Practicum 7

Amino Acid Analysis

Ion exchange chromatography

Outline
Learning objectives 1
Introduction 1
Basic Principle 2
Work Procedures 2
Quiz 4
Case study 4

Learning objectives
1. Understand the principle of amino acid analysis
2. Enable to calculate, analyze and interpret data of amino acid analysis

Introduction
https://www.youtube.com/watch?v=1l-R05spRuk

Ion-exchange chromatography is defined as the reversible adsorption between charged

molecules and ions in solution and a charged solid support matrix. Ion-exchange
chromatography is the most commonly used protein separation technique and results in an
average eightfold purification. A positively charged matrix is called an anion exchanger
because it binds negatively charged ions or molecules in solution. A negatively charged matrix
is called a cation exchanger because it binds positively charged ions or molecules. The most
commonly used exchangers for protein purification are anionic diethylaminoethyl derivatized
supports, followed by carboxymethyl and phosphor cation-exchangers.

The protein of interest is first adsorbed to the ion exchanger under buffer conditions (ionic
strength and pH) that maximize the affinity of the protein for the matrix. Contaminating
proteins of different charges pass through the exchanger unabsorbed. Proteins bound to the
exchangers are selectively eluted from the column by gradually changing the ionic strength or
pH of the eluting solution. As the composition of the eluting buffer changes, the charges of
the proteins change and their affinity for the ion-exchange matrix is decreased.
Basic Principle
Amino acid analysis is used to quantitatively determine the amino acid composition of a
protein. The protein sample is first hydrolyzed to release the amino acids. Amino acids are
then separated using chromatographic techniques and quantified. Ion-exchange
chromatography, reversed-phase liquid chromatography, and gas–liquid chromatography are
three separation techniques used. This practicum will describe the use of ion-exchange
chromatography.

Work Procedures
In general, a protein sample is hydrolyzed in constant boiling 6 N HCl for 24 h to release amino
acids prior to chromatography. Accurate quantification of some amino acids is difficult
because they react differently during hydrolysis. Consequently, special hydrolysis procedures
must be used to prevent errors. Tryptophan is completely destroyed by acid hydrolysis.
Methionine, cysteine, threonine, and serine are progressively destroyed during hydrolysis;
thus, the duration of hydrolysis will influence results. Asparagine and glutamine are
quantitatively converted to aspartic and glutamic acid, respectively, and cannot be measured.
Isoleucine and valine are hydrolyzed more slowly in 6 N HCl than other amino acids, while
tyrosine may be oxidized. In general, losses of threonine and serine can be estimated by
hydrolysis of samples for three periods of time (i.e., 24, 48, and 72 h) followed by amino acid
analysis. Compensation for amino acid destruction may be made by calculation to zero time
assuming first-order kinetics. Valine and isoleucine are often estimated from a 72-h
hydrolysate.

Cysteine and cystine can be converted to the more stable compound, cysteic acid, by
hydrolysis in performic acid and then hydrolyzed in 6 M HCl and chromatographed.
Tryptophan can be separated chromatographically after a basic hydrolysis or analyzed using a
method other than amino acid analysis. In the original method developed by Moore and Stein
and later revised by Stein and colleagues, amino acids were separated by cation-exchange
chromatography using a stepwise elution with three buffers of increasing pH and ionic
strength. In a procedure called post column derivatization, amino acids eluting from the
column were derivatized and quantified by reaction with ninhydrin (reacts with primary amino
group of amino acids) to produce a colored product that was measured
spectrophotometrically.

The method was automated in the late 1970s and adapted for use with high-performance
liquid chromatographs in the 1980s, as new ion-exchange resins were developed that could
withstand high pressures and extremes of pH, ionic strength, and temperature. Amino acids
eluting from the column also may be derivatized with o-phthalaldehyde (OPA) (reacts with
primary amino group of amino acids), then measured with a fluorescence detector. (Note: The
ninhydrin and OPA methods can be used not only for amino acid analysis but also to monitor
hydrolysis of proteins and to assay for protease activity, since these result in an increase of
free primary amino groups.) Other methods were developed in the 1980s using precolumn
derivatization of the amino acids followed by reversed-phase HPLC. The hydrolyzed amino
acids are derivatized prior to chromatography with phenylthiocarbamyl (reacts with primary
amino group of amino acids) or other compounds, separated by reversed-phase HPLC, and
quantified by UV spectroscopy. Methods using precolumn derivatizations can detect picomole
quantities of amino acids. Chromatographic runs usually take 30 min or less. A chromatogram
is a record to show the separation of amino acids in.

The quantity of each amino acid in a peak is usually determined by spiking the sample with a
known quantity of internal standard. The internal standard is usually an amino acid, such as
norleucine, not commonly found in a food product. Results are usually expressed as mole
percent. This quantity is calculated by dividing the mass of each amino acid (determined from
the chromatogram) by its molecular mass, summing the values for all amino acids, dividing
each by the total moles, and multiplying the result by 100.

