Amino Acid Analysis

Analysis of Amino Acids by HPLC
Rita Steed
Agilent Technologies, Inc.
800-227-9770 opt 3/opt3/opt 2
Page 1
Amino Acid Analysis - Agilent

Restricted
June 24, 2010
Outline
Amino Acids Structure, Chemistry
Separation Considerations
Challenges
Instrumentation
Derivatization OPA, FMOC

Overview of Separations
Examples
Amino Acid Analysis - Agilent Restricted

Page 2
June 24, 2010
Amino Acids Structure, Chemistry
CH3
Alanine ((Ala))
Glutamic Acid ((Glu))

Page 3
June 24, 2010
Amino Acids Zwitterionic

Page 4
June 24, 2010
Separation Considerations
Zwitterions - poor solubility near isoelectric
electric point
Most have poor UV absorbance
Derivatization OPA,
OPA FMOC
Reduce polarity increases retention in reversed-phase
chromatography
g p y
Improve sensitivity UV, Fluorescence
Detector; DAD, FLD, MS, ELSD

Page 5
June 24, 2010
Ortho Phthalaldehyde (OPA) and Fluorenylmethoxy

chloroformate (FMOC) Reactions with Amines
OPA
SR
H
H
+RNH2
RSH
NR
Room Temperature
Non-fluorescent
Non
fluorescent
Does not absorb at 338nm
FMOC
- HCl
RRNH
+ or
RNH2 Room Temperature
Fluorescent
Absorbs at 262nm and
Fluorescences at 324nm
Fluorescence: Ex 340nm, Em 450nm

DAD: 338,
338 10nm; Ref.
Ref 390,
390 20nm
NRR
or
NHR
Fluorescence: Ex 260nm, Em 325nm

DAD: 262, 16nm; Ref. 324,8nm
Group/Presentation Title
Agilent Restricted
Month ##, 200X
Names and Order of Elution for OPA and FMOC

Derivatives of Amino Acids
Peak #
Peak #
AA Name
AA Abbreviation
Derivative Type
AA Name
AA Abbreviation
Derivative Type
Agilent Restricted
Month ##, 200X
Agilent AAA Methods - Theyve Evolved

Automated Amino Acid Analysis AminoQuant I & II (1987)
1090
1100,, Pub. No. 5968-5658E
Eclipse AAA
Columns
993400
993400-902
902, 4
4.6x150mm,
6x150mm 5um
963400-902, 4.6x150mm, 3.5um
966400-902, 3.0x150mm, 3.5um
Pub. No. 5980-1193E
Eclipse Plus
Column Options
Application
pp
Notes
5989-6279EN
5990-4547EN
5989-6297EN
5990-3283EN
Agilent Restricted
Month ##, 200X
Agilents Solutions for Amino Acid Analysis

A Closer Look
Agilent has one basic type of chemistry for amino acid analysis
OPA/FMOC derivatization.
We have 3 columns and methods that can be used to achieve
separations of amino acids.
AminoQuant this is our oldest methodology
It will go out of support due to supply inconsistencies
Eclipse
p AAA this method uses a specially
p
y tested Eclipse
p XDB-C18
column and modified mobile phase for improved resolution
This method is not set-up for anything other than 4.6mm ID columns
Gua
Guard
d columns
co u s a
are
e needed
eeded for
o maximum
a
u lifetime
et e
Eclipse Plus AAA
Has solutions for all column dimensions and works on multiple LCs
Kits have not been created yet
Confidentiality Label
9
June 24, 2010
AminoQuant Amino Acid Method

Based on Hypersil ODS material that is specially
treated.
treated
Currently we have to adjust the mobile phase and
provide directions to customers with each batch
batch.
This is not good for regulated methods.
These columns will be made obsolete in the future.
10
June 24, 2010
AminoQuant Method
AminoQuant
serial no.: USOE007668
part no.: 79916AA-572
batch no.: 51205964
L x i.d. = 200 x 2.1 mm; dp = 5 m
Eluent:A: 500 ml 20 mM Sodium acetate + 2 mg EDTA
+ 0.018% Triethylamine(v/v) adjusted to pH 7.2 with
Acetic acid + 0.3% Tetrahydrofuran
y
B:100 ml 20 mM Sodiumacetate adjusted to pH7.2 with
Acetic acid + 200 ml Methanol + 200 ml Acetonitrile
Gradient:
%A
%B
flow
Time (min)
0
17.0
100
40
0
60
0.45
18.0
18
0
18.1
23.9
0
0
0
100
100
100
0.45
0
45
0.8
24.0
25.0
0
100
100
0
0.8
0.45
Sample:
Amino acid standard [part no. 5061-3330]
name
abbr.
L-Aspartaic
L
Aspartaic acid
ASP
L-Glutamic acid
GLU
LSerine
SER
L-Histidine
HIS
Glycine
GLY
L Th
L-Threonine
i
THR
LArginine
ARG
L-Alanine
ALA
L-Tyrosine
TYR
L-Cystine
y
CY2
L-Valine
VAL
L-Methionine
MET
L-Phenylalanine
PHE
L-Isoleucine
ILE
L Leucine
L-Leucine
LEU
l-Lysine
LYS
L-Proline
PRO
Flow:
0 45 ml/min
0.45
Temperature: 40 oC
Injection:
injection program, including derivatisation
steps with OPA and FMOC.
injected mixture contains 1 l AA sample.
11
June 24, 2010
Example of Amino Acid Separation on AminoQuant

