CHM 120: Introductory to Organic Chemistry

Purification of Organic Compounds

Introduction
Crystallization
Organic compounds can be purified by several methods. The applications of
these methods depend on some differences in the physical properties of
the components of the impure mixtures. Fig 1 shows some common
purification methods for purifying impure organic compounds.

1. Crystallization
Pure
2. Sublimation
organic
Impure
3. Distillation compound
organic
compound 4. Solvent extraction
5. Chromatography

6. Drying

Fig. 1 purification/separation techniques

Crystallization
Uses differences in solubility in a solvent
• The impure solid dissolved in a minimum amount of a suitable hot
solvent found by trial and error
• The hot solution is filtered (to remove impurities)
• The filtrate is allowed is allowed to cool gradually
• Crystals of the pure substance separate and are obtained by filtration;
recrystallization may be carried out to purity the compound further E.g.
sugar contaminated with charcoal with sand (hot water is used as
solvent).
Sublimation
Sublimation
Changing from the solid state direct to the gaseous state or changing from
the gaseous state direct to the solid state. This can be used to purify:
• impure iodine
• Impure naphthalene
• Impure camphor
• Impure cardice (solid CO2)
• Impure benzoic acid
• Impure ammonium chloride

Distillation
The physical properties exploited is differences in boiling point. There are
various types of distillations:
• Simple distillation
• Fractional distillation
• Distillation under pressure
• Steam distillation

Simple Distillation
Simple distillation is used to purity a liquid containing solid impurities.
• The mixture is boiled in a flask
• The vapour from it is passed through a condenser to the condense
vapour
• The distillate is collected in a receiver (beaker or flask)
• The impurities remain in the distillation flask.
Fractional distillation
It is used when two or more volatile miscible liquids with different boiling
points are to be separated.
The compound with the lowest boiling point distils first. The receiver is
changed, and the other liquid is collected.
A fractionating column is incorporated in a set up like that of simple
distillation.er
reduced pressure

acuum Distillation
Since the boiling point is dependent on the atmospheric pressure, the liquids will boil at a temperature
lesser than their boiling points if they were distilled in an atmosphere having lower pressure. This is
achieved by using a vacuum pump. Since the atmospheric pressure is reduced, the liquids also boil
faster and hence the whole process of distillation is made fast.
Steam distillation
A steam generator is incorporated. The steam helps to build up the pressure
in the set up. The liquid boils and its vapour together with water vapour are
passed over the condenser and collected as the distillate (the water can be
removed from the distillate using a separating funnel). In this variant, steam is
passed into the flask containing the liquids to be separated. The principle here is that the liquids will
boil faster because of aqueous tension (vapour pressure of water) helps in equalising the atmospheric
pressure.
Total pressure = Aqueous tension + vapour pressure of liquid components

This method is used

for separating a high boiling liquid that is immiscible with (does not dissolve
in) water. E.G. aniline and water
V

Solvent Extraction
It is application from the extraction of :
• Oils from seeds (n-hexane solvent)
• Dyes or pigments from materials
• Drugs from plant materials
• Chemicals
A good extraction solvent should possess the underlisted properties. It
should:
• Be safe to handle
• Be relatively low boiling so that it can easily be removed by distillation
at the end of the extraction process
 • Not react chemically with the substrate (i.e. the material being
extracted
• Not react chemically with the solvent in which the substrate dissolved
• Not soluble in the solvent containing the substrate
• Have a high partition coefficient (Kp>5) in favour of the solvent, i.e., the
solubility of the substrate in the solvent containing the substrate
• Be insoluble in the solvent containing the substrate
• Have low to moderate toxicity.
Typical solvents include: pentane b.p 69oC, Chloroform b.p 61oC, diethyl
ether b.p 35oC, petroleum ether b.p 30 -60 oC, ethyl acetate b.p 77oC and
methlene chloride b.p 40oC.

