Determination Of Seven Anions Biology Essay

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In this study, the standard analysis methods were used to determine the contents of anions in seven different types of tea. Ion Chromatography DIONEX ICS-1000 Ion Chromatography was used to identify and quantify the content of anions of the respective tea samples (100-fold dilution), where the results of the analyses showed that the fluoride content ranged from 0.52 mg/L to 32.82 mg/L, chloride content ranged from 7.84 mg/L to 78.15 mg/L, phosphate content ranged from 24.03 mg/L to 114.50 mg/L, and the sulphate content ranged from 2.43mg/L to 66.88 mg/L. Bromide, nitrate and nitrite were not detectable. Fluoride, chloride, bromide and nitrate Ion Selective Electrodes (Oakton) were also used to determine the anions content of tea samples (50-fold dilution), where the results showed that the concentration of fluoride ions ranging from 0.07 ± 0.007 mg/L to 0.18 ± 0.016 mg/L, and concentration of chloride ions ranging from 2.6 ± 0.100 mg/L to 7.8 ± 0.158 mg/L. Bromide and nitrate were not detectable. Furthermore, reflectoquant (RQ Flex 10) was also used to determine the chloride, nitrite and nitrate ions content of the respective tea samples, the analyses showed that the chloride content ranged from 0.5 mg/L to 1.2 mg/L. Chloride content in Jasmine tea, nitrite and nitrate in respective tea samples were not detectable. Ion Chromatography is more reliable.

1.0 Introduction

Tea is one of the most popular beverages in the world after water. The world annual production of tea is about 3.5 million tons. Tea is originated in China and Southeast Asia. It has been cultivated and consumed for thousand years (Yang, Malaikal & Meng, 2002). All teas are derived from the evergreen plant which its scientific name is Camellia Sinesis. Evergreen plant refers to the plant that leaves all the seasons; it would not undergo defoliation no matter what seasons it was. Tea can be divided into five basic categories which are black, green, oolong, white and pu-erh. It was produced in different types of oxidation and methods (Perry, 2008). Besides, there is also a type of tea named flowering tea.

Black tea usually undergoes full oxidation. The characteristic of black tea includes the dark brown and black leaf, robust and pronounced flavors. Black tea is allowed to wither, which precedes a process called oxidation during which water evaporates out of the leaf and the leaf absorbs more oxygen from the air. Green tea undergoes the least amount of oxidation. It tends to produce more subtle flavors with many undertones and accents. Green tea is essentially plucked, withered, rolled, and then immediately heated to halt the oxidation process. Oolong tea usually undergoes partial oxidation. The flavor of oolong tea is typically not as robust as black tea or as subtle as green tea but has its own extremely fragrant and intriguing tones. It is often compared to the taste and aroma of fresh flowers or fresh fruit. White teas, the most delicate of all teas, are appreciated for their subtlety, complexity and natural sweetness. Like green tea, white tea undergoes the least amount of oxidation. White tea undergo long withering cycle and never rolled and shaped. Tea is then gently air dried. Pu-erh is an aged black tea from China prized for its medicinal properties and earthy flavor. Flowering teas, also known as Artisan Teas which have great tasting. These are made by binding tea leaves and flowers together into a bulb and then are set to dry. The processing of white tea, green tea, oolong tea and black tea is shown in Figure 1.

Figure 1: The Processing of White tea, Green tea, Oolong tea and Black tea

Tea composition varies with climate, season, horticultural practices, variety and the age of leaves (Graham, 1992). Thus, the anions content of tea may differ. Appropriate dietary intake of anions makes up some serious health benefits. However, negative effects can also be resulted when exposure of anions exceed the safety guidelines. Hence, the quality control during the tea production is very important. Safety guidelines for each of the anions will be discussed starting from next paragraph.

Fluoride, F- prevents the acid produced by the bacteria in plaque from dissolving, or demineralising, tooth enamel, the hard and shiny substance that protects the teeth (Fawell et al., 2006). Fluoride also allows teeth damaged by acid to repair, or re-mineralize, themselves. Small amount of fluoride also helps maintain bone structure and the addition of fluoride to tap water can helps reduce cavities in children. Deficiency of fluoride may leads to increased of cavities, and weak bones and teeth. Infants who get too much of fluoride before their teeth have broken through the gums have changes in enamel that covers the teeth. Faint white lines or streaks may appear but are usually not easy to be seen. Daily dietary intake of fluoride range between 2mg - 4mg.