DAD1 A, Sig=338,10 Ref=390,16, TT (C:\HPCHEM\1\DATA\030818\011-0103.D)
1.398 - glutamic accid
14.739 - lysine
14.057 - leucine
13.185 - phenylalanine
13.404 - isoleucine
11.610 - valine
11.828 - methionine
11.209 - cystine
9.700 - tyrosine
200
6.231 - histidine
6.369 - g
glycine
5.155 - serine
300
8.093 - alanine
8.372 - arginine
1.274 - as
spartic acid
400
These 3 amino acid pairs are difficult to separate.

Aspartic acid/glutamic acid separation can be improved with
Eclipse AAA or Eclipse Plus AAA method.
Histidine/glycine can also be improved.
Alanine/Arginine is sensitive to ionic strength of mobile phase.
6.752 - threonine
mAU
10.900
100
0
0
10
12
14
16
min
12
June 24, 2010
Eclipse AAA Method

Primary goal of Eclipse AAA method was to provide better resolution of the
critical pairs in the AminoQuant method.
Secondary goal was to provide options for a faster separation.
An application/technical note (Pub no. 5980-1193) and detailed instructions
(Agilent ZORBAX Eclipse AAA Instructions for Use
Use, Pub no
no. 5980
5980-3088EN,
3088EN
June 2008) are available.
The method has been in place for a number of years and is reliable.
It was originally written for an 1100 and the autosampler derivatization
protocol has been rewritten for the 1200.
Assumes a binary instrument for optimum results.
results
13
June 24, 2010
Eclipse AAA Method

Column:
C
l
ZORBAX E
Eclipse-AAA;
li
AAA 3.5m;
35
L x i.d.=150
i d 150 x 4.6
4 6 mm [USXH001289]
Eluent: A: 40 mM Phosphate buffer pH 7.8
B: Methanol /Acetonitrile/Water = 45/45/10
Gradient:
Time (min)
%B
0
0
1.9
0
18.1
57
18.6
100
22 3
22.3
100
23.2
0
26
0
Flow:
2.0 ml/min
T
Temperature:
t
40 C
Injection: injection program, including derivatisation steps with OPA and FMOC, injected mixture
contains 0.5 l AA sample.
Detection:
DAD
signal A: = 338 nm,
nm bw = 10 nm; reference = 390 nm
nm, bw = 20 nm (for OPA
OPA-amino
amino acids)
signal B: = 262 nm, bw = 16 nm; reference = 324 nm, bw = 8 nm(for FMOC-amino acids)
FLD
time = 0 min: Ex/Em = 340/450 nm, gain = 10
time = 15 min: Ex/Em = 266/305 nm, gain = 9
14
June 24, 2010
Amino Acids in Eclipse AAA Method

Amino acid standard [part no. 5061-3331] + supplement amino acid kit [part no. 5062-2478]
name
abbr.
Concentration (nmol/l)
Aspartate
ASP
0.9
Glutamate
GLU
0.9
Asparagine
ASN
1.8
Serine
SER
09
0.9
Glutamine
GLN
1.8
Histidine
HIS
0.9
Glycine
GLY
0.9
Threonine
THR
0.9
Arginine
ARG
0.9
Alanine
ALA
0.9
Tyrosine
TYR
0.9
Cystine
CY2
0.9
Valine
VAL
0.9
Methionine
MET
0.9
Norvaline
NVA
0.5
Tryptophan
TRP
1.8
Phenylalanine
PHE
0.9
Isole sine
Isoleusine
ILE
09
0.9
Leusine
LEU
0.9
Lysine
LYS
0.9
Hydroxiproline
HYP
1.8
Sarcosine
SAR
0.5
Proline
PRO
0.9
15
June 24, 2010
10
16
0
2
20
4
6
8
10
12
14
14.125
16
17.654 - PRO
17.060 - SAR
14
16.650
14.586
12.895
12
14.959 - HYP
2.745
12
13.119
13.300
13.493
12.441
10
11.257
11.494
11.662
11.887
12.006
12.092
10.02
27
8.704
8.887
0
2
4
6
DAD1 B, Sig=262,16 Ref=324,8 (AMINO3\AA6-0003.D)
8.2
236
10
11.49
94 - CY2
10
0.027 - TYR
8.889 - ALA
8
8.239 - ARG
7.166 - HIS
7.503 - GLY
7
7..699 - THR
14.586
6 - LYS
14.12
25 - LEU
13.300 - PHE
13.4
493 - ILE
12.895
5 - TRP
11.887 - VAL
12.092 - MET
12.437 - NVA
6.936 - GLN
6
6.038 - ASN
6
6.304
- SER
3.790 - GLU
40
6.940
7.167
7.498
7.699
9
3.314
3
1.792 - ASP
30
5.276
5.455
5.684
5.851
6.038
6.303
mAU
1.077
1
20
3.786
1.793
30
0.806
Eclipse AAA Method Chromatograms (UV and FLD)

mAU
DAD1 A
A, Si
Sig=338,10
338 10 R
Ref=390,20
f 390 20 (AMINO3\AA6
(AMINO3\AA6-0003.D)
0003 D)
0
16
40
0
18
min
18
min
June 24, 2010
Eclipse AAA Method

