chem assignment 11

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Experiment 11: CLASSIFICATION OF CARBOHYDRATES

Purpose: The differences in the molecular structure of some typical carbohydrates are utilized to distinguish one carbohydrate from another. An unknown carbohydrate is to be identified based on the three chemical tests described.

Introduction: The term carbohydrate (hydrate of carbon) refers to a class of organic, biochemical compounds with the general formula Cn(H2O)m, also known as sugars. It was thus named because heating these compounds produced C and H2O molecules. In actuality, there are no water molecules in the structure. Instead, the “H2O” is attached to the C atoms as –H and −OH groups, and these molecules are best considered as either a polyhydroxy aldehyde (aldose) or polyhydroxy ketone (ketose). D-glucose is an example of an aldose, and D-ribulose is an example of a ketose. Since glucose contains 6 C atoms, it is an aldohexose, and ribulose, with 5 C atoms, is a ketopentose.

The structures are shown above as Fischer projections. The location of the hydroxyl group on the second carbon from the bottom in the Fischer projection determines whether the structure is the D- or the L-isomer. When the −OH is on the right, it is the D-isomer. When it is on the left, it is the L-isomer. Thus, both glucose and ribulose shown above are the D- isomers. In this experiment we will stick with only D-isomers. The Fischer projections are also useful in identifying a monosaccharide as being an aldose or ketose.

In actuality, these molecules exist in a cyclic form, where they are less easily identified as an aldose or ketose. For example, D-glucose exists as either β-D-glucose or α-D-glucose.

aldehyde functional group

ketone functional group

CH2OH

H O

OHH HOH OHH OHH

CH2OH

OHH OHH

D-glucose (an aldohexose) D-ribulose (a ketopentose)

CH2OH

H O

OHH HOH OHH OHH

O CH2OH

H H

OH OH

H OH

H H

H O

O CH2OH

H H

OH OH

H OH

H H

OH O

CH2OH

H H

OH OH

H OH

1 23

D-glucose in Fischer projection

β−D-glucose in Haworth projection

α−D-glucose in Haworth projection

β-hyroxy group

α-hydroxy group

2 EXPERIMENT 11: CLASSIFICATION OF CARBOHYDRATES

In this cyclic form, the two anomers (α- and β-isomers) are distinguished by the position of the hydroxyl group attached to the anomeric C. In the β-anomer, the –OH is up; whereas, in the α-anomer, the −OH is down, as shown in the Haworth projections below.

O CH2OH

H H

OH H

H OH

O CH2OH

H H

OH OH

H OH

β−D-glucose α−D-glucose

α-hydroxy group

β-hyroxy group

anomeric C

In addition you should be familiar with four more terminologies related to carbohydrates: hemiacetal, acetal, hemiketal, and ketal. The hemiacetal and acetal are derived from an aldehyde, and the hemiketal and ketal are derived from a ketone.

H R OH

OR'

R OR' OR'

R OH OR'

R OR' OR'

R"R

hemiacetal

+ R'OH + R'OH

acetal

hemiketal

+ R'OH + R'OH

ketal

aldehyde

ketone The cyclic glucose is a hemiacetal and not a hemiketal because it is derived from an aldehyde and not a ketone (see the Fischer projection). Furthermore, it is a hemiacetal and not an acetal. Its anomeric C is bonded to −OH and −OR' rather than two −OR' (see Haworth projection below for D-glucose).

O CH2OH

H H

OH H

H OH

O H

H H

OH OH

CH2OH

OH H

D-glucose a hemiacetal

-OR

-OH

-H

-R' D-fructopyranose a hemiketal

-OR

-R'

-OH

-R

anomeric C

The key to recognizing whether a structure is a hemiacetal or hemiketal is to examine the Haworth projection and locate the O that is part of the ring. Then examine the C atoms attached to either side of that O. If one of these C atoms has a −OH and −H attached to it, the molecule is a hemiacetal. If one of these C atoms has a −OH and −R attached to it, the molecule is a hemiketal. For D-fructopyranose (see Haworth projection shown above), one of the C atoms is attached to −OR, −OH, −R' and −R", and therefore it is considered a hemiketal. Note that the C on the other side of O in the ring is not attached to an −OH and

EXPERIMENT 11: CLASSIFICATION OF CARBOHYDRATES 3

therefore is not under consideration. If the ring O is not connected to a C atom with an −OH group, the molecule is neither a hemiacetal nor a hemiketal. THE SELIWANOFF’S TEST In this experiment, you will use the Seliwanoff’s Test to distinguish between an aldose and a ketose. The reagents consist of resorcinol and concentrated hydrochloric acid. Ketoses are dehydrated (H2O removed) and then reacted with resorcinol to produce a deep cherry red color. Formation of this red color indicates the presence of a ketose.

O CH2OH

OH H

OH OH

CH2OH

O CH2OH

H H

- 3 H2O

+ HCl

α-D-fructose (a ketose)

dehydrated fructose

resorcinol

RED DYE Carbohydrates can be classified as monosaccharides, disaccharides, or polysaccharides. The monosaccharide (simple sugar) is a single unit of aldose or ketose. The disaccharide is composed of two monosaccharides bonded together, and the polysaccharide is composed of a long chain of units of monosaccharides bonded together. Lactose and sucrose are examples of disaccharides: Note that since sucrose is made of a glucose and a fructose unit, and fructose is a ketose, this disaccharide will test positive in the Seliwanoff’s Test.

