Flow Injection Fourier Transform Infrared Determination of Caffeine in

A fully automated procedure has been developed for FT-. IR determination of caffeine in soft drinks. Samples are first degasified by filtration and th...
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Anal. Chem. 1997, 69, 1086-1091

Flow Injection Fourier Transform Infrared Determination of Caffeine in Soft Drinks Yasmina Daghbouche,† Salvador Garrigues, M. Teresa Vidal,‡ and Miguel de la Guardia*

Department of Analytical Chemistry, University of Valencia, 50 Dr. Moliner Street, 46100 Burjassot, Valencia, Spain

A fully automated procedure has been developed for FTIR determination of caffeine in soft drinks. Samples are first degasified by filtration and then directly injected into a flow manifold and passed through a 100 mg C18 SPE cartridge, conditioned with methanol and water. After the cartridge has been cleaned with water, the caffeine is eluted with CHCl3 and stabilized with ethanol. The flow injection (FI) recording is obtained by measuring the absorbance at 1658 cm-1 with a baseline established at 1800 cm-1. Area values for the FI recording obtained between 0.4 and 1.4 min after sample injection are interpolated in the calibration graph of a series of aqueous standards of caffeine, which were loaded and eluted in the same way as the samples. Preliminary studies were carried out using off-line SPE and off-line elution and online extraction and on-line elution, and the parameters involved in both the retention and elution of caffeine in solid phase extraction cartridges were evaluated, together with the measurement conditions for FT-IR determination of caffeine in CHCl3 solutions and the flow injection parameters. The procedure developed provides a limit of detection of 10 µg mL-1 of caffeine for a sample injection of 1 mL, a sampling frequency of 30 h-1, and a relative standard deviation of 3.5% for eight independent measurements at a concentration level of 100 µg mL-1. Fourier transform infrared spectrometry (FT-IR) is an excellent multiwavenumber detector for use in flow injection analysis (FI), and over the last 10 years it has clearly been demonstrated that FI-FT-IR provides a synergistic combination, since use of the FI strategy involves a dramatic reduction of reagents and solvents1while also allowing on-line recovery of the carrier,4 thus providing an environmentally friendly analytical technique. It allows fast and easy filling and cleaning of the liquid cells and provides an excellent sampling frequency. Moreover, FT-IR improves the performance of flow injection analysis, allowing continuous monitoring of the spectral baseline and simultaneous determination of several species in the same sample, without requiring complex pretreatments.5,6 †Permanent

address: University of Constantine, Algeria. Permanent address: Universidad Polite´cnica de Valencia, Spain. (1) de la Guardia, M.; Garrigues, S.; Gallignani, M.; Burguera, J. L.; Burguera, M. Anal. Chim. Acta 1992, 261, 53-57. (2) Curran, D. J.; Collier, W. G. Anal. Chim. Acta 1985, 177, 259-262. (3) Guzman, M.; Ruzicka, J.; Christian, G. D.; Shelley, P. Vib. Spectrosc. 1991, 2, 1-14. (4) Sa´nchez-Dası´, J.; Cervera, M. L.; Garrigues, S.; de la Guardia, M. First Mediterranean Basin Conference on Analytical Chemistry, Cordoba, Spain, 5-10 November, 1995. ‡

