CHEM 127.1, Advanced Analytical Chemistry Laboratory Page 1 of 5 Experiment No. 8: Determination of Caffeine in Beverages Using High Performance Liquid Chromatography BARBA, Bin Jeremiah D.
Group No. 1, Chem 127.1, MAD, Prof. Jireh Sacramento October 3, 2011
I. Abstract
High performance liquid chromatography is technique for the separation of the components of a sample by means of a high pressure pump forcing the eluent through a column. Polar, dispersive or ionic forces working on the samples allow its components to either be retained in the stationary phase or eluted out by the mobile phase thereby producing responses with different retention times. The area under each response (peak) can be used to obtain its concentration. HPLC is used extensively for analysis of drugs, food and beverage analysis, and in routine analysis of a wide range of analytes. In this experiment, reverse phase HPLC was used to determine the caffeine content of tea, coffee and cola products. External calibration was done to generate the standard curve where the respective concentrations of caffeine in the samples were intrapolated from. The concentrations calculated were 0.173M, 0.009M 0.003M and 0.005M for Great Taste Coffee, Lipton Tea, Pop and Zesto Cola. When compared to the US standard caffeine content of such beverage, the percent error was found to be extremely high showing inefficiency of the chromatographic system which may be due to error in the calibration, measurement of chemicals, or column conditioning.
II. Keywords: high performance liquid chromatography, retention time, stationary phase, mobile phase III. Introduction
Chromatographic separations are based on the forced transport of a liquid mobile phase carrying the analyte through the porous particles of the stationary phase, and the retention of the analyte in either phases resulting in different migration rates.
Liquid chromatography (LC) uses capillary forces and/or electroosmotic flow to analyze liquid samples. Before, LC was done using large columns and large matrix particles under a gravity feed which was highly time-consuming and required manual operation almost all throughout the chromatographic run.
In 1964, the American chemist J. Calvin Giddings summarized the necessary conditions that would give liquid chromatography the
resolving power achievable in gas
chromatograph. He predicted that very small particles with a thin film of stationary phase in small-diameter columns will increase the efficiency and resolution of the separation. This paved the way for the development of the technique now termed high-performance liquid chromatography (HPLC) which primarily depended on the development of pumps that would deliver a steady stream of liquid at high pressure to the column to force the liquid through the narrow interstitial channels of the packed columns at reasonable rates, and detectors that would sense the small sample sizes mandated. The first High Performance Liquid Chromatograph was built by Professor C. Horvath who further defined the term “performance” in HPLC as “an aggregate of the efficiency parameters.”
A typical HPLC is composed of the reservoir which contains the solvent/s to be used in the chromatographic runs; the pump which
forces the mobile phase through the column, containing the stationary phase, at a specific flow rate; the injector which introduces the liquid sample; the detector which detects (i.e. by UV, MS, refractive index, fluorescence) the eluted component and monitors the response of the analyte in the form of a chromatogram; and the integrator or computer which allows the visualization of the chromatogram as well as the measurement of certain parameters such as retention time and peak area. Some of the more recent machines also include autosampler which allow batch chromatographic runs; mixing vessels for solvent gradients and degassers which reduce dissolved gas content that may introduce errors in separation.
HPLC allows the separation and/or purification of analytes in a given sample by utilizing the different forces acting on the analytes allowing for their partition between the mobile and stationary phase. For example, in reverse phase, dispersive forces act on the sample so that the more polar analyte will be retained in the mobile phase and eluted earlier, while non-polar analytes are retained in the stationary phase and eluted much later.
HPLC assays are powerful tools to gather qualitative and quantitative data, to understand and monitor chemical reactions and subsequent product isolation. They are primarily used to quickly access conversion, relative reaction purity profiles and purity of isolated products. Caffeine can be qualitatively and quantitatively detected by HPLC in beverage samples and pain relievers.
The objective of this experiment is to apply the principles of reverse phase high performance liquid chromatography to determine the concentration of caffeine in beverages such as teas, coffees and soda.
CHEM 127.1, Advanced Analytical Chemistry Laboratory Page 2 of 5 IV. Methodology
Caffeine from beverage samples was determined using High Performance Liquid Chromatography.
External calibration was done by preparing the following quantities of caffeine: 2.5, 5.0, 7.5 and 10 mg. They were diluted in a 100 mL volumetric flask using the solvent used for the mobile phase (2:8 methanol to water adjusted to approximately pH 3.5 with phosphoric acid). The solutions were shaken to ensure dissolution and then subsequently degassed for 5 minutes and filtered using 0.45 m filter membrane.