Chloride, Cl- is a chemical that human body requires for metabolism. Deficiency of chloride may leads to excessive sweating, vomiting or diarrhea. Excessive intake of chloride will increase blood pressure and cause a buildup of fluid in people with congestive heart failure, cirrhosis or kidney failure. Daily dietray intake of chloride range between 1.8 - 2.3g (WHO, 1996)

Bromide, Br - has a low degree of toxicity; thus, bromide is not of toxicological concern in nutrition. Limited findings shows that bromide may be nutritionally beneficial; for example, insomnia exhibited by some haemodialysis patients has been associated with bromide deficiency (WHO, 2009). Daily dietary intake of bromide is about 2-8 mg. Excess bromine might acts as a depressant and narcotic, particularly to the brain and nervous systems.

Excessive exposure to nitrate, NO3 - and nitrite, NO2 - can lead to acute acquired methemoglobinemia, a serious health condition (ATSDR, 2011). Both nitrate and nitrite alter the iron in hemoglobin to the Fe3+ state. This forms methemoglobin, an abnormal form of hemoglobin which the blood loses its ability to carry oxygen to tissues. Methemoglobinemia can cause cyanosis (blue skin) of limbs or trunk, weaknesses and rapid heart rate. Severe methemoglobinemia can cause lethargy, brief loss of consciouness, irregular heartbeat, shock, convulsions, coma and even death. Besides, nitrate and nitrite able to react with molecules in organism to form N-nitroso compound, some of which may cause cancer. Daily dietary intake of nitrate and nitrite are 7mg/kg body weight and 0.33mg/kg body weight.

Phosphates, PO43- are chemical compounds that are found in the human body and throughout the natural world (Demi, 2011). Phosphorus is a key component of phosphates which are essential to human, plant and animal life. In addition to being critical components of human's bones and teeth, phosphate also aids in maintenance of tissues and cells, storage and usage of energy, and also help to make up the genetic materials that comprise DNA. Inadequate levels of phosphorous might leads to anxiety, fragile bones, fatigues, joint stiffness, loss of appetite and fluctuations in weight. Excess dietary intake of phosphates will lead to osteoporosis, heart disease, cancer and soft tissue calcification. The normal daily recommended intakes are generally between 800-1200mg.

Sulfate, SO42- is generally considered to be non-toxic. Sulfate is needed for formation of proteins in joints, start the cascade of digestive enzymes released from the pancreas, essential in forming the mucin proteins which line the gut walls and necessary for formation of brain tissue. The consumption of drinking water containing high amounts of magnesium or sodium sulphate may result in intestinal discomfort, diarrhea and consequently dehydration. This laxative effect is often observed when someone drinks water that contains greater than 500 milligrams per litre (mg/L) of sulphate. Over time, individuals appear to develop a tolerance to higher concentrations of sulphate. Diarrhea and dehydration are often observed when individuals accustomed to drinking water with low concentrations of sulphate consume water with high amounts of sulphate. There is no recommended intake was set.

2.0 Literature Review

Tea analysis is a process that plays an important role in determining the anion contents of the tea samples which are used for nutritional labeling. Many anions such as fluoride are very important micronutrients at low concentration but when they are in high concentrations, they are toxic to the body. For example. exposure to high levels of fluoride can cause the sympton ranging from discoloration of teeth in children to severe osteoarthritis in adults. According to the World Health Organisation (WHO), the optimum fluoride intake for human ranges from 2 to 4 mg per day. The total daily intake of fluoride that causes fluorosis in adults is over 13.0 to 14.5 mg per day. Thus, tea analysis also used as a powerful tool in food quality control.

