/ Parallel Combinatorial Synthesis of Azo Dyes w A Combinatorial Experiment Suitable for Undergraduate Laboratories

Benjamin W. Gung* and Richard T. Taylor Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056; *gungbw@muohio.edu

Combinatorial chemistry has become an increasingly imponant tool in the search for compounds with desired properties, with broad applications in science and engineer­ing (1-6). Its introduction into our classroom and teaching activities at the undergraduate level is important for several reasons. The paradigm of combinatorial chemistry is a pow­erful research technique that also readily accommodates other desirable educational outcomes. A suitably designed labora­tory experience in combinatorial chemistry emphasizes the relationship of structure to molecular properties. It reinforces the concept that data acquisition must often precede a theo­retical framework. Finally, it allows each student to work in­dependently, yet leads them to share data and interact collaboratively ro reach conclusions. We have been engaged in a program to design laboratory experiments that are com­patible with a wide variety of student populations and equip­ment inventories, yet retain the flavor of the combinatorial approach ro doing science.

The first combinatorial experiment suitable for second­semester organic laboratory was reported several years ago (7). A combinatorial synthesis of esters using the traditional

+N::NCI-

6 X

diazonium ion

aromatic compound azo coupling an electron-rich !

an azo dye

Scheme I. Generalized synthesis of an azo dye.

Fischer esterification experiment was employed, which cre­ates diversity by using differenr combinations of alcohols and carboxylic acids. The distinct smell of each ester produced served as a biochemical assay for the experiment (7). More recently, a combinatorial synthesis ofhydrazones was reported to be suitable for high school and undergraduate laborato­ries (8) . The principle of deconvolution of libraries of mix­rures was demonstrated by screening for antibiotic activity against Escherichia coli (8).

We have developed an experiment in the parallel syn­thesis of azo dyes that illustrates the concepts of strucrure­activiry relationships and chemical diversity with vivid colors. Since the compounds are obtained in relatively pure form and the screening of "activity" is visual, the experiment can be readily transported to most laboratories. Both chemistry majors (16 students per lab) and nonmajors (100 students in a one-semester organic chemistry course) have carried out this experiment and both groups were able to become ac­quainted with combinatorial chemistry. The principle of com­binatorial chemistry is illustrated by generating a relatively large number of colorful dyes using only one common reac­tion, the diazo coupling, and rwo common reactants with small variations. Each student station is turned into an indi­vidual "well" in terms of combinatorial chemistry. At tl1e con­clusion of this experiment, students were asked to discuss the relationship berween structure and function when compar­ing the dye structures and rhe multifiber strips dyed with their own azo dyes. The pedagogical value of this experiment lies in that the structure-function relationship is demonstrated in vivid colors. Instructor-led discussions can be expanded to combinatorial reactions where structure variations lead to improvement in other functions of an organic compound, such as antimicrobial activity or catalytic capacity, et cetera.

Combinatorial Synthesis of Azo Dyes

Various dyes were considered for a combinatorial experi­ment that would show a spectrum of colors. A.zo dyes are prepared from the coupling of aryl diazonium ions with an activated aromatic compound (Scheme I). Diazotization re­actions are discussed in second-semester organic chemistry courses. The preparation of dyes is commonly performed in organic chemisny laboratories (9, 1 0). Azo dyes can be pre­pared easily in one laboratory period. This includes the dye­ing of the multifiber strip at the end of the lab period. A.zo dye preparation turns out to be an excellent reaction to illus­trate diversity oriented synthesis.

Diversification can be derived from both arenes (Scheme I). Both the substitution pattern (ortho, meta, or para) and the substituents can be varied to produce different coupling products. Furthermore, the starting aromatic compounds are available commercially and are inexpensive.

