HPLC lab - High performance liquid chromatography lab PDF

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High performance liquid chromatography lab...

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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 tea and caffeinated energy drink using high performance liquid chromatography against a set of caffeine sample with set standards at different concentrations, and preparing calibration curves to determine the concentration of caffeine in the unknown solutions of tea and pepsi.

Background: In many previous experiments, the main method of separation of molecules was gas chromatography, in which a small sample amount is put into a mobile phase and passed through a column that contains a stationary phase. In this experiment, high performance liquid chromatography was used. It works similarly to gas chromatography where the mobile phase can be composed of either polar or nonpolar molecules depending on the polarity of the molecule being evaluated. The stationary phase may also be made up of polar or nonpolar particles. To determine the caffeine in beverages, we used a C 18 column which contains n-octadecane and silica, this means that it attracts non-polar molecules and polar molecules travel through the column and elute out first. Water and methanol make up the mobile phase so that caffeine is eluted and is then passed onto the UV detector. The purpose of the UV detector is to analyze the sample by measuring the absorbance levels at different wavelengths. Attached below is the setup of apparatus used with HPLC and the UV detector.

Caffeine is a stimulant that is a large component in teas, coffees, energy drinks, as well as over the counter pain relievers. Caffeine stimulates the nervous system and prevents tiredness or drowsiness, as well as being used for treatment of parkinsons, and preventing premature infant breathing disorders. However, while it has benefits it can also cause sleep disruption and creates a risk for addiction. The molecular structure of caffeine contains a purine, ketones, and nitrogen which makes it a polar molecule thus requiring the use of a non polar stationary phase and a polar mobile phase to elute the caffeine first.

Experimental procedure: 1. Prepare solvent A by making 500 mL of 20% methanol per volume by diluting 100 mL of methanol with ultra-purified water to 500 mL in a volumetric flask 2. Prepare a 125 mg/L caffeine standard by dissolving caffeine standard in the 500 mL of solvent A 3. Prepare the caffeine dilutions by diluting 0.020,0.015,0.010, and 0.005 mg of caffeine to 25 mL using solvent A in volumetric flasks to get dilutions of caffeine at 100,75,50, and 25 mg/L. 4. A unknown tea sample was prepared by boiling 400 mL of distilled water with a tea bag and taking 10 mL of the tea sample after 1 minute, 5 minutes, and 10 minutes , and taking 5 mL of the extracted sample and diluting them to 25 mL with solvent A in a volumetric flask. 5. Unknown pepsi samples were prepared by transferring 10 mL of each sample to a 25 mL volumetric flask and diluting it to the mark with solvent A.

6. All of the standards and samples were degassed using nitrogen gas and filtered with μm filters to transfer to containers. 7. Each sample was analyzed three times using 20 μL of samples.

Data:

Calculation: Calculation of caffeine concentration in diet pepsi samples from peak height: At 254 nm: 758.76=1.0925x-0.1383, x=694.64 mg/L At 275 nm: 1251.315=1.0925x-0.1383, x=1145.49 mg/L Calculation of caffeine concentration in Cherry pepsi samples from peak height: At 254 nm: 808.89=1.0515x-0.0793, x=769.35 mg/L At 275 nm: 1371.79=1.0185x-0.0291, x=1346.90 mg/L Calculation of caffeine concentration in diet pepsi samples from peak area: At 254 nm: 4505.23=1.0896x-0.134, x=4134.87 mg/L At 275 nm: 7041.77=1.0214x-0.0305, x=6894.26 mg/L Calculation of caffeine concentration in Cherry pepsi samples from peak area: At 254 nm: 4747.005=1.0579x-0.089, x=4487.28 mg/L At 275 nm: 7734.36=1.0436x-0.0672, x=7411.29 mg/L Calculation of caffeine concentration in tea sample from peak height: At 254 nm: 1 min: 676.51=6843.8x-9710, x= 1.517 mg/L 5 min: 908.3466667=-14045x+22252, x= 1.519 mg/L 10 min: 1020.646667= -41240x+63650, x=1.518 mg/L At 275 nm: 1 min: 1422.693333=17825x-25648, x=1.518 mg/L 5 min: 1919.686667=-30474x+48261, x=1.521 mg/L 10 min: 2081.85= -72883x+112839, x=1.520 mg/L Calculation of caffeine concentration in tea sample from peak area: At 254 nm: 1 min: 4263.88=-22358x+38195, x=1.518 mg/L 5 min: 5686.72=-22198x+39420, x=1.519 mg/L 10 min: 1.22E+04=-435714x+674307, x=1.520 mg/L At 275 nm: 1 min: 8190.906667=100847x-144963, x=1.518 mg/L 5 min: 1.12E+04=-128571x+206714, x=1.521 mg/L 10 min: 1.22E+04=-435714x+674307, x=1.519 mg/L Best calibration curve: Avg peak height vs concentration at 254 nm based on highest R 2 value. 1 min: 4263.88=29.593x-316.99, x=154.79 mg/L 5 min: 5686.72=29.593x-316.99, x=202.87 mg/L 10 min: 1.22E+04= 29.593x-316.99, x=422.97 mg/L

