You are on page 1of 33

Subscriber access provided by UNIV OF UTAH

Article
Rapid Acid-Base Titrations Using Microfluidic Paper-Based Analytical Devices
Shingo Karita, and Takashi Kaneta
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac5039384 • Publication Date (Web): 25 Nov 2014
Downloaded from http://pubs.acs.org on December 1, 2014

Just Accepted

“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth


Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.
Page 1 of 32 Analytical Chemistry

1
2
3
4
5
6 Rapid Acid-Base Titrations Using Microfluidic Paper-Based
7
8
9
Analytical Devices
10
11
12 Shingo Karita and Takashi Kaneta*
13
14
15 Department of Chemistry, Graduate School of Natural Science and Technology, Okayama
16 University, Okayama, 700-8530 Japan
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59 1
60
ACS Paragon Plus Environment
Analytical Chemistry Page 2 of 32

1
2
3
4 ABSTRACT: Rapid and simple acid-base titration was accomplished using a novel
5
6
7 microfluidic paper-based analytical device (µPAD). The µPAD was fabricated by wax
8
9
10 printing and consisted of ten reservoirs for reaction and detection. The reaction
11
12
13 reservoirs contained various amounts of a primary standard substance, potassium hydrogen
14
15
16
phthalate (KHPth), whereas a constant amount of phenolphthalein was added to all the
17
18
detection reservoirs. A sample solution containing NaOH was dropped onto the center of
19
20
21 the µPAD, and was allowed to spread to the reaction reservoirs where the KHPth
22
23
24 neutralized it. When the amount of NaOH exceeded that of the KHPth in the reaction
25
26
27 reservoirs, unneutralized hydroxide ion penetrated the detection reservoirs, resulting in a
28
29
30 color reaction from the phenolphthalein. Therefore, the number of the detection
31
32
33 reservoirs with no color change determined the concentration of the NaOH in the sample
34
35
36 solution. The titration was completed within 1 min by visually determining the end point,
37
38
39 which required neither instrumentation nor software. The volumes of the KHPth and
40
41
42 phenolphthalein solutions added to the corresponding reservoirs were optimized to obtain
43
44
45
reproducible and accurate results for the concentration of NaOH. The µPADs determined
46
47
the concentration of NaOH at orders of magnitude ranging from 0.01 to 1 M. An acid
48
49
50 sample, HCl, was also determined using Na2CO3 as a primary standard substance instead
51
52
53 of KHPth. Furthermore, the µ-PAD was applicable to the titrations of nitric acid, sulfuric
54
55
56 acid, acetic acid, and ammonia solutions. The µPADs were stable for more than 1 month
57
58
59 2
60
ACS Paragon Plus Environment
Page 3 of 32 Analytical Chemistry

1
2
3
4 when stored in darkness at room temperature, although this was reduced to only five days
5
6
7 under daylight conditions. The analysis of acidic hot spring water was also demonstrated
8
9
10 in the field using the µPAD and the results agreed well with those obtained by classic
11
12
13 acid-base titration.
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59 3
60
ACS Paragon Plus Environment
Analytical Chemistry Page 4 of 32

1
2
3
4 Recent advances in micro- and nano-scale analytical devices have resulted in excellent
5
6
7 performance, which cannot be achieved by classic techniques. For example, the
8
9
10 miniaturization of analytical devices has permitted a high degree of sensitive detection,1
11
12
13 rapid separation,2 and enhancement in the rate of chemical reactions.3 In particular,
14
15
16
microfluidic devices consisting of glass and polymer substrates are widely employed for
17
18
miniaturizing several analytical devices including chemical sensors, separation channels,
19
20
21 and manipulation channels of micro-sized particles and biological cells.4
22
23
24 Conversely, microfluidic paper-based analytical devices (µPADs) have recently
25
26
27 attracted a great deal of interest due to a simple structure, easy fabrication, lightness of
28
29
30 weight, inexpensive materials, and rapid analysis. The first µPAD used a
31
32
33 photolithographic method to determine blood constituents such as protein and glucose.5
34
35
36 Thereafter, several improvements in the fabrication of µPADs have been explored using
37
38
39 wax printing,6,7 ink-jet printing,8 screen printing,9 laminate film,10,11 and movable-type
40
41
42 printing.12 Among these, wax printing and ink-jet printing are the simplest and most
43
44
45
precise ways to fabricate microfluidic channels on paper since conventional drawing
46
47
software can be employed for designing µPADs.
48
49
50 Using µPADs, some detection techniques have been applied for the determination of
51
52
53 analytes, such as colorimetry for metal ions,13-16 biomarkers,17 DNA,18 and foodborne
54
55
56 pathogens.19 Other techniques include enzyme assays20 and electrochemistry.21-24
57
58
59 4
60
ACS Paragon Plus Environment
Page 5 of 32 Analytical Chemistry

1
2
3
4 Colorimetry is perhaps the most popular since only a scanner (or a digital camera) and
5
6
7 software for image processing are necessary for quantitative analysis, i. e., both the devices
8
9
10 as well as the detection system are small, inexpensive, and easy to use. A limitation of
11
12
13 µPADs that has been reported in past publications is the need to calibrate using standard
14
15
16
solutions to quantify an analyte. The intensity of the color for the sample must be
17
18
compared with those of standard solutions, and calibration curves must be constructed for
19
20
21 quantitative analysis using a scanner and image processing software. Only a few
22
23
24 exceptions have been demonstrated without the use of either a scanner or a digital camera.
25
26
27 Cate and coworkers used the distance of color development to quantify analytes,25 and
28
29
30 Lewis and coworkers employed time as a quantitative readout.26,27 In the µPAD based on
31
32
33 the distance of color development, as flowing analyte forms precipitation by the reaction
34
35
36 with reagents, color develops along the flow channel until all of the analyte is consumed.
37
38
39 When using the µPAD, however, the distances in color development still must be
40
41
42 calibrated to the concentrations of the analyte using the standard solutions.
43
44
45
Conversely, compared to modern instrumental analysis, classic analytical methods such
46
47
as titrimetry and gravimetry need no calibration curve since the analyses are based on the
48
49
50 absolute amounts of titrant and the weight of the precipitation. For example, in acid-base
51
52
53 titration, if we use a primary standard solution as a titrant, or titrand, the concentration of
54
55
56 an acid or a base solution is directly calculated from the volume required to reach the end
57
58
59 5
60
ACS Paragon Plus Environment
Analytical Chemistry Page 6 of 32

