ANALYTICAL BIOCHEMISTRY
HIGH-PERFOMANCE LIQUID CHROMATOGRAPHY
REPORT
PRACTICAL 8
L.T Shezi 22314912
B.Z Sibisi 22321182
N.F Shezi 2233668
S.N Gwala 22143914
L Mlambo 22313044
S.A Mthethwa 22314930
N.P Mkhwanazi 22215725
S.P Mkhwanazi 22300523
S.L Zondi 22248838
S Ngubo 22301415
High Performance Liquid Chromatography (HPLC)
INTRODUCTION
High Performance Liquid Chromatography (HPLC) is a very sophisticated
analytical method used for component separation, identification, and
quantification in complicated mixtures. It is essential to many industries,
such as clinical diagnostics, food safety, medicines, and environmental
monitoring. High pressure, small particle sizes, and several separation
modes are the key components of HPLC’s effectiveness and accuracy,
which makes it a fundamental technique in analytical biochemistry (Peter
et al., 2019). HPLC will be utilized to determine the caffeine content in
various beverages.
Caffeine is a natural stimulant found in coffee, tea, cola beverages, and
energy drinks. It is known for its ability to increase alertness and improve
concentration. However, excessive consumption of caffeine can have
negative effects on health, such as insomnia, restlessness, and increased
heart rate.
The core of HPLC lies in the use of high-pressure pumps to push a liquid
mobile phase through a column packed with small particles (the stationary
phase). Different compounds in the sample interact with the stationary
phase to varying extents, causing them to elute from the column at
different times, a process known as retention. These differences in
retention times are used to separate and quantify the components.
The separation efficiency is enhanced by use of pressures up to 6000 psi
accelerates the movement of the mobile phase through the packed
column, smaller stationary phase particles increase surface area for
interactions, resulting in better resolution (Gritti & Guiochon, 2020).
Various modes of chromatography (e.g., reversed-phase, normal-phase,
ion-exchange) allow for tailored separations based on the analyte’s
chemical properties.
The aim of this experiment is to use High Performance Liquid
Chromatography (HPLC) to separate, identify, and quantify the
concentrations of key active ingredients, such as caffeine, taurine, and
vitamins, in various commercial energy drinks. By analysing these
components, the experiment will determine whether the energy drinks
meet their labelled specifications for caffeine and other ingredients, as
well as assess variations between different brands or formulations. This
analysis highlights the utility of HPLC in the quality control of beverages,
ensuring product consistency and safety for consumers.
METHODS AND MATERIALS
Took 500ul of Bioplus and added 1ml of mobile phase solution which is 1:2
dilution. Filtered the sample with a syringe and transferred 1.5 ml of
sample to glass vial for HPLC system.
First, the sample was introduced onto the column through injection,
utilizing either an injector or autosampler. Next, the mobile phase flowed
through the column, separating the analytes based on their interactions
with the stationary phase, a process known as chromatography. The
separated analytes were then detected by a detector, which measured
their signal intensity, often using techniques such as ultraviolet (UV) or
fluorescence spectroscopy. The chromatographic data was then collected
and processed using specialized software. Finally, the data was analyzed,
involving peak integration, retention time calculation, and quantification
of the analytes.
RESULTS
DISCUSSION
The effectiveness of HPLC is rooted in its capacity to utilize various
stationary and mobile phases, allowing for tailored separation based on
the unique properties of the analytes. The method is particularly
advantageous for analysing small quantities of compounds due to its
sensitivity and accuracy, making it ideal for detecting trace levels of
substances like caffeine in beverages. HPLC’s adaptability to different
sample types and its compatibility with various detectors further enhance
its applicability, making it a staple in laboratories focused on chemical
analysis and quality control.
The results obtained from the HPLC analysis of caffeine in bioplus
demonstrated the method's effectiveness in separating and quantifying
caffeine within the beverage. The chromatograms presented clear peaks
corresponding to various compounds, with caffeine exhibiting a prominent
peak at a retention time of approximately 4.5 minutes. The distinctiveness
of this peak confirms the successful separation of caffeine from other
components present in the bioplus sample, showcasing the high resolution
and sensitivity of the HPLC technique (Almatar et al., 2023).
The peak area obtained for the bioplus sample was 1,501,150 mAU,
indicating a substantial concentration of caffeine. When compared to the
standard caffeine solutions (150 ppm and 200 ppm), the peak area
suggests that the caffeine content in bioplus exceeds these values,
reaffirming the drink's identity as an energy beverage with significant
stimulant properties. By correlating the peak area with known
concentrations from the standard curve, an accurate quantification of
caffeine concentration in bioplus can be achieved. This relationship
exemplifies the Beer-Lambert Law, which states that absorbance is
directly proportional to the concentration of the absorbing species (Cohen
et al., 2019). The experiment's results align with this principle, validating
the reliability of the method for quantifying caffeine in beverage samples.