Preparation of the samples

The samples were homogenized in a blender, dried at 50°C for 12 hours, defatted using a
petroleum ether extraction for 16 h, and then ground in a mill with an 80 mesh. The total
nitrogen in the samples was determined by the Kjeldhal method. Amino acid hydrolisis
Duplicate samples of each food (0.1 g) were hydrolyzed using 6 N HCL (Merck) for 18 hours in
an autoclave at 15 psi. Sodium thioglycolate (Sigma Chemical Co.) was added to the sample to
prevent oxidation. Cysteine was determined by the performic acid oxidation method of Moore
with the following modifications: 20 mL of cold performic acid, freshly prepared, was added
to 0.1 g of the sample; after incubating overnight at 0-5°C, 3 mL of cold 47% HBr (Merck) was
added along with a few drops of 1-octanol as an antifoaming agent; the acid was evaporated
at 40°C using a rotary evaporator; the amino acids were then hydrolyzed according to the
above described method. Tryptophan was determined colorimetrically according to the
method of Vollmer, using 4 N sodium hydroxide for 14-16 hours at 110°C.

Amino acid analysis by HPLC

A liquid chromatograph was used for the determination of amino acids using a fluorescence
detector. The sensitivity was set at a 1 µA full scale. The sample was introduced using a valve
equipped with a 10 µl loop. Amino acid separations were performed on a column (4.6 x 100
mm) packed with 3 µm reversed-phase C-18 octadecyl dimethylsilane particles and connected
to a 4.5 x 30 mm precolumn packed with the same material. Pure amino acid standards were
obtained from PIERCE Chemical Company. The dried hydrolyzates were diluted in sodium
citrate buffer (pH 2.2) (Pierce Chemical Co.) and filtered through a glass microfiber filter
(Whatman 934-AH). Subsequently, 100 ml of the hydrolyzate were mixed with 40 ml of α-
aminobutyric acid as an internal standard and brought to 1 mL with sodium citrate buffer. For
the sample derivatization, an orthophthalaldehyde (OPA) solution was prepared as follows: to
10 mg OPA dissolved in 250 µL of methanol, 37.5 µL 30% Brij 35, 25 µL of 2-mercaptoethanol
and 3 mL of 0.5 M potassium borate buffer pH = 10.4 were added. This solution was diluted
to 10 mL with borate buffer and mixed well. It was then stored under refrigeration in the dark
and allowed to stand for 24 h before use. The preparation was made one day prior to use.
Immediately prior to loading the injection loop, a combination of 0.5 mL OPA solution and 0.5
mL of sample containing internal standard was prepared in a small tube and mixed.
The sample was injected in triplicate and was eluted at 1.5 mL/min with a mixture of 0.1 M
sodium acetate buffer (pH = 7.2):methanol:THF 900:90:10 (v/v/v) as solvent A and methanol
as solvent B. The following gradient was used: 20% B (at 0 min), 30% B (5-8 min), 50% B (10-
15 min), 80% B (18-22 min) and 20% B (at 25 min). The amino acids were completely eluted
at 22 minutes, and the column was equilibrated for 10 minutes. The amino acids were
monitored using a Varian 430020-02 Fluorichrom fluorescence detector at excitation and
emission wavelengths of 350 and 455 nm, respectively. The method was sensitive with
detection limits of approximately 50 femtomoles.

Amino acid score

The amino acid score was calculated using the ratio of a gram of the limiting amino acid in the
food to the same amount of the corresponding amino acid in the reference diet multiplied by
100. The scoring patterns suggested by the FAO/WHO for children of 1-2 years of age were
used for this purpose.

Quiz
The quiz can be accessed via the following link: https://forms.gle/2HBNXvTZ4VdBhMEQA

Case study
The simulation video can be accessed via the following link:
https://www.youtube.com/watch?v=5bBeXvo7ScA
https://www.youtube.com/watch?v=FwU1jB0D3BQ

Table 1. Result of amino acid analysis of soy bean and soy tempe
Soy bean Soy tempe
Crude protein %wb 34.79 36.88 48.11 49.21
Amino acids mg/kg
1 L-Histidin 22398.38 22315.46 5741.57 5729.37
2 L-Serin 47389.52 48128.84 10783.16 10901.63
3 L-Arginin 58257.99 59754.98 12811.18 12854.69
4 Glisin 35971.16 37039.67 9607.53 9688.11
5 L-Asam aspartat 96578.6 99459.94 22407.3 22691.26
6 L-Asam glutamat 169552.1 174929.4 36907.59 37246.32
7 L-Treonin 36190.51 37187.24 9392.62 9485.25
8 L-Alanin 36013.94 37118.77 10802.79 10930.28
9 L-Prolin 40703.41 41927.76 10337.55 10453.94
10 L-Sistin 2890.86 2874.4 498.55 493.84
11 L-Lisin 59779.67 61804.59 11726.39 11806.56
12 L-Tirosin 26169.7 26925.12 6099.66 6118.05
13 L-Metionin 5927.06 5979.31 0.00 0.00
14 L-Valin 42720.07 44150.02 12225.11 12297.66
15 L-Isoleusin 40229.85 41397.29 11720.89 11845.73
16 L-Leusin 65904.36 67672.32 17910.46 18064.21
17 L-Fenilalanin 44942.99 46203.83 11624.14 11722.06
18 L-Triptofan 662.67 654.96 946.37 922.68
Amino acids score ? ?
Table 2. Amino acid standard for calculating amino acid score
Amino acids
Isoleucine 28 mg/g crude protein
Leucine 66 mg/g crude protein
Lysine 58 mg/g crude protein
Total sulfur amino acids 25 mg/g crude protein
Total aromatic amino acids 63 mg/g crude protein
Threonine 34 mg/g crude protein
Tryptophan 11 mg/g crude protein
Valine 35 mg/g crude protein

1. Identify the key points in the procedure of amino acid analysis that need to be
considered to assure the data quality!
2. How to check the data quality?
3. According to the above result please check the data quality and interpret the result!