Chocolate determination

Several standard methods for the quantitative analysis of food samples are based on measuring the sample s mass following a selective solvent extraction. For example, the crude fat content in chocolate can be determined by extracting with ether for 16 h in a Soxhlet extractor. After the extraction is complete, the ether is allowed to evaporate, and the residue is weighed after drying at 100 °C. This analysis has also been accomplished indirectly by weighing a sample before and after extracting with supercritical GO2. 

The theobromine and caffeine contents of several finished chocolate products as determined by hplc at Hershey s laboratories are presented in... 

Theobromine was determined by GC in various foods (bitter chocolate, milk chocolate, chocolate cake, cocoa powder, chocolate milk), and results are given in graphs and tables.27 Homogenized samples were boiled in alkaline aqueous media, then fat was extracted with n-hexane. The aqueous layer was acidified with diluted HC1 and NaCl was added. Theobromine was extracted from this treated aqueous solution with dichloromethane and the extract was evaporated to dryness. The residue was redissolved in dichloromethane containing an internal standard. GC analysis was performed on a column packed with 1% cyclohexane dimethanol succinate on Gaschrom Q, with FID. Average recoveries were 99 to 101%, coefficient of variation was less than 3% and the limit of detection for theobromine in foods was about 0.005%. 

HPLC allows a quantitative determination with relatively simple extractions. In many cases, extraction only involves a heating of the commodity with water, followed by filtration and injection onto an HPLC column. In the determination of caffeine, theobromine, and theophylline in cocoa, coffee, or tea, as well as in other foods, there is scarcely a month that passes without a new paper on this assay. Kreiser and Martin provide typical conditions for analysis.28 In their studies, samples were extracted in boiling water and filtered prior to injection onto the HPLC column. The HPLC conditions used a Bondapak reversed phase column and a mobile phase of water methanol acetic acid (74 25 1) with detection at 280 nm. This method is accurate, precise, and conserves time. It has also been adopted by the AOAC as an official method for the determination of theobromine and caffeine in cocoa beans and chocolate products.29... 

Zoumas et al.30 presented work on the use of this method in the determination of caffeine and theobromine in various chocolate-containing products, while Blauch and Tarka31 reported the use of a similar method for the determination of caffeine and theobromine in various beverages containing these methylxanthines. 

Kreiser, W.R. and Martin, R.A. Jr., High pressure liquid chromatographic determination of theobromine and caffeine in cocoa and chocolate products, JAOAC, 61,1424,1978. 

Chocolate liquor is the solid or semiplastic food prepared by finely grinding the nib of the cacao bean. It is commonly called baking chocolate, unsweetened chocolate, or bitter chocolate and, in Europe, is frequently referred to as chocolate mass or cocoa paste. Chocolate liquor is essentially the starting point from which all chocolate products are produced. Table 5 lists the theobromine and caffeine content of 22 various chocolate liquor samples determined by high pressure liquid chromatography (HPLC). The liquors averaged 1.22% theobromine and 0.214% caffeine.27- 28 The ratio of theobromine to caffeine ranged from 2.5 1 to 23.0 1. 

It is necessary to determine the methylxanthine content of chocolate foods, as well as beverages, in order to obtain an accurate assessment of the total amount of theobromine and caffeine that is ingested via the diet. This area of analysis has received little attention, and only scant published data exist on the methylxanthine content of chocolate foods. 

Chemical analysis of the finished food product is a more accurate determination of the methylxanthine content. In studies performed at Hershey Foods Corporation, the methylxanthine content of a large variety of commercially available chocolate foods was measured by HPLC methods.38 These results have been compiled together with other literature values in Table 11. Large methylxanthine variations can be seen among the chocolate foods, as well as within different brands of the same item. 

Kreiser, W., Martin, R., Cacao products — high pressure liquid chromatographic determination of theobromine in cocoa and chocolate products, J. Assoc. Off. Anal. Chem., 61, 1424, 1978. 

Kiefer, B. A., Martin, R. A., Determination of theobromine and caffeine in chocolate products by HPLC. Unpublished data, 1987. 

Brereton, P., Hague, M., Wood, R., The determination of theobromine in cocoa and chocolate products, /. Assoc. Publ. Analysts, 30, 23, 1994. 

Rein D, Lotito S, Holt R, Keen C, Schmitz H and Fraga C. 2000. Epicatechin in human plasma in vivo determination and effect of chocolate consumption on plasma oxidation status. J Nutr 130 2109S—2114S. 

A variety of xanthines including caffeine, theobromine, and theophylline have been found from food materials including.coffee, chocolate, and tea (419-420). Theophylline determination in sera has been much studied. The technique allows the determination of theophylline at serum levels of 1.5-20 mg/liter theophylline with sample sizes ranging from 50 to 10 /xl (42 -425). Hill (426) assayed theophylline using 50 /xl of serum and an analysis time of 8 min with good interbatch precision and accuracy. Alternative methods which allow the determination of as little as 0.1 mg/ml (427) or 20 ng theophylline in 10 ml serum have been described (428). 

