Microchemical Journal 149 (2019) 104072

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Microchemical Journal
journal homepage: www.elsevier.com/locate/microc

A smartphone-based colorimetry after dispersive liquid–liquid T

microextraction for rapid quantification of calcium in water and food
samples
Bo Penga, , Juanjuan Zhoua, Jiamei Xua, Mimi Fana, Yongjun Maa, Min Zhoua, Tingting Lib,
⁎

Shengguo Zhaoc
a
Key Laboratory of Bioelectrochemistry & Environmental Analysis of Gansu Province, Key Laboratory of Eco-Environment-Related Polymer Materials, Ministry of
Education, College of Chemistry and Chemical Engineering, Northwest Normal University, Lanzhou 730070, China
b
Food Test Center of Jiuquan, Jiuquan 735000, China
c
Gansu Entry-Exit Inspection and Quarantine Bureau, Lanzhou 730020, China

ARTICLE INFO ABSTRACT

Keywords: A simple method on dispersive liquid–liquid microextraction combining with smartphone colorimetric analysis
Calcium was developed to determine trace calcium. The colored complex of calcium and glyoxal bis(2-Hydroxyanil)
Microextraction (GBHA) can be extracted into a mixture solvent of chloroform and 1,2-dichloromethane in the presence of
RGB tetradecyldimethylbenzylammonium chloride (zephiramine). The R, G and B value of organic phase after DLLME
Smartphone
was caught by Android app Color Grab. The quantitative relationship between the color intensity and calcium
Colorimetry
content was constructed based on G channel as the analytical signal. The calibration curve was linear in the
range of 0.06–1.5 μg mL−1 of calcium ion with a limit of detection (LOD) of 0.017 μg mL−1 and limit of
quantification (LOQ) of 0.056 μg mL−1. The proposed method was successfully applied to analyze calcium in
water and food samples and validated with FAAS method and CRM material.

1. Introduction chromomeric reagents such as glyoxal bis(2-hydroxyanil) (GBHA)

Calcium is one of the most important elements in human body and
widely exists in environmental water bodies. Calcium is closely related
to body function and metabolism. At present, many people don't have
enough calcium intakes, especially for the elderly and adolescents.
Excessive intake of calcium in human body can lead to excessive blood
calcium and endanger human health. The content of calcium in water is
one of the regular items of industrial water, such as precipitation,
groundwater, and oilfield water injection and so on. Different mature
instrumental techniques have been developed for the analysis of cal-
cium ion, such as atomic absorption spectrometry [1], atomic emission
spectrometry [2], X-ray fluorescence [3], inductively coupled plasma
mass spectrometry (ICP-MS) [4], etc.. Although these instrumental
analyses are accepted for very good accuracy and accuracy in chal-
lenging detection scenes, most of these them need expensive purchase
and maintenance cost, time-consuming manipulation steps and special
training for measurement accuracy, precision and sensitivity.
The UV–Vis spectrophotometric determination of calcium is gen-
erally based on the complexation reaction between calcium and some
[5,6], bromopyrogallol red [7], calmagite [8], methylthymol blue
(MTB) [9], Eriochrome Black T (EBT) [10], calmagite [8], etc. The
UV–Vis method is widely employed in many small factories, routine
works or base laboratory in China because the colorimetric method has
advantages including low cost, fast reaction, enough accuracy and re-
liability suitable for resource limited settings. However, most of these
conventional colorimetric analyses are still limited due to lack of sta-
bility, adequate sensitivity and selectivity or suffering complex ma-
nipulation. Therefore, an accurate, simple and cost-effective routine
detecting method for trace calcium ion is also important.
Many household color-recording devices, such as office scanners
[11], digital cameras [12], smartphones [13] have been introduced for
rapid quantitative chemical analysis [14], chemical and biosensing
[15], bioanalytical diagnosis [13], food diagnostics [16,17] and water
analysis [18,19]. Smartphone-based spectrometer and colorimetry have
been gaining continuous attention, popularization and fast application
due to their low cost, fast response, simple analysis process [20], real-
time communicate on [21], field detection [22], and compatibility with
data acquisition and processing for “lab-on-a-chip” systems [23]

⁎
Corresponding author.
