BIO / EXPERIMENT Separation of Amino Acids by Paper Chromatography

Slides:



Advertisements
Similar presentations
Identifying the components of a mixture
Advertisements

Paper Chromatography 1Dr. Nikhat Siddiqi. Paper chromatography and TLC are examples of adsorption chromatography. 2Dr. Nikhat Siddiqi.
Chromatography Forensic Science T. Trimpe
Analysis of Analgesic Tablets by Thin Layer Chromatography (TLC) Organic Chemistry Lab I Fall 2008 Dr. Milkevitch Oct 12 & 14, 2009.
Lab Activity 7 IUG, Fall 2012 Dr. Tarek Zaida IUG, Fall 2012 Dr. Tarek Zaida 1.
Paper and Thin layer Chromatography
Chromatography.
Prepared by : Mona Abo Hasera
Thin Layer Chromatography of Lipids
What is Chromatography?
Dr. Samah Kotb Nasr Eldeen.  Amino acids: are the building blocks of peptides and proteins.  They possess two functional groups, the carboxylic acid.
What is Chromatography? Chromatography is a technique for separating mixtures into their components in order to analyze, identify, purify, and/or quantify.
Chromatography Chromo: color Graph: to write
What is Chromatography? Derived from the Greek word Chroma meaning colour, chromatography provides a way to identify unknown compounds and separate.
Paper and Thin Layer Chromatography (TLC) Experiment 4 BCH 333[practical]
What is Chromatography? Chromatography is a technique for separating mixtures into their components in order to analyze, identify, purify, and/or quantify.
Chromatography Lab # 5.
What is Chromatography? Chromatography is a technique for separating mixtures into their components in order to analyze, identify, purify, and/or quantify.
Experiment.13 Amino acid analysis by adsorption thin layer chromatography (adsorption TLC)
Transamination.
© SSER Ltd.. General principles Substances are separated according to their differential solubility between the stationary phase, represented by the water.
Paper and Thin Layer Chromatography (TLC) Experiment 6 BCH 333 [practical]
T. Trimpe What is chromatography? From Wikipedia... Chromatography (from Greek word for chromos for colour) is the collective.
Thin-Layer Chromatography (TLC) Uses: To separate the components of a mixture To determine the purity of a compound To see if two compounds are identical.
Chromatography A technique for separating and identifying components in a mixture. From the Greek: chroma = color and graph = writing. Involves the interaction.
S EPARATION BY CHROMATOGRAPHY Describe chromatography as an analytical technique that separates components in a mixture. State that the mobile phase may.
Chromatographic Separation of Sugar
Chromatography Chapter Dr Gihan Gawish. 1. Paper Chromatography Dr Gihan Gawish  Paper chromatography is a technique that involves placing a small.
Separating plant pigments
ANALYSIS AND IDENTIFICATION OF ABNORMAL METABOLITES IN URINE USING : CHROMATOGRAPHY Paper and Thin layer Chromatography.
INTRODUCTION TO CHROMATOGRAPHY DR. Harrizul Rivai, m.s.
Chromatography Homogeneous mixtures of a solvent and one or more solutes (dissolved substances) are often separated by chromatography. Chromatography works.
General Tests for Chemistry 101
Lab Activity 7 IUG, Fall 2012 Dr. Tarek Zaida IUG, Fall 2012 Dr. Tarek Zaida 1.
Using chromatography to identify amino acids
Cellular Biochemistry and Metabolism (CLS 333 ) Dr. Samah Kotb Nasr Eldeen Identification of free amino acids by Thin layer chromotography (TLC) using.
PAPER CHROMATOGRAPHY.
By Susan McCullough With Thanks to Lori Olson at SRI International.
Principles of chromatography
Chromatography.
PLANAR CHROMATOGRAPHY Ferosekhan. S FNB-41 Chromatography Chromatography is a technique for separating mixtures into their components in order to analyze,
THIN LAYER CHROMATOGRAPHY
PAG6 TLC. Need to book Repro – Example results or Page 19 of delivery guide – Example method – FLIPPED learning sheet for start of nucleotides Applied.
Biochemical instrumental analysis - 9 Dr. Maha Al-Sedik 2016 CLS 332.
Separating Mixtures Differences in physical properties can be used to separate mixtures What are some physical properties we can use to separate mixtures?
PAPER CHROMATOGRAPHY.
What is Chromatography?
Paper Chromatography.
Chromatography.
BASED ON POLARITY.
Lab Activity 4 IUG, Fall 2017 Dr. Tarek Zaida.
What is Chromatography?
Grab a whiteboard and pen
Organic Instrumentation
What is Chromatography?
What is Chromatography?
Let us understand the basic process of chromatography….
Dnyanasadhana College, Thane. Department of Chemistry T. Y. B. Sc
SEPARATION TECHNIQUES
Paper and Thin Layer Chromatography (TLC)
What is Chromatography?
What is Chromatography?
1/18 Objective: Explain the purpose and process of chromatography.
Lab Activity 4 IUG, Fall 2017 Dr. Tarek Zaida.
Paper title-Analytical techniques in chemistry
What is Chromatography?
What is Chromatography?
What is Chromatography?
Chromatography Forensic Science.
Presentation transcript:

BIO 306.01/02 EXPERIMENT 5 Separation of Amino Acids by Paper Chromatography A. NAZLI BAŞAK

The methodology of chromatography includes now several procedures. Separation of Amino Acids by Paper Chromotography Chromatography was first developed in the 19. century as a means of studying and separating plant pigments (hence the name “chroma”). The methodology of chromatography includes now several procedures. All separate on the same general principle; uninterrupted flow of a moving phase thru a region of stationary phase (substance) which contains the sample to be analyzed.

Separation of Amino Acids by Paper Chromotography Compounds in the sample to be analyzed interact to different degrees with the stationary and moving phases, thus affecting the separation. The type of interaction between the stationary phase and the compounds in the sample is what distinguishes the different procedures. The interaction is based on one of the following principle: ion exchange; solubility; adsorption; affinity or sieving. Some procedures utilize a combination of factors.

Paper chromatography: Separation of Amino Acids by Paper Chromotography Paper chromatography: One of the early designs in the development of chromatography techniques was the use of a sheet of filter paper as an inert stationary support for a liquid. The paper absorbs water vapor, and the stationary phase is a polar liquid (the absorbed water) and not the paper itself. Another solvent with more non-polar constituents, such as n-butanol / water, is allowed to migrate up or down the paper by capillary action.

Separation of Amino Acids by Paper Chromotography When this so-called developing solvent reaches the location, where the sample was applied onto the paper(origin), the sample will partition to different extents between the moving, non-polar phase and the stationary polar phase. This partioning will continue as long as the developing solvent moves along the paper.

Separation of Amino Acids by Paper Chromotography Origin of application

The extent of migration is expressed by the Rf value; Separation of Amino Acids by Paper Chromotography Substances that are more hydrophilic will move less than those that are more hydrophobic. The extent of migration is expressed by the Rf value; Both distances measured from the origin of sample application

Experiment Procedure: Separation of Amino Acids by Paper Chromotography Experiment Procedure: In paper chromatography substances are distinguished by their relative position in the paper after solvent has moved a given distance. The sample is placed at a marked spot on the paper and allowed to dry; this is the origin.

The paper is then placed into the chromatography tank (closed chamber) Separation of Amino Acids by Paper Chromotography The paper is then placed into the chromatography tank (closed chamber) One end is immersed in the mobile phase (n-butanol / water also called developing solution) Capillary draws the solvent thru paper, dissolves the sample as it passes the origin, and moves the components in the direction of flow.

Separation of Amino Acids by Paper Chromotography After the “solvent front” has reached a point near the top end of the paper, the sheet is removed and dried. The ratio of distances traveled by the sample and by the solvent is the Rf value. Value of Rf depends on the substance, the paper and the solvent. The Rf value is always < 1.

Detection of samples by Ninhydrin reactions Separation of Amino Acids by Paper Chromotography Detection of samples by Ninhydrin reactions A very widely used reaction of the α-amino group is the Ninhydrin reactions, which is utilized to estimate amino acids quantitatively in very small amounts. An α-amino acid reacts with two molecules of Ninhydrin on heating to yield an intensively colored product. Amino acids and peptides with a free α-NH2 group give a purple color, whereas Proline with its NH group (imino) yields a yellow color.

Separation of Amino Acids by Paper Chromotography Oxidative decarboxylation

Separation of Amino Acids by Paper Chromotography

Separation of Amino Acids by Paper Chromotography Ninhydrin brings about the oxidative decarboxylation of the amino acid. The ammonia and Hydrindantin so formed react with a second molecule of Ninhydrin to yield a purple pigment of which only the nitrogen atom arises from the amino acid. designed and prepared by alburse