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2556

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สมุดกลาง

THE DEVELOPMENT OF NITRATE TEST KIT USING COUPLING REGENTS

By

Mrs. Prasartporn Kittiyayam

An Independent Study Submitted in Partial Fulfillment of the Requirements for the Degree

Master of Science Program in Environmental Science

Department of Environmental Science

Graduate School, Silpakorn University

Academic Year 2013

Copyright of Graduate School, Silpakorn University



“

”

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. )

.......... .................... . . ..........

.

....................................................

( . )

............/......................../..............

....................................................

( . )

............/......................../..............

....................................................

( . )

............/......................../..............



52311308 :

: / /Dopamine/MBTH

: .

: . . . 61 .

Dopamine

MBTH (3-methyl-2-benzothiazolinone hydrazone hydrochloride)

2

60

4 1, 3, 5 7

5, 10, 15 20

(0 )

1.0 10

........................................ 2556

........................................



52311308: MAJOR: (ENVIRONMENTAL SCIENCE)

KEY WORDS: TEST KIT/NITRATE/DOPAMINE/MBTH

PRASARTPORN KITTIYAYAM: THE DEVELOPMENT OF NITRATE TEST KIT

USING COUPLING REGENTS: INDEPENDENT STUDY ADVISOR: ASST.PROF.NATDHERA

SANMANEE, Ph.D. 62 pp.

The purpose of this study was to develop test kit for nitrate using a coupling reagent.

Dopamine and 3-methyl-2-benzothiazolinone hydrazone hydrochloride (MBTH) were employed as a

coupling agent in this spectrophotometric study. The results were divided into two steps: color band

determination and comparative study with series salt waters.

More than sixty percent of the fifty observers separated four distinct color bands

representing 1, 3, 5 and 7 mg/L of nitrate, respectively. These color bands called standard color

bands were applied to the next comparative study. The series of salt waters—5, 10, 15 and 20 ppt

of sodium chloride were tested against the control (0 ppt). The results showed that the higher of

salt concentration illustrated the less absorbency. Although higher concentrations of

the coupling reagent gave the stronger color, the effect of salt was greater. The standard color

bands could be used properly for only nitrate concentration was not higher than 1.0 mg/L in

which salt concentration did not exceed 10 ppt. At higher nitrate concentrations, the effects of salt

interruption were too high and not good for eye sensitivity. Only spectrophotometer could be used

to separate the difference among them. Nevertheless, this test kit was still useful for most of fresh

waters where the salt concentration was usually low.

Department of Environmental Science Graduate School, Silpakorn University

Student's signature ........................................ Academic Year 2013

An Independent Study Advisor’s signature .........................................



.

.

.



……………………………………………………………………..............

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……………………………………………………………….……………..

…………………………………………………………………………………..

………………………………………………………………………….……...

1 ……………………………………………………….…………………………… 1

………………………………………......... 1

……………………………….…...... 3

………………………………………….……................... 3

……………………………………………………................ 3

………………………………………………………………....... 5

…………………………………………………............ 6

2 ………………………………………………………….. 5

…………………………………………………………………..…........ 5

……………………………………………...…..…..... 5

…………………………..….......... 6

…………………………………………………………..…................... 6

…………………………………………..…...................... 6

..…………….........…………..…............ 6

..………………………..…...................... 6

……………………………………………………….....………… 6

……………………………………………......................... 7

………………………………………....……….…........ 7

………………………………………………....................... 7



………....................................... 7

………………………………………........................... 7

……………………………………………………................... 8

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.......…………………………......................... 9

………....…………………….…............ 9

…………………………........................... 10

………………………………......………..... 10

……….....…….... 11

…................................................................................................. 12

3 ……………………………………………......................................... 14

…………........................................ 14

(Chemical and Reagents) …………........................................ 17

(Instrumentation) ......................................................... 17

…........................................................................................................ 17

…............................................................................................................. 18

