This blog is based on a true story.....
Rachel loves to drink bubble tea ,especially with bubble tea pearls. She drinks it at least 7 times a week.
One day while reading the newspaper she came across a shocking report about bubble tea!
“Agri-Food & Veterinary Authority of Singapore (AVA) has recalled 11 food products from sale after maleic acid was found to be present."
She gasped in disbelief! "No wonder they tasted so good!"
Dropping the tea Rachel went to analyse bubble tea samples immediately, she is a BioMed student after all!
What is Maleic acid?
IUPAC name: But-2-enedioic acid
Molecular weight: 116.0722
Molecular formula: C4H4O4
Boiling poin: 157.8 °C
Physical state: White odourless solid crystal
Non-flammable, Non-explosive
Tasteless in solid and aqueous form
Solubility: 478.8g/L at 20 °C Tasteless in solid and aqueous form
Maleic acid is a dicarboxylic acid. It is mainly used as a detergent in surface cleaning! It’s applications include being used as sizing agents for the textile industry, photographical chemicals for quick-photo processing, making maleate salts in the pharmaceutical industry, dying auxiliary, and as an oil and fat preservative. It is also used for manufacturing polyester resins, pesticides, tartaric acid, and succinic acid!
Maleic acid is found typically in industrial settings as a raw material and there workers are advised to keep it away from their skin, eyes, and to avoid ingestion.
It is not an approved food additive, and long term usage can harm our kidneys!
So if Maleic acid is so dangerous, what is it is it doing in bubbletea?
The bubble tea pearls do not actually produce maleic acid. The maleic acid is only formed when maleic anhydride comes into contact with water, and is hydrolysed to form the acid. The maleic anhydride is thought to have been esterified in the starch used to make the pearls, and so that the pearls become more chewy. This causes the pearls to be saturated with maleic anhydride and will result in formation of maleic acid when exposed to water.
What is Maleic Anhydride?
IUPAC: Furan-2,5-dione
Molecular Weight: 98.06
Molecular Weight: 98.06
Molecular Formula: C4H2O3
Melting Point: 52.8 °C
Boiling point: 202 °C
Boiling point: 202 °C
Physical state: Colorless acrid smelling needles and pellet
Solubility in water: 491mg/L at 25°
Now that we know that the cause of maleic acid is actually due to chemical reaction in the pearls, we should analyse the bubble tea pearls for the presence and quantity of Maleic anhydride.
Here is a list of methods used to detect and quantify Maleic anhydride and Maleic acid.
Current Available Methods
1) High Performance Liquid Chromatography (HPLC)
This is probably the simplest and most used method to detect and analyse Maleic Anhydride samples. This method should be able to effectively seperate maleic anhydride and its hydrolysed form maleic acid from other acids such as fumaric acid, malic acid, acetic acid etc...
However we found that there is more than one way for HPLC to be used depending on the type of detector and the phase used.
a) Reverse phase HPLC-Derivatization of Maleic Anhydride
Maleic anhydride is first derivatized by p-Anisidine before being chromatographed. The collection and derivatization of Maleic Anhydride occurs on a sampling train consisting of two absorption tubes connected in series.
The first tube contains XAD-2 coated with p-anisidine, and the second contains untreated XAD-2. The derivative is then released from the tube coating surface using methanol, and then analyzed using Reverse phase HPLC.
- Matrix: Air with a volume of 20L & flow rate of 0.1L/min.
- The detection and quantitation limit of the procedure is 97 ng of maleic anhydride per sample, making it a sensitive method for detecting our compound.
- The recovery of derivative from the tubes has to be greater than 75% for the method to be effective though.
- There is also a downside to this method though, in that that p-anisidine is a suspected carcinogen, and that the capacity of the absorption tubes is limited.
Maleic anhydride is first derivatized by p-Anisidine before being chromatographed. The collection and derivatization of Maleic Anhydride occurs on a sampling train consisting of two absorption tubes connected in series.
The first tube contains XAD-2 coated with p-anisidine, and the second contains untreated XAD-2. The derivative is then released from the tube coating surface using methanol, and then analyzed using Reverse phase HPLC.
- Matrix: Air with a volume of 20L & flow rate of 0.1L/min.
- The detection and quantitation limit of the procedure is 97 ng of maleic anhydride per sample, making it a sensitive method for detecting our compound.
- The recovery of derivative from the tubes has to be greater than 75% for the method to be effective though.
