Solubility Of Sa And Potassium Sorbate

Published Date: 02 Nov 2017

BY

SEOW YIT LONG

C:\Users\ALEN\Desktop\Untitled.png

SCHOOL OF ACT AND SCIENCE

TUNKU ABDUL RAHMAN COLLEGE

KUALA LUMPUR

2012/2013

DETERMINATION OF BENZOIC ACID AND SORBIC ACID IN JAM BY USING HIGH PERFORMANCE LIQUID CHROMATOGRAPHIC

BY

SEOW YIT LONG

A project report submitted to the School of Arts and Science in partial fulfillment of the requirement for the Bachelor of Science, Campbell University, U.S.A. and Advanced Diploma in Science, Tunku Abdul Rahman College

2012/2013

DECLARATION ON PLAGIARISM

Name: SEOW YIT LONG Student’s ID: 11WAB11101

Title of Thesis: DETERMINATION OF BENZOIC ACID AND SORBIC ACID IN JAM BY USING HIGH PERFORMANCE LIQUID CHROMATOGRAPHIC

Declaration

I confirm that I understand what plagiarism entails and am aware of the College’s policy in this regard.

I confirm that I have not committed plagiarism when completing the attached piece of work.

I did not make use of another student’s previous work and submitted it as my own.

I did not allow and will not allow anyone to copy my work with the intention of presenting it as his or her own work.

I declare that this thesis is my own, original work.

Signature: ……………………………………...

Date: …………………………………………

ACKNOWLEDGEMENTS

Throughout the past few months, there is a lot of contribution given from my friends and my supervisor Ms. Chen to the completion of my final year project. Now I would like to express my thankful those my treasure people helps and contribution to my final year project.

First of all, I would like to thank the most important person is my supervisor, Ms. Chen valuable advices regarding this project. She was always listen to my problems patiently whenever I facing obstacle in my experiment. Besides that, I would also like to thank to my lecturers Dr.Yue Chen Son and Dr. Chong Kian Wei who have provided me knowledge and information about how to operate the High Performance Liquid Chromatography.

Secondly, I would like to express my appreciation to all my fellow friends especially Ong Hui Kian, Teoh Chin Hui, Chan Kar Men and Ong Seng Wee, who have supported, and offered ideas and commented to my error in the preparation of experiment. Besides that, they were also giving me inspiration whenever I had failed to get the result from my project experiment.

Lastly, I would like to give my grateful to my family who have provide encouragement, inspiration and motivation to allow me to possess until completed my final year project such a heavy task.

CONTENTS

ACKNOWLEDGEMENTS

LIST OF TABLES

LIST OF FIGURES

LIST OF SYMBOLS AND ABBREVIATIONS

ABSTRACT

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1.0 INTRODUCTION

Preservative

Benzoic acid and Sorbic Acid

Antimicobial Action of Weak Organic Acid

LITERUTURE REVIEW

The Effect of Preservative for Human Body

The Analytical Methods for Determination of BA and SA

Method Extraction for UV/Vis Spectrophotometer Analysis

Multivariate Calibration

Method of Methanol Extraction of BA, SA, MP and PP in Jams by HPLC

Comparison between UV Spectrophotometer Method and HPLC method for the Analysis of Sodium Benzoate and Potassium Sorbate in Food Products

Effect of Concentration of samples solution on the maximum absorption

Effect of Microbiology Activity on SA Determination in Samples Solution.

Objective

METHODOLOGY

Chemicals and Reagents

Food samples

Preparation of samples analyzed with UV/Vis Spectrophotometer method and HPLC method

Chromotographic Condition

Instrumental Setting for HPLC

Instrumental Setting for UV/Vis Spectrophotometer

RESULT AND DISCUSSION

Determination of BA and SA by Using UV/Vis spectrophotometer

Beer-Lambert Law

Effect of Temperature on The Absorption Spectrum

Effect of pH on the Absorption Spectrum

Molecular Structure and absorption spectrum

Determination of BA with HPLC

Comparison Method of Determination of BA with HPLC and UV/Vis Spectrophotometer.

CONCLUSION

REFERENCES

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LIST OF TABLES

Table

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Information of BA used in the project

Information of SA used in the project

Solubility of SA and potassium sorbate.

Result of analysis on standard samples of sodium benzoate.

Repeatability of benzoate analysis with standard samples of sodium benzoate.

Sodium benzoate levels (in PPM) in sample of canned pickled cucumbers produced in food producing factories. Mean concentration calculated was 3 ppm. (Delavar et al., 2012).

Sodium benzoate level (in PPM) in samples of canned pickles produced in food. Mean concentration was calculated which is 2.9 PPM. (Delavar et al., 2012)

Sodium benzoate levels (in PPM) in samples of bulk cargo pickled cucumbers. (Delavar et al., 2012)

Statistical variables for benzoate concentration in the studied samples. (Delavar et al., 2012)

Concentration of potassium sorbate (mg/L) in cookie sample with UV and HPLC method. (Marjan et al.,2010)

Concentration of sodium benzoate (mg/L) in yogurt with UV and HPLC method. (Marjan et al.,2010)

Effect of microbiology activity on SA determination in cider. The sorbated-cider and its blank contained same amount were inoculated with spoilage organism fermenting cider. The cider was of late season and had a pH 3.95.

Maximum absorption of standard solutions of preservative BA.

Maximum absorption indicates BA presence in.jam samples.

Concentration of benzoic acid analyzed from the jam samples.

Maximum absorption of sample preservative SA and sample (jams) particularly.

Is the exact concentration of benzoic acid analyzed from the jam samples.

Concentration of benzoic acid and sorbic acid analyzed from 1g of the particular jam the jam sample respectively.

Absorption band under the effect of temperature on BA and SA.

Absorption band under the effect of different pH on the wavelength for BA and SA.

Standard calibration of BA by external method which increasing the concentration of the standard such as 0.2x10-6 mol/ml, 0.3x10-6 mol/ml, 0.4x10-6 mol/ml, 0.5x10-6 mol/ml and 0.6x10-6 mol/ml.

Area [µV.s] of peak obtained from the samples (jam) at the retention of 7.0 to 8.0 mins

Amount of benzoate acid analyzed in the jam sample respectively by using HPLC

Comparison between the UV/vis method and HPLC method in determination of BA. Percentage of variation is calculated by [(A-B)/(A+B)] x 100%.

