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CHROMATOGRAPHY ( HPLC ) LAB REPORT

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 CHROMATOGRAPHY ( HPLC )

LAB REPORT

By :

1. Sakullapat Homkhao M6030063

2. Pongsatorn Poopisut M5930333
3. Dyah Wulandari D5930166
1. Purpose
1.1.Study principles of chromatography
1.2.Study how to use high performance liquid chromatography (HPLC)

2. Introduction

2.1. Chromatography

Chromatography’ is an analytical technique commonly used for separating a mixture of

chemical substances into its individual components, so that the individual components can be
thoroughly analyzed. There are many types of chromatography e.g., liquid chromatography,
gas chromatography, ion-exchange chromatography, affinity chromatography, but all of these
employ the same basic principles.

Chromatography is a separation technique that every organic chemist and biochemist is

familiar with. I, myself, being an organic chemist, have routinely carried out chromatographic
separations of a variety of mixture of compounds in the lab. In fact, I was leafing through my
research slides and came across a pictorial representation of an actual chromatographic
separation that I had carried out in the lab. I guess that picture would be a good starting point
for this tutorial!

2.1.1. Principles of chromatography

Let’s first familiarize ourselves with some terms that are commonly used in the context
of chromatography:
 Term Definition

Mobile phase or carrier solvent moving through the column

Stationary phase or
adsorbent substance that stays fixed inside the column

Eluent fluid entering the column

Eluate fluid exiting the column (that is collected in flasks)

the process of washing out a compound through a column using

Elution a suitable solvent

mixture whose individual components have to be separated and

Analyte analyzed

Now let’s try to understand the principle of chromatography. Let us draw a pictorial
representation of a column chromatographic separation set up.

As depicted above, the analyte is loaded over the silica bed (packed in the column) and
allowed to adhere to the silica. Here, silica acts as the stationary phase. Solvent (mobile phase)
is then made to flow through the silica bed (under gravity or pressure). The different
components of the analyte exhibit varying degrees of adhesion to the silica (see later), and as a
result they travel at different speeds through the stationary phase as the solvent flows through
it, indicated by the separation of the different bands. The components that adhere more strongly
to the stationary phase travel more slowly compared to those with a weaker adhesion.
Analytical chromatography can be used to purify compounds ranging from milligram to gram
scale.

2.1.2. Different types of chromatography

Throughout this article we are dealing with what we refer to as normal-

phasechromatography, implying that our stationary phase is polar (hydrophilic) in nature and
our mobile phase is non-polar (hydrophobic) in nature. For special applications, scientists
sometimes employ reverse-phase chromatographic techniques where the scenario is reversed
i.e. the stationary phase is non-polar while the mobile phase is polar.

There are several types of chromatography, each differing in the kind of stationary and mobile
phase they use. The underlying principle though remains the same: differential affinities of the
various components of the analyte towards the stationary and mobile phases results in the
differential separation of the components. Again, the mode of interaction of the various
components with the stationary and mobile phases may change depending on the
chromatographic technique used. The commonly used chromatographic techniques are
tabulated below.

2.2. High performance liquid chromatography (HPLC)

High performance liquid chromatography is a powerful tool in analysis. This page looks
at how it is carried out and shows how it uses the same principles as in thin layer
chromatography and column chromatography.

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 400 atmospheres. That makes it much faster.

It also allows you to use a very much smaller particle size for the column packing material
which gives a much greater surface area for interactions between the stationary phase and the
molecules flowing past it. This allows a much better separation of the components of the
mixture.

The other major improvement over column chromatography concerns the detection
methods which can be used. These methods are highly automated and extremely sensitive.

2.2.1. The column and the solvent

Confusingly, there are two variants in use in HPLC depending on the relative polarity of
the solvent and the stationary phase.

2.2.2. Normal phase HPLC

This is essentially just the same as you will already have read about in thin layer
chromatography or column chromatography. Although it is described as "normal", it isn't the
most commonly used form of HPLC.

The column is filled with tiny silica particles, and the solvent is non-polar - hexane, for
example. A typical column has an internal diameter of 4.6 mm (and may be less than that), and
a length of 150 to 250 mm.

Polar compounds in the mixture being passed through the column will stick longer to the
polar silica than non-polar compounds will. The non-polar ones will therefore pass more
quickly through the column.

2.2.3. Reversed phase HPLC

In this case, the column size is the same, but the silica is modified to make it non-polar
by attaching long hydrocarbon chains to its surface - typically with either 8 or 18 carbon atoms
in them. A polar solvent is used - for example, a mixture of water and an alcohol such as
methanol.

