determination of caffeine content in beverages by fourier
TRANSCRIPT
Determination of caffeine content in beverages by Fourier transform Infra-red spectroscopy
Clarissa S. Gold Lab Partner: Aimee Johnson TA: Kevin Fischer Dates lab performed: 4/17/18 Date report submitted: 5/1/18
Abstract Caffeine is known to experience anharmonic stretches of its carbonyl groups when subjected to
photons with frequencies on the order of 5x1013 Hz This makes Fourier transform Infra-red
spectroscopy a promising candidate for molecular absorbance analysis of caffeine. In this
experiment, the concentration of caffiene was quantified in extracts of decaffeinated espresso,
Kazaal espresso, cold brew coffee, and black tea using absorbance measurements collected
with a Shimadzu IRAffinity spectrometer equipped with a PIKE MIRacle attenuated total
refection (ATR) attachment. Based on the linear relationship between absorption and
concertation outlined by the Beer-Lambert law, a calibration curve was developed using peak
absorbance measurements obtained from FTIR spectra of standard caffeine samples with
concentrations of, 2.57x10-5M (5.00 ppm,) 7.72x10-5M (15.0 ppm,) 1.54x10-4M (30.0 ppm,) and
2.06X10-4M (40.0 ppm.) Absorbance measurements were corrected using a two-point algorithm
to mitigate the effects of a sloping spectral baseline. Least squares regression analysis was
performed to derive the calibration curves line of best fit, y= [144.95(43.0) x (Abs. Int. Corrected)]
-0.02419 (0.006), after which baseline corrected absorbance values from FTIR spectra of
beverage extracts were used to calculate their corresponding concentration of caffeine. The
concentration of caffeine in Kazaal espresso, cold brew and black tea was determined to be, -
4.99x10-4 0.005 M (~96.8 ppm,) 4.45x10-4 0.005 M (~86.3 ppm,) and 8.45x10-4 0.005 M
(~164 ppm) respectively. The concentration of decaffeinated espresso could not be determined
as its corrected absorbance intensity yielded a concentration that was below the 23 ppm
detection limit. The accuracy of the caffeine concentrations calculated for beverage extracts
was limited by a high degree of uncertainty associated with the calibration curve, which had a
large standard deviation about the regression (0.005) and a one decade linear dynamic range.
Replicate trials should be conducted gather more conclusive results as to the true
concentration of caffeine within the analyzed beverages; however, a poor sensitivity to small
analyte concentrations and a variable baseline restricts the quantitative power of IR absorbance
spectroscopy.
Introduction
Infra-red (IR) absorbance spectroscopy is one of the most versatile methods of optical
molecular analysis. This versatility may in large part be attributed to the universal abundance of
IR active vibrational motions. As a qualitative method, IR absorbance spectroscopy is frequently
employed for use in compound and functional group identification, and may be used to
elucidate the key components of chemical structures within bulk materials. IR absorbance
spectroscopy has also seen extensive use as a quantitative method, particularly in online and
real-time applications. (1)
In its provenance as an instrumental method, Infra-Red spectroscopy utilized
spectrometers that were equipped with conventional dispersive devices. Current IR methods
however, have shifted almost entirely towards the use of non-dispersive elements like those
present in Fourier transform instruments. The benefits of Fourier transform (FT) infrared
spectroscopy (FTIR) are multi-fold. One of the most important assets of FT is its capability of
enhancing signal-to-noise ratios. Unlike those methods which depend on dispersive techniques,
FTIR spectroscopy allows for a higher optical throughput, greater speeds of spectral collection,
and simultaneous measurement of relevant wavelengths. (1)
The utility of IR absorbance spectroscopy as an analytical tool is further strengthened by
the fact that it works well for analytes in the solid, liquid, or gas phase. For solid analyte, the
most common FTIR sampling technique is Attenuated Total Reflection (ATR.) ATR operates by
measuring changes in the attenuation of an internally reflected source beam following
interaction with a solid analyte surface. The result is an IR spectrum that depicts peaks which
correspond to both the quantity and frequency of photons that were absorbed during distinct
rovibrational transitions. (2)
ATR-FTIR spectroscopy is very prevalent for industrial analysis of caffeine.
