Agilent 1100 User Manual
Series diode array detector (DAD)
is below 5 pmol with a signal-tonoise ratio of 2. For the Agilent
1100 Series fluorescence detector
(FLD) the limit of detection is
below 100 fmol except for Cys-SSCys. Due to better selectivity we
recommend the use of the FLD
below 100 pmol. The linearity correlation factor for well resolved
amino acids is between 0.99998
and 0.99999 in the range from
10 pmol up to 1000 pmol for DAD.
A special amino acid report is
shown and hints for maintenance
and troubleshooting are included.
Ordering information about
columns, standards, chemicals
and capillaries, and Agilent Application Services is also included.
Angelika Gratzfeld-Huesgen
Sensitive and Reliable Amino Acid
Analysis in Protein Hydrolysates using
the Agilent 1100 Series HPLC
Pharmaceutical
Technical Note
Agilent Technologies
Innovating the HP Way
Abstract
This technical note demonstrates
the performance of amino acids
analysis on the Agilent 1100 Series
modules and systems for LC.
Detailed information regarding
instrumental and chromatographic
conditions and performance are
given. The precision for retention
times over 10 runs is as low as
0.2 % RSD, and the RSD of areas is
between 0.6 and 5 %. The limit of
detection for the Agilent 1100
Introduction
Since the introduction of automated amino acid analysis on the
HP 1090 Series HPLC system
using a two step precolumn
derivatization, this technique has
become a well accepted and routine method for the analysis of primary and secondary amino acids
in protein hydrolysates. This technical note will demonstrate that
amino acid analysis can be done
on the Agilent 1100 Series modules and systems for LC using the
binary pump to obtain even better
performance than was achieved
with the HP 1090 Series. The
conditions used are the same,
that were used on the HP AminoQuant 2 based on the HP 1090
Series system.
Experimental
HPLC Instrumentation:
The following Agilent 1100 series
modules were used:
• high pressure gradient pump
order number: G1312A
• online vacuum degasser
order number: G1322A
• autosampler
order number: G1313A ( or
thermostatted autosampler
order number: G1327A)
• thermostatted column
compartment
order number: G1316A
• diode array detector for
concentrations above 100 pmol
order number: G1315A
• fluorescence detector for
concentrations below 100 pmol
order number: G1321A
• Agilent ChemStation
order number: G1319A
• Software for amino acid reports
part number G1300-10013
(user contributed software).
One modification was made for
the standard high pressure
gradient pump. The solvent mixer
(part number G1312-87330) was
replaced by a capillary (part
number 1090-87610) or Upchurch
mixer to reduce delay volume.
Amino acid Standards
Five different concentrations of
amino acid standards were used
for evaluating the precision of
retention times and areas, and the
limit of detection and linearity.
These were 10, 25, 100, 250 and
1000 pmol/µl. The standards
contained the following
compounds:
Asp Aspartic acid
Glu Glutamic acid
Ser Serine
His Histidine
Gly Glycine
Thr Threonine
Ala Alanine
Arg Arginine
Tyr Tyrosine
Cys-SS-Cys Cystine
Val Valine
Met Methionine
Phe Phenylalanine
Ile Isoleucine
Leu Leucine
Lys Lysine
Pro Proline
Derivatization Reagents
The online derivatization was
performed using ortho-phthalaldehyde (OPA) for the primary amino
acids and 9-fluorenylmethyl
chloroformate (FMOC) for the
secondary amino acids. A 0.4 N
borate buffer was used with pH
10.4.
Preparing mobile phases,
standards and derivatization
reagents
Mobile phase A:
1. Weigh 1.36 ± 0.025 g of sodium
acetate tri-hydrate and transfer
it into a 800 ml glass beaker.
2. Add 500 ml of purified water
and stir until all crystals are
completely dissolved.
3. Add 90 µl of triethylamine
(TEA) and mix.
4. Adjust the pH to 7.20 ± 0.05 by
adding a few drops of 1–2 %
acetic acid.
5. Add 1.5 ml of tetrahydrofuran
(THF) and mix.
FMOC reagent
1. Place several microvials in the
microvial rack.
2. Open one ampule of the FMOC
reagents. This is the approximate amount needed for ten
days.
3. Pipette between 50 and 100 µl
into each microvial.
4. Cap all vials. Store vials which
are not directly used in the
refrigerator.
Borate buffer
Place 1 ml of borate buffer into
a 2 ml vial.
Water
Place 1 ml purified water into a
2 ml vial.