Some people prefer dark chocolate. Others go for the gooey chocolate-covered cherries. Many don t care for coconut in their sweets. And most people are happy with any mixture of any kind. Different kinds of candies have different prices, depending on the ingredients. When you combine different candies in packages, the quality or price of each type is multiplied by the quantity or weight to determine the price of the mixture. 

These laboratory robots bear no resemblance to C3P0 and R2D2 of Star Wars fame, but rather they are complex computer controlled units specifically manufactured for use in analytical chemistry and are capable of a large number of tasks. They can be obtained commercially or can be laboratory manufactured (1 2). The initial application in our laboratory was to automate the preparation of samples for a final HPLC determination of sorbate in chocolate syrup. 

The initial application of the unit was for the automation of a sample preparation method for the determination of sorbate in chocolate syrup. Table I summarizes the data in this study. Figure 3 outlines the sample preparation scheme for this assay. 

Dr. Joe Vinson of the University of Scranton believes that there is something to the chocolate effect, and he came to Belmont to tell us about his intriguing research. Vinson has determined the total polyphenol content of various chocolates and has also found a way of measuring how effective these mixtures are in preventing the oxidation of human ldl in a test tube. In other words, he has calculated a phenol antioxidant index, which takes into account both the quantity and the quality of these desirable substances. At the symposium Vinson reported that cocoa powder and dark chocolate are the best, followed by milk chocolate. Instant cocoa mixes trail the field. Then Vinson delivered the kicker chocolate has more, and better, polyphenols than fruits or vegetables and more than red wine. A forty-gram bar of dark chocolate has as many polyphenols as a cup of that widely promoted antioxidant cocktail we call tea. But there is still the matter of chocolate s fat content. Researchers tell us, though, that at least half of it is stearic acid, which does not raise blood cholesterol. 

N. Jalbani, T. G. Kazi, M. K. Jamali, M. B. Arain, H. I. Afridi, S. T. Sheerazi and R. Ansari, Application of fractional factorial design and Doehlert matrix in the optimisation of experimental variables associated with the ultrasonic-assisted acid digestion of chocolate samples for aluminium determination by atomic absorption spectrometry, J. AO AC Int., 90(6), 2007, 1682-1688. 

Two students, Denby and Scott, began their quest at the library with a computer search for analytical methods. Searching with the key words caffeine and "chocolate," they uncovered numerous articles in chemistry journals. Reports titled High Pressure Liquid Chromatographic Determination of Theobromine and Caffeine in Cocoa and Chocolate Products 5 described a procedure suitable for the equipment in their laboratory.6... 

The first step in any chemical analysis is procuring a representative sample to measure—a process called sampling. Is all chocolate the same Of course not. Denby and Scott bought one chocolate bar in the neighborhood store and analyzed pieces of it. If you wanted to make broad statements about caffeine in chocolate, you would need to analyze a variety of chocolates from different manufacturers. You would also need to measure multiple samples of each type to determine the range of caffeine in each kind of chocolate. 

These include mainly crystallised fruits, preserved fruits, jams, chocolates, sweetmeats, biscuits, effervescent citrate of magnesia, honey, condensed milk, liqueurs and sweet wines. Certain of these products are dealt with in other places, condensed milk, liqueurs and sweet wines, for instance, in the chapters dealing respectively with milk, spirits and liqueurs, and wines. The others are treated below, special attention being paid to the determination of the sugars. 

The analysis of chocolate comprises mainly the tests and determinations indicated below. With chocolates containing inclusions of extraneous substances (whole walnuts, almonds, etc., or liqueurs), it is advisable to separate mechanically the outer layer of chocolate from the included substance and to examine it separately.1... 

It may happen, especially in liqueur chocolates, that such separation is not possible. In such case the whole sample is reduced to a homogeneous mass and analysed, the alcohol also being determined by distillation after dilution. The -water is then represented by the difference between the loss on drying and the alcohol (by weight) found. 

With chocolate to which starch or flour has been added, since only a small part (usually 1-2%) of the starch piesent consists of that contained in the cacao, the quantity of the starch or fioui added may often be determined sufficiently approximately from the starch content Wien ordinary staich is added, it may be assumed that this contains on the average about 80% of actual starch, the lest being mostly moisture... 

Alkaloids.—These rarely occur in chocolate but, when necessary they may be determined as follows (Savun) 12 grams of the powdered chocolate are introduced mto a -litre flask and boiled with 70 c c of light petroleum on a water-bath for 15 minutes, the solvent is then decanted on to a small filter and the operation repeated twice... 

Where sweets rich in insoluble substances, such as starch, almonds, etc., are to be examined, the volume of the insoluble matter is determined as in the case of chocolates, i.e., by making two solutions of the normal weight in 100 and 200 c.c. respectively. 

Fats.—These are determined by extraction with a suitable solvent, e.g., ether or carbon tetrachloride (see Chocolate). The necessary constants of the fat are then determined and its character established. 

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