**3.1 HAP formation**

A series of caffeine studies on crystallization research required more than several years. Thus, a simple in vitro study was conducted to see whether other xanthine family members could reveal if any, other different crystallization value(s) from caffeine.

*The Contrasting Effects between Caffeine and Theobromine on Crystallization… DOI: http://dx.doi.org/10.5772/intechopen.101116*

All the solutions contained 0.01 molar CaCl2 and Na3PO4. Several sets of experiments were conducted with the addition of each of methylxanthine at low concentrations, 50 mg, and 200 mg/L. The effect of the xanthine compounds was compared with a control solution containing CaCl2 and Na3PO4 only. Solutions were mixed at 25°C, and pH adjusted to 9–9.5 with 0.1 molar NaOH and left to crystallize for 20 days. The crystalline was washed five times with distilled water and prepared for X-ray diffraction (**Figure 6**).

All the data from the various members of the xanthine family on the effects of crystallization were already reported [26, 27].

To our surprise, the value of theobromine was much lower than caffeine. Specifically, 1-methylxanthine values were lower than the caffeine group but not as low as the theobromine group.

Caffeine (small crystal size) and theobromine (large crystal size) with the lack of one position of the methyl group, crystal size was opposite. Therefore, it should be further investigated how the methyl position 1 alone can influence the crystallization.

#### **Figure 6.**

*The (300) reflection was scanned to investigate crystallinity. FWHM (full width-half maximum peak height) divided by maximum peak height (FWHM/M) and for the (300) reflection is given in* **Table 2***.*


#### **Table 2.**

*Hydroxylapatite which was grown in vitro of three xanthine families, caffeine, theobromine and 1-methylxanthine are shown.*

The crystal size of position 1 alone did not have much effect on the crystal size. See the chemical formulae (**Figure 7**).

The crystal size of the theobromine group taken by an electron microscope was shown below and the size was four times bigger than that of the control group (**Figure 8**).
