The journal homepage www.jpacr.ub.ac.id p-ISSN : 2302 – 4690 | e-ISSN : 2541 – 0733
This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
(http://creativecommons.org/licenses/by-nc/4.0/)
Flow Injection–Indirect Spectrophotometry for Hydroquinone Analysis Based on the Formation of Iron(II)-Phenanthroline
Complex
Mariam Mohamed Omar Albhibani1, Hermin Sulistyarti1,2*, Akhmad Sabarudin1
1Chemistry Department, Faculty of Science, Brawijaya University, Jl. Veteran Malang 65145 Indonesia
2Research Center for Low Cost and Automated Method and Instrumentation Analysis (LCAMIA), Brawijaya University, Indonesia
*Corresponding email: [email protected]
Received 16 May 2019; Accepted 25 October 2019
ABSTRACT
Hydroquinone is an organic antioxidant widely used for skin lightening products which can cause negative impact in excessive use. This study is focused on the development of fast method for the determination of hydroquinone using flow injection-indirect spectrophotometry based on the formation of red complex iron(II)-phenanthroline. In this method, hydroquinone reduced iron(III) to iron(II) which in the presence of phenanthroline formed iron(II)-phenanthroline complex detected at maximum wavelength of 510 nm. The common operational and chemical conditions were optimized and the effect of several interfering compounds was also studied to achieve the highest sensitivity with acceptable analysis time. The optimum method performance was obtained under the conditions of 100 µL sample volume, 50 cm mixing coil-1and 75 cm mixing coil-2, 5 ml/min flow rate, 100 mgL-1 Iron(III) concentration, and 0.15 % phenanthroline. Under these conditions the proposed FI-spectrophotometry gave results to linear calibration over the concentration range from 2-100 mgL-1 (y = 0.028x and R2 of 0.999). The method was not interfered in the presence of vitamin C 1 mgL-1 and resorcinol up to 10 mgL-1. However, the higher concentration of vitamin C ≥10 ppm and resorcinol ≥ 20 ppm gave significant error of measurements. Method validation using standard additions gave results to average recovery value of 97.02 %, which indicates that the FI-spectrophotometry method can be used as an alternative method for determining hydroquinone in cosmetic.
Key words: Hydroquinone, flow injection, spectrophotometry, iron, phenanthroline.
INTRODUCTION
Hydroquinone is a drug used to treat hyperpigmentation that occurs in the skin.
Hyperpigmentation is the darkening of parts of the skin that commonly occurs after inflammation, such as acne scars, scars, or black spots due to sun exposure. In addition to inflammation, darkening of the skin can also occur due to the influence of hormones in pregnant women, the use of birth control pills, hormone therapy, or skin injury [1]. In contact with skin, hydroquinone acts as an alternate substrate of tyrosinase. In the place of tyrosine, which should be transformed into melanin, hydroquinone metabolizes into benzoquinone and free radicals. These radicals can then attack melanocyte membranes exerting a cytotoxic effect [2]. The use of cosmetic with hydroquinone exceeding 5 % can cause redness and burning sensation on the skin. Excessive use can cause skin irritation, skin redness, burning, kidney abnormalities, blood cancer and liver cancer.
The journal homepage www.jpacr.ub.ac.id
p-ISSN : 2302 – 4690 | e-ISSN : 2541 – 0733 209
Several HQ analysis techniques such as electrometry [3], HPLC [4], GC/MS [5], and SPE/GC/HPLC [5] have been reported. However, spectrophotometry is the most preferred method because it is available in most of laboratories with simple technique and accurate.
Spectrophotometry for hydroquinone was reported using redox reaction based on the use of ammonium molybdate, Mo(VI), in the acid medium as an oxidizing agent for the conversion of hydroquinone to p-benzoquinone, and Mo(VI) to Mo(V) which is detected at 580 nm. This method is linear in the range of 10-100 mgL-1 (r2 = 0.9999) and has been applied to skin lightening creams [6]. Other spectrophotometric method for the determination of hydroquinone is based on the use of KMnO4 as an oxidizing hydroquinone to p- benzoquinone [7]. Nevertheless, these batch methods consumed large amounts of reagents and samples as well as time consuming. The combination of spectrophotometry with flow injection technique offer considerable advantageous as this very efficient in term of reagent, sample, and time with high reproducibility and [8,9].
