Measurement of caffeine in soft drinks using HPLC PDF

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Abstract Caffeine is common chemical that can be found in various beverages like coke, tea, etc. In this experiment, different concentrated standard caffeine solutions were prepared to analyze the concentration of caffeine present in different beverages. The HPLC data obtained for standard caffeine solution was unsatisfactory to establish the standard curve so HPLC data from file V were used. The concentration of caffeine was highest for red bull followed by coke and tea.

Introduction Caffeine has chemical formula of C8H10N4O2 which can be found in coffee, coke, tea, etc. Caffeine has medicinal effect such as to improve metal alertness and stimulant among athletics. Also, most commonly known effect of caffeine is preventing sleep. Due to these beneficial effects, some beverages like Red bull™ and Monster™ have higher concentration of caffeine compared to common source, coffee to increase the beneficial effects of caffeine. In this experiment, high performance liquid chromatography (HPLC) was used to analysis concentration of caffeine present in different beverages such as green tea, coffee, Coca-Cola™ and red bull™ by using standard caffeine solution. HPLC can separate identify and quantify the components present in a mixture which is soluble in solvent, mobile phase. In this experiment, methanol was used as solvent since caffeine is soluble in methanol to quantify the caffeine present in different beverages.

Materials required 5ml of Coca-Cola™, 3ml of red bull™, 0.5g of green tea, 5ml of coffee, 0.01g of caffeine, ionized water, methanol, injection filter

Experiment no. 5

Procedure Preparation of standard solution of caffeine 0.01g of caffeine was added into 100ml volumetric flask and ionized water was added up till 100ml mark in the flask to make concentrated caffeine solution. The flask was shaken well and 4 different concentration of caffeine solution was prepared in 10ml volumetric flask. Below are 4 different concentration of caffeine solution, A) 2ml of caffeine solution with 8ml of ionized water B) 4ml of caffeine solution with 6ml of ionized water C) 6ml of caffeine solution with 4ml of ionized water D) 8ml of caffeine solution with 2ml of ionized water 1ml of four solutions of A, B, C and D were added into 4 different flat amber bottle using syringe and injection filter. The flat amber bottles were taken for HPLC analysis. Preparation of solution of Coca-Cola™ 5ml of commercial Coca-Cola™ was added into 10ml volumetric flask and ionized water was added until it filled 10ml mark in the flask. 1ml of Coca-Cola™ solution was added into flat amber bottle using syringe and injection filter. The flat amber bottle was taken for HPLC analysis. Preparation of solution of Red Bull™ 3ml of commercial Red Bull™ was added into 10ml volumetric flask and ionized water was added until it filled 10ml mark in the flask. 1ml of Red Bull™ solution was added into flat amber bottle using syringe and injection filter. The flat amber bottle was taken for HPLC analysis. Preparation of solution of coffee 5ml of prepared coffee was added into 10ml volumetric flask and ionized water was added until it filled 10ml mark in the flask. 1ml of coffee solution was added into flat amber bottle using syringe and injection filter. The flat amber bottle was taken for HPLC analysis.

Experiment no. 5

Preparation of solution of tea 0.53g of tea was added to 30ml of ionized water and was boiled for 20minutes. Then, the solution was filtered using funnel. 5ml of filtered tea solution was added into 10ml volumetric flask and ionized water was added until it filled 10ml mark in the flask. 1ml of tea solution was added into flat amber bottle using syringe and injection filter. The flat amber bottle was taken for HPLC analysis.

Results The standard caffeine solutions that were prepared couldn’t be analyzed by the HPLC machine. Failure of getting standard curve from standard caffeine solution resulted into failure of analyzing concentration of caffeine present in different beverages even though, HPLC data for different beverages were satisfactory. Therefore, the data provided was used to demonstrate how the calculation of concentration of caffeine in different beverages are calculated using standard curve obtained from standard caffeine solutions. In this experiment, data from V file were used because it gave the most linear standard curve for standard caffeine solutions.

Table 1. Data for different concentration of standard caffeine solutions for V

Concent

Retention time (min)

Area (mAU∙s)

ration (μg∙ml-1)

Trial 1

Trial 2

Trial 3

Average

Trial 1

Trial 2

Trial 3

Average

20

3.238

3.239

3.239

3.239

116.47987

115.94524

115.59039

116.0052

40

3.241

3.234

3.237

3.237

231.65169

231.68469

231.78845

231.7083

60

3.233

3.232

3.233

3.233

351.15976

350.94308

350.78421

350.9624

80

3.233

3.234

3.230

3.232

465.72705

465.58508

466.30908

465.8737

From table 1, we can know that the retention time of caffeine is ≈ 3.23 minutes .