1630 Journal of Chemical Education • Vol. 81 No. 11 November 2004 • www.JCE.DivCHED.org

In the laboratory, the positions of the students are di­vided into columns headed with letters (A-D, in Table 1) and rows labeled with numbers (1-4, in Table 1). Different columns of the lab bench positions are given different aro­matic amines while each row is assigned a unique aromatic compound to couple with the diazonium ion generated from the amine. Each student produces a unique azo dye, whose structure is coded according to his or her lab bench position (Al-D4, Table l). Some of the azo dyes are known certified colors in the United States. The unlmown combinations pro­vide opportunities for "discovery'' of new azo dyes with dif­ferent shades of colors. At the end of the experiment, a multifiber strip is dyed using the student's own synthetic dye. A sample collection of the dyed strips from this experiment is shown in Figure 1. A total of ca. 60 students from two laboratory classes and a NSF-sponsored workshop for col­lege teachers have performed this experiment. This combi­natorial experiment uses a color assay, unlike a previously reported undergraduate laboratory experimem, which uses

Figure 1 . A collection of dyed multifiber strips from the combinato­rial experiment. (This image appears in color on page 1539.)

odor of the products (7). It is a much safer process using color assay for obvious reasons. This experiment is best suited for second-semester organic chemistry laboratory since the diazo coupling reaction is commonly covered in the second semes­ter. However, the simple operation of the experiment and the colorful end results allow this experimem to be used for a one-semester organic laboratory as well.

Table 1. Illustration of the Parallel Combinatorial Synthesis of Azo Dyes

A B c D

9" qso,H

~so,H 9 NH2

NH2 NH2 NH2

1 roOH

A 1 (orange 11)0 B1 C1 01 (American flag red)

OH

2 c6 A2 B2 C2 02 (magneson II)

3 A3 B3 C3 03 (solochrome orange M)

4 A4 B4 C4 04 (Easter purple)

°Colors in parentheses are certified colors in the United States.

www.JCE.DivCHED.org • Vol. 81 No. 11 November 2004 • Journal of Chemical Education 1631

Hazards

4-Nitroaniline is a highly roxie compound. Students should be instructed to avoid skin contact with arylamines. Diazonium salts are explosive in the solid state and should be kept in solurion and used immediately after preparation. A.:zo dyes are skin irritanr. Wear gloves when dyeing fabrics. Sodium hydroxide is caustic. Avoid skin contacr. Hydrochloric acid is highly corrosive. Handle it with care. Sodium niuite is a roxie oxidizer. Naphthol derivarives are irritants. 1-Naphthol is toxic.

Acknowledgment

RTT thanks the National Science Foundation for a grant (0127205) supporting undergraduate instruction in combi­natorial chemisuy. We also thank graduate teaching assistants, Lizhi Zhu, Godwin Kumi, and the CHM254 (class 2002) and CHM 255 (class 2003) for their enthusiastic participa­tion in this experiment.

wsupplemental Material

Instructions for the students, notes for the instructor, and a sample lab report are available in this issue of ]CE Online.

Literature Cited

1. Liu, D. R.; Schultz, P. G. Angew. Chern., Int. Ed. Engl. 1999, 38,36-54.

2. Schreiber, S. L. Science 2000, 287, 1964-1969. 3. Truran, G. A.; Aiken, K. S.; Fleming, T. R.; Webb, P. J.;

Markgraf, J. H.] Chern. Educ. 2002,79, 85-86. 4. Hof, F.; Nuckolls, C.; Rebek, ].] Am. Chem. Soc. 2000, 122,

4251-4252. 5. Jandeleir, B.; Schaefer, D. J.; Powers, T. S.; Turner, H . W.;

Weinberg, \Y!. H. Angew. Chern., Int. Ed. Engl. 1999, 38, 2495-2532.

