Abstract A sustainable and environmentally friendly pro-cedure has been developed for the FTIR determination ofcaffeine in tea leaf samples. The method is based on theextraction with ammonia and CHCl3 and direct determi-nation of caffeine on the chloroform extracts using peakheight absorbance measurements at 1658.5 cm1 and ex-ternal calibration. The method provides a sensitivity of0.2142 absorbance units mg1 mL and a limit of detectionof 1 mg L1, corresponding to 0.002% m/m caffeine in tealeaves. As compared with a reference procedure, based onUV absorbance measurement at 276 nm after low pres-sure column chromatography, the developed procedure re-duces the consume of CHCl3 by a factor of 10, that ofNH4OH by a factor of 20 and avoids the use of diethylether and Celite. The time required to do the analysis of asample is 15 minutes as compared with the 6 hours for thereference one.

Keywords Caffeine Tea FTIR Solvent extraction

Introduction

Caffeine is the most important alkaloid present in tea andthus, the determination of this stimulatory effect alkaloidis an important factor in the quality control of tea.

Caffeine concentrations vary from one to another typeof tea with typical concentration levels of 3.2% (m/m) forblack tea and concentrations varying from 1 to 3% (m/m)in green tea [1].

Caffeine has been determined in drinks by different an-alytical techniques such as UV-VIS spectrophotometry [2,

3, 4] or amperometry [5] and most frequently by usingseparation methods like high performance liquid chro-matography (HPLC) [6, 7, 10], ion chromatography [11],capillary electrophoresis [12, 13, 14], as well as high per-formance thin layer chromatography (HPTLC) [15, 16,17] and micellar capillary electrophoresis [18, 19, 20].The official method for caffeine determination in tealeaves [21] is based on low-pressure column chromatog-raphy and spectrophotometric determination at 276 nm.This method involves a sample preparation time of morethan 6 hours and the use of 200 mL diethyl ether and 50 mL CHCl3 for each sample. So it is a costly and te-dious methodology, making the development of a fast,simple and less contaminant procedure to determine caf-feine in tea necessary.

Caffeine has been determined in other beverages likecoke [22] and coffee samples [23, 24] by the use of FTIRspectrometry, because this technique offers an excellentsensitivity and selectivity for this kind of analysis beingpossible to do a practically direct determination after asimple solid phase [22] or a solvent extraction [23, 24].

The main object of this work was the development ofan FTIR method for caffeine determination in tea samplesin order to reduce the time and the solvent volume re-quired as much as possible for this determination.

Experimental

Apparatus and reagents

A Magna FTIR 750 system from Nicolet (Madison, USA)equipped with a temperature-stabilised DGTS detector with aGe/KBr beamsplitter and precise digital signal processing (DSP)was employed for all the FTIR measurements, with a nominal res-olution of 4 cm1, accumulating 25 scans and using a micro flow-through cell with ZnSe windows and a pathlength of 0.457 mm.

A stopped-flow system was employed for sample introductioninside the IR cell with a Gilson Minipuls P2 (Villiers-le-Bel,France) peristaltic pump. For sample transport, Viton (Isoversinic)3 mm o.d. and 1 mm i.d. pump tubing and PTFE 0.8 mm i.d. con-necting tubes were used. For processing the FTIR absorbance data,the 2.1 version of the Omnic software of Nicolet Corp. was em-ployed.

Janina Ohnsmann Guillermo Quints Salvador Garrigues Miguel de la Guardia

Determination of caffeine in tea samples by Fourier transform infrared spectrometry

Anal Bioanal Chem (2002) 374 :561565DOI 10.1007/s00216-002-1503-8

Received: 30 April 2002 / Revised: 17 June 2002 / Accepted: 24 June 2002 / Published online: 10 September 2002

ORIGINAL PAPER

Janina Ohnsmann is on leave from the University of Mainz (Germany)J. Ohnsmann G. Quints S. Garrigues () M. de la GuardiaDepartment of Analytical Chemistry, University of Valencia, Edifici Jeroni Muoz, 50th Dr. Moliner, 46100 Burjassot, Valencia, Spaine-mail: salvador.garrigues@uv.es

Springer-Verlag 2002

A Hewlett-Packard (Palo Alto, USA) 8452A UV-VIS DADsystem was employed for the UV-Vis determination of caffeine.