Notwithstanding its potentially adverse effects on human health as an alkaloid stimulant,
caffeine is present in wide variety of consumer beverages including coffee, tea, and soft drinks
(molecular structure of caffeine depicted in Figure 1.) Caffeine displays a region of maximum
absorbance in IR spectra at approximately 1655cm-1, which emanates from stretches of the
carbonyl groups. (3) The intensity of the absorption at this characteristic peak is inherently
related to the quantity of the caffeine through the Beer-Lambert law. In this experiment
caffeine concentration was determined for samples of decaffeinated espresso, Kazzal espresso,
cold brew coffee, and black tea using ATR-FTIR absorbance spectroscopy.
Figure 1. Molecular structure of caffeine (4)
Experimental
Theory
Molecules can be made to transition between quantized vibrational states when
exposed to infra-red radiation. For excitation to occur, incident photons must possess a
frequency that is equivalent to the energy difference between vibrational modes. In IR
absorbance spectroscopy, attenuation of incident light as a consequence of molecular
vibrational transitions is measured as a decrease in transmittance of the IR source beam. The
intensity of transmitted light can be readily converted into a corresponding measure of
absorbance using the equation, 𝐴 = −log(𝑃𝑇
𝑃0), where PT and Po are respective measures of
radiant power for the transmitted and incident light. (5)
Intensity of absorption by an analyte at a discrete photonic frequency is related to the
concentration of the absorber through the Beer-Lambert Law, 𝐴 = 𝜀𝑏𝑐, where 𝜀 is the molar
absorptivity of the analyte (M-1cm-1) at the vibrational transition frequency, 𝑏 is the path length
(cm), and c is the concentration (M) of analyte in the sample. (5)
Due to the linear dependence of absorbance on analyte concentration, calibration
curves can be developed from a series of standard samples for which the exact concentration of
analyte is known. Using these calibration curves, analyte concentrations within unknown
samples can be interpolated by substituting measured values of absorbance derived from IR
spectra. (6)
Instrumentation
IR spectra were collected over the 1500- 1800cm-1 range, with absorbance
measurements being collected every 0.48cm-1. All measurements were obtained on a Shimadzu
IRAffinity spectrometer equipped with a PIKE MIRacle single reflection ATR attachment
containing a zinc-selenide crystal sample deposition platform.
Procedures
A primary 0.00515 M stock solution (SS1) of caffeine was prepared in a 250mL
volumetric flask by solvating 0.2488 grams of powdered caffeine in reagent grade chloroform. A
second 0.000515M stock solution was prepared by diluting a 10mL aliquot of SS1 by a factor of
ten. Four standard caffeine samples with concentrations of 2.57x10-5M (5.00 ppm,) 7.72x10-5M
(15.0 ppm,) 1.54x10-4M (30.0 ppm,) and 2.06X10-4M (40.0 ppm,) were prepared from SS2. The
volumes of SS2 and chloroform used to prepare standard sample solutions are outlined in Table
A1 of the appendix.
Caffeine was extracted from previously prepared samples of decaffeinated espresso,
Kazaal espresso, cold brew coffee, and black tea, using 3mL of caffeine and a seperatory funnel.
A 1mL aliquot of each sample extract was placed in 10mL volumetric flask and diluted to
volume with chloroform.
Data Analysis
Raw FTIR spectra were smoothed to filter out noise using a moving average approach.
