HPLC

Analysis of Amino Acids

Composition in Proteins

SCHEMATIC OF HPLC ANALYSIS

OF AMINOACIDS

Acid
precipitati
on of
protein

Sample
Preparation

Hydrolysis
of Proteins
Acid Hydrolysis
Alkaline
Hydrolysis
Enzymatic
hydrolysis

K2B4O7

Amino acid
Hydrolysates

Vial
1.2 % Benzoic acid (in saturated
K2B4O7)
Saturated K2B4O7
Ethanolamine (10M, Internal
Standard)
HPLC Grade water

1)0.1M
1)0.1M Sodium
Sodium
acetate
acetate buffer(pH
buffer(pH
7.2),
7.2), 0.5%
0.5%
tetrahydrofuran,9%
tetrahydrofuran,9%
methanol(Solvent
methanol(Solvent A)
A)
2)Methanol(Solvent
2)Methanol(Solvent B)
B)

Gradient1.1 ml/min

Derivatization with Ophthaldialdehyde in

autosampler for 1 min

ReversedPhase HPLC
Guard
column
40-m c18,
504.6mm ID

Reversed-Phase
HPLC Analytical
column
Fluorescence
Detection at
Ex.340 nm &
Em.455 nm
Computer
workstation
(Peak integration
& Data
processing)

3m c18,
1504.6 mm
ID

HPLC method for analysis of

Amino acids
Derivatizing Reagent

O-pthaldialdehyde

HPLC Column

150mm 4.6 mm
3-m(c18), reversed-phase

Mobile Phase

0.1M sodium acetate (pH7.2) &

Methanol

Sample Preparation

Requirement of deproteinization only

Derivatization

Pre-column at Room temperature

Removal of reagents or dryness

No

Detection Wavelength

Ex. 340 nm
Em. 455 nm

Sensitivity

fmol

ADVANTAGES
Simple procedures for preparation of samples
Rapid pre column formation of OPA derivatives within 1
minute
High sensitivity of detection at fmol level
Efficient chromatographic separation at room temperature
Easy automation on the HPLC apparatus
Rapid regeneration of guard and analytical columns
Few interfering side reactions
Stable chromatography baseline & absence of irregular
shape peaks

Potential problems
1)Use of OPA
solution within
36 hours of its
preparation
3)Analysis of
one set of
samples within
12.5 h

2)Autosampler
for both
sampling and
in line
derivatization
4)Rapid injection of
the derivatives into
the HPLC column
exactly 1 min after
mixing of OPA with
AA

Acidic hydrolysis of proteins for

analysis of AA except Glu, Gln, Asp,
Asn and Trp
Place 0.5 mL of sample or protein pellet (obtained from 0.5
mL sample), or 0.5 mL H2O (blank) into a 15-mL Kimax glass
tube.
Add 10mL of 6.3M HCl to the sample or 10mL of 6M HCl to
the protein pellet.
Gas the tube with N2 for 1 min and cap the tube.
Place all the tubes in an oven with an inside temperature of
110C.
Two hours later, gently shake the tubes to ensure that the
sample is completely in the solution.
Five hours before the end of a 24-h period hydrolysis, gently
shake the tubes to ensure that the precipitate is suspended
in the solution.
At the end of the 24-h hydrolysis, transfer the whole solution
to a 100-mL flask and make up to the final volume of 100 mL
with H2O. Mix thoroughly the solution, which is stable at 80
C for 6 months. Dilute this solution further by a factor of 10

Alkaline hydrolysis of proteins in foods for

Trp analysis
Place 0.5 mL of sample or protein pellet or 0.5 mL H2 O (blank) into
a 15-mL Kimax glass tube.
Add 10 mL of 4.41 M NaOH and 0.105 mL thiodiglycol into the tube.
Cap the tube. Gently shake the tube.
Place all the tubes in an oven with an inside temperature of 105C.
Two hours later, gently shake the tubes to ensure that the sample is
completely in the solution.
Five hours before the end of a 20-h period hydrolysis, gently shake
the tubes to ensure that the precipitate is suspended in the solution.
At the end of the 20-h hydrolysis, transfer the whole solution to a
50-mL flask and make up to the final volume of 50 mL with H 2O to
obtain Trp concentration within the range of 2.550 M.
Mix thoroughly the solution, which is stable at 80 C for 6 months.
Transfer 5 mL of the solution to a plastic tube and centrifuge it at
600 g for 10 min. Use the supernatant fluid directly for HPLC
analysis of Trp .

