INSTRUMENT ANALYSIS

ANALYSIS CARBOHYDRATE CONTAINED IN PALM SUGAR BY HIGH

PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)

CHEMISTRY DEPARTMENT OF EDUCATION

FACULTY OF MATHEMATICS AND NATURAL SCIENCES

GANESHA UNIVERSITY OF EDUCATION

SINGARAJA

2014
ANALYSIS CARBOHYDRATE CONTAINED IN PALM SUGAR (Borassus flabellifer
Linn.) BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)
By:
Ni Putu Nia Repli Suandewi (1113031084)
Chemistry Education Department, Faculty of Mathematic and Sciences, Universitas
Pendidikan Ganesha, Singaraja

1. Objectives
a. Students are able to understand analysis method by HPLC.
b. Students are able to analysis data that obtained from HPLC.
2. Introduction
Chromatography is a separation method of complex components based on the
interaction between components, stationary phase and mobile phase. High Performance
Liquid Chromatography (HPLC) is analysis method based on separation of analyte
component on chromatography column by using high pressure. High Performance Liquid
Chromatography (HPLC) is common used for separation analytical technique, because
this method is very sensitive so very good for qualitative analysis, for nonvolatile
compound and other compound which is sensitive with heat. Compounds that usually
separated by using High Performance Liquid Chromatography (HPLC) is such as amino
acids, protein, nucleic acid, hydrocarbon, carbohydrate, medicine, terpenoid, pesticide,
antibiotic, steroid, and other inorganic compounds (Muderawan, 2009).
In HPLC, a liquid sample, or a solid sample dissolved in a suitable solvent, is
carried through a chromatographic column by a liquid mobile phase. Separation is
determined by solute/stationary-phase interactions, including liquid–solid adsorption,
liquid–liquid partitioning, ion exchange and size exclusion, and by solute/mobile-phase
interaction (Harvey, 2000). In each case, however, the basic instrumentation is essentially
the same. For compounds that has molecular weight more than 10.000 can be separated
by gel chromatography, while for ion compound with low molecular weight can be
separated by ion exchange chromatography. For the non-ionic polar compound with low
molecular weight can be separated by partition chromatography (Muderawan, 2009).
Partition chromatography is the chromatography technique mostly used between
all of the liquid chromatography procedures. This type of chromatography is used to
separate anionic polar compounds that have low until medium of relative molecular
weight usually Mr < 3.000. Partition chromatography can be divided into two such as
liquid-liquid chromatography and bonded-phase chromatography. The difference
between both of them is placed in the stationary phase that binds in the supported
particles in column. For liquid-liquid chromatography, its stationary phase is liquid that
binds by physical adsorption on the surface of supported particles. For bonded-phase
chromatography, its stationary phase is chemically bind on the surface of supported
particles. Firstly, partition chromatography is the type of liquid-liquid, but now it is
dominated by bonded- phase chromatography. It is caused by the possibility the losing of
phase chromatography in mobile phase in the liquid- liquid chromatography
(Muderawan, 2009).
Adsorption chromatography or liquid- solid is classical form of liquid
chromatography that was firstly introduced by Tswett in the beginning of 20 th century.

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 Stationary phase used to liquid- solid chromatography is only sillica in alumina. This
type of chromatography is also can be used for various compound. The retention time of
compound follows the increasing of polarity of compound namely elefine < aromatic
hydrocarbon < halide, sulfide < ether < nitro compound < ester ~ aldehyde ~ ketone <
alcohol ~ amine < sulfone < sulfoxide < amide < amide < carboxylic acid (Muderawan,
2009).
In ion-exchange chromatography, retention is based on the attraction between
solute ions and charged sites bound to the stationary phase. Ions of the same charge are
excluded. This form of chromatography is widely used in purifying water, Ligand-
exchange chromatography, Ion-exchange chromatography of proteins, High-pH anion-
exchange chromatography of carbohydrates and oligosaccharides, etc (Muderawan,
2009).
Size exclusion chromatography (SEC), also called as gel permeation
chromatography or gel filtration chromatography mainly separates particles on the basis
of size. It is also useful for determining the tertiary structure and quaternary structure of
proteins and amino acids. This technique is widely used for the molecular weight
determination of polysaccharides (Muderawan, 2009).
Adsorption chromatography mostly used to separate nonpolar compounds,
structural isomer and group compound of aliphatic hydrocarbon from aliphatic alcohol.
In this experiment, it was done the analysis of palm sugar carbohydrate by using HPLC.
Borassus flabellifer Linn. (Arecaceae) is a tall tree (palm) growing in sandy soil
and attaining a height of about 20-30 meters with a straight trunk. The fruits are large and
fibrous, containing usually three nuts like portions each of which encloses a seed.
Flowers and fruits during December to August. The plant has been used traditionally as a
stimulant, anti-leprotic, diuretic and antiphlogistic. The fruits are stomachic, sedative,
laxative and aphrodisiac in nature useful in hyperdipsia, dyspepsia, flatulence, skin
diseases, haemorrhages, fever and general debility. The roots and juice of the plant are
useful in inflammatory reactions. The ash obtained by burning the inflorescence is a good
antacid antiperiodic, and is useful in heart burn, splenomegaly (H. J., Yadav, V.N.,
Mohite, & Wadkar, 2013).