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LIST OF FIGURES

Figure

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Structure of BA

Structure of SA

A mechanism of the translocation of weak organic acid over plasma membrane. Consisting three separated kinectic steps which are insertion into the outer hemi-leaflet of the membrane (kon), flip-flop of fatty acid between outer and inner leaflets (kff), and the dissociation from the inner leaflets (kff). Concepts and drawing taken from (Antonenko et al., 1993)

Absorption spectra of four preservative at (A) conventional UV spectra and (B) first-derivative spectra. BA, MP, PP, SA and regent blank. Concentration 2.0 μg mL- 1 for BA, MP, PP, and 1.0 μg mL- 1 for SA at pH 2.21.

Typical chromatogram of standard mixture pf preservative, mgL-1; BA is 48.2, SA, MP and PP, 5.0 each.

Different concentration of SA has been used for determination in cider. A) cider blank; B) cider + 23 ppm SA; C) cider + 46 ppm SA; D) cider + 91 ppm sorbic acid; E) cider +182 ppm SA

UV spectrum of sorbated-cider distillate; dilution fastor = 100. A) cider blank; B) cider + 180ppm SA ; C) cider + 96 ppm SA.

Absorption spectra of BA is 0.858 at 226 nm

Absorption spectra of SA is 0.084 at 250 nm.

Standard calibration of various concentration of benzoic acid, the concentration of samples (jam) is calculate by using the linear line plotted by using 10ml of known of benzoic acid of 1x10 -5mol/L, 1x10 -6 mol/L, 5x10 -6 mol/L, 1x10 -7 mol/L, which is prepared by dilution of 0.122±0.005g of BA dissolve in 10 ml of ethyl ether given 1x10-2 mol/L and then under dilution.1 gram of sample is extracted by ether and diluted to 200 ml.

Standard calibration of various concentration of sorbic acid, the concentration of samples (jam) is calculated by using 10ml of known concentration of SA : 1.0x10-8, 2.5x10-7, 5.0 x10-7, 1.0 x10-6, 2.5 x10-6, 5.0 x10-6. Which is prepared by dilution of 0.112±0.005g of BA dissolve in 10ml of ethyl ether given 1x10-2 mol/ml and then under further dilution.1 gram of sample is extracted by ether and diluted to 200ml. Due to higher concentration of SA content are not able to be detected accurately by the spectrophotometer, the absorbance are exceed a range from 0 to 1, the extracts were then diluted 10 times factor in order to get a more precise value of the absorbance.

Effect of temperature on BA

Effect of temperature on SA

Effect of pH on BA

Effect of pH on SA

The standard testing result shown the mixture of BA and SA having a retention particularly at which BA is 7.467 mins and SA is 9.508 mins.

Standard calibration line that has been draw above used to determine the unknown concentration of the samples (jam).

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LIST OF SYMBOLS AND ABBREVIATIONS

°C

µg

mg

g

BA

SA

MP

PP

nm

ml

g L ̄ ¹

kg

%

HPLC

UV-vis

FA

Degree Celsius (unit)

Ultragram (unit)

Miligram (unit)

Gram (unit)

Benzoic acid

Sorbic Acid (unit)

Methylparaben

Propylparaben

Nanometer (unit)

Mililiter (unit)

Gram per liter (unit)

kilogram

Percentage (unit)

High Performance Liquid Chromatographic

Ultraviolet visible

Fatty Acid

ABSTRACT

Food manufacturers use preservatives to prevent spoilage of the product during transportation and shelf life. Chemicals such as benzoic acid and sorbic acid are preservative act as food additive was determined by HPLC and UV/ Vis spectrophotometer method. UV/Vis spectrometer method was done by using ethyl ether is an organic solvent to extract preservative from jams. Separation funnel was use to filter the unwanted substance such as sugar and others coloring enhancer by using acid and base. While, HPLC method was used methanol for the extraction of preservative from the jams and placed the mixture into an ultra-sonicator with 50oC for 30 minutes. Determination result of HPLC and UV/Vis methods has a matching percentage at 92.25 %. The concentration of benzoic acid content for orange jam analyzed result is 2.477 mg g-1 which has excess the permissible amount of preservative in foods. Preservative such as benzoic acid and sorbic acid have carcinogen property which may affecting consumer health with a higher doses excising the permissible amount.

1.0 INTRODUCTION

Preservatives

Preservative are essential to prevent microbial spoilage of all aqueous-based hydrocarbon-rich products. Nowadays, commercial products are typically loaded with preservative to allow them to remain fresh during the long span between the products and help to keep them free from bacteria. There were different methodologies that were used from olden-age till today for food preservative. Some of the prior used techniques to preserve food are boiling, freezing, pasteurization and dehydration. However, modern technologies today using chemical efficiently such as preservative used are benzoate such as benzoic acid, nitrates like sodium nitrate (NaNO3), sulphites such as sulphur dioxide .

Antioxidant or antimicrobial they used as a function to kill bacteria. Besides that, preservative also prevent losing of some essential vitamin as well as amino acid which is a condition that bacterial like source to grow, thus preservative play a very effective role in the process of the food to keep food fresh. There is actually committed a fresh and healthy food to the consumers. (Mahanti.et al., 2007). Although benzoic acid has been acknowledged as permitted food additives but they are harmful to consumer health. Therefore, the level of benzoic acid used is regulated in routine of food analysis. In order to reduce the contamination of microbe growing to the food, benzoic acid is widely used to because it is lower price. According to the information from (Banerjee et al., 2004), BA recommended dose is 0.2 mg g-1 -1.5 mg g-1 the maximum are allowable. Sorbic acid is preferred to others because it’s physiological harmlessness and organoleptic neutrality. Maximum concentration report of sorbic acid for food preservative purpose was in the range of 1.0 mg g-1 and 2.0 mg g -1.

Benzoic acid and sorbic acid

Table 1. Information of BA used in the project

Benzoic acid

Production identification

CAS NO.

EINECS NO.

FORMULA

MOL WT.

H.S. CODE

TOXICITY

SYNONYMS

Smiles

Classification

65-85-0

200-618-2

C6H5COOH

122.12

2916.31.1105

Oral rat LD50: 1700mg/kg

Benzenemathanoic acid; carboxybenzene : acide benzoique (French) ; Acido benzoic: Benzenecarboxylic acid; benzeneformic acid: Benzoate: Benzoesaeure; Carboxybenzene: Dracylic acid;

C1(ccccc1)C(=O)O

Carbocylic Carboxylic acid. Preservative, antifungal agent.

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Benzoic acid is an aromatic carboxylic acid containing carboxyl group bonded directly to benzene ring, is a white, crystalline organic compound; melting at 122 °C (starting sublime at 100 °C; boiling at 249 °C; slightly soluble in water, and it is soluble in ethanol, very slightly soluble in benzene and acetone. BA is harmful ingestion and inhalation and it may cause irritant. The most of the commercial benzoic acid is prepared by the reaction of toluene with oxygen at temperature around 200 °C in the liquid phase and in present of cobalt and manganese salt as catalyst. Besides that, it can be also prepared by oxidation of benzene with concentration sulphuric acid or carbon dioxide in the presence of catalyst.