Many researchers had performed research on the tea varieties. For example, Kumar, Narayan and Hassarajani (2008) had done a study on the determination of anionic minerals in black and kombucha tea using ion chromatography. In their research, a one step sample pretreatment procedure was used to eliminate the matrix interference which caused by the presence of large concentration of interfering organic compounds in tea. Thus, making it possible to analyze the anion contents in tea directly by using ion chromatography in suppressed mode using sodium carbonate and bicarbonate mixture as eluent, and conductometric method of detection. Similar ion chromatograms are obtained from both tea brew samples. The overall results showed that the anionic mineral components identified in black tea were 1.20±0.06mg g-1 fluoride, 3.12±0.13mg g-1 chloride, 0.04±0.01mg g-1 bromide, 0.34±0.02mg g-1 nitrate, 0.08±0.01mg g-1 phosphate, 4.20±0.17mg g-1 sulphate and 0.44±0.04mg g-1 iodide, respectively. On the other hand, kombucha tea was found to contains 3.20±0.16mg g-1 fluoride, 0.96±0.04mg g-1 chloride, 0.04±0.01mg g-1 bromide, 0.18±0.01mg g-1 nitrate, 0.04±0.01mg g-1 phosphate, 1.02±0.04mg g-1 sulphate and 1.04±0.08mg g-1 iodide, respectively.

Besides that, Alcazar et al. (2003) had done a study on determination of chloride and phosphate in tea samples using ion chromatography. In their study, three different varieties of tea samples were used which are non-fermented (green tea), fermented (black tea) and semi-fermented (oolong tea). An anion chromatography using an isocratic elution with phtalate or phtalic acid (0.6 mM)/4% acetonitrile as mobile phase and conductimetric detection was applied for determination of organic acids and inorganic anions in precipitation samples. There is no pretreatment procedure used. The chromatogram of the green tea was found to contain 0.5mg g-1 chloride and 1.91mg g-1 phosphate respectively. Conversely, the chromatogram of oolong tea and black tea were found to contain 0.55mg g-1, 0.6mg g-1 chloride and 2.34mg g-1, 2.93mg g-1 phosphate, respectively.

In addition, some researcher such as Tokalioglu, Kartal and Sahin (2004) also had done a review study on the determination of fluoride ions in Turkish tea, sage tea and imported tea using a fluoride ion selective electrode instead of ion chromatography. In their study, a standard analysis method was being used to determine the content of anions in tea samples. The tea samples were infused and the fluoride levels were determined in liquor taken at 20 min of infusion using ion the ion selective electrode. The values obtained were 55 ± 6 and 127 ± 8 µg g−1 for two Turkish tea samples; 0.30 ± 0.03 µg g−1 for sage tea; and 142 ± 2, 56.4 ± 0.8 and 195 ± 12 µg g−1 for three imported tea samples.

Fluoride ions content in black tea had been studied by Cao et al. (2004). Fluoride ion selective electrode was used to determine the fluoride ions content in 20 tea samples, in various black tea commodities, namely stick-shaped, granular, canned, or bottled tea beverage, originally produced in India, Sri Lanka, Japan, China and UK, and milk-tea after addition of milk and sugar. The fluoride ions content of five stick-shaped black teas was ranging from 96.9 - 148 mg/kg, that of eight granular black tea was ranging from 139 - 223 mg/kg, and the three canned or bottled black tea beverages was ranging from 0.70 - 0.96 mg/L, respectively.

Further study of fluoride levels in various black tea commodities had also been done by Cao et al. (2006). The fluoride content of various products of black tea were determined by using the standard fluoride selective ion electrode method. The fluoride content was found to be 0.95-1.41 mg/L in black tea sticks, 0.70-2.44 mg/L in black tea granules and 1.15-6.01 mg/L in black tea bags. Of the products that tested, the fluoride content was greatest in black tea bags. This is because black tea bags are made of low cost and older tea leaves.