1
2
3
4 point. In addition, titration permits on-site measurement using no instrumentation since
5
6
7 the human eye determines the end point. However, this type of analysis requires a large
8
9
10 amount of glassware, large volumes of solutions, technical skills, and a long period of
11
12
13 time.
14
15
16
Using a similar principle for titration, we developed a novel µPAD that needs no
17
18
calibration curve to accomplish acid-base titration. This µPAD consists of reaction
19
20
21 reservoirs and detection reservoirs that contain varying amounts of a primary standard
22
23
24 substance and a constant amount of a visible indicator. A sample solution is dropped onto
25
26
27 the center of the µPAD, and is then allowed to spread to each reaction reservoir where it is
28
29
30 neutralized by different amounts of the primary standard substance. The neutralized
31
32
33 sample changes the color of the indicator in the detection reservoir only when the primary
34
35
36 standard substance is insufficient to neutralize the sample solution. The titration is
37
38
39 completed within one minute, including the time required for sample application and for
40
41
42 determining the end point. Therefore, this µPAD is more advantageous than classic
43
44
45
titrimetry in terms of speed, portability, and disposability, although the number of
46
47
significant figures is less. The design of this µPAD, and the volumes of the primary
48
49
50 standard, indicator, and sample solutions to be added were optimized to obtain
51
52
53 reproducible results. In addition, the stability of this µPAD was evaluated by storage
54
55
56 under various conditions of temperature and light exposure. We also used this µPAD for
57
58
59 6
60
ACS Paragon Plus Environment
Page 7 of 32 Analytical Chemistry

1
2
3
4 the on-site analysis of acidic water obtained from a natural hot spring.
5
6
7
8
9
10 EXPERIMENTAL SECTION
11
12
13 Materials. All reagents were of analytical grade and were used as received. Deionized
14
15
16
water was prepared by means of an Elix water purification system (Millipore Co. Ltd.,
17
18
Molsheim, France). Potassium hydrogen phthalate (KHPth), sodium hydroxide,
19
20
21 hydrochloric acid, nitric acid, sulfuric acid, 1 M sodium hydroxide solution (factor, 1.000),
22
23
24 1 M hydrochloric acid (factor, 1.001), phenolphthalein, orange I, and ethanol were
25
26
27 purchased from Wako Pure Chemical Industries (Osaka, Japan). Sodium carbonate,
28
29
30 acetic acid, and ammonia were obtained from Kanto Chemical (Tokyo, Japan).
31
32
33 Bromocresol purple was purchased from Tokyo Kasei Kogyo (Tokyo, Japan). Stock
34
35
36 solutions of 1 M KHPth and 1 M sodium carbonate were prepared by dissolving the
37
38
39 appropriate amounts in water. Stock solutions of 1(w/w)% phenolphthalein and
40
41
42 bromocresol purple were prepared by dissolving 1 g each of phenolphthalein and
43
44
45
bromocresol purple in separate containers of 99 g of ethanol.
46
47
µPAD design and fabrication. We used Microsoft Office Power Point 2010 to design a
48
49
50 µPAD with a sample reservoir located at the center and ten reaction and detection
51
52
53 reservoirs each arranged radially in a 30 × 30 mm square. Details of the design of the
54
55
56 µPAD are provided in the Supporting Information (Figure S1). According to the method
57
58
59 7
60
ACS Paragon Plus Environment
Analytical Chemistry Page 8 of 32

1
2
3
4 reported by Carrilho and coworkers,7 the designed µPADs were printed on a sheet of filter
5
6
7 paper (200 × 200 mm, Chromatography Paper 1CHR, WhatmanTM, GE Healthcare
8
9
10 Lifesciences, United Kingdom) by a wax printer (ColorQube 8570N, Xerox, CT), followed
11
12
13 by heating at 150 ºC for 2 min in a drying machine (ONW-300S, AS ONE Corporation,
14
15
16
Osaka, Japan). The back side of the printing surface was covered with clear packing tape
17
18
to prevent solution from leaking out underneath the µPAD, as reported by Mentele and
19
20
21 coworkers.13 The areas of the channels and reservoirs were measured using the public
22
23
24 domain software Image J (National Institutes of Health). Each µPAD was cut to a 30 ×
25
26
27 30 mm piece, and then was prepared by applying appropriate volumes of primary standard
28
29
30 solutions and an indicator solution to the reaction reservoirs and detection reservoirs via a
31
32
33 micropipette. After drying, the µPAD was sandwiched by an acrylic plate holder that was
34
35
36 composed of two 30 × 30 mm plates. The bottom plate had four stops that fix the top
37
38
39 plate, whereas the top plate had a hole (3 mmφ) to introduce a sample solution into the
40
41
42 µPAD (Supporting Information, Figure S2). An aliquot of a sample solution was gently
43
44
45 introduced into the µPAD through the hole in the top plate by dropping 30 µL of a sample
46
47
solution via a micropipette. Between titrations, the acrylic plate holder was flushed with
48
49
50 deionized water and then was wiped with disposable paper wiper.
51
52
53 Analysis of acidic hot spring water. An acidic hot spring water sample was obtained at
54
55
56 the source of the Tsukahara Onsen hot spring, Yufu, Oita, Japan. Hot spring water was
57
58
59 8
60
ACS Paragon Plus Environment
Page 9 of 32 Analytical Chemistry