Benzoic acid is converted to its salts and ester for the use of preservative application in foods, drugs and personal products. For example, Sodium benzoate, sodium salt of benzoic acid which function used preferably as one of the principal anti-microbial preservative in foods and beverage. However, due to the toxicity toward human body, its concentration is limited usually not exceeding 0,1%, salt or ester form is about 200 times more soluble than benzoic acid form. Hence the ester form of BA which normally is sodium benzoate are always been used to applied in food due to higher solubility instead of just BA.

Table 2. Information of SA used in the project

Sorbic Acid

Production identification

CAS NO..

FORMULA

MOL WT.

TOXICITY

SYNONYMS

Smiles

Classification

110-44-1

C6H8O2

112.1265

Oral rat LD50; 7360 mg/kg

(E,E)-2,4-Hexadienoic acid; 2-Propenylacrylic acid; alpha- trans-gamma-trans-sorbic acid.

C(=C\C=C\C)\C(O)=O

Preservative, antifugal agent, antibacterial agent.

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Sorbic acid is a white crystalline powder, is also chemically call 2,4-hexadienoic acid, is an unsaturated fatty acid which consist of two double bond conjugation. Sorbic acid is also a well-known preservative used such as number E200, Sorbic salt including calcium sorbate (E203), potassium sorbate (E202), and sodium sorbate (E201). Sorbic acid is an antimicrobial, antifungal preservative normally used in prepared foods and drinks. Sorbic aicd is normally occur in fruit, it inhibit the fungal growth but allow the activity of bacterial.

Sorbic acid normally in the salt form such as potassium sorbate; calcium sorbate; its salt are used according to different solubility can be explained by (Murk et al., 1975), potassium sorbate will release back sorbic acid if dissolve in water, the effectiveness of solubility of salt form of potassium sorbate is higher than the sorbic acid.

Table 3. Solubility of SA and potassium sorbate.

Solvent

%solubility SA

%Solubility Potassium Sorbate

Water

20°C (68°F)

0.16

58.20

50°C (112°F)

0.55

61.00

100°C (212°F)

4.00

64.00

Ethanol

5%

0.15

57.40

100%

12.90

2.00

Sucrose

10%

0.15

58.00

40%

0.10

45.00

60%

0.08

28.00

Obviously potassium sorbate is more high soluble in water compare to sorbic acid. In formation source was came from (Gooding et.al.1995). Sorbic acid is used as a mold, bacterial and yeast inhibitor and as a fungistatic agent in food.

Figure 1. Structure of BA

Figure 2. Structure of SAC:\Users\ALEN\Desktop\Benzoic_acid.png C:\Users\ALEN\Pictures\Sorbic_acid.jpg

1.3 Antimicrobial action of weak organic acid

Benzoic acid and sorbic acid are the weak organic acid play important role in alternative proposition for the mode of action in cellular energy depletion and membrane perturbation for the microbe organism (Stanley et al., 2007) .SA and BA are polyunsaturated molecule. They consist of multi- conjugated double fatty acid (FA), a hydrophobic tail and hydrophilic head provide the solubility in either water phase or enter microbial cells. In the uncharged form of weak organic acid, BA and SA are able to enter intercellular membrane of the microbe organism that presence in jam, undissociated acid concentration is lower than outside of the cell, noted that in order to pass through the hydrophobic plasma membrane barrier, weak acid must presence in protonated form. This process is a mechanism called ‘flip-flop’, which can be explained clearer in Figure 3 below.

Figure 3. A mechanism of the translocation of weak organic acid over plasma membrane. Consisting three separated kinectic steps which are insertion into the outer hemi-leaflet of the membrane (kon), flip-flop of fatty acid between outer and inner leaflets (kff), and the dissociation from the inner leaflets (kff). Concepts and drawing taken from (Antonenko et al., 1993)

Distortion of normal exchange molecule across the permeable plasma membrane cause membrane perturbation (Antonenko et al.,1993) Membrane perturbation would subsequently lend to direct uncoupling of membrane-located essential transport processes and thus constitutes an alternative antimicrobial action of the compound. FA and the level of FA saturation determine the strength of hydrophobic interaction between membrane and the tail of a weak acid.

This accumulation of weak acid inner the cell able to damage normally cell mechanism and organization caused by mentioned perturbation of plasma membrane. In particular, it may affect uptake of substrates. Moreover, in fungi, oxidation stress can occur if the weak acid accumulation in the mitochondrial membrane, hence compromising the function of the respiratory chain of the cell. During normal physiological of a cell, the total H+-ATPase activity uses approximately10-15% of the total ATP flux produced in the cell. However, the insertion of the weak-organic acid or weak organic acid stressed cells, the ATP assumption of cells increase can be as high as 60%. Sorbate (anion) present inner of cell. Henriques et al. provided evidence of an efflux system that removes the accumulated of the anion which is weak-organic acid from inside the cell. This theory was then corroborated and reinforced by Piper et al., who demonstrate the existence of a large membrane protein 170kDa in sorbic –acid-adapted cells of Saccharamyces cerevisiae.

The protein is known as ATP-binding cassette (ABC) transporter Pdr12, which is believed to confer resistance by actively extruding preservative anion from the cell. Pdr12 is rapidly induced upon weak-acid stress, ultimately reaching cellular level comparable to the H+- ATPase. By make used of S.aerovisiae as the target cell, sorbate and benzoate stressing the cell as a result increase energy (ATP) demand. Bacterial or fungal are die due to energy deficiency although energy is keeping producing but that is not enough for cell respiratory after immersed by the preservative.

2.0 LITERUTURE REVIEW

2.1 Effect of preservative on human body

Preservatives are categorized as food additives, today additives are strictly regulated and go through an authorization procedure. Authorized food additives are clearly defined are fixed to protect the consumer.

From the available data, a maximum level of additive that has no demonstrable toxic effect is determined. This is call the no-observed-adverse effect level (NOAEL) and is used to determine the Acceptable Daily Intake (ADI) figure for each food additive. Permitted food additive are broadly classified into several categories according to the function they perform. Safety assessment of food additives is based on review of all the toxicological data, including in humans and in animal models. The NOAEL is then divided by a safety factor, usually 100 m which result in a large margin of safety. This is because NOAEL is determine in animal but not in human. This purpose is assuming that man is more sensitive than the most sensitive test animal. Another reason is because the reliability of toxicity test is limited by the number of animals test. Such test cannot represent the diversity of the human population, subgroup of which may show different sensitivities. For example, children, the old and the ill. Again it is prudent to adjust for these differences.