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Recent studies developed by coupling an on-line separation step with FT-IR determination, using FI, are very useful for avoiding spectral interference from complex matrices in direct analysis by on-line generation of a vapor phase7,8 and for improving the limit of detection of FT-IR by on-line preconcentration using solid phase extraction (SPE).9,10 Caffeine is one of the legally permitted additives in the manufacture of soft drinks, with a tolerated upper limit of 0.015% (w/w).11 Analytical control of this parameter is, therefore, required in order to safeguard the health of consumers. The normal caffeine content in cola drinks is around 100 mg L-1,12 and because of the complex matrices of these types of samples (containing large quantities of sugars, dyes, sweeteners, acids, and so on), the determination of caffeine must be carried out by means of chromatographic techniques.13-16 Caffeine is highly soluble in organic solvents, which present a low absorbance in the infrared range, and provides characteristic bands in the middle infrared range which are useful for the quantitative determination of this compound.17 The main objective of the present paper is, therefore, to develop an appropriate procedure for the determination of caffeine in soft drinks by means of on-line solid phase extraction (SPE) and on-line elution, using FT-IR spectrometry for fast and accurate flow injection determination. The use of SPE permits preconcentration of the caffeine and sample cleanup and also allows a change of solvent in order to obtain appropriate solutions for working in the middle infrared range. (5) Garrigues, S.; Gallignani, M.; de la Guardia, M. Analyst 1992, 117, 18491853. (6) Garrigues, S.; Gallignani, M.; de la Guardia, M. Talanta 1993, 40, 17991807. (7) Lo´pez-Anreus, E.; Garrigues, S.; de la Guardia, M. Anal. Chim. Acta 1995, 308, 28-35. (8) Pe´rez-Ponce, A.; Garrigues, S.; de la Guardia, M. Anal. Chim. Acta, in press. (9) Garrigues, S.; Vidal, M. T.; Gallignani, M.; de la Guardia, M. Analyst 1994, 119, 659-664. (10) Daghbouche, Y.; Garrigues, S.; de la Guardia, M. Anal. Chim. Acta 1995, 314, 203-212. (11) Ministerio de Agricultura, Pesca y Alimentacio´n. Recopilacio´n Legislativa Alimentaria, Capitulo 29, bebidas no alcoho´licas, Madrid, 1982. (12) Halvax, J. J.; Wiese, G.; Van Bennekom, W. P.; Bult, A. Anal. Chim. Acta 1990, 239, 171-179. (13) Uppal, M.; Massan, M. M. A.; Al-Meshal, I. A. In Analytical Profiles of Drug Substances;Florey, K., Ed.; Academic Press Inc.: London, 1986; Vol. 15. (14) Thompson, C. O.; Trenerry, V. C.; Kemmery, B. J. Chromatogr. 1995, 694 (2), 507-514. (15) Hawthorne, S. B.; Miller, D. J.; Pawliszyn, J.; Arthur, C. L. J. Chromatogr. 1992, 603 (1-2), 185-191. (16) Gennaro, M. C.; Abrigo, C. Fresenius’ J. Anal. Chem. 1992, 343 (6), 523525. (17) Baucells, M.; Ferrer, N.; Lacort, G.; Roura, M. Quim. Anal. 1991, 10, 211219. S0003-2700(96)00693-2 CCC: $14.00

© 1997 American Chemical Society

Figure 1. Manifold employed for on-line solid phase extraction and on-line elution of caffeine.

EXPERIMENTAL SECTION Apparatus and Reagents. A Magna IR 750 system from Nicolet (Madison, WI) equipped with a temperature-stabilized detector with a KBr beam splitter and precise digital signal processing (DSP) was employed to carry out all the IR measurements, using a microsize flowthrough cell (Spectra Tech, Orpington, UK) with ZnSe windows and a 0.5 mm lead spacer. Using a spectral resolution of 4 cm-1 and setting the speed of the moving mirror of the interferometer to 0.6329 cm s-1, a time of 1.03 s is sufficient to obtain and store the complete interferograms for all the samples. For processing the FT-IR absorbance data in both stoppedflow and flow injection modes, version 2.1 of the Omnic software (developed by Nicolet Corp.) was employed. For the preconcentration of the caffeine, commercially available Bond Elut C18 octadecyl 3 mL cartridges from Varian (Harbor City, CA) were employed, containing 500 mg of solid phase with an average particle size of 35 µm and a specific area of 420 m2 g-1. To perform the off-line preconcentration and on-line elution of the caffeine, a manifold similar to that previously reported for carbaryl and naphthol determination was employed,10 including a Spetec Perimax 12 peristaltic pump (Erding, Germany) equipped with 2.79 mm i.d. poly(vinyl chloride) tubes for sample loading and 1 mm i.d. Viton (isoversinic) tubes for on-line elution with CHCl3. For on-line extraction and elution of caffeine, the manifold depicted in Figure 1 was employed. It consists of two Gilson P2 Minipuls peristaltic pumps (Villiers-le-Bel, France). Pump A uses 1 mm i.d. Viton tubes to transport chloroform and methanol, and pump B is equipped with poly(vinyl chloride) tubes with internal diameters of 1.85 and 1.52 mm to transport the samples and the water carrier, respectively. The manifold includes two six-way Rheodyne type 50 injection valves (Coati, CA), one equipped with a 1 mL sample loop and the other connected to the C18 cartridge mounted in the sample loop. Two T-pieces permit sequential passing of methanol and water through the SPE cartridge to condition it. All connecting tubes and loops are made of PTFE and have an internal diameter of 0.8 mm.