Enzymatic hydrolysis of proteins in foods for

analysis of Glu, Gln, Asp, and Asn
Add 0.1mL of sample , or 0.5mL H 2O (blank) into a 15-mL
Kimax glass tube.
Add 2 mL of the porcine gastric fluid.
Incubate the solution at 37 C for 2 h.
Neutralize the solution with 0.2 mL of 0.1 M NaOH.
Add 4 mL of the porcine small-intestinal luminal fluid to
the neutralized solution and incubate the solution at 37
C for 5 h.
At the end of the 4-h incubation, add 0.5 mL of 1.5 M
HClO4 to the solution, followed by slow addition of 0.25mL
of 2M K2CO3 to the solution. The neutralized solution is
centrifuged at 600 g for 10 min, and the supernatant
fluid is analyzed for Glu, Gln, Asp, and Asn using our HPLC
method. This solution is stable at 80 C for 6 months.

Analysis of protein-derived AA by HPLC and

fluorescence detection as OPA derivatives
Mobile phase A (0.1 M sodium acetate, pH 7.2;
Solvent A): Add 27.3 g sodium acetate trihydrate
to 1.6 L H2O a glass bottle, fol- lowed by
sequential addition of 96 L of 6 M HCl, 180 mL
methanol, and 10 mL tetrahydrofuran. Mixing is
performed after each addition. Adjust the final
volume of the solution to 2 L with H2O.
Mobile phase B (Solvent B): 100% Methanol in a
brown glass bottle.

Performance of HPLC
analyses
Preparation of standard or sample vials: To a 4-mL glass vial, add the
following:

100 L of 1.2% benzoic acid

100 L of saturated K2B4O7.

100 L of AA standard (10 M) or sample.

100 L of EA (10 M; internal standard).

1.4mL of HPLC H2O.

Cap the tubes and vortex them before placing them onto an autosampler
(one vial containing a reference standard at both the start and the end, with
samples in the middle). For a 39-min running program, the number of
samples should not exceed 15 in a sample set to ensure the stability of both
OPA and AA.
Condition HPLC columns. Before running a sample set, the guard column and
the connecting analytical column are washed sequentially with water,
methanol, and Solvent A for 15, 20, and 20 min, respectively, at a flow rate
of 1.1 mL/min.
The autosampler is programmed to mix 15 L of a standard or sample
solution with 15 L of the OPA reagent solution for 1 min in the
derivatization loop.

 After the AA-OPA mixtures are injected into the HPLC column, a
solvent gradient (a total running time of 39 min, including the time
for column regeneration between samples) is started, with the
combined flow rate for Solvents A and B being 1.1mL/min. The
percentage (%) of Solvent A is as follows: 0 min, 86; 14 min, 80;
18 min, 70; 18.1 min, 55; 24 min, 50; 26 min, 30; 3032 min, 0;
and 32.139min, 86. This gradient program is also used for Trp
analysis. The change in the mobile-phase solutions between 32
and 32.1min helps reduce the time for column regeneration and
has no adverse effect on the HPLC system. Helium sparge for
Solvents A and B is 100 at 0min and 30 at 18min to save helium.
After each sample set is completed, the HPLC columns are washed
sequentially with water, watermethanol (50:50; v/v), and
methanol (at least 20 min with each solvent), at a flow rate of 1.1
mL/min.
Integration of AA peaks. All AA peaks are identified and integrated
by the workstation software.

Thank You