6. Kuntz, K. W.; Snapper, M. L.; Hoveyda, A. H. Cm·r. Opin. Chern. Bioi. 1999,3, 313-319.

7. Birney, D. M.; Starnes, S.D.] Chern. Educ. 1999,76, 1560-1561.

8. Wolkenberg, S. E.; Su, A. I. J Chern. Educ. 2001, 78, 784-785 .

9. Palleros, D. R. Experimental Organic Chemistry, 1st ed.; John \XTiley & Sons: New York, 2000.

10. Lehman, J. \Y!. Operational Organic Chemistry, 4th ed.; Pren­tice Hall: Upper Saddle River. NJ, 2002.

The structures of a number of the molecules discussed in this article are available in fully

manipulable Chime format as JCE Featured Molecules in JCE Online (see page 1680).

1632 Journal of Chemical Education • Vol. 81 No. 11 November 2004 • www.JCE.DivCHED.org

Lab Documentation

Parallel Combinatorial Synthesis of Azo Dyes

A Combinatorial Experiment Suitable for Undergraduate Laboratories

Benjamin W. Gung* and RichardT. Taylor

Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056,

gungbw@muohio.edu

Student Handout

The following directions were provided to the students.

Combinatorial Chemistry

Parallel Combinatorial Synthesis of Azo Dyes

Background

Arguably, the most notable development in synthetic organic chemistry in the last decade

is probably the so called combinatorial chemistry. The goal of combinatorial chemistry is to

prepare a large number of structurally diversified but related compounds efficiently. A new

journal has emerged that is devoted entirely to combinatorial chemistry. The pharmaceutical

industry has embraced this new development and invested millions of dollars into the area. The

products from a combinatorial synthesis are usually called a library, which must be screened for a

desired activity. This desired activity could range from anti-tumor or anti-HIV properties to

effective catalytic properties.

In this experiment, the principle of combinatorial chemistry is shown through preparing

azo dyes using the combinatorial approach. The coupling reactions involve an aromatic diazo

compound and an electron-rich, water-soluble aromatic compound as the coupling partners. The

so-called "point of diversification" involves the structure variation on each reactants. Each

aromatic ring can be diversified by substitution pattern. Each student is assigned a unique

coupling reaction on the basis of his/her position in the lab (see Figure on next page) by the

diversification of the aromatic coupling partners. The entire class will perform the same reaction

while each student will synthesize a distinct product. Assay and the identification of individual

compound are a straightforward process for this parallel experiment. A fabric dying experiment

follows the coupling experiment. The color of the dyed multi-fiber strip serves as the final assay

for this experiment. Each student should compare the structure of the coupling reaction product

with other students in the lab. At the end of this experiment, the colors of the dyed multi-fiber

strips from the entire class should be compared. Any conclusions from the correlation of the

product structures to the colored strips should be discussed in the lab report.

In the laboratory, the positions of the lab benches are divided into columns headed with

letters (A-D, see Figure) and rows labeled with numbers (1-4, see Figure). Different columns of

WOH 1 ~

OH

2 c6 .

OH

3 aCO,H

I

65NH, 4

~

A B

Orange II ; A1 B1

·- ----- --- -- -----r----- ---- ---- --- -'

A2 B2 ' -- -- - - ---------T------- -- -- ---- -0

' ' ' '

A3 B3 ---- - - - - -------~------- - -- -----

' ' ' ' '

A4 B4

c

C1 '

0

¢' NH2

American Flag Red

01 ·--------- -------.------ ----- ------

C2

;Magneson H

02 - - - -- -----T----- -- ----

0

; Solochrome :Orange M

C3 ' 03

C4

Easter Purple

0 4

the lab bench positions will be using different aromatic amines while each row has a unique

aromatic compound to couple with the diazonium ion generated from the amine. You need to

first find out your bench position in the lab according to the Figure. Based on your bench

position, you will be able to decide which two reactants to use for this lab. Your combination of

starting materials should be unique and should produce a unique azo dye (Al-D4, see Figure).

Double check with your lab instructor before you start the experimental procedures.

After you have completed the synthesis of the dye, proceed to dye a multi-fiber strip using

your own synthetic dye following the instructions in the experimental procedure. Following your

teacher's instructions on comparison of the dyed color strips among your lab mates and on lab

write-up. Consult your instructor for the exact format to use in your lab report.