The ADI provides a large margin of safety and refers to the amount of a food additive that can be taken daily in the diet, over a lifetime span, without any negative effect on health. The ADI is compared with average and extreme consumption estimates in the population as whole or in particular subgroups of the population. Provided that intakes for average and extreme consumers are within the ADI, it is unlikely that any harm will result because the ADI is based on a no-observed adverse effect level, to which a large safety margin has been applied. To ensure that consumers are not exceeding the ADI by consuming too much or too many products containing a particular additive.

Oral ingestion of benzoic acid and sodium benzoate in experimental human may cause the rapid absorption of the undissociated BA from the gastrointestinal tract. The substances are metabolized in the liver mainly by conjugation with glycine, resulting in the formation of hippuric acid which is rapidly excreted via the urine. However, benzoate can be penetrating through the skin, the excretion or their metabolites of benzoate are not to be expected. Besides, BA is slightly irritating to the skin and eye, whereas sodium benzoate is not irritating to the skin but irritating to the eye. Other than that, in short-term inhalation study, BA can cause the problem in breathing such as asthma.

The effect of ingestion of SA also providing a bad healthy result in the human. Skin irritation may also be caused by SA ( Humphrey et al., 2011). It is said that skin contact with the SA can cause a rash known as contact urticaria. Besides that, SA may also cause eye and irritation, for the contact can affect other organs with what are called extracutaneous reactions. These symptoms are rare in SA exposure, since SA contact urticaria is a non- allergic reaction, unlike other sources of the condition, such as latex or antibiotics. However, if you are exposed to SA, there is a small chance to experience watery eyes, wheezing runny nose.

2.2 Analytical Methods for determination of BA and SA.

There are many analytical methods to determine the BA and SA usually in foods. Spectrophotometer is usually used to determine the electronic absorption in the standard calibration, there have the other method to determine BA abs SA by using high performance liquid chromatographic (HPLC), gas chromatographic mass spectrophotometer (GCMS).

However, HPLC will be the more effective and easy to be used in determination of BA and SA. Because of the process of operation, machine that use to test the samples preservative is not that complex compared to the GCMS. Besides, the samples extraction routine for HPLC is more simpler compare to the GCMS, However, HPLC has its found out that, machine usually appear problem if a very small error have been carried out. This is because the chromatographic column for HPLC is easily to be blocked by the present of impurity either in the samples or mobile phases that used. Hence, ultra-filtration must be done for the samples analysis and the mobiles phases that will be used. Furthermore, column of chromatographic should be flashed continuously with mobile phases to wash the column to avoid salts or samples that coagulate inside the column of HPLC. UV/Vis may be also used to analysis for the sample preservation.

UV/Vis spectrophotometer provide a easier and faster way to determine the preservatives in foodsuch as Benzoic acid (BA), methylparaben (MP), propylparaben (PP) and (SA), and they have well defined UV spectra. However, their spectra overlap seriously, and it is difficult to determine them individually from mixture pre-separation. Different chemometric approaches were applied to resolve the overlapping spectra and to determine these compound simultaneously. With respect to the criteria of % relative prediction error (RPE) AND % recovery, principal component regression (PCR) and radial basis function-artificial neural network (RBF-ANN) were the preferred methods. These two methods were successfully applied to the analysis of some commercial samples.

2.3 Method Extraction for UV/Vis spectrophotometer Analysis

Anecdotal information has suggested that sodium benzoate is used with more than permissible doses during production step of food products especially pickles and pickled cucumbers in food producing factorized in Markaza province and other food producing factories (Delavar et al., 2012). The present study was done to evaluate factual concentration of sodium benzoate in these products. The samples were categorized into canned pickles and pickled cucumbers in food producing factories in Markazi. Overall, 33 samples from canned pickled cucumbers. In this experiment, UV/Vis spectrophotometer was used for determination of sodium benzoate level in the studied samples.

To verify the analyzing method, 2 and 4 mg/l sodium benzoate of standard samples was performed. The result showed the validity and the precision of the method (table 4). The RSD was less then 4% and the samples were keeping not more than 3 days in analysis. Table 2 has shown analyzing the method of the study has had satisfactory repeatability.

Filtration was done after the extraction was performed by petroleum ether solvent in the presence of hydrochloric acid 0.6 N. UV absorption at wavelength of 228 nanometers was determined and relevant calibration curve was drawn. The result were analyzed by N-par test using SPSS software version 16.0. (Delavar et al., 2012)

Table 4. Result of analysis on standard samples of sodium benzoate.

Table 5. Repeatability of benzoate analysis with standard samples of sodium benzoate.

Canned pickles and pickled cucumbers produced in food producing factories in Markazi province and other producing factories which production sold in Arak city were analyzed in table 3 and 5. Each of the samples were repeated to analysis was reported in the tables. Descriptive statistical variable for benzoate concentration in the samples are shown in table 6.

Table 6. Sodium benzoate levels (in PPM) in sample of canned pickled cucumbers produced in food producing factories. Mean concentration calculated was 3 ppm. (Delavar et al., 2012).

Table 7. Sodium benzoate level (in PPM) in samples of canned pickles produced in food. Mean concentration was calculated which is 2.9 PPM. (Delavar et al., 2012)

Table 8. Sodium benzoate levels (in PPM) in samples of bulk cargo pickled cucumbers. (Delavar et al., 2012)

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Table 9. Statistical variables for benzoate concentration in the studied samples. (Delavar et al., 2012)

Benzoate level presence in the samples of food was nearly to zero, but its level in bulk cargo pickled cucumbers products was above the permission level. The benzoate level was also measured by HPLC which in the study conducted by USA in 2000, test samples including fruit juice, sodas, ketchup, soy sauce, peanut, butter and cream cheese. The result varies from the using of UV/VIS is between 0.015 and 0.1% according to (Grether.M.T et al 2000).

2.4 Multivariate calibration

An experiment have been done by (Yan et al., in 2008).Benzoic acid (BA), methylparaben (MP). Propylparaben (PP) and sorbic acid (SA) have been analyzed my using UV/VIS spectrophotometer. Seven different chemometric approaches were applied to resolve the overlapping spectra and to determine these compound simultaneously.

Full-size image

Figure 4. Absorption spectra of four preservative at (A) conventional UV spectra and (B) first-derivative spectra. BA, MP, PP, SA and regent blank. Concentration 2.0 μg mL- 1 for BA, MP, PP, and 1.0 μg mL- 1 for SA at pH 2.21.

The absorption spectra over the 200-320 nm and the peak maxima of the conventional spectra of the four preservative were at 229 nm, 256 nm and 263 nm for BA, MP, PP and SA, respectively. The spectra of these compound overlapped one another can be observed in Figure 1. Consequently, chemometric method was applied to quantitatively resolve the overlapped UV spectra.