The same 3 pairs of amino acids that are poorly separated or
have p
problems due to mobile p
phase ionic strength
g on the
AminoQuant column are better separated by the Eclipse AAA
method.
The pH of the mobile phase is higher for this separation.
Therefore a guard column is strongly recommended for
maximum
i
lif
lifetime
ti
with
ith thi
this method.
th d
It should be changed every 100 injections and a total expected
lifetime will be about 500 injections
injections.
17
June 24, 2010
Eclipse Plus AAA Method

Goal of the Eclipse Plus AAA method is to offer
more column options.
This includes sub 2um column choices.
Gives more options to choose between resolution
and speed.
This is more critical with more complex
p
samples
p
(possibly more amino acids) and matrices.
18
June 24, 2010
Amino Acid Analysis on Eclipse Plus-C18, 2.1, 3.0 or 4.6 x

50mm, 1.8 Column: Experimental Conditions
AAA on production 1200SL using Eclipse Plus-C18, 2.1 or 4.6 x 50mm, 1.8u
Mobile phase A: 10mM Na2HPO4 10mM Na2B4O7, pH 8.2
5.6gm anhydrous Na2HPO4 + 15.2gm Na2B4O7 10H2O in 4L water + 32mg NaN3
Adjust to ~pH 9 with 6ml concentrated HCl and then small drops until pH 8.2. Be cautious with strong acids!
g 0.45regenerated
g
cellulose membranes ((Agilent
g
P/N 3150-0576))
Filter through
Stable for ~ 2 weeks at room temperature
Mobile phase B: ACN: MeOH: H2O 45:45:10 by volume
Injection diluent: 1ml Mobile phase A + 15l concentrated H3PO4 in a 1 ml vial. Make this in 100ml batches.
Instrument config:
Pump: no mixer, no pulse dampener, bypass used (at 0.1min after inject command), compressibility settings used: A= 35, B= 80
Flow rate: 0.420ml/min for 2.1mm ID; 0.85ml/min for 3.0mmID; 2.00ml/min for 4.6mm ID
Gradient Timetable:
Time (min)
%B
0.0
2.0
1.0
2.0
7.0
57.0
7.1
100.0
84
8.4
100 0
100.0
8.6
2.0
Stop time 8.7
DAD: PW 0.01min; slit 4nm; Stop time 7min (adjust as needed) Cell = 5l, 6mm flow path (Agilent P/N G1315-60025
338, 10nm; Ref 390, 20nm
262, 16nm; Ref 324, 8nm
338 10nm; Ref 390,
338,
390 20nm
230, 16nm; Ref 360,100nm
Timetable Signal C): 0.00 min 338, 10nm; Ref 390, 20nm
5.53 min 262, 16nm; Ref 324, 8nm (adjust as needed; 4.6mm ID transition at ~5.4min)
FLD: PW 0.01min, Stop time 7 min (adjust as needed), never use this detector before another due to fragility of flow cell
Ex 340nm; Em 450nm; Filter 390nm (Default filter)
Ti t bl Si
Timetable
Signal:
l 0
0.00
00 min
i Ex
E 340nm,
340
E 450nm;
Em
450
PMT Gain
G i 10 (as
( needed)
d d)
5.53 min Ex 260nm, Em 325nm; PMT Gain 10 (as needed; 4.6mm ID transition at ~5.4min)
.
.
.
Agilent Restricted
Month ##, 200X
Amino Acid Analysis on Eclipse Plus-C18, 2.1, 3.0

or 4.6 x 50mm, 1.8 Column: Conditions cont
contd
d
TCC: used with low dispersion kit installed; T = 40C for column side, 35C for exit side. Low dispersion kit
used on both sides.
WPS: Def. vol set to 0.5ul, def speed used throughout injector program is 200ul/min
Injector program:
1) Draw 2.5l from Borate vial 1(Agilent P/N 5061-3339)
2) Draw 0.5l from Sample vial
3) Mix 3l in washport 5X
4) Wait 0.2min
5) Draw
a 0
0.5l
5 from
o O
OPA vial
a 2 ((Agilent
g e P/N
/ 506
5061-3335)
3335)
6) Mix 3.5l in washport 6X
7) Draw 0.4l from FMOC vial 3 (Agilent P/N 5061-3337)
8) Mix in 3.9l in washport 10X
9) Draw 32l from Diluent vial 4
10) Mix 20l in seat 8X
11) Inject
12) Wait 0.10 min
13) Valve bypass
Tubing is 0.12mm ID throughout. Maximum sensitivity is obtained with 2.1mm columns. To
properly integrate the first peak,
peak set the integrator to detect negative peaks.
peaks If you wish to minimize
the degradants in the DAD chromatogram use new reagents and mobile phase. OPA and FMOC are
not stable left open to the atmosphere at room temperature. All modules are 1200SL where
available; the autosampler is a standard 1200 SL WPS.
Agilent Restricted
Month ##, 200X
Amino Acid Analysis on Eclipse Plus-C18, 4.6 x

