Tips & Tricks to Achieve Fast, Sensitive, and Reproducible Separation of Amino Acids
Anne Blackwell, Ph.D.
Biocolumns Product Support Scientist
September 12, 2017
1
For Research Use Only. Not for use in diagnostic procedures.
Some Basics
Amino Acids are the building blocks of proteins
Amino Acids require derivatization to be detected by UV or FL
- OPA/FMOC, Ninhydrin, Dansyl chloride, and PITC are common reagents used
Derivatization can be done pre-column or post column
- OPA/FMOC, Dansyl chloride, and PITC are common reagents used for pre-column
- Ninhydrin is common for post column methods
Analysis of AA can be done by several methods:
- GC, CE, HPAE-PAD
- LC/UV/FL, LC/MS
2
For Research Use Only. Not for use in diagnostic procedures.
Why is Amino acid analysis important?
• Important for protein and peptide identification and quantitation
• Part of reverse-phase characterization in biopharma
• Required by the FDA
• Important for monitoring cell culture media
• Used for the analysis of metabolic intermediates - “Bound vs. Free”
3
For Research Use Only. Not for use in diagnostic procedures.
The Agilent Amino Acid Analysis solution
4
Ready to use
AdvanceBio AAA kit
(Standards and
Reagents)All Agilent LC
systems including
Infinity II systems
AdvanceBio AAA
Columns
Fast and rugged
2.7µm
0.5µm
1.7µm
For Research Use Only. Not for use in diagnostic procedures.
Agilent AdvanceBio AAA
Previous Agilent AAA Method
Agilent has a well established solution for Amino Acid
Analysis
- based on automated pre-column derivatization
capabilities of Agilent Autosamplers
- Uses ZORBAX Eclipse AAA column
- Well established method using reagents and
standards from Agilent
What’s New?
- All reagents conveniently kitted together under a
single part number
- Introduced an HpH chemistry on a Poroshell
particle for improved column lifetime
- Traditional silica columns dissolve above neutral
pH, but HpH chemistry stabilizes column
• AA derivatization and separation are most efficient
at higher pH
- Poroshell column with 2 µm frits is less
susceptible to clogging
5
For Research Use Only. Not for use in diagnostic procedures.
Pre- vs Post-Column DerivatizationPost Column Derivatization - The historic Gold Standard of dedicated Amino Acid Analyzers
DerivatizationPump Autosampler Detector
Cation exchange
Ninhydrin or
Fluorescamine or
OPA
UV/Vis
FLD
6
Pre Column Derivatization - Offline:
Pump Detector
Reversed Phase
UV or FLD
Derivatization
Autosampler
Derivatization done offline, either
manually or with separate automation,
and samples are transferred to
autosampler
Pre Column Derivatization - Online:
Pump
Autosampler
Derivatization
Detector
Reversed Phase
Derivatization done online, in the autosampler – eliminates error
associated with manual sample handling for highly consistent,
reproducible results
UV or FLD
For Research Use Only. Not for use in diagnostic procedures.
AdvanceBio AAA Reagent Kit
Part Number Material
5061-3339 100mL Borate Buffer
5061-3337 FMOC reagent - 10 ampoules, 1 mL each
5061-3335 OPA reagent, 10 mg/mL, 6 ampoules
5062-2479 Dithiodiproprionic acid (DTDPA)
5061-3330 AA, standard 1nmol 10/PK
5061-3331 AA standards, 250 pmol 10/PK
5061-3332 AA standards, 100 pmol 10/PK
5061-3333 AA standards, 25 pmol 10/PK
5061-3334 AA standards, 10 pmol 10/PK
5062-2478 AA supplements, 1g each
7
Order components
individually, or together as
part of a kit with a single
part number (5190-9426)
For Research Use Only. Not for use in diagnostic procedures.