2.5 Method of methanol extraction of BA, SA, MP and PP in jams by HPLC

According to information given by Bahruddin Saad et al. The extraction method has been developed was applied to the determination of preservative in foods such as soft drinks, jams, sauces, canned fruits/vegetables and others.

Solid food samples were finely ground prior to the extraction. About 1g of the samples is acutely weighed in a screw-capped test tube. A 25ml of the methanol is added to the samples test tube and place in a sonicator (ULTRAsonik Model 28X, Ney Dental,Yucaipa, California) that maintained at 50°C for about 30mins.The test tube was then subjected to vortex mixing (KIKA Works, Model MS1. Malaysia) for 2 min. Filtration is done by using a 0.45 micro meter nylon membrane. This extraction is actually supported by the method of HPLC and UV/VIS.

Full-size image

Figure 5. Typical chromatogram of standard mixture pf preservative, mgL-1; BA is 48.2, SA, MP and PP, 5.0 each.

Under the experiment condition have been followed, the baseline resolution of four component were achieved. The retention times for BA, SA, PP and MP are about 7.5, 8.5, 11.2 and 21.0 min respectively showed in Figure 1. The use of methanol-acetate buffer (35:65) was effective to be used for separation to determine BA, SA, and MP.

2.6 Comparison between UV Spectrophotometer Method and HPLC method for the Analysis of Sodium Benzoate and Potassium Sorbate in Food Products.

Table 10. Concentration of potassium sorbate (mg/L) in cookie sample with UV and HPLC method. (Marjan et al.,2010)

Cookie samplese samples

C(mg/l)UVa8 (mg/l)UV

C(mg/l)HPLCbg/l)HPLC

1

561.6

209.0

2

451.6

340.5

3

671.8

517.7

4

767.8

523.0

5

180.0

132.2

6

522.6

427.5

7

695.2

450.3

8

834.8

507.6

9

761.6

517.9

10

1169.4

652.4

11

213.2

141.7

12

1117.0

518.6

13

213.2

282.5

14

731.4

373.6

15

1266.2

358.6

16

1182.6

570.4

aConcentration of potassium sorbate (mg/L) in cookie samples with UVmethod.

bCocentration of potassium sorbate (mg/L) in cookie samples with HPLC method

Table 11. Concentration of sodium benzoate (mg/L) in yogurt with UV and HPLC method. (Marjan et al.,2010)

Yogurt diluted with water sample

C(mg/L)UVa

C(mg/L)HPLCb

1

74.5

N.D.

2

61.0

N.D.

3

120.4

117.0

4

108.1

11.6

5

68.1

13.4

6

148.8

135.8

7

57.0

18.0

8

67.6

N.D.

9

63.7

39.0

10

88.2

52.9

aConcentration of sodium benzoate (mg/L) in cucumber pickle samples with UV method

bConcentration of sodium benzoate (mg/L) in cucunmber pickle samples with HPLC method.

The table 9 and 10 above are shown the result of determination of potassium sorbate and sodium benzoate by using UV and HPLC method. By comparing the data achieved from the UV and HPLC method. It is clear that the amount of obtained concentration by HPLC is lesser than those obtained by UV. The data showed that in these food products, interfering compounds, such as vanilla in cookies and flavoring agents in yogurt diluted with water and cucumber nickel samples are present.

2.7 Effect of Concentration of samples solution on the maximum absorption

An experiment experiment was determination of sorbic acid in apple cider. Spectrophotometric determination of sorbic acid in apple cider is based on the degree of ultraviolet absorbance of SA at the wave length of maximum absorption, where the maximum absorption occur at 262 nm and absorption is increase when the concentration of the solution increase which is obeyed with the Beer’s Law ( Harrington et al.,2006).

Figure 6. The different concentration of SA has been used for determination in cider. A) cider blank; B) cider + 23 ppm SA; C) cider + 46 ppm SA; D) cider + 91 ppm sorbic acid; E) cider +182 ppm SA.

Figure 6 illustrates application of the dilution method for SA determination at various levels in cider. Curve A was set as 1$ cider blank, while curves B, C, D, and E respectively represent cider sample containing 23, 46, 91, and 182 ppm of SA. The result of the application of different level of concentration samples solution, gives the interfere in the maximum absorption peak at wavelength about 262 nm.

2.8 Effect of microbiological activity on SA determination in sample solution

Based on the result that has been done by (Harrington et al.,1997), many SA determination procedures are based on its recovery by distillation. The method that used in this experiment applied to cider has under some condition, irrelevant material interfere with the absorbance readings. Problem that are affecting the absorption band have been clarified.

Table 12. The effect of microbiology activity on SA determination in cider. The sorbated-cider and its blank contained same amount were inoculated with spoilage organism fermenting cider. The cider was of late season and had a pH 3.95.

The result that has been recorded is that the microorganism are able to ferment either the sorbated cider or blank cider. With the procedure of dilution, the concentration of SA (ppm) content reduce when the temperature increase which as a manipulated variable in these experiment. The UV absorbance at 262 nm reduced slightly. The method of using distillation gives a similar result with the method of dilution. However, the concentration of SA (ppm) content calculated is increased. The concentration of SA content was calculated by using these formulae: [(sorbated-cider) – (blank-cider)] x100 / 0.23 = ppm SA

Figure 7. UV spectrum of sorbated-cider distillate; dilution fastor = 100. A) cider blank; B) cider + 180ppm SA ; C) cider + 96 ppm SA.

When SA is distilled from cider (B and C curves), peak absorption (between 262 nm to 282 nm) is shifted to the SA ratios. This effect is especially larger at low SA level because of superimposition of the spectra of the interfering cider distillate materials on that of SA.

Hence, the dilution method showed more microorganism fermentation take place in the cider compare to the method of distillation which is a prevention of microbial growth. However, the accuracy for the calculation the amount of recovered SA will be interfered by microbial growth which alters the cider quality. Distilled cider also are not able to be calculated the amount of SA content, this uncertainty is due to either blank or sorbated-cider which are changing in quality.

This problem are be answered by (Wolfrom et al., 1948), whose has provided an explanation about the effect of heating on the sugars. Spectra of heated sugar solution were compared with the interfering substances observed in cider distillates. A 1% of D-frutose, the predominant and least-stable sugar that has been corroborated by (Smock et al., 1948).

The unheated sample has given a lower absorption at 282 nm, whereas the heated sample has given a higher absorption at 282 nm which undergoes the distillation process. This is because effect of heat result partly from sugar decomposition, which is accentuated by longer heating periods, higher temperatures, and addition of acid, but diminish if fermentation occurs.

