QUALITY IN ANALYTICAL MEASUREMENT (CHM561)

ASSIGNMENT 1

NAME: AHMAD ZAKWAN BIN KASSIM

AUTHORS
Chi Gao, David G. Cunningham, Haiyan Liu, Christina Khoo and Liwei Gu.

TITLE
Development of a Thiolysis HPLC Method for the Analysis of
Procyanidins in Cranberry Products.

TYPES OF ANALYTES
Procyanidins is being the analytes for thiolysis HPLC analysis.

SAMPLE PRE-TREATMENT
For the sample pre-treatment, firstly we dissolve ten milligrams of partially purified cranberry
procyanidins in 1.0 mL of 95% ethanol to create a solution with a concentration of 10 mg/mL.
This step ensures that the procyanidins are evenly distributed in the solvent. Next, we adjust
the acidity of the cranberry procyanidins solution to 0.4 M using hydrochloric acid (HCl). This
adjustment creates the right conditions for the subsequent thiolysis reaction, allowing it to
proceed efficiently. In the thiolysis reaction, we mix 80 μL of the cranberry procyanidins
solution with 80 μL of cysteamine dissolved in ethanol. This mixture is then heated in a water
bath at 60 °C for 20 minutes. The thiolysis reaction breaks down the procyanidins into smaller
fragments, which are easier to analyze. After the thiolysis reaction, we immediately transfer
the mixture to a freezer set at -20 °C. Freezing stops any further chemical reactions and helps
preserve the procyanidin fragments for analysis. By keeping them at a low temperature, we
prevent degradation and maintain the integrity of the compounds. Before analysis, we pass the
solution through a filter with small pores (0.45 μm) to remove any particles or impurities. This
ensures a clean sample, free from any substances that could interfere with the analysis.

METHODS/EXTRACTION PROCESS/SAMPLE
PREPARATION
To start the extraction, we use craisin dried cranberries and cranberry concentrate dietary
supplements. First, we take one gram of ground sample and place it in a capped test tube. Then,
we add a solution of 70% (v/v) aqueous acetone, which is made slightly acidic with 0.5% (v/v)
acetic acid. This solution helps extract the procyanidins from the cranberry products. We mix
the sample and solvent vigorously for one minute using a vortex mixer to ensure thorough
mixing. This step helps release and dissolve the procyanidins from the cranberries. After
mixing, we subject the test tube to sonication for 10 minutes at room temperature. Sonication
involves using ultrasonic waves to enhance the extraction process by breaking down cell walls
and increasing contact between the sample and solvent. Next, we let the tube rest in darkness
for 20 minutes at room temperature. This allows for further extraction of procyanidins. To
optimize the extraction, we perform an additional 5 minutes of sonication. Finally, we separate
the solid components by centrifuging the tube for 15 minutes. The resulting solution contains
the extracted procyanidins.
The next step is purifying the extracted procyanidins. We transfer the extracted solution to a
larger tube and remove the solvent (acetone) by evaporating it under partial vacuum at room
temperature using a SpeedVac concentrator. This step leaves behind a dried residue containing
concentrated procyanidins. To further purify the procyanidins, we disperse the dried residue in
a solution of 30% (v/v) aqueous methanol. Then, we load the sample onto a solid-phase
extraction (SPE) column. This column contains a special material called Sephadex LH-20,
which helps purify procyanidins. Before loading the sample, we equilibrate the column with
30% aqueous methanol for 4 hours. For cranberry juice samples, we directly load the juice onto
the SPE column without prior extraction. The samples, whether extracted solution or cranberry
juice, are loaded onto the column at a controlled flow rate. This allows the procyanidins to
interact with the column material, separating them from unwanted compounds. During column
elution, we use a solution of 30% aqueous methanol to remove sugars and other phenolic
compounds that are not procyanidins. This solution is passed through the column at a specific
flow rate. Finally, we recover the purified procyanidins from the column by eluting with a
solution of 70% (v/v) aqueous acetone. The eluents containing the purified procyanidins are
collected and further processed for analysis or other applications. Any remaining solvents are
removed by evaporating the eluents, resulting in residues containing concentrated
procyanidins. These residues are then dissolved in ethanol, creating a suitable solution for
subsequent thiolysis reactions.

METHOD DEVELOPMENT
The primary objective of the method development process is to optimize the thiolysis reaction.
To accomplish the objective, several factors are considered during the method development
process. Temperature, reaction time, acidity, and fold excess of thiolytic reagents are
recognized as critical variables that can influence the thiolysis reaction. Each of these factors
has the potential to impact the efficiency, selectivity, and overall success of the reaction. The
method development follows the One Factor at a Time (OFAT) approach. This systematic
approach involves varying one factor at a time while keeping the others constant. By isolating
each factor, researchers can observe its individual effect on the thiolysis reaction, providing
valuable insights into the optimal conditions.
Temperature is a vital parameter that significantly affects reaction kinetics. In the method
development process, the temperature is varied within a specific range, such as 20°C to 60°C.
The duration of the reaction, known as the reaction time, is another critical factor under
investigation. Different reaction times are tested, ranging from shorter durations (e.g., 1 hour)
to longer durations (e.g., 24 hours). Acidity plays a pivotal role in catalyzing thiolysis reactions.
In the method development process, acidity is varied by using different acids or adjusting the
pH of the reaction mixture. This allows for the evaluation of the effect of acidity on the thiolysis
reaction. The fold excess of thiolytic reagents, which refers to the amount of reagents used
relative to the reactants, is another factor of interest. The method development process involves
testing different fold excesses of thiolytic reagents to determine their impact on the efficiency
of the thiolysis reaction. The experimental results obtained from varying each factor are
subjected to rigorous data analysis. Statistical techniques and data visualization methods are
employed to identify trends, correlations, and optimal conditions. The factors that contribute
to the desired reaction outcome, such as high yield, selectivity, or efficiency, are determined.
Furthermore, the results from individual factor variations can be combined to establish the
optimized reaction conditions that provide the best overall performance.

INSTRUMENT
High-Performance Liquid Chromatography (HPLC) was used for analyzing procyanidins
before and after thiolysis. Separation was performed on a Luna silica column, and the peaks
were monitored by fluorescence detection.

CONCLUSION
In conclusion, the study developed a thiolysis HPLC method using cysteamine as a substitute
for toluene-α-thiol. The optimized conditions for depolymerization of cranberry procyanidins
were determined, and the method was validated for quantifying total procyanidins, average
degree of polymerization, ratio of A-type linkages, and A-type procyanidin equivalents in
cranberry products. The article focused on investigating the effects of different factors on the
reaction efficiency. By varying factors such as reaction time, temperature, solvent, and catalyst
concentration, the researchers were able to determine the optimum conditions for the thiolysis
reaction. The results of the study indicated that 4 hours of reaction time, 60°C of temperature,
the use of dichloromethane as the solvent, and concentration of 10 mol% of a catalyst were
found to be the optimum conditions for achieving high yield and efficiency in the thiolysis
reaction.

REFERENCES
UiTM Library e-Resources. (n.d.). Online Database.
https://pubs-acs-org.ezaccess.library.uitm.edu.my/doi/10.1021/acs.jafc.7b04625