Differential extraction
This method is used for immiscible liquids, that is, liquids that do not mix
together. For example, oil and water are immiscible.
The immiscible liquids are taken in a separating funnel and left undisturbed.
After a while, they separate out according to their specific gravities, with the
heavier liquid at the bottom. Then they are later collected.
Substances can also be separated according to their preferential solubilities
in the liquid. For example, if phenol is to be extracted, it can be preferentially
extracted using NaOH solution as one of the liquids used

Chromatography
The following are the various types of chromatography
• Column chromatography
• Paper chromatography
• Thin layer chromatography
• Gas liquid chromatography
• High performance liquid chromatography
The principle behind chromatography is the distribution of substances
between two phases:
Stationary phase (this phase is not in motion) - adsorbent
Mobile phase (moving phase) – liquid/solvent

Column (adsorption) chromatography

• he adsorbent (stationary phase) is a finely powdered solid, e.g.
alumina (Al2O3), silica gel (SiO2 XH20)
• The separated bands along the column are known as the
chromatogram
• Separation occurs because some components of the mixtures adsorb
more tightly on the adsorbent while others have more affinity for the
liquid mobile phase and therefore move faster down the column with
the liquid.
• Example: separation of chlorophylls A abd B from leaf extracts.
Paper chromatography is one of the chromatographic techniques used
to separate the mixture of colored components using the paper on
which each component moves at a different speed and get separated.
The component with a greater speed moves fast compared to one with
lesser speed.

The principle of paper chromatography is partition. In paper

chromatography there are two phases one is the stationary phase and
the other is the mobile phase. Here, water trapped in the paper acts as
the stationary phase and solvent acts as the mobile phase. Thus, as the
solvent moves the components in the mixture also moves depending
on their speed. In this way, the component is distributed between the
mobile and stationary phases.

Paper chromatography is specially used for the separation of a mixture

having polar and non-polar compounds.

For separation of amino acids.

It is used to determine organic compounds, biochemicals in urine, etc.

In the pharma sector, it is used for the determination of hormones, drugs,

etc.

Sometimes it is used for evaluation of inorganic compounds like salts and

complexes.

Retention Factor

After a separation is complete, individual compounds appear as spots separated

vertically. Each spot has a retention factor (Rf) which is equal to the distance migrated
over the total distance covered by the solvent. The Rf formula is

Rf=distance traveled by sample / distance traveled by solvent

The Rf value can be used to identify compounds due to their uniqueness to each
compound. When comparing two different compounds under the same conditions, the
compound with the larger Rf value is less polar because it does not stick to the
stationary phase as long as the polar compound, which would have a lower Rf value.

Thin layer chromatography is a kind of chromatography used to separate and

isolate mixtures that are non-volatile in nature. Just like other chromatography
processes, this one consists of a mobile phase and a stationary phase.

The latter one here is a thin layer of absorbent material, such as aluminium oxide,
silica gel, or cellulose. This layer is applied to plastic, glass, or aluminium foil sheets
called an inert substrate. The mobile phase in the TLC procedure is a solvent or a
mixture of it.

If you want to learn more about the thin layer chromatography procedure, you have
landed at the right place. Here we will be discussing its principle, process, and
applications in different industries.

Thin Layer Chromatography Principle

The separation principle of the TLC procedure is based on the given compound’s
relative affinity towards the mobile and the stationary phase. The process begins here
by moving the mobile phase over the stationary phase’s surface. During this
movement, the higher affinity compounds gain less speed as compared to the lower
affinity compounds. This results in their separation.

Once the procedure gets completed, different spots can be found on the stationary
surface at distinct levels, reflecting various elements of the mixture. Basically, the
compounds that are more attracted towards the stationary phase secure their position
at lower levels while others move towards the higher levels of the surface. So their
spots can be seen accordingly.
Thin Layer Chromatography Procedure

From the information mentioned above, it must be clear to you how TLC works.
However, you still need to learn about its complete procedure to see its entire
functioning. A few components involved in the TLC procedure are as follows.