3.0 Materials and Methods

3.1 Determination of Anions Content by Using Ion Chromatography

3.1.1 Principle and Application of Ion Chromatography

Ion Chromatography is a method used for separating mixtures of substances by using stationary and mobile phases. In Ion chromatography, a stoichiometric chemical reaction occurs between ions in a solution and a solid substance carrying functional groups that can fix ions as a result of electrostatic forces. There are three types of Ion Chromatography which are Ion-Exchange Chromatography (simply known as IC), Ion-Pair Chromatography (IPC) and Ion-Exclusion Chromatography (IEC). Ion-Exchange Chromatography is further divided into Anion-Exchange Chromatography and Cation-Exchange Chromatography. For anion chromatography, the functional groups that carried in stationary phase was quaternary ammonium groups while the functional groups that carried in cation chromatography is either sulfonic acid groups or carboxylic acid groups. In principle, ions with the same charge are exchanged completely reversibly between the two phases. The process of ion exchange leads to the condition of equilibrium, the side to which the equilibrium lies depends on the affinity of the participating ions to the functional groups of the stationary phases. Ion chromatography is a simple, with high-sensitivity detection and high signal-to-noise ratio instrument. In this study, the anions in tea samples were identified and quantitated by using Anion-Exchange chromatography to measure the conductivity of the solution.

3.1.2 Materials

Tea samples that bought from local market ( Jasmine tea, Chrysanthemum tea, Lipton tea, Sabah tea, Japanese green tea, Chinese black tea, and Pu-erh tea).

3.1.3 Chemicals

Dionex seven anion standard II that bought from DIONEX company, 0.5M sodium bicarbonate concentrate and 0.5M sodium carbonate concentrate.

3.1.4 Apparatus

Beakers, measuring cylinder, dropper, volumetric flask, micro-pipette, spatula, pestle and mortar, Buchner funnel, Millipore filter and syringe.

3.1.5 Equipment

Electronic weighing machine (Sartorius), hot plate (Snijders) and vacuum suction pump.

3.1.6 Instrument

Ion Chromatography (Dionex ICS-1000).

3.1.7 Preparation of Seven Anions Standard Solution and Tea Samples for Ion Chromatography Analyses

A series of seven anions standard solution was prepared by diluting Dionex seven anions standard II solution with the dilution factor of 1000, 500, 200, 100, 50, 20 and 10 respectively with ultrapure water. The analysis was started with the least concentrated seven anion standard solution (5 ml), which was loaded into the sample loop of ion chromatography and analyzed. A chromatogram consisted of the seven standard anions was obtained. Similar steps were repeated for the higher concentration of the seven anion standard solution.

Tea brew samples were prepared by weighing exactly 1g of the grinded tea sample using a electronic weighing machine. It was then transferred to a beaker which has been filled with 50ml of hot boiling water in order to prepare a 0.02% (w/v) concentration of tea brew sample. The tea leaves were allowed to seep for 20mins. Then, the tea brew sample was filtered with the use of strainer, Buchner funnel and Millipore filter. 1ml of the filtrate was pipetted into a volumetric flask and made up to 100ml with ultrapure water. Similar steps were repeated for the other 6 types of tea samples. Results of the analyses were recorded. The chromatogram of tea brew samples that obtained were used for the identification of the anions content. It was done by comparing the chromatogram of the tea samples with the chromatogram of seven anions standard solutions. The quantification of the anions content in the tea samples were done by refering the calibration curve obtained from the seven anions standard solutions.

3.2 Determination of Anions Content by Using Ion Selective Electrode

3.2.1 Principle and Application of Ion Selective Electrode

Ion selective electrode is a sensor that the activity of specific ion will be converted into a voltage, which then can be measured by an Ion meter. The voltage is theoretically dependent on the logarithm of the activity of ion and as described by the Nernst

equation (). Ion Selective Electrode is simple to use, relatively inexpensive, has no consumption of analyte, short response time and have an extremely wide range of applications and wide concentration range. In this study, the anions content in the tea samples were determined and quantitated by measuring the specific ion activity using ion selective electrode.

3.2.2 Materials

Tea samples bought from local market ( Jasmine tea, Chrysanthemum tea, Lipton tea, Sabah tea, Japanese green tea, Chinese black tea, and Pu-erh tea).

3.2.3 Chemicals

Reference solution (KNO3 and KCl) , 1000ppm fluoride standard solution, 1000ppm chloride standard solution, 1000ppm bromide standard solution, 1000ppm nitrate standard solution.

