What Do We Need, What Do We Do.

Materials:

1.    Decaffeinated coffee from different companies

2.    Pure Caffeine 

3.    Mobile phase (80.0% Water and 20.0% Methanol, buffered to pH 3.50) 

4.    0.2-mm syringe type disposable nylon filter cartridge for standards and samples

5.    100-ml volumetric flasks (dry)

6.    10-ml volumetric flasks (dry)

7.    5-ml bulb pipette  

8.    Sample vials, 1.5-ml   

9.    HPLC column 

 
Method:

Preparation of Mobile phase:

1. The mobile phase is a mixture of 20.0% Methanol and 80.0% Water, buffered to pH 3.50. 
2. Measure 200ml of Methanol 
3. Measure 800ml of Distilled water
4. Mix the two mixture 
5. Buffer solution to pH 3.50

6. Filter the solvent with Solvent Filtration Apparatus 

7. Transfer to solvent bottle

Preparation of Caffeine Standards:

1. Prepare 10 ml of caffeine standards ranging from 1 to 140mg/L by dissolving appropriate amount 

    of caffeine in mobile phase.

2. Mix properly

3. Filter these solutions with disposable 0.2-mm nylon filter cartridges into labelled sample vials.

Sample Solution Preparation:

1. Dilute 5.0 ml of each sample to 10 ml with mobile phase. (If sample does not have a high level of

    caffeine present, no dilution is required)

2. Mix well.

3. Take note of dilution factor for each sample (if any)

3. Filter this solution with a disposable 0.2-mm nylon filter cartridge into a sample vial. 

4. Label this vial as (Sample Name) Solution. 

Sample Analysis

1. Set up the HPLC system with the following conditions:

    Mobile phase = 20:80 methanol:water

    Flow rate = 1.5mL/min

    Stationary phase = C-18

    Absorbance detection = 254 nm 

2. Place the Standards and the Samples onto the rack in the HPLC autosampler

3. Program autosampler to inject 20microliter of the standards and samples in separate runs into
    HPLC column

4. Determine peak area signals of standard solutions and sample solutions 

5. Using the standard's values, plot a calibration graph of peak area signal against concentration

6. Determine concentration of caffeine using the calibration plot