Experimental Procedures1

(A) Couplings using the Diazonium Salt from Aminobenzenesulfonic acid

( 1) Diazonium Salt Preparation

In a test tube place 0.49 g (2.8 mmol) of an aminobenzenesulfonic acid, 0.13 g of sodium

carbonate, and 5 mL of water. A clear solution is obtained by warming the test tube in a water

bath. Remove the test tube from the water bath and add a solution of 0.2 g of sodium nitrite in

0.5 mL of water. In a second test tube place 0.53 mL of concentrated HCl and 3 g of ice. The

solution from the first test tube is added dropwise with a Pasteur pipet to the second test tube.

The resulting mixture is placed in ice-water bath to induce precipitation of the diazonium salts.

The suspension is used in the next step.

(2) Coupling reaction

In a 25 mL Erlenmeyer flask place 2.6 mmol of one aromatic coupling reagent (1- or 2-

naphthol, salicylic acid, or 8-anilino-1-naphthalenesulfonic acid ammonium salt depending on

your bench position in the lab) and add 2 mL of a 2.5 M aqueous solution of sodium hydroxide.

Place the Erlenmeyer flask in a ice-water bath. The suspension of the diazonium salts prepared in

the first step is added portionwise with a Pasteur pipet to the Erlenmeyer flask. The reaction

mixture is stirred with a glass rod after each addition. The color of the solution should change

during this period of reaction. Let the reaction proceed for about 10 min with occasional stirring.

Then heat the suspension on a hot plate till the solid dissolves. Add 1 g ofNaCl and continue

heating to dissolve the it. Cool the Erlenmeyer flask at room temperature first. Then cool it in

an ice-water bath. Using a Hirsch funnel to vacuum filter the solid. Wash the solid with 2 mL of

saturated NaCl solution and let it dry in the air. Weigh the product of azo dye.

(3) Dying a fiber strip

Disposable gloves should be worn in this experiment. Dissolve 50 mg of the azo dye

prepared in the previous step in 20 mL of water in a 100-mL beaker. Put a strip of the multi-fiber

(see key to multi-fiber below) in the solution of the azo dye and keep it immersed and boil the

solution for about 5 minutes. Remove from heat with a tweezers and rinse the fiber wifh tap

water. Pat dry the dyed fiber with a paper towel and compare the color with your lab mates.

(B) Couplings using tbe Diazonium Salt from 4-Nitroaniline

(1) Diazonium Salt Preparation

In a 0.5 x 4" test tube, place 1.5 mL of concentrated hydrochloric acid and 1.5 mL of

water. Place the test tube in an ice-water bath. In another test tube of the same size, place 0.7 g

(5 mmol) of 4-nitroaniline, 0.38 g (5 .5 mmol) of sodium nitrite, and 1.5 mL of water and mix the

content well by using a touch mixer. Add the suspension to the HCl solution at 0 oc using a

pipet. Stir the reaction mixture during the addition with a glass rod and occasionally thereafter

for about 10 min. Remove any solid particles by gravity filtration using a small glass funnel and

a small cotton plug. Collect the filtrate in another test tube.

(2) Coupling reaction

Dissolve 5.1 mmol of one of the four aromatic coupling reagent (1- or 2-naphthol,

salicylic acid, or 8-anilino-1 -naphthalenesulfonic acid ammonium salt) in 10 mL of a 2.5 M aq.

sodium hydroxide solution in a 25-mL Erlenmeyer flask and place it in an ice-water bath. Add

the diazonium solution from the previous step to the Erlenmeyer flask dropwise with stirring.

Leave the flask in ice-water bath for about 10 min and record any color change. Slowly add

about 1.5 mL of concentrated hydrochloric acid to the mixture till the pH is between 3 and 4.