The pump and detector (UV-Vis) were turned on, with the pump flow rate at 2.3 mL/min and detector sensitivity at 0.08 AUFS (absorbance unit full scale). The recorder was turned on and set at a slow speed rate. The mobile phase was passed through the column (C18) for 5-10 minutes for equilibration and record
detector response to ensure the column was washed and no residual analytes from previous runs were retained.
With a glass syringe, 25 L of caffeine standards were injected into the column starting with the least concentrated and doing two trials.
The samples to be analyzed for caffeine content were prepared next. Coffee and tea samples, 5 mL and 0.5 mL respectively, were diluted into 25 mL volumetric flask using the mobile phase solvent. Cola beverage, on the other hand, was first poured back and forth between beakers to cease bubbling. An alternative method for this was done by placing the sample in an ultrasonicator for about 5 mines. 10 mL of the decarbonated cola was also diluted in a 25 mL volumetric flask. The samples were injected and run in the same manner as the standards. The samples used were Great Taste Coffee, Lipton Tea and Zesto and Pop Cola.
After the last chromatographic run, the column was flushed with 50 mL of the solvent (not adjusted to pH 3.50).
V. Results a. Raw Data
Table 1. Retention time and peak area of caffeine standards
Sample Retention Time (min) Peak Area
1A 3.32 1424257 2A 3.383 2170162 3A 3.32 3008474 4A 3.223 3629426 1B 3.398 1273650 2B 3.382 2216944 3B 3.31 3076734 4B 3.215 3761981
Table 2. Retention time & peak area beverage samples
Sample Retention Time
(min) Peak Area
Great Taste 1 3.146 2885639 Great Taste 2 3.11 2553966 Lipton 1 3.33 1729109 Lipton 2 3.716 1762299 Pop 1 3.409 1427172 Pop 2 3.347 1328449 Zesto 1 3.4 1893026 Zesto 2 3.401 1865263
b. Pertinent Equations, Sample
Computations
Concentration of Standards (for 25 mg Caffeine):
Table 3. Concentration of Standards
Sample Concentration
Standard 1 0.00128733
Standard 2 0.00257467
Standard 3 0.00386200
Standard 4 0.00514933
Concentration of Samples (Great Taste 1): (Eq. 1) (Eq. 2) Table 4. Concentration of caffeine in various samples of coffee, tea and soft drinks (intrapolated from the average of regression values from standard A and B)
Sample [Caffeine] (M) [Caffeine]*DF (M)
Great Taste 0.00378405 0.1892026 0.00317447 0.15872371 Lipton 0.00178663 0.00893317 0.00195025 0.00975126 Pop 0.00126517 0.00316291 0.00127935 0.00319838 Zesto 0.00206973 0.00517433 0.00210947 0.00527368
Table 5. Average concentration of caffeine in the samples analyzed
Samples Ave Conc (M)
Great Taste 0.173121951
Lipton 0.009364794
Pop 0.003181626
CHEM 127.1, Advanced Analytical Chemistry Laboratory Page 3 of 5 c. Plots/Graphs
Figure 1. Standard curves for caffeine (A: above, B: below)
Table 6. Regression values for standard curve of caffeine
Std y-intercept Slope R
A 694625 579012660 0.99843758
B 501131.5 646669143 0.99754715
Average 597878.25 612840902 n/a
VI. Discussion
In HPLC, important to consider are the nature of the stationary phase (or column), the mobile phase which controls the partitioning or separation of the sample analyte and their rate and order of elution.
The nature of the stationary phase will determine whether the column can be used for normal or reverse phased chromatography. For normal phase, a polar stationary phase and non-polar mobile phase so that more non-polar compounds will be eluted later than non-polar compounds generally. In reverse phase chromatography, on the other hand, uses a non-polar stationary phase and non-polar mobile phase causing polar peaks to generally elute earlier than non-polar ones.
There are different types of matrices for support of the stationary phase. Examples include silica, polymers, alumina and zirconia. Silica, most commonly matrix, are robust, easily derivatized, manufactured to consistent sphere size and do not tend to compress under pressure. The disadvantage of silica is that it is unstable at
high pH where silica can dissolve. Although in recent years, there have been silica supported columns developed for use at high pH. The nature, shape and particle of silica affect separation. Smaller particles result in greater number of theoretical plates thereby increasing separation efficiency. Use of smaller particles, however, also result in increased backpressure during chromatographic run causing column clogging which is why 5 Å columns are more frequently used then the 3 Å. Narrower particle size distribution of the particles also result in better resolution.