Furthermore, the consistency of retention times across different samples
is indicative of the method's reproducibility and stability, which are critical
parameters in HPLC analyses. The observed retention times for caffeine in
both standard and sample analyses were consistent, reflecting that the
chromatographic conditions were appropriately maintained throughout the
experiment. This stability is vital for ensuring accurate and dependable
results in analytical chemistry, particularly in food and beverage testing,
where regulatory compliance is essential (Dahl et al., 2020).
The significance of accurately quantifying caffeine in beverages extends
beyond analytical interest. As caffeine consumption increases among
consumers, understanding its concentration in popular beverages like
bioplus becomes increasingly relevant for public health. The ability to
determine caffeine levels helps consumers make informed decisions about
their intake, especially for vulnerable populations such as pregnant
women and individuals with certain health conditions (Gritti et al., 2020).
HPLC thus serves not only as a method of analysis but also as a means of
promoting consumer safety and awareness in the food and beverage
industry.
Components in complex beverage matrices may affect the analysis,
leading to potential interferences. Method development is crucial to
minimize these effects. Ensuring consistency in sample preparation and
chromatographic conditions is essential for accurate results. Adhering to
safety standards and regulations, such as those set by the FDA or other
health authorities, is vital for products that contain caffeine (Smith et al.,
2023). There are challenges faced with the intake of caffeine; to help
maintain these challenges the following things are done as precaution.
There is regular monitoring of caffeine levels in various beverages to
maintain label accuracy and compliance with health regulations. Studying
the impact of processing methods on caffeine content, flavour profile
development, and consumer health implications. Assessing caffeine intake
in dietary studies and its effects on health (Johnson et al., 2022).
To enhance the accuracy, sensitivity, and efficiency of HPLC determination
of caffeine, several approaches can be implemented like using Solid Phase
Extraction (SPE)to concentrate caffeine from the beverage matrix,
removing interferences and improving detection limits. For complex
matrices, this technique can improve purity and concentration of caffeine
prior to HPLC analysis. Samples must be filtered or centrifuged before
injection to remove particulates that may clog the column or interfere with
analysis. Using a column with appropriate stationary phase (C18, C8, or
phenyl columns) can enhance. By applying these enhancements, you can
improve the overall effectiveness of HPLC for the determination of caffeine
in various beverage samples, leading to more accurate and reproducible
results (Harris et al.,2023).
CONCLUSION
In conclusion, the use of HPLC is brought to light as one of the most
essential techniques in quantification of caffeine in beverage samples due
to its high sensitivity, accuracy and repeatability. Due to its flexibility, the
method can optimise the separation process depending on the stationary
and mobile phase of the system for the analytes, as in the case of
caffeine. HPLC thus affirms its position in quality assurance and food
safety regulation in the food and beverage industry due to its accurate
separation of caffeine and production of clear distinct chromatographic
peaks. In addition, due to its versatility it is capable of analysing samples
with complicated matrices and interferences by applying dilution methods
and optimally extracting samples with high matrix effects for instance
Solid Phase Extraction improving on its ability to deliver accurate results
on such samples.
The caffeine content analysis in bioplus shows the efficiency of HPLC in
giving quantitative data of caffeine content of product which is now highly
required due to increase in awareness level of health hazards and
stringencies of food laws. HPLC sustains the consumer’s choices on intake
of caffeine as a helpful aid in public health. With the ongoing refinements
in the chromatographic methods, HPLC does not cease to be the
fundamental building block of the comprehensive analytical chemical
industry, which acts as the cornerstone in providing key solutions that
contribute to determining the quality and safety of products amid the
constantly changing and increasingly complex market scenarios.
REFERENCES
Gritti, F. & Guiochon, G., 2020. High-Performance Liquid
Chromatography: Fundamentals and Applications. Journal of
Chromatography A, 1616, pp. 1-12
Johnson, M. & Lee, T., 2022. Method development for the
quantification of caffeine in energy drinks by HPLC. Journal of Food
Chemistry, 350, pp. 128-135.
Smith, A., Roberts, K. & Chen, Y., 2023. Caffeine content in
commercially available energy drinks: A survey of analytical results.
Food Research International, 147, pp. 110-117.
Thompson, P. & Harris, R., 2023. Health implications of caffeine
consumption: A review focusing on young adults. Journal of
Nutritional Health & Food Engineering, 10(1), pp. 22-28.
Dosa, M., El-Habashy, H. & Ahmad, A., 2021. High-Performance
Liquid Chromatography: A powerful tool for caffeine analysis in
energy drinks. Asian Journal of Chemistry, 33(2), pp. 430-436.
Johnson, M. & Lee, T., 2022. Method development for the
quantification of caffeine in energy drinks by HPLC. Journal of Food
Chemistry, 350, pp. 128-135.