All the reagents employed in this study were of analyticalgrade. Chloroform, stabilised with 150 ppm of amylene, was ob-tained from Scharlau (Barcelona, Spain), ammonium hydroxide30% from Panreac (Barcelona, Spain), and caffeine from Fluka(Buchs, Switzerland). The tea samples were obtained from theSpanish market.

Reference procedure

A 1-g ground sample was accurately weighed inside a 100-mLbeaker, 50 mL NH4OH (4 mol/L) was added and the mixturewarmed on a boiling H2O bath for 2 min. After cooling, transferredquantitatively to a 100-mL volumetric flask and diluted to the vol-ume with H2O. Then, 5-mL aliquots of the turbid solution werecarefully mixed with 6 g Celite 545 and the mixture was placed in-side a 25250 mm tube column to obtain a homogenous and com-pact layer above a glass wool plug and a mixture of 3 g Celite 545and 2 mL NaOH 2 mol/L. This basic column was mounted abovethe acid column prepared from well mixed 3 mL 2 M H2SO4 and 3 g Celite 545 placed between glass wool layers inside a 25250 mm tube. A 150-mL aliquot of H2O-saturated ether was se-quentially passed through the basic and acidic columns and dis-carded. A 50-mL aliquot of H2O-saturated ether was then passedthrough the acidic column and discarded. A 48-mL aliquot of H2O-saturated CHCl3 was also passed through the acidic column. Thefirst portions of the eluent were then passed through the basic col-umn and the caffeine solution recovered in a 50-mL volumetricflask. The column eluate was diluted to the mark with H2O-satu-rated CHCl3 and the absorbance read at 276 nm against an H2O-saturated CHCl3 blank, using caffeine CHCl3 solutions as cali-brants.

Recommended Procedure

A 250-mg sample of a blended tea was accurately weighed in aglass tube, 5 mL NH4OH 2 mol/L was added and then the mixturewas mechanically shaken for 2 min. Then 5 mL CHCl3 were addedand the mixture mechanically shaken for an additional 2 min. Af-ter that, the mixture was centrifuged for 10 min.

The chloroform solution was introduced into a microflow-through IR cell using a peristaltic pump and the FTIR spectra ob-tained between 4000 and 600 cm1 at 4 cm1 resolution and accu-mulating 25 scans for spectra. The background spectrum was es-tablished from the cell filled with a blank extract solution. For the

determination of caffeine peak height values at 1658.5 cm1, cor-rected using a baseline at 1800 cm1, obtained for samples wereinterpolated in a calibration line obtained from caffeine calibrantsolutions directly prepared in CHCl3 and measured under the sameconditions as the sample extracts and using a background ofCHCl3.

Results and Discussion

FTIR spectra of caffeine and tea extracts

Figure 1 shows the FTIR spectra of the caffeine calibrantsolution in chloroform and a tea extract. Both spectra arevery similar and have a characteristic absorption that canbe used for the determination of caffeine the main differ-ence being the presence at 1730 cm1 of an overlappedband probably due to the absorption of other extractedcomponents present in tea samples. The carbonyl bands at1704.8 cm1 and 1658.5 cm1 are the most intense bands,but additionally there are others present at 1554.4 cm1,1415.5 cm1 and 1361 cm1. So that could be useful forthe analysis of CHCl3 extracts obtained from tea leaves.

In Figure 2, the spectra of three different kinds of teapresent the shoulder at 1730 cm1 but demonstrate that ispossible to identify the main characteristic bands of caf-feine in every tea sample extracted with CHCl3. So differ-ent studies were carried out in order to find the appropri-ate conditions for the quantitative extraction of caffeineand its determination in the simplest way possible .