2.9 OBJECTIVE

The aim is to determine the amount of benzoic acid and sorbic acid in jams by using HPLC and UV/Vis spectrophotometer. In this experiment, the results of the amount of benzoic acid and sorbic acid analyzed are used to compare with the legislation amount of BA and SA in jams (Orange, blackberry, blueberry and strawberry) from produced from individual company A, B, C and D.

3.0 METHODOLOGY

3.1 Chemicals and reagents

Chemicals and the reagents that have been used were BA (99%), SA (99%), ammonium acetate (98%), sodium hydroxide and acetic acid (99%), pure methanol (grade for HPLC). Ethyl ether, 0.01% hydrochloric acid, 0.01% ammonium hydroxide.

3.1.1 Food samples

Various types of jam like orange jam, blackberry jam, blueberry jam and strawberry jam which had been used as the samples for this experiment were from branded company A, B, C, and D. Those jams were purchased from supermarket (Carrefour) located at Kuala Lumpur, Malaysia.

3.1.2 Preparation of samples analyzed with UV/Vis Spectrophotometer method and HPLC method

For the preparation of samples analyzed with UV/Vis spectrophotometer. Small amount of sample was grinded until the solidified jam is grinded become viscous-like-liquid. 1g of sample was weighed into a 100 ml of beaker. 50 ml of ethyl ether was added and mixed well about 5 minutes. The ether extracted sample was filtered and pour into a separating funnel, 25ml of 0.01% of hydrochloric acid was added and mixed (Noted that: 0.01% of HCl is enough to used to for extraction of benzoic acid and sorbic acid from 50ml of ethyl ether to aqueous solution)(Arnnok,P, R et.al). Aqueous phase was taken and 25ml of 0.01% of ammonium hydroxide was added to neutralize the hydrochloric acid (Noted that: Weak acid which is ammonium hydroxide is used to neutralize the strong acid in order to slightly increase the pH of the sample solvent in order to make BA and SA to be extracted by ethyl ether). The sample extracted in the acidified solution was then extracted by 50 ml of ether. The extracted solution was poured into a volumetric flask and diluted to 200 ml.

For the preparation of samples analyzed with HPLC. Small amount of sample was grinded until the solidified jam is grinded become viscous-like-liquid. 1g of sample was weighed into a 45ml of centrifuge tube and twenty-five milliliters of methanol was added, and finally place it in a ultrasonicator (JAC 2010) with 50°C for about 30 minutes. The samples were then place on the shaker machine (Heidolph Unimax) for about 10 minutes. For the testing sample, filtration was done by using special titan membrane filter for HPLC. The 20 µL of the clear filtrate sample was injected into the HPLC column.

3.2 Chromatographic condition

Analytical separation was carried up on a Perkin Elmer HPLC using ChromSep HPLC Column SS 150x4.6mm. Spectrophotometer was set at 245nm and volume of injected was 20 µL. The aqueous phase was prepared by weighing 3.8g of ammonium acetate and dissolved in 1 litter of eluents-deionizer water, and its pH was adjusted at 4.4 using acetic acid. The mobile phase was used which is pure methanol. Both of aqueous phase and mobile phase were filtered before running for the HPLC column. Concept was taken and modified from ( Bahruddin Saad et al).

3.2.1 Instrumental Setting for HPLC

Initial flow time before analyzing samples: 0.5

Samples flow time: 25min

Flow: 1.0 min/ml

Mobile phase labeled A : ammonium acetate

Mobile phase labeled B: 100% methanol.

Injection 20µL portion each of sample extracts and mixed standard

3.2.2 Instrument setting for UV/Vis spectrophotometer

Cuvette that used was quartz call. Two quartz cells were used and filled 70% full with ethyl ether. Program of the spectrophotometer was set the user baseline as blank before used to test the samples.

The absorption spectrum over the 200- 800 nm of the individual preservative solution and reagent blank are shown in figure 1 and 2. Figure 1 and 2 shown that the maxima of the conventional spectra of the two preservative have been determine at 226 and 250nm for BA and SA respectively.

4.0 RESULT AND DISCUSSION

4.1 Determination of BA and SA by using UV/Vis spectrophotometer

Figure 8. Absorption spectra of BA is 0.858 at 226 nm

Figure 9. Absorption spectra of SA is 0.084 at 250 nm.

Various concentrations (g/L) were calibrated shown in Figure 8 for the BA and Figure 9 for the SA. A lower concentration is used to plot a graph this is due to accuracy; the absorbance must not able to exceed 1.000 ABS for a spectrophotometer. Besides that, the concentration that needed to be used to calibrate for determination of the concentration of samples jam were very small amount. This is because the preservative content that obtained were also in very low level.

Due to higher concentration of BA contain are not able to be detected by the spectrophotometer, the absorbance are exceed a range from 0.000 to 1.000, the extract was then diluted to 10 time factor in order to get a more precise value of the absorbance.

Table 13. Maximum absorption of standard solutions of preservative BA.

Concentration of BA (mol/L)

Absorbance

Average

Test 1

Test 2

Test 3

1x10-7

0.026

0.022

0.020

0.023

1x10-6

0.033

0.027

0,022

0.027

5 x10-6

0.075

0.072

0.070

0.072

1 x10-5

0.123

0.125

0.129

0.126

Table 14. Maximum absorption indicates BA presence in.jam samples.

Samples (jam)

Absorbance

Average

Concentration of BA (mol/L) of 200ml of sample concentration in 10ml of Standard solution.

Test 1

Test 2

Test3

Orange

0.138

0.140

0.134

0.137

2.028x10-3

Black berry

0.052

0.052

0.058

0.054

7.997x10-4

Blue berry

0.072

0.071

0.079

0.074

1.095x10-3

Strawberry

0.033

0.039

0.035

0.036

5.331x10-4

Figure 10. Standard calibration of various concentration of benzoic acid, the concentration of samples (jam) is calculate by using the linear line plotted by using 10ml of known of benzoic acid of 1x10 -5mol/L, 1x10 -6 mol/L, 5x10 -6 mol/L, 1x10 -7 mol/L, which is prepared by dilution of 0.122±0.005g of BA dissolve in 10 ml of ethyl ether given 1x10-2 mol/L and then under dilution.1 gram of sample is extracted by ether and diluted to 200 ml.

Table 15. Concentration of benzoic acid analyzed from the jam samples.

Samples (jam)

Concentration of BA (mol/g)

Orange

2.028x10-5

Black berry

7.997x10-6

Blue berry

1.095x10-5

Strawberry

5.331x10-4

Base on the line of calibration, using sample absorbance detected. Orange has obviously highest the absorption band of orange that has been detected among the other jam samples such as black berry, blue berry and strawberry.

