereochemical configurations, the so-called α-anomer and the β-anomer:
P a g e 4 When in equilibrium, in an aqueous environment, approximately 60% of Glucose molecules exist as the β-anomer, 40% exist as the α-anomer and less than 1% exist as the free aldehyde. However, it is the free aldehyde form that is important in terms of the redox chemistry of sugars. When treated with an oxidizing agent, such as Benedict's Reagent, the aldehyde is oxidized to a carboxylic acid. The copper of Benedict's Reagent is reduced to Cu +1 ; causing the color of the Reagent to change from the blue of the Cu 2+ form to a reddish/green of the Cu +1 form. RCHO + 2 Cu 2+ + 4 OH - blue RCOOH + Cu 2 O + 2 H 2 O red In this case, Glucose is said to be Reducing, because it causes the reduction of Benedict's Reagent. And, because of its color change, Benedict's makes for a nice test for Reducing sugars. If the hemiacetal had been converted to a glycosidic, or acetal, linkage, it could not exist in equilibrium with the free aldehyde and it would give a negative Benedict's test. Such sugars are said to be non-reducing. In this laboratory we will isolate lactose from non-fat powdered milk. We will then compare our results for the amount recovered to those listed on the nutrition label of the milk. In order to better understand the nature of the acetal and hemiacetal linkages which exist in a molecule of Lactose, we will also build a models of the simple sugars which comprise Lactose. These models will then be connected via an appropriate Glycosidic Linkage to form a model of a molecule of Lactose. Finally, we will perform a Benedict's Test on Lactose to determine if it is reducing or non-reducing. For comparative purposes, we will also perform the Benedict's Test on several other sugars.
P a g e 5 Pre-Lab Questions 1. Draw the α-anomeric form of Galactose. (The β-anomer is used in forming Lactose.) 2. Provide a mechanism for the acid catalyzed conversion of a hemiacetal into an acetal. 3. Do you expect Lactose to be reducing or non-reducing. Explain.
P a g e 6 Procedure Isolation of Lactose from Milk 1. Weigh out 10g of non-fat powdered milk in a weighing boat. Dissolve the milk in 100mL of warm water in a 250mL beaker. 2. Heat the milk solution to 40 o C. 3. Add 6mL of 10% Acetic Acid to precipitate out the Casein, and stir the mixture slowly and briefly. Avoid adding excess Acetic Acid. Work the Casein into a mass and remove it with a stirring rod or spoon. Place this in a trash can. 4. Immediately add 2.5g of powdered Calcium Carbonate. Stir the mixture thoroughly. 5. Heat the mixture almost to boiling for about 10 minutes; stirring continuously. This should precipitate the remaining proteins. 6. Filter the hot mixture, collecting the filtrate in a 250mL beaker. 7. Stir the filtrate continuously while boiling it down to about 10mL, and then add 50mL of 95% Ethanol. 8. Carefully heat the solution to 70 o C. (Ethanol boils at 78 o C.) 9. Filter the warm Ethanol solution, collecting the filtrate in a 125mL Erlenmeyer flask. Stopper the flask and place it in your lab drawer until the next lab period. (During the Next Lab Period) 10. Filter off the crystals of Lactose. 11. Allow them to dry for one hour. 12. Weigh the crystals. 13. Compare your percentage recovery of Lactose with the percentage carbohydrate as reported on the nutritional label of the powdered milk you used.
P a g e 7 Model of Lactose As has been noted, Lactose is a Complex Sugar comprised of the Simple Sugars Glucose and Galactose A Fisher Projection of each monosaccharide is provided below. 1. Build a model of the Open Chain form of both of these molecules. Make sure they are stereochemically correct. 2. Convert these to the Pyranose form for each Sugar. Observe both the α and β Anomers for each. 3. Form the Glycosidic Linkage between these two Simple Sugars to make Lactose. Recall, Lactose has a β-1,4 Glycosidic Linkage between the Galactose and Glucose. Reducing vs. Non-Reducing Sugars 1. For each of the following sugars: Glucose Lactose Fructose Maltose Sucrose test the sugar with Benedict's Solution to determine if it is a reducing or nonreducing sugar. In a 6" test tube, place 3mL of Benedict's Solution and heat to a gentle boil. Add 4-6 drops of the 2% sugar solution and continue to boil gently for a minute or two. Observe the results. A yellow, green or red color indicates the presence of a reducing sugar. If no reducing sugar is present, the solution remains clear.
P a g e 8 2. Examine the structure for each sugar for which you performed the Benedict's Test and determine if it is consistent with your test results. Comment.
P a g e 9 Post Lab Questions 1. What is the nature of the glycosidic linkage in Sucrose? 2. What is the carbohydrate composition of honey? 3. The carbohydrate Trehalose is a naturally occurring disaccharide. a) What are some natural sources of this disaccharide? b) What monosaccharides comprise this compound? c) What is the nature of the glycosidic linkage between the monosaccharide subunits? d) What is the biological purpose of this compound? 4. Solanine is a glycoalkaloid derived from plants of the nightshade family. Examine the structure of this compound. What is the anomeric configuration (α or β) of the sugar constituents of this compound?