O CH2OH

O O

CH2OH

H OH

H H

H OH

lactose

galactose unit glucose unit

O CH2OH

H H

H OH

O CH2OH

CH2OH H

OH H

sucrose

glucose

fructose

4 EXPERIMENT 11: CLASSIFICATION OF CARBOHYDRATES

THE BARFOED’S TEST In this experiment, you will use the Barfoed’s Test to distinguish between monosaccharides and disaccharides. The Barfoed reagent contains copper(II) ions in a slightly acidic solution. The solution oxidizes monosaccharides only. In this reaction, the copper(II) ion is reduced to copper(I) ion, which appears as the brick red copper(I) oxide, Cu2O. The solution turns various shades of green, yellow or orange before the red appears. The monosaccharide is acting as the reducing agent, and is itself oxidized (from aldehyde to carboxylic acid).

CH2OH

H O

OHH HOH OHH OHH

CH2OH

OH O

OHH HOH OHH OHH

Cu2+ blue

Cu+ red

RCHO (aldehyde) ⎯⎯⎯⎯⎯⎯⎯→ RCOOH (carboxylic acid) + 2 Cu2+ + 2 H2O + Cu2O + 4 H+ THE BENEDICT’S TEST The Benedict Test distinguishes between a reducing sugar and a nonreducing sugar. A reducing sugar is a carbohydrate that, under alkaline condition, forms an aldehyde or ketone that reacts as a reducing agent. It is similar to the Barfoed Test in that a positive test involves the reduction of blue Cu2+ to brick red Cu+ in the form of Cu2O. The difference is that the reagent is alkaline and some disaccharides will give a positive test as well. All monosaccharides will test positive, but disaccharides with hemiacetal or hemiketals will also test positive. This is why it is important to be able to distinguish between a hemiacetal and hemiketal from examining the structures. Benedict's reagent can be used to test for the presence of glucose in urine. Glucose found to be present in urine is an indication of diabetes mellitus. Once a reducing sugar is detected in urine, further tests have to be undergone in order to ascertain which sugar is present. You will be given an unknown which is one of the following: α-D-glucose, α-D-fructose, sucrose or lactose By doing the three tests on the unknown, alongside the knowns, you will be able to identify your unknown.

EXPERIMENT 11: CLASSIFICATION OF CARBOHYDRATES 5

FLOWCHART FOR THE IDENTIFICAITON OF UNKNOWN CARBOHYDRATES

UNKNOWN positive Barfoed’s Test negative MONOSACCAHRIDES DISACCHARIDES Seliwanoff Test Benedict’s Test

positive negative positive negative KETOSE ALDOSE REDUCING NONREDUCING SUGAR SUGAR Equipment/Materials Test tubes (15), 400-mL beaker, 10-mL graduated cylinder, Seliwanoff reagent, Barfoed’s reagent, Benedict’s reagent Procedure (Using a pen or pencil, record by hand all of your data and results on the Data Collection and Results Pages.) Before beginning, complete the Pre-lab Preparations, #1-3, on your Data Collection and Results Pages. Remember to record the Unknown # of your sample in the table. Prepare a hot, boiling water bath by filling a 400-mL beaker ½ full of hot tap water and placing it on a hotplate. Add a couple of boiling chips. Watch the level of the water. It is likely you have to add more water during the experiment. THE SELIWANOFF’S TEST 1. Label five test tubes near the top in such a way that the label won’t come off in the water

bath. G = glucose F = fructose S = sucrose L = lactose U = unknown 2. Using a 10-mL graduated cylinder, measure 5 mL of the Seliwanoff reagent into one of

the test tubes. Using the level in your first test tube as a guide, fill the other labeled test tubes with the Seliwanoff reagent.

6 EXPERIMENT 11: CLASSIFICATION OF CARBOHYDRATES

3. Next add 2 drops of the appropriate carbohydrate solution to each test tube. Mix the contents by rocking the tube back and forth. Be sure the reagents mix otherwise the test may not work.

4. Place the test tubes in the beaker of boiling water for 3 full minutes. 5. Record your observations in your notebook and draw a conclusion as to whether your

unknown is a ketose or aldose. THE BARFOED’S TEST Check to see whether the hot water bath needs refilling and that it is boiling hot. 6. Label five clean test tubes as before. This time you are to fill the test tubes with the

carbohydrate solutions first. Using a 10-mL graduated cylinder measure 5 mL of glucose solution in the test tube. With this as the fill guide, fill the other labeled test tubes with their appropriate carbohydrate solutions.

7. Add 3 mL of Barfoed’s reagent to the first test tube. Using this level as a guide, add 3

mL of the reagent to each of the other labeled test tubes. Mix the contents by rocking the tube back and forth. Be sure the reagents mix otherwise the test may not work.

8. Place the test tubes into the boiling water bath for 5 full minutes. 9. Record your observations into your lab notebook and draw a conclusion as to whether

your unknown is a monosaccharide or not. THE BENEDICT’S TEST Check to see whether the hot water bath needs refilling and that it is boiling hot. 10. Label 5 test tubes as before and fill them as in Step 6 with the appropriate carbohydrate

solution. 11. Using a 10-mL graduated cylinder, measure 4 mL of Benedict’s reagent to the first test

tube and once again use this as a guide to fill the rest. Mix the contents by rocking the tube back and forth. Be sure the reagents mix otherwise the test may not work.

12. Place the test tubes into the boiling water bath for 3 full minutes. 13. Record your color changes and record your observations in your lab notebook. Draw a

conclusion as to the meaning of these color changes.