Automated Derivatization in the Autosampler
RR’NH
+ or
RNH2
- HCl
Room TemperatureNRR’
or
NHR
Fluorescence: Ex 260nm, Em 325nm
DAD: 262, 16nm; Ref. 324,8nm
+RNH2
R’SH
Room Temperature
Fluorescence: Ex 340nm, Em 450nm
DAD: 338, 10nm; Ref. 390, 20nm
H
H
O
O
NR
SR
Non-fluorescent
Does not absorb at 338nm
Fluorescent
Absorbs at 262nm and
Fluorescences at 324nm
’
Optimal pH for reaction with AA: ~10.0
1. Allows visualization by UV or FL
2. Helps retain very polar compounds
Ortho Phthalaldehyde (OPA)
Fluorenylmethoxy
chloroformate
(FMOC)
8
For Research Use Only. Not for use in diagnostic procedures.
Online derivatization/Injection program
• Draw 2.5 μL from borate vial (Agilent p/n 5061-3339)
• Draw 1.0 μL from sample vial
• Mix 3.5 μL in wash port 5 times
• Wait 0.2 min
• Draw 0.5 μL from OPA vial (Agilent p/n 5061-3335)
• Mix 4.0 μL in wash port 10 times default speed
• Draw 0.4 μL from FMOC vial (Agilent p/n 5061-3337)
• Mix 4.4 μL in wash port 10 times default speed
• Draw 32 μL from injection diluent vial
• Mix 20 μL in wash port 8 times
• Inject
• Wait 0.1 min
• Valve bypass
9
1290 Infinity II Multisampler
1260 Infinity II Vialsampler
Method can be programmed into ANY
Agilent autosampler –
- Eliminates manual labor and variability
- Enables highly precise data
For Research Use Only. Not for use in diagnostic procedures.
Online derivatization/Injection program
10
1290 Infinity II Multisampler
1260 Infinity II Vialsampler
Method can be programmed into ANY
Agilent autosampler –
- Eliminates manual labor and variability
- Enables highly precise data
For Research Use Only. Not for use in diagnostic procedures.
OpenLab ChemStation C.01
Robust Columns for AAA
11
• 2.7 µm particles, 110 Å pore size
• Two dimensions available: 3.0 x 100 mm, 4.6 x 100 mm
• Guard columns also available in each i.d.
• Each individual column is tested for efficiency
• Each batch is tested with amino acid standards to ensure performance
A robust, high efficiency Fast LC column with resistance to elevated pH and
temperature offering users performance comparable to that of sub-2 µm alternatives
but with up to 50% less back pressure.
Core P120 Particle Treated P120
Utilizes a proprietary
technology for particle synthesis
For Research Use Only. Not for use in diagnostic procedures.
Chromatographic Method
• Flow rate – 1.5 mL/min for 4.6 mm and 0.62 mL/min for 3
mm i.d.
• Injection volume – 1µL with needle wash at the wash port
for 7s
• Column temperature – 40 ºC
• Detection wavelength – 338 and 262nm
• Samples- Agilent AAA standards, media samples and
protein hydrolysate standards
12
Time (min) %B
0 2
0.35 2
13.4 57
13.5 100
15.7 100
15.8 2
18 stop
For Research Use Only. Not for use in diagnostic procedures.
Fast and Rugged Amino Acids Separation
13
DAD = 338 nm DAD = 262 nm
For Research Use Only. Not for use in diagnostic procedures.
Order of Elution for OPA and FMOC derivativesPeak # AA Name AA Abbreviation Derivative Type
1 Asparic Acid ASP OPA
2 Glutamic Acid GLU OPA
3 Aspraragine ASN OPA
4 Serine SER OPA
5 Glutamine GLN OPA
6 Histidine HIS OPA
7 Glycine GLY OPA
8 Threonine THR OPA
9 Arginine ARG OPA
10 Alanine ALA OPA
11 Tyrosine TYR OPA
12 Cysteine CYS-CYS OPA
13 Valine VAL OPA
14 Methionine MET OPA
15 Norvaline* NVA OPA
16 Tryptophan TRP OPA
17 Phenylalanine PHE OPA
18 Isoleucine ILE OPA
19 Leucine LEU OPA
20 Lysine LYS OPA
21 Hydroxyproline HYP FMOC
22 Sacrosine (IS) SAR FMOC
23 Proline PRO FMOC
14
Primary AA
Secondary AA
For Research Use Only. Not for use in diagnostic procedures.