The operation of the manifold depicted in Figure 1 involves four steps: (1) cleaning of the flow cell; (2) sample loading and conditioning of the cartridge; (3) sample preconcentration and cleaning of the cartridge; and (4) elution of the caffeine and FTIR determination. During the first step, pump B is stopped, and with valve A in the injection position, pump A is used to clean the flow cell with a flow of CHCl3, which is employed to determine the background, and at the same time 0.5 mL of methanol is loaded in the loop located outside valve B. In the second step, pump A is stopped, and pump B is used to fill the sample loop, with valve B in the loading position, and to condition the cartridge with methanol and water, with injection valve A in the loading position. The third step involves passing the sample through the cartridge, with valve A in the loading position and valve B in the injection position. To elute the caffeine, pump A is used to pass a flow of CHCl3 through the cartridge, with valve A in the injection position, while pump B is stopped. At the same time, 0.5 mL of methanol is again confined in the loop outside valve B, to condition the cartridge after elution of the preceding sample or standard. This operation is repeated for each of the samples analyzed, apart from the first step, which is needed onlyto establish the initial background. To analyze additional samples, only steps 2-4 are required. All the reagents employed throughout this study were of analytical grade. Chloroform, stabilized with 1% (v/v) ethanol or with 50 mg L-1 2-methyl-2-butene (β-amylene), were obtained from Scharlau (Barcelona, Spain), methanol from Panreac (Barcelona, Spain), and caffeine from Fluka (Buchs, Switzerland). Aqueous standard solutions of caffeine were prepared from a stock solution of 200 mg L-1 in a concentration range varying from 25 to 200 mg L-1, and the chloroform standards were prepared in chloroform stabilized with amylene or ethanol in a concentration range varying from 0.05 to 1 mg mL-1. General Procedure. The samples are degasified by filtration and then loaded in SPE C18 cartridges, which are first conditioned Analytical Chemistry, Vol. 69, No. 6, March 15, 1997

