Discussion:

In this experiment, the quatifying individual sugars were determined by using High
Performance Liquid Chromatography (HPLC). Sugars are the major components of citrus juice
soluble solids and sweetness of orange juice is intrinsic to its sugar composition. This
experiment was started by preparing four different concentrations of standard solutions of
20ppm, 100ppm, 300ppm, and 500ppm. Fruit juice used in this experiment are apple, mango
and dates. For fruit juice sample, sample was degassed or filtered through minimum 0.25 or
0.45 µm nylon filter size and diluted by ratio 1:10 before being analyzed. By the appearance
of the peak, it showed that the sample contained sugar. Based on the result obtained, standard
curve is plotted and based on the R2 value obtained it showed that the graph plotted.

High Performance Liquid Chromatography (HPLC) is one mode of chromatography

which is also the most widely used analytical technique. Chromatography processes can be
defined as separation techniques involving mass transfer between stationary and mobile
phase. It is used to separate compounds that dissolved in solution. Compounds are separated
by injecting sample mixture into column. It pumps sample mixture or analytes in a solvent
which also known as mobile phase at high pressure through a column with chromatographic
packing material which is stationary phase (Linde, 2018). As for the mobile phase, liquid is
used as the carrier for HPLC and gas is used as the carrier for Gas Chromatography (Bhanot,
2014). GC analyze compounds that dissolved in gas or also known as volatile while for HPLC,
it analyze compound from non volatile sample. The other differences of HPLC and GC is on
their application. The application of HPLC is wider as it can pharmaceutical, foods,
environment and clinical tests (Bhanot, 2014) GC is mainly used to analyse small molecules
and simple solvents.Coke was used in this experiment.

In this experiment, the compound that going to be separated from the sample solution
is sugar. HPLC used is from Shimadzu. The content of individual sugar was analyze in
different concentration of glucose sample. Sugars were identified by comparing the retention
times shown in the peaks of the chromatographed samples with those of authentic sugar
samples. After being analyzed, standard curve is plotted based on the peak areas obtained.
Quantitation of the sugars present in the samples was obtained from the measured peak areas
and the corresponding standard curves. Based on the graph plotted, an equation of y =
205.63x + 0.9608 is obtained.
 Next, for the sample of fruit juice, before the experiment was conducted, the sample
has been degassed to avoid any bubble occurred during the experiment because HPLC is a
sensitive instrument. If the sample is not being degassed, it will affect the pumps and detector.
This is because air in the ump will cause the pump to stop delivering mobile phase to the
column (Dolan, 2014). if air bubble is present in the pump, it will affect on the flow rate which
then will cause to the retention time problems (Dolan, 2014). These cases can be avoided by
degassing the sample before being analyzed.

Sugars are the major components of citrus juice soluble solids and the sweetness of o
juice is intrinsic to its sugar composition. The main portion of carbohydrates in citrus fruits are
the three simple sugars: sucrose, glucose, and fructose. Together, they represent about 80%
of the total soluble solids of orange juice, and the ratios of sucrose:glucose:fructose are
generally about 2:1:1.1 An earlier study2 indicated that sucrose and invert sugars (fructose
and glucose) are the principal sugars in orange juice. Besides free sugars, galactose,
arabinose, xylose, mannose, and fucose have been reported as sugar units of soluble
polysaccharides in orange juice. (H. S. Lee et, al)

Bhanot, D. (2014). What are the Differences between GC and HPLC?. Lab-Training.com.
Retrieved 15 April 2018, from http://lab-training.com/2014/04/02/what-are-the-
differences-between-gc-and-hplc/

Dolan, J. (2014). Mobile-Phase Degassing: What, Why, and

How. Chromatographyonline.com. Retrieved 13 April 2018, from
http://www.chromatographyonline.com/mobile-phase-degassing-what-why-and-how

Lee, H. S., & Coates, G. A. (2000). Quantitative Study Of Free Sugars And Myo-Inositol In
Citrus Juices By Hplc And A Literature Compilation. Journal of Liquid
Chromatography & Related Technologies,23(14), 2123-2141. doi:10.1081/jlc-
100100476