3.2.4 Apparatus

Beakers, measuring cylinder, dropper, volumetric flask, micro-pipette, spatula, pestle and mortar, Buchner funnel, Millipore filter and syringe.

3.2.5 Equipment

Electronic weighing machine (Sartorius), hot plate (Snijders) and vacuum suction pump.

3.2.6 Instrument

Fluoride ion selective electrode, chloride ion selective electrode, bromide ion selective electrode, nitrate ion selective electrode (Oakton), ion 700 meter (Oakton).

3.2.6 Preparation of Standard Solutions and Tea Samples for Ion Selective Electrode Analyses

Exact 1g of grinded tea sample was weighed by using the electronic weighing machine. The tea sample was then transferred to a beaker which has been filled with 50ml of hot boiling water to make up a 0.02% (w/v) concentration of tea sample. The tea leaves were allowed to seep for 20mins. Then, the tea brew sample was filtered with the use of strainer, Buchner funnel and Millipore filter. 2ml of the filtrate was pipetted into a volumetric flask and made up to 100ml with ultrapure water.

0.1 ppm, 1 ppm, 10ppm and 100 ppm of standard solution were prepared by undergoing serial dilution of the 1000ppm of standard solution. Calibration of each types of Ion Selective Electrode was automatically done by the instrument ion 700 meter, just dipping the probe directly into the series of standard solution according to the sequence from the lowest concentration to the highest concentration. Once the calibration was done, the concentration of anions were quantitated by dipping the probe into the diluted tea brew samples.

3.3 Determination of Anions Content by Using Reflectoquant

3.3.1 Principle and Application of Reflectoquant

The principle of operation of Reflectoquant to determine the anions was based on the theory of reflectometry (remission photometry) in which the reflected light from the strip was measured. Just as in the classical photometry, the difference in intensity of emitted and reflected light allows the quantitative determination of the concentration of specific analytes. In this study, the anions content in the tea samples were identified and quantitated by measuring the reflected light from the strip using a Reflectoquant RQflex 10.

3.3.2 Materials

Tea samples bought from local market ( Jasmine tea, Chrysanthemum tea, Lipton tea, Sabah tea, Japanese green tea, Chinese black tea, and Pu-erh tea).

3.3.3 Apparatus

Beakers, measuring cylinder, dropper, spatula, Buchner funnel, Millipore filter and, pestle and mortar.

3.3.4 Equipment

Electronic weighing machine (Sartorius), hot plate (Snijders), vacuum suction pump, reflectoquant strips(calibration, chloride, nitrate and nitrite), test- and batch-specific bar-code strip(calibration, chloride, nitrate and nitrite).

3.3.5 Instrument

Reflectoquant (Merck RQflex 10).

3.3.6 Preparation of Tea Samples for Analyses using Reflectoquant

Exact 1g of grinded tea sample was weighed by using the electronic weighing machine. The tea sample was then transferred into a beaker which has been filled with 50ml of hot boiling water to make up a 0.02% (w/v) concentration of tea sample. The tea leaves were allowed to seep for 20mins. Then, the tea brew sample was filtered with the use of strainer, Buchner funnel and Millipore filter.

Calibration of ions were done by inserting the bar-code strip and reflectoquant strip into the bar-code reader and measurement chamber. No standard solution were required to prepare for calibration. "CAL" was displayed on the LED when calibration was in progress and once the calibration was done, it will automatically shut down. Then, chloride bar code strip was inserted into the bar code reader. 3 digits of the chloride bar code number was then showed on display. Tea samples can now be tested by immersing the test strip into the samples for certain time as mentioned on the display. Test strip was inserted quickly into the measurement chamber 5 seconds before the end of reaction time. Excess liquid was allowed to run off. Concentration of chloride ions in unit of ppm was displayed directly on the screen. Similar steps were repeated by using nitrate and nitrite batch bar-code strip and test strip.