min1 2 3 4 5 6 7 8 9
mAU
0
100
200
300
400
*DAD1 A, Sig=338,10 Ref=390,20 (AAA FINAL\STD WITH NAN3\1BE-0201.D)
*DAD1 A, Sig=338,10 Ref=390,20, TT (AAA FINAL\STD WITHOUT NAN3\1BG-0401.D)
Glu
tam
ic a
cid
Asp
arag
ine
Seri
ne
Glu
tam
ine
His
tid
ine
Gly
cin
e
Arg
inin
e
Tyro
sin
e
Ala
nin
e
Cys
tin
e
Val
ine
Me
thio
nin
e
No
rval
ine Tryp
top
han
Ph
en
ylal
anin
e
Iso
leu
cin
e
Leu
cin
e
Lysi
ne
Asp
arti
c ac
id
Thre
on
ine
Elution Profile with and without Sodium Azide
15
• Historically NaN3 has been
added to aqueous mobile
phase to reduce bacterial
growth.
• NaN3 is highly toxic.
• No effect on the
separation.
• Highly recommend filtering
mobile phases (0.45 or 0.2
µm) to reduce bacterial
growth.
For Research Use Only. Not for use in diagnostic procedures.
Amino Acids RT RSD (%) Area RSD (%)
1. Aspartic acid 1.270 1.066
2. Glutamic acid 0.973 1.85
3. Asparagine 0.605 1.79
4. Serine 0.629 1.82
5. Glutamine 0.470 1.56
6. Histidine 0.430 1.22
7. Glycine 0.477 1.92
8. Threonine 0.440 1.95
9. Arginine 0.251 2.15
10. Alanine 0.280 3.06
11. Tyrosine 0.128 1.65
12. Cystine 0.067 1.9
13. Valine 0.084 2.47
14. Methionine 0.073 1.82
15. Norvaline 0.073 1.72
16. Tryptophan 0.054 1.57
17. Phenylalanine 0.051 1.66
18. Isoleucine 0.047 1.72
19. Leucine 0.03 1.7
20. Lysine 0.028 1.66
21. Hydroxyproline 0.021 4.13
22. Sarcosine 0.026 1.15
23. Proline 0.021 4.36
Reproducible Separations
16
min2 4 6 8 10 12
mAU
0
50
100
150
200
250
300
350
400
DAD1 A, Sig=338,10 Ref=390,20,
Glu
tam
ic a
cid
Asp
arag
ine
Seri
ne Glu
tam
ine
His
tid
ine Gly
cin
e
Arg
inin
e
Tyro
sin
e
Ala
nin
e
Cys
tin
e
Val
ine
Me
thio
nin
e
No
rval
ine Tr
ypto
ph
an
Ph
en
ylal
anin
e
Iso
leu
cin
e
Leu
cin
e
Asp
arti
c ac
id
Thre
on
ine
Lysi
ne
Hyd
roxy
pro
lin
e
Sarc
osi
ne
Pro
lin
e
DAD = 338 nm DAD = 262 nm
1 nmol amino acid standards
4.6 x 100 mm column
• Retention time %RSD mostly under 1%
• Peak area %RSD mostly under 3%
For Research Use Only. Not for use in diagnostic procedures.
System suitability as per European Pharmacopoeia (Ph.Eur)
The European Pharmacacopoeia (Ph. Eur.) defines requirements for the qualitative
and quantitative composition of amino acids and mixtures of amino acids. The
requirements for allowed impurities are also defined. Manufacturers of amino acids
are legally bound to prove that their amino acids meet these specifications before
they can distribute their products in Europe.
Leucine (Leu) is a branched-chain α-amino acid and is produced by the fermentation
process. During this process, isoleucine can be produced as a by-product. The
European Pharmacopoeia states that leucine and isoleucine should have a
resolution of not less than 1.5
17
LeucineIsoleucine
≥ 1.5
Ref: Ph.Eur.9.0 (2.2.56) Amino Acid Analysis
For Research Use Only. Not for use in diagnostic procedures.