Experiment no. 5

Sample calculation of average retention time of caffeine at 20 μg∙ml-1,

average result= ¿

x1 +x 2+...+ xn n

3.238 + 3.239 + 3.239 3

¿ 3.239 minutes

Sample calculation of average area of caffeine at 20 μg∙ml-1,

average result= ¿

x1 +x 2+...+ xn n

116.47987 +115.94524 + 115.59039 3

¿ 116. 0052 mAU ∙ s

Table 2. Percentage relative standard deviation (RSD) of standard caffeine solutions for V

Concentration (μg∙ml-1) 20 40 60 80

% RSD of retention time 0.02 0.11 0.02 0.07

% RSD for area 0.39 0.03 0.05 0.08

Sample calculation of % relative standard deviation (RSD) of retention time at 20 μg∙ml-1,

% RSD=

standard deviation (S) ×100 acverageresult

Where average result is,

average result= ¿

x1 +x 2+...+ xn n

3.238 + 3.239 + 3.239 3

¿ 3.239 minutes

Experiment no. 5

Where standard deviation is,

S=

√

2

2

2

(x 1−average result ) +(x 2−average result ) +...+( xn −average result ) n−1

√

2

2

2

(3.238−3.239) +( 3.239−3.239 ) +( 3.239−3.239 ) ¿ 3−1 ¿ 7.07 ×10−4 Therefore % RSD is,

% RSD=

¿

standard deviation (S) ×100 acverageresult

7.07 × 10−4 ×100 3.239

¿ 0.02 %

From table 1, we can draw average concentration versus average area curve Graph 1. Average concentration versus average area curve for standard caffeine solutions.

Average area (mAU∙s)

Average concentration VS Average area 500 450 400 350 300 250 200 150 100 50 0 10

f(x) = 5.84 x − 1.08 R² = 1

20

30

40

50

60

70

80

90

Average concentration (μg∙ml-1)

Equation of line for graph 1. is as follows,

y =5.8443 x −1.0776(equation1) Where y is area and x is concertation of caffeine in the solution

Experiment no. 5

Table 3. Retention time of caffeine, area and average concentration in coke, tea and red bull

Retention time of caffeine (min) Trial Trial

Trial 1

2

3

Averag

Trial

e

1

Area

Average

(mAU∙s) Trial Trial

concentratio

2

Average

3

n of caffeine present

Coke

3.24

3.23

3.23

3.238

314.1561

312.8931

312.9916

313.347

(μg∙ml-1) 107.60

Redbul

0 3.23

5 3.23

8 3.23

3.233

6 419.7167

0 419.0835

1 418.8038

0 419.201

239.71

l Tea

1 3.23

7 3.23

2 3.23

3.235

1 561.8248

6 558.0062

0 557.4587

4 559.096

106.45

5

7

3

9

3

4

6

We can rearrange equation 1, as the following below to determine the concentration of caffeine present in different beverages.

y =5.8443 x −1.0776 x=

y+1.0776 5.8443

Where y is area and x is concertation of caffeine in the solution Calculation of average concentration of caffeine present in coke,

x= ¿

y+1.0776 5.8443

313. 3470 +1.0776 5.8443 −1

¿ 53.80 μ g ∙ ml

Since coke solution was diluted from 5ml to 10ml, original concentration of caffeine in coke is,

c 1 v 1= c 2 v 2

c 1=

¿

c2 v2 v1

53.80 × 10 5 −1

¿ 107.60 μ g ∙ ml

Experiment no. 5

Calculation of average concentration of caffeine present in red bull,

x= ¿

y+1.0776 5.8443

419.2014 +1.0776 5.8443 −1

¿ 71.91 μ g ∙ ml

Since red bull solution was diluted from 3ml to 10ml, original concentration of caffeine in red bull is,

c 1 v 1= c 2 v 2

c 1=

¿

c2 v2 v1

71.91 × 10 3 −1

¿ 239.71 μ g ∙ ml

Calculation of average concentration of caffeine present in tea,

x= ¿

y+1.0776 5.8443

559. 0966 +1.0776 5.8443

¿ 95.85 μ g ∙ ml−1 The amount of tea used and the volume of tea solution used in this experiment was different with that from lab manual. In lab manual, 0.2g of tea was used to extract 90ml of tea solution by boiling 30ml of distilled water with 0.2g of tea three times and collecting the tea solution. It was then diluted in 100ml of volumetric flask. Therefore, the calculation below was done according to the procedure in lab manual. Since tea solution was diluted from 90ml to 100ml, original concentration of caffeine in tea is,

c 1 v 1= c 2 v 2

Experiment no. 5

c 1=

¿

c2 v2 v1

95.85 × 100 90

¿ 106.45 μ g ∙ ml−1 To determine the concentration of caffeine present in tea in μg∙g-1, following calculation can be done, Since there was 90ml of tea solution used, total amount of caffeine can be calculated in 90ml of tea solution by,

Amount of caffeine=concentration× volume ¿ 106.45 × 90

¿ 9580.5 μ g Since 0.2g of tea leaves were used,

¿

9580.5 0.2 −1

−1

¿ 47902.5 μ g ∙ g ≈ 47.90 m g ∙ g

Table 4. Percentage relative standard deviation (RSD) of different beverages for V

