QUALITY IN ANALYTICAL MEASUREMENT (CHM561)

ASSIGNMENT 1

NAME: AHMAD ZAKWAN BIN KASSIM

STUDENT ID: 2021886994
CLASS: AS2534A2
DATE OF SUBMISSION: 26 MAY 2023
NAME OF LECTURER: DR. ROSSURIATI BINTI DOL HAMID
 NAME OF JOURNAL, VOLUME, PAGE AND PUBLISHER
Journal of Agricultural and Food Chemistry (2018), Volume 66, Page 2159 – 2167, publish
by American Chemical Society in 2018.

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