Determination of Caffeine in Beverages by High Performance Liquid Chromatography James E. DiNunzio Wright State University, Dayton, OH 45435 In rhe last st!\wul years caffeine and the role it plays in human health have received considerahle atlention. Caffeine is one of t h e most widely consumed drugs worldwide. Its stimulant effects are well known, and n o doubt contribute t o its popularity. T h e average American consumes caffeine in a wide variety of products. T h e widespread use of caffeine has prompted considerahle study of its effect on humans. Caffeine has been linked t o everything from "nerves" and heart palpitations through heart attacks and birth defects. While these links remain t o b e conclusively proven they have generated substantial interest for many persons with regards to sources and levels of caffeine in commerciallv available nroduds. In fact. manv individuals are actively limiting their'caffeine intake and are interested in knowine t h e amount of caffeine in t h e products they conHigh performance liquid chromatography is a method used for the determination of caffeine, and it seemed ideal t o develop an experiment for caffeine determination a s a means of introducing the technique. This paper describes just such a n experiment. T h e procedure is simple, fast, and accurate. Bec a k e sample preparation is minimal, the experiment is well suited for use in a teaching laboratory. Experimental
All solvents and samoles were filtered throueh 0.5-um filters. (Rainin Instrument ('o.,'\vohurn. MA! prior to use. ~ a k p l e ucr" s liltrred using l%mm filter holdrrsuifh 1.uer ronnectionsnnd 3-mI. diipoiahle svringes. 'This was accc,mpl~shrdby filling the s)Tlnae mfh the sample, attaching the filter holder to the syringe, and forcing the sample through the filter. The filtered samples were collected in small testtuhes. Laboratory-grade (USP) caffeine (Fisher Scientific Co.) was used for the preparation of standards. Methanol, HPLC grade (Fisher Scientific Co.), was used for the preparation of the mobile phase. Procedure Standard sdutions are prepared h y dilution of a caffeine stock solution (lInX)ppm).Atleast four standads covering the rangeof 1-10 ppm rafieine in water should be prepared. The caffrine should be dried at llO°C for 1h prior to use. Each student should ohtain at least three samples of similar type (i.e., soda pop, tea, coffee, etc.). Sample preparation will be somewhat different depending on the type of heverages chosen. The basic idea is to obtain initially a sample which represents a "typical serving" of the beverage under investigation. For liquids this usually is as ohtained. For solids that need to he dissolved (coffees,teas, etc.) the "typical serving" must be ~ r e ~ a r eThis d . is done by weighing some of the sample and dissolving it in an exactly known volume of water to simulate a "typical serving." Once the "typical serving" of beverage is obtained samples can he prepared for analysis. All carbonated beverages must be degassed either under vacuum or ultrasound. Filter each beveraee throueh - i..t 4 2 tiltrr paper. Prepare the ianlple ior nnnlyils 1,s diluting the lilfrrrd brwrage I:lO uith water. Triplicntrs of csch beverage should he prt-parcdThi~rciult~ ina n,i!litnum t m d d I4 a h t i u n , ~~~~~~~~~is: ~~
Apparatus and Materials A Waters Associates liquid chromatograph, consisting of an M6000A solvent delivery system, a U6K Universal Injector, and a Model 440 fixed wavelength UV detector (Waters Assnciates, Milford, MA) was used. Separations were achieved using an Alltech C18,lOfim, 25-cm X 4.6-mm i.d. column (Alltech Associates, Deerfield, IL). A pre-column, 5-cm X 4.6.id. packed with 37-fim C18 pellicular particles was coupled to the analytical column.
~
0 Figure 1. Chromatogramsof some coffees and teas: (1) 40 ppm caffeinestandard, (2) instant decaffeinatedcoffee,(3)brewed coffee, (4) blank. (5)brewed tea. 1caffeinepeak.
446
Journal of Chemical Education
10 20 Time (min
30
Figure 2. Chromatagrams of some soda pops: (I nan-cola ) soda pop. (2) cola soda pop. (3)eaffeina-free wla soda pop, 1caffeine peak.
Accuracv for Caffeine Determination Sample
Reported Valuea
Determined Value
Coffee 1 Coffee 2 Coffee 3 Coffee 4 Soda Pop 1 Sada Pop 2
5 52 68 2 52 37
4.9 54 65 2.1 56 38
Repaned V* (1981).
given as mglrerving ( C M M M B T Repans. 44. 56%(191%).46, 595
4 standards, 9 beverage solutions,and 1reagent blank (which has been treated as the beverages). Analysis of the samples can he performed in the following manner. cellulose Each solution must be filtered throueh a 0.5-urn . reeenerated .. filre~prior1c injectiminw rhr chnmnroyraph. Sc,lutionssh~uldhr injrr~edin random order. Chn~marographivcc~nd~rionr consiswd of a ;Id 711 I W V I methanol water moh~lrphait nr 2.0 nlL mm. Sample size ranged from 5-20 FL and the absorbance of the sample was monitored at 254 nm.
Results and Discussion
The purpose of designing this experiment was to give students artual experience in the tprh&ue of high perfknancc liquid chromarogrnphy. I1 was hoped that the analysis of
beverages that were actually consumed by the students would increase their interest in the exweriment and helw them to understand the technique. In this regard the experiment is very successful. Typical chromatograms obtained from a variety of beveraces are shown in Figures 1and 2. Caffeine. which aonears as .. t i e last peak in allthe chromatograms, elutes in approximately 7 min. This fast analysis time allows a significant number of samples to be analyzed during a single laboratory period. Quantitation of caffeine in the bewrngn samplrs is achieved ~hruugha cnlihration curve. Brcause the cntfei~welutes as u ~ h a r p - ~ e athe k peak height is linearly proportional to the concentration of caffeine in the solution. A calibration curve covering the range 1-40 ppm was adequate for all the heverages examined. The nrecision obtainable was measured in two wavs. First. the preksion was determined by replicate injection of one o i the prepared standards. The relative standard deviation for this was about 2% for most students. Next, the precision of the overall determination was found by the analysis of triplicate samples of the beverages under investigation. The relative standard deviation for this ranged from 3%11%. The table shows the accuracy obtainable for the determination of caffeine in beverages by high performance liquid chromatography. As can be observed excellent agreement between reported values can be obtained with this method.
Volume 62
Number 5 May 1985
447