................................................................................................... 18

Dopamine MBTH

0.5:0.5 ......................................................................................................... 19

4 ..…………………………………….......................... 21

…………………………………………....................... 21

0.5:0.5

1.0:1.0 ….................................................................................................... 26

1.0:1.0 ………........... 33



………………………….................................. 43

5 …………………………………………………......... 45

………………………………………........................... 45

…........... 46

………………....……........... 46

…………….................... 48

…………………………………………………………………………….…..... 49

……………………………………………………………………………….…....... 56

........................………………………………...................... 57

…………………………………………………………………………….……... 61



1 ........................................................... 10

2 A 1.0-4.0 ........... 22

3 B 5.0-8.0 ................................ 23

4 C 9.0-12.0 ....... 24

5 4 1.0, 3.0, 5.0

7.0 ......................................................................................... 26



1 .............................................................................................. 5

2 Dopamine MBTH ................. 12

3 ................................................................................................ 15

4 .................................................................................................. 16

5 1.0-12.0 ................................... 19

6 ............................................................................................... 20

7 1.0 0.5:0.5

) ) 5, 10, 15 20

.................................................................................................. 27

8 1.0

0.5:0.5 ........................................ 28

9 3.0 0.5:0.5

) ) 5, 10, 15 20

.................................................................................................. 29

10 3.0

0.5:0.5 ....................................... 30

11 5.0 0.5:0.5

) ) 5, 10, 15 20

................................................................................................. 32

12 5.0

0.5:0.5 ........................................ 33

13 7.0 0.5:0.5

) ) 5, 10, 15 20

............................................................................................... 34



14 7.0

0.5:0.5 ............................ 35

15 1.0 1.0:1.0

) ) 5, 10, 15 20

............................................................................................... 36

16 1.0

1.0:1.0 ............................. 37

17 3.0 1.0:1.0

) ) 5, 10, 15 20

................................................................................................. 38

18 3.0

1.0:1.0 ............................. 39

19 5.0 1.0:1.0

) ) 5, 10, 15 20

................................................................................................ 40

20 5.0

1.0:1.0 .............................. 41

21 7.0 1.0:1.0

) ) 5, 10, 15 20

............................................................................................... 42

22 7.0

1.0:1.0 ............................ 43

23 0.5:0.5 1.0:1.0 .............. 44



1

1

1.1

(NO3-)

(NH3)

( , 2549; Atlas and Bartha, 1998)

(Eutrophication)

( , 2545; , 2548; Saad,

1980)

(Methaemoglobinaemia)

(Blue Babies Syndrome)

(Cytochrome)

( , 2547; . 2549;

Shuval and Gruener, 1974; Wasik et al., 2001; Mizuta et al., 2004)

8 (2537)

5.0 ( , 2556 )

10

( , 2556 )

( , 2546; , 2549; APHA AWWA and WPCF, 1995)



2

(

, 2556)

( , 2552)

- (NO3-N-mg/L)

450 500 (

, 2556)

300 (

, 2550)

250 (

, 2556)

Nagaraj and Kumar (2002) (2552)

(Coupling Reagent) Dopamine (3-

Hydroxytyramine) MBTH (3-Methyl-2- Benzothiazolinone Hydrazone Hydrochloride)



3

1.2

1.

2. Dopamine

MBTH

3.

1.3

1.0 - 12.0

1.0–12.0

1.4

1.0–12.0

(5.0 ) (

, 2556 ) Nagaraja and

Kumar (2002) (2552)

50 15-35

4

4 5, 10, 15 20

Dopamine MBTH

2 0.5:0.5 1.0:1.0



4

1.5

1.

2.

3.

4.

5.

6.

1.6

1.

2. Dopamine MBTH

3.