TLC Plates: These are used for applying the thin layer of stationary phase. They are
inert or stable in nature. The layer of stationary phase is kept even throughout these
plates for better analysis. Usually, ready-to-use plates are preferred by the people
conducting experiments.
Mobile Phase: This comprises a solvent (or solvent mixture). The taken solvent
needs to be chemically inert, of the highest possible purity, and particulate-free. Only
then can the TLC spots be able to develop.

TLC Chamber: This is where the thin layer chromatography procedure takes place.
It keeps the dust particles away from the process and does not let the solvent
evaporate. In order to develop the spots appropriately, a uniform environment is
maintained inside this chamber.

Filter Paper: This gets placed inside the chamber after being moistened with the
mobile phase solution. It ensures that the mobile phase rises uniformly throughout the
TLC plate’s length.

After collecting all these components, the process begins. Here are the steps followed
in it:

• The process starts by making a thin mark on the TLC plate’s bottom with a
pencil. It helps in the application of sample spots. These spots are kept at equal
distances.
• The sample is then applied to these spots made on the line.
• Then the TLC chamber is filled with the mobile phase up to a few centimetres
of its bottom.
• After pouring the mobile phase, the moistened filter paper is placed along with
the inside of the chamber wall. This helps to avoid the edge effect by
maintaining equal humidity.
• Finally, the prepared stationary phase plate is put inside the chamber. At this
point, the sample spots are kept on the mobile phase’s side.
• The chamber is then closed after placing the plate into it.
• Once enough time has elapsed for the process, the plate is taken out and
allowed to dry.
• At last, the sample spots get analyzed through a suitable method for the sample,
such as UV light, KMnO4 stain, and iodine staining.

This way, the TLC procedure gets completed. After analyzing the compound, it gets
described in its relative mobility’s terms, i.e., its Rf value is calculated. This value
changes for each compound, even under the same circumstances.
Usually, relative Rf comes into use here because keeping all the TLC factors constant
may not be possible. These aspects include adsorbent, temperature, adsorbent
thickness, spotted material’s amount, and solvent system. The formula used for Rf
value calculation is:

Rf = (distance covered by the sample) / (distance covered by the solvent)

Rf values and reproducibility can be affected by a number of different factors such as

layer thickness, moisture on the TLC plate, vessel saturation, temperature, depth of
mobile phase, nature of the TLC plate, sample size, and solvent parameters. These
effects normally cause an increase in Rf values. However, in the case of layer thickness,
the Rf value would decrease because the mobile phase moves slower up the plate.

If it is desired to express positions relative to the position of another substance, x,

the Rx (relative retention value) can be calculated:

Rx=distance of compound x from origin / distance of solvent travelled from origin

While Rf can never be greater than 1, Rx can be (i.e., faster than the reference
compound x

Thin Layer Chromatography Applications

Just understanding the principle and procedure of TLC is not enough. You also need
to learn about thin layer chromatography uses to see how and where it works in the
real world. Some standard TLC applications include:

• Being a separation process, TLC proves to be highly effective for separating

pharmaceutical formulations that consist of multiple components.
• The process can be used to examine a given product’s purity.
• Medicines like local anaesthetics, analgesics, sedatives, hypnotics,
anticonvulsant tranquilizers, and steroids go through the TLC procedure for
their qualitative testing.
• The cosmetic industry also uses TLC for checking the presence of preservatives
in the products.
• A given compound can be purified using TLC and then compared with a
standard sample.
 • TLC also finds its use in Biochemical analysis. Here, it can be used for
biochemical metabolites’ separation from urine, blood plasma, serum, and body
fluids.
• Just like the cosmetic industry, the food industry also utilizes TLC for the
detection of preservatives, artificial colours, and sweetening agents.
• A reaction’s progress can also get tracked with TLC to see whether it is
complete or not.

These were some typical thin layer chromatography applications that you can find at
different places. However, the procedure is not limited to these, and you can also see
its use in a lot of other industries.