E-mail address: pengbo.nwnu@hotmail.com (B. Peng).

https://doi.org/10.1016/j.microc.2019.104072
Received 31 May 2019; Received in revised form 4 July 2019; Accepted 4 July 2019
Available online 04 July 2019
0026-265X/ © 2019 Elsevier B.V. All rights reserved.
B. Peng, et al.
especially in resource-limited settings [15]. The digital colorimetry
based on digital camera has been applied to calcium water hardness
analysis [6] using GBHA as an indicator in which Factorial Analysis was
employed to explore RGB color intensity data. However, GBHA‑calcium
complex will easily breakdown exposure to air [24–26], especially
during transition and placement in the spectrometer. The chelates
change to be stable if they exist in an organic solvent such as chloro-
form [26]. Consequently the underlying organic layer in matched round
spectrophotometer cells instead of rectangular cells was ever employed
without removal of the aqueous layer [27].
In this paper, a method on DLLME combining with Android smart-
phone colorimetric analysis in-stu centrifuge tube was developed to
determine trace calcium. The free Android app Color Grab [28] was
used to pick RGB data of the colored solvent layer in a homemade
closed box for quantitative analysis. No extra image exporting, im-
porting and processing with computer software [29–31] was required.
The current method has been successfully applied to the determination
of calcium in real samples with simple operation, high accuracy and
lower detection limit. The smartphone colorimetry box is also readily to
deploy on site. The aim of this work is to develop a fast and potential
portable method for calcium analysis alternative to traditional re-
ference methods at very low cost.
2. Material and methods
2.1. Instruments
A pH-meter (PHS-3C, Jinke, Shanghai, China) was used to measure
pH. A hotplate used for heating during sample digestion. A vortex
agitator was used to vigorously mix the solution in centrifuge tube. A
centrifuge (80–1, Guohua, Shanghai, China) with 15 mL calibrated
centrifuge tube for accelerating the process of the phase separation.
A Xiaomi 4G smartphone was used. Parameter: Android 4.4, CPU
Snapdragon 625 OctaCore 2.0 GHz, GPU Adreno 506, Screen (5.0 in.,
1920 × 1080 (FHD), LCD IPS, Colors 16 M), Camera Rear 13.0 MP,
3 GB RAM, Internal storage 32 GB, Lithium Polymer 4100 mA h Battery.
A homemade smartphone colorimetric box (7.0 cm × 10.0 cm × 5 cm)
with a low wattage LED light was designed to catch the color of organic
phase at the bottom of centrifuge tube without removal of the aqueous
layer and sampling transfer, or dilution either (Fig. 1).
A 3 W 6500 K white LED light is embedded in a polystyrene block
(2.0 cm × 3 cm × 5 cm) to obtain uniform scattered light on top of
sample. The light block is put beside the centrifuge tube as the light
source, 1 cm from the tube, 9 cm from the bottom of tube. The top light
layout can efficiently eliminate any effect from reflected light occurring
in glass tubes, and avoid consequent lensing effect resulting from
bottom light [32]. The condition to capture and measure colors in a
Fig. 1. Smartphone colorimetric box.
2
Microchemical Journal 149 (2019) 104072
reproducible way is crucial to the study, especially for RGB color space-
based models whose value of intensity are highly susceptible to ambient
light source and other factors [33]. A preliminary experiment in the
present study has shown the above size and layout was the optimal to
capture color by directly using the bottom of centrifuge tube as mea-
suring place in stu, no sample dilution and transfer needed. The in-
tensity of the color product inside centrifuge tube is picked using Color
Grab under automatic focusing and automatic shooting mode. Ambient
light does not affect color capturing because of using the closed box
[34–36]. The closed box in the experiments is made of inexpensive and
flexible polystyrene (EPS) with the white internal mist surface without
reflection effect in order to obtain uniform internal illumination. All
measurements were uniformly carried out at room temperature.