1
2
3
4 taken from a pool near a spa (40 ºC). The hot spring water was passed through a
5
6
7 cation-exchange solid phase extraction (SPE) cartridge (HyperSep™ SCX Strong Cation
8
9
10 Exchanger SPE Cartridge, 100 mg, Thermo Scientific, Thermo Fisher Scientific, MA) to
11
12
13 remove metal ions and then was determined by the µ-PADs in the field. To exchange H+
14
15
16
with Na+ before use, the cation-exchange SPE cartridge was pretreated with 1 mL of 1 M
17
18
NaCl. After the pretreatment, 0.25 mL of the hot spring water was added to the
19
20
21 cation-exchange SPE cartridge for flushing out the residual NaCl solution, and then 1.5
22
23
24 mL of the hot spring water was introduced to collect a sample for determination using the
25
26
27 µ-PAD. The cation-exchange SPE cartridge could be regenerated by flushing
28
29
30 successively with 1 mL of 5 M NaCl and 1 mL of 1 M NaCl and could be used at least five
31
32
33 times. The hot spring water was also brought back to our laboratory for classic acid-base
34
35
36 titration. In the laboratory, the hot spring water was pretreated by the same procedure as
37
38
39 that carried out in the field analysis. A 1-mL aliquot of the collected sample was titrated
40
41
42 with 0.01 M NaOH (factor, 1.000) using a 25-mL burette.
43
44
45
46
47
RESULTS AND DISCUSSION
48
49
50 Applied volumes and concentrations of reagents. A µPAD for acid-base titration
51
52
53 was prepared by applying primary standard solutions of acid or base in varying
54
55
56 concentrations and a solution of an indicator, phenolphthalein, to the reaction and detection
57
58
59 9
60
ACS Paragon Plus Environment
Analytical Chemistry Page 10 of 32

1
2
3
4 reservoirs. The volumes needed to fill the reaction and detection reservoirs were initially
5
6
7 estimated in order to optimize the applied volumes of the reagents. In the preliminary
8
9
10 study, we confirmed that the volume needed for wetting the whole channel was 30 µL,
11
12
13 according to a method reported by Dungchai and coworkers wherein different volumes of
14
15
16
a colored solution were applied to the µPADs to fill the whole channel.28 Then, the
17
18
volumes used to fill the reaction and detection reservoirs were estimated by measuring the
19
20
21 area of the whole channel, and those of the reaction and detection reservoirs. The areas
22
23
24 were measured using Image J software (shown in Supporting Information, Table S1).
25
26
27 The volumes of the reaction and detection reservoirs were obtained by multiplying the
28
29
30 volume required to fill the whole channel by the area ratio of each reservoir in the whole
31
32
33 channel. The volumes needed to occupy the reaction and detection reservoirs were
34
35
36 calculated as 0.98±0.08 and 0.54±0.001 µL, respectively. Therefore, 1 µL each of the
37
38
39 primary standard solutions was added to each reaction reservoir so as to be equivalent to
40
41
42 the volume of the sample solution needed to fill the reaction reservoir, and 0.5 µL of a
43
44
45
phenolphthalein solution was applied to the detection reservoirs to detect the excess
46
47
amounts of acid or base that were not neutralized by the primary standard substance in the
48
49
50 reaction reservoirs.
51
52
53 It should be noted that the amount of phenolphthalein was an important factor in a
54
55
56 clear visualization of a color change. Therefore, concentrations of phenolphthalein that
57
58
59 10
60
ACS Paragon Plus Environment
Page 11 of 32 Analytical Chemistry

1
2
3
4 varied from 0.01 to 1% were added to the detection reservoirs without the addition of acid
5
6
7 to the reaction reservoirs, and 0.1 and 0.01 M NaOH solutions were applied to the center
8
9
10 of the µ-PAD in order to determine the optimal concentration for a clear visualization of
11
12
13 color change, as shown in Figure 1. Figure 1a shows the immediate color change with
14
15
16
different intensities in all the detection reservoirs that the phenolphthalein underwent when
17
18
0.10 M NaOH solution reached each of them. However, as the concentration decreased,
19
20
21 the color gradually disappeared (Figure 1b). A concentration higher than 0.25% was
22
23
24 needed for 0.1 M NaOH in order to determine a visible color change after the µ-PAD had
25
26
27 dried. Initially, the color could be seen in the detection reservoirs containing more than
28
29
30 0.25% of phenolphthalein for 0.01 M NaOH (Figure 1c), although it had almost
31
32
33 disappeared in all the detection reservoirs after drying (Figure 1d). Compared to Figure
34
35
36 1a, the color intensity in Figure 1c was obviously reduced, so that an increase in the
37
38
39 concentration of phenolphthalein seemed better when using 0.01 M NaOH. Consequently,
40
41
42 0.25% of phenolphthalein was employed for the titration of 0.1 to 1 M NaOH whereas the
43
44
45
concentration of phenolphthalein was increased to 0.5% in the µPAD for measuring the
46
47
samples containing from 0.01 to 0.1 M NaOH.
48
49
50 With this system, we also could estimate the excess amounts of NaOH when the
51
52
53 concentration of NaOH was higher than that of the primary standard added to the reaction
54
55
56 reservoirs. Assuming that the solution in the reaction reservoir was roughly
57
58
59 11
60
ACS Paragon Plus Environment
Analytical Chemistry Page 12 of 32