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with 0.5 mL of methanol and 0.5 mL of water. The SPE preconcentration and FT-IR caffeine determination were carried out by three different procedures: (i) off-line extraction and elution, (ii) off-line extraction and on-line elution, and (iii) on-line extraction and on-line elution. (i) FT-IR Determination Using Off-Line Solid Phase Extraction. A 10 mL aliquot of sample is loaded in a 200 mg C18 cartridge, using a carrier flow of 9 mL min-1. The cartridge is then dried with a flow of N2 for 5 min, and the caffeine is eluted with four 1 mL portions of CHCl3 stabilized with ethanol, making the caffeine extract up to a total volume of 5 mL. This solution is then placed inside the measurement cell and the flow stopped. FT-IR spectra are collected by accumulating 10 scans with a resolution of 4 cm-1, and the wavenumber range from 2000 to 1250 cm-1 is selected for absorbance measurement. Previously, the background of the cell was established for the pure solvent in the same conditions. The maximum absorbance value of caffeine is determined at 1658 cm-1, corrected with a baseline established at 1800 cm-1 in order to avoid interference from the water band at 1602 cm-1. The caffeine concentration is determined using standards prepared in CHCl3 stabilized with ethanol. (ii) FT-IR Determination Using Off-Line Solid Phase Extraction and On-Line Elution. A 100 mg C18 cartridge, previously loaded with 1 mL of sample and cleaned with 0.5 mL of water, is inserted in the manifold described in a previous paper10 and then eluted on-line with a 0.58 mL min-1 CHCl3 carrier flow. The corresponding FI recording is obtained from the absorbance values at 1658 cm-1 as a function of time, corrected with a baseline established at 1800 cm-1. The caffeine concentrations are determined by interpolating the recorded FI area values in a calibration curve obtained from aqueous standards treated in the same way as the samples, with the background in this case being measured for a CHCl3 solution passed through an SPE cartridge previously conditioned and loaded with distilled water. (iii) On-Line SPE and FT-IR Determination. Using the manifold shown in Figure 1, a 100 mg C18 cartridge is conditioned with 0.5 mL of methanol and 0.5 mL of water and then loaded with 1 mL of sample, using a carrier flow of 1.88 mL min-1. After the loaded cartridge is cleaned with 0.5 mL of water, the caffeine is eluted with CHCl3 stabilized with ethanol. The analytical measurements are carried out using the area values of the FI recording obtained from the absorbance data at 1658 cm-1, corrected with a baseline established at 1800 cm-1 and measured in the time interval between 0.4 and 1.4 min after sample injection. The background is established from the absorbance of the solvent carrier solution passed through the solid cartridge, which is previously loaded with 1 mL of distilled water. For standardization, it is recommended to use aqueous standards, loaded and eluted in the same way as the samples. RESULTS AND DISCUSSION FT-IR Determination of Caffeine Dissolved in CHCl3. As can be seen in Figure 2, caffeine solutions in CHCl3 stabilized with either β-amylene or ethanol present two intense absorbance bands at 1658 and 1705 cm-1. CHCl3 is almost completely transparent in the middle IR range and provides a good method for caffeine determination. The band at 1602 cm-1 present in the solution stabilized with amylene corresponds to a residual water content in this solvent. 1088 Analytical Chemistry, Vol. 69, No. 6, March 15, 1997

Figure 2. FT-IR spectra of a 0.2 mg mL-1 caffeine solution in CHCl3 stabilized with ethanol (s) or with amylene (- - -).

Peak height absorbance measurements at 1658 cm-1 and area measurements in the wavenumber range from 1685 to 1620 cm-1 were performed for a series of standard solutions of caffeine, ranging from 0.05 to 1 mg mL-1, dissolved in CHCl3 and stabilized with β-amylene or ethanol, and the data obtained were used to establish the corresponding calibration equations reported in Table 1, where the main analytical features of FT-IR caffeine determination are also indicated. From the data summarized in Table 1, it can be concluded that CHCl3 is an appropriate solvent for FT-IR determination of caffeine, providing a low limit of detection, of the order of 2-5 µg mL-1, an extremely good variation coefficient of the absorbance signals from 0.4 to 1.9%, and good linearity of the calibration lines. Selection of Conditions for Solid Phase Extraction of Caffeine. A series of experiments was carried out for the preconcentration of 25 mL of an aqueous solution of 25 mg L-1 of caffeine, using different SPE cartridges: CH (cyclohexyl-), C2 (ethyl-), C8 (octyl-), C18 (octadecyl-), PH (phenyl-), and CN (cyanopropyl-). The retention elution process was applied to the standard solutions with ultraviolet measurements at 276 nm, both in the aqueous phase passed through the column and in the organic elution phase. It was previously observed that C18, C8, C2, and CH cartridges retain caffeine from aqueous solutions quantitatively, while C18 cartridges have the additional advantage that the solid phase can easily be regenerated several times after performing sample extraction. In this context, it must be noted that UV spectrometry was useful only for the evaluation of the process with standard solutions and would not be at all appropriate for real samples containing sugars, flavoring agents, and dyes. To select the most appropriate solvent for elution of caffeine from the C18 loaded cartridges, CHCl3 and CH2Cl2 were assayed. The average yield obtained from the solid phase extractionelution process was 93.2 ( 0.7% for CHCl3 and 34 ( 4% for CH2Cl2 (in both cases using a sample volume of 25 mL with a concentration of 100 mg L-1, eluted with five portions of 1 mL of each solvent). This indicates that chloroform is a suitable solvent for both FT-IR determination and SPE of caffeine. To test the effect of CHCl3 stabilizers (β-amylene and ethanol) and of the moisture which may remain after elution of the solid cartridges, a series of FT-IR measurements was made using 10 mL of an aqueous solution containing 100 mg L-1 caffeine, preconcentrated in a 500 mg C18 cartridge, dried with a N2 flow at 1 bar pressure for 10 min, and eluted with CHCl3 to a final volume of 5 mL.

