Determination of caffeine content in beverages by Fourier

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

-0.005

0

0.005

0.01

0.015

0.0214

99.

6666

4415

08.

8285

9415

17.

9905

4515

27.

1524

9515

36.

3144

4615

45.

4763

9615

54.

6383

4715

63.

8002

9815

72.

9622

4815

82.

1241

9915

91.

2861

4916

00.

4481

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)

-0.006

-0.004

-0.002

0

0.002

0.004

0.006

0.008

1450 1500 1550 1600 1650 1700 1750 1800 1850Ab

sorb

ance

Wavenumber (cm-1)

Absorbance Sloping Baseline

Caffine Peak 1666 wavenumbers Linear (Sloping Baseline )



-0.035

-0.03

-0.025

-0.02

-0.015

-0.01

-0.005

0

0.005

0.01

0.015

0.02

149

9.66

6644

150

8.34

6386

151

7.02

6129

152

5.70

5872

153

4.38

5614

154

3.06

5357

155

1.74

5099

156

0.42

4842

156

9.10

4585

157

7.78

4327

158

6.46

407

159

5.14

3813

160

3.82

3555

161

2.50

3298

162

1.18

304

162

9.86

2783

163

8.54

2526

164

7.22

2268

165

5.90

2011

166

4.58

1754

167

3.26

1496

168

1.94

1239

169

0.62

0982

169

9.30

0724

170

7.98

0467

171

6.66

0209

172

5.33

9952

173

4.01

9695

174

2.69

9437

175

1.37

918

176

0.05

8923

176

8.73

8665

177

7.41

8408

178

6.09

815

179

4.77

7893

Ab

sorb

ance

Wavenumber (cm-1)

-0.015

-0.01

-0.005

0

0.005

0.01

0.015

1450 1500 1550 1600 1650 1700 1750 1800 1850

Ab

sorb

ance

Wavenumber (cm-1)AbsorbanceSloping BaselineCaffeine Peak 1660 wavenumbers



-0.01

-0.005

0

0.005

0.01

0.015

0.02

0.025

0.03

149

9.66

664

4

150

8.34

638

6

151

7.02

612

9

152

5.70

587

2

153

4.38

561

4

154

3.06

535

7

155

1.74

509

9

156

0.42

484

2

156

9.10

4585

157

7.78

4327

158

6.46

407

159

5.14

381

3

160

3.82

355

5

161

2.50

329

8

162

1.18

304

162

9.86

278

3

163

8.54

252

6

164

7.22

226

8

165

5.90

201

1

166

4.58

175

4

167

3.26

149

6

168

1.94

123

9

169

0.62

098

2

169

9.30

072

4

170

7.98

046

7

171

6.66

020

9

172

5.33

995

2

173

4.01

969

5

174

2.69

9437

175

1.37

918

176

0.05

8923

176

8.73

866

5

177

7.41

840

8

178

6.09

815

179

4.77

789

3

Ab

sorb

ance

Wavenumber (cm-1)

-0.01

-0.005

0

0.005

0.01

0.015

0.02

0.025

1450 1500 1550 1600 1650 1700 1750 1800 1850

Ab

sorb

ance

Wavenumber (cm-1)


Caffeine peak 1660 wavenumbers Linear (Sloping Baseline )



-0.02

-0.01

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

149

9.66

6644

150

8.34

6386

151

7.02

6129

152

5.70

5872

153

4.38

5614

154

3.06

5357

155

1.74

5099

156

0.42

4842

156

9.10

4585

157

7.78

4327

158

6.46

407

159

5.14

3813

160

3.82

3555

161

2.50

3298

162

1.18

304

162

9.86

2783

163

8.54

2526

164

7.22

2268

165

5.90

2011

166

4.58

1754

167

3.26

1496

168

1.94

1239

169

0.62

0982

169

9.30

0724

170

7.98

0467

171

6.66

0209

172

5.33

9952

173

4.01

9695

174

2.69

9437

175

1.37

918

176

0.05

8923

176

8.73

8665

177

7.41

8408

178

6.09

815

179

4.77

7893

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

0.035

0.04

1450 1500 1550 1600 1650 1700 1750 1800 1850

Ab

sorb

ance

wavenumber (cm-1)


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

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

149

9.66

6644

150

8.34

6386

151

7.02

6129

152

5.70

5872

153

4.38

5614

154

3.06

5357

155

1.74

5099

156

0.42

4842

156

9.10

4585

157

7.78

4327

158

6.46

407

159

5.14

3813

160

3.82

3555

161

2.50

3298

162

1.18

304

162

9.86

2783

163

8.54

2526

164

7.22

2268

165

5.90

2011

166

4.58

1754

167

3.26

1496

168

1.94

1239

169

0.62

0982

169

9.30

0724

170

7.98

0467

171

6.66

0209

172

5.33

9952

173

4.01

9695

174

2.69

9437

175

1.37

918

176

0.05

8923

176

8.73

8665

177

7.41

8408

178

6.09

815

179

4.77

7893

Ab

sorb

ance

wavenumber (cm-1)

-0.02

-0.01

0

0.01

0.02

0.03

0.04

0.05

0.06

1400 1450 1500 1550 1600 1650 1700 1750 1800 1850

Ab

sorb

ance

Wavenumber (cm-1)


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

0

0.005

0.01

0.015

0.02

149

9.66

6644

150

8.34

6386

151

7.02

6129

152

5.70

5872

153

4.38

5614

154

3.06

5357

155

1.74

5099

156

0.42

4842

156

9.10

4585

157

7.78

4327

158

6.4

640

7

159

5.14

3813

160

3.82

3555

161

2.50

3298

162

1.1

830

4

162

9.86

2783

163

8.54

2526

164

7.22

2268

165

5.90

2011

166

4.58

1754

167

3.26

1496

168

1.94

1239

169

0.62

0982

169

9.30

0724

170

7.98

0467

171

6.66

0209

172

5.33

9952

173

4.01

9695

174

2.69

9437

175

1.37

918

176

0.05

8923

176

8.73

8665

177

7.41

8408

178

6.0

981

5

179

4.77

7893

Ab

sorb

ance

Wavenumber (cm-1)

-0.001

0

0.001

0.002

0.003

0.004

0.005

0.006

0.007

0.008

0.009

1450 1500 1550 1600 1650 1700 1750 1800 1850

Ab

sorb

ance

Wavenumber (cm-1)



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)


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)



Documents

Determination of caffeine content in beverages by Fourier