Thin Layer Chromatography Benefits and Drawbacks

Finally, let’s explore some of the benefits and drawbacks of using the thin layer
chromatography procedure.

Advantages of TLC

These include:

• The separated spots of TLC can be further visualized without any trouble.
• This chromatography process is cost-effective as compared to other methods.
• It can be used for a number of compounds, and it does not take much time
because it is quicker.
• The process is much more straightforward than other methods.
• TLC makes it simple to analyze any given compound’s purity standards.
• Several compounds can easily get isolated through TLC.

Disadvantages of TLC

The drawbacks of the process are:

• The TLC procedure can not be used for lower detection limit experiments
because it has a high detection limit.
 • The plates used in TLC do not possess a more extended stationary phase.
• Result reproduction is challenging in TLC.
• TLC is limited to qualitative analysis, and it can not be used for quantitative
analysis.
• The separation length is also restricted as compared to other chromatography
methods.
• The process here does not take place in a closed system. Therefore, aspects like
temperature and humidity can affect the results, making them inaccurate.

Introduction
High-performance liquid chromatography (HPLC) is a powerful separation
technique used for the analysis of ions, proteins, and organic molecules.
HPLC is based on the mechanisms of adsorption, partition and ion
exchange, which depend on the type of stationary phase used. The
technique involves a solid stationary phase, usually enclosed in a stainless-
steel column, and a liquid mobile phase. The molecules of interest are
separated based on the difference in the relative distribution ratios of the
solutes between the two phases. HPLC is widely used to assess the purity
and to determine the content of potential pharmaceutical molecules. It can
also be employed for the determination of enantiomeric composition, using
the modified mobile phases or chiral stationary phases.
In the 1970s, the term High-performance liquid chromatography (HPLC)
was introduced to distinguish the modern high-performance technique
from the classical low-pressure column chromatography. The HPLC has
many applications in the fields of forensics, biochemistry, clinical science,
nutraceuticals, environmental science, and pharmaceuticals. The high
performance liquid chromatography could be used as an analytical
technique for drug trials or the quantitation of biological components for
clinical studies.

Principle
The high-performance liquid chromatography works on the principle that
some components of the sample take longer than others to pass through
the chromatography column. The time taken by the molecules depends on
the affinity of the molecule with the mobile phase (liquid or gas) and the
stationary phase (solid or liquid). The ones with greater affinity with the
stationary phase take longer to pass through the column and vice versa.

Apparatus
The apparatus consists of a pumping system, an injection valve, a
chromatographic column, stationary and mobile phases, connecting tubes,
a detector, and a data collecting device. The pump is used to ensure an
adjustable, constant, and reproducible flow of 1.0 ml/min to the analytical
column. The injector introduces a narrow stream of variable and
reproducible sample volume onto the top of the analytical column. The
optimum level of the analyte is maintained in the analytical column at an
acceptable pressure drop. The detector provides the system with high
sensitivity, linearity, and long term stability to detect the signals from the
chromatographic column. The data collecting device acquires, stores data,
and perform calculations. These calculations are used to control and
adjust the operational parameters and separation conditions.

Protocol
Mobile phase preparation
1. Add 400 mL of acetonitrile to approximately 1.5 liters of purified
deionized water.
2. Add glacial acetic acid (2.4 mL) to this solution.
3. Dilute the solution with purified deionized water to make a total
volume of 2.0 L in a volumetric flask. The pH of the resulting
solution should be between 2.8 to 3.2.
4. Add 40% sodium hydroxide to adjust the pH to 4.2.
5. Filter the mobile phase under vacuum to remove the gas and the
solids that could clog the chromatographic column from the
solution.

Component solution preparation

Make the solution of the sample as per the requirements of the
components need to be separated.

Making the standard solutions

1. Pipet the appropriate amount of each component in a 50-mL
volumetric flask.
 2. Dilute the stock solutions to the 50-mL mark on the volumetric
flasks with the mobile phase.
3. Pour each standard solution in small vials.
4. Store the samples in a refrigerator, along with the remaining
solutions.

