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Activity 5 Post Lab

The document discusses various chromatography techniques. It defines chromatography as a separation technique involving two phases, mobile and stationary, that move relative to each other while maintaining contact. The key objectives of chromatography are resolution of mixtures, determination of homogeneity, purification, and identification. The document then describes various types of chromatography techniques in detail, including column chromatography, thin layer chromatography, gas chromatography, high pressure liquid chromatography, and ion-exchange chromatography.

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Shaira Sta Cruz
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28 views

Activity 5 Post Lab

The document discusses various chromatography techniques. It defines chromatography as a separation technique involving two phases, mobile and stationary, that move relative to each other while maintaining contact. The key objectives of chromatography are resolution of mixtures, determination of homogeneity, purification, and identification. The document then describes various types of chromatography techniques in detail, including column chromatography, thin layer chromatography, gas chromatography, high pressure liquid chromatography, and ion-exchange chromatography.

Uploaded by

Shaira Sta Cruz
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PDF, TXT or read online on Scribd
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_____________________________________________

ORGANIC CHEMISTRY LABORATORY

Name: Date:____________________

Year & section: __________________________Instructor: Melvin Reyes,RPh.

ACTIVITY NO. 5: CHROMATOGRAPHIC TECHNIQUES

Objectives:
• At the end of the activity the student must be able to:

1. Define chromatography
2. Enumerate and describe the different types of chromatography

NOTE:

Chromatography is a separation technique with extraordinarily


versatile variants. In all chromatographic separations, there are two
phases: a mobile phase and a stationary phase, which move relative to
one another while maintaining contact. The sample is introduced into
the mobile phase, and the sample components are distributed between
the stationary and mobile phases. The components of the mixture to
be separated spend different times in each phase, and therefore
separation occurs. If a component spends most of the time in the
mobile phase, the product moves quickly, whereas if it spends the
main time in the stationary phase, the product is retained and its
output is much slower

The principal objectives of obtainable through the use of


chromatography are:

1. Resolution of mixtures into constituent parts


2. Determination of homogeneity
3. Comparison of substances suspected of being identical
4. Purification
5. Concentration of substances from dilute solution
6. Identification and control of technical products
7. Qualitative separation from complex mixtures
8. Indication of molecular structure
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ORGANIC CHEMISTRY LABORATORY

COLUMN CHROMATOGRAPHY (CC)

A method frequently used for purifying substances and separating


mixtures at a preparative scale. Use a chromatography column as the main
device. The column is filled with an adsorbent that acts as a stationary phase,
usually silica gel or alumina. The sample is introduced into the column and
a solvent or solvent mixture (eluent) is passed to separate the impurities of
desired product or the components of a mixture, due to the different retention
time on the stationary phase to separate. The eluent can be run throughout
the column either by gravity or by exerting pressure with an inert gas or by
an air pump. This type of CC is called flash chromatography.

Simplest type of chromatographic column consists of a suction flask


and a cylindrical glass or quartz tube considered at one end.
_____________________________________________
ORGANIC CHEMISTRY LABORATORY

GEIGER MULLER COUNTER-


determine the position of the
component that are radioactive.

ELUTION CHROMATOGRAPHY
- The column is washed with suitable solvent called Eluants until each
compound is successively obtained in the effluent fluid known as the
Eluate.

ADSORPTION CHROMATOGRAPHY
- Separation of mixtures through a competitive process in which the
molecules of the mobile phase compete with analyte molecules for
polar adsorption sites on the adsorbents.

- LIQUID SOLID CHROMATOGRAPHY


Mobile phase: LIQUID

PARTITION CHROMATOGRAPHY

- MOBILE AND STATIONARY is liquid


- LIQUID – LIQUID CHROMATOGRAPHY

PAPER PARTITION CHROMATOGRAPHY

- Differences in partition coefficients of substances between two


immiscible liquids.
- If the solid adsorbent is filter paper (CELLULOSE) the process is
referred PPC

Three main methods or procedures for the preparation of paper


partition paper chromatogram

1. DESCENDING CHROMATOGRAPHY:
- accomplished by allowing the mobile phase to flow downward on
the paper strip.
_____________________________________________
ORGANIC CHEMISTRY LABORATORY

2. ASCENDING CHROMATOGRAPHY:

- Mobile phase is allowed to rise upward on the paper by capillary


attraction.