50mm, 1.8 Column: DAD 125pMole on column
mAU
1 ASP
2 GLU
3 ASN
4 SER
5 GLN
6 HIS
7 GLY
8 THR
9 ARG
10 ALA
11 TYR
12 CYS-CYS
13 VAL
14 MET
15 NVA
16 TRP
17 PHE
18 ILE
19 LEU
20 LYS
21 HYP
20
10
1
2
Degradation peaks from old reagents
12
9
4
67
22 SAR
23 PRO
10
13 15
14
16 1718
11
19 20
21
22
23
min
Agilent Restricted
Month ##, 200X

50mm, 1.8 Column: FLD 125pMole on column
LU
22
1 ASP
2 GLU
3 ASN
4 SER
5 GLN
6 HIS
7 GLY
8 THR
9 ARG
10 ALA
11 TYR
12 CYS-CYS
13 VAL
14 MET
15 NVA
16 TRP
17 PHE
18 ILE
19 LEU
20 LYS
21 HYP
300
250
1
200
2
150
100
21
9
11
1718 19
10
7
4
3
16
23
22 SAR
23 PRO
50
13 15
14
20
12
0
0
min
Agilent Restricted
Month ##, 200X
Amino
Acid Analysis on Eclipse Plus-C18, 2.1 x
25
50mm, 1.8 Column: DAD 50pMole on column
mAU
20
1 ASP
2 GLU
3 ASN
4 SER
5 GLN
6 HIS
7 GLY
8 THR
9 ARG
10 ALA
11 TYR
12 CYS-CYS
13 VAL
14 MET
15 NVA
16 TRP
17 PHE
18 ILE
19 LEU
20 LYS
21 HYP
15
1
10
Degradation
g
peaks
p
from old reagents
g
9
8
4
10
11
12 13
14
15
18 19
21
17
20
23
16
5
22
22 SAR
23 PRO
-5
0
min
Agilent Restricted
Month ##, 200X

22
LU
21
200
1 ASP
2 GLU
3 ASN
4 SER
5 GLN
6 HIS
7 GLY
8 THR
9 ARG
10 ALA
11 TYR
12 CYS-CYS
13 VAL
14 MET
15 NVA
16 TRP
17 PHE
18 ILE
19 LEU
20 LYS
21 HYP
175
150
2
125
100
75
50
13
14
10
11
19
17
15
16
22 SAR
23 PRO
25
23
18
20
12
0
0
7 min
Agilent Restricted
Month ##, 200X

LU
13
18
14
14
1 ASP
2 GLU
3 ASN
4 SER
5 GLN
6 HIS
7 GLY
8 THR
9 ARG
10 ALA
11 TYR
12 CYS-CYS
13 VAL
14 MET
15 NVA
16 TRP
17 PHE
18 ILE
19 LEU
20 LYS
21 HYP
12
10
19
10
8
11
15
17
22
3
6
21
16
23
20
22 SAR
23 PRO
12
0
0
7 min
Agilent Restricted
Month ##, 200X
Many Column and Instrument Options