Table 1. Analytical Features of FT-IR Determination of Caffeine in CHCl3 Solutions chloroform stabilized with amylene

chloroform stabilized with ethanol

parameter

absorbance peak height at 1658 cm-1

peak area between 1685 and 1620 cm-1

absorbance peak height at 1658 cm-1

peak area between 1685 and 1620 cm-1

regression line correlation coefficient RSD% (n ) 10) for 0.2 mg mL-1 limit of detection(µg mL-1) concn range (mg mL-1)

0.0005 + 0.4877Ca 0.9996 0.5 1.8 0.05-1

0.13 + 10.73C 0.9996 1.9 4.5 0.05-1

-0.0004 + 0.4728C 0.9999(8) 0.4 1.7 0.05-1

-0.01 + 10.58C 0.9999 0.5 4.8 0.05-1

a Concentration in mg mL-1. The limit of detection was established for k ) 3 and expressed in µg mL-1 in the organic solution. In all cases, the baseline was established at 1800 cm-1. Measurements were carried out at a nominal resolution of 4 cm-1. Ten scans were accumulated, and a spectral wavenumber range from 2000 to 1250 cm -1 was selected.

The results obtained in these experiments showed that CHCl3 stabilized with ethanol provides a quantitative recovery of caffeine from aqueous solutions. However, when CHCl3 stabilized with β-amylene was employed, a poor recovery, of the order of 3050%, was obtained. The presence of water can also affect absorbance measurements of SP extracts of caffeine, especially the area values when the background is established with a water-saturated solvent. Therefore, in this study, absorbance measurements at 1658 cm-1 are recommended, corrected with a baseline established at 1800 cm-1. Using C18 cartridges and CHCl3 stabilized with ethanol as the elution solvent, additional parameters for SPE of caffeine were tested, such as the effect of the ethanol concentration in CHCl3, the amount of the solid phase employed, and the drying time required after loading the cartridges. An ethanol concentration of 1% or more (v/v) improves the quantitative recovery of caffeine preconcentrated and eluted from the C18 cartridges. The normal ethanol concentration employed to stabilize CHCl3 is of the order of 1% (v/v).7,18 Drying the solid phase, after loading the sample, seems to be important to obtain reproducible results, and it is also necessary to monitor the baseline carefully in order to avoid the effect of the remaining moisture on the caffeine absorbance data. The amount of the C18 solid phase required for the preconcentration of caffeine depends on the caffeine concentration and the volume of the sample, as can be seen in Figure 3, from which it appears that 200 mg of solid phase is sufficient for quantitative retention of 1 mg of caffeine, even if diluted to 10 mL. In the conditions established, for a loading volume of 10 mL of sample, using 200 mg of a C18 solid phase and eluting the caffeine with four portions of 1 mL of CHCl3 stabilized with 1% (v/v) ethanol, the typical calibration line found was A ) 0.0014 + 0.4513C, A being the absorbance of the spectra at 1658 cm-1 with a baseline correction at 1800 cm-1, and C the concentration of caffeine in the CHCl3 expressed in milligrams per milliliter, with a correlation coefficient of 0.9998. This equation compares well with that obtained for caffeine standard dissolved in CHCl3, which is A ) -0.0001 + 0.4708C, thus showing that an average recovery percentage of 95.2% can be obtained for the retention elution process of caffeine in the concentration range assayed, from 50 to 500 µg mL-1 in the final organic phase. However, in order to achieve high accuracy, the use of aqueous standards treated in the same way as the samples must be recommended. (18) Lo´pez-Anreus, E.; Garrigues, S.; de la Guardia, M. Fresenius’ J. Anal. Chem. 1995, 351, 724-728.

