HPLC - determination of caffeine concentration in beverages by HPLC PDF

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determination of caffeine concentration in beverages by HPLC ...

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Hanna Thomson Chem421 HPLC TA: Patrick

CHEMISTRY 421

EXPERIMENT NAME: HPLC

STUDENT: HANNA THOMSON

EXPERIMENT PREFORMED ON: 3/3/2020

TA’S NAME: PATRICK FISHER

DATE OF SUBMISSION: 4/6/2020

DUE DATE OF EXPERIMENT: 4/7/2020

NAME OF LAB PARTNER: ADNAN ALHABISS

Hanna Thomson Chem421 HPLC TA: Patrick

High Performance Liquid Chromatography (HPLC) for Determination of Caffeine in Beverages

Abstract The purpose of this lab was to determine the amount of caffeine in popular tea and coke drinks using high performance liquid chromatography (Agilent 1260 HPLC) against a set of caffeine standards at varying concentrations measured with a UV detector and the Chem Station software controls, and preparing a set of calibration curves to determine the concentration of caffeine in the unknown solutions of coke and tea. Background High performance liquid chromatography is one of the most commonly used instruments for separation of molecules. It works similarly to gas chromatography in which a small amount of sample is inserted in to a moving stream of liquid also known as the mobile phase, and passed through a column containing the stationary phase. The mobile phase can compose of either molar or non-polar molecules depending on the molecule you are evaluating, as well as the stationary phase can be interchangeable between polar or non-polar coating particles. In this case, we are working with a C18 column which is composed of n-octadecane and silica, attracting non-polar molecules and allowing the polar molecules to elute out first. The mobile phase in this experiment consists of water and methanol in order to elute caffeine at certain times. Once the sample passes through the stationary phase, it is passed in to a detector, in this case a UV detector. The UV detector utilizes light to analyze samples by measuring the samples absorbance at different wavelengths [ CITATION HPL \l 1033 ]. The set up of the apparatus is shown below in figure 1 [ CITATION Edi20 \l 1033 ].

Hanna Thomson Chem421 HPLC TA: Patrick

Figure 1: HPLC apparatus set-up. Image obtained from https://laboratoryinfo.com/hplc/

Caffeine is a desired component of many teas, coffees, carbonated sodas, and over the counter pain relievers. It’s a central nervous system stimulant of the methylxanthine class, and works by inhibiting adenosine on it’s receptors which prevents the feeling of tiredness or drowsiness. Caffeine can also be used for various other things such as treating and preventing premature infant breathing disorders and treatment of Parkinson’s disease, however it could also cause sleep disruption and is at risk for dependency and can lead to mild withdrawal symptoms. Due to the molecular structure of caffeine being a purine and structurally similar to adenine and guanine, as well as containing ketones and nitrogen, caffeine is a polar molecule and will require the use of a non-polar stationary phase and a polar mobile phase in order to be eluted through the column first and analyzed by the UV detector. The molecular structure of caffeine is pictured below in figure 2.

Hanna Thomson Chem421 HPLC TA: Patrick

Figure 2: molecular structure of caffeine. Image obtained from https://en.wikipedia.org/wiki/Caffeine

According to the USDA (U.S Department of Agriculture) the average amount of caffeine in 1 fluid oz of coke or 30.7 g, is around 39 mg, and the average amount of tea in one serving (tea bag) 8 fluid oz or 238 g, is around 26 mg. So after preparing a set of calibration curves we should expect to calculate the average amount of caffeine to be of similar ratio respectively. Experimental Procedure Solvent A was prepared by making 500 mL’s of 20% methanol per volume by diluting 100 mL of HPLC grade methanol with ultra-purified water to 500 mL in a volumetric flask. A 125 mg/L caffeine standard was prepared by dissolving 0.0625 g of caffeine standard in the 500 mL of solvent A. Dilutions of caffeine at 100, 75, 50, and 25 mg/L were prepared by diluting 0.020 0.015, 0.010, and 0.005 mg of caffeine to 25 mL using solvent A in volumetric flasks. An unknown tea sample was prepared by boiling 400 mL of distilled water with a tea bag, taking 10 mL of tea sample after 1 minute, 5 minutes, and 10 minutes and taking 5 mL of the tea samples extracted and diluting them to 25 mL with solvent A using volumetric flasks. Unknown coke samples were prepared by transferring 10 mL of each sample of coke to 25 mL volumetric flasks and diluting to the mark with solvent A.

