Theobromine separation from caffeine

Sample cleanup refers to the removal of undesirable components of an unknown that interfere with measurement of analyte. In chocolate analysis, polar molecules such as sugars that are not retained by C g-silica wash right through the column and are separated from caffeine and theobromine. Fats that could interfere are probably retained on the C g column and therefore also separated from caffeine and theobromine (Figure 22-33). 

Theobromine can be separated from caffeine by the formation of insoluble silver-theobromine, C7H702N4Ag, on addition of silver nitrate or, in a mixture of the two, caffeine can be extracted in alkaline solution with chloroform, theobromine being left as the sodium salt in aqueous solution. 

Figure 18-7. Effect of temperature on the separation of caffeine derivatives on a Hypercarb column (1 mm x 100mm). (a) Column at 100°C, mobile phase acetonitrile (b) Colnmn at 180°C, mobile phase water/acetonitrile 70/30. Samples 1, hypoxantine 2, theobromine 3. theophylline 4, caffeine 5, P-hydroxyethyltheophylline. (Reproduced from reference 35, with permission.)...

Nine xanthines (xanthine, I-, 7-, and 3-methylxanthine, isocaffeine [IS], theobromine, paraxanthine, theophylline, caffeine) were extracted from serum and urine separated on a 32°C Cg column (A = 270 run) using a 20-min 90/10 70/30 water... 

S.CO2 extraction has been mentioned by Calame and Steiner [13]. There is a US patent on the S.CQz extraction of cocoa nibs at 320 bar and 45° with subsequent separation at 56 bar and 38° to recover 74% of the fat [111]. A UK patent to remove the stimulants theobromine, xanthine and caffeine without cocoa butter, from wet cocoa nibs with S.CO2 at 300 bar and 90°C was granted [112]. 

HPLC with thermospray MS was reported by Hurst et al.40 where residues from an archeological site were analyzed for caffeine and theobromine using reversed phase HPLC coupled to a thermospray MS interface. Samples were extracted in water and separated on a reversed phase column. The presence of theobromine in this sample was confirmed by monitoring the MH+ ion at 181 for theobromine. 

Chocolate milk samples prepared from sweetened cocoa powders averaged 58 mg per serving of theobromine and 5 mg per serving of caffeine.28 Analysis of a "home-style" recipe resulted in higher methylxanthine values — 94 mg theobromine and 10 mg caffeine per serving. However, the authors noted that this recipe also had a stronger chocolate flavor. The lower values reported by Zoumas et al. and Blauch and Tarka compared to others was attributed to the inability of older methods to separate theobromine and caffeine, and the lack of precision and accuracy of the older methods. A compendium of theobromine and caffeine values reported for chocolate beverages from both published and unpublished studies has been compiled in Table 8. 

The adjustment of die pH is critical for the separation of 8-chlorodieophylline. UV detection is at 280 nm. Other xandiines and xandiine metabolites also elute from this system. Retention times relative to theophylline are 3-methylxanthine, 0.49 theobromine, 0.63 paraxanthine, 0.88 caffeine, 1.36 ... 

Fig. 11.1. Separation of some xanthine derivatives Column pBondapak C18 (300x4 rim ID), mobile phase acetonitrile - 0.01 M sodium acetate buffer (pH 4.0) (7 93), flow rate 2.0 ml/min, detection UV 254 nm. Peaks 1, 1-methyluric acid 2, 3-methyl xanthine 3, 1,3-dimethyl-uric acid 4, theobromine 5, theophylline 6, B-hydroxy-ethyltheophylline 7, phenobarbital 8, caffeine 9, 8--chlorotheophyl1ine. (reproduced with permission from ref. 56, by the courtesy of Clinical Chemistry)...

Xanthines. It was found that the capacity factors and the separation ratios were both functions of the identity of the modifier as well as of the quantity of the modifier in the carbon dioxide. In Figure 6, it can be seen that with 9-5% 2-methoxyethanol in carbon dioxide the first two components of a xanthine mixture, caffeine and theophylline, are not well-separated but the elution order is caffeine, theophylline, theobromine, and xanthine. At 6.5f 2-methoxyethanol in carbon dioxide, there is essentially baseline resolution of the first three components in less than one minute xanthine (not very soluble in liquid 2-methoxyethanol and not at the usual 1 mg/mL sample concentration but at some undetermined saturation concentration) at a k of over 5 has begun to tail and to disappear into the baseline. While it is obvious that the capacity factors, k, increase with a decrease in modifier concentration from 9-5 to 6.5% 2-methoxyethanol in C02, less obviously the separation ratios (for example, with respect to caf-... 

Precipitation of the extract is achieved by pressure reduction and/or absorption of the extracted compounds in an absorbing liquid. For the extraction of xanthines (caffeine, theobromine and theophylline) the absorbing liquid is water. The supercritical gas. loaded with the extract, enters the precipitator at the lower end and flows upwards against the absorbing water, which is sprayed from the top of the vessel. The regenerated gas leaves the vessel at the top after passing a liquid/gas separator for removing droplets. 

Figure 0-4 provided an example of chromatography used to separate caffeine from theobromine in a chemical analysis. Absorption of light and electrochemical reactions... 

Figure 22-33 Solid-phase extraction separates caffeine and theobromine from sugars (more polar) and fats (less polar) found in chocolate.

Figure 20 Separation of some xanthines in tea extract by HPLC. 1. theophylline 2, caffeine 3, theobromine each 1 pg/10 pi eluent flow rate, 0.75 cm3/min. (a) Using direct injection sample volume, 10 pi. (b) Using preseparation in a tank and analyte transfer by means of the OPLC interface sample volume 10 pi, applied on to the layer, developed twice with ethyl acetate prior to analysis. (Reproduced by permission from Ref. 71.)...

FIGURE 17 Reversed-phase separation of beverage additives on 10-fjim C-18 silica (Partisil-10 ODS-2). Column 4.6 mm x 25 cm, mobile phase 50 50 methanol-water, flow rate 0.6 ml/min., pressure 529 psi, UV detection at 254 nm. Peaks (a) Saccharin, (b) Theobromine, (c) Theophylline, and (d) Caffeine. [Reprinted by permission from Whatman, Inc.]... 

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