Caffeine Content Determination with HPLC

Starting the day without a cup of coffee is unthinkable for many people. Coffee has become a global commodity not only because of the taste, but also because of the stimulating effect of caffeine. In addition to caffeine, coffee contains many other ingredients such as hydroxycinnamic acid and chlorogenic acid. However, due to the roasting process and other structural changes in the natural substances, it is difficult to accurately identify all the ingredients.

Determination of caffeine content even in small quantities

Several chromatographic techniques can be used to determine caffeine content. These techniques must ensure that even very small quantities, such as those found in decaffeinated coffee, are detected with all the other ingredients. This is performed with a technique that incorporates a diverter valve and a guard column using a mixed-mode phase.

Example: Caffeine Determination with Primesep Column

The amount of caffeine can be determined using a Primesep SB column, which consists of a hydrophobic chain with basic functional groups as an anion exchanger. This method shows good selectivity and resolution with a caffeine retention time of 12.5 minutes. Using an isocratic method or gradient elution system may result in very long run times, with the last components only eluted after about an hour. In contrast, results obtained with RP columns without a diverter valve exhibit prolonged retention times and minimal resolution and selectivity.

Sample preparation for shorter throughput times

To avoid long throughput times, it is essential to implement an optimal sample preparation method. This can be achieved through either solid phase extraction (SPE) or the use of a diverter valve, as previously described. The guard column is integrated into the diverter valve, eliminating the need for it to be placed directly in front of the main column. Once caffeine reaches the separation column after sample injection, the valve can be switched to reverse the flow of the remaining substances (backflush) to prevent them from sticking to the separation column. To facilitate the switch, the guard column is placed behind the detection unit (UV detector), which requires a certain pressure stability. The detector cell of the Knauer Smartline UV detector utilized in this method has a pressure resistance of up to 400 bar.

Determining Retention Times

It is crucial to accurately determine retention times to ensure that caffeine is eluted on the separation column while other components are flushed back to the guard column. This allows for the caffeine content to be determined promptly, and late elutes can be disregarded.