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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
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
O-pthaldialdehyde
HPLC Column
150mm 4.6 mm
3-m(c18), reversed-phase
Mobile Phase
Sample Preparation
Derivatization
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
Performance of HPLC
analyses
Preparation of standard or sample vials: To a 4-mL glass vial, add the
following:
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