In this case, there will be a strong attraction between the polar solvent and polar
molecules in the mixture being passed through the column. There won't be as much attraction
between the hydrocarbon chains attached to the silica (the stationary phase) and the polar
molecules in the solution. Polar molecules in the mixture will therefore spend most of their
time moving with the solvent.

Non-polar compounds in the mixture will tend to form attractions with the hydrocarbon
groups because of van der Waals dispersion forces. They will also be less soluble in the solvent
because of the need to break hydrogen bonds as they squeeze in between the water or methanol
molecules, for example. They therefore spend less time in solution in the solvent and this will
slow them down on their way through the column.

That means that now it is the polar molecules that will travel through the column more
quickly.

Reversed phase HPLC is the most commonly used form of HPLC.

2.2.4. Injection of the sample

Injection of the sample is entirely automated, and you wouldn't be expected to know how
this is done at this introductory level. Because of the pressures involved, it is not the same as
in gas chromatography

2.2.5. Retention time

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 a maximum peak height for that compound.

Different compounds have different retention times. For a particular compound, the retention
time will vary depending on:
  the pressure used (because that affects the flow rate of the solvent)

 the nature of the stationary phase (not only what material it is made of, but also particle
size)

 the exact composition of the solvent

 the temperature of the column

That means that conditions have to be carefully controlled if you are using retention times as a
way of identifying compounds.

2.2.6. The detector

There are several ways of detecting when a substance has passed through the column. A
common method which is easy to explain uses ultra-violet absorption.

Many organic compounds absorb UV light of various wavelengths. If you have a beam of UV
light shining through the stream of liquid coming out of the column, and a UV detector on the
opposite side of the stream, you can get a direct reading of how much of the light is absorbed.

The amount of light absorbed will depend on the amount of a particular compound that is
passing through the beam at the time.

You might wonder why the solvents used don't absorb UV light. They do! But different
compounds absorb most strongly in different parts of the UV spectrum.

Methanol, for example, absorbs at wavelengths below 205 nm, and water below 190 nm. If you
were using a methanol-water mixture as the solvent, you would therefore have to use a
wavelength greater than 205 nm to avoid false readings from the solvent.
2.2.7. Interpreting the output from the detector

The output will be recorded as a series of peaks - each one representing a compound in
the mixture passing through the detector and absorbing UV light. As long as you were careful
to control the conditions on the column, you could use the retention times to help to identify
the compounds present - provided, of course, that you (or somebody else) had already measured
them for pure samples of the various compounds under those identical conditions.

But you can also use the peaks as a way of measuring the quantities of the compounds present.
Let's suppose that you are interested in a particular compound, X.

If you injected a solution containing a known amount of pure X into the machine, not only
could you record its retention time, but you could also relate the amount of X to the peak that
was formed.

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 green in the (very simplified) diagram.

If the solution of X was less concentrated, the area under the peak would be less - although the
retention time will still be the same. For example:

This means that it is possible to calibrate the machine so that it can be used to find how much
of a substance is present - even in very small quantities.

Be careful, though! If you had two different substances in the mixture (X and Y) could you say
anything about their relative amounts? Not if you were using UV absorption as your detection
method.
In the diagram, the area under the peak for Y is less than that for X. That may be because there
is less Y than X, but it could equally well be because Y absorbs UV light at the wavelength
you are using less than X does. There might be large quantities of Y present, but if it only
absorbed weakly, it would only give a small peak.

2.2.8. Coupling HPLC to a mass spectrometer

This is where it gets really clever! When the detector is showing a peak, some of what is passing
through the detector at that time can be diverted to a mass spectrometer. There it will give a
fragmentation pattern which can be compared against a computer database of known patterns.
That means that the identity of a huge range of compounds can be found without having to
know their retention times.

3. Material and Method

3.1. Material
- Micropipette
- Tip
- HPLC Machine
- Syringe
- Filter 0.2µm
- Eppendorf tube
- Vial tube and cap

3.2. Reagent
- Sucrose 10mg/ml
- Samples 24.25.1 & 24.25.2
- H2SO4 0.005N liquid as mobile phase
- DI water
3.3. Method
3.3.1. Sample preparation
1. Sucrose standard:
The sucrose standard was done by the dilution from the sucrose stock in
concentration 2.5mg/ml, 5 mg/ml, 7.5mg/ml and 10mg/ml.
2. Sample preparation
The samples have to dilute in 20X and 50X
V total
Calculation = 20𝑥 = V sample, so if the V total 1000µl, the volume sample we

use 50µl, and add the DI water 950 µl

V total
50𝑥 = V sample, so if the V total 1000µl, the volume sample we use 20µl and

add the DI water 980 µl

3. After the sucrose standard and the samples already prepared, then we do the
filtration by the filter paper connect with the syringe with the d = 0.2µm, then
put in the vial then close with the caps and labelled the samples
4. After all preparation finish, run the samples with HPLC machine.
5. Before use, set the condition of HPLC machine first with the injection volume
10ul, the flow 0.5 ml/min, pressure bar 35.85, and temperature 55ºC

6. Placed the samples into the tray and don’t forget to type sample’s name. After
all sett then run the machine. Each samples takes time around 50minutes to
detect the chemical inside the samples.
 7. After finish, collect the chromatogram data and calculate the concentration of
the samples by compare with the standard curve.