Figure 3. Effect of the amount of the C18 phase on the SPE of caffeine by loading: 1 mL of an aqueous solution containing 1000 mg L-1 caffeine (s) and 10 mL of a 100 mg L-1 solution (- - -).

In these conditions, the limit of detection of the method is 1.5 µg mL-1, and a typical relative standard deviation value for 10 independent analyses of a sample containing 0.2 mg mL-1 is 0.5%. Experimental Conditions for On-Line Elution of Caffeine from C18 Cartridges. For on-line elution of caffeine, the loaded cartridges must be inserted in a FI manifold and eluted with the minimum amount of CHCl3, thus improving sensitivity and sampling throughput. For this process, 100 mg C18 cartridges were employed, and the preliminary drying step was omitted. The background was established by passing CHCl3, stabilized with ethanol 1% (v/v), through a cartridge previously activated with methanol and water and washed with distilled water in order to obtain a comparable moisture level to that obtained after SPE of caffeine in real samples. This corrects the overlapping of the water band at 1602 cm-1 and its effect on the caffeine determination measurements. Figure 4 shows the profiles of two FI recordings obtained by off-line SPE and on-line elution of a real cola soft drink. The analytical measurements were performed at 1658 cm-1 with a baseline correction established at 1800 cm-1 and using a carrier flow of 0.58 mL min-1. As Figure 4A shows, when loaded cartridges are eluted directly, the presence of sugars affects the FI recording profiles dramatically. To avoid sugar interference during the measurement step, preliminary cleaning of the samples loaded on the C18 cartridge is necessary. To evaluate this on-line, different volumes of water were employed (from 0 to 2 mL), and the results of the area of the FI recording obtained for an aqueous standard solution (containing 5% (w/v) of fructose) and a real sample are sumAnalytical Chemistry, Vol. 69, No. 6, March 15, 1997

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Table 3. Recovery of Caffeine from Spiked Soft Drink Samples Analyzed by SPE-FT-IR Using Off-Line Preconcentration and On-Line Elution caffeine concn (mg mL-1) sample

previousa

added

found

recovery (%)

cola light 1 cola light 2 cola classic low-caffeine drink high-caffeine drink

53 ( 8 54 ( 3 46 ( 2 20 ( 3 57 ( 2

25 85 60 100 80

76 ( 3 137 ( 5 105 ( 6 122 ( 6 140 ( 7

92 98 98 102 104

a

Figure 4. FI recordings obtained for a real sample of a cola drink, preconcentrated off-line in a C18 cartridge and eluted on-line with a 0.58 mL min-1 carrier flow of CHCl3 stabilized with ethanol (1% v/v), (A) without cleaning the cartridge and (B) cleaning the cartridge with 0.5 mL of distilled water. Table 2. Effect of the Loading and Elution Parameters on the FI Peak Recording Area Measured at 1658 cm-1 with a Baseline Established at 1800 cm-1, Using SPE-FT-IRa volume of water used to wash cartridge (mL)b

area of a standard solution

area of a real sample

0 0.25 0.5 1.0 1.5 2.0

0.181 ( 0.006 0.172 ( 0.05 0.186 ( 0.01 0.175 ( 0.002 0.169 ( 0.003 0.168 ( 0.007