2

2.1

2.1.1

(Nitrification)

( , 2545;

, 2547) 1

1

: (2548)

5

6

2.1.2

2.1.2.1

( , 2550; , 2551; ,

2553)

2.1.2.2

( , 2554)

2.1.2.3

( , 2535; , 2542)

2.1.2.4

( , 2538; , 2539; , 2552)

2.2

( , 2541)

2.2.1

picric acid

7

2.2.2

2.2.3

2.2.4

2.3

(Organic Nitrogen) (Inorganic Nitrogen)

( , 2537;)

(NH4+)

(Nitrifyingbacteria Bacteria)

( Autotroph Bacteria) 2 ( Nitrosomonas Bacteria)

(Nitrobacter Bacteria)

( , 2542; , 2552)

( Dentrification)

(Biological Molecules)

8

( , 2541)

2.4

( , 2547; Escobal, 1996)

(2556 )

5.0

(Algae Bloom)

( , 2533) (Red Tide)

–

–

( , 2540; , 2547; , 2549)

2.5

2.5.1

( , 2550;

, 2551; , 2553) 6

9

(Methemoglobin)

( , 2531;

, 2550; , 2552; , 2555 )

(2548)

11

2.5.2

( , 2536; , 2541)

20 2556

4 3 30-60

12-15

10

(

, 2556)

2.5.3

( , 2540; , 2549)

2.6

1

1

4.0

61

. . 2524

2-4

5.0

8 ( . . 2537)

11

1 ( )

- 4 5

. . 2551

-

500

501

6

-

60 -

27 . . 2549

: 1)

2-4 1 5

2) 6

2.7

Naggaraja and Kumar (2002) (2552)

Dopamine MBTH

2

530 Nagaraja and Kumar

12

(2002) (2552)

2 Dopamine MBTH

: Nagaraja and Kumar (2002)

2.8

Nagaraja and Kumar (2002)

Dopamine MBTH

530

0.04-1.0 Molar Absorptivity ( ) 1.073×104 L mol-1 cm-1

0.5 (SD)

(RSD) 0.01 0.203 %

(2552) Nagaraja and Kumar (2002)

4 0.092, 0.92, 3.0, 9.2

13

(2545)

Flow infection analysis (FIA)

Sulfanilamide N-(1-napthy)

Ethylenediamine dihydrochloride

520

0 .2 -20

(R2) 0.9999 0.01

1.0 8.0 (%RSD)

2.01 0.73 (%Recovery)

0.2, 1.0 4.0 90, 89 95 %

120

14

3

2

1.0-12.0

4 5, 10, 15 20

Dopamine MBTH

0.5:0.5 1.0:1.0

3 4

3.1

Dopamine MBTH


(2552)

1.0-12.0

1.

4.0 ( , 2556 )

2.

5.0 (

, 2556 )

3.

10 ( , 2556 )

15

3

4

-

530

6

Nagaraja and Kumar

0.5:0.5

16

4

0.5:0.5

Nagaraja and Kumar (2002) (2552)

1.0:1.0

0, 5, 10, 15 20

530

6

17

3.2 (Chemical and Reagents)

Chemical Abstracts

Service (CAS) number

1. (Potassium nitrate: KNO3) CAS: 7440-09-7 Ricdel-de Haen

2. (Sulfuric acid: H2SO4) CAS: 7664-93-9 Merck

3. Dopamine hydrochloride (Dopamine: C8H11NO2ClH) CAS : 62-31-7 Sigma

Aldrich

4. 3-Methyl-2-benzothiazolinone hydrazone hydrochloride hydrate (MBTH : C8H9N3S.HCl)

CAS: 149022-15-1 Aldrich

5. (NaCl) CAS: 7647-14-5 Merck

6. (Deionized water)

3.3 (Instrumentation)

1. 25

2.