Name of beverage

% RSD of retention time

% RSD for area

Coke Red bull Tea

of caffeine 0.08 0.10 0.06

0.22 0.11 0.43

Sample calculation of % relative standard deviation (RSD) of retention time of caffeine for coke,

% RSD=

standard deviation (S) ×100 acverageresult

Where average result is,

average result=

x1 +x 2+...+ xn n

Experiment no. 5

¿

3.240 + 3.235 + 3.238 3

¿ 3.238 minutes Where standard deviation is,

√

(x 1−average result )2+( x 2−average result)2 +...+( xn −average result)2 S= n−1

√

(3.240−3.238)2 +( 3.235 −3.238 )2+( 3.238−3.238 )2 ¿ 3−1 −3

¿ 2.55 ×10

Therefore % RSD is,

% RSD=

¿

standard deviation (S) ×100 acverageresult

2.55 ×10−3 ×100 3.238

¿ 0.08 %

Analysis and discussion of the result In this experiment, even though the HPLC data that were obtained for different beverages were satisfactory, HPLC data for standard caffeine solutions were not good for the analysis. Unavailability of standard curve for standard caffeine solutions made analysis of concentration of caffeine present in different beverages not possible. In addition, from the obtained HPLC data, the absorbance by caffeine was different when different wavelengths/signals were used. Therefore, using the standard curve of standard caffeine solution from file V was meaningless because the absorbance by caffeine will be different leading to different concentration. Wavelength used in file V was different with that of one used in this experiment. Thus, in this experiment, the HPLC data provided, file V were used to determine the concentration of caffeine present in different beverages which were coke, red bull and tea. Among different files such as V, Z and ZXX, the results from file V were taken because this gave most linear curve for the standard caffeine solutions. This is important because more linear the curve it is less error it will have. From the standard curve for standard caffeine solution, value of R 2 was 1 which means variation in y value is 100% by variation in x values. In addition, % relative standard

Experiment no. 5

deviation of the data obtained had less than 0.5% which is very small. From file V, the retention time of caffeine was around 3.23 minutes. And this was used to determine the peak of caffeine in different beverages. The graph 1. was plotted using table 1. And using the equation of line from graph 1, the average concentration of caffeine in different beverages were determined. According to the caffeine chart from center for science in public interest, coke contains 95 μg∙ml-1 and red bull contains 322 μg∙ml-1. However, from the result, coke had similar concentration with theoretical one by actual value of 107.60 μg∙ml-1 but red bull had 239.71 μg∙ml-1 which was lower than that of theoretical value. Concentration of tea in μg∙ml-1 was 106.45 μg∙ml-1 and in μg∙g-1 was 47902.5 μg∙g-1. Concentration of caffeine in tea was similar in concentration with that of coke. The list of factors which contribute to the error of measurements are,  Irregular pressure Applying irregular pressure would result into different flow rate of mobile phase. This will affect the retention time.  Baseline irregularity Electrical interferences, detector faults, solvent impurities, column contamination, air bubbles, cause irregularity in baseline. If the baseline is irregular, the height and area of the peak will be inaccurate.  Preparation of mixed solvents or system leaks and build-up of contaminants Build-up of contaminants and poor preparation of solvents cause change in retention time.  Temperature and injection volume of sample This would change the retention time because HPLC is sensitive so if too much sample is injected, then some of samples might not separate properly.

Conclusion This experiment was quantitative analysis of caffeine present in different beverages. The experiment that was performed didn’t have satisfactory result that could be used to determine the concentration of caffeine in different beverages. From the data provided in file V, red bull had highest concentration of caffeine (239.71 μg∙ml-1) followed by coke (107.60 μg∙ml-1) and tea (106.45 μg∙ml-1, 47902.5 μg∙g-1). It was observed that concentration of caffeine in coke and tea was very similar. This is because concentration of caffeine

Experiment no. 5

present in tea varies according to different types of tea used. Also, concentration of caffeine in a tea solution varies for how long the tea was boiled for. The retention time of caffeine was determined by 3.23 minutes and according to this range of retention time, the caffeine peak in HPLC data of different beverages were selected. Each component has different retention time if same environment such as temperature, stationary phase, mobile phase, etc. are used. Therefore, this would allow qualitative analysis of different components in a mixture. However, there must be references for different components to determine which peaks belong to which components. It is also possible for different components to have same or similar retention time and this would result into single peak when combined. Hidden peak or peaks in the same or similar retention time resulted into higher concentration at retention time.

References WebMD Online, https://www.webmd.com/vitamins/ai/ingredientmono-979/caffeine, (accessed November 2019). Chemistry LibreTexts Online, https://chem.libretexts.org/Bookshelves/Analytical_Chemistry/Supplemental_Modules_(Analytical_C hemistry)/Instrumental_Analysis/Chromatography/High_Performance_Liquid_Chromatography, (accessed November 2019). Center for science in the public interest Online, https://cspinet.org/eating-healthy/ingredients-ofconcern/caffeine-chart, (accessed November 2019)....