Add one gram ofNaCl and heat the Erlenmeter flask on a hotplate till it is boiling. Cool the

mixture to room temperature and then in ice-water bath. Vacuum-filter the solid in a small

Buchner funnel (The solution can be used to dye the fabrics directly if no solid is formed). Wash

the solid with 2-5 mL of water and leave it dry. Weigh your product.

(3) Dying a fiber strip

In a 100-mL beaker, dissolve 50 mg of the azo dye prepared in the previous step in 20 mL

of water. Add 3 mL of a 2.5 M sodium hydroxide solution to the beaker and heat the mixture on

a hot plate and stirring it with a glass rod. Put a strip of the multi-fiber (see key to multi-fiber

below) in the solution of the azo dye and keep it immersed and boil the solution for about 3

mmutes. Remove from heat with a tweezers and rinse the fiber with tap water. Pat dry the dyed

fiber with a paper towel and compare the color with your lab mates.

....

Warning

• 4-Nitroaniline is a highly toxic compound. A void skin contact with arylamines.

• Sodium hydroxide is caustic. A void skin contact.

• Hydrochloric acid is highly corrosive. Handle it with care.

• Sodium nitrite is a toxic oxidizer.

• Naphthol derivatives are irritants. 1-Naphthol is toxic.

• Diazonium salts are explosive in the solid state and should be kept in solution and used

immediately after preparation.

• Azo dyes are irritant. Wear gloves when dying the fabrics .

Key to multi-fiber strip

Black thread

1. acetate rayon,

2. SEF (acrilan),

3. Amel,

4. cotton,

5. Creslan 61,

6. Dacron 54,

7. Dacron 64,

8. nylon 6.6,

9. Orion 75,

10. silk,

11. polypropylene

12. VISCOSe rayon

13. wool

1. Experimental procedures are based on the lab manual: Daniel R. Palleros, Experimental

Organic Chemistry, John Wiley & Sons, Inc., New York, 2000, pp. 627-628.

Sample Lab Report

Eexperiment #

Lab Section:

9

A

Today's Date: 3-15-03

TA's Name: Zhu

Title of the Experiment: Parallel Combinatorial Synthesis of Azo Dyes

Introduction

A parallel combinatorial synthesis of azo dyes will be performed by the lab section, which

uses distinct colors to illustrate the concept of diversity and structure-function relationships. In

the experiment, the positions of the laboratory are divided into a grid. Each bench position

produces a unique azo dye, whose structure is coded according to the lab bench position. At the

end of the experiment, a multi-fiber strip is dyed using the synthetic dye. A colorful spectrum of

azo dyes will be produced collectively by the class. The reaction involved is the diazo coupling

between a diazonium ion and an electron rich aromatic compound.

Reaction Equation:

9 NaN02 , HCI, H20

N02

MW.138

N:N~ocr~ OH

¢ ~I// I

Azo coupling

N02

Diazonium ion

144

Reagents Equivalents mmol

4-nitroaniline 1 5.1

NaN02 1.1 5.5

HCl (concentrated) 3.5 18

2-naphthol 1 5.1

NaOH(2.5 M) 4.9 25

NaCl 3.4 17

Multi-fiber strip (1 )

s-OH ___/\__ N=N~N02

;;

An azo dye

weight/volume

0.7 g

0.38g

1.5mL

0.74 g

10mL

1.0 g

293

Teacher's Notes continued

(\Supplementary Information

f

S03Na S03Na

OCC~~H(C,Hs)CHr-Q

~ so3N•

N(C,Hs)CH,~O

0

0

Figure 1. FD&C Blue No. 1 Figure 2. FD&C Blue No. 2

NaO

Figure 4. FD&C Red No.3

. ~S03Na

HO ~ N•S03-o-N=N{~ ~

S03Na Figure 5. FD&C Red No. 40 ·

Na03S-o-N=N-Q

L(

Na02C

Figure 6. FD&CYellow No. S

S03Na

Figure 7. FD&C Yellow No. 6

-8-

© 2011 Flinn Scientific. Inc. All Rights Reserved.

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