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Pre-Lab Study Questions 33
1. Which two functional groups are in all amino acids?
2. How does an R group determine if an amino acid is acidic, basic, or nonpolar?
3. In a chromatography experiment, a student calculated an fR value for alanine of 0.70 and 0.91
for leucine. Which amino acid traveled higher on the chromatography paper? Explain your
easoning.
4. In a chromatography experiment, a student calculated that the solvent front was 6.8 cm above the
starting line. Arginine traveled a distance of 4.9 cm and glycine traveled 3.4 cm. What fR values
would the student calculate for arginine and glycine?
Date Name
Section Team
Instructo
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REPORT SHEET
Amino Acids
LAB
33
A. Amino Acids
1. Condensed structural formulas (ionized) of amino acids
Glycine Alanine Serine
2. Polar or nonpolar?
3. Condensed structural formulas of amino acids in an acidic solution
4. Condensed structural formulas of amino acids in a basic solution
Date Name
Section Team
Instructor
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LABORATORY GOALS
ill be nonpolar (hydrophobic), polar (hydrophilic),
acidic, or basic.
fR values for amino acids.
LAB INFORMATION
Time: 2
Comments: Ninhydrin spray causes stains. Use it carefully.
Tear out the report sheets and place them next to the matching procedures.
Related Topics: Amino acids
Dispose of all chemicals as directed by your lab instructor.
CHEMICAL CONCEPTS
A. Amino Acids
In our bodies, amino acids are used to build tissues, enzymes, skin, and hair. Essential amino acids,
about half of the naturally occu
ing amino acids, must be obtained from the proteins in the diet because
the body cannot synthesize them. Amino acids are similar in structure because each has an ionized
amino group 3 and an ionized ca
oxylate group ( COO ). Individual amino acids have
different organic groups (R groups) attached to the alpha ( ) ca
on atom. Variations in the R groups
determine whether an amino acid is nonpolar (hydrophobic), polar (hydrophilic), acidic, or basic.
Some R groups contain ca
on and hydrogen atoms only, which makes the amino acids nonpolar and
OH or SH atoms and provide a polar area
that makes the amino acids soluble in water; they
amino acids contain R groups that are ca
oxylic acids (acidic) or amino groups (basic). The R groups of
some amino acids used in this experiment are given in Table 33.1.
Amino Acids 33
374 Laboratory Manual for General, Organic, and Biological Chemistry
TABLE 33.1 Amino Acids Found in Nature
Amino Acid Symbol Polarity Reaction to Wate
Glycine Gly. G Nonpolar Hydrophobic
Alanine Ala, A Nonpolar Hydrophobic
Phenylalanine Phe, F Nonpolar Hydrophobic
Serine Ser, S Polar Hydrophilic
Aspartic acid Asp, D Acidic Hydrophilic
Lysine Lys, K Basic Hydrophilic
Ionization of Amino Acids
In acidic solutions (low pH), the ionized amino acid accepts a proton (H ) to form an ion with a
positive charge. When placed in a basic solution (high pH), the ionized amino acid donates a proton
(H ) to form an ion with a negative charge (see Table XXXXXXXXXXThis is illustrated using alanine.
Amino Acids 375
TABLE 33.2 Ionized Forms of Nonpolar and Polar Neutral Amino Acids
B. Chromatography of Amino Acids
Chromatography is used to separate and identify the amino acids in a mixture. Small amounts of amino acids
and unknowns are placed along one edge of Whatman #1 paper, which makes the chromatogram. The
chromatogram is then placed in a container with solvent. With the paper acting like a wick, the solvent flows
up the chromatogram, ca
ying amino acids with it. Amino acids that are more soluble in the solvent will
move higher on the paper. Those amino acids that are more attracted to the paper will remain closer to the
origin line. After removing and drying the chromatogram, the amino acids can be visualized by spraying the
dried chromatogram with ninhydrin. The distance each amino acid travels from the starting line is measured and
the fR values calculated (see Figure 33.1).
f
distance traveled by an amino acid
R
distance traveled by the solvent
To identify an unknown amino acid, its fR value and color with ninhydrin are compared to the fR
values and colors of known amino acids. In this way, the amino acids present in an unknown mixture of
amino acids can be separated and identified.
FIGURE 33.1 A developed chromatogram f(R values calculated for A, B, and C).