0.141 0.169 0.176 0.171 0.139 0.128

loading flow (mL min-1)c

area of a standard solution

area of a real sample

1.24 1.88 2.54 3.18 3.84

0.199 ( 0.006 0.186 ( 0.007 0.195 ( 0.016 0.201 ( 0.013 0.169 ( 0.011

0.181 ( 0.002 0.187 ( 0.004 0.174 ( 0.016 0.167 ( 0.009 0.178 ( 0.008

flow of elution (mL min-1)d

area of a standard solution

area of a real sample

0.40 0.58 0.76 0.94 1.12 1.30

0.213 ( 0.003 0.183 ( 0.003 0.171 ( 0.005 0.101 ( 0.012 0.118 ( 0.023 0.099 ( 0.010

0.216 ( 0.010 0.186 ( 0.004 0.182 ( 0.019 0.148 ( 0.003 0.136 ( 0.002 0.131 ( 0.002

volume of sample loaded (mL)e

area of a standard solution

area of a real sample

0.5 0.7 1.0 1.2 1.5

0.109 ( 0.006 0.140 ( 0.003 0.186 ( 0.005 0.202 ( 0.013 0.244 ( 0.012

0.109 ( 0.003 0.143 ( 0.007 0.187 ( 0.004 0.213 ( 0.017 0.262 ( 0.020

a Caffeine concentration for both standard and sample, 100 mg L-1. Elution flow rate, 0.58 mL min-1 (standard contains 5% (w/v) of fructose). c Elution flow rate, 0.58 mL min-1. Volume of sample loaded, 1 mL. d Loading flow rate, 1.88 mL min-1. Volume loaded, 1 mL. e Loading flow rate, 1.88 mL min-1, elution flow rate, 0.58 mL min-1. b

marized in Table 2, which shows that, to obtain good comparability between samples and aqueous standards treated in the same way, a water volume between 0.25 and 1 mL can be recommended. In 1090 Analytical Chemistry, Vol. 69, No. 6, March 15, 1997

These data correspond to 50% (v/v) diluted samples.

this study, accurate results were obtained by using 0.5 mL of water to wash the C18 loaded cartridge (see the FI recording in Figure 4B obtained after carrying out this cleaning operation). In the conditions indicated, the regression equation between the area of the FI recording and the concentration of caffeine in the original aqueous solutions can be described by A ) 0.009 + 1.78C, A being the area of the FI recording obtained at 1658 cm-1 as a function of time, and C the concentration of caffeine in the aqueous solutions expressed in milligrams per milliliter, thus providing a sensitivity 4 times higher than that found by off-line elution, with a regression coefficient of 0.9991 in the concentration range from 25 to 200 mg L-1 (considered in the original aqueous phase). The limit of detection, for a probability level of k ) 3, is 4.7 mg L-1, and the relative standard deviation of five independent measurements of a sample containing approximately 100 mg L-1 of caffeine is 4.3%. Studies on the recovery of caffeine concentrations ranging from 25 to 100 mg L-1, added to real samples of soft drinks of different types and determined by FT-IR using off-line preconcentration and on-line elution, provided recovery percentage values from 92 to 104%, as can be seen in Table 3. Effect of FI Parameters on the On-Line SPE and FT-IR Determination of Caffeine. Complete automation of FT-IR determination of caffeine in soft drinks involves the development of an appropriate manifold for the conditioning of the SPE cartridges, loading of the samples, cleanup, and on-line elution of the caffeine. The manifold depicted in Figure 1 enables us to carry out all these steps very simply. Based on preliminary studies performed with off-line preconcentration and elution and on-line elution, for the method 100 mg of a C18 phase and CHCl3 stabilized with 1% (v/v) ethanol were used, together with a cleanup step of the loaded cartridges with water. Additional experiments were carried out using both real samples and standard aqueous solutions, in order to test the effect of the loading and eluting carrier flow rates and the sample injection volume. The sample loading flow rate was evaluated in the range between 1.24 and 3.84 mL min-1 (see Table 2), which gave the best repeatability and agreement between data found for samples and standards containing 100 mg L-1 for a loading flow of 1.88 mL min-1. This also provides a good compromise in terms of speed of measurement and high yield from the retention elution processes. The elution carrier flow was evaluated in the range between 0.4 and 1.3 mL min-1 by measuring the area under the FI peaks obtained for both a real sample and a standard solution containing