Peaks associated with caffeine’s carbonyl stretch were observed over the 1610- 1685cm-1
range. Maximum absorbance intensities of these peaks were corrected with respect to their
sloping baseline, which was calculated using a two-point algorithm. (3) A calibration curve was
generated by plotting the value of maximum corrected absorbance intensity as a function of
standard sample concentration. Linear regression analysis was performed to calculate the most
accurate line of best fit. The concentration of caffeine in the four beverage sample extracts
were determined by solving a rearranged form of the linear regression equation using baseline
corrected absorbance values obtained from each sample’s smoothed IR spectrum. The
uncertainty in calculated analyte concentrations was said to be proportional to the standard
deviation about the regression, where uncertainty is introduced only a consequence of the
variability in corrected absorbance measurements. (7) A value for this uncertainty was
calculated using the equation:
𝑠𝑟 = √∑ (𝑦𝑖−�̂�𝑖)2𝑖
𝑛−2
where 𝑦𝑖 is the experimental value of corrected absorbance for a given standard, �̂�𝑖 is the
corresponding value predicted by the regression line, and n is the total degrees of freedom (for
this regression n=3.) After determining the slope (m) and standard deviation (𝑠𝑟) of the
regression the LOD was calculated with the expression, 𝐿𝑂𝐷 =3𝑥𝑠𝑟
𝑚. (5)
Results
Raw and smoothed FTIR spectra used to obtained absorbance data for standard samples
and caffeine extracts of beverage samples are provided in the appendix (Figures A2-A17.) A
calibration curve was developed using corrected absorbance intensities of standard samples 1,
3, and 4 (Figure 2.) Corrected absorbance and concentration values used to construct the
caffeine standard calibration curves are presented in Table A2 of the appendix. Standard
sample 2 was omitted from the calibration curve following analysis of residual plots that
deemed it to be an outlier (Figure A1.) Confidence intervals were placed about the regression in
an effort to gauge whether the function defined by the calibration curve was a statistically
reasonable projection of the relationship between corrected absorbance and predicted
concentration (Figure 3.) From Figure 3, it can be seen that the true corrected absorbance
values for standards 1,3, and 4, lie outside of the 95% confidence interval. Values of the slope,
intercept, correlation coefficient and 95% confidence intervals acquired from least-squares
regression analysis of the calibration curve are reported in Table 1. Uncertainty in the slope and
intercept are reported as the standard error. The standard deviation about the regression,
which manifests as uncertainty within calculated caffeine concentrations was determined to be
0.005 M (1096 ppm.) The limit of detection defined by this calibration curve was 1.2x10-4 M
(22.7 ppm.)
Caffeine extracts from beverage samples displayed peak absorbance for the carbonyl
stretch at 1660.72cm-1 (Figures A10-A17.) The calculated concentration of caffeine in
decaffeinated espresso, Kazaal espresso, cold brew coffee, and black tea were -7.69x10-5
0.005 M (-14.9 ppm,) 4.99x10-4 0.005 M (96.8 ppm,) 4.45x10-4 0.005 M (86.3 ppm,) and
8.45x10-4 0.005 M (164ppm) respectively. A summary of corrected absorbance inputs and
resultant values of caffeine quantity for beverage sample extracts are presented in Table 2.
Figure 2. Caffeine calibration curve developed from concentration and corrected absorbance values of standards 1, 3, and 4.
Figure 3. Caffeine calibration curve featuring 95% confidence intervals estimated by linear regression analysis. Error bars depict upper (0.06) and lower (-0.11) confidence bands.
Table 1. Least squares regression analysis of calibration curve Slope 144.95 (43.0) Intercept -0.02419 (0.006)
Upper 95% Confidence Limit 0.06
Lower 95% Confidence Limit -0.11
Correlation Coefficient (R2) 0.91 Standard Deviation of Regression (𝑠𝑟) 0.005
Limit of Detection (LOD) 0.00011 (M), 23 (ppm)
Table 2. Baseline corrected absorbance intensity and calculated concentration of caffeine in extracts of decaffeinated espresso, Kazaal espresso, cold brew coffee, and black tea
Beverage Extract Baseline Absorbance
Intensity
Concentration (M) Concentration (ppm)
Decaffeinated espresso
-0.03534 -7.69x10-5 (0.005) -14.9
Kazaal espresso 0.04812 4.99x10-4 (0.005) 96.8
Cold brew coffee 0.04025 4.45 x10-4 (0.005) 86.3
Black tea 0.09834 8.45x10-4 (0.005) 164
Discussion
Black tea was found to have the highest concentration of caffeine (164ppm,) followed
by Kazaal espresso (96.8 ppm) and cold brew coffee (86.3 ppm.) A negative value for the
caffeine concentration of decaffeinated espresso (-14.9 ppm) indicated that the true
concentration was below the 23 ppm LOD. A relative error for calculated caffeine
concentrations could not be determined as there are no previously reported values of the exact
caffeine concentration present within these samples. Nonetheless, the accuracy of caffeine
concentrations predicted using the calibration curve are extremely limited.
There are several indicators of the inherent inaccuracy of the calibration curve, with the
most obvious being the large standard deviation about the regression (𝑠𝑟=0.005.) Ultimately,
this means there is significant uncertainty regarding where the absolute best fit of the
regression lies. Hence, the estimated function used in this analysis most likely does not reflect
the relationship between corrected absorbance intensity and caffeine concentration. Further
evidence for this claim is presented in Figure 3., where it is shown that all measures of
corrected absorbance intensity fall outside of the 95% confidence interval. Again, this suggests
that the calibration curve is not a statistically significant representation of corrected
absorbance as a function of caffeine concentration. An effective way to minimize the
uncertainty in the calibration curve would be to acquire a greater number of replicate
absorbance measurements for the standards.