4.0 Results and Discussions

4.1 Determination of Anions Content by Using Ion Chromatography

In this study, 1g of tea sample was used to determine the anions content by using Ion Chromatography. The concentrations of seven anions standard solutions that used for calibration purpose were ranged between 0.002 to 10 ppm. The series of seven anions standard solutions (standard 1 - 7) were prepared from the stock solution which called DIONEX seven anion standard II. The stock solution contains 20 ppm of fluoride, 100ppm of chloride, nitrite, bromide, nitrate and sulfate, and 200ppm of phosphate. Table 1 shows the concentrations of anions of the series of seven anions standard solutions that prepared from DIONEX seven anion standard II.

Table 1: Concentrations of anions prepared from the stock solution (DIONEX seven anion standard II).

STD

Concentration, mg/L (ppm)

F-

Cl-

NO2-

Br-

NO3-

PO42-

SO42-

1

0.02

0.10

0.10

0.10

0.10

0.20

0.10

2

0.04

0.20

0.20

0.20

0.20

0.40

0.20

3

0.10

0.50

0.50

0.50

0.50

1.00

0.50

4

0.20

1.00

1.00

1.00

1.00

2.00

1.00

5

0.40

2.00

2.00

2.00

2.00

4.00

2.00

6

1.00

5.00

5.00

5.00

5.00

10.00

5.00

7

2.00

10.00

10.00

10.00

10.00

20.00

10.00

STD = Standard solution

Ion chromatograms were obtained after the analysis of seven anions standard solutions and were shown in Figures 2, 3, 4, 5, 6, 7 and 8.

Figure 2: Ion chromatogram of standard solution 1.

IMAG13391.jpg

Figure 3: Ion chromatogram of standard solution 2.

IMAG13341.jpg

Figure 4: Ion chromatogram of standard solution 3.

IMAG13351.jpg

Figure 5: Ion chromatogram of standard solution 4.

1ppm.jpg

Figure 6: Ion chromatogram of standard solution 5.

2PPM.jpg

Figure 7: Ion chromatogram of standard solution 6.

5 ppm.jpg

Figure 8: Ion chromatogram of standard solution 7.

10pp.jpg

Based on these Figures 2 to 8, the retention time of each anions has been recorded and shown in Table 2.

Table 2: Retention time for each type of anions which including fluoride, chloride, nitrite, bromide, phosphate and sulfate.

Anion

Retention time, tR

Fluoride

3.180 - 3.280

Chloride

4.600 - 4.730

Nitrite

5.377 - 5.630

Bromide

6.757 - 7.033

Nitrate

7.540 - 7.897

Phosphate

9.930 - 10.250

Sulfate

11.720 - 12.213

Sometimes, retention time may shift from one to another due to the contamination of column. This problem can be solved by washing the columns with specific solutions for removing contaminants with different properties such as acid or base-soluble, organic ions, etc (Dionex, 2002).

Calibration curves were plotted according to the peak area obtained from the ion chromatography system versus the concentration of the seven anions standard solution. It is shown in Figures 9, 10, 11, 12, 13, 14 and 15.

Figure 9: Calibration curve of peak area versus concentration of fluoride.

Figure 10: Calibration curve of peak area versus concentration of chloride.

Figure 11: Calibration curve of peak area versus concentration of nitrite.

Figure 12: Calibration curve of peak area versus concentration of bromide.

Figure 13: Calibration curve of peak area versus concentration of nitrate.

Figure 14: Calibration curve of peak area versus concentration of phosphate.

Figure 15: Calibration curve of peak area versus concentration of sulphate.

The calibration curves of the seven anions standard solutions show that the peak areas of the anions are directly proportional to the concentrations of the anions.

The analytical method was validated by determining the linear range, limits of detection (LOD) and quantification (LOQ), precision and recovery. The results are shown in Table 3. Good linearity was obtained between the peak areas and the anions concentrations. The precision of the system was tested with the standard solution 1 for 3 times. Peak areas of each anions that obtained from the test were used to calculate the relative standard deviation.

Calculation relataive standard deviation (%RSD),

%RSD = 100S/mean

S = Standard deviation

Table 3: Analytical characteristic of Ion Chromatography.