Ample Resolution of Leucine & Isoleucine
Baseline resolution of isoleucine and leucine (Rs = 4.35)
meeting the regulatory requirements for these components.
min2 4 6 8 10 12
mAU
0
100
200
300
400
500DAD1 A, Sig=338,10 Ref=390,20,
Aspart
ic a
cid
Glu
tam
ic a
cid
Serine
Try
osin
e
His
tidin
e Gly
cin
eT
hre
onin
e
Ala
nin
e
Cyste
ine
Valin
eM
eth
ionin
e
Phenyla
lanin
e
Iso
leu
cin
e
Leu
cin
eLysin
e
. P
rolin
e
Rs = 4.35Rs = 2.0
Competition
data
Arg
inin
e
18
Column Leu/Ile Rs
( ≥ 1.5)
Agilent AdvanceBio AAA
4.6 × 100 mm4.5
Agilent AdvanceBio AAA
3 × 100 mm4.6
Protein hydrolysate
sample
For Research Use Only. Not for use in diagnostic procedures.
Linearity and Limits of Detection & Quantitation
19
min4 6 8
mAU
0
20
40
60
80
100
Aspara
gin
e
Glu
tam
ine
Try
pto
phan
min2.4 2.6 2.8
mAU
0
20
40
60
80
100
min3.2 3.4 3.6
mAU
0
10
20
30
40
50
60
min8.2 8.4 8.6
mAU
0
10
20
30
40
50
60
70
Asparagine Glutamine Tryptophan
For Research Use Only. Not for use in diagnostic procedures.
y = 2.0654x + 2.6479R² = 0.9993
0
500
1000
1500
2000
2500
0 200 400 600 800 1000 1200
Are
a
Concentration (pmol)
Linearity for Aspargine
y = 1.0107x + 0.3716R² = 0.9997
0
200
400
600
800
1000
1200
0 200 400 600 800 1000 1200
Are
a
Concentration (pmol)
Linearity for Glutamine
y = 1.6446x + 6.0767R² = 0.9999
0
200
400
600
800
1000
1200
1400
1600
1800
0 200 400 600 800 1000 1200
Are
a
Concentration (pmol)
Linearity for Tryptophan
Linearity and Limits of Detection & Quantitation
20
Concentration (pmol) S/N ratio
Asparagine
0.9 (LOD) 5.3
1.9 (LOQ) 10.8
Glutamine
0.9 (LOD) 3.0
3.8 (LOQ) 13.8
Tryptophan
0.9 (LOD) 4.5
3.8 (LOQ) 20.5
S/N >3.0 = LOD
S/N > 10 = LOQ
For Research Use Only. Not for use in diagnostic procedures.
min2 3 4 5 6 7 8 9
mAU
100
50
0
50
100
150
Glu
tam
ic a
cid
Asp
arag
ine
Seri
ne
Glu
tam
ine
His
tid
ine
Gly
cin
e
Thre
on
ine
Arg
inin
e
Tyro
sin
e
Ala
nin
e
Cys
tin
e
Val
ine
Me
thio
nin
e
No
rval
ine
Ph
en
ylal
anin
e
Iso
leu
cin
e
Leu
cin
e
Lysi
ne
Glu
tam
ic a
cid
Glu
tam
ine
His
tid
ine
Thre
on
ine
Arg
inin
e
Tyro
sin
e
Ala
nin
e Cys
tin
e
Val
ine
Me
thio
nin
e
Ph
en
ylal
anin
e
Iso
leu
cin
e
Leu
cin
e
Lysi
ne
MEM Eagle , Sig=338,10 Ref=390,20,
250pmol AA std , Sig=338,10 Ref=390,20,
AAA of Cell Culture Media – MEM
21
L-Arginine, L-Cystine, L-Glutamine, L-Histidine, L-Isoleucine, L- Leucine, L-Lysine, L-Methionine, L- Phenylalanine, L-
Threonine, L-Tryptophan, L- Tyrosine and L-Valine, L-Glutamic acid
For Research Use Only. Not for use in diagnostic procedures.
