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2556
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2556
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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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“
”
……...........................................................
. )
.......... .................... . . ..........
.
....................................................
( . )
............/......................../..............
....................................................
( . )
............/......................../..............
....................................................
( . )
............/......................../..............
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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
........................................
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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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.
.
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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
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………....................................... 7
………………………………………........................... 7
……………………………………………………................... 8
……………….…............................. 8
.......…………………………......................... 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
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………………………….................................. 43
5 …………………………………………………......... 45
………………………………………........................... 45
…........... 46
………………....……........... 46
…………….................... 48
…………………………………………………………………………….…..... 49
……………………………………………………………………………….…....... 56
........................………………………………...................... 57
…………………………………………………………………………….……... 61
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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
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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
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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
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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)
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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)
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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
สำนกัหอ
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4
1.5
1.
2.
3.
4.
5.
6.
1.6
1.
2. Dopamine MBTH
3.
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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
Nagaraja and Kumar (2002)
(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
Nagaraja and Kumar (2002)
(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.
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58
(The Development of Nitrate
Test kit Using Coupling Regents)
1.
1.
2. Dopamine
MBTH
3.
2. 3 3
1
/
2
(The Development of Nitrate Test kit Using Coupling Regents)
/
3
59
1
1.
2.
15 - 20 21 - 25
26 - 30 31 – 35
3.
4.
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5.
60
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1. 1 – 12
( 1 )
A B C
3
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5 6 1 7 8 9 11 131 10 2 4 5
1 6 1 7 8 9 11 123
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-
Mrs. Prasartporn Kittiyayam
72/1 4
76000
K_prasartpron@hotmail.com
2536
2555
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2554
2543 1) 2543
2)
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2555 “ ” 2555
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