Anion

Linear range, mg/L

R2

LOD, mg/L

LOQ, mg/L

% RSD (n= 3)

Fluoride

0.02 - 2

0.9992

0.06

0.60

10.64

Chloride

0.1 - 10

0.9993

0.30

3.0

7.37

Nitrite

0.1 - 10

0.9985

0.30

3.0

21.21

Bromide

0.1 - 10

0.9994

0.30

3.0

37.78

Nitrate

0.1 - 10

0.9991

0.30

3.0

4.38

Phosphate

0.2 - 20

0.9995

0.60

6.0

22.41

Sulfate

0.1 - 10

0.9985

0.30

3.0

0.99

Ion chromatograms of 100-fold dilution of seven different types of tea samples were also obtained and the peak areas of each anions in these seven different types of tea samples were shown in Table 4. Tea samples were required to undergo certain process of dilution due to the high concentration of anions might contaminate or spoil the guard column of Ion Chromatography.

Table 4: The peak areas of the seven anions that quantitated in seven different types of tea sample with 100-fold dilution.

Anions

Peak area, µm*min

JT

CT

PT

LT

ST

CBT

JGT

Fluoride

0.0066

0.0031

0.0181

0.0539

0.0627

0.0645

0.0426

Chloride

0.0206

0.1318

0.1184

0.0139

0.0217

0.0404

0.0260

Nitrite

ND

ND

ND

ND

ND

ND

ND

Bromide

ND

ND

ND

ND

ND

ND

ND

Nitrate

ND

ND

ND

ND

ND

ND

ND

Phosphate

0.0432

0.0302

0.0560

0.0142

0.0160

0.0528

0.0340

Sulphate

0.0111

0.0197

0.0243

0.0221

0.0346

0.0161

0.0371

JT = Jasmine tea CT = Chrysanthemum tea

PT = Pu-erh tea LT = Lipton tea

ST = Sabah tea CBT = Chinese black tea

JGT = Japanese green tea

ND = Not detected

The anions content in the tea samples were quantitated by comparing the peak area of the anions of the tea samples with the calibration plot obtained from the seven anions standard solutions. The anions content in seven different types of tea sample is shown in Table 5.

Table 5: Determination of anions content in seven different types of tea sample (100-fold dilution) by using Ion Chromatography.

Anions

Concentration, mg/L or ppm

JT

CT

PT

LT

ST

CBT

JGT

Fluoride

0.0234

0.0052

0.0830

0.2688

0.3145

0.3282

0.2102

Chloride

0.1742

0.7815

0.7084

0.1376

0.1802

0.2824

0.2037

Nitrite

ND

ND

ND

ND

ND

ND

ND

Bromide

ND

ND

ND

ND

ND

ND

ND

Nitrate

ND

ND

ND

ND

ND

ND

ND

Phosphate

0.8680

0.5866

1.1450

0.2403

0.2792

1.0756

0.6688

Sulphate

0.0243

0.0988

0.1386

0.1196

0.2279

0.0676

0.2496

JT = Jasmine tea CT = Chrysanthemum tea

PT = Pu-erh tea LT = Lipton tea

ST = Sabah tea CBT = Chinese black tea

JGT = Japanese green tea

ND = Not detected

Based on Table 4 and Table 5, the results show that nitrite, bromide and nitrate ions were not detected. This may due to the extremely low concentration of nitrite, bromide and nitrate ions in respective tea samples below the detection limit of Ion Chromatography cannot detect.

4.2 Determination of Anions Content by Using Ion Selective Electrode

In this study, 1g of tea sample was used to determine the anions content by using Ion Selective Electrode. The concentrations of fluoride, chloride, bromide and nitrate standard solutions used were 10 mg/L, 100 mg/L and 1000 mg/L respectively. The individual anion was measured by using the respective ion selective electrodes. Each electrode was calibrated with the respective standard anion solution before the measurement of tea samples. The concentrations of fluoride and chloride ions in the seven different types of tea sample with 50 fold-dilution were quantitated and shown in Tables 6 whereas nitrate and bromide were not detected. Standard deviation was also calculated.

Calculation of standard deviation,

standard-deviation-formula.gif

N = set of the values

Xi = values obtained from experiment

µ = average of values obtained from experiment

Table 6: Determination of the concentration of fluoride ions in seven different types of tea sample with 50-fold dilution by using fluoride, chloride, bromide and nitrate Ion Selective Electrode method.