Table 16. Maximum absorption of sample preservative SA and sample (jams) particularly.

Concentration of SA (mol/L)

Absorbance

Average

Test 1

Test 2

Test 3

1.0 x10-8

0.025

0.022

0.017

0.02

2.5 x10-7

0.055

0.039

0.035

0.043

5.0 x10-7

0.067

0.058

0.054

0.059

1.0 x10-6

0.092

0.082

0.079

0.093

2.5 x10-6

0.141

0.164

0.157

0.154

5.0 x10-6

0.151

0.177

0.171

0.166

Samples (jam)

Absorbance

Average

Concentration of SA (mol/L) of 200ml of sample concentration in 10ml of Standard solution.

Test 1

Test 2

Test 3

Orange

0.043

0.038

0.039

0.040

1.645x10-5

Black berry

0.114

0.118

0.111

0.114

4.689x10-5

Blue berry

0.060

0.057

0.066

0.061

2.509x10-6

Strawberry

0.121

0.123

0.121

0.122

5.018 x10-5

Base on the line of calibration, using sample absorbance detected. Orange has obviously highest the absorption band of strawberry that has been detected among the other jam samples such as black berry, blue berry and orange.

Figure 11. Standard calibration of various concentration of sorbic acid, the concentration of samples (jam) is calculated by using 10ml of known concentration of SA : 1.0x10-8, 2.5x10-7, 5.0 x10-7, 1.0 x10-6, 2.5 x10-6, 5.0 x10-6. Which is prepared by dilution of 0.112±0.005g of BA dissolve in 10ml of ethyl ether given 1x10-2 mol/ml and then under further dilution.1 gram of sample is extracted by ether and diluted to 200ml. Due to higher concentration of SA content are not able to be detected accurately by the spectrophotometer, the absorbance are exceed a range from 0 to 1, the extracts were then diluted 10 times factor in order to get a more precise value of the absorbance.

Table 17. Is the exact concentration of benzoic acid analyzed from the jam samples.

Samples (jam)

Concentration of SA (mol/g)

Orange

1.645x10-7

Black berry

4.689x10-7

Blue berry

2.509x10-8

Strawberry

5.018 x10-7

Table 18. Concentration of benzoic acid and sorbic acid analyzed from 1g of the particular jam the jam sample respectively.

Samples (1 gram)

benzoic acid content( mg g -1 )

Orange

2.477

Black berry

0.977

Blue berry

1.337

Strawberry

0.651

Samples (1 gram)

sorbic acid content (mg g-1)

Orange

0.018

Black berry

0.052

Blue berry

0.003

Strawberry

0.054

.

4.1.1 Beer-Lambert Law

Base on Beer-Lambert law, absorbance of a mixture at certain is the sum of each component absorbance at the considered wavelength used to determine the absorbance is equal with the number of the constituent from mixture. The absorption band that obtained from the jams sample was chosen that wavelength at which each component has the maximum of absorption.

The amount of radiation absorbed may be measured by a formulae according to the Beer-Lambert Law which is A=epsilon.b.c.

lo- the intensity of the incident light

l – the intensity of the emerging light

b – length of light path through the sample

c – sample concentration

E – molar absorptivity, [E]SI = 1/mol.cm

If all the light passes through a absorbance is zero, and present transmittance is 100 percent. If all the light is absorbed, then percent transmittance is zero, and absorption is infinite. The absorption for sample for mixture is present of two peak that are about a range 210nm to 230 wavelength for BA and another peak is at the range of 240 to 260nm.for SA. Absorption of curve is influenced by temperature stat that higher the temperature monomers are predominant. Meaning that, higher the absorption curves that are able to obtain

4.1.2 Effect of Temperature on the absorption spectrum

F:\DCIM\115_PANA\P1150511.JPG F:\DCIM\115_PANA\P1150512.JPG

Figure 12. Effect of temperature on BA. Figure 13. Effect of temperature on SA

Table 19. Absorption band under the effect of temperature on BA and SA

BA

Temperature °C

Absorption (ABS)

20

0.179

30

0.332

40

0.102

SA

Temperature °C

Absorption (ABS)

24

0.629

30

0.754

40

0.974

Figure 10.1 and 10.2 shown effect of temperature to the level of absorption curves of BA and SA. Temperature °C have been controlled and designed at 20°C, 30°C and 40°C for BA and 24°C, 30°C and 40°C for SA. High temperature may result the high absorbance value.

In experiment, temperature was set up at room temperature for BA and SA which are 20°C and 24°C, this is because temperature is not the main objective for analysis purpose. However, lower concentration have been adjusted and builded up a calibration for determination of samples preservative content. Besides that, pH has been proven by (YanQ C and Yong N N et el 2009) who corroborate that the degree of protanation would be changed with the pH. BA and SA has constant absorbance, and their maximum absorbance started to decrease and peak wavelength shift from 229nm and 263nm to 223nm and 254nm. Sample extracts of pH have been tested which is slightly acid about 5.30 for BA and 5.06 for SA by using the AB204-3 Mettler Toledo as pH detector. The reason of why the peak obtained that at a lower wavelength is suspected because of the degree ionization of weak acid would change with the changing pH, and both BA and SA would be existed in nearly complete neutral form.

4.1.3Effect of pH on absorption spectrum

F:\DCIM\115_PANA\P1150514.JPGF:\DCIM\115_PANA\P1150513.JPG

Figure 14. Effect of pH on BA Figure 15. Effect of pH on SA

Table 20. Absorption band under the effect of different pH on the wavelength for BA and SA.

BA

Number of peak

pH

Wavelength (nm) obtained

1

5.30

224

2

6.00

221

3

7.00

207

SA

Number of peak

pH

Wavelength (nm) obtained

1

5.06

251

2

6.00

248

3

7.00

247

Various pH level haven been adjusted by using NaOH and HCl solution. Result has shown increase the pH value for BA, the wavelength of the maximum absorption band decrease from 224 nm to 207 nm, while the wavelength of the maximum absorption change from 251 nm to 247 nm when increasing the pH from 5.06 to 7.00

4.1.4 Structural molecule and absorption spectrum

Absorption spectrum consists of absorption bands corresponding to structural group of molecules. Structural groups are also called chromophores that are responsible for electronic absorption. For example, chromosphores can give the color of substance or carbonyl, carboxyl, alkenes that are also play in role for the electronic absorption. The color of the BA and SA dissolved in organic solvent give a colorless solution. However, due to the structure of BA and SA, UV/Vis spectrophotometer is able to use to determine the wavelength and electronic absorption. BA is consisting of benzene and carboxyl group, while SA has two conjugated double bond and one carboxyl group. Different of structure determine different wavelength in the electronic absorption. When the ultraviolet /visible light passed through the sample solution, excitation occurred. The emission and excitation spectrum is produced by the functional group and the conjugation double bond that present on the molecules of the sample either BA or SA.