Calcium quantifications in real samples were carried out using a Z-
2000 (Hitachi, Japan) atomic absorption spectrophotometer equipped
with standard air–acetylene flame, single element hollow cathode
lamps and Zeeman background correction. The flame composition was
always the same for all analytes according to the manufacturer's re-
commendations. Operating conditions used were: air flow rate,
10 L min−1; acetylene flow rate, 1.8 L min−1; burner height, 5.5 mm;
wavelengths, 422.7 nm; slit widths, 0.2 nm; lamp currents, 10 mA; so-
lution aspiration rate, 4.5 mL min−1; measurement time, 3 s; and the
number of replicates, 3.
2.2. Chemicals and reagents
All Chemicals and reagents used in this work were of analytical-re-
agent grade unless otherwise stated. Ultra-pure water (resistivity of
18.2 MΩ cm) was used throughout the work. Stock standard solution of
Ca2+ (1000 μg mL−1) was supplied by National Non-ferrous Metal &
Electronic Material Analytical and Testing Center (Beijing, China). The
working solution of Ca2+ (2.5 × 10−4 mol L−1) was freshly prepared
and diluted from the stock solution. Glyoxal bis (2-Hydroxyanil) (GBHA)
was purchased from Beichen Fangzheng reagent factory of Tianjin
(Tianjin, China). GBHA working solution (1.25 × 10−3 mol L−1) was
prepared in absolute ethanol every day and stored in the dark at 4 °C
until used. Zephiramine was purchased from Sigma Aldrich cargo Co.,
Ltd. (Shanghai, China), and 5 × 10−4 mol L−1 zephiramine aqueous
solution was used. Chloroform, absolute alcohol, 1, 2-dichloromethane,
sodium borate(stored in plastic bottles), sodium hydroxide, calcium
carbonate, sodium chloride, nitric acid (70%), hydrochloric acid (37%),
hydrogen peroxide (30%) were purchased from Tianjin Kaitong
Chemical Reagent Co., Ltd. (Tianjin, China). All glassware containers
were previously immersed with 2 mol L−1 nitric acid for 24 h and sub-
sequently washed with Ultra-pure water.
2.3. Dispersive liquid–liquid microextraction procedure
In DMLLE procedure, 0.5 mL 1.25 × 10−3 mol L−1 glyoxal bis (2-
Hydroxyanil) (GBHA), 1.5 mL absolute alcohol, 0.5 mL Ca2+ solution
and 3.0 mL of borax buffer solution were added into a 15 mL centrifuge
tube and diluted to 10.0 mL. Afterward, 1.4 mL 5 × 10−4 mol L−1
zephiramine solution was added and 300 μL of mixed solvent chloro-
form and 1, 2-dichloromethane (1:3, v/v) was rapidly injected into the
solution by a micro syringe to induce the formation of cloudy solution.
By following this strict sequence of addition, it has been possible to
obtain satisfactory extraction efficiency and reproducibility [5]. The
mixture solution was vigorously agitated using a vortex agitator for
1 min at 3000 rpm until the droplets were finely dispersed and a cloudy
solution further formed. Then the turbid mixture was centrifuged at
4000 rpm for 5 min. The dispersed fine droplets of the extraction phase
were settled at the bottom of centrifuge tube giving an intense orange
red color. Then color value RGB was caught by using a smartphone with
Color Grab. The reagent blank solution was also run and recorded under
the same procedure and conditions. A schematic diagram of DLLME-
smartphone colorimetric procedure is roundly shown in Fig. 2.
B. Peng, et al.
Fig. 2. Schematic diagram of DLLME-smartphone colorimetry procedure.
2.4. Sample collection and pre-treatment
Tap water samples were collected from public water supplies in our
lab. The bottle mineral water and milk powders were purchased from
local market (Lanzhou, China). The certified reference materials (CRM)
water sample was purchased from the Standard Sample Research
Institute of the Ministry of Environmental Protection (Beijing, China).