A very sensitive spectrophotometric method for the determination of hydroquinone was reported based on the reaction of iron(II) which forms a colored complex with phenanthroline [10]. This method can be used for the determination of hydroquinone 6.0-0.1 mgL-1, and none of researcher used this principle of reaction in flow technique. Therefore, this spectrophotometric method was combined with flow injection technique to develop a ne method for analyzing hydroquinone in cosmetics. The determination of hydroquinone is based on the reduction of iron(III) by hydroquinone to iron(II), which in the presence of phenanthroline forming a red complex of iron(II)-phenanthroline detected at a 510 nm. Flow injection performance is strongly influenced by operational factors including sample volume, mixing coil, and flow rate as well as chemical factors including the concentration of Fe(II) reagent and phenanthrolin reagent. Therefore, the proposed FI-spectrophotometry was optimized to those factors in order to achieve the greatest analytical performance.
EXPERIMENT
Chemicals and instrumentation
Hydroquinone were purchased from Sigma Aldrich (China), phloroglucinol and sodium hydroxide (99%) were purchased from Merck (Germany) and 95% ethanol were purchased from sigma Aldrich (China).
Flow injection analyzer (Ismatecs peristaltic pump, Rheodyne injector, silicon tubing, sample loop and mixing coil (PTFE i.d 0.75 mm)), UV-Vis Spectrophotometer 1601/Shimadzu, and UV-Probe 2.21 Application.
Procedure
Preparation of solutions
The stock standard solutions of 1000 mgL-1 hydroquinone were prepared by dissolving 100 mg hydroquinone in 100 mL demineralized water, and the lower concentrations of hydroquinone solutions were made by diluting the stock solution into the appropriate volume using demineralized water.
The stock solutions of 1000 mgL-1 iron(III) was prepared by dissolving 0.290 iron(III) chloride in 100 mL 0.01 M hydrochloric acid (pH 2), and the lower concentrations of Iron(III) solutions were made by diluting the stock solution using 0.01 M hydrochloric acid solution.
Phenantroline stock solutions of 0.25 % was prepared by dissolving 250 mg in 10 mL ethanol, transferred quantitatively into a volumetric flask, then dilute with demineralized
The journal homepage www.jpacr.ub.ac.id
p-ISSN : 2302 – 4690 | e-ISSN : 2541 – 0733 210
water up to 100 mL. The lower levels of phenanthroline solutions were made by diluting the stock solution into demineralized water.
Flow Injection Procedure
The FI-Spectrophotometry system to determine hydroquinone is depicted in Figure 1.
Figure 1. The schematic flow injection spectrophotometry for hydroquinone analysis Optimization of Operational Factors
Wavelength of measurement
Determination of λ max was done by injecting 100 µL 10 mgL-1 hydroquinone in 100 ppm iron(III) stream and reacted with 0.25 % phenanthroline in stream-2, and the absorbance of the red solution of iron(II)-phenanthroline complex was read at 490 nm. The procedure was repeated using various λ from 490-530 nm. The λ that gives maximum absorbance will be used as λ max.
Flow rate
The optimization of flow rate was done by injecting 100 µL 10 mgL-1 hydroquinone into 100 mgL-1 Fe(III) stream at pH 2 and reacted with 0.25 % phenanthroline in stream-2 under flow rate of 2.5 mL/minute (of each stream) with mixing coil-1 and mixing coil-2 of 50 cm, and read the absorbance of iron(II)-phenanthroline complex at maximum wavelength of 510 nm. The procedure was repeated using different flow rate of each stream from (0.5-2.5 mL/minute). The peak that gives maximum absorbance with acceptable analysis time was considered as optimum.
Sample volume
The procedure was done similarly using optimum flow rate, but sample loop was varied from 50 to 150 µL. The optimum sample volume was determined by the peak which provides highest absorbance and fast analysis.