1
2
3
4 homogeneous when neutralization progressed, the concentration of the NaOH solution
5
6
7 penetrating into the detection reservoir was simply given by subtracting the concentration
8
9
10 of the acid solution added to each reaction reservoir from that of the NaOH since the
11
12
13 volume of the acid solution was equal to the sample volume that was needed to fill the
14
15
16 reaction reservoir. For example, when the µ-PAD contained 0.1 to 1 M of acid solution in
17
18
the reaction reservoirs, we can determine NaOH solutions with concentrations of 0.1−1 M.
19
20
21 In the µ-PAD, the concentrations of hydroxide ion in the reaction reservoirs were more
22
23
24 than 0.1 M after neutralization if the acid concentration in the reaction reservoir was lower
25
26
27 than that of the NaOH solution; COH, detect = COH, sample − CH, react ≥ 0.1, where COH, detect, COH,
28
29
30 sample, and CH, react are the concentrations of OH− penetrating into the detection reservoir,
31
32
33 OH− in the sample solution, and the acid in the reaction reservoir, respectively. Thus, the
34
35
36 concentration of the NaOH was sufficient to change the color of the phenolphthalein once
37
38
39 it had penetrated the detection reservoir. The situation was similar when using the µ-PAD
40
41
42 for 0.01 to 0.1 M NaOH; namely, the concentrations of NaOH were higher than 0.01 M
43
44
45
when the concentrations of the acid were lower than that of NaOH.
46
47
Acid-base titration. To determine the concentrations of NaOH solutions, 1 µL of KHPth
48
49
50 solutions that varied from 0.1 to 1 M were applied to the ten reaction reservoirs in intervals
51
52
53 of 0.1 M, whereas the detection reservoirs contained 0.5 µL of 0.25% phenolphthalein,
54
55
56 which were the optimal conditions, as shown in Figure 1. Thus, the µPADs permitted the
57
58
59 12
60
ACS Paragon Plus Environment
Page 13 of 32 Analytical Chemistry

1
2
3
4 determination of solutions with concentrations of 0.1-1 M NaOH. The results for 0.4, 0.6,
5
6
7 and 0.8 M NaOH solutions are shown in Figure 2. The numbers on the µPAD indicate
8
9
10 the molar concentration of KHPth being added to the reaction reservoirs, as seen in Figure
11
12
13 2. Each titration was completed within 1 min, which includes the time needed to apply
14
15
16
the sample solution (Supporting Iinformation, Video S1). The detection reservoirs turned
17
18
to a red color when the concentrations of KHPth were lower than that of NaOH.
19
20
21 Therefore, a clear red color was observed in detection reservoir numbers 0.3, 0.5, and 0.7
22
23
24 for 0.4, 0.6, and 0.8 M NaOH, respectively, as shown in Figures 2a, 2c, and 2e after the
25
26
27 whole channel was occupied by the sample solution. As the results show, the
28
29
30 concentrations of NaOH were determined directly with no calibration since each reaction
31
32
33 reservoir contained a known amount of the primary standard substance, which neutralized
34
35
36 the NaOH. In practice, the concentrations of NaOH ranged from 0.31-0.40, 0.51-0.60,
37
38
39 and 0.71-0.80 M, as shown in Figures 2a, 2c, and 2e, respectively. Reproducibility of the
40
41
42 µPAD was excellent since the same results were obtained for five independent
43
44
45
measurements for each sample.
46
47
The color is clearly shown in Figures 2a, 2c, and 2e where the µ-PADs were still wet,
48
49
50 whereas the color change was unclear after drying, as shown in Figures 2b, 2d, and 2f.
51
52
53 Furthermore, some detection reservoirs underwent an incorrect color change after drying
54
55
56 for 60 min due to an unfavorable diffusion of excess hydroxide ion. These results
57
58
59 13
60
ACS Paragon Plus Environment
Analytical Chemistry Page 14 of 32

1
2
3
4 indicate that the end point should be found as soon as the sample solution occupies the
5
6
7 whole channel.
8
9
10 To determine lower concentrations of NaOH solutions, the standard solutions of
11
12
13 KHPth were diluted 10-fold. The solutions of KHPth with concentrations of 0.01 to 0.1
14
15
16
and 0.5% phenolphthalein were applied to the reaction and the detection reservoirs, and
17
18
then 30 µL of 0.04, 0.06, and 0.08 M NaOH solutions were applied to the µPADs. Clear
19
20
21 end points were found for dilute alkaline solutions with concentrations ranging from 0.01
22
23
24 to 0.1 M (Supporting Information, Figure S3). According to these results, the µPADs are,
25
26
27 in fact, useful for the determination of NaOH at concentrations ranging from 0.01 M to 1
28
29
30 M since it is too difficult to measure these concentrations via conventional pH test paper
31
32
33 (Supporting Information, Figure S4). We also fabricated the µPAD for measuring 0.005
34
35
36 to 0.05 M NaOH by adding 1% phenolphthalein to the detection reservoirs. The results
37
38
39 showed that the µPAD was applicable to the determinations of 0.005 to 0.05 M NaOH at
40
41
42 intervals of 0.005 M.
43
44
45
Furthermore, by coupling two types of µPADs containing 0.1 to 1 M KHPth and 0.01
46
47
to 0.1 M KHPth, the concentrations of NaOH were determined more precisely at intervals
48
49
50 of 0.02 M. Initially, a sample was applied to the µPAD for 0.1 M to 1 M (high
51
52
53 concentration) to determine a rough concentration. Then, the sample was mixed with an
54
55
56 equal volume of KHPth with the concentration determined by the first titration, followed
57
58
59 14
60
ACS Paragon Plus Environment
Page 15 of 32 Analytical Chemistry