repeatability of different injections of the same solution. From this figure it can be concluded that the sampling frequency provided by the procedure developed is of the order of 30 h-1. Analysis of Real Samples by FT-IR. Real samples of soft drinks were analyzed by the procedure developed, and the results obtained, in milligrams per liter, are 110 ( 6 and 114 ( 6 for two different samples of cola light, 94 ( 4 for cola classic sample 1, 96 ( 3 for cola classic sample 2, and 115 ( 4 for a coffee drink, which shows that a relative standard deviation below 5% can be obtained in this type of determination. It must also be noted that these data are in good agreement with those found by off-line preconcentration and elution, which were 88 ( 2 for cola classic sample 1, 96.3 ( 0.6 for cola classic cola sample 2, and 117.4 ( 0.5 for the coffee drink. These data also correspond well with those declared by the manufacturer and reported in previous studies. Figure 5. FI recording obtained for different standard solutions of caffeine by on-line SPE-FT-IR determination. Data were obtained from absorption at 1658 cm-1 with a baseline established at 1800 cm-1. The inset corresponds to eight independent loadings of standard solution of 100 mg L-1 of caffeine. All data were obtained with a loading flow rate of 1.88 mL min-1 and elution flow rate of 0.58 mL min-1. Volume of sample, 1 mL.

100 mg L-1 of caffeine. It can be seen from the data in Table 2 that, when elution flow rates higher than 0.58 mL min-1 are used, it is impossible to obtain a good comparison between samples and aqueous standards. However, the use of extremely low elution flows reduces sampling frequency and creates additional problems for the area determination of lengthy FI recording. Accordingly, a 0.58 mL min-1 flow was chosen for this study. Increasing the volume of sample loaded also increases the area of the FI recording. However, as Table 2 shows, the use of loaded volumes higher than 1 mL can cause problems connected with the comparability of the area of the FI recording obtained for samples and standards. Consequently, a sample volume of 1 mL was chosen. In the conditions described, a typical calibration curve obtained with previously treated aqueous standards is A ) 0.015 + 1.72C, A being the area of the FI recording obtained at 1658 cm-1 as a function of time, and C the concentration of caffeine in the aqueous solution expressed in milligrams per milliliter, with a regression coefficient of 0.998, a limit of detection of 10 µg mL-1 (for k ) 3), and a relative standard deviation of 3.5% for eight independent measurements at a concentration level of 100 mg L-1. By way of example, Figure 5 shows the FI recordings obtained for a series of caffeine standards and an indication of the

CONCLUSION The studies carried out have shown that caffeine in soft drinks can be determined by FT-IR by means of previous degassing of the samples by filtration followed by direct injection into a flow manifold equipped with a C18 SPE cartridge in which the caffeine is preconcentrated and, after the cartridge has been washed with water, eluted on-line with CHCl3 stabilized with ethanol. The procedure developed is fully automated. The same manifold can be used for all the various steps of (1) conditioning the cartridge, (2) loading the sample, (3) washing the solid phase, and (4) eluting the caffeine. This procedure can be used for quality control of caffeine concentration in soft drinks with a sampling frequency of 30 h-1 and a limit of detection of 10 µg mL-1, giving good recovery values for the concentration of caffeine added to real samples and precise results (a relative standard deviation less than 5%) for independent analysis of real samples. ACKNOWLEDGMENT The authors acknowledge the financial support of the Conselleria de Cultura Educacio´n y Ciencia de la Generalitat Valenciana (Project GV 1021/93) and of the Spanish DGICYT (Project PB 92/0870).

Received for review July 18, 1996. Accepted January 6, 1997.X AC960693V X

Abstract published in Advance ACS Abstracts, February 15, 1997.

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