3. (1, 3 5 )

4. (Volumetric flask) 10, 25, 50, 100, 500, 1,000

5. UV-VIS Spectrophotometer JASCO V-530 530

6. Glass cell Quartz Cell 1

7. 4

3.4

3.4.1 100 (NO3 –N)

100 (NO3 –N)

0.7218 1

(Serial dilution)

1.0–12.0 (NO3 –N)

18

3.4.2 14.4 (14.4 M sulfuric acid)

14.4 (14.4 M sulfuric acid) 500

125

3.4.3 Dopamine MBTH 0.5:0.5 1.0:1.0

Dopamine MBTH Dopamine MBTH 0.5

1.0:1. 0 100

3.4.4 5, 10, 15 20

5, 10, 15 20

4.5625, 9.1249, 13.6874 18.2498

1,000

3.5

1

10 14.4 5 Dopamine

MBTH 3 15

10

530

3.6


(2552)

15–35

19

3

1 2 3

3 1 1.0-3.0

2 3.1-7.0 3 7.1-12.0

3

3.7 Dopamine MBTH 0.5:0.5

Dopamine MBTH 0.5:0.5

25 25

50 15-35

6 4

60

5 1.0-12.0

1 10 2 41

5 6 7 8 9 11 1 12 13

20

A B C

6

21

4

Dopamine MBTH

2 1) 2)

4.1

50 15-35

3 1

2 3

4 1 1.0-4.0 2 2

5.0-8.0 3 3 9.0-12.0

4

1.0-4.0 2

A 50 1) 1.0

A 1 62 A 3, A 2, A 5 A4

14, 12, 8 4 2) 2.0 A 1

A 3 30 A 2 A 4 28

12 A 5 3) 3.0 A 3

64 A 2 A 1 28 8 A 4 A 5

4) 4.0 A 3 A 4

22

2 A 1.0-4.0

1.0

2.0

3.0

4.0

A 1

31

62

15

30

4

8

0

0

A 2

6

12

14

28

14

28

0

0

A 3

7

14

15

30

32

64

22

44

A 4

2

4

6

12

0

0

22

44

A 5

4

8

0

0

0

0

6

12

22

3 B 5.0-8.0

5.0

6.0

7.0

8.0

B 1

35

70

23

46

0

0

0

0

B 2

3

6

2

4

0

0

0

0

B 3

9

18

23

46

1

2

17

34

B 4

3

6

2

4

37

74

16

32

B 5

0

0

0

0

12

24

17

34

23

4 C 9.0-12.0

9.0

10.0

11.0

12.0

C 1

0

0

3

6

0

0

0

0

C 2

8

16

9

18

7

14

5

12

C 3

34

68

19

38

7

14

23

46

C 4

8

16

19

38

36

72

22

44

C 5

0

0

0

0

0

0

0

0

24

25

44 A 5 12 A 1 A 2

A 1 A 3 1.0 3.0

5.0-8.0 3

B 50 1) 5.0

B 1 70 B 3

18 B 2 B 4 6 B 5

2) 6.0 B 1 B 3 46

B 2 B 4 4 B 5 3)

7.0 B 4 74 B 5

B 3 24 2 B 1 B 2 4)

8.0 B 3 B 5 34 B 4

32 B 1 B 2 B 1 B 4

5.0 7.0

9.0-12.0 4

C 50 1)

9.0 C 3 68 C 2 C 4

16 C 1 C 5 2) 10.0

C 3 C 4 38 C 2

C 1 18 6 C 5 3) 11.0

C 4 72 C 2 C 3

14 C 1 C 5 4) 12.0

C 3 46 C 4 C 2 44

12 C 1 C 5 C 3 C 4

9.0 11.0

4 1.0,

3.0, 5.0 7.0 5

26

5 4 1.0, 3.0, 5.0

7.0

1.0

3.0

5.0

7.0

31 62 32 64 35 70 37 74

4.20

0 0.5:0.5

4.2.1 Dopamine MBTH

0.5:0.5 1.0

4 5, 10, 15 20

Dopamine MBTH

0.5:0.5 1.0

7

0 > 5 > 10 >15 > 20

8

Y = -0.0034×+ 0.2006 , r2 = 0.9701

4 5

5 10

1.0 15 20

7

27

) 1.0

) 1.0 5, 10, 15 20

7 1.0 0.5:0.5 )