A second major issue with the calibration curve was that its linear dynamic range only
spanned one order of magnitude. Moreover, the regression did not encompass corrected
absorbance values greater than 0.0028. As a result, concentration values of caffeine in
beverage sample extracts were extrapolated rather than interpolated, since they all displayed
corrected absorbance intensities that were above the 0.0028 upper absorbance limit of the
curve. In future experiments, standards should be prepared at concentrations that are more
likely to mirror those present in analyzed samples.
Apart from the considerable levels of uncertainty that arose from a lack of replicate
trials and a small linear dynamic range, the most fundamental source of error in this
experiment was introduced by fluctuations in spectral baselines. A lack of resolution in FTIR
spectra for samples with a low caffeine concentrations made it difficult to apply the two-point
algorithm, as the baseline peak width was not apparent. Thus, maximum absorbance values for
standard one and the decaffeinated espresso were corrected to a limited degree of accuracy
which may account for their negative corrected absorbance values and observed deviations
from Beer’s law.
The overwhelmingly high levels of uncertainty associated with this analysis suggests that
the calibration method utilized was inadequate for accurate quantification of caffeine in
beverage sample extracts. Given the limited sensitivity of IR methods it would be appropriate to
use an alternate form of molecular absorption spectroscopy, such as Uv-vis absorbance, to
perform quantitative analysis on caffeine analytes.
Works Cited
(1) Shaikh, T. N.; Agrawal, S.A. Qualitative and Quantitative Characterization of Textile Material by Fourier Transform Infra-Red: A Brief Review. J. Eng. Tech. 2014, 3, 8496-8501.
(2) Bradley, M. FTIR Sample Techniques: Attenuated Total Reflection (ATR). ThermoFisher: Waltham, MA. Technical Bulletin [Online] (accessed Apr. 28, 2018).
(3) G. Petrucci, FTIR of Caffeine in Beverages. CHEM 219 Lab Manual, University of Vermont, 2018.
(4) National Center for Biotechnology Information. PubChem Compound Database; CID=2519,
https://pubchem.ncbi.nlm.nih.gov/compound/2519 (accessed Apr. 30, 2018). (5) Skoog, D. A.; Holler, F.J.; Crouch, S.R. Principles of Instrumental Analysis. 2007. 6th edition. (6) Singh, B. R.; Wechter, M. A.; Hu, Y.; Lafontaine. C. Determination of caffeine content in
coffee using Fourier transform infra-red spectroscopy in combination with attenuated total reflectance technique: a bioanalytical chemistry experiment for biochemists. Biochem. Ed. 1998, 26, 243-247.
(7) Harvey, D. Linear Regression and Calibration Curves [Online] October 14, 2016.Expanded
Academic Index. http:/chem.libretexts.org (accessed Apr. 28, 2018).
Appendix
Table A1. Volumes of SS2 (0.000515M) used for preparation of standard caffeine samples
Standard Volume of SS2 Final Concentration (ppm)
1 0.5 4.998
2 1.5 14.994
3 3 29.998
4 4 39.984
Table A2. Baseline corrected absorbance intensities and exact concentration of caffeine standard
samples. Sample 2 was omitted from calibration curve.
Standard Sample Concentration (M) Corrected Absorbance
Intensity
1 2.57x10-5 -0.0217 2 7.72x10-5 0.00068 3 1.54x10-4 0.00267
4 2.06x10-4 0.00245
Figure A1. Residual error in corrected absorbance measurements for standards 1,2,3 and 4, as a
function of standard sample concentration in linear regression model.
Figure A2. Raw FTIR spectrum of caffeine standard 1
Figure A3. Smoothed FTIR Spectrum of caffeine standard 1
-0.02
-0.015
-0.01
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99.