- There is also a downside to this method though, in that that p-anisidine is a suspected carcinogen, and that the capacity of the absorption tubes is limited.
b) HPLC analysis using UV-vis detector
2) Quantification of maleic acid using chemical titrations and Fourier transform infrared spectroscopy (FTIR)
Maleic anhydride samples obtained by drawing air through glass fiber filters coated with 2 mg of 3,4-dimethoxybenzylamine forming derivative of Maleic anhydride-veratrylamine derivative
Samples are extracted with 90:10 (v/v) acetonitrile/dimethylsulfoxide, then analyzed by HPLC using a UV detector. Detection and quantitation limit of the technique is 1.99 µg per sample.
Samples are extracted with 90:10 (v/v) acetonitrile/dimethylsulfoxide, then analyzed by HPLC using a UV detector. Detection and quantitation limit of the technique is 1.99 µg per sample.
c) HPLC analysis using PhotoDiode Array (PDA ) detector
Maleic anhydride sample prepared using methanol, 0.5M potassium hydroxide, 5M hydrochloric acid, then analyzed by HPLC using a PDA detector. Limit of quantitation is 10 ppm. Useful for providing trace analysis and quantitation of maleic anhydride.
2) Quantification of maleic acid using chemical titrations and Fourier transform infrared spectroscopy (FTIR)
Maleic anhydride is grafted onto polypropylene through reactive processing.The samplesRE are analysed to estimate the amount of Maleic Anhydride. This is done by the titration of the acid groups. further confirmation is done by Fourier Transform infrared spectroscopy ( FTIR). Acid base titration is a more commonly used method for quantifying the concentration of anhydride groups onto polyolefins.
Acid Base titrations
Acid base titration is usually done in aromatic compounds such as xylene or toluene . Bromothymol blue is often use as the color indicator and titration is usually carried out until it meets the visual end point of the coloured indicator ,which indicates the neutralisation of the grafted functions.
If the solvent used is saturated with water, this will cause the anhydride groups to hydrolyse . Hence quaitfication becomes easier
If the solvent used is saturated with water, this will cause the anhydride groups to hydrolyse . Hence quaitfication becomes easier
FTIR
The polyolefins samples are first process in l00 μm films, then the films are dried under vacuum at 110 °C for 24 hours.
The FTIR spectra is recorded on a Perkin–Elmer FTIR Spectrometer 1760-X from 4000 to 400 cm−1 with a 0.5 cm−1 resolution.
The disappearance of the characteristic absorption band at 720 cm−1 is related to the CH out-of-plane deformation of maleic anhydride, and by the disappearance of the absorption band at 1715 cm−1, assigned to the carboxylic acid . This helps to ensure the sublimation and cyclisation of the unreacted maleic anhydride.
The FTIR spectra is recorded on a Perkin–Elmer FTIR Spectrometer 1760-X from 4000 to 400 cm−1 with a 0.5 cm−1 resolution.
The disappearance of the characteristic absorption band at 720 cm−1 is related to the CH out-of-plane deformation of maleic anhydride, and by the disappearance of the absorption band at 1715 cm−1, assigned to the carboxylic acid . This helps to ensure the sublimation and cyclisation of the unreacted maleic anhydride.
Limitations of grafting Maleic Acid onto Polypropylene
Polymerization of maleic anhydride onto polypropylene can cause serious degradation of the polypropylene. According to several studies, maleic anhydride that is grafted onto polypropylene has shown difficulties in analyzing the reacted maleic anhydride. The presence of unreacted maleic anhydride in the reaction mass makes it difficult for the direct measurement of bound maleate.
Materials and Methods
Out of all these methods, we decided to choose one, which is HPLC analysis using PDA detector.
Below are the reasons why...
1) It is the method available that we are more familiar with
2) It has been established as an effective method to quantify Maleic anhydride in starch/ food
3) Due to its chemical structure, maleic anhydride is able to fully dissolve in the solvent we are using as mobile phase, methanol
4) Using this method, we do not need to do much processing on our sample before analysis such as chemical derivatizations
5) PDA detector is able to give a complete spectrum in a very short time (0.1 sec) , so the experimental time is not prolonged, it is also useful for trace analysis of substances, so in this case it can help us detect even small quantities of maleic anhydride
Instrument
HPLC Conditions
-Analysis was done on Agilent1200 High Performance Liquid Chromatogram using a PhotoDiode Array (PDA) detector at UV214nm.
