Determination Of Caffeine In Beverages By High Performance Liquid Chromatography (hplc).docx

TITLE Determination

of

Caffeine

in

Beverages

by

High

Performances

Liquid

Chromatography (HPLC)

OBJECTIVE 1. To determine amount of caffeine in sample of beverages by using HPLC.

INTRODUCTION Caffeine is a bitter, white crystalline purine, a methylxanthine alkaloid, and is chemically related to the adenine and guanine bases of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). It is found in the seeds, nuts, or leaves of a number of plants native to South America and East Asia and helps to protect them against predator insects and to prevent germination of nearby seeds. The most well known source of caffeine is the coffee bean, a misnomer for the seed

of Coffea plants.

Beverages containing caffeine are ingested to relieve or prevent drowsiness and to improve performance. To make these drinks, caffeine is extracted by steeping the plant product in water, a process called infusion. Caffeine-containing drinks, such as coffee, tea, and cola, are very popular.

Caffeine can have both positive and negative health effects. It can treat and prevent the premature infant

breathing disorders bronchopulmonary dysplasia of

maturity and apnea

prematurity. Caffeine citrate is on the WHO Model List

of

of Essential Medicines. It may confer a modest protective effect against some diseases, including Parkinson's disease. Some people experience insomnia or sleep disruption if they consume caffeine, especially during the evening hours, but others show little disturbance. Evidence of a risk during pregnancy is equivocal; some authorities recommend that pregnant women limit consumption to the equivalent of two cups of coffee per day or less. Caffeine can produce a mild form of drug dependence – associated

with withdrawal symptoms such as sleepiness, headache, and irritability

– when an individual stops using caffeine after repeated daily intake Tolerance to the

autonomic effects of increased blood pressure and heart rate, and increased urine output, develops with chronic use. High-performance liquid chromatography (HPLC) is developed to increase speed and efficiency in liquid chromatography. Decreasing the size of the solid support material increased efficiency.

Figure 1 : Structure of caffein

MATERIALS AND PROCEDURES Chemicals and Materials Caffeine Magnesium oxide Methanol Beverages sample

Apparatus HPLC with UV detector and isocratic system Biker Volumetric flask Syringe

Part A : Preparations of standard solutions 1. 100 ml, 1000 ppm of caffeine stock solution prepared. 2. The stock solution diluted to 100 ml of desire concentration of 20 ppm, 40 ppm, 60 ppm, 80 ppm and 100 ppm caffeiene with distilled water.

Part B : Sample preparation 1. 0.5 g beverages sample and 5g MgO mixed in 200 ml water in beaker and stirred for 20 minutes at 90C in water bath. The solution cooled to room temperature. 2. The solution sample degased by placing it in sonicator for 5 minutes. 3. All the samples filtered through filter papers. 4. The filtered samples diluted 10 times prior for injection.

Part C : HPLC method 1. The sample loop cleaned by flushing 250 µL of mobile phase to clean loading passages. 2. The syringe refilled, wipe cleaned, reinsert and flushed in another 250 µL, making sure not to inject any air bubbles. 3. The syringe rinse with several aliquots of the sample to be injected. Before injected 100 µL, the syringe was checked to make sure no air bubbles. 4. The data recorded.

RESULTS AND DISCUSSION A. Standard Caffeine Sample Retention time of standard caffeine sample :

Table 4.1 : Peak area or peak height of standard caffeine sample Concentration of

Peak Area

Peak Height

20

2775.79688

179.60068

40

5991.54883

385.86853

60

8834.93457

564.23248

80

1.25160e4

774.64056

100

1.46333e4

895.24420

Caffeine (ppm)

B. HPLC Result

Types of Beverages

Retention Times of

Peak Area

Caffeine

Peak Height

Concentration

% of

of Caffeine

Caffeine

Present

in Sample

1.030

10.97531

1.68316

100

5.8785

1.218

9.11339

1.51511

100

4.8812

1.318

11.97022

1.76196

100

6.4114

1.509

10.85414

1.30932

100

5.8136

2.329

6.96240

1.09867

100

3.7291

6.028

136.82666

8.83598

100

73.2861

Tea Leaves

HPLC is chromatographic technique used to separate the components in a mixture, used mainly to identify each component, and to quantify each component. HPLC is considered as an instrumental technique of analytical chemistry. Separation is based on the analyte’s relative solubility between two liquid phases. The method involves a liquid sample being passed over a solid adsorbent material packed into a column using a flow of liquid solvent. Each analyte in the sample interacts slightly

differently with the adsorbent material, thus retarding the flow of the analyte. If the interaction is weak, the analyte’s flow off the column in a short amount of time, and if the interaction is strong, then the elution time is long.

In this experiment, reverse phase HPLC is used. Reversed phase HPLC (RP-HPLC) used for the separation of molecules based on their hydrophobic interaction between the solute molecule in the mobile phase and the immobilised hydrophobic ligand in the stationary phase. For reverse phase HPLC, the mobile phase is more polar than stationary phase. The process of chromatography starts by injecting a sample into the instrument. The mobile phase moves it through the column chromatography which is the stationary phase.

The mobile phase moves particles through the column while the stationary phase allows particles to remain in the column. On the other hand, the caffeine standards provide a frame of reference to which the data obtained from the soft drinks can be compared.

A calibration curve for peak area vs. concentration is made. One can

determine the amount of caffeine in the soft drinks by plotting data on this curve.

Lastly, the syringe have to be carefully rinsed before used to remove any contaminents from the syringe that basically can cause errors occured in the results. Thus, to make sure a certain a peak in the soda was caffeine and not another substance with a similar retention time The mobile phase needed to be change and inject the caffeine and then the sample. Both peaks should be affected similarly if they are both due to the presence of caffeine. CONCLUSION In conclusion, this experiment have determined the average retention time of standard solution of caffeine which is 2.239 minutes. Then, the caffeine average peak area in the tea leaves has identified which is 31.11702 of peak area while the average peak height is 2.70070. Next, the average percentage of caffeine in tea leaves sample is 16.6667%.

QUESTIONS 1. Briefly explain how HPLC is used as a separation technique. 

HPLC is chromatographic technique used to separate the components in a mixture, used mainly to identify each component, and to quantify each component. HPLC is considered as an instrumental technique of analytical chemistry. Separation is based on the analyte’s relative solubility between two liquid phases.



The method involves a liquid sample being passed over a solid adsorbent material packed into a column using a flow of liquid solvent. Each analyte in the sample interacts slightly differently with the adsorbent material, thus retarding the flow of the analyte. If the interaction is weak, the analyte’s flow off the column in a short amount of time, and if the interaction is strong, then the elution time is long.

2. What is the purpose of the mobile phase? Of the stationary phase? 

The mobile phase moves particles through the column while the stationary phase allows particles to remain in the column.

3. What is the purpose of the caffeine standards? 

The standards provide a frame of reference to which the data obtained from the soft drinks can be compared. A calibration curve for peak area vs. concentration is made. One can determine the amount of caffeine in the soft drinks by plotting data on this curve.

4. Why does the syringe have to be carefully rinsed before each use?



Contaminents must be removed from the syringe.

5. How could you be certain a peak in the soda was caffeine and not another substance with a similar retention time? 

Change the mobile phase and inject the caffeine and then the sample. Both peaks should be affected similarly if they are both due to the presence of caffeine.

REFERENCES Clark,

J.

(2016).

Paper

Chromatography.

Retrieved

from

https://www.chemguide.co.uk/analysis/chromatography/paper.html HPLC.

(n.d).

Retrieved

https://www.omicsonline.org/hplc-scholarly-open-access-journals.php

from