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9061
Ab
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Wavenumber (cm-1)
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Wavenumber (cm-1)
Absorbance Sloping Baseline
Caffine Peak 1666 wavenumbers Linear (Sloping Baseline )
Figure A4. Raw FTIR spectrum of caffeine standard 2
Figure A5. Smoothed FTIR Spectrum of caffeine standard 2
-0.035
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Ab
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Wavenumber (cm-1)
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Wavenumber (cm-1)AbsorbanceSloping BaselineCaffeine Peak 1660 wavenumbers
Figure A6. Raw FTIR spectrum of caffeine standard 3
Figure A7. Smoothed FTIR Spectrum of caffeine standard 3
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Wavenumber (cm-1)
Absorbance Sloping Baseline
Caffeine peak 1660 wavenumbers Linear (Sloping Baseline )
Figure A8. Raw FTIR spectrum of caffeine standard 4
Figure A9. Smoothed FTIR Spectrum of caffeine standard 4
-0.02
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Wavenumber (cm-1)
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wavenumber (cm-1)
Absorbance Sloping Baseline
Caffeine Peak 1663 wavenumbers Linear (Sloping Baseline )
Figure A10. Raw FTIR Spectrum of cold brew caffeine extract
Figure A11. Smoothed FTIR Spectrum of cold brew caffeine extract
-0.02
-0.01
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Wavenumber (cm-1)
Absorbance Sloping Baseline
Caffiene peak 1660 wavenumbers Linear (Sloping Baseline )
Figure A12. Raw FTIR Spectrum of decaffeinated espresso caffeine extract
Figure A13. Smoothed FTIR Spectrum of decaffeinated espresso caffeine extract
-0.01
-0.005
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6.4
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1.1
830
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9.86
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8.54
2526
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7.22
2268
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2011
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4.58
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167
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1496
168
1.94
1239
169
0.62
0982
169
9.30
0724
170
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0467
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6.66
0209
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5.33
9952
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9695
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9437
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1.37
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8923
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8.73
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7.41
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981
5
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4.77
7893
Ab
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ance
Wavenumber (cm-1)
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Wavenumber (cm-1)
Absorbance Sloping Baseline
Caffeine peak 1660 wavenumbers Linear (Sloping Baseline )
Figure A14. Raw FTIR Spectrum of Kazaal espresso caffeine extract
Figure A15. Smoothed FTIR Spectrum of Kazaal espresso caffeine extract
-0.02
-0.01
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
149
9.66
6644
150
8.82
8594
151
7.99
0545
152
7.15
2495
153
6.31
4446
154
5.47
6396
155
4.63
8347
156
3.80
0298
157
2.96
2248
158
2.12
4199
159
1.28
6149
160
0.44
81
160
9.61
005
161
8.77
2001
162
7.93
3951
163
7.09
5902
164
6.25
7853
165
5.41
9803
166
4.58
1754
167
3.74
3704
168
2.90
5655
169
2.06
7605
170
1.22
9556
171
0.39
1506
171
9.55
3457
172
8.71
5408
173
7.87
7358
174
7.03
9309
175
6.20
1259
176
5.36
321
177
4.52
516
178
3.68
7111
179
2.84
9061
Ab
sro
ban
ce
Wavenumber (cm-1)
-0.02
-0.01
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
1450 1500 1550 1600 1650 1700 1750 1800 1850
Ab
sro
ban
ce
Wavenumber (cm-1)
Absorbance Sloping Baseline
Caffiene peak 1660 wavenumbers Linear (Sloping Baseline )
Figure A16. Raw FTIR Spectrum of black tea caffeine extract
Figure A17. Smoothed FTIR Spectrum of black tea caffeine extract
-0.02
-0.015
-0.01
-0.005
0
0.005
0.01
0.015
0.02
0.025
0.03
149
9.66
6644
150
8.82
8594
151
7.99
0545
152
7.15
2495
153
6.31
4446
154
5.47
6396
155
4.63
8347
156
3.80
0298
157
2.96
2248
158
2.12
4199
159
1.28
6149
160
0.44
81
160
9.61
005
161
8.77
2001
162
7.93
3951
163
7.09
5902
164
6.25
7853
165
5.41
9803
166
4.58
1754
167
3.74
3704
168
2.90
5655
169
2.06
7605
170
1.22
9556
171
0.39
1506
171
9.55
3457
172
8.71
5408
173
7.87
7358
174
7.03
9309
175
6.20
1259
176
5.36
321
177
4.52
516
178
3.68
7111
179
2.84
9061
Ab
sorb
ance
Wavenumber (cm-1)
-0.015
-0.01
-0.005
0
0.005
0.01
0.015
0.02
0.025
0.03
1450 1500 1550 1600 1650 1700 1750 1800 1850
Ab
sro
ban
ce
Wavenumber (cm-1)
Absorbance Sloping Baseline
Caffeine peak 1663 wavenumbers Linear (Sloping Baseline )