Calcium and zinc gluconates oral liquid samples were purchased from
Hubei Midday Pharmaceutical Ltd. (Hubei, China). Calcium‑zinc oral
liquid samples were purchased from Harbin Pharmaceutical Group
Sixth Pharmaceutical Factory (Harbin, China). Limestone was pur-
chased from Tianjin Kaixin Chemical Industry Co., Ltd. (Tianjin, China).
All water samples were filtered through the ultrafiltration membrane
with aperture 2–4 μm before analyzing.
Limestone sample (0.05 g) was put into 100 mL conical flask and
dissolved with appropriate amount of 2 mol L−1 hydrochloric acid. The
conical flask was heated on a hotplate below 110 °C for further diges-
tion in fume hood until completion of sample decomposition. Repeat
the procedure if the solution was not transparent. Finally, the tem-
perature was slightly elevated until almost dry the digested remnants
for eliminating most of the residual acid. The solution was finally
transferred into a 50 mL volumetric flask and made up to the mark with
water.
Milk powder sample (0.4 g) was laid into 100 mL conical flask,
10 mL concentrated nitric acid was added to wet up the sample for
about 12 h at room temperature. It was followed by addition of 10 mL
concentrated nitric acid and adding dropwise 30% hydrogen peroxide
and heating on a hotplate below 130 °C for further digestion in fume
hood until completion of sample decomposition. Repeat the procedure
if the solution was not transparent. Finally, the temperature was
slightly elevated until almost dry the digested for eliminating most of
the residual acid. The solution was finally transferred into a 50 mL
volumetric flask and made up to the mark with water (pH = 2.0–4.0).
All digested samples were filtered through the ultrafiltration membrane
with aperture 2–4 μm and stored in the refrigerator until the time of the
analysis. Reagent blank solutions were prepared similarly to evaluate
some possible contamination.
2.5. Data processing
Color Grab is a powerful, easy operation of free color acquisition
software that one touch shot can obtain RGB, HSV, Lab, CMYK and
other more than a dozen color space values with self-defined Region of
Interest (ROI). The raw RGB values (IR, IG, and IB) were directly read
out by the App on smartphone for further quantitative analysis [37,38]
without additional computational digital image transformation and
process. Calibration curves for calcium quantification employing cur-
rent method were first built to evaluate the channels, R, G, and B.
3
Microchemical Journal 149 (2019) 104072
According to preliminary statistical analysis, linear relationships be-
tween calcium and color variables deduced from the G basic color, such
as log (G), G/R, or G/Itot ratios [6] and other mathematical models
[34]. ∆EL⁎a⁎b⁎ and ∆ELuv based on other color spaces [39] were also
studied and compared. In all cases, results were not better than using
the single G channel. The G channel showed a good linear response with
satisfactory coefficient of determination because the green color is just
the complementary color of calcium-GBHA complex. Therefore, the
individual G channel value was deduced and employed as the vertical
ordinate (analytical response) for the further experiments. To build
calibration curve in this study, horizontal ordinate is the concentration
of calcium and the vertical ordinate (instrumental response) is the re-
lative intensity of G channel which is normalized as follow:
I = (IG blank IG)/255 (1)
where IG represents the G channel color value of sample solution, IG
blank represents the G channel color value of reagent blank solution. The
intensity with normalization has proved to be linearly related to the
calcium concentration. The linear relationship between calcium con-
centration and the relative intensity I is constructed under the optimum
conditions.
It is worth noting that the confirmation of an appropriate and rea-
sonable instrumental response is probably the key problem when ap-
plying smartphone colorimetry to a certain color Hue series. Different
studies revealed that the conversion of the device-dependent non-uni-
form RGB signals into the corresponding device-in-dependent, uniform
color spaces signals [19,38–40] can greatly enhance the calibration
linearity and sensitivity. However, no significant superiority was ob-
served when employing other color spaces instead of RGB color space in
current study. It can be considered that the homemade closed box could
provide a uniform and stable measuring place with constant conditions.
The differences in smartphone hardware and quantitative models re-
sulting from different color space were weakened. Therefore, the
comparison of analytical signals derived from same color space or de-
rived from various color spaces should be carried out carefully if the
determination and calculation depend on image color quantification
using camera or smartphone.