1
2
3
4 by an application to the µPAD at 0.01 M to 0.1 M (low concentration). Examples are
5
6
7 shown in Figure 3 where 0.25, 0.27, and 0.29 M NaOH were determined using the µ-PADs
8
9
10 for both high and low concentrations. The results shown in Figure 3a were obtained by
11
12
13 the µ-PAD for high concentrations and show that the sample contained NaOH in
14
15
16
concentrations ranging from 0.21 to 0.30. Then, we added an equal volume of 0.20 M
17
18
KHPth and applied the mixture to the µ-PAD for low concentrations. The results shown
19
20
21 in Figure 3b, which were obtained by the µPAD for low concentrations, suggest that the
22
23
24 concentration of the mixture was from 0.021 to 0.030 M. Consequently, it is apparent
25
26
27 that the concentration of NaOH in the sample ranged from 0.242 to 0.260, since the sample
28
29
30 solution was diluted two-fold before being applied to the µ-PAD for low concentrations.
31
32
33 For 0.27 M and 0.29 M NaOH, the µPAD for high concentrations (0.1-1 M) showed the
34
35
36 same results (Figure 3c and 3e) as that of 0.25 M with good reproducibility using triplicate
37
38
39 measurements, as expected, whereas the mixtures with 0.2 M KHPth were determined to
40
41
42 be from 0.031 to 0.040 M for 0.27 M NaOH (Figure 3d) and from 0.041 to 0.050 M for
43
44
45
0.29 M NaOH (Figure 3f). Therefore, we should be able to discriminate these
46
47
concentrations by adding a certain amount of KHPth to the sample, followed by a
48
49
50 measurement using the µPAD for low concentrations (0.01-0.1 M)
51
52
53 The same titration method is applicable to the determination of HCl by using Na2CO3
54
55
56 as the primary standard substance instead of KHPth. In this case, when the reaction
57
58
59 15
60
ACS Paragon Plus Environment
Analytical Chemistry Page 16 of 32

1
2
3
4 reservoirs contain Na2CO3 at concentrations higher than that of the sample solution (acid),
5
6
7 a red color is observed in the corresponding detection reservoir. The results for HCl
8
9
10 solutions for concentrations of 0.4, 0.6, and 0.8 M are shown in Figure 4. The
11
12
13 performances of µPADs for HCl were similar to those for NaOH, so both acid and base
14
15
16
were determined with only the use of an appropriate primary standard substance.
17
18
We also determined nitric acid, sulfuric acid, acetic acid, and ammonia since the
19
20
21 µ-PAD is expected to be applicable to the determination of other acids and bases. Strong
22
23
24 acids (nitric acid and sulfuric acid) and a weak acid (acetic acid) were successfully
25
26
27 determined as well as HCl with good reproducibility. The µ-PAD also worked well with
28
29
30 a clear end point in the determination of ammonia solutions with the concentrations of 0.1
31
32
33 to 1 M, although the intensity of the red color was lower than that obtained by NaOH due
34
35
36 to the weak basic property of ammonia. However, the end point for 0.01 to 0.1 M
37
38
39 ammonia was undetectable as shown in Figure 5a where 0.07 M of an ammonia solution
40
41
42 was determined using KHPth and phenolphthalein as the acid and indicator. As Figure 5a
43
44
45
shows, we could not find the correct end point due to the weak intensity of the color,
46
47
although the detection reservoirs of 0.01 to 0.05 seemed to be slightly red. However, the
48
49
50 detection reservoir for 0.06 M must turn a definite red color since the concentration of
51
52
53 ammonia is 0.07 M. In addition, the slight red color had completely disappeared within a
54
55
56 few minutes after completing the titration. Ammonia is a weak base with a pKb of 4.76
57
58
59 16
60
ACS Paragon Plus Environment
Page 17 of 32 Analytical Chemistry

1
2
3
4 so that the pH of 0.01 M ammonia solution was estimated to be 10.6. A pH of 10.6 was
5
6
7 much lower than that of 0.01 M NaOH (pH=12), resulting in a difficulty of visual
8
9
10 detection.
11
12
13 To detect the end point for diluted ammonia solutions, a suitable indicator should be
14
15
16
selected instead of phenolphthalein. Methyl orange may be a candidate for detecting an
17
18
excess amount of ammonia since its indicator range is acidic (3.1~4.4). However, it was
19
20
21 difficult to find the color change from orange to yellow on the µ-PAD with the naked eyes.
22
23
24 Consequently, to detect ammonia at a low concentration, we employed bromocresol purple
25
26
27 as the indicator, because it has an indicator range (5.2~6.8) that is lower than that of
28
29
30 phenolphthalein (8.3~10) and the color change from yellow to purple is clearer than that
31
32
33 from orange to yellow.
34
35
36 Figure 5b shows the results of 0.07 M ammonia using bromocresol purple as the
37
38
39 indicator. A clear end point at 0.07 M was found in Figure 5b where the end point
40
41
42 became more visible than that in Figure 5a. These results indicate that several weak acids
43
44
45
and weak bases can be determined by selecting an appropriate indicator to detect the end
46
47
point.
48
49
50 Stability of µPADs. The developed µPADs can definitely be employed for point-of-use
51
52
53 and rapid measurements of acid and base concentrations for field analysis. However, the
54
55
56 µPADs must have a long lifetime for conventional use in the field where no
57
58
59 17
60
ACS Paragon Plus Environment
Analytical Chemistry Page 18 of 32

1
2
3
4 instrumentation is available. Therefore, we examined the stability of the µPADs under
5
6
7 conditions of different temperature and light exposures.
8
9
10 When the µPADs were stored at room temperature under light, they only worked for
11
12
13 five days. After five days, the phenolphthalein showed an unclear color change.
14
15
16
Conversely, when we kept the µPADs at room temperature and a low temperature of 4 ºC
17
18
in the dark, they were effective for more than a month. Therefore, the µPADs should be
19
20
21 stored in the dark before use in order to obtain reproducible results. The instability can
22
23
24 obviously be attributed to the susceptibility of phenolphthalein to light, as indicated in the
25
26
27 safety data sheet.
28
29
30 On-site analysis. To demonstrate the utility of the µ-PAD in on-site analysis, acidic hot
31
32
33 spring water was determined at the site of the Tsukahara Onsen hot spring. The hot
34
35
36 spring water contained a high concentration of iron ion which formed precipitation of
37
38
39 hydroxide by consuming hydroxide ion, resulting in a positive error. So, in the analysis,
40
41
42 the iron ion was removed using a cation-exchange SPE cartridge before the titration.
43
44
45 Figure 6a shows the tools used for the titration by the µ-PAD in the field. It should be
46
47
noted that no glassware was needed for the titration by the µ-PAD. The results obtained
48
49
50 in the field by the µPAD for 0.005 to 0.05 M are shown in Figure 6b. The acid
51
52
53 concentration of the hot spring water was determined to be 0.020 M (practically ranging
54
55
56 from 0.020 to 0.024 M) with good reproducibility in triplicate measurements by the µPAD.
57
58
59 18
60
ACS Paragon Plus Environment
Page 19 of 32 Analytical Chemistry