Mixing coil
Mixing coil-1 optimization was done similarly using optimum flow rate and sample volume obtained from previous experiment, but length of mixing coil-1 was varied from 25 to 125 cm. The length of the mixing coil-1 optimum is shown by the highest absorbance value and the best peak of iron(II)-phenanthroline complex. This procedure was repeated for mixing coil-2 under optimum mixing coil-1.
Optimization of Chemical Factors
Concentration of Iron(III) and phenanthroline
The optimization of the other parameters was done similarly to the optimization of operational parameters, but it used optimum flow rate, sample volume, and mixing coils. In
The journal homepage www.jpacr.ub.ac.id
p-ISSN : 2302 – 4690 | e-ISSN : 2541 – 0733 211
this optimization, the variation of iron(III) concentration was 50-300 mgL-1, phenanthroline was from 0.05-5 %. The optimum each parameters was chosen based on the highest absorbance of iron(II)-phenanthroline complex.
Linearity of Measurements
The linearity of measurement was done similarly to the optimization procedure under the obtained optimum conditions of operational and chemicals. However, the hydroquinone concentration was varied from 0-100 mgL-1. The range of concentrations which give linear correlation with absorbance was determined as linearity of measurement.
Sensitivity of Measurement
The sensitivity of measurement was done by injecting water (blank solution) into the FI-system under the optimum conditions of operational and chemicals. The sensitivity of the method was calculated as LOD (three times of signal noise) and LOQ (ten times of signal noise).
Application to cosmetics
The application of the proposed FI-spectrophotometry was done by injecting extract cosmetics sample into the FI-system under the optimum conditions of operational and chemicals and the validity was conducted using standard addition followed by calculating the percent recoveries.
RESULT AND DISCUSSION
Hydroquinone can be detected by indirect spectrophotometry based on its capability to reduce iron(III) to iron(II) in acidic condition, which in the presence of phenanthroline will produce a red complex of iron(II)-phenanthroline detected at 510 nm. The absorbance of iron(II)-phenanthroline complex is proportional to the concentration of hydroquinone. Thus, the concentration of hydroquinone can be determined based on the absorbance of ferroin. In this method, reduction of iron(III) by hydroquinone is done in the stream-1 (Reaction-1) and reaction of iron(III) with phenanthroline to form iron(II)-phenanthroline complex is done in stream-2 (Reaction-2) (Figure 2).
Figure 2. Schematic reactions for complex formation iron(II) phenantroline
HO
OH
O
O
+ 2 Fe3+ + 2 Fe2+
N
N
+ Fe2+
3
N
N N
N N N Fe
2+
reaction 1
reation 2
The journal homepage www.jpacr.ub.ac.id
p-ISSN : 2302 – 4690 | e-ISSN : 2541 – 0733 212
Optimization of Flow Rate
Flow rate is one of factors to control the dispersion in flow injection analysis because increasing flow rate trigger turbulent flow in tubular reactor causes decrease in the dispersion and increase the peak signal [12]. The effect of flow rate was evaluated in the range of 0.9 to 2.5 mL min-1 and the results showed that the peak height increased with flow rate (Figure 3).
The flow rate of 2.5 ml min-1 gave the highest absorbance, however the signal showed as a splitting peak which indicates the reaction between hydroquinone and phenanthroline is not well accomplished. The splitting peak also leads to the irreproducible measurement. A flow rate of 2.8 ml/min was selected for the most adequate sensitivity and analysis time with nice peak shape and efficiency of chemicals.
.
Figure 3. The effect flow rate on absorbance of ferroin
Figure 4. Effect of mixing coil-1 (a), and mixing coil-2 (b).
