# Computer Percolation Models for Espresso Coffee: State of the Art, Results and Future Perspectives
## 3 Results and Discussion
### Results of Chemical Analyses
#### 3.1.2 Bioactive Compounds Analysis by HPLC-DAD: Results  
The analysis of nine bioactive compounds such as alkaloids (caffeine and trigonelline), chlorogenic acids (3,5-dicaffeoylquinic acid--3,5-dicCQA, 3-caffeoylquinic acid--3-CQA and 5-caffeoylquinic acid--5-CQA), phenolic acids (ferulic acid) and organic acids (acetic, citric and tartaric acid) was performed by HPLC-DAD instrument in both EC and R&G coffee. Before investigation of coffee samples, the analytical method was validated by studying linearity, limit of detection (LOD), limit of quantification (LOQ) and repeatability. Linearity was evaluated by injecting seven different concentrations of the target analytes then plotting and calculating calibration curves with the respective determination coefficients (R2). All target compounds showed good linearity because the R2 was higher than or equal to 0.9953. LODs and LOQs were calculated by injecting low concentrations of reference standards and the concentration with the signal-to-noise ratio (SN) of 10 was assigned to LOQ while that with SN of 3 to LOD. LODs ranged from 0.09 to 8.5 mg/L (L denotes litre) while LOQs were 0.3-28 mg/L for all analytes. Repeatability has been calculated by injection of different standard concentrations five times in the same days (intra-day repeatability) and over three consecutive days (inter-day repeatability). All the repeatability measurements were expressed as Relative Standard Deviations (% RSD). The intra-day repeatability ranged from 2.1 to 4.3% while the inter-day repeatability was 3.6-6.7% for all target compounds. The content of bioactive compounds in EC samples prepared by varying three variables is presented in Tables 4 and 5. The average contents of caffeine, chlorogenic acids, ferulic acid, organic acids and trigonelline are 5.18, 8.35, 0.28, 24.77 and 3.39 g/L which correspond to 103.5, 166.9, 5.7, 495.3 and 67.8 mg per cup of Arabica; while in Robusta samples the average contents of the same compounds are 7.48, 6.09, 0.40, 32.33 and 2.74 g/L which coincide to 149.6, 121.8, 8.0, 646.7 and 54.8 mg per cup. Similar levels are reported in the literature [8,20]. For instance, in [8], in ECs prepared by changing perforated disk height, filter basket design and amount of R&G coffee, caffeine and 5-CQA levels go from 3.2 to 5.7 g/L and from 1.8 to 5.0 g/L for Arabica, while from 4.8 to 12.1 g/L and 2.1-6.1 g/L for Robusta. In [20], which analysed 20 commercial coffee samples, the caffeine amount goes from 116.9 to 199.7 mg per cup, while trigonelline from 28.2 to 65.1 mg per cup. On the other hand, higher levels of organic acids are found in the current work with respect to [8,36] but these variations can be attributed to the complexity of the coffee supply chain. In fact, several factors related to species and plant cultivation, harvesting and processing methods, roasting, grinding and preparation techniques play an important role in coffee quality and its content of volatile and non-volatile compounds [10,37]. As for total solids and lipids, the content of bioactive compounds increases when finer particles are used. For instance, the averages of caffeine content obtained in all ECs prepared with coarse, optimal and fine granulometry are 4.82, 5.24 and 5.58 g/L in Arabica while 6.86, 7.63 and 7.98 g/L in Robusta. The same behaviour has been observed for all analytes and this inverse increment of compounds levels, with respect to particle sizes, were found also in other scientific papers [32,34,38]. Instead, the contribution of water temperature and pressure on the extraction of bioactive compounds is extremely complex and it is not possible to describe it as a linear increment or decrement. However, some small variations in compounds contents have been found. For instance, preparing ECs at 9 bar with optimal particle sizes, higher contents of caffeine, chlorogenic acids and organic acids are obtained at 88 and 93.4 \({}^{\circ}\)C. Some authors reported that 92 \({}^{\circ}\)C is an ideal water temperature for Arabica and Robusta natural blends [39].