1
2
3
4 The hot spring water was also determined at the laboratory by classic titration, and the
5
6
7 results showed a concentration of 0.0222±0.0005 M (n=5) which was consistent with
8
9
10 those of the on-site analysis. Therefore, the µ-PAD permitted acid-base titration in the
11
12
13 field without using glassware.
14
15
16
17
18
CONCLUSIONS
19
20
21 A simple and rapid titration method was developed using µ-PADs fabricated by a wax
22
23
24 printer. The newly designed µ-PADs were successfully applied to the titrations of both
25
26
27 acids and bases using KHPth and Na2CO3 as the primary standard substances, respectively.
28
29
30 Titration was completed within one minute by dropping 30 µL of a sample solution via a
31
32
33 micropipette. The µ-PAD has advantages in portability and disposability since it is small,
34
35
36 light, and inexpensive. It should be emphasized that the method needs no calibration
37
38
39 curve since the concentration of the sample is determined directly by the known amounts
40
41
42 of a primary standard substance that has been added to the reaction reservoirs. The
43
44
45 µPAD permitted acid-base titration in the field as demonstrated in the analysis of an acidic
46
47
hot spring water sample. A similar strategy could be applicable to the other titration
48
49
50 methods including chelate titration, redox titration, and precipitation titration by using an
51
52
53 appropriate primary standard substance and an indicator. Consequently, the proposed
54
55
56 µ-PAD is a viable alternative to classic titration methods in the analysis of several
57
58
59 19
60
ACS Paragon Plus Environment
Analytical Chemistry Page 20 of 32

1
2
3
4 chemical species in the field at sites where glassware and large volumes of solutions are
5
6
7 difficult to obtain.
8
9
10
11
12 Corresponding Author
13 *E-mail: kaneta@okayama-u.ac.jp. Tel: +81-86-251-7847. Fax: +81-86-251-7847.
14
15
16 Notes
17
18 The authors declare no competing financial interest.
19
20
21
22
23 ACKNOWLEDGMENTS
24
25
26 This research was supported by The Yakumo Foundation for Environmental Science and
27
28
29 Grants-in-Aid for Scientific Research, Scientific Research (B) (No. 26288067). We
30
31
would like to thank Ms. M. Sakurai, Mr. T. Shinohara, Mr. T. Abe, and Mr. H. Takahata of
32
33
34 Beppu Hakudo Kogyo Corp. for their kind help in the sampling of the hot spring water.
35
36
37
38
39
40 SUPPORTING INFORMATION AVAILABLE
41
42
43 Estimated volumes of solution needed to fill the µ-PAD, the design of the µPAD, a photo
44
45
46 of an acrylic plate holder, titration of NaOH solutions with concentrations of 0.04, 0.06,
47
48
49 and 0.08 M, color change of conventional pH test paper, and a video of titration. This
50
51
52 information is available free of charge via the Internet at http://pubs.acs.org/.
53
54
55
56
57
58
59 20
60
ACS Paragon Plus Environment
Page 21 of 32 Analytical Chemistry

1
2
3
4 REFERENCES
5
6
7 (1) Lagally, E. T.; Medintz, I.; Mathies R. A. Anal. Chem. 2001, 73, 565–570.
8
9
10 (2) Jacobson, S. C.; Hergenroder, R); Koutny, L. B.; Ramsey, J. M. Anal. Chem. 1994, 66,
11
12
13 1114–1118.
14
15
16
(3) Lee, J. H.; Song, Y.-A.; Tannenbaum, S. R.; Han, J. Anal. Chem. 2008, 80, 3198–3204.
17
18
(4) Qin, D.; Xia, Y.; Rogers, J. A.; Jackman, R. J.; Zhao, X.; Whitesides, G. M. Top. Curr.
19
20
21 Chem. 1998, 194, 1−20.
22
23
24 (5) Martinez, A. W.; Phillips, S. T.; Butte, M. J.; Whitesides, G. M. Angew. Chem., Int. Ed.
25
26
27 2007, 46, 1318−1320.
28
29
30 (6) Lu, Y.; Shi, W.; Jiang, L.; Qin, J.; Lin, B. Electrophoresis 2009, 30, 1497–1500.
31
32
33 (7) Carrilho, E.; Martinez, A. W.; Whitesides, G. M. Anal. Chem. 2009, 81, 7091−7095.
34
35
36 (8) Maejima, K.; Tomikawa, S.; Suzuki, K.; Citterio, D. RSC Adv. 2013, 3, 9258−9263.
37
38
39 (9) Dungchai, W.; Chailapakul, O.; Henry, C. S. Analyst 2011, 136, 77−82.
40
41
42 (10) Cassano, C. L.; Fan, Z. H. Microfluid. Nanofluid. 2013, 15, 173−181.
43
44
45
(11) Liu,W.; Cassano, C. L.; Xu, X,; Fan, Z. H. Anal. Chem. 2013, 85, 10270−10276.
46
47
(12) Zhang, Y.; Zhou C.; Nie, J.; Le, S.; Qin, Q.; Liu, F.; Li, Y.; Li, J. Anal. Chem. 2014, 86,
48
49
50 2005−2012.
51
52
53 (13) Mentele, M. M.; Cunningham, J.; Koehler, K.; Volckens, J.; Henry, C. S. Anal. Chem.
54
55
56 2012, 84, 4474−4480.
57
58
59 21
60
ACS Paragon Plus Environment
Analytical Chemistry Page 22 of 32