Optimization of Mixing Coil Length
This FI technique involved 2 mixing coils (reactors) to facilitate better mixing of the two reactions (Reaction-1 and Reaction-2). Mixing coil-1 accommodated the reduction of Iron (III) to Iron (II) by hydroquinone, while mixing coil-2 responsible for providing better mixing for reaction of iron(II) with phenanthroline to form iron(II)-phenanthroline complex. The
The journal homepage www.jpacr.ub.ac.id
p-ISSN : 2302 – 4690 | e-ISSN : 2541 – 0733 213
length of both of mixing coils was optimized by monitoring the peak height (absorbance) of iron(II)-phenanthroline complex as optimization factor. The reactor length was evaluated in the range from 25 to 150 cm. The results (Figure 4) showed an increment of peak height with reactor length-1 up to 50 cm and reactor length-2 up to 75 cm. This effect was attributed to the reaction rate, in which longer reactor increased the reaction time and consequently the analytical signal. However, further increase of reactor length increased diffusion on the reactor and dilution of the iron(II)-phenanthroline complex, thus a decreasing on the analytical signal was observed [13]. The reactor length of 50 cm and 75 cm were selected as mixing coil-1 and mixing coil-2 for the most appropriate signal with acceptable analysis time.
Figure 5. Effect of sample volume on absorbance of iron(II)- phenanthroline complex
Optimization of Sample Volume
The amount of sample volume influenced the peak height of the FI-signal. The increase sample volume will impact on the dispersion of the sample solution. Thus, this optimization is conducted using various sample volume from 50-150 µL in order to obtain the highest peak (maximum absorbance) with minimum dispersion. Kolev and Mckelvie [14]
explain the higher sample volume will produce higher peaks. The higher sample volume affects the physical dispersion and produces a widened peak; thus results in longer analysis time. Results of the effect of sample volume examination are depicted in Figure 5. Based on Figure 4, the absorbance increase with the highest absorbance attained using sample volume of 125 µL. However, sample volume 125 µL gave double peak due to the incomplete overlapping between sample and reagent zones. Besides, the signal obtained from 100 µL sample volume is a single peak and the peak height did not show significant difference to that obtained from 125 µL. Therefore, 100 µL was selected as the optimum sample volume as it gave single peak with high absorbance and less time of analysis. Therefore, the sample volume is not only chosen by the highest signal but also peak shape and analysis time.
Optimization of Iron(III) Concentration
The concentration of Iron(III) in stream-1 needs to be optimized to ensure the adequate amount of Iron(III) to be completely reduced ton Iron(II) by hydroquinone (Reaction-1).
Thus, the availability of Iron (III) indirectly determined the formation of iron(II)- phenanthroline complex in stream-2, when iron(II) solution reacted with phenanthroline
The journal homepage www.jpacr.ub.ac.id
p-ISSN : 2302 – 4690 | e-ISSN : 2541 – 0733 214
under Reaction-2. The maximum iron(II) will produce maximum iron(II)-phenanthroline complex which is proportional to the hydroquinone. The effect of varying concentration of Iron(III) between 50 to 300 mgL-1 was examined with results shown in Figure 6. As shown in Figure 6, the absorbance of iron(II)-phenanthroline complex increased with concentration of iron (III) up to 100 mgL-1, but higher concentration gave relatively constant absorbance. The highest peak of 100 ppm means that the formation of iron(II)-phenanthroline complex was optimal at 100 ppm, and this concentration was used for further experiment.
Figure 6. Effect of iron(III) concentration on the formation of the complex iron(II)-phenanthroline
Figure 7. Effect of phenanthroline concentration on the formation of iron(II)-phenanthroline complex
Optimization of Phenanthroline Concentration
In order to obtain the optimum concentration of phenanthroline used to complex with Iron(II) to produce iron(II)-phenanthroline complex (Fe(II)-Phen) which proportional to hydroquinone concentration, the effect of phenanthroline concentration was investigated in the range of 0.05-0.25 %. The correlation of phenanthroline concentration to the absorbance of iron(II)-phenanthroline complex is depicted in Figure 7. It shows that the absorbance of
The journal homepage www.jpacr.ub.ac.id
p-ISSN : 2302 – 4690 | e-ISSN : 2541 – 0733 215
Fe(II)-Phen (peak height) increased as concentration of phenanthroline increasing up to 0.15
%, and relatively constant at higher concentration up to 0.25 %. Therefore, considering the highest peak which gave best sensitivity of the measurement, the 0.15 % phenanthroline was chosen for further study of hydroquinone analysis.