Preparing internal standards of
5 nmol and 500 pmol
The secondary amino acids were
quantified using sarcosine as an
internal standard. The primary
amino acids can be quantitated by
either using external standard
procedures or by using norvaline
as an internal standard. If you are
working with DTDPA to convert
cystine and cysteine to the stable
Cys-MPA during hydrolysis, then
make up your ISTD stock solutions in borate buffer instead of
HCl to avoid solubility problems.
Mobile phase B:
1. Weigh 1.36 g ± 0.025 g of
sodium acetate trihydrate and
transfer it into a 200 ml glass
beaker.
2. Add 100 ml of purified water
and stir until all crystals are
dissolved.
3. Adjust pH to 7.20 ± 0.05 by
adding a few drops of 1–2 %
acetic acid.
4. Add this solution into a
mixture of 200 ml acetonitrile
and 200 ml methanol and mix.
Derivatization reagents:
For convenience use microvial kit
from Agilent (part number 9301-
1388)
OPA reagent
1. Place several microvials in the
microvial rack.
2. Open one ampule of the OPA
reagents. This is the approximate amount needed for ten
days. Preparing more could
cause problems with oxidation.
3. Pipette between 50 and 100 µl
into each microvial.
4. Cap all vials. Store vials which
are not directly used in the
refrigerator.
1. 5 nmol ISTD standard:
Weigh 22.3 mg sarcosine
(optional 29.3 mg norvaline)
and dissolve in 50 ml
0.1 N HCl.
2. 500 pmol ISTD standard:
Take 5 ml of 5 nmol ISTD and
dilute with 50 ml 0.1N HCl.
Preparing the calibration
standards
For standard sensitivity eight
different concentration were used:
1. 900 pmol/µl amino acid standards with 500 pmol/µl internal
standards.
Place 900 µl from standard
with 1000 pmol amino acids
and 100 µl from ISTD (5 nmol)
in a 2 ml vial. Mix and place
100 µl each in microvials
2. 225 pmol/µl amino acid standards with 500 pmol/µl
internal standards. Use 250
pmol amino acid standard
and follow the same procedure
as under 1.
3. 90 pmol/µl amino acid
standards with 500 pmol/µl
internal standards.
Use 100 pmol amino acid
standard and follow the same
procedure as under 1 of this
section.
For high sensitivity three
different concentrations were
used:
1. 90 pmol/µl amino acid
standards with 50 pmol/µl
internal standards.
Place 900 µl from standard
with 100 pmol amino acids and
100 µl from ISTD (500 pmol) in
2 ml vial. Mix and place 100 µl
each in microvials.
2. 22.5 pmol/µl amino acid
standards with 50 pmol/µl
internal standards.
Use 25 pmol amino acid
standard and follow the same
procedure as under 1.
3. 9 pmol/µl amino acid standards
with 50pmol/µl internal standards.
Use 10 pmol amino acid
standard and follow the same
procedure as under 1.
Extended Amino Acids (EAA)
In addition to the previously
analyzed 17 amino acids which
can be found in hydrolysates (see
figure 1), the amino acid supplement kit contains the following
amino acids as solids, which are
of interest for amino acid analysis
in food:
norvaline
sarcosine
asparagine
glutamine
tryptophan
and hydroxyproline
Table 1 shows the stock solutions
used for different purposes.
Use these stock solutions in place
of the 5 nmol ISTD or 500 pmol
ISTD stock solutions previously
described, to prepare your calibration standards.
Preparing internal standards of
10 nmol and 1 nmol
The secondary amino acids were
quantitated using sarcosine as an
internal standard. The primary
amino acids can be quantitated by
either using external standard
procedures or by using norvaline
as an internal standard. If you are
working with DTDPA to convert
cystine and cysteine to the stable
Cys-MPA during hydrolysis, then
make up your ISTD stock
solutions in a borate buffer
instead of HCl to avoid solubility
problems.
1. 10 nmol ISTD standard:
Weigh 44.6 mg sarcosine
(optional 58.6 mg norvaline)
and dissolve in 50 ml
0.1 N HCl.
2. 1 nmol ISTD standard:
Take 5 ml of 10 nmol ISTD and
dilute with 50 ml 0.1 N HCl.
Standard sensitivity
9 nmol/µl extended amino acid with 5 nmol/µl internal standards
2.25 nmol/µl extended amino acid with 5 nmol/µl internal standards
900 pmol/µl extended amino acid with 5nmol/µl internal standards
High sensitivity
900 pmol/µl extended amino acid with 500 pmol/µl internal standards
225 pmol/µl extended amino acid with 500 pmol/µl internal standards
90 pmol/µl extended amino acid with 500 pmol/µl internal standards
Table 1
Stock solutions
Loading...