2.6. Statistical analysis
All experiments were carried out three times and the data was ex-
pressed as mean ± SD (standard deviation). The student's t-test was
used for the statistical evaluation of the results. Treatment of data and
statistical analysis (average value and recovery) were performed using
datasheets prepared in Microsoft Excel 2013 and P-values of < 0.05
were considered statistically significant.
B. Peng, et al.
3. Results and discussion
3.1. Effect of type, ratio and amount of extraction solvent
Chloroform, 1, 2-dichloromethane and carbon tetrachloride are
three kinds of most commonly-used extractants in DLLME. It was found
that the nature of the organic extractants tremendously influenced the
extractability of the calcium-GBHA complex. The phase ratio and the
total volume of organic extractant would also seriously affect enrich-
ment factor and sensitivity. Therefore, single or combinations of these
solvents were investigated in the study.
The result indicated that the extraction efficiency and color stability
were greatly improved in the presence of 1, 2-dichloromethane as a
synergistic solvent. It was very difficult to reach the maximum extrac-
tion efficiency and color intensity using any of these solvents alone.
However, the extract color faded fairly quickly after removal of the
aqueous layer [5]. Therefore, the combination of chloroform and 1, 2-
dichloromethane mixed solvent was chosen.
Different volume ratios of chloroform to 1,2-dichloromethane
mixed solvent (1:6–5:1) were subjected to the same DLLME procedure.
The DLLME extraction efficiency was found to be highly dependent
upon the volume ratio of chloroform to 1,2-dichloromethane. When the
volume ratio of chloroform to 1, 2-dichloromethane was 1: 3, the color
intensity of the organic phase reached the maximum (Fig. S1). Thus the
mixed solvent with a volume ratio of 1:3 was maintained throughout in
the experiments.
The volume of mixed solvent (200–550 μL) was determined. The
result in Fig. S2 indicated the DLLME was incomplete if the total vo-
lume of organic phase was lower than 280 μL. While the color intensity
began to decrease as the volume was higher than 320 μL due to dilution
and decreasing in enrichment factor. Thus 300 μL of mixed solvent of
chloroform and 1, 2-dichloromethane was selected as the optimum for
balancing both high enrichment factor and lower detection limit.
3.2. Effect of type and volume of disperser solvent
The injection of the disperser solvent along with mixed solvent
followed by vortex agitating made a stable cloudy solution.
Consequently, the extraction equilibrium was achieved greatly by in-
creasing the contact area between the organic phase and aqueous
phase. However, the calcium-GBHA chelate was non-extractable even
though GBHA had a superior selectivity towards calcium [40]. It was
important to control exactly the volumes and types of the reactive so-
lutions, including extraction solvent and disperser agent in DLLME.
Several common hydrophilic dispersive solvents such as acetonitrile,
ethanol, methanol and acetone were tested at first with 300 μL ex-
tracting solvent (chloroform and 1, 2-dichloromethane). However no
efficient extraction phenomenon was observed with these dispersive
solvents.
Then different surfactants or emulsifiers such as TritonX-114, Octyl
phenol polyoxyethylene (10) ether (OP-10), methyl three oxygen am-
monium chloride, Aliquat336 (N235), polyvinyl alcohol, twelve alkyl-
benzalkanoate, polyvinylpyrrolidone, n-chloro sixteen alkyl pyrrole,
zephiramine were also tried along with disperser solvent. The results
showed that only zephiramine [41], a kind of quaternary ammonium
base, could prompt the DLLME extraction efficiency in the presence of
ethanol. The similar active synergistic effect coming from zephiramine
was also found in other UV–vis detection scenes [42]. Practically, the
color of the organic phase keeps very deep and stable at the bottom of
the centrifuge tube. This phenomenon could be a kind of “self-syner-
gism” induced by the presence of zephiramine molecule. Zephiramine
has an positive effect of changing a coordination unsaturated chelate to
extractable coordination-saturated chelate by replacing water mole-
cules with the same reagent as the ligand GBHA [41]. No active effect
on the DLLME was found using other surfactants or emulsifiers. Thus
zephiramine played an indispensable role in the current process of
4
Microchemical Journal 149 (2019) 104072
DLLME. The volume optimization result of zephiramine was shown in
Fig. S3.
