EXPERIMENT NO: 5

TITLE: Detection and determination of carbohydrate concentration using High

Performance Liquid Chromatography (HPLC)

Aim: 1. Detection of presence of carbohydrate in a sample using HPLC.

2. Determination of carbohydrates concentration using HPLC.

Apparatus: Solvents, samples, HPLC system, computer system.

Theory And Working Principle:

Chromatography involves a sample (or sample extract) being dissolved in a mobile

phase (which may be a gas, a liquid or a superficial fluid).The mobile phase is then
forced through an immobile, immiscible stationary phase .The phases are chosen
such that components in the sample having different solubilities in each phase. A
component which is quite soluble in the stationary phase will take longer to travel
through it than a component which is not very soluble in the stationary phase but
very soluble in the mobile phase. As a result of these differences in mobilities,
sample components will become separated from each other as they travel through
the stationary phase.

Techniques such as HPLC(High Performance Liquid Chromatography) and GC(Gas

Chromatography) use columns-narrow tubes packed with stationary phase, through
which the mobile phase is forced. The sample is transported through the column by
continuous addition of mobile phase. This process is called elution. The average rate
at which an analyte moves through the column is determined by the time it spends
in the mobile phase.

High performance liquid chromatography is basically a highly improved form of

column chromatography. Instead of a solvent being allowed to drip through a
column under gravity, it is forced through under high pressures of up to
400atmospheres. That makes it much faster.

The time taken for a particular compound to travel through the column to the
detector is known as its retention time. This time is measured from the time at
which the sample is injected to the point at which the display shows the maximum
peak height for that compound. HPLC techniques are highly automated and
extremely sensitive.

Procedure and description of HPLC system:

The HPLC use for the assay of carbohydrate is from Agilent (1200 series)
constituting of some parts described as following:

1) Solvent Rack: In the solvent rack, the bottles filled with solvents are kept
without disturbing. Solvents act as de-mobile phases. For the detection and
measurement of acetic acid, we are having mobile phase (acetonitrile75%
and 25% de-ionized water) at a flow rate o f 1.4ml/min.
2) Degasser: The degasser unit helps in degasification of the solvent to enter
inside the column, if some water bubbl live mixed with the solvent which is
purged to the column, may cause severe hamper and damage to the column.
Hence this unit lives being an essential one while removing gas bubbles
from solvent stream.
3) Pump: A highly sensitive and high pressure generating quaternary pump
was used in this purpose which can create up to 400atms pressure. The
pump is functionally automatic and a flow rate of maximum 1.5ml/min was
used where during the process generally the flow rate was maintained at
1.4ml/min.
4) Sampler: The sampler of Agilent (1200 series) HPLC is an automatic one.
The samples are collected from the vials with the help of a robotic hand and
a needle to draw the solution from vials and then to push it to the column.
5) Column: The carbohydrate concentration was measured by Ultron ES-OVM
Chairal Carbohydrate Column (Agilent Technologies). The column has 3
parts:
a) Inlet end fitting,
b) Column guard,
c) Outlet end fitting. The column guard is mainly made of ceramic and
protects the column from passing of harmful or insoluble particles which
may cause hamper to the column. Inside the column, the stationary
phase like silica particles present.
6) Detector: Refractive Index Detector (RID) is used for the detection of non-
polar compounds like carbohydrates (fructose).
7) Recording Device: A computer was attached with this HPLC device to
control and analyze, and integrate and keep record of each and every data
generated during the process. We can perfectly control the system
temperature, flow rate, stop time of pump by using the software installed
for HPLC suggested by Agilent.
Carbohydrate Analysis:

1. For the purpose of analysis, we need to make some samples of known

concentration like 5,10,15,20g/L and they are passed through the column
along with the solvents.
2. The output will be recorded as a series of peaks. The peaks were integrated
and thus the areas under the curve were found out .
3. The area under the peak is proportional to the amount of X which has passed
the detector, and this area can be calculated automatically by the computer
linked to the display. The area it would measure is shown in the diagram.

If the solution of X was less concentrated, the area under the peak would be less-
although the retention time will start is the same.
This means that it is possible to calibrate the machine so that it can be used to find
how much of a substrate is present-even in very small quantities.

4. Then at calibration, we need to deliver the actual concentrations used during

calibration and it plots the concentration Vs area of the peak in a chart. If the
calibration is correct, then we obtain a straight line curve.
5. When the calibration is over, then we are allowed to pass the solution where
acetic acid concentrations are unknown. Similarly, the peaks generated
during this process possess a definite area, by the use of which is shown in
computer display.

Results:

The retention time of the unknown sample is 4.216 min.

The area of the peak of unknown sample is 3.39486*e^5Mau*s

The concentration of sample from calibration curve is 3.19971g/L.

Discussion:

Analytical instruments such as GC-MS, AAS, or HPLC can provide a lot of

information about the contents of such a sample. They can tell us what is in a
mixture and how much is there. Determining the identities of components is
referred to as qualitative analysis. Determining the amounts of those compounds is
called quantitative analysis. HPLC is a separation technique consisting of a solid
stationary phase and a liquid mobile phase. The sample is injected through an
injector and carried into the column by the mobile phase, the components are
separated on the stationary phase and pass through the detector in succession, a
chromatogram is recorded.

Conclusion: The concentration of sample from calibration curve is 3.19971g/L.