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Experiment 4 Separation of Amino Acids by TLC

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organic- bio chemistry (CHEM153L)

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Experiment 4

Separation of Amino Acids by Thin Layer

Chromatography

Chromatographic methods revolutionized experimental organic chemistry. These methods are by far the most powerful techniques for separating mixtures and isolating pure substances, either solids or liquids. Chromatography is the resolution (separation) of a multi-component mixture (several hundred components in some cases) by distribution between two phases, one stationary and one mobile. The various methods of chromatography are categorized by the phases involved: column, thin-layer, and paper (all solid-liquid chromatography); partition (liquid-liquid chromatography); and vapor-phase (gas-liquid chromatography, or simply gas chromatography). The principal mechanism of these separations depends on the differential solubility, or adsorptivity, of the mixture components in the two phases involved. That is, the components must exhibit different partition coefficients.

Thin-layer chromatography (TLC) uses a thin layer (100μm) of stationary phase spread over a flat surface. A microscopic glass slide or a sheet of plastic can be used as support for the thin layer of stationary phase. A mobile liquid phase (solvent system) flows through the thin layer of stationary phase. TLC, just like column chromatography uses polarity differences to separate materials. A sample on a TLC plate is subjected to two opposing forces: (1) the solubility of the sample in the solvent system, and (2) the adsorption forces binding the sample to the solid phase.

Alumina (aluminum oxide, Al 2 O 3 ) and silica gel (SiO 2 ) are typical materials for stationary phase in TLC. Many common organic solvents are used as the liquid (sometimes called eluent) that act as the mobile phase and elute (wash) materials through the plate.

All 20 of the common amino acids (standard amino acids) are α-amino acids. They have a carboxyl group and an amino group bonded to the same carbon atom (the α- carbon). They differ from each other in their side chains, or R groups, which vary in structure, size, and electric charge. The interaction of the amino acids with the stationary phase like silica varies depending on their 'R' groups. The amino acid that interacts strongly with silica will be carried by the solvent to a small distance, whereas the one with less interaction will be moved further. By running controls (known compounds) alongside, it is possible to identify the components of the mixture.

C H E M 153 L

O R G A N I C - B I O C H E M I S T R Y (Laborator

y)

PROCEDURE

A. PREPARATION OF THE TLC DEVELOPING CHAMBER AND THE PLATE

  1. Pour the solvent mixture n-nutanol, acetic acid and water in the ratio 12:3:5 by volume in to the TLC chamber and close the chamber.

  2. The chamber should not be disturbed for about 30 minutes so that the atmosphere in the jar becomes saturated with the solvent.

  3. Using a pencil (never ever use a pen) gently draw a straight line across the adsorbent side of the plate approximately 1 cm from the bottom.

B. APPLICATION OF THE SAMPLE (Test samples: 2% solution of: Glycine, Tyrosine, Tryptophan, Unknown)

  1. Fill a capillary micropipette with the clear solution of the test samples.

  2. On the straight line drawn on the plate, spot a minute drop of the sample by gently touching the tip of the capillary micropipette to the plate.

  3. Allow the spot to dry.

  4. Spot the second and third amino acid on the plate (enough space should be provided between the spots).

  5. Repeat the above step for spotting the unknown amino acid.

C. DEVELOPMENT OF THE CHROMATOGRAM

  1. Place the spotted TLC plate in the chamber as evenly as possible and lean it against the side. Position the plate such that the spots are above the solvent level.

C H E M 153 L O R G A N I C - B I O C H E M I S T

R Y (Laboratory)

Expt. 4 Separation of Amino Acids by Thin Layer

Chromatography

I. Objectives

II. Apparatus: III. Symbols of Chemicals:

IV. Procedure: (Note: Write schematic diagram on the sheet provided.)

V. Data and Observations

Group Members: 1. 2.

Group No. Section: Date Submitted:

Member 1 Member 2 Pre-lab Data Data and Observations Post-lab Questions Sub-total Online Act Participation Pre-lab Online Quiz Total Score

G W Y Unknown

Rf values of amino acids and unknown. Distance Traveled Rf

  1. Glycine

  2. Tryptophan

  3. Tyrosine

  4. Unknown

  5. Solvent (__________________)

Identity/Composition of the Unknown

POST-LAB QUESTIONS
  1. What is the principle behind TLC? (5 points)
  1. What is/are the limitation/s of TLC? (5 points)
  1. How can one apply the methods of TLC in the identification of an unknown amino acid? (5 points)

####### Schematic Diagram of Procedure

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Experiment 4 Separation of Amino Acids by TLC

Course: organic- bio chemistry (CHEM153L)

145 Documents
Students shared 145 documents in this course

University: Xavier University - Ateneo de Cagayan

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For the exclusive use of Chem 153L class in Xavier-Ateneo EXPERIMENT 4
THIS MATERIAL IS OWNED BY THE CHEMISTRY DEPT OF XAVIER-ATENEO
1
Experiment 4
Separation of Amino Acids by Thin Layer
Chromatography
Chromatographic methods revolutionized experimental organic chemistry. These methods are by
far the most powerful techniques for separating mixtures and isolating pure substances, either
solids or liquids. Chromatography is the resolution (separation) of a multi-component mixture
(several hundred components in some cases) by distribution between two phases, one stationary
and one mobile. The various methods of chromatography are categorized by the phases involved:
column, thin-layer, and paper (all solid-liquid chromatography); partition (liquid-liquid
chromatography); and vapor-phase (gas-liquid chromatography, or simply gas chromatography).
The principal mechanism of these separations depends on the differential solubility, or
adsorptivity, of the mixture components in the two phases involved. That is, the components must
exhibit different partition coefficients.
Thin-layer chromatography (TLC) uses a thin layer (100µm) of stationary phase spread over a flat
surface. A microscopic glass slide or a sheet of plastic can be used as support for the thin layer of
stationary phase. A mobile liquid phase (solvent system) flows through the thin layer of stationary
phase. TLC, just like column chromatography uses polarity differences to separate materials. A
sample on a TLC plate is subjected to two opposing forces: (1) the solubility of the sample in the
solvent system, and (2) the adsorption forces binding the sample to the solid phase.
Alumina (aluminum oxide, Al2O3) and silica gel (SiO2) are typical materials for stationary phase in
TLC. Many common organic solvents are used as the liquid (sometimes called eluent) that act as
the mobile phase and elute (wash) materials through the plate.
All 20 of the common amino acids (standard amino acids) are α-amino acids. They have a carboxyl
group and an amino group bonded to the same carbon atom (the α- carbon). They differ from
each other in their side chains, or R groups, which vary in structure, size, and electric charge. The
interaction of the amino acids with the stationary phase like silica varies depending on their 'R'
groups. The amino acid that interacts strongly with silica will be carried by the solvent to a small
distance, whereas the one with less interaction will be moved further. By running controls (known
compounds) alongside, it is possible to identify the components of the mixture.
C H E M 153 L O R G A N I C - B I O C H E M I S T R Y (Laboratory)

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