Hanna Thomson Chem421 HPLC TA: Patrick All standards and unknown samples were degassed using nitrogen gas, and filtered with 0.45 μm filters to transfer to plastic test tube containers. Each sample was analyzed 3 times using 20 μL of sample. Data and Calculations Table 1: Peak height and areas for 254 and 275 nm wavelengths of all the samples sample 25 mg/L std

50 mg/L std

75 mg/L std

100 mg/L std

trial

wavelengt h

peak height

ave peak height

1 2 3

254 254 254

18.4673 19.0955 18.75432

18.8 18.8 18.8

1 2 3

275 275 275

18.1035 18.6506 18.3257

18.4 18.4 18.4

1 2 3

254 254 254

46.92348 46.56432 47.01376

46.8 46.8 46.8

1 2 3

275 275 275

53.65432 53.12765 54.75216

53.8 53.8 53.8

1

254

89.98573

89.4

2

254

88.73254

89.4

1 2

275 275

1

254

2

254

1

275

2

275

119.2563 118.8921 135.8437 8 134.5478 2 291.8872 3 290.8932 1

119.1 119.1

135.3

std dev 0.31448 9

0.27515 2

0.23774 7

0.82882 1

0.88613 9

0.25752 8

0.91638 2

135.3 291.4 291.4

0.70287 8

peak area

ave peak area

11.61065 10.90776 13.03289

11.9 11.9 11.9

25.7805 24.18137 26.63713

25 25 25

25.14545 27.29235 22.18172

25 25 25

47.71021 50.26462 44.56924

48 48 48

39.08675 3 38.87644 2

39

71.57365 71.22807

71.4 71.4

52.6843

52.74

52.70234

52.74

91.87362

93

94.26931

93

39

std 1.08266 6

1.24644 8

2.56617 1

2.85272

0.14871 2

0.24436 2

0.01275 6

1.69400 9

Hanna Thomson Chem421 HPLC TA: Patrick 1 minute tea 5 minute tea 10 minute tea

coke sample

1 1

254 275

19.45281 21.98321

x x

13.14395 25.35421

x x

1 2

254 275

27.2498 28.27435

x x

17.56782 35.15398

x x

1 2

254 254

54.3297 52.8594

53.6 53.6

27.27563 28.43562

27.9 27.9

1 2

275 275

58.19078 55.1513

57 57

53.23649 53.89471

53.1 53.1

1

254

133.2

52.10983

52

2

254

132.298 133.4378 2 289.2984 3 290.3456 2

51.85432

52

92.87221

92.3

91.76342

92.3

1

275

2

275

1.03965 9 2.14923 7 0.80597 4

133.2 0.74047 5

289.8 289.8

Standard graphs:

Average Peak Height vs. Concentration at 275 nm Wavelength 350 300

peak height

250 200

f(x) = 3.54 x − 100.4 R² = 0.89

150 100 50 0 20

30

40

50

60

70

concentration (mg/L)

80

90

100

110

0.82023 7 0.46543 2 0.18067 3

0.78403 3

Hanna Thomson Chem421 HPLC TA: Patrick

Average Peak Height vs. Concentration at 254 nm wavelength 160 140

peak height

120 100

f(x) = 1.57 x − 25.45 R² = 0.99

80 60 40 20 0 20

30

40

50

60

70

80

90

100

110

concentration (mg/L)

peak area

Average Peak Area vs. Concentration at 275 nm Wavelength 100 90 80 70 60 50 40 30 20 10 0 20

f(x) = 0.91 x + 2.5 R² = 1

30

40

50

60

70

80

90

100

110

concentration (mg/L)

Average Peak Area vs. Concentration at 254 nm Wavelength 60

Peak Area

50 40

f(x) = 0.55 x − 1.97 R² = 1

30 20 10 0 20

30

40

50

60

70

concentration (mg/L)

80

90

100

110

Hanna Thomson Chem421 HPLC TA: Patrick Unknown Graphs

Peak Height Caffiene in Tea 10 Minutes vs. 254 nm Wavelength 60

peak height

50

f(x) = 3.85 x + 12.87 R² = 0.94

40 30 20 10 0 0

2

4

6

8

10

12

time (min)

Peak Height in Tea vs. Wavelength 275 nm 60

peak height

50

f(x) = 3.97 x + 14.59 R² = 0.92

40 30 20 10 0 0

2

4

6

8

10

Time (min)

Calculation of caffeine concentration in coke samples from peak height: At 254 nm: 133.2=1.5684x-25.45, x= 101.15 mg/L At 275 nm: 289.8=3.5372x-100.4, x=110.31 mg/L Calculation of caffeine concentration in coke samples from peak area: At 254 nm: 52=0.5461x-1.97, x=98.82 mg/L At 275 nm: 92.3=0.9096x+2.5, x=98.72 mg/L Calculation of caffeine concentration in tea sample from peak height: At 254 nm: 1min: 19.45=1.5684x-25.45, x=28.63 mg/L 5min: 27.24=1.5684x-25.45, x=33.65 mg/L