#
MethodCategory
Traditional
Column/MethodName
AnalysisTimew/re TypicalMinimum ApproximatemL

equilibration
ResolutionFactor Solvent/Analysis*
AgilentHPLC
Instrument
4.6x250,5m
40min
2.4
64
1200or1200SL
3.0x250,5m
40min
2.4
28
1200or1200SL
4.6x150,3.5m
25min
42
1200or1200SL
3 0 150 3 5
3.0x150,3.5m
25 i
25min
18
1200 1200 SL
1200or1200SL
2.1x150,3.5m
25min
12
1200or1200SL
4.6x100,1.8m
16min
2.4
28
1200SL
2.1x100,1.8m
16min
2.4
1200SL
4.6x50,1.8m
9min
1.5
23
1200SL
3.0x50,1.8m
9min
1.5
10
1200SL
10
2.1x50,1.8m
9min
1.5
1200SL
2
3
RapidResolution
RapidResHT
*includesinjectorprogramandprerunDADautobalancing (2.42min),andreequilibrationtime.
Agilent Restricted
Month ##, 200X
Instrument Options
Online automated derivatization will change depending on your
autosampler
G1376C well plate automatic liquid sampler (WPALS), with injection program:
1) Draw 2.5 L from Borate vial (Agilent P/N 5061-3339)
2) D
Draw 1
1.0
0 L
L ffrom S
Sample
l vial
i l
3) Mix 3.5 L in washport 5X
4) Wait 0.2 min
5) Draw 0.5 L from OPA vial (Agilent P/N 5061-3335)
6) Mix 4.0 L in washport 10X max speed
7) Draw 0.4 L from FMOC vial (Agilent P/N 5061-3337)
9) Draw 32 L from Injection Diluent vial
10) Mix 20 L in washport 8X
11) Inject
12) Wait 0.1 min
13) Valve bypass
G1329A automatic
t
ti li
liquid
id sampler
l (ALS)
(ALS), with
ith injection
i j ti program:
2) Draw 1.0 L from Sample vial
3) Mix 3.5 L in air, max speed 5X
4) Wait 0.2 min
6) Mix 4.0 L in air, max speed 10X max speed
8) Mix 4.4 L in air, max speed, 10X max speed
10) Mix 20 L in air, max speed 8X
11) Inject
12) Wait 0.1 min
13) V
Valve
l b
bypass
Agilent Restricted
Month ##, 200X
Linear Gradients
Traditionalhighresolutionmethodgradients,5m
4.6x250, 5m
3.0x250, 5m
PN959990902
PNcustom
time(min.)
%B
%B
0
2
2
0.84
2
2
33.4
57
57
33.5
100
100
39.3
100
100
39.4
2
2
40
end
d
end
d
flow(mL/min.)
1.5
0.64
RapidResolutionHighThroughputmethodgradients,
1.8m,100 mm
time(min.)
0
0.35
13.4
13 5
13.5
15.7
15.8
16
flow(mL/min.)
4.6x100, 1.8m
2.1x100, 1.8m
PN959964 902
PN959964902
PN959764 902
PN959764902
%B
2
2
57
100
100
2
end
1.5
%B
2
2
57
100
100
2
end
0.42
RapidResolutionmethodgradients,3.5 m
4.6x150, 3.5m 3.0x150, 3.5m
PN959963902
time(min.)
0
0.5
20
20.1
23.5
23.6
25
flow(mL/min.)
%B
2
2
57
100
100
2
end
1.5
PN959963302
%B
2
2
57
100
100
2
end
0.64
RapidResolutionHighThroughputmethodgradients
1.8m,50mm
3.0x50,1.8m
4.6x50,1.8m
PN959941902
time(min.)
0
0.2
7 67
7.67
7.77
8.3
8.4
9
fl ( L/ i )
flow(mL/min.)
%B
2
2
57
100
100
2
end
20
2.0
PN959941302
%B
2
2
57
100
100
2
end
0 85
0.85
2.1x150, 3.5m
PN959763902
%B
2
2
57
100
100
2
end
0.42
2.1x50,1.8m
PN959741902
%B
2
2
57
100
100
2
end
0 42
0.42
Agilent Restricted
Month ##, 200X
Agilent LC Flow Paths

Traditional(5m)
Hi h R l ti
HighResolution
Methods
Methodname
RapidResolution(3.5m)
R
id R l ti (3 5 )
Methods
4.6x250,5m
3.0x250,5m
4.6x150,3.5m
3.0x150,3.5m
2.1x150,3.5m
1100
1200
1200SL
1200SL
1200SL
G1312A
G1311Aquat
G1312B
G1312B
G1312B
yes
n/a
yes
yes
bypassed
G132887600(green500mm)?
G132887600(green500mm)?
50211823(red400mm)
50211823(red400mm)
50211823(red400mm)
G1367A
G1329A
G1367C
G1367C
G1367C
G136787101(green)
G131387201(green)
G136787201(red)
G136787201(red)
G136787201(red)
G131387305(green180mm)
0109087611(red105mm)
0109087611(red105mm)
0109087611(red105mm)
0109087611(red105mm)
G1316A3L
G1316A3L
G1316800031.6L
G1316800031.6L
G1316800031.6L
50211817(green150mm)
50211816(green105mm)
50211820(red105mm)
50211820(red105mm)
50211820(red105mm)
8209509364pk,4.6id
8211259364pk,2.1id
8209509364pk,4.6id
8211259364pk,2.1id
8211259364pk,2.1id
959990902
959990
902
custom
959963902
959963
902
959963302
959963
302
959763902
959763
902
Postcolumntounion
50659931(200mmgreen)
50659931(200mmgreen)
n/a
n/a
n/a
ZDVuniontoflowcell
50222184
50222185
n/a
n/a
n/a
G1315B
G1315D
G1315C
G1315C
G1315C
2LG131560024
2LG131560024
2LG131560024
2LG131560024
2LG131560024
2 1 x 50 1 8 m
2.1x50,1.8m
LCModel
Pump
Dampener/staticmixer
PurgevalvetoALS
ALS
Needleseat
ALStoheatexchanger
Heatexchanger
Heatexch.tocolumnorguard
Optionalguardcartridge
Column
Detector
Flowcell
RRHT1.8mMethods(100
mm)
Method name
Methodname
RRHT1.8mMethods
(50mm)
4 6 x 100 1 8 m
4.6x100,1.8m
2 1 x 100 1 8 m
2.1x100,1.8m
4 6 x 50 1 8 m
4.6x50,1.8m
3 0 x 50 1 8 m
3.0x50,1.8m
LCModel
1200SL
1200SL
1200SL
1200SL
1200SL
Pump
G1312B
G1312B
G1312B
G1312B
G1312B
Dampener/staticmixer
PurgevalvetoALS
ALS
Needleseat
ALStoheatexchanger
Heatexchanger
Optionalguardcartridge
ColumnPN
Columntoflowcell
yes
bypassed
yes
bypassed
bypassed
50211823(red400mm)
50211823(red400mm)
50211823(red400mm)
50211823(red400mm)
50211823(red400mm)
G1367C
G1367C
G1367C
G1367C
G1367C
G136787201(red)
G136787201(red)
G136787201(red)
G136787201(red)
G136787201(red)
0109087611(red105mm)
0109087611(red105mm)
0109087611(red105mm)
0109087611(red105mm)
0109087611(red105mm)
G1316800031.6L
G1316800031.6L
G1316800031.6L
G1316800031.6L
G1316800031.6L
none
none
none
none
none
959964902
959764902
959941902
959941302
959741902
directlyconnected
directlyconnected
directlyconnected
directlyconnected
directlyconnected
Detector
G1315C
G1315C
G1315C
G1315C
G1315C
Flowcell
l
ll
2LG131560024
2LG131560024
2LG131560024
2LG131560024
2LG131560024
Agilent Restricted
Month ##, 200X
Increase Sensitivity
Sensitivity increases when a longer flow cell path is used
mAU
30
25
20
3 mm flow cell G1315-60024