376 Laboratory Manual for General, Organic, and Biological Chemistry
EXPERIMENTAL PROCEDURES GOGGLES REQUIRED!
A. Amino Acids
Materials: Organic model kits or prepared models
1. Use an organic model kit or observe models of glycine, alanine, and serine. Draw their condensed
structural formulas in the ionized form.
2. Indicate whether each of the amino acids is polar or nonpolar.
3. Draw the condensed structural formula of glycine, alanine, and serine in an acidic solution.
4. Draw the condensed structural formula of glycine, alanine, and serine in a basic solution.
B. Chromatography of Amino Acids
Materials: 600-mL beaker; plastic wrap; plastic gloves; Whatman chromatography paper #1 (12 cm
24 cm); toothpicks or capillary tubing; drying oven (80 °C) or hair dryer; metric ruler;
stapler; amino acids (1% solutions): phenylalanine, alanine, glycine, serine, lysine, aspartic
acid, and unknown amino acid; chromatography solvent: isopropyl alcohol, 40.5 M NH OH;
0.2% ninhydrin spray
Keep your fingers off the chromatography paper because amino acids can be transfe
ed from the
skin. Do not use pen, the ink will dissolve in the solvent and ruin your results.
Using forceps, plastic gloves, or a paper towel, pick up a piece of Whatman #1 chromatography
paper that has been cut to a size of 12 cm 24 cm.
Draw a pencil line about 2 cm from the long edge of the paper or plate. This will be the starting
line (origin). With a pencil, mark off 7 equally spaced points about 3 cm apart along the line (see
Figure XXXXXXXXXXLabel each with the a
eviation of one of the amino acids.
Place your name or initials in the upper corner with the pencil.
Use the toothpick applicators or capillary tubes provided in each 1% amino acid solution to make
a small spot (the size of the letter o) by lightly touching the tip to the paper or plate. Always return
the applicator to the same amino acid solution. Dry the spot either with a hair dryer or by allowing
it to air dry. After the spot dries, repeat the application of the amino acids twice more, for a total
of three applications.
FIGURE 33.2 Preparation of a chromatogram.
Amino Acids 377
Prepare the solvent by mixing 10 mL of 40.5 M NH OH and 20 mL of isopropyl alcohol.
Pour the solvent into a 600-mL beaker to a depth of about 1 cm but not over 1.5 cm. The height of
the solvent must not exceed the height of the starting line on your chromatography paper.
Cover the beaker tightly with plastic wrap. This is your chromatography tank.
Label the beaker with your name and leave it in the hood.
Roll the paper with the amino acids into a cylinder and staple the edges without overlapping. The
edges should not touch.
Slowly lower the cylinder into the solvent of the chromatography tank with the row of amino acids
at the bottom. Make sure that the paper does not touch the sides of the beaker (see Figure 33.3).
FIGURE 33.3 Chromatogram in a solvent tank.
Cover the beaker with the plastic wrap and leave the tank and paper undistu
ed.
Do not let the solvent run over the top of the paper.
Carefully remove the paper from the tank. Take out the staples and spread the chromatogram out on
a paper towel. Immediately, use a ruler and pencil to mark the solvent line.
Allow the chromatogram to dry completely. A hair dryer or an oven at about 80 °C may be used to
speed up the drying process.
Dispose of the solvent as directed by your instructor.
Working in the hood, spray the paper lightly, but evenly, with a ninhydrin solution. Distinct,
colored spots will appear as the ninhydrin reacts with the amino acids.
Caution: Do not
eathe the fumes or get spray on your skin.
Calculations:
1. Draw the chromatogram on the report sheet, or staple the original to the report sheet.
2. Outline each spot with a pencil. Place a dot at the center of each spot. Record the color of each
spot on the drawing or original.
3. Measure the distance, in centimeters, from the starting line to the solvent line to obtain the
distance traveled by the solvent. Record.
4. Measure the distance, in centimeters, from the starting line to the center dot of each spot. Record.
5. Calculate and record the fR values for the known amino acid samples and the unknown.
f
distance traveled by an amino acid
R
distance traveled by the solvent
6. Compare the color and fR values produced by the unknown amino acids to those of the known
samples. Identical amino acids will give similar fR values and form the same color with
ninhydrin.
Identify the amino acid(s) in the unknown.