Anion

Concentration, mg/L or ppm

JT

CT

PT

LT

ST

CBT

JGT

Fluoride

0.12 ± 0.000

0.10 ± 0.007

0.09 ± 0.007

0.18 ± 0.016

0.14 ± 0.012

0.07 ± 0.007

0.10 ± 0.012

Chloride

3.4 ± 0.000

7.8 ± 0.158

4.0 ± 0.158

2.6 ± 0.100

2.7 ± 0.071

4.2 ± 0.000

3.5 ± 0.071

Bromide

ND

ND

ND

ND

ND

ND

ND

Nitrate

ND

ND

ND

ND

ND

ND

ND

JT = Jasmine tea CT = Chrysanthemum tea

PT = Pu-erh tea LT = Lipton tea

ST = Sabah tea CBT = Chinese black tea

JGT = Japanese green tea

ND = Not detected

Based on Table 6, bromide and nitrate ions were not detectable due to the extremely low concentration that Ion Selective Electrode cannot detect. From Table and Table 7, the experimental values obtained by using fluoride Ion Selective Electrode was in close proximity with the experimental values obtained by using Ion Chromatography. However, there was a large difference between the concentration of chloride ions that quantitated by using Ion Chromatography and Ion Selective Electrode. Chloride ions that quantitated by using Ion Selective Electrode has higher value. The difference was due to the temperature since Ion Selective Electrode does not have thermally controlled conductivity cell as Ion Chromatography that permits measurement without unaffected by temperature. Measurement of ions were fluctuated and become inaccurate under different temperature. Besides that, it might also caused by the interference from the matrix elements such as OH- and I- (Kumar, Narayan & Hassarajani, 2008).

4.3 Determination of Anions Content by Using Reflectoquant

In this study, 1g of tea sample was used to determine the anions content by using a Reflectoquant RQflex 10. No standard solutions were used for calibration but with the use of test- and batch-specific bar-code strip.The specific anion was measured by using different test- and batch- specific bar-code strip with different reaction time. In this study, only chloride, nitrate and nitrite were tested. The results of the anions that were quantitated are shown in Table 7.

Table 7: Determination of the concentration of chloride, nitrate and nitrite ions in seven different types of tea (without dilution).

Anions

Concentration, mg/L or ppm

JT

CT

PT

LT

ST

CBT

JGT

Chloride

Low

0.7

1.0

1.2

0.5

0.7

0.5

Nitrite

Low

Low

Low

Low

Low

Low

Low

Nitrate

Low

Low

Low

Low

Low

Low

Low

JT = Jasmine tea CT = Chrysanthemum tea

PT = Pu-erh tea LT = Lipton tea

ST = Sabah tea CBT = Chinese black tea

JGT = Japanese green tea

Low = Concentration are too low that instrument cannot detect. In other words, not detected.

Based on Table 7, concentration of chloride in Jasmine tea and concentration of nitrate and nitrite in the seven different types of tea were too low to be detected. Based on the comparison of fluoride's concentration that was quantitated by using Ion Selective Electrode and Ion Chromatography, Ion Chromatography is much more reliable and by comparing Ion Chromatography method with Reflectoquant method, the experimental values were not acceptable.

5.0 Conclusion

The analyses results from Ion Selective Electrodes and Reflectoquant were used to support the analyses results from Ion Chromatography. However, due to the inherent limitations of Ion Selective Electrode, only fluoride ions could be determined. The values obtained from fluoride Ion Selective Electrode and Reflectoquant were in close proximity to the values obtained from Ion Chromatography. Thus, Ion Chromatography was more reliable compared to Ion Selective Electrode and Reflectoquant anions content in seven different types of tea sample. The results of the analyses showed that the fluoride content ranged from 0.52 mg/L to 32.82 mg/L, chloride content ranged from 7.84 mg/L to 78.15 mg/L, phosphate content ranged from 24.03 mg/L to 114.50 mg/L, and the sulphate content ranged from 2.43mg/L to 66.88 mg/L. In this study, all these teas were safe to be consumed and would give lot of benefits to health.

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