Methods Available:
UV-Visible Spectrophotometry enables the analysis and detection of compounds that absorb in the UV-visible light region. A beam of UV-visible light is passed through the sample and the absorption of light is measured by a detector in the spectrophotometer. In particular, this method can only be used for compounds containing chromophores. Chromophores are functional groups that are able to absorb UV and/or visible light. When a beam of UV/Visible Light is passed through the sample in a cuvette, compounds absorb light as it absorbs energy for its electrons to undergo rearrangement.
The signal produced is read as Absorbance. Detection of caffeine present in the sample will be displayed as peak(s) in the UV spectrum at the expected wavelength(s) of absorption. The identity can be confirmed by Peak Identification. This is done by comparing the sample's spectrum to the stamdard UV spectrum of caffeine and the position of maximal absorption.
Quantification of caffeine by UV-visible spectrophotometry can be done by measuring the absorbance at the wavelength where maximal absorption takes place. If multiple standards are available, it is calculated by plotting a calibration graph. In an event where only one standard is available, simultaneous equations using Beer-Lambert's law is used to find the concentration of the caffeine in the sample.
The most common technique used to determine caffeine concentration in beverages is High Performance Liquid Chromatography (HPLC). HPLC is widely used because it is able to analyse multi-component samples such as commercial beverages. It will efficiently separate caffeine from the other substances in the coffee, thus allowing the detection and quantification of caffeine present.
Gas Chromatography-Mass Spectrometry (GCMS) is one of the techniques that can be used to determine the amount of caffeine in coffee and beverages. This is a technique that combines 2 methods. Gas Chromatography allows the separation of components in a multi-component sample whereas Mass Spectrometry allows the identification and quantification of the components. Hence, this is an extremely powerful analytical method that can be used to separate the components in coffee and allow precise analysis of the caffeine present.
Voltammetry can also be used to determine the amount of caffeine present in the beverages. It has been used by people and is found to be relatively sensitive and good to detect caffeine. The 3-electrode system can be used to detect caffeine and different types of electrodes can be utitlised. For instance, one of the method is the utilisation of Glassy Carbon Electrode for caffeine detection by differential-pulse voltammetry. However, this method has a shortcoming. In order to increase sensitivity and peak resolution, an acid-methanol needs to be used as both a solvent and supporting electrolyte. However, the volatile methanol gives a difficulty for quantitative determination. Another improvised method is to use Square-Wave voltammetric method to determine the caffeine quantity. This method uses a different electrolyte known as Nafion-ruthenuim oxide pyro-chlore chemically modified electrode (CME). This method have been proven to be relatively rapid and sensitive.
- UV-Visible Spectrophotometry
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| UV Spectrophotometer |
UV-Visible Spectrophotometry enables the analysis and detection of compounds that absorb in the UV-visible light region. A beam of UV-visible light is passed through the sample and the absorption of light is measured by a detector in the spectrophotometer. In particular, this method can only be used for compounds containing chromophores. Chromophores are functional groups that are able to absorb UV and/or visible light. When a beam of UV/Visible Light is passed through the sample in a cuvette, compounds absorb light as it absorbs energy for its electrons to undergo rearrangement.
Understanding
that UV-visible Spectrophotometry is able to be used on
chromophore-containing compunds, it is thus able to be used to detect
and quantify the amount of caffeine found in coffee and beverages. This
is because caffeine contains chromophores. The presence of chromophores allow caffeine in the sample to absorb UV light at
~205nm and ~273 nm. Analysis is typically made by making a UV light of
260nm pass through the coffee sample.The caffeine should be purified before the analysis to produce a single-component sample.
 |
| UV Spectrum of Caffeine |
The signal produced is read as Absorbance. Detection of caffeine present in the sample will be displayed as peak(s) in the UV spectrum at the expected wavelength(s) of absorption. The identity can be confirmed by Peak Identification. This is done by comparing the sample's spectrum to the stamdard UV spectrum of caffeine and the position of maximal absorption.
 |
| Beer Lambert's Law |
Quantification of caffeine by UV-visible spectrophotometry can be done by measuring the absorbance at the wavelength where maximal absorption takes place. If multiple standards are available, it is calculated by plotting a calibration graph. In an event where only one standard is available, simultaneous equations using Beer-Lambert's law is used to find the concentration of the caffeine in the sample.