Linearity of Measurement
The linearity of the proposed FI-spectrophotometry method was determined by injecting various concentration of hydroquinone from 0-100 mgL-1, under optimum conditions of operational (2.5 mlmin-1, mixing coil-1 50 cm, mixing coil-2 75 cm, 100 µL) and optimum chemical parameters (0.25 % phenanthroline and 100 mgL-1 Iron(III)). As illustrated in Figure 8, the FI-spectrophotometry gave wide linear concentration range for hydroquinone measurement from 2-100 mgL-1 with linear equation of y = 0.028 [hydroquinone] and coefficient of determination, R2, close to 1 (0.999). The LoD and LoQ of this method were respectively 0.04 1n3 0.1 mgL-1.
Figure 8. Linear calibration of hydroquinone
Determination of hydroquinone in cosmetics
As an example for application of the method, the system was applied for determination of hydroquinone in whitening cosmetics. Under this experiment, using standard addition, the FI-method was also examined the recoveries of the samples after adding hydroquinone standard. The results are shown in Table 1, with satisfactory recoveries ranging from 95-97 %.
Table 1. Application of FI-Spectrophotometry recovery by standard addition Sample Hydroquinone added (mg/L) Measured
Hydroquinone (mg/L) % Recovery
Cosmetic A 0 39.11 -
5 42.52 95.93
10 48.09 97.39
15 53.23 97.74
The journal homepage www.jpacr.ub.ac.id
p-ISSN : 2302 – 4690 | e-ISSN : 2541 – 0733 216
CONCLUSION
Based on the results obtained in this study, it can be concluded that the FI- spectrophotometry provide fast, simple, precise, and accurate technique for determination of hydroquinone. The proposed method is quite useful for the determination of hydroquinone in cosmetics. The rapid analysis of the FI-spectrophotometry offered a suit technique for cosmetics hydroquinone monitoring which involve large number of cosmetics samples.
ACKNOWLEDGMENT
The author is grateful to Chemistry Department, Faculty of Science, Brawijaya University for facilitating research and the Ministry of Research, Technology and Higher Education of Indonesia for financial support.
CONFLICT OF INTEREST
Authors declare no competing interests.
REFERENCES
[1]. Amponsah, D., Levels of Mercury and Hydroquinone in Some Skin- Lightening Creams and Their Potential Risk to The Health of Consumers in Ghana, Department of Chemistry, Kwame Nkrumah University of Science and Technology, 2010.
[2]. Gillbro, J. M., Olsson, M. J, Int. J. Cosmet. Sci, 2011, 33 (3), 210–221.
[3]. Safitri, A., Mulyasuryani, A., Tjahjanto, R.T., J. Pure App. Chem. Res. 2015, 4(1), 1–4.
[4]. Siddique, S., Parveen, Z., Ali Z., Zaheer, M., J. Cosmet. Dermatol. Sci. Appl., 2012, 2, 224-228.
[5]. Jurica K, Karačonji IB, Šegan S, Opsenica DM, Kremer D., Arh. Hig. Rada Toksikol.
2015, 66(3):197-202.
[6]. Bielicka, K. D., Hadzicka, M., Voelkel, A., ISRN Chromatogr., 2012, 680929.
[7]. Seliem, A.F., and Khalil, H.M, Ultra Chemistry, 2013, 9(2), 221-228.
[8]. Qassim, B.B. and Omaish, H.S., J. Chem. Pharm. Res., 2014, 6(3),1548-1559.
[9]. Fahmi, M.I., Sulistyarti, H., Mulyasuryani, A., Wiryawan, A. J. Pure App. Chem. Res., 2019, 8(1), 53-61.
[10]. Sulistyarti, H., Kolev, S. D. Microchem. J, 2013, 111, 103–107.
[11]. Belcher, R., and West, T.S., Anal. Chim. Acta, 1951, 5, 599-602.
[12]. Ruzicka, J.; Hansen, E.H. Flow Injection Analysis. Wiley, 1988.
[13]. Karlberg, B., Pacey, G, Flow Injection Analysis: A Practical Guide, 1st ed., 1989, vol.
10, Elsevier.
[14]. Kolev, S. D. and McKelvie, I. D., Advance in flow injection analysis and related techniques, 2008, vol 54, 1st edition, Elsevier.