1
2
3
4 (14) Rattanarat, P.; Dungchai, W.; Cate, D. M.; Siangproh, W.; Volckense, J., Orawon
5
6
7 Chailapakul, O.; Henry, C. S. Anal. Chim. Acta 2013, 800 50– 55.
8
9
10 (15) Cate, D. M.; Nanthasurasak P.; Riwkulkajorn, P.; L’Orange, C.; Henry, C. S.;
11
12
13 Volckens, J. Ann. Occup. Hyg. 2014, Vol. 58, 413–423.
14
15 (16) Yamada, K.; Takaki, S.; Komuro N.; Suzuki, K.; Citterio, D. Analyst 2014, 139,
16 1637–1643.
17
18
(17) Li, X.; Tian, J.; Shen, W. Anal. Bioanal. Chem. 2010, 396, 495–501.
19
20
21 (18) Wang, Y; Wang, S.; Ge, S.; Wang, S.; Yan, M.; Zang, D.; Yu, J. Monatsh. Chem.
22
23
24 2014, 145, 129–135
25
26
27 (19) Jokerst, J. C.; Adkins, J. A.; Bisha, B.; Mentele, M. M.; Goodridge, L. D.; Henry, C.
28
29
30 S. Anal. Chem. 2012, 84, 2900−2907.
31
32
33 (20) Cheng, C.-M.; Martinez, A. W.; Gong, J.; Mace, C. R.; Phillips, S. T.; Carrilho, E.;
34
35
36 Mirica, K. A.; Whitesides, G. M. Angew. Chem., Int. Ed. 2010, 122, 4881−4884.
37
38
39 (21) Rattanarat, P.; Dungchai, W.; Siangproh, W.; Chailapakul, O.; Henry, C. S. Anal.
40
41
42 Chim. Acta 2012, 744, 1−7.
43
44
45
(22) Lankelma, J.; Nie, Z.; Carrilho, E.; Whitesides, G. M. Anal. Chem. 2012, 84,
46
47
4147−4152.
48
49
50 (23) Santhiago, M.; Wydallis, J. B.; Kubota, L. T.; Henry, C. S. Anal. Chem. 2013, 85,
51
52
53 5233−5239.
54
55
56 (24) Santhiago, M.; Kubota, L. T. Sens. Actuators, B 2013, 177, 224– 230.
57
58
59 22
60
ACS Paragon Plus Environment
Page 23 of 32 Analytical Chemistry

1
2
3
4 (25) Cate, D. M.; Dungchai, W.; Cunningham, J. C.; Volckens J.; Henry C. S. Lab Chip
5
6
7 2013, 13, 2397−2404.
8
9
10 (26) Lewis, G. G.; Robbins, J. S.; Phillips, S. T. Anal. Chem. 2013, 85, 10432–10439.
11
12
13 (27) Lewis, G. G.; Robbins, J. S.; Phillips, S. T. Chem. Commun. 2014, 50, 5352–5354.
14
15
16
(28) Dungchaia, W.; Chailapakul, O; Henry, C. S. Anal. Chim. Acta 2010, 674, 227–233.
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59 23
60
ACS Paragon Plus Environment
Analytical Chemistry Page 24 of 32

1
2
3
4 FIGURE LEGENDS
5
6
7 Figure 1. Optimization of the concentration of phenolphthalein added to the detection
8
9
10 reservoirs.
11
12
13 The concentration of phenolphthalein; Reservoir number, 1=0.01% , 2=0.025%, 3=0.050%,
14
15
16
4=0.075%, 5=0.10%, 6=0.25%, 7=0.50%, 8=0.75%, 9=1%, 10=0% (blank); volume added,
17
18
0.5 µL. (a) Immediate image after the addition of 0.1 M NaOH, (b) image of (a) after
19
20
21 drying, (c) immediate image after the addition of 0.01 M NaOH, (d) image of (c) after
22
23
24 drying.
25
26
27
28
29
30 Figure 2. Titration of NaOH solutions with different concentrations. (a) 0.4 M NaOH
31
32
33 (immediately after addition), (b) 0.4 M NaOH (after drying), (c) 0.6 M NaOH
34
35
36 (immediately after addition), (d) 0.6 M NaOH (after drying), (e) 0.8 M NaOH
37
38
39 (immediately after addition), (f) 0.8 M NaOH (after drying). The numbers of the
40
41
42 reservoirs indicate the concentrations of KHPth solutions added to the reaction reservoirs.
43
44
45 Detection reservoirs contained 0.5 µL of 0.25% phenolphthalein.
46
47
48
49
50 Figure 3. Titration of 0.25, 0.27, and 0.29 M NaOH solutions. The numbers of the
51
52
53 reservoirs indicate the concentrations of KHPth solutions added to the reaction reservoirs.
54
55
56 (a) Detection reservoirs, 0.5 µL of 0.25% phenolphthalein; sample, 0.25 M NaOH. (b)
57
58
59 24
60
ACS Paragon Plus Environment
Page 25 of 32 Analytical Chemistry

1
2
3
4 Detection reservoirs, 0.5 µL of 0.5% phenolphthalein; sample, a mixture of equal volumes
5
6
7 of 0.25 M NaOH and 0.20 M KHPth that should contain 0.025 M of hydroxide ion. (c)
8
9
10 Detection reservoirs, 0.5 µL of 0.25% phenolphthalein; sample, 0.27 M NaOH. (d)
11
12
13 Detection reservoirs, 0.5 µL of 0.5% phenolphthalein; sample, a mixture of equal volumes
14
15
16
of 0.27 M NaOH and 0.20 M KHPth that should contain 0.035 M of hydroxide ion. (e)
17
18
Detection reservoirs, 0.5 µL of 0.25% phenolphthalein; sample, 0.29 M NaOH. (f)
19
20
21 Detection reservoirs, 0.5 µL of 0.5% phenolphthalein; sample, a mixture of equal volumes
22
23
24 of 0.29 M NaOH and 0.20 M KHPth that should contain 0.045 M of hydroxide ion.
25
26
27
28
29
30 Figure 4. Titration of HCl solutions with different concentrations.
31
32
33 The numbers of the reservoirs indicate the concentrations of Na2CO3 solutions added to the
34
35
36 reaction reservoirs. Detection reservoirs contained 0.5 µL of 0.5% phenolphthalein.
37
38
39 (a) 0. 4 M HCl, (b) 0.6 M HCl, (c) 0.8 M HCl.
40
41
42
43
44
45
Figure 5. Titration of 0.07 M ammonia solution.
46
47
Indicator, (a) 0.5% phenolphthalein, (b) 0.5% bromocresol purple. The reaction
48
49
50 reservoirs contained 1 µL of 0.01 to 0.1 M KHPth solutions. Detection reservoirs
51
52
53 contained 0.5 µL of the indicator.
54
55
56
57
58
59 25
60
ACS Paragon Plus Environment
Analytical Chemistry Page 26 of 32