) 5, 10, 15 20

10

15

20

5

28

8 1.0

0.5:0.5

10

4.2.2 Dopamine MBTH

0.5:0.5 3.0

4 5, 10, 15 20

Dopamine MBTH

0.5:0.5 3.0

9

0 > 5 > 10 > 15 > 20

10 Y = -0.0141x

+ 0.7447, r2 = 0.5898

y = -0.0034x + 0.2006

r² = 0.9701

0

0.05

0.1

0.15

0.2

0.25

0 5 10 15 20 25 ( )

29

) 3.0

) 3.0 5, 10, 15 20

9 3.0 0.5:0.5 )

) 5, 10, 15 20

10

15

20

5

30

10 3.0

0.5:0.5

4 5

5, 10, 15 20

7.0

20

9

4.2.3 Dopamine MBTH

0.5:0.5 5.0

4 5, 10, 15 20

y = -0.0141x + 0.7447

r² = 0.5898

0.00

0.20

0.40

0.60

0.80

1.00

0 5 10 15 20 25( )

31

Dopamine MBTH

0 . 5 :0 . 5 5. 0

11

0 > 5 > 10 >15 >

20 12

Y = -0.0207× + 0.8768, r2 = 0.9817

4 5

5 10

15 20

11

4.2.4 Dopamine MBTH

0.5:0.5 7.0

4 5, 10, 15 20

Dopamine MBTH

0.5:0.5 7.0

13

0 > 5 > 10 >15 >

20 14

Y = -0.0306× + 1.6344, r2 = 0.9156

4 5

13

32

) 5.0

) 5.0 5, 10, 15 20

11 5.0 5:0.5 )

) 5, 10, 15 20

10

15

20

5

33

12 5.0

0.5:0.5

4.3

1.0:1.0

4.3.1 Dopamine MBTH

1.0:1.0 1.0

4 5, 10, 15 20

Dopamine MBTH

1.0:1.0 1.0

15

0 > 5 > 10 >

15 > 20

y = -0.0207x + 0.8768

r² = 0.9817

0.00

0.20

0.40

0.60

0.80

1.00

0 5 10 15 20 25 ( )

34

) 7.0

) 7.0 5, 10, 15 20

13 7.0 0.5:0.5 )

) 5, 10, 15 20

10

15

20

5

35

14 7.0

0.5:0.5

16 Y=-0.0035×+0.2384,

r2 = 0.9903

4 5

15

4.3.2 Dopamine MBTH

1.0:1.0 3.0

4 5, 10, 15 20

y = -0.0306x + 1.6344

R² = 0.9156

0.00

0.50

1.00

1.50

2.00

0 5 10 15 20 25

( )

36

) 1.0

) 1.0 5, 10, 15 20

15 1.0 1.0:1.0 )

) 5, 10, 15 20

10

15

20

5

37

16 1.0

1.0:1.0

Dopamine MBTH

1.0:1.0 3.0

17

0 > 5 > 10

>15 >20 18

Y = -0.0195× + 1.0201, r2 = 0.9827

4 5

5 10

15 20

17

y = -0.0035x + 0.2384

r² = 0.9903

0.00

0.10

0.20

0.30

0 5 10 15 20 25 ( )

38

) 3.0

) 3.0 5, 10, 15 20

17 3.0 1.0:1.0

) ) 5, 10, 15 20

10

15

20

5

39

18 3.0

1.0:1.0

4.3.3 Dopamine MBTH

1.0:1.0 5.0

4 5, 10, 15 20

Dopamine MBTH

1.0:1.0 5.0

19

0 > 5 > 10 >

15 > 20 20

Y = -0.027× + 1.264, r2 = 0.9721

4 5

19

y = -0.0195x + 1.0201

r² = 0.9827

0.00

0.50

1.00

1.50

0 5 10 15 20 25

( )