Study of the extraction conditions

Caffeine extraction from an aqueous solution to a chloro-form phase is a well known quantitative process. For thisreason, in the determination of caffeine in tea samples, themain problem is to guarantee the extraction of the caffeinefrom the solid to an aqueous phase. Previous studies [23,24] proved that caffeine can be easily extracted by using

562

Fig.1 FTIR spectra of a caffeinestandard of 1.5 mg mL1 (), a sam-ple extract (--) and a blank solution (--). Every spectrum was obtainedfor 25 cumulated scans at a resolutionof 4 cm1. For the sample extract thespectrum was obtained using a blanksolution as background, the caffeinestandard was measured in the sameconditions as the sample extract butusing a background of CHCl3

alkaline solutions such as ammonia. So the shaking mode,shaking time and the volume and concentration ofNH4OH were studied in order to obtain the best conditionsfor a quantitative extraction of caffeine from tea leaves.

Jasmine tea samples and peak height measurements at1658.5 cm1, corrected using a baseline at 1800 cm1,were employed in order to search for the best extractionconditions.

Two different shaking modes, mechanical and ultra-sonic, were assayed. For both modes, shaking times from1 to 5 minutes were evaluated. From the results obtainedit can be concluded that there is no significant (at p=0.05)difference between sonication and mechanical shaking,the simplest method being the latter procedure. Using themechanical mode, caffeine was quantitatively recoveredafter a 2-min shaking time.

The increase in NH4OH concentration from 0.25 to 4 mol/L does not influence the caffeine extraction. So aNH4OH 2 mol/L concentration was selected in order toguarantee a precise and robust procedure.

The effect of the volume of the NH4OH was evaluatedfrom 5 to 10 mL, it was found that 5 mL was enough toobtain a quantitative recovery of caffeine from tea. Theuse of reduced volumes of NH4OH and CHCl3 permittedus to carry out all the different steps of the extraction,such as solid-liquid extraction with ammonia, liquid-liq-uid extraction with chloroform and phase separationthrough centrifugation.

Selection of the appropriate bands for FTIR determination of caffeine

Figure 1 shows that at least two bands, namely 1704.8 and1658.5 cm1, could be used for the quantitative determina-tion of caffeine in tea extracts. For these bands, peakheight measurements were considered. When applyingtwo alternative baseline criteria for the carbonyl band at1704,8 cm1, it is possible to compensate for an eventual

matrix interference during the extraction step (see Table 1).In order to choose between the criteria, the height ratiobased on the most intense bands was calculated for thecalibration solution, sample extracts and spiked samples.In fact, a constant proportion between bands considered inall types of solutions indicates a good comparability be-tween natural sample extracts, external standards andstandard addition solutions.

Table 2 shows the peak height ratio between the threebands considered working with a fixed baseline criterion(at 1800 cm1) and with corrected values at 1883 cm1 forthe carbonyl bands at 1704.8 and 1658.5 cm1.

As it can be seen, the bands at 1704 and 1658.5 cm1are those which provided the best sensitivity and the bestcomparability when considering pure CHCl3 solutions,extracts obtained from natural samples and those found

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Fig.2 FTIR spectra of a Jasmine,Green and Ceylon tea samples ex-tracted in CHCl3

Table 1 Calibration lines obtained for FTIR determination of caf-feine using different bands

Band Calibration line R2 b RSDc LODd (Baseline) % (mg L1)1704.8 cm1 y =0.0017+0.1333Ca 0.9984 0.3% 1.5(1800 cm1)1704.8 cm1 y =0.0005+0.1053C 0.9995 0.3% 2.2(1683 cm1)1658.5 cm1 y =0.0016+0.2142C 0.9993 0.1% 1(1800 cm1)1554.4 cm1 y =0.0001+0.0361C 0.961 1.3% 4.8(1800 cm1)1415.5 cm1 y =0.0009+0.0177C 0.8995 5.1% 2.1(1800 cm1)Notes: Calibration lines were established in the concentrationrange from 0 to 4 mg mL1 caffeine using 5 standard solutions; aC: Concentration of caffeine in mg mL; bR2 : Regression coefficient; cRSD: Relative standard deviation for five independent measure-ments of a sample containing 1.45 mg mL1 caffeine; dLOD Limit of detection established for a probability level of99.6% (k=3).

for spiked samples, thus indicating that a method based onthe aforementioned bands could be a robust alternative forcaffeine determination in tea leaves. However, the band at1704.8 cm1 could be affected by that at 1730 cm1 whichappears in some sample extracts and, because of that peakheight measurements at 1658.5 cm1 established with abaseline at 1800 cm1, seems the best alternative for FTIRdetermination of caffeine in tea.