1
2
3
4 Figure 6. Titration of water samples from the source of a hot spring in the field.
5
6
7 (a) Tools for on-site analysis. 1=disposable syringe for applying pressure to a
8
9
10 cation-exchange SPE cartridge, 2=cation-exchange SPE cartridge, 3=sample tube,
11
12
13 4=µ-PAD, 5=acrylic plate holder. (b) Titration by the µ-PAD. The reaction reservoirs
14
15
16 contained 1 µL of 0.005 to 0.050 M Na2CO3 solutions. Detection reservoirs contained
17
18
0.5 µL of 1% phenolphthalein. The insert shows the sampling from the source of a hot
19
20
21 spring.
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59 26
60
ACS Paragon Plus Environment
Page 27 of 32 Analytical Chemistry

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
Figure 1. Optimization of the concentration of phenolphthalein added to the detection reservoirs.
47
The concentration of phenolphthalein; Reservoir number, 1=0.01% , 2=0.025%, 3=0.050%, 4=0.075%,
48 5=0.10%, 6=0.25%, 7=0.50%, 8=0.75%, 9=1%, 10=0% (blank); volume added, 0.5 µL. (a) Immediate
49 image after the addition of 0.10 M NaOH, (b) image of (a) after drying, (c) immediate image after the
50 addition of 0.01 M NaOH, (d) image of (c) after drying.
51 926x1190mm (96 x 96 DPI)
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment
Analytical Chemistry Page 28 of 32

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
Figure 2. Titration of NaOH solutions with different concentrations. (a) 0.4 M NaOH (immediately after
47
addition), (b) 0.4 M NaOH (after drying), (c) 0.6 M NaOH (immediately after addition), (d) 0.6 M NaOH
48 (after drying), (e) 0.8 M NaOH (immediately after addition), (f) 0.8 M NaOH (after drying). The numbers of
49 the reservoirs indicate the concentrations of KHPth solutions added to the reaction reservoirs. Detection
50 reservoirs contained 0.5 µL of 0.25% phenolphthalein.
51 926x1190mm (96 x 96 DPI)
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment
Page 29 of 32 Analytical Chemistry

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
Figure 3. Titration of 0.25, 0.27, and 0.29 M NaOH solutions. The numbers of the reservoirs indicate the
47
concentrations of KHPth solutions added to the reaction reservoirs. (a) Detection reservoirs, 0.5 µL of 0.25%
48 phenolphthalein; sample, 0.25 M NaOH. (b) Detection reservoirs, 0.5 µL of 0.5% phenolphthalein; sample,
49 a mixture of equal volumes of 0.25 M NaOH and 0.20 M KHPth that should contain 0.025 M of hydroxide
50 ion. (c) Detection reservoirs, 0.5 µL of 0.25% phenolphthalein; sample, 0.27 M NaOH. (d) Detection
51 reservoirs, 0.5 µL of 0.5% phenolphthalein; sample, a mixture of equal volumes of 0.27 M NaOH and 0.20 M
52 KHPth that should contain 0.035 M of hydroxide ion. (e) Detection reservoirs, 0.5 µL of 0.25%
53 phenolphthalein; sample, 0.29 M NaOH. (f) Detection reservoirs, 0.5 µL of 0.5% phenolphthalein; sample, a
54 mixture of equal volumes of 0.29 M NaOH and 0.20 M KHPth that should contain 0.045 M of hydroxide ion.
381x457mm (96 x 96 DPI)
55
56
57
58
59
60
ACS Paragon Plus Environment
Analytical Chemistry Page 30 of 32

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
Figure 4. Titration of HCl solutions with different concentrations.
47
The numbers of the reservoirs indicate the concentrations of Na2CO3 solutions added to the reaction
48 reservoirs. Detection reservoirs contained 0.5 µL of 0.5% phenolphthalein. (a) 0. 4 M HCl, (b) 0.6 M HCl,
49 (c) 0.8 M HCl.
50
51 381x457mm (96 x 96 DPI)
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment
Page 31 of 32 Analytical Chemistry

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
Figure 5. Titration of 0.07 M ammonia solution.
47
Indicator, (a) 0.5% phenolphthalein, (b) 0.5% bromocresol purple. The reaction reservoirs contained 1 µL
48 of 0.01 to 0.1 M KHPth solutions. Detection reservoirs contained 0.5 µL of the indicator.
49
50 381x457mm (96 x 96 DPI)
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment
Analytical Chemistry Page 32 of 32

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
Figure 6. Titration of water samples from the source of a hot spring in the field.
47
(a) Tools for on-site analysis. 1=disposable syringe for applying pressure to a cation-exchange SPE
48 cartridge, 2=cation-exchange SPE cartridge, 3=sample tube, 4=µ-PAD, 5=acrylic plate holder. (b) Titration
49 by the µ-PAD. The reaction reservoirs contained 1 µL of 0.005 to 0.050 M Na2CO3 solutions. Detection
50 reservoirs contained 0.5 µL of 1% phenolphthalein. The insert shows the sampling from the source of a hot
51 spring.
52
53 381x457mm (96 x 96 DPI)
54
55
56
57
58
59
60
ACS Paragon Plus Environment