The color intensity of the organic phase reached the highest when
volume of zephiramine was 1.4 mL. Therefore, 1.4 mL of zephiramine
was selected as the most effective volume. The experiment showed that
choosing extra 1.5 mL of alcohol as a disperser solvent on the basis of
using zephiramine could improve the extraction efficiency. Therefore,
the combination use of 1.5 mL ethanol and 1.4 mL zephiramine solution
was employed throughout the experiment.
3.3. Effect of pH
The chromogenic reaction of calcium and GBHA is usually carried
out under alkaline conditions, thus pH has main influence on the ex-
traction efficiency. The result plotted in Fig. S4 showed that the max-
imum color intensity was at pH = 12.5–13.0 with borax sodium hy-
droxide buffer. Thus buffer solutions with pH = 12.8 were finally
chosen.
3.4. Effect of GBHA
The reaction of GBHA with calcium in alkaline solution to give a
bright red-colored chelate has served as a classically sensitive and se-
lective chromomeric reagent for calcium [26,40] detection. As shown in
Fig. S5, the color intensity increased with the increasing of GBHA
concentration and reached maximum when GBHA volume was at
0.5 mL, and then the intensity became slightly reduced with increasing
volume. GBHA was prepared in ethanol due to its limited solubility and
stability in water. The total amount of ethanol would contribute to the
dispersive effect in the DLLME. Thus this is the second sources of
ethanol introduced (the other one is in Section 3.2). The optimum
GBHA volume was chosen as 0.5 mL in all studies.
3.5. Effect of salt
The influence of ionic strength was evaluated by adding different
amounts of NaCl (0–1.0 mol L−1). The results indicated that the ex-
traction efficiency had not obvious changes with increasing NaCl ad-
dition compared with no salt added. Thus, salt was not employed in all
experiments.
3.6. Effect of extraction time and centrifugation time
The extraction time in DLLME procedure refers to the interval time
between injections the dispersant and the extractant into the aqueous
sample and starting to centrifuge. According to the obtained results, the
equilibrium state was obtained quickly within 1 min with vortex agi-
tating in the presence of zephiramine. The variations of longer vortex
agitating time were not significant. The time-consuming step was the
centrifuging of sample solution. Fig. S6 indicated that the sample
mixture was centrifuged for 5 min at 4000 rpm to reach a clear phase
separation.
3.7. The color stability of organic phase
The red complex of calcium with GBHA remains stable only 6 h
when it is extracted with 1,2-dichloroethane [40], while it fades easily
once the solution is exposed to air in basic media within a few minutes
[5,26]. In current study, the color of chelates in organic phase could
keep constant up to 20 h because the complexes in organic phase were
sealed at the bottom of the centrifuge tube after phase separation.
DLLME procedure provides a more favorable environment to stabilize
the colored analyte in the organic phase, avoiding air oxidation and
facilitating the color-picking in stu. All the measurement 30 min of
standing time was selected.
B. Peng, et al.
3.8. Interferences
The potential interference from other species on the determination
of 1 μg mL−1 Ca2+ was researched. Solutions containing Ca2+ and
various amounts of interfering ions were treated according to the re-
commended procedure. The tolerance limit of various ions was defined
as the largest amount making a variation of less than ± 5%. GHBA
chelates calcium at a high pH value so interferences are generally
limited [5]. The result showed that up to 2000-fold amounts of K+,
MoO42−, NH4+ did not interfere, up to 500-fold amounts of SCN− and
thiourea did not interfere, 200-fold amounts of SO42−, Ba2+, Mn2+,
Cr3+, Fe2+, and PO43− ions did not interfere. 50-fold amounts of some
transition metal ions Ni2+, Fe3+, Cu2+, Sr2+, Co2+, Zn2+ and Mg2+
had positive interference. EDTA could be used as masking agent for
Sr2+, Co2+, Zn2+. Thiourea was used to mask Cu2+, Hg2+, Ni2+, Fe2+.