40

) 5.0

) 5.0 5, 10, 15 20

19 5.0 1.0:1.0

) ) 5, 10, 15 20

0

10

15

20

5

41

20 5.0

1.0:1.0

15

4.3.4 Dopamine MBTH

1.0:1.0 7.0

4 5, 10, 15 20

Dopamine MBTH

1.0:1.0 7.0

21

0 > 5 > 10 >

15 > 20 22

Y = -0.0389× + 2.0322, r2 = 0.9147

y = -0.027x + 1.264

r² = 0.9721

0.00

0.50

1.00

1.50

0 5 10 15 20 25 ( )

42

) 7.0

) 7.0 5, 10, 15 20

21 7.0 1.0:1.0

) ) 5, 10, 15 20

0

10

15

20

5

43

22 7.0

1.0:1.0

4 5

21 15

4.4

23

0.5:0.5 1.0, 3.0, 5.0 7.0 0.0034, 0.0141, 0.0207

0.0306

Y = -0.0034×+ 0.2006 r2 = 0.9701,

Y = -0.0141×+ 0.7447 r2 = 0.5898,

y = -0.0389x + 2.0322

r² = 0.9147

0.00

1.00

2.00

3.00

4.00

0 5 10 15 20 25

( )

44

Y = -0.0207×+ 0.8768 r2 = 0.9817

Y = -0.0306×+ 1.6344 r2 = 0.9156

1.0:1.0

1.0, 3.0, 5.0 7.0 0.0035, 0.0195, 0.0270 0.0389

Y = -0.0035×+ 0.2384 r2 = 0.9903,

Y = -0.0195×+ 1.0201 r2 = 0.9827,

Y = -0.027×+ 1.264 r2 = 0.9721

Y = -0.0389×+ 2.0322 r2 = 0.9147

23 0.5:0.5 1.0:1.0

45

5

Dopamine MBTH Nagaraja and Kumar (2002)

(2552)

2 1) 2)

5.1

50 15-35

3 4

1 1.0-4.0 2 5.0-8.0

3 9.0-12.0

60 1)

1.0 A 1 31 62 2)

3.0 A 3 32 64 3)

5.0 B 1 35

70 4) 7.0 B 4 37

74 4 1.0, 3.0, 5.0 7.0

46

5.2

0.5:0.5 4

1.0, 3.0, 5.0 7.0 4 5, 10, 15 20

1.0 5 10

1.0

10.0

3.0, 5.0, 7.0

1.0:1.0 4 1.0,

3.0, 5.0 7.0 4 5, 10, 15 20

1.0

3.0 5.0

7.0

5.3

5.3.1

4 1.0, 3.0, 5.0 7.0

47

5.3.2

0.5:0.5 1.0:1.0

5.3.3

Y= -0.0034×+

0.2006, Y = -0.0141×+ 0.7447, Y = -0.0207×+ 0.8768 Y=-0.0306×+ 1.6344

Dopamine MBTH 0.5:0.5 Y= -0.0035×+ 0.2384, Y = -0.0195×+ 1.0201,

Y = -0.027×+ 1.264 Y = -0.0389×+ 2.0322 Dopamine MBTH

1.0:1.0 1.0

-0.0034

-0.0035 3.0, 5.0 7.0

Dopamine MBTH 1.0:1.0 -0.0195, -0.0270

-0.0389 Dopamine MBTH 0.5:0.5

-0.0141, -0.0207 -0.0306

5.4

Dopamine MBTH 0.5:0.5

48

5.5

49

. 2549. .

. 4.

, .

. 2541. - . 2.

, .

______2556 . .

. : http://www.pcd.go.th/download/regulation.cfm?task=s3, 5

2556.

______ 2556 . . .

: http://www.pcd.go.th/info_serv/reg_std_water01.html#s1, 5 2556.

______ 2556 . .

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