AAA of Cell Culture Media – NEAA cell culture supplement
22
L-Alanine, L-Asparagine, L-Aspartic acid, L-Glutamic acid, Glycine, L-Proline and L-Serine
min2 4 6 8 10 12
mAU
80
60
40
20
0
20
40
60
Glu
tam
ic a
cid
Asp
arag
ine
Seri
ne
Glu
tam
ine
His
tid
ine
Gly
cin
eTh
reo
nin
e
Arg
inin
e
Tyro
sin
e
Ala
nin
e
Cys
tin
e
Val
ine
Me
thio
nin
e
No
rval
ine
Ph
en
ylal
anin
eIs
ole
uci
ne
Leu
cin
e
Lysi
ne
Hyd
roxy
pro
lin
e
Sarc
osi
ne
Pro
lin
e
Asp
arti
c ac
id
Asp
arti
c ac
id
Ala
nin
eAsp
arag
ine
Glu
tam
ic a
cid
Gly
cin
e
Pro
lin
eSeri
ne
NEAA cell culture supplement , Sig=338,10 Ref=390,20,
250pmol AA std , Sig=338,10 Ref=390,20,
For Research Use Only. Not for use in diagnostic procedures.
AAA of Cell Culture Media – RPMI 1640
23
L-Arginine, L-Glutamic acid, L-Asparagine, L-Cystine, Glycine, L-Histidine, Hydroxy-L-Proline, L-Isoleucine, L-Leucine,
L-Lysine, L-Methionine, L-Phenylalanine, L-Proline, L-Serine, L-Threonine, L-Tryptophan, L-Tyrosine and L-Valine
min2 4 6 8 10 12
mAU
150
100
50
0
50
100
150
200
Glu
tam
ic a
cid
Asp
arag
ine
Seri
ne
Glu
tam
ine
His
tid
ine
Gly
cin
eTh
reo
nin
e
Arg
inin
e
Tyro
sin
e
Ala
nin
e
Cys
tin
e
Val
ine
Me
thio
nin
e
No
rval
ine
Tryp
top
han
Ph
en
ylal
anin
e
Iso
leu
cin
e
Leu
cin
e
Lysi
ne
Hyd
roxy
pro
lin
e
Sarc
osi
ne
Pro
lin
e
Asp
arti
c ac
id
Pro
lin
e
Arg
inin
eAsp
arag
ine
Cys
tin
e
Gly
cin
e
His
tid
ine
Hyd
roxy
pro
lin
e
Iso
leu
cin
e
Leu
cin
e
Lysi
ne
Me
thio
nin
e
Ph
en
ylal
anin
eSeri
ne
Thre
on
ine
Tryp
top
han
Tyro
sin
e
Val
ineG
luta
mic
aci
d
Asp
arti
c ac
id
RPMI 1640 Media , Sig=338,10 Ref=390,20,
250pmol AA std , Sig=338,10 Ref=390,20,
For Research Use Only. Not for use in diagnostic procedures.
Lifetime of SPP columns in phosphate buffer, pH 8, at elevated temperature. Mobile phase:
Premixed 60% 30 mM sodium phosphate buffer at pH 8 and 40% acetonitrile; Flow rate 0.4
mL/min; UV absorbance 254 nm; 65 °C; Columns: 2.1 x 50 mm, 2.7 µm; Analyte: Naphthalene.
50
60
70
80
90
100
110
0 1000 2000 3000
% In
itia
l Ef
fici
en
cy
mL Stress Buffer
Non-HPH silica column
Poroshell HPH-C18
Poroshell HPH C18 last longer in phosphate buffer
24
For Research Use Only. Not for use in diagnostic procedures.
Traditional Lifetime Testing
For Research Use Only. Not for use in diagnostic procedures.
25
Using conventional column testing Eclipse Plus C18 column test looks great and will perform well for most customers. However it is not a realistic test for most lab workflow.
Improvements in Lifetime Testing for AA Analysis
Past lifetime testing consisted of 500 injections without stopping
• This test, while it could differentiate long life columns, was not indicative of a customer workflow
Customers will typically run ~20 to 50 or 100 samples, then stop, and resume a day
or more later
• This is much more stressful on the column, and will cause columns to fail sooner versus running 500
injections straight through.
The lifetime testing for the Poroshell HPH-C18 was adapted to more customer
focused workflow, with breaks between series of injections to give a true indicator of
the lifetime ni the customers’ hands
26
For Research Use Only. Not for use in diagnostic procedures.