4.6 x 100 mm, 1.8 m
15
10
0
2
mAU
30
25
10
12
min
10
12
min
6 mm flow cell G1315-60011

4 6 x 100 mm
4.6
mm, 1
1.8
8 m
m
20
15
10
0
2
Agilent Restricted
Month ##, 200X
ZORBAX Eclipse Plus C18 250 mm, 5 m

Traditional High
g Resolution Options
p
1.
2.
3
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14
14.
15.
16.
17.
18.
19
19.
20.
21.
22.
23.
Aspartic acid
Glutamic acid
Asparagine
Serine
Glutamine
Histidine
Glycine
Threonine
Arginine
Alanine
Tyrosine
Cystine
Valine
M thi i
Methionine
Norvaline
Tryptophan
Phenylalanine
Isoleucine
Leucine
Lysine
Hydroxyproline
Sarcosine
Proline
mAU
25
4.6 x 250 mm, 5 m

PN 959990-902
959990 902
15
Rs= 2.4
12
20
10
34 5 7
6 8
11
10
13 14
1718 19 20
15 16
21
22
23
0
5
mAU
25
10
3.0 x 250 mm, 5 m
15
20
25
30
min
30
min
PN custom
Rs= 2
2.3
3
20
15
10
0
5
10
15
20
25
Agilent Restricted
Month ##, 200X
ZORBAX Eclipse Plus C18 150 mm, 3.5 m

Rapid Resolution (RR) Options
1.
2.
3
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14
14.
15.
16.
17.
18.
19
19.
20.
21.
22.
23.
Aspartic acid
Glutamic acid
Asparagine
Serine
Glutamine
Histidine
Glycine
Threonine
Arginine
Alanine
Tyrosine
Cystine
Valine
M thi i
Methionine
Norvaline
Tryptophan
Phenylalanine
Isoleucine
Leucine
Lysine
Hydroxyproline
Sarcosine
Proline
mAU
40
30
4.6 x 150 mm, 3.5 m

PN 959963-902
2
12
3
20
4 56
9 10
8
11
Rs= 2.6 20
Rs
13
14 16
15
19
10
21
22 23
0
2.5
mAU
AU
40
7.5
3.0 x 150 mm, 3.5 m
10
12.5
15
17.5
20
min
15
17.5
20
min
15
17.5
20
min
R 2
Rs=
2.0
0
PN 959963-302
30
20
10
0
2.5
mAU
40
30
7.5
10
2.1 x 150 mm, 3.5 m
12.5
Rs= 2.0
PN 959763-902
20
10
0
2.5
7.5
10
12.5
Agilent Restricted
Month ##, 200X

Rapid Resolution High Throughput (RRHT) Options
1.
2.
3
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14
14.
15.
16.
17.
18.
19
19.
20.
21.
22.
23.
Aspartic acid
Glutamic acid
Asparagine
Serine
Glutamine
Histidine
Glycine
Threonine
Arginine
Alinine
Tyrosine
Cystine
Valine
M thi i
Methionine
Norvaline
Tryptophan
Phenylalanine
Isoleucine
Leucine
Lysine
Hydroxyproline
Sarcosine
Proline
mAU
50
12
4.6 x 100 mm, 1.8 m

PN 959964-902
959964 902
40
3 4
30
20
7
6 8
10
Rs= 2.6
1314 16
19
11
20
21
15
10
22 23
0
2
)
mAU
50
10
12
14
2.1 x 100 mm, 1.8 m

PN 959764-902
min
Rs= 2.4
40
30
21
20
23
22
10
0
2
10
12
14
Agilent Restricted
Month ##, 200X
min

Rapid
p Resolution High
g Throughput
g p (RRHT)
(
) Options
p
1.
2.
3
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14
14.
15.
16.
17.
18.
19
19.
20.
21.
22.
23.
Aspartic acid
Glutamic acid
Asparagine
Serine
Glutamine
Histidine
Glycine
Threonine
Arginine
Alinine
Tyrosine
Cystine
Valine
M thi i
Methionine
Norvaline
Tryptophan
Phenylalanine
Isoleucine
Leucine
Lysine
Hydroxyproline
Sarcosine
Proline
mAU
80
60
40
4.6 x 50 mm, 1.8m