Both triethanolamine and tartrate could be used to mask Fe3+, Al3+.
However, excessively large amount of interfering ions should be sepa-
rated at first by controlling pH.
3.9. Analytical features
Under the optimized conditions, the analytical parameters were
determined. The calibration curve was linear in the range of
0.06–1.5 μg mL−1 of calcium ion with a correlation coefficient (R2) of
0.9998 (Regression equation, I = 0.4896C - 0.0188). The LOD and LOQ
of the method were calculated from the ratio of 3 and 10 times of
standard deviation of the measurement blanks (n = 10) to the slope of
the linear section of calibration curve was 0.017 μg L−1 and
0.056 μg L−1. The precision of the method for 0.5 μg mL−1 calcium
detection was calculated in terms of relative standard deviation (RSD)
was 2.71% for the intra-day precision (n = 9) and 3.93% for inter-day
(n = 9).
The same concentration of calcium was measured by using 10 dif-
ferent centrifuge tubes to evaluate the repeatability resulting from the
shape difference of centrifuge tubes, the precision was calculated in
terms of RSD was 1.07%. The same centrifuge tube was continuously
determined by rotation angle 36 degrees of each rotation. The precision
of 10 times determinations was calculated in terms of RSD was 1.01%.
The precision experiments suggest that the color measurement is not
affected by a slight displacement of the centrifuge tube when each in-
serting. It can be also found the subtle difference of the bottom of
centrifugal tubes in shape or glass thickness does not affect the color
catching either. Such small deviation indicates that the disturbance
caused by the irregularity of glass material processing and the aniso-
tropy of centrifuge tube can be negligible in color-picking. The sample
insert/injection in current method has satisfactory reproducibility due
to the well-designed dark box with a constant illumination conditions
which are the key problem when adopting RGB color space as a
quantitative model.
4. Application
The practical samples were subjected to the DLLME procedure. The
suggested method was validated by comparing determination results
with Flame Atomic Absorption Spectrophotometry (FAAS) method. All
values of t value calculated according to the paired t-test are smaller
than t95%, 4 = 2.78 (t-test for a confidence level of 95%, repeated 5
times). The data revealed that the determination between FAAS and the
proposed method did not present statistically significant differences.
The recovery experiments were conducted and all samples were spiked
with Ca2+ standard solutions in three levels. The recoveries were found
ranging from 96.0%–103%. The determination results were showed in
Table S1. The proposed method was also applied in the determination
of calcium in water quality sample Certified Reference Material
(GSB07–1192-2000). The results were given in Table S2. Student's t-test
data showed there was no statistically significant difference at a 95%
5
Microchemical Journal 149 (2019) 104072
confidence level. It turns out that the developed method can be suc-
cessfully apply to quantitative analysis of Ca2+ in real samples with
accurate results.
5. Conclusion
A DLLME procedure combined with smartphone colorimetric de-
termination based on RGB color space in stu was proven to be an effi-
cient, fast, reproducible and sensitive methodology to analyze trace
amount of calcium in water and food simples. The current method was
optimized and validated showing low LOQ, good repeatability and
linearity. The developed method has lower sensitivity and detection
limit in comparing with digital camera method [6]. Android app Color
Grab facilitates color catching in real time. DLLME technique combing
one touch color picking in-stu greatly enhances the sensitivity and
convenience of analysis. The analytical method using smartphone App
is interesting and attractive because the whole procedures require re-
latively inexpensive equipment, simple to operate, with satisfactory
accuracy, sensitivity. Most chromogenic reactions suitable for extrac-
tion spectrophotometry could be easily simplified as current metho-
dology accompanying improvement of sensitivity. The developed
method is prospect to be applicable and provides employment of usual
laboratory equipment as a screening method for trace level ions.
Declaration of Competing Interest
The authors declare that there are no conflicts of interest.
Acknowledgement
The authors would like to thank the National Natural Science
Foundation of China (21767026, 21862019) for financial support.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.microc.2019.104072.
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