4.2 Determination of BA with HPLC

C:\Users\ALEN\Desktop\standarf.pngFigure 16. The standard testing result shown the mixture of BA and SA having a retention particularly at which BA is 7.467 mins and SA is 9.508 mins.

However, the samples that contains with the SA are almost in a very small amount. Base of the LOD of the HPLC method for benzoic acid and sorbate acid was obtained in 0.3mg/g, SA present inside the jam samples are difficult to recognize the retention about 9.508mins for the SA. While BA was the preservative which gives a stronger signal for detection. Hence, BA in the jam samples was analyzed and the results were used to compare with the UV/Vis Spectrophotometer method.

Table 21. Standard calibration of BA by external method which increasing the concentration of the standard such as 0.2x10-6 mol/ml, 0.3x10-6 mol/ml, 0.4x10-6 mol/ml, 0.5x10-6 mol/ml and 0.6x10-6 mol/ml.

Concentration (1x10-6 mol/ml)

Standard Calibration of BA (Area [µV.s] )

Average

Test 1

Test 2

0.2

16119.56

16457.79

16288.68

0.3

28902.98

29843.22

29373.10

0.4

44485.60

45635.70

45060.65

0.5

144400.00

144677.10

144538.55

0.6

187447.30

177400.81

182424.06

Table 22. Area [µV.s] of peak obtained from the samples (jam) at the retention of 7.0 to 8.0 mins

Samples

Area [µV.s]

Orange

419274.22

Blackberry

367776.79

Blueberry

145613.52

Strawberry

79856.18

Figure 17. Standard calibration line that has been draw above used to determine the unknown concentration of the samples (jam).

Table 23. Amount of benzoate acid analyzed in the jam sample respectively by using HPLC

Samples (jam)

mg g-1

Orange

2.344

Blackberry

1.099

Blueberry

1.282

Strawberry

0.982

4.3 Comparison method of determination of BA with HPLC and UV/vis spectrophotometer.

Table 24. Comparison between the UV/vis method and HPLC method in determination of BA. Percentage of variation is calculated by [(A-B)/(A+B)] x 100%.

Samples (1 gram)

BA detection by UV/vis (mg/g) [A]

BA determination by HPLC(mg/g) [B]

Varies (A-B)

Percentage of different (%)

Orange

2.477

2.344

±0.133

2.758

Black berry

0.977

1.099

±0.122

5.876

Blue berry

1.337

1.282

±0.055

2.100

Strawberry

0.651

0.982

±0.331

20.269

Average varies

7.751%

According to the result obtained by the UV/vis method and the HPLC method, the difference between two methods obtained result 7.751%. Some of the reason may be suspected due to the problem in the sample extraction and preparation. Besides that, background interference would also been suspected to be another problem that affecting the result in the analysis, which substance within having absorption wavelength in a range between 300nm – 200nm in the detection for the UV/Vis spectrophotometer.

BA recommended dose is 0.2-1.5 mg g-1 in food which is maximum allowable and the maximum concentration report of SA for food preservative purpose was in the range of 1.0 and 2.0 mg g -1. (Banerjee et al., 2004)

Therefore, the addition of the concentration of preservative such as BA and SA are applied in the jam samples that have been analyzed the BA content for orange: 2.477 mg/g, blueberry: 0.977 mg/g blackberry: 1.337 mg/g and strawberry: 0.651 mg/g and the SA content for orange: 0.018 mg/g, blueberry: 0.052 mg/g, blackberry: 0.003 mg/g and strawberry: 0.054 mg/g. the BA content reported for the orange jam has over the permissible level which is 1.5 mg/g. These may cause dangerous to the consumer safety. Excess the safety level of BA intake for the human body most probability getting stomach irritating or aching, asthma, or inhalation symptom.

Besides that, benzoic acid is also has carcinogen property, higher intake level of BA may cause cancer disease throughout the body due to BA is a very penetrating molecule that can be absorbed from the stomach and sending this molecule by the bloodstream and affecting the other cells or organs even though some of the BA molecule may be successfully excreted by the body metabolism. There were some problem in the analysis is that the effect of concentration, temperature and the pH value can affect the experiment result from the detection curve. The concentration of the sample have diluted to 200 ml in a volumetric flask in order to obtain the absorption band with zero to one. .

Besides the concentration of the samples, the temperature and the pH of the solution were also may affecting the curves obtained. This is because higher the temperature or lower the pH, the sugar which contained inside the sample solution may cause breaking the structure of the sugar especially D-fructose which can convert to 5-(hydroxymethyl)-2-furaldehyde (Smock et al., 1948), this would like to absorb more light from the hollow charter lamp inside the UV/Vis spectrophotometer (Hitachi 2000). Other than that, the solvent which has used was methanol must be for the HPLC grade which is 100 percent pure. This is because from the failure experiment in obtaining the result of the absorption peaks, the impure methanol would like to affect the absorption bands as well as the location curves at the wavelength which is 206 nm - 230 nm for BA and 240 nm -260 nm for SA

Base on UV spectrophotometer method, impurity was minimized following the separation procedure. Colorless of the benzoic acid and sorbic acid are able to be detected by using UV spectrophotometer due to they are present of conjugated double bond which helps to absorb light from the lamp source. However, HPLC are highly recommended to be used to for preservative detection. This is because the column for HPLC are able to separate the component present in the testing sample base on their retention time, this may actually time saving compare to the UV method which separation and extraction procedure are needed to be carried out before analyze using UV-spectrophotometer. This is because jam are present of sugar, flavor, and other coloring food additive besides the benzoic acid or sorbic acid. Fortunately, benzoic acid and sorbic acid are present of carboxylic acid group which is –COOH which is acting as hydrophilic behaviors that enhance the effectiveness in extraction.

5.0 CONCLUSION

The HPLC method us better than the method if UV/Vis spectrophotometer in determine the preservative from the food samples. HPLC more realizable because it can be used to separate chemical substance present inside the extracted samples and determine the specific molecule such as BA base on the retention time. The preservatives applied in the orange jam which is the production from the company A was analyzed shown excess the legislation level which is over 2.0mg/g. While, the preservatives applied in blackberry, blueberry and strawberry were analyzed which at the safety level for the consumer. This is important to test the concentration of preservative either BA or SA used for food preservation. The excess doses of preservative added product may not secure healthy of the consumer. Companies that did not fulfill of permissible usage amount of preservative can be sued and summoned.