PN 959941-902
959941 902
2
1
3 4
5 78
9 10
20
11
Rs 1.9Rs= 1.9
Rs=
20
12
16
19
15
21
22 23
3.0 x 50 mm, 1.8m

1
mAU
AU
80
min
min
min
Rs= 1
1.6
6Rs=
R 1
1.6
6
PN 959941-302
60
40
20
0
2.1 x 50 mm, 1.8m

1
mAU
80
Rs= 1.5
Rs= 1.5
PN959741-902
Rs= 1.5Rs= 1.5
60
40
20
0
1
Agilent Restricted
Month ##, 200X
Amino Acid Analysis on 1290 Infinity UHPLC

1200SL
1290 Infinity
Agilent Restricted
Month ##, 200X
17 Amino Acid Analysis on 1290 Infinity

time(min.)
0
0.35
13.4
13.5
15.7
15 8
15.8
16
mAU
50
RRHT Eclipse Plus C18

3 0 x 100 1.8
3.0
1 8 um
Pmax=474 bar
F=0.86 mL/min
0
0
%B
2
2
57
100
100
2
end
Original 1200 SL injection program

100 uL sample loop
3) Mix 3.5 L in washport 5X
5) Mix 4
4.0
0 L in washport 10X max speed
9) Mix 20 L in washport 8X
j
10)) Inject
10
12
min
i
Original 1290 Infinity injection program

20 uL sample loop (40 uL available)
3) Mix 1.8 L in location P1C1 5X default speed, offset
5)) Mix 2.3 L
in location P1C1 5X default speed,
p
, offset
7) Mix 2.5 L in in location P1C1 5X default speed, offset
8) Draw 7.4 L from Injection Diluent vial
9) Mix 9.9 L in location P1C1 5X default speed, offset
10) Inject
Agilent Restricted
Month ##, 200X
Double Flow Rate and Halve Gradient Time to

Double Throughput
RRHT Eclipse Plus C18
3.0 x 100
mAU
50
Pmax=474 bar
F=0.86 mL/min
0
0
mAU
50
0
0
)
mAU
100
80
60
40
20
time(min.)
2
0
0.35
13.4
13 5
13.5
15.7
15.8
16
time(min.)
0
.18
6.7
6.8
7.8
7.9
8
%B
2
2
57
100
100
2
end
%B
2
2
57
100
100
2
end
Rs6,5=2.90
=0.036 min
10
Rs14,13=2.34
=2 34
12
min
RRHD Eclipse Plus C18

3.0 x 100
Pmax=528 bar
F=0.86 mL/min
Rs6,5=2.77
w
=0.023 min
.Rs
10
12
min

3.0 x 100
12,11=2.06
Rs14,13
2.12
12
14 13=2
Pmax=1000bar
F=1.72 mL/min
min
Agilent Restricted
Month ##, 200X
Summary of Method
Use a programmable autosampler to do on-line derivatization
- OPA reacts with 1 amino acids
- FMOC reacts
t with
ith 2 amino
i acids
id
Use a gradient to separate the derivatized amino acids
Use UV and FLD to detect derivatized amino acids

Page 38
June 24, 2010
Examples
Samples from AOAC methods using SampliQ
..QuEChERS
QuEChERS kits
Fruits & Vegetables
Apple
Spinach
Beverages
Protein Hydrolysates
y
y
Agilent Restricted
Month ##, 200X
Amino acids in Apple

from QuEChERS tube ((AOAC Method 2007.01))
2 1 x 100
2.1
F=0.42
mAU
35
30
25
20
15
10
5
0
Acetonitrile Fraction
Organic
Layer
12
14
min
Pulp
Salt
solids
14
min
10
PRO
225 pmol/uL
SAR
LEU
U
LYS
L
12
27 AA Standard
HYP
20
10
NVA
TRP
PHE
ILE
CY2
ALA
10
A layer
Aq.
l
VA
AL
MET
30
Aqueous Fraction
TY
YR
GLU
U
40
ASN
SER
S
GLN
G
HIS
S
GLY
TH
HR
mAU
50
TAU
U
ARG
ALA
A
-ALA
GAB
BA
TH
HE
ASP
SER
GL
LU
100
80
60
40
20
0
GLN
120
GABA
ASN
mAU
ASP
P
0
0
10
12
14
min
Includes: GABA, -alanine, taurine, theanine, norvaline, hydroxyproline, sarcosine

Agilent Restricted
Month ##, 200X
Amino acids in Spinach leaf

from QuEChERS tube ((AOAC Method 2007.01))
2.1 x 100
mAU
30
Acetonitrile Fraction
F=0.42
F
0.42 mL/min
20
10
Organic
Layer
0
-10
10
12
14
PHE
P
ILE
LEU
L
Aqueous Fraction
min
Pulp
LY
YS
50
TY
YR
100
ARG
A
ALA
GA
ABA
150
SE
ER
GLU
200
THR
T
250
ASN
mAU
GLN
ASP
A layer
Aq.
l
mAU
50
30
10
12
14
27 AA Standard
TAU
40
ARG
AL
LA
-ALA
GAB
BA
TH
HE
Salt
solids
min
20
10
0
0
10
12
14
min
Agilent Restricted
Month ##, 200X
Amino Acid Profile Comparison