- Reverse Phase HPLC
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| HPLC |
The most common technique used to determine caffeine concentration in beverages is High Performance Liquid Chromatography (HPLC). HPLC is widely used because it is able to analyse multi-component samples such as commercial beverages. It will efficiently separate caffeine from the other substances in the coffee, thus allowing the detection and quantification of caffeine present.
There are many different types of HPLC methods that can be used - Normal Phase and Reverse Phase HPLC. Since coffee is an aqueous sample, it is not able to be conducted through Normal Phase HPLC as the high water content is likely to affect the polar stationary phase, producing poor analysis. Hence, in the analysis of caffeine in coffee, a non-polar stationary phase is used instead. This is known as the Reverse Phase HPLC.
During HPLC, a high pressure is applied to force the sample and mobile phase through a packed column. There are particles in the column which are coated with a liquid phase (stationary phase) that help to separate the components present in the sample, allowing the analysis of each individual component before passing on to the detector and being read as a signal output. This signal is then plotted on a graph against time for further interpretations.
Due to the different properties of the components, components will be eluted at different timings. Hence, the detection of caffeine can be done by comparing the retention times of standards and the sample. Also, the quantification of caffeine can be done by plotting a calibration graph. The standard's peak area and concentration is plotted. This allows the determination of caffeine in the sample of interest by extrapolating the sample's caffeine peak area.
- Gas chromatography-Mass spectrometry
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| Schematic Diagram of GC/MS |
In GC-MS, the components are first separated and eluted at different timings to the mass spectrometer. As the components are eluted into the mass spectrometer, they are bombarded by electrons and ions to be fragmented into charged fragments. The signal will pick this charged fragments as signals and plot them onto a graph. Thus, a Mass Spectrum is produced. It is plotted with Abundance against M/Z ratio. M/Z ratio is mass-to-charge ratio.Since most of the charge is usually +1, M/Z ratio typically indicates the molecular weight of the component. The mass spectrum is typically the same for each given chemical component, hence it is like a fingerprint for the molecule. By using the mass spectrum produced, qualitative and quantitative analysis can be done.
For the analysis of caffeine, m/z ratio of 194 and 109 ions are selected to identify caffeine in the sample.
Caffeine was qualitatively identified by comparing with the caffeine standard of retention time and m/z ratio. Quantitative analysis can be done by plotting a calibration graph with the use of multiple standards. The calibration graph would be constructed by plotting peak area against concentration. The peak area of the caffeine in the sample of interest can then be extrapolated on the calibration graph.
- Voltammetry
 |
| Schematic Diagram of 3-Electrode System |
Voltammetry can also be used to determine the amount of caffeine present in the beverages. It has been used by people and is found to be relatively sensitive and good to detect caffeine. The 3-electrode system can be used to detect caffeine and different types of electrodes can be utitlised. For instance, one of the method is the utilisation of Glassy Carbon Electrode for caffeine detection by differential-pulse voltammetry. However, this method has a shortcoming. In order to increase sensitivity and peak resolution, an acid-methanol needs to be used as both a solvent and supporting electrolyte. However, the volatile methanol gives a difficulty for quantitative determination. Another improvised method is to use Square-Wave voltammetric method to determine the caffeine quantity. This method uses a different electrolyte known as Nafion-ruthenuim oxide pyro-chlore chemically modified electrode (CME). This method have been proven to be relatively rapid and sensitive.
Caffeine quantification can be achieved by measuring the current of the oxidation peak after subtraction of the background. Different standards will be used to plot a calibration graph of Current against Concentration. The sample of interest will be analysed using the same method and its value is extrapolated to obtain the concentration. In an event that the values do not fall in the linear range, the samples are diluted to a suitable dilution factor and quantified again.
There are also many novel techniques that chemical analysts have developed. Currently, there are a wide range of analytical methods that can be used to detect caffeine in samples like commercial beverages. For example, there are Ion Chromatography, Flow Injection Fourier Transform Infrared Spectrocopy, NIR Spectroscopy and Mircroemulsion Electrokinetic Chromatography.
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