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CHROMATOGRAPHY

Chromatography is a separation technique that uses the interactions between compounds and two phases, a stationary phase and a mobile phase, as the compounds travel through a supporting medium. The stationary phase is coated on the supporting medium and interacts with analytes, while the mobile phase is a solvent that flows through the supporting medium. Chromatography can be used to check sample purity, identify compounds, and evaluate reaction processes. Thin-layer chromatography (TLC) is a common chromatography technique used for rapid, inexpensive qualitative analysis using silica or alumina plates and a mobile phase solvent.

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851 views

CHROMATOGRAPHY

Chromatography is a separation technique that uses the interactions between compounds and two phases, a stationary phase and a mobile phase, as the compounds travel through a supporting medium. The stationary phase is coated on the supporting medium and interacts with analytes, while the mobile phase is a solvent that flows through the supporting medium. Chromatography can be used to check sample purity, identify compounds, and evaluate reaction processes. Thin-layer chromatography (TLC) is a common chromatography technique used for rapid, inexpensive qualitative analysis using silica or alumina plates and a mobile phase solvent.

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Brian Paguia
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© © All Rights Reserved
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Introduction to Chromatography

Definition
Chromatography is a separation technique based on the different
interactions of compounds with two phases, a mobile phase and a
stationary phase, as the compounds travel through a supporting
medium.

Components:
mobile phase: a solvent that flows through the supporting medium

stationary phase: a layer or coating on the supporting medium that


interacts with the analytes

supporting medium: a solid surface on which the stationary phase is


bound or coated
Applications

1.To check the purity of the given samples.


2.Identification of compounds like acids, alcohols,
proteins, alkaloids, amines, antibiotics, and more.
3.To evaluate the reaction process by assessment
of intermediates, reaction course, and so forth.

Being a semi-quantitative technique, TLC is used more


for rapid qualitative measurements than for quantitative
purposes. But due to its rapidity of results, easy handling,
and inexpensive procedure, it finds its application as one
of the most widely used chromatography techniques.
System Components
TLC system components consist of
1.TLC plates, preferably ready-made with a stationary phase:
These are stable and chemically inert plates, where a thin layer of
stationary phase is applied on its whole surface layer. The
stationary phase on the plates is of uniform thickness and is in fine
particle size.
2.TLC chamber. This is used for the development of the TLC
plate. The chamber maintains a stable environment inside for
proper development of spots. It also prevents the evaporation of
solvents and keeps the process dust-free.
3.Mobile phase. This comprises of a solvent or solvent mixture.
The mobile phase used should be particulate-free and of the
highest purity for proper development of TLC spots. The solvents
recommended are chemically inert with the sample, a stationary
phase.
4.A filter paper. This is moistened in the mobile phase, to be
placed inside the chamber. This helps develop a uniform rise in a
mobile phase over the length of the stationary phase.
STEPS IN TLC
1. TLC involves spotting a dilute solution (1%) of sample on one end of
a small sheet that has been coated with silica gel (SiO2) or alumina
(Al2O3), known as the stationary adsorbent phase.

2. The sheet is placed upright inside a jar in a small pool of solvent. As


the solvent rises up the sheet by capillary action, the components
travel at different rates based on competing interactions with the mobile
(solvent) and adsorbent phases.

SiO2 is used for separation of more polar compounds while Al2O3 is


used in the separation of non-polar compounds.

3. Once separation occurs, the individual components are visualized as


spots at a respective level of travel on the plate.

- iodine or sulphuric acid is used for most organic mixture


- Ninhydrin is used for amino acid
- 2,4- Dinitrophenylhydrazine is used for aldehudes and ketones
Thin-Layer Chromatography (TLC)
• A polar solvent will carry a polar compound
farther while a non-polar solvent will carry a
non-polar compound farther.
• Rf value is the ratio of the distance the spot
travels from the origin to the distance the
solvent travels.
The analytes interacting most
strongly with the stationary
phase will take longer to pass
through the system than those
with weaker interactions.

These interactions are usually


chemical in nature, but in some
cases physical interactions can
also be used.
Theory of Chromatography

1.) Typical response obtained by chromatography (i.e., a chromatogram):

chromatogram - concentration versus elution time

Wh

Wb

Inject

Where:
tR = retention time
tM = void time
Wb = baseline width of the peak in time units
Wh = half-height width of the peak in time units
Note: The separation of solutes in chromatography depends on two factors:

(a) a difference in the retention of solutes (i.e., a difference in their time or volume of
elution
(b) a sufficiently narrow width of the solute peaks (i.e, good efficiency for the separation
system)

Peak width & peak position


determine separation of peaks

A similar plot can be made in terms of elution volume instead of elution time. If volumes
are used, the volume of the mobile phase that it takes to elute a peak off of the column is
referred to as the retention volume (VR) and the amount of mobile phase that it takes to
elute a non-retained component is referred to as the void volume (VM).
2.) Solute Retention:

A solute’s retention time or retention volume in


chromatography is directly related to the strength of the
solute’s interaction with the mobile and stationary phases.

Retention on a given column pertain to the particulars of


that system:
- size of the column
- flow rate of the mobile phase
resolution (RS) – resolution between two peaks is a second measure of how well two
peaks are separated:
tr2 – tr1
RS =
(Wb2 + Wb1)/2
where:
tr1, Wb1 = retention time and baseline width for the
first eluting peak
tr2, Wb2 = retention time and baseline width for the
second eluting peak

Rs is preferred over a since both


retention (tr) and column efficiency
(Wb) are considered in defining
peak separation.

Rs $ 1.5 represents baseline


resolution, or complete separation
of two neighboring solutes  ideal
case.

Rs $ 1.0 considered adequate for


most separations.

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