On the other hand, Figure 3 shows the FTIR spectra ofthe solid residue obtained after evaporation, onto a BaF2plate, of a few microliters of the chloroform extracts ofdifferent tea samples considered and a calibrant solutionof caffeine. On comparison with the caffeine standard,one can see differences in the relative intensity of theband at 1704.8 cm1 especially for Jasmine tea samples;which confirms the selection of the band at 1658.5 cm1for quantification of caffeine in the organic extracts of teasamples.

Analysis of natural samples of tea

As can be seen in Table 3, caffeine concentrations foundby FTIR in three types of tea samples are comparable tothose found in the literature and data found are of thesame order to those obtained by the official method.

In fact, it seems that the results from the referencemethod based on UV detection at 276 nm are a little bitlow and that the simple liquid-liquid extraction and FTIRmeasurement gives errors a little too high compared withFTIR determinations carried out after the AOAC prepara-tion procedure.

In order to verify the accuracy of the developed proce-dure, recovery experiments were carried out by spikingCeylon and Jasmine tea samples with amounts of caffeine

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Table 2 Peak height ratio through bands presented by caffeine at 1704.8 cm1, 1658.5 cm1 and 1554.4 cm1 in different types of sam-ple extracts and standards

H1704/H1658 sa H1704/H1554 sa H1658/H1554 sa H1704/H1658 sb H1704/H1554 sc H1658/H1554 sd

Standards 0.632 0.002 3.80.3 6.10.4 0.5650.004 3.00.1 5.40.2Ceylon Tea 0.6340.002 3.610.05 5.690.07 0.5680.005 2.730.07 4.810.09Jasmine Tea 0.6320.002 3.730.09 5.90.1 0.5790.006 3.00.1 5.20.2Green Tea 0.6300.002 3.410.05 5.420.08 0.5580.003 2.530.05 4.540.07Spiked Ceylon Tea 0.6330.002 3.90.1 6.10.1 0.5730.002 3.00.1 5.30.2Spiked Jasmine Tea 0.6340.001 3.770.03 5.940.06 0.5740.002 2.930.03 5.100.06aUsing a common baseline established at 1800 cm1 for all the bands.b,c,dConsidering a baseline correction at 1883 cm1for the bands at 1704.8 and 1658.2 cm1, and corrected at 1800 cm1 for the band at1554.4 cm1.

Table 3 Results obtained for the determination of caffeine in teasamples by different procedures

Sample Caffeine % (m/m)FTIR determination Reference

methodLLEa AOACbpreparation preparation

Ceylon Tea 2.670.03 2.590.02 2.10.4Green Tea 2.700.02 2.210.02 2.10.2Jasmine Tea 2.290.04 2.190.07 1.90.1aLiquid-liquid extraction;bFTIR determination after the official AOAC preparation method.Data reported are the average of 3 independent determination thecorresponding standard deviation.

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Fig.3 FTIR spectra from the residueremaining after evaporation onto aBaF2 plate of 100 L of caffeine stan-dard solution and the chloroform ex-tracts of tea samples. The spectra havebeen shifted to avoid band overlap-ping

ranging from 2.5 to 7.35 mg. Data in Table 4 show thataverage recovery percentages vary from 97 to 98% thusindicating the absence of systematic errors of the FTIRmeasurements carried out after solvent extraction of sam-ples with ammonia and CHCl3.

Analytical performance of the developed procedure

The methodology developed for caffeine determination intea leaves by FTIR measurement of CHCl3 extracts ob-tained after extraction of the solid samples with ammoniagives a sensitivity of 0.2142 absorbance units per mgmL1 caffeine with a limit of detection of 1 mg mL1, cor-responding to a 0.002% m/m concentration in the solidsample and a limit of quantification of 3.4 mg L1 thus ofthe order of 0.007% m/m caffeine in tea leaves (takinginto account the extraction of 250 mg sample with a finalvolume of 5 mL CHCl3).