The stationary phase for reverse phase chromatography can be created by reacting free silanols in the silica support with a chlorosilane with hydrophobic functionality to introduce the non-polar surface. Due to steric constraints, only about one third of the surface silanols are modified allowing the remaining free silanols to interact with analytes and cause peak tailing. As a remedy, the column is further reacted with chlorotrimethylsilane to end cap remaining free silanols and improve column efficiency. Common stationary phases are C4 (butyl), C8
(dimethyloctylsilane), C18 (octadecylsilane), nitrile
(cyanopropyl) and phenyl (phenyl propyl) columns. In general, longer alkyl chains, higher phase loading and higher carbon loads provide greater retention of non-polar analytes. C18
columns are the most widely used and tend to be the most retentive for non-polar analytes. Examples include Zorbax SB-C18,
YMC-Pack-ODS and Luna C18.
The mobile phase for reverse phase is composed of an aqueous buffer and a water miscible organic solvent that has little to no absorption above 200 nm. Organic solvents are added to adjust the polarity of the mobile phase and allow the elution of non-polar analytes after the polar ones. Also, standard C18 columns and
similar stationary phases will undergo phase collapse at highly aqueous mobile phases, typically less than 5-10% organic composition, decreasing analyte-stationary phase interaction. Choice of organic solvents can affect selectivity and resolution. The common organic solvents used are methanol, acetonitrile and tetrahydrofuran arranged in increasing ability to retain analyte in the mobile phase.
The mobile phase is usually buffered to control the pH, an important parameter to consider in HPLC. At low pH, the mobile phase protonates free silanols on the column and reduces peak tailing. Also, at sufficiently low pH, basic analytes are protonated or ionized thereby allowing it them to elute more quickly with improved peak shape. On the other hand, acid analytes will remain uncharged, increasing retention. Conversely, at higher pH, basic compounds will be more retained and ionized acidic compounds will elute earlier. Peak splitting may occur when the pKa of the compound is y = 6E+08x + 694625 R² = 0.9969 0 1000000 2000000 3000000 4000000 0 0.002 0.004 0.006 Peak Ar e a Concentration (M) y = 6E+08x + 501132 R² = 0.9951 0 1000000 2000000 3000000 4000000 5000000 0 0.002 0.004 0.006 Peak Ar e a Concentration (M)
CHEM 127.1, Advanced Analytical Chemistry Laboratory Page 4 of 5 similar to that of the buffer allowing the elution of
both the charged and uncharged species. The pH of the buffer will not greatly affect the retention of non-ionizable sample components.
Before chromatographic analysis of the sample, calibration is done to establish a relationship between signals or the response detected and the concentration of the analyte. External calibration, which was the one used in the experiment, involves the construction of a standard curve by injecting different concentrations of a known standard and intrapolating from there the concentration of subsequent runs. The samples must have no variation in injection volume all throughout because the calibration parameters will be specific to the chromatographic system established. Injection of accurate volumes can be tricky and prone to human errors. A technique that overlooks the problem with volume constancy is the internal standard calibration. Internal standard calibration involves the comparison of the instrument responses from the target compounds in the sample to the responses of reference standards added to the sample or sample extract before injection. An internal standard becomes an additional component of the sample to be run and concentration can be obtained from a ratio of the peak area and concentration of the internal standard and those of the sample, or by plotting the ratio of the peak areas and concentrations of the sample and standard to generate a standard curve. This method accounts for routine variation in the response of the chromatographic system and for the variations in the exact volume of sample or sample extract introduced into the chromatographic system since whether smaller or larger injection volume is used, the ratio of peaks will be the same hence the calibration is still valid. The internal standard must have similar analytical behaviour as that of the sample and yet not found in the sample. It must be soluble in the same solvent and not a degradation product of the sample, and it should not be affected by target analytes, surrogates or matrix interferences. The difficulty of finding an appropriate internal standard that meets the criteria is the main drawback of this method. There can also be problems in purification or isolation of the analyte if the standard could not be separated from the sample. For the determination of caffeine, some internal standards used are salicylic acid and nicotinic acid.