Figure 01. Whole plant of B. flabellifer Linn.

(Source: H. J., Yadav, V.N., Mohite, & Wadkar, 2013)
3. Instrument
HPLC (High Performance Liquid Chromatography)
4. Experimental
1. Sample Preparation
Standard sample solution was made from fructose, glucose, maltose, and sucrose
by concentration for each 0.5 % (w/w) in water.

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 Palm sugar sample was made from nira Palm (400 mL) by evaporating the nira
water until concentrated, then it was heated in oven by various temperatures at 90oC,
110oC, 120oC, and 130oC until the sugar formed. Sugar palm was out from oven and
stirrer so it becomes powder. Sample of palm sugar solution was made by diluting 0.5
gram aren sugar powder in water until reach 50 mL of solution.
2. HPLC Analysis
Composition of each palm sugar carbohydrate was analysis by using HPLC.
Standard solution and each sugar palm sample solution was injected into HPLC with
condition: column of Restex Pinnacle II Amino with particle size 5 µm, mobile phase
of asetonitril mixture with water (80 : 20), by rate of water 1mL/minute, temperature of
column at 40oC, and detector was refraction index detector (RID).
5. Result and Discussion
(1) Chromatogram Carbohydrate Standard Solution
Table 01. Retention time, area, and concentration of standard solution

No Concentration Name Carbohydrate

Time Area
% (w/w)

1 0.067 6322 -

2 2.133 5460470 - Solvent

3 4.317 447294 0.50 Fructose

4 5.042 377412 0.50 Glucose

5 5.333 549231 0.50 Maltose

6 7.742 448153 0.50 Sucrose

(2) Chromatogram Sample Solution

i. Chromatography Palm Sugar (90oC)
Table 02. Retention time, area, and concentration of standard solution

No Concentration Name Carbohydrate

Time Area
% (w/w)

1 0.065 6405 -

2 2.130 5064924 - Solvent

3 4.292 17938 2 Fructose

4 4.998 14044 1.86 Glucose

5 7.705 778001 86.80 Sucrose

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ii. Chromatography Palm Sugar (100oC)
Table 03. Retention time, area, and concentration of standard solution

No Concentration Name Carbohydrate

Time Area
% (w/w)

1 0.053 6415 -

2 2.127 5385004 - Solvent

3 4.280 16583 1.85 Fructose

4 4.998 15163 2.10 Glucose

5 7.680 768147 85.70 Sucrose

iii. Chromatogram Palm Sugar (110oC)

Table 04. Retention time, area, and concentration of standard solution

No Concentration Name Carbohydrate

Time Area
% (w/w)

1 0.060 6116 -

2 2.132 5151615 - Solvent

3 4.283 16677 1.86 Fructose

4 4.978 16544 2.19 Glucose

5 7.682 768062 85.69 Sucrose

iv. Chromatogram Palm Sugar (120oC)

Table 05. Retention time, area, and concentration of standard solution

No Concentration Name Carbohydrate

Time Area
% (w/w)

1 0.062 6537 -

2 2.128 5410195 - Solvent

3 4.280 17936 2.00 Fructose

4 4.980 17548 2.60 Glucose

5 7.672 765636 85.42 Sucrose

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 v. Chromatogram Palm Sugar (130oC)
Table 05. Retention time, area, and concentration of standard solution

No Concentration Name Carbohydrate

Time Area
% (w/w)

1 0.060 6998 -

2 2.137 5454836 - Solvent

3 4.277 20628 2.30 Fructose

4 4.977 19247 2.55 Glucose

5 7.665 756293 84.38 Sucrose

6. Conclusion
Based on the discussion above it can be concluded that;

7. References
Muderawan, I. W. (2009). Analisis Instrumen. Singaraja: Universitas Pendidikan
Ganesha Press.
Harvey, D. (2000). Modern Analytical Chemistry. North America: United States of
America.