12

Hanna Thomson Chem421 HPLC TA: Patrick 10min: 53.6=1.5684x-25.45, x=50.40 mg/L At 275 nm: 1min: 21.98=3.5372x-100.4, x=34.59 mg/L 5min: 28.27=3.5372x-100.4, x=36.38 mg/L 10min: 57=3.5372x-100.4, x=44.49m mg/L Calculation of caffeine concentration in tea sample from peak height: At 254 nm: 1min: 13.14=0.5461x-1.97, x=27.67 mg/L 5min: 17.56=0.5461x-1.97, x=35.76 mg/L 10min: 27.9=0.5461x-1.97, x=54.69 mg/L At 275 nm: 1min: 25.35=0.9096x+2.5, x=25.12 mg/L 5min: 35.15=0.9096x+2.5, x=41.39 mg/L 10min: 53.1=0.9096x+2.5, x=55.63 mg/L T-Tests: Actual coke caffeine concentration vs. experimental concentration: μ= X +/- (t x S)/sqrt(n), μ= 10.4 + (2.92 x 1.0115) / sqrt (2) = 9.944 Peak area vs peak height coke caffeine concentration: (110.31-98.72)+2.92 (0.74052/2 + 0.7842/2)=12.978 Peak area at 254 vs 275 nm coke caffeine concentration: = 1.04513 Peak height at 254 vs 275 nm coke caffeine concentration: = 10.9098 Discussion The purpose of this lab was to determine the caffeine concentration in unknown samples of coke and tea using a set of standards with varying caffeine concentrations by using HPLC and a UV/Vis detector. The calibration curves chosen to determine the concentration of the unknowns were the peak area, and peak height calibration curves at 254 and 275 nm, because they showed a strong linear relationship between the standard concentrations and the peak area or peak height it was deemed a good fit in determining the concentration of the unknown coke and tea samples. The t-test results show there is a statistical difference between peak height and peak area of the caffeine concentration in coke because the t-obtained > t-95%. However there was not a statistical difference between the concentrations of the peak area at 254 and 275 nm because t-obtained < t-95%. There is a statistical difference between the peak height at 254 vs 275 nm since t-obtained > t-95%. Therefore, the most probably concentration of the unknown comes from the peak area at 254 or 275 nm.

Hanna Thomson Chem421 HPLC TA: Patrick There was only one coke sample analyzed during this experiment so determination of caffeine concentrations between diet and regular coke could not be determined. There should be a difference in intensity for peak heights or peak areas for caffeine at the different wavelengths of detection, because at different wavelengths of detection the UV absorbance will differ as well [ CITATION Sho \l 1033 ]. Questions 1: explain the rationale for using a reverse phase C18 column with the given mobile phase for determination of caffeine. -

We used a reverse phase C18 column for the determination of caffeine because we were interested in analyzing caffeine, a polar molecule, so we wanted a stationary phase that would attract non-polar molecules allowing the polar molecules such as caffeine for detection to elute out of the column first, and a polar mobile phase such as water and methanol to help carry the polar molecules through the column allowing them to elute.

2: we use two channel tunable detector in this experiment. How does this detector work? -

The two channel tunable detector allows us to measure absorption at multiple wavelengths, and are therefore more sensitive. We are able to measure absorption at the 254 and 275 nm wavelengths for detection of caffeine in various samples during this experiment to obtain more accurate results.

3: C18 reverse phase chromatography is used in this experiment. Describe another type of chromatography that can be used with HPLC and which kind of molecules the described chromatography would be appropriate for. -

Another type of chromatography that can be used with HPLC would be normal phase, such as silica gel which is very polar, attracting polar molecules to the stationary phase and allowing nonpolar molecules to elute our first for detection. This method is used for things such as phospholipids, when the non-polar hydrophobic chains of phospholipids are the desired component for analysis and the phosphate group attracts to the stationary phase.

References HPLC UV detector (UV/Visible HPLC detectors). (n.d.). Retrieved from biocompare: https://www.biocompare.com/Lab-Equipment/13036-HPLC-UV-Detector-UV-Visible-HPLCDetectors/ Shodex. (n.d.). Lesson 6: Detectors for HPLC. Retrieved from Shodex: https://www.shodexhplc.com/lessons/lesson-6-detectors-for-hplc/ Team, E. (2020, January 11). High Performance Liquid Chromatography (HPLC) : Principle, Types, Instrumentation and Applications. Retrieved from LaboratoryInfo: https://laboratoryinfo.com/hplc/ USDA. (2019, 4 1). Food Data Central. Retrieved from USDA Agricultural Research Service: https://fdc.nal.usda.gov/fdc-app.html#/food-details/175093/nutrients

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