On comparing the solvent extraction FTIR procedurewith the reference one using low pressure chromatogra-phy and UV determination, the consumption of CHCl3was reduced by a factor of 10 and that of NH4OH by afactor of 20, thus providing a cheaper alternative. The useof diethyl ether is also avoided and as well as that ofCelite, thus providing an economic sustainable procedurewhich drastically reduces the environmental impact. Inaddition, the time required for each determination is ap-proximately 15 minutes, compared with 6 hours neededfor the reference procedure. So, the new procedure notonly increases the productivity of the laboratory but alsoreduces the cost of each determination.

Acknowledgements. The authors acknowledge the financial sup-port of the Generalitat Valenciana Project GV01249. G. Quintsacknowledge the grant provided by the Laboratorio de HigieneLaboral y Ambiental of the Universitat de Valencia and J. Ohns-mann acknowledge the Erasmus grant of the European Union.

References

1.Fernandez PL, Martin HJ, Gonzalez AG, Pablos F (2000) Ana-lyst 125:421425.

2.Capeda A, Paseiro P, Simal J, Rodrguez JL (1990) Alimenta-ria 211:2327.

3.Capeda A, Paseiro P, Simal J, Rodrguez JL (1991) An Broma-tol 42:241249.

4.Lau O, Luk S, Cheng O, Chiu TPY (1992) Analyst 117:777783.

5.Meyer A, Ngiruwonsanga T, Henze G (1996) Fresenius J AnalChem 356:284287.

6.Muhtadi F, El-Hawary S, Hifnawy MS (1990) J Liq Chro-matogr 13:10131028.

7.Arlorio M, Coisson JD, Martelli A (2000) Chromatographia52:579583.

8.Rojo-de Camargo MC, Teledol MCF (1999) J Sci Agric 79:18611864.

9.Shi YL, Han JT, Liu QP, Cheng JK (1996) Fenxi Shiyanshi15:1518.

10.Gennaro MC, Abrigo C, (1992) Fresenius J Anal Chem 343:523525.

11.Chen QC, Mou SF, Hou XP, Ni ZM (1998) Anal Chim Acta371:287296.

12.Hurst WJ, Martin RA (1993) Analusis 21:389391.13. Jin WR, Yu DQ, Dong Q, Ye XY (2000) J Chromatogr Science

38:1115.14.Swinney KA, Bornhop DJ (1999) J Microcolumn Sep 11:

596604.15.Sarin RK, Sharma GP, Vrshney KM, Rasool SN (1998) J Chro-

matogr A 822:332335.16.Vovk I, Golc-Wondra A, Prosek M (1997) J Planar Chro-

matogr Mod TLC 10:416419.17.Walker JC, Zaugg SE, Walker EB (1997) J Chromatogr A 781:

481485.18.Zhao Y, Lunte CE (1997) J Chromatogr Biomed Appl 688:

265274.19.Sun BG, Hou PL, Lin YF (1999) Fenxi Kexue Xuebao 15:

124127.20.Thompson CO, Trenerry VC, Kemmery B (1995) J Chro-

matogr A 694:507514.21.Herrlich K (1990) Association of Official Analytical Chemists,

Official Methods of Analysis, Arlington, 769781.22.Daghbouche Y, Garrigues S, Vidal MT, de la Guardia M

(1997) Anal Chem 69:10861091.23.Bouhsain Z, Garrigues JM, Garrigues S, de la Guardia M

(1999) Vibr Spectroscopy 21:143150.24.Garrigues JM, Bouhsain Z, Garrigues S, de la Guardia M

(2000) Fresenius J Anal Chem 366:319322.

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Table 4 Recovery of caffeine from tea samples

Sample Caffeine Caffeine Recovery Mean added (mg) found (mg) (%)a recovery (%)

Ceylon Tea 2.50 2.62 104.70.2 9865.00 4.75 94.90.27.35 6.95 94.60.2

Jasmine Tea 2.51 2.51 100.20.6 9734.92 4.82 98.00.17.33 6.84 93.30.3

aValues correspond to the average recovery the standard devia-tion of 3 independent determinations