Task3: How could HPLC be developed.
Discuss how a HPLC method could be developed to determine the amount of a specific sugar molecule in a sugar mixture
Task 4: How to optimize separation.
Discuss how a HPLC method could be developed to determine the amount of a specific sugar
molecule in a sugar mixture
Your discussion will be based upon research into various methods that could be adapted (you will
need to include standard referencing of your sources of information and research). You will examine
whether the methods identified would work for your selected sugar compound. As part of your
discussion, you will need to:
o Consider appropriate solvents and columns that could be used
o Consider appropriate methods of detecting components
o Propose initial experiments that you would conduct to test the method
o Propose any calibration and refinements that could be made
Method for Separation, Identification, and Quantification of Fructose, Glucose, and Sucrose using
HPLC
Introduction:
The aim of this experiment is to examine the use of the HPLC method for the separation,
identification, and quantification of fructose, glucose, and sucrose in a Goji Berries. The
concentrations of these sugars may vary due to the size of the fruit chosen. The experiment aims to
determine the sugar content in Goji berries. Additionally, berries from different shops (supermarket
and pharmacists) will be analysed to know if there is any variations in sugar composition based on
the source. The Goji berries used in this study are derived from the Lycium Barbarum plant.
Sample preparation: A handful of goji berries will be collected from various sources such as a
supermarket and a pharmacy. To ensure fairness, the same quantity of goji berries from each source
will be kept in a dry, dark place with climate control to preserve their integrity. The berries will be
crushed and homogenized to obtain the sample that will be used in this experiment.
Why
This is done in order to maintain it integrity ( it will not got Bad/rot and that the berry will not be
bruised ). The goji berries need to be crushed and homogenized to break down its structure and
create a homogeneous sample. This ensures that the analysis captures an accurate composition of
sugar in the berries and allows for consistent and reliable results in the experiment and the reason
why we picked from different sources is because to see if there is any difference in sugar composition
Extraction: We will be using an appropriate solvent as the mobile phase in this experiment. The
combined use of chloroform and acetonitrile will be use. Chloroform will serve as a source of
dichlorocarbene while Acetonitrile will be chosen for its miscibility with chloroform and having the
polarity which will serve as an excellent mobile phase for the experiment.
, Why
This step is need because we need a solvent that can carry the sample throughout the system. And
not just any solvent, but it has to be an suitable solvent. The mobile phase can effect result of the
experiment for example ; the wrong mobile phase can cause the sample to dissolve or denature and
may not properly separate the component. This could lead to wrong result . so picking and choosing
the suitable mobile phase is need .Acetonitrile is a polar solvent which work well with sugar (sugar is
a polar molecule). Acetonitrile is also compatible with various detection techniques, such as UV-Vis
spectroscopy. chloroform is non-polar but aids in separation.
Column Separation: The prepared sample will be injected into the HPLC system equipped with a
suitable column. The column to be chosen will be the Grace HPLC Column Platinum Amino. The
reason why this column will be chosen will be because the stationary phase will be compatible with
the mobile phase thus ensuring optimal interactions. The high polarity of amino acids will enhance
the efficiency of the separation.
Why?
This step is need because this is what separate the component of the component and what wase
then to leave the column at different times. This can affect the result of the experiment quit
tremendously. By using the wrong stationary phase, the stationary phase could interact with the
mobile phase or the stationary phase could wrongly affect how the sample should supposed to
separate thus leading to incorrect analysis. When carrying out this step it is important to choose the
correct one
Detection: As the separated compounds will elute from the column, they will pass through a UV
detector. Deionized water will be used in the UV detector to provide further analysis and identify
each component based on their UV absorption characteristics. The detector will generate signals
corresponding to the different sugar compounds present in the sample. The reason why this type of
detector will be used will be because UV detectors can analyze compounds that absorb UV light,
including the sugar compounds in the Goji Berry samples, thus allowing for their detection and
quantification at specific wavelengths. Deionized water will be used as a solvent to ensure the
absence of contaminants or impurities that could interfere with the analysis, thereby maintaining the
integrity of the separation and quantification.
Why
This step is also important because this is how we can detect the component then use it to produce a
chromatogram. Without this we can’t analysis the data we got. The data retrieved will come in form
of a peak. The detection step identifies and quantify the data gotten after separation. Choosing the
right detector is important as it can affect how we can interpret the data gotten. The reason chose
uv detector was because it very sensitivity and can detect even the smallest amount of sugar , plus
different types sugar have it own distinct uv characteristic which can all we caught with the uv
detector
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