-Column used is GL Sciences InertSustain C18, 4.6 mm i.d. × 25 cm for 5 μm particles.
-Flowrate: 1mL/min
-Mobile Phase: methanol : 0.1% phosphoric acid (98:2, v/v), Isocratic elution
Sample
We used a popularly used brand of tapioca balls which is known to be contaminated with Maleic anhydride Sunright brand tapioca balls. (Used by the shop Rachel frequents)
Standard
We used pure Maleic anhyrdide as standard, to compare the peaks of Maleic anhydride in the balls and that of pure Maleic anhydride.
Method
After extraction and basic hydrolysis, we collected Maleic anhydride from the pearls to determine the concentration.
A) preparation of standard
We dissolved 100mg pure maleic anhydride in 100-ml volumetric flask with methanol, and then diluted to the mark. This is our reference standard.
B) Sample preparation
Transfer about 1 g of Sunright brand sample to a centrifuge tube, add 25 mL of
50% Methanol, then shake for 30 min to allow Maleic anhydride to dissolved into methanol. Add 20 mL of 0.5N potassium hydroxide and mix. After standing for 2 hrs, acidify with 3 mL of 5N Hydrochloric acid, dilute with HPLC grade water to 50 mL, and stand for several minutes. This is our stock solution. Dilute 100 μL of the mixture with water to 1mL, then mix and filtrate with membrane filter. This is our sample solution.
Both standard solutions and sample solubles are then analysed, and injected into HPLC via autosampler.
While waiting for our HPLC results to come out, we also conducted another test to confirm the identity of Maleic acid.
Confirmation test
Preparing Test solution
Preparing Test solution
We extracted maleic acid from the bubble tea pearls by soaking them in distilled water and stirring to simulate effects of when pearls are in the tea, then we used this as the stock solution of maleic acid.
Dilute 5mL of the stock solution (supposedly contain maleic acid) to 10mL with water.(Test solution)
Testing
Then dissolve 35mg of resorcinol in 10mL of sulfuric acid (resorcinol solution). Then to 3mL of our
Testing
Then dissolve 35mg of resorcinol in 10mL of sulfuric acid (resorcinol solution). Then to 3mL of our
test solution, we add 1mL of bromine, and heat in a water bath for 15mins to remove bromine, then heat to boiling, then cool mixture. Then to 0.2mL of this solution, add 3mL of resorcinol solution, and heat for another 15mins. If maleic acid is present, we should see a violet-pink color developing.
Source: British Pharmacopeia
Risk assessment Source: British Pharmacopeia
Results
Qualitative analysis
We identified the presence of Maleic anhydride in the tapioca balls by comparing the retention times of both the standard solution and that of the sample solution. They match each other with similar retention times of around 6 mins. Also, for our confirmation test, we observed a violet-pink color. This shows that Bubble tea tapioca balls indeed contain Maleic anhydride!
Quantitative analysis
After comparing peak area of sample solution and that of standard solution, we obtained the values, and in order to obtain total amount of Maleic acid and Maleic anhydride in sample solution applied the following formula: ＝ CVF / M
where
C: the concentration of maleic anhydride in the sample solution calculated by the peak area of sample solution
V: the volume of sample (mL)
M: the weight of sample (g)
F: the dilution factor
The resulting verdict we got from our calculations is that there is indeed presence of maleic anhydride in the tapioca balls, but there is only trace amounts of it,about 15µg/mL, this is in safe levels below the recommended limit of maleic anhydride/acid consumption mandated by US Environmental Protection Agency at 10mg/kg of body weight per day. As long it it is consumed in small quantities.
Discussion
Thus our analysis for the the tapioca pearls in Rachel’s bubble tea is that there is not a dangerously high enough concentration in the pearls to pose as an immediate health hazard. We conducted the same tests for several other tapioca ball brands from Taiwan such as Full Free Green Tea Tapioca Ball, Unbranded Starch Ball, and Redman Tapioca Ball with similar results. Further research showed that while it is not an approved food additive, and that prolonged consumption can caused kidney damage, Maleic acid is safe in small quantities, and does not posed as a health hazard if one consumes a small quantity once in awhile.
Conclusion
Now knowing that her favorite drink does not posed as a health risk, Rachel felt relieved...
But she has been drinking Bubble tea everyday hasn’t she?
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