For questions 3-7, t-tests were not able to be conducted due to the lack of data. A second replicate is needed in order to find the standard deviations between the two samples to compare with the actual pepsi caffeine concentrations for number 4. The same situation applies to 5-7 because the data given does not provide if the concentrations have equal variance.

Discussion: Final table: Sample

Trial

Wavelength

Concentration

Diet pepsi

Peak height

254

694.64

Diet pepsi

Peak height

275

1145.49

Diet pepsi

Area

254

4134.87

Diet pepsi

Area

275

6894.26

Cherry pepsi

Peak height

254

769.35

Cherry pepsi

Peak height

275

1346.9

Cherry pepsi

Area

254

4487.28

Cherry pepsi

Area

275

7411.29

Tea (1 min)

Peak height

254

1.517

Tea (5 min)

Peak height

254

1.519

Tea (10 min)

Peak height

254

1.518

Tea (1 min)

Peak height

275

1.518

Tea (5 min)

Peak height

275

1.521

Tea (10 min)

Peak height

275

1.520

Tea (1 min)

Peak area

254

1.518

Tea (5 min)

Peak area

254

1.519

Tea (10 min)

Peak area

254

1.520

Tea (1 min)

Peak area

275

1.518

Tea (5 min)

Peak area

275

1.521

Tea (10 min)

Peak area

275

1.519

The calibration curves that were chosen to determine the concentration of the unknowns was the average peak height versus concentration at 254 nm wavelength, because it had a strong linear relationship and the R2 value was the largest. Between the standard concentration and the peak area it was a good fit in determining the concentration of the unknown pepsi samples. T-tests were not able to be performed on this set of data because only one set of data was given, there must be two sets of data in order to find the statistical difference between the concentrations of the peak areas. There was not much a difference between the concentrations of caffeine between cherry pepsi and diet pepsi. I did not expect the caffeine concentrations to change between these drinks because the main difference in them is sugar levels. There was not much difference between the peak heights or areas, if there was a difference the UV absorbance would differ as well. This shows that the caffeine concentration between the two drinks does not vary. The intensities for peak heights and peak areas for caffeine should be different at different wavelengths because the different wavelengths cause different UV absorbance levels. (Intro. to Signal Processing:Integration and Peak Area Measurement)

Questions: 1. Explain the rationale for using a reverse phase C18 column with the given mobile phase for the determination of caffeine. a. The reverse phase C18 column was used for the determination of caffeine because the end goal was to analyze caffeine which is a polar molecule. The stationary phase attracts nonpolar molecules which allows polar molecules (caffeine) to elute from the column first. The polar mobile phase with water and methanol allowed the polar molecules to be carried through the column and elute. 2. We use two channel tunable detectors in this experiment. How does the detector work? a. The detector allows the measurement of absorption at multiple wavelengths. This means that the detector is more sensitive to these wavelengths so it yields more accurate results. The absorption measured at 254 nm and 275 nm wavelengths for the detection of caffeine in several samples with the detector will 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. a. The other type of chromatography that can be used with HPLC is a normal phase such as

silica gel. This is a polar gel that will attract polar molecules to the stationary phase and it allows non-polar molecules to elute out the column first. Using a normal phase can be used to analyze phospholipids, the non-polar hydrophobic chains of phospholipids are analyzed and the phosphate group of the phospholipid is attracted to the stationary phase.

Citations: “Principle of HPLC (Liquid Chromatography).” Pharmaceutical Guidelines, www.pharmaguideline.com/2013/07/principle-of-hplc-liquid-chromatography.html. Intro. to Signal Processing:Integration and Peak Area Measurement, terpconnect.umd.edu/~toh/spectrum/Integration.html. Advances in Chromatography and Lab Automation, “Chromatography Review, SpectraPhysics, June 1978. L.C. Column Report, “Nonaqueous Reversed- Phased Chromatography,” DuPont Instruments, 1978. Altex High-Performance Chromatography Catalogue, Altex Instruments, 1978....