4. Results & Discussions

Table 1. retention time of standard sugars

retention time retention time retention time

avg. 1 avg. 2 avg. 3
arabinose 14.691 - -
glucose 12.608 - -
xylose 13.59075 - -
fructose 13.66025 - -
maltose 10.623 - -
sucrose 10.693 12.3846 13.0716

From Table 1. retention time of standard sugars , the retention time of sucrose should
have a peak but this analysis sucrose from HPLC have 3 peaks . it is error because we use
sulfuric acid 0.005 N for mobile phase , sucrose can be dehydrated with sulfuric acid and maybe
produce some carbon and SO3. Thus analysis of standard sucrose have 3 peaks . We should
change the solution for mobile phase.

Figure 1 . analysis sample 1(dilute 20 times and 50 times) from HPLC

Table 2. the retention time of sample 1 (dilute 20 times and 50 times)

retention time retention time retention time retention time retention time
1 2 3 4 5
result1x50 10.694 12.668 13.662 18.18 -
result1x20 10.673 12.595 13.611 17.295 20.344
Retention
10.6835 12.6315 13.6365 17.7375 20.344
time avg.

When we compare retention time of sample From Table 2. the retention time of sample
1 (dilute 20 times and 50 times) with standard sugars from Table 1. retention time of standard
sugars and calculate sugar’s concentration from standard curves.

1. the retention time avg. 1 (10.6835 min) is nearly the retention time avg. of sucrose
(10.693 min)
2. the retention time avg. 2 (12.6315 min) is nearly the retention time avg. of glucose
(12.608 min)
3. the retention time avg. 3 (13.6365 min) is nearly the retention time avg. of fructose
(13.66025 min)
4. the retention time avg. 4 & 5 we don’t know because the standard of sugars don’t
show peaks at retention time around 17 and 20 min.

Table 3 . the results of sample 1 (dilute 20 times and 50 times)

sucrose conc. glucose conc.

fructose conc.(mg/l)
(mg/l) (mg/l)
sample 1(dilute 50
7.05702 0.12836 0.843763
times)
sample 1(dilute 20
6.45041 0.097733 0.254314
times)
avg. 6.753715 0.1130465 0.5490385
 Figure 2 . analysis sample 2 (dilute 20 times and 50 times) from HPLC

Table 4. the retention time of sample 2 (dilute 20 times and 50 times)

retention time Retention time retention time retention time

retention time 5
1 2 3 4
result2x50 10.621 12.58 13.567 - -
result2x20 10.614 12.58 13.363 17.2 20.307
Retention
10.6175 12.58 13.465 17.2 20.307
time avg.

When we compare retention time of sample From Table 4. the retention time of sample
2 (dilute 20 times and 50 times) with standard sugars from Table 1. retention time of standard
sugars and calculate sugar’s concentration from standard curves.

1. the retention time avg. 1 (10.6175 min) is nearly the retention time avg. of
maltose(10.623 min)
2. the retention time avg. 2 (12.58 min) is nearly the retention time avg. of glucose
(12.608 min)
3. the retention time avg. 3 (13.465 min) is nearly the retention time avg. of xylose
(13.59075 min)
4. the retention time avg. 4 & 5 we don’t know because the standard of sugars don’t
show peaks at retention time around 17 and 20 min.
 table 5 . the results of sample 2 (dilute 20 times and 50 times)

maltose conc. glucose conc. xylose conc.

(mg/ml) (mg/ml) (mg/ml)
sample 2
1.844376 0.863133 0.42084
(dilute 50 times)
sample 2
1.574105 0.312792 0.137708
(dilute 20 times)
avg. 1.7092405 0.5879625 0.279274

5. Conclusion

1. the first sample have sucrose 6.754 mg/ml , glucose 0.549 mg/ml and fructose
0.113 mg/ml
2. the second sample have maltose 1.071 mg/ml , glucose 0.588 mg/ml and xylose 0.279
mg/ml

Reference

https://www.khanacademy.org/test-prep/mcat/chemical-processes/separations-
purifications/a/principles-of-chromatography

http://www.chemguide.co.uk/analysis/chromatography/hplc.html

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