Separation, Identification and Analysis of the Amino Acid Components of Gluten by Paper Chromatography C.A.M. Dimatatac, M.J.T. Dy, *R.D.V. Figuerroa, J.M.T. Geotina, V.D.V Lazatin Abstract…Full description
Identification of Amino Acids-Paper Chromatography
4 Amino Acids This chapter introduces you to the structure and chemistry of amino acids. The chapter begins by discussing the zwitterionic character of amino acids at physiological pH, fol…Full description
Titration Curve of Amino Acids
Chem 31.1 Experiment 4 ATQ
Experiment Amino AcidsFull description
CTTO. Not Mine.
Exercise 10 (Amines, Amino Acids and Proteins)Full description
Amino acid descriptions and structure for biology olympiadsFull description
Characterized proteins and amino acids through different tests namely Biuret Test, Ninhydrin Test, Hopkins-Cole Test, Lead Acetate Test, Sakaguchi Test and Xanthoproteic Test.Full description
constitutional lawFull description
The test bank from the publisher for the 4th edition ofFull description
The test bank from the publisher for the 4th edition ofDescripción completa
Separation of Oil, GasDescription complète
Experiment 2 Separation of Amino Amino Acids Acids by Paper Chromatography Magbanua, Dianne B. Olabre, Ian Gabriel N.
proper application of the techniques of paper chromatography
the computation and the comparison of the Rf values of the given amino acids
the identification of the unknown amino acid by comparison of Rf values.
KEYWORDS: Retention Factor (RF) ratio of the time spent by the analyzed compound in the stationary phase relative to the time it spent in the mobile hase •
a physical method of separation in which the components to be separated are distributed between two phases:
stationary phase: solid or a liquid supported on a solid which is fixed in place mobile phase: liquid or gas which moves in a definite direction.
Paper chromatography • type of partition chromatography • based on the rate of migration of the
compounds being analyzed while they are in simultaneous contact with the stationary phase.
stationary phase: water bound to the cellulose matrix of the filter paper mobile phase: solvent mixture
RESULTS Amino Acid Distance Distance Standards Travelled by Travelled by the Amino the Solvent Acid (cm) Mixture (cm)
12. 5 12. 5
Unknown Glycine Leucine
Table I. The Rf Values of the Amino
THEORETICAL Rf VALUES OF AMINO ACIDS Amino Acids
Theoretical Rf Values
The separation and migration of the amino acids are based on their affinities to the stationary and mobile phases.
Factors that affect the affinity of a substance: ü
DISCUSSION Higher affinity to the stationary phase
Higher affinity to the mobile phase •Stick to the paper •Unimpeded by the paper •Travel more slowly •Travel with the solvent front •Smaller Rf values •Larger Rf values •Polar compounds •Nonpolar compounds •Bond to the cellulose of the paper •Remain dissolved in the mobile more quickly phase •
Glycine: most polar Leucine: least polar Unknown: Glycine + Leu
Filter paper - made up of cellulose (non polar) fibers which attract and adsorb water vapor to its surface.
20 different amino acids - linked together by peptide bonds to form proteins.
Different proteins vary in the order and number of amino acids in their polypeptide chains.
Amino acids with nonpolar, neutral hydrocarbon side chains: hydrophobic, lower water solubility
Amino acids with polar but neutral R groups: hydrophilic, promote water solubility
Amino acids with polar and basic and acidic R groups: promote water solubility
Solubility of all amino acids in water depends on the pH of the solution.
Rate of migration of each amino acid also depends on the pH of the mobile phase.
Hydrophobic, aliphatic, neutral, non-polar
Aminoethanoic acid Aminoacetic acid
non-essential amino acid with a polar side group
Aromatic, hydrophilic, neutral
basic essential amino acid
Polar, hydrophilic, charged (+)
the amino acids visible
ammonia, or primary and secondar amines
Identify the stationary and mobile phases in paper chromatography. stationary phase – water mobile phase – mixture of butanol, glacial acetic acid and water
GUIDE Explain briefly the differences in Rf QUESTIONS values of the amino acid component of your mixture.
~depends on the polarity or affinity of each substance to the mobile and stationary phases. less polar amino acid mobile phase
GUIDE What are the factors that could affect the QUESTIONS Rf value of a solute? ü
polarity or affinity of the solute to the mobile and stationary phases
nature of stationary and mobile phase
GUIDE Give reasons for the following procedure: QUESTIONS a. The diameter of the amino acid spots should
be about 1mm only. ~spots will usually be larger than the original spots after drying
GUIDE b. The solvent mixture should be QUESTIONS allowed to saturate the chromatography chamber.
~allows for more effective development of the chromatograms ~prevents the evaporation of the solvent
d. The chromatography paper
GUIDE A mixture of amino acids was separated QUESTIONS into its components by two-dimensional chromatography using solvents S-1 and S2. Amino Acid A
S1 (cm) 6.1
S2 (cm) 5.8
Amino Acid Ala Phe Lys Leu Glu His Trp
3.7 9.14 6.15 2.0 2.3 9.0 5.9
6.5 4.9 1.3 9.6 7.5 2.2 6.0
Amino Acid A Amino Acid B Amino Acid C Amino Acid D
Tryptophan Histidine Lysine Phenylalanine
GUIDE Discuss briefly the basic principles of QUESTIONS the following chromatographic
- solvent moves up the plate due to capillary action - components of the sample mixture get separated based on their attraction to
-liquid-solid chromatography - form of adsorption chromatography -different substances will adsorb or adhere onto the surface of fine particles of a solid adsorbent
GUIDE Gas Chromatography QUESTIONS
-stationary phase is a high-boiling liquid -mobile phase is an inert gas -boiling point of the compound (but not always) that determines how fast a component travels through the column ü High
-the separation of compounds is carried out on the basis of their characteristic polarities
GUIDE QUESTIONS ü Reversed
-results from the adsorption of hydrophobic molecules onto a hydrophobic solid support in a polar mobile phase
Chromatography is a set of laboratory techniques used for the separation of mixtures: ü
high performance chromatography,
reversed phase chromatography.
Paper chromatography involves a solvent moving along a filter paper.
The interaction of the analyzed compounds, the filter paper, and the solvent mixture determines the rate of separation and the distances travelled by the substances.
Since the amino acids are colorless, ninhydrin is necessarily applied on the filter paper to react with the amino acids
The difference in affinities of the amino acids leads to their separation on the filter paper and to the formation of a column of spots on the direction of the movement of the mobile phase.
The retention factor (Rf) values of the amino acids is calculated by dividing the distance travelled by the amino acid by the distance travelled by the solvent mixture.
The Rf values may be affected by the
Leucine (Rf=0.7990) is the least polar and has the highest affinity to the mobile phase.
Glycine (Rf=0.2626) is the most polar and has the highest affinity to the stationary phase.
The higher the RF value the less polar is the amino acid.
The unknown is a mixture of lysine and
RECOMMENDATION For more effective development of the S chromatogram, the solvent mixture must be covered and allowed to stand overnight.
The students must wear gloves and be very careful in handling and preparing the filter paper to be used.
The drying time should be prolonged to further reduce the chances of contamination and the overlapping of amino acid spots.