Checking the settings of the HPLC system

1. Ensure that the waste line is in the waste container.
2. Set the flow rate of the mobile phase to 0.5 mL/min.
3. Set the minimum and maximum pressure and the flow rate to the
correct values on the front panel of the delivery pump.
4. Set the minimum pressure at 250 psi.
5. Set the maximum pressure at 4,000 psi.
6. Press “zero” on the detector’s front panel to set at the pure mobile
phase.
7. Rinse a 100-µL syringe with deionized water, then wash it with
several volumes of component solutions to be analyzed, and fill
the syringe.

Sample injection and data collection

1. Slowly inject 100 µL of the solution with the injector handle in the
load position through the septum port.
2. Set the data collection program to collect data for 300 seconds, to
get the 3 peaks to elute through the detector.
3. Before starting the trial, rotate the injector handle to the inject
position and click “Start Trial” on the computer data collection
program immediately.
4. Once 300 seconds have passed, the data collection sends a signal
to save the data file.
5. Note the time in seconds for the peak of each trial to identify each
component.
6. Remove the syringe from the septum and perform the process for
each of the remaining working standards.

Calculations
1. Calculate the concentration of all the components in the sample
solution.
 2. Determine the peak areas on the chromatograms for each
standard and the unknown samples by the triangular method,
which equals peak height times the width at ½ height
3. Determine the time taken by the components to reach the peaks.
4. Draw calibration curves of peak area vs. concentration (mg/L) for
all components.
5. Calculate the least-squares fit for calibration curves.

Applications
• Quantitation of antibiotics

• Analysis of detergent-solubilized integral membrane proteins

• Analysis of urine for cysteine, cysteinylglycine, and homocysteine

Precautions
• Use HPLC grade water and reliable solvents for the procedure.
• Use the same mixing procedure for multiple times to prevent errors
caused by the heat of mixing.
• After mixing the solvents, vacuum filter the mobile phase to
remove any suspended impurities that could block the column
thereby affecting the flow rate of the mobile phase.
• Degas the HPLC mobile phase before use to prevent air bubble
formation.
• Avoid excessive degassing as this could cause the loss of volatile
components of the mobile phase mixture.
• Wash the column and pump with water to prevent deposition of
solid crystals which could lead to column blockages and faster
wear of pump components.

Strengths and limitations

• The high-performance liquid chromatography is extremely quick
and efficient as compared to the other chromatographic
techniques.
• The process takes 10 to 30 minutes on an average and delivers a
high resolution. It is a highly accurate and reproducible separation
technique for organic molecules.
• The HPLC requires minimal training and includes automated
equipment.
• HPLC is a versatile and extremely precise analytical method to
identify and quantify the chemical components.
• The advantages of the HPLC include the requirement of small
sample size, modification of the depending on the level of
quantification needed, and the reliability of the results.
• Despite its advantages, it could be costly, requiring large quantities
of expensive organics.

References
1. Berridge., R. Chalk., N. D.’Avanzo., L. Dong., D. Doyle., J. Kim., . . .
Gileadi., O. (2011). High-performance liquid chromatography
separation and intact mass analysis of detergent-solubilized
integral membrane proteins. Anal Biochem, 410(2), 272-280.
2. I. N. Ehle., T. T. Yoshikawa., M. C. Schotz., & Guze., a. L.
(1978). Quantitation of Antibiotics by High-Pressure Liquid
Chromatography: Cephalothin. Antimicrob Agents Chemother,
13(2), 221-227.
3. Kuśmierek., R. Głowacki., & Bald., E. (2006). Analysis of urine for
cysteine, cysteinylglycine, and homocysteine by high-performance
liquid chromatography. Anal Bioanal Chem, 385(5), 855-60.
4. E. Petrova., & Sauer., K. (2017). High-performance liquid
chromatography (HPLC)-based detection and quantitation of
cellular c-di-GMP. Methods Mol Biol, 1657, 33-43.