Apple
Spinach
High in aspartic acid,

asparagine,
p g , alanine,, GABA
High in aspartic acid,

glutamic acid,, asparagine,
g
p g ,
arginine, GABA
250
Low in glutamine, arginine

others
High in glutamine, arginine

others
200
Poor in 8 of 8 essential aa
Rich in 6 of 8 essential aa
*APPLE fruit
*SPINACH Leaf
ASP
GL
LN
mAU
GLU
ASN
300
LYS
T
TRP
TYR
PHE
ILE
LEU
ARG
ALA
GABA
100
THR
SER
150
50
0
0
10
ug/mL
Apple
Spinach
ASP
99
195
ASN
77
220
ALA
31
46
GABA
43
22
12
14
min
Agilent Restricted
Month ##, 200X
Comparison of the Amino Acid Content of a

Variety of Bottled Beers
Agilent Restricted
Month ##, 200X
Underivatized AAs
Reversed-Phase
Ion Exchange
Ion pair reversed-phase LC
Detectors
ELSD
MS
CLND
UV-Vis
Agilent Restricted
Month ##, 200X
Underivatized Amino Acids on Bonus-RP
Pub. No. 5989-5838EN

Agilent Restricted
Month ##, 200X
Characteristic Fragments of 22 AAs and Ions Used

in SIM Mode for Quantification
Agilent Restricted
Month ##, 200X
Determination of Underivatized Free Amino Acids

using LC/APCI-MS
LC/APCI MS
MS signals for specific m/z of 22 amino acids in green peas using a single injection
Agilent Restricted
Month ##, 200X
22 Free Amino Acids in Various Foods*
*as mg/100g FW
Agilent Restricted
Month ##, 200X
Conclusions
There are a number of separation challenges when analyzing
amino acids
A flexible, automated online derivatization method for amino
acids using ZORBAX Eclipse Plus C18 can be customized and
optimized
p
The variety of column choices with Eclipse Plus lets you
choose between high
g resolution,, high
g speed,
p
, reduced solvent
consumption, or a combination that bests suits your needs
y
without derivatization
Amino acids can be analyzed
Instrumentation plays a key role in successful amino acid
analysis
y
Agilent Restricted
Month ##, 200X

Amino Acid Analysis

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Amino Acid Analysis

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Analysis of Amino Acids by HPLC

Amino Acid Analysis - Agilent

Derivatization OPA, FMOC

Amino Acid Analysis - Agilent Restricted

June 24, 2010

Amino Acids Structure, Chemistry

Glutamic Acid ((Glu))

June 24, 2010

Amino Acids Zwitterionic

Amino Acid Analysis - Agilent Restricted

June 24, 2010

Detector; DAD, FLD, MS, ELSD

Amino Acid Analysis - Agilent Restricted

June 24, 2010

Ortho Phthalaldehyde (OPA) and Fluorenylmethoxy

Fluorescence: Ex 340nm, Em 450nm

Fluorescence: Ex 260nm, Em 325nm

Names and Order of Elution for OPA and FMOC

Agilent AAA Methods - Theyve Evolved

Agilents Solutions for Amino Acid Analysis

June 24, 2010

AminoQuant Amino Acid Method

June 24, 2010

June 24, 2010

Example of Amino Acid Separation on AminoQuant

These 3 amino acid pairs are difficult to separate.

June 24, 2010

Eclipse AAA Method

June 24, 2010

Eclipse AAA Method

June 24, 2010

Amino Acids in Eclipse AAA Method

June 24, 2010

Eclipse AAA Method Chromatograms (UV and FLD)

Eclipse AAA Method

June 24, 2010

Eclipse Plus AAA Method

June 24, 2010

Amino Acid Analysis on Eclipse Plus-C18, 2.1, 3.0 or 4.6 x

Amino Acid Analysis on Eclipse Plus-C18, 2.1, 3.0

Amino Acid Analysis on Eclipse Plus-C18, 4.6 x

Degradation peaks from old reagents

Amino Acid Analysis on Eclipse Plus-C18, 4.6 x

Amino Acid Analysis on Eclipse Plus-C18, 2.1 x

Amino Acid Analysis on Eclipse Plus-C18, 2.1 x

Many Column and Instrument Options

AnalysisTimew/re TypicalMinimum ApproximatemL

Agilent LC Flow Paths

3 mm flow cell G1315-60024

6 mm flow cell G1315-60011

ZORBAX Eclipse Plus C18 250 mm, 5 m

4.6 x 250 mm, 5 m

3.0 x 250 mm, 5 m

ZORBAX Eclipse Plus C18 150 mm, 3.5 m

4.6 x 150 mm, 3.5 m

3.0 x 150 mm, 3.5 m

2.1 x 150 mm, 3.5 m

ZORBAX Eclipse Plus C18 100 mm, 1.8 m

4.6 x 100 mm, 1.8 m

2.1 x 100 mm, 1.8 m

ZORBAX Eclipse Plus C18 50 mm, 1.8 m

4.6 x 50 mm, 1.8m

3.0 x 50 mm, 1.8m

2.1 x 50 mm, 1.8m