3. RADIAL CHROMATOGRAPHY:

- Mobile phase moves out in concentric circle from the center of a circle
piece of paper.

REVERSED PHASE CHROMATOGRAPHY

- Is also based on the partition phenomenon but as the name implies,


the on polar solvent is fixed to the paper or solid column and act as
the stationary phase and polar is used as the mobile phase.

MOLECULAR EXCLUSION CHROMATOGRAPHY

- AKA: GEL FILTRATION OR GEL PERMEATION


CHROMATOGRAPHY
- Separation procedure in which differential migration of solute
molecules is based on molecular size.

GAS CHROMATOGRAPHY

- Mobile phase an inert gas commonly called carrier gas.

HIGH PRESSURE LIQUID CHROMATOGRAPHY

The advantages of using HPLC as compared to classical


chromatographic methods are greater speed, precision, accuracy and
ease of operation.

THIN-LAYER CHROMATOGRAPHY (TLC)

This is one of the most popular solid-liquid chromatography variants,19


and its most common uses are:
• To identify the components of a mixture.
• To identify two substances.
• To follow the course of a reaction.
• To determine the appropriate conditions for the separation of mixtures
by Column chromatography
• To follow the development of a Column chromatography
_____________________________________________
ORGANIC CHEMISTRY LABORATORY

a) Adsorption process

The sample is applied to the layer and is adsorbed on the material surface
by the action of electrostatic forces (van der Waals forces, hydrogen bonds,
inductive effects, etc.). Later, when the plate is exposed to a flow of solvent
by capillarity, the substance undergoes a competition between the active site
of the adsorbent and the solvent.

b) Adsorbents

The most common adsorbents in TLC are:

• Silica gel (SiO2 ) (used in 80% of separations).

• Alumina (Al2O3 ) (acid, neutral, or basic).

• Siliceous earth or Kieselguhr.

• Cellulose (native or microcrystalline).

• Polyamid

a) Chromatographic plates: These are used as the adsorbent-carrier sheets


of glass, plastic, or aluminum. The sizes (TLC foils) of conventional TLC plate
are 20×20, 10×20 and 5×2 (in cm). There are plates containing a fluorescent
indicator (F254 or F366).

b) Capillaries and Pasteur pipettes: The sample is applied dissolved onto


the plate whether the target is preparative or analytical. For preparative
purposes, the sample is dissolved and applied with a Pasteur pipette; this is
called band application. If the aim is to perform the technique for analytical
purposes, the sample dissolved is applied using a glass capillary. In this case,
it is called point or spot application. The sample should be applied at a
distance from the edge of the plate. The height and the precise location where
the sample is placed can be marked with a pencil to avoid damaging the
adsorbent
_____________________________________________
ORGANIC CHEMISTRY LABORATORY

c). Choice of eluent: Choosing a solvent in which the components of the


mixture present an Rf average around 0.3–0.5 is recommended. The search
for a suitable eluent requires testing several solvents of different polarities or
mixtures

d) Elution tank (chromatography tanks): This is a closed container used


for the development of TLC plates. Its atmosphere is saturated with eluent
vapors. There are various types of tanks on the market (see Figure 4.34). Flat-
bottom tanks with different designs, dual compartment tanks (with
advantages the former of less solvent consumption and no waste, allowing
the environment to be saturated with different vapor-eluting solvent), etc.
Closed bottles and even a beaker with a watch glass is also used. The tank is
filled with the eluent to a height that does not reach the area where the
products are located. Usually, it is a good practice to place in the elution tank
a piece of filter paper that reaches the mouth of the tank so that the eluent
rises by capillarity, and the tank becomes saturated with eluent vapors. This
facilitates proper development of the plate. When the solvent front has
reached the upper edge of the plate, this tank is removed and then revealed

ION-EXCHANGE CHROMATOGRAPHY

Ion-exchange chromatography (or ion chromatography) allows the separation


of ions and molecules with a net charge. Ionic resins are used as stationary
phase. Depending on the load of the sample to separate or purify, two types
of anion and cation exchange are distinguished. Buffered aqueous solutions
are used as a mobile phase

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