From the chromatogram generated for each chromatographic run, some descriptors can be obtained which will help to analyze the samples. The peak area (PA) is basically, the area under each detected response which can be correlated to the concentration of the analyte. The retention time (tR) – the distance of the peak
maxima from the injection point expressed in time units – serves as the identifier for a given analyte
on that particular system. Void time (t0) is the
retention of the non-rained analyte (i.e. thiourea, uracil, NaNO3) or basically the part of the total
analyte retention time that the analyte spends in the mobile phase going through the column. It serves as the correction factor to especially adjust in case of retention drifting, as well as a validation measure for the flow rate. The retention factor or capacity factor (k), which is characteristic of a particular chromatographic system, ensures the reproducibility of the runs. It can be obtained from the ratio of the reduced retention time (tR-t0) to the
void time. The selectivity and sensitivity of the chromatographic system may be obtained by calculation of the selectivity factor (), resolution, limits of detection (LOD) and limits of quantification (LOQ). Efficiency is measured using the number or height of theoretical plates. The column is considered to be divided into a number of hypothetical plates. Each plate has a finite height and the analyte spends a finite time in that plate where retention, separation or partition occurs. The smaller the plate height and the greater number of plates ensure the efficiency of separation of the analytes in the sample. It also measures chromatographic band broadening (i.e. the narrower the band, the more efficient).
In this experiment, concentration levels of caffeine in tea, coffee, and cola beverages are determined using HPLC. A chromatogram was obtained for each analysis, i.e. the response is displayed on a graph where the x-axis is the retention time and the y-axis is a measure of the intensity of the response. The size of the peak is proportional to the concentration of the analyte and can be obtained after calibration and necessary computations have been made. When calculated, the percent error of the results obtained from probable theoretical caffeine content of the beverages resulted were very high, exceeding a 100% (not shown). Main sources of error in the HPLC analysis are in the inefficiency of calibration, measurement of chemicals (i.e. in the preparation of standards or in the injection volume which is very vital in external calibration) or in column conditioning. For the latter, it can be the column was not adequately washed before the start of the experiment or that it was not allowed to equilibrate sufficiently. The inefficiency could also come from the column such as ineffective capping of silanol residues, packing of silica particles, uniformity of C18 coating, etc. This
is particularly evident seeing as the peak produced in the chromatogram was broad and it showed tailing instead of producing a narrow peak. It can be noted that the experiment did not specify the use of a substance for the void time therefore drifts in the retention time of the analyte (3.1 – 3.7) was not accounted for. Also, degassing should be done before filtration because the latter step may still cause bubbles to form in the solvent which causes clogging of the column and errors in response detection.
CHEM 127.1, Advanced Analytical Chemistry Laboratory Page 5 of 5 VII. Conclusion and Recommendations
High performance liquid chromatography is a powerful tool for separation, purification and analysis of analytes in the sample. It is relatively simple to use and the necessary computations needed for analysis are fairly basic.
HPLC was used in the experiment to determine the concentration of caffeine present in various samples of beverages. External calibration was done and the resulting regression values were used to compute the concentrations which were 0.173M, 0.009M 0.003M and 0.005M for Great Taste Coffee, Lipton Tea, Pop and Zesto Cola respectively. However, the results were found to highly deviate from standard caffeine concentration of the products. The errors were attributed to problems in the calibration, measurement of chemicals (human error) and inefficiency of the chromatographic system itself, especially of the column.
It is therefore recommended that the procedure be re-evaluated and optimized for the determination of caffeine in the beverage samples. The void time should be measured to correct for drifting. The mobile phase should also be filtered first and then degassed finally to avoid bubble formation and gas dissolution which may affect the run. The column should be washed and equilibrated thoroughly as well as assessed for its efficiency prior to analysis. Internal standard calibration may also be used by adding salicylic acid or nicotinic acid as the internal standard to account for injection volume variation.
VIII. References
Hearn, G.M. (2009). A Guide to Validation in HPLC. Massachusetts, Perkin Elmer Corp. Guinn, R. & Zarzycki-Siek, J. (2007) The Determination of Caffeine in Beverages by High Performance Liquid Chromatogrpahy.
Jones, S. & Quay, L. (2009). Making an HPLC Calibration Work. Chromatography Focus. Retrieved from laserchrom.com.
Levin, S. (2010). High Performance Liquid Chromatography (HPLC) in the pharmaceutical analysis. Medtechnica.
Skoog D. A., Holler F. J., & Crouch S. R. (2007). Determination of Caffeine in Beverages by High Performance Liquid Chromatography. Harcourt Brace College Publishers.
Teledyne Technologies Company. (n.d.). High Perfromance Liquid Chromatography. Syringe Pump Application Note. Nebraska: Teledyne Company.
Wagaw, S., Tedrow, J., Grieme, T., Bavda, L., Wang, W., Viswanath, S., Barnes, D. & McLaughlin, M. (2011). HPLC Guide. Retrieved from chemgroups.northwestern.edu.