Test like a customer runs: 100 injections (3 days), Store (4 days)
min0 2 4 6 8 10 12
mAU
0
20
40
60
80
DAD1 A, Sig=338,10 Ref=390,20, TT (AALIFETIME\AALIFE 2014-09-05 10-21-37LFS-RDWS022\WJL00010000025.D)
1.0
76
1.6
37
3.4
42
4.1
07
4.3
66
4.5
32
4.7
93
5.1
23
5.4
86
6.4
52
7.5
02
7.6
03 7.8
84
8.0
51
9.0
08
9.1
66
9.6
71
10.1
04
11.1
53
min0 2 4 6 8 10 12
mAU
0
20
40
60
80
DAD1 A, Sig=338,10 Ref=390,20, TT (AALIFETIME\AALIFE 2014-09-15 16-13-54LFS-RDWS022\WJL00010000225.D)1.0
81
1.6
56
3.4
21
4.0
91
4.3
42
4.5
03
4.7
63
5.1
05
5.4
48
6.4
17
7.4
72
7.5
68
7.8
42
8.0
11
8.9
65
9.1
16
9.6
22
10.0
70
11.1
20
min0 2 4 6 8 10 12
mAU
0
20
40
60
80
DAD1 A, Sig=338,10 Ref=390,20, TT (AALIFETIME\AALIFE 2014-09-18 14-02-17LFS-RDWS022\WJL00010000400.D)
1.0
61
1.5
89
3.4
03
4.0
73
4.3
19
4.4
81
4.7
42
5.0
79
5.4
15
6.3
88
7.4
41
7.5
39 7.8
07
7.9
76
8.9
32
9.0
84
9.5
86
10.0
41
11.0
83
min0 2 4 6 8 10 12
mAU
0
20
40
60
80
DAD1 A, Sig=338,10 Ref=390,20, TT (AALIFETIME\AALIFE 2014-09-25 12-56-57LFS-RDWS022\WJL00010000500.D)
1.0
70
1.5
46
3.3
81
4.0
49
4.2
90
4.4
51
4.7
15
5.0
60
5.3
85
6.3
64
7.4
20 7
.509
7.7
71
7.9
42
8.8
87
9.0
39
9.5
33
9.9
71
11.0
23
Injection 25
Injection 225
Injection 400
Injection 500
Stops @
300 and 400
Stop @
100 and 200
Stop @
525
Most labs run a batch of samples (25-100) and then shut off for a few days so we did too
Looks great after 500 injections run 100 injections shut down 4 days repeat
27
For Research Use Only. Not for use in diagnostic procedures.
Amino Acid Analysis in Fermentation Applications
min1 2 3 4 5 6 7
mAU
0
25
50
75
100
125
150
175
DAD1 A, Sig=338,10 Ref=390,20, TT (WJL_HPH_AA\WJL_AA2 2015-03-29 13-52-07\AA_HPH0000008.D)
0.4
84 0.7
05
1.1
23
1.2
75
1.8
93
2.2
25
2.5
79
2.7
75
2.8
63
2.9
20
2.9
93
3.1
92
3.4
00
4.0
62
4.7
92
4.9
78
5.2
24
5.5
38
5.6
62
5.7
59
5.8
46
6.0
65
6.6
65
min1 2 3 4 5 6 7
mAU
0
10
20
30
DAD1 B, Sig=262,16 Ref=324,8, TT (WJL_HPH_AA\WJL_AA2 2015-03-29 13-52-07\AA_HPH0000008.D)
0.3
68
0.4
33
0.4
86
0.5
31
0.5
47
0.5
61
0.5
98
0.6
73
0.6
93
1.1
23
1.1
86
Soy Sauce diluted 1/100
Many other foods (such as soy
sauce) and pharmaceuticals that
are produced using fermentation
processes are monitored by AAA
28
For Research Use Only. Not for use in diagnostic procedures.
Amino Acids Analysis for Batch Comparison
Old Dominion Cherry Blossom Lager
Kronenburg Blanc
Blue Moon Belgian Style Wheat
min0 1 2 3 4 5 6 7
mAU
-50
0
50
100
150
200
DAD1 A, Sig=338,10 Ref=390,20, TT (WJL_HPH_AA\WJL_AA2 2015-03-29 13-52-07\AA_HPH0000002.D)
0.6
88
1.1
04
2.1
70
2.2
28
2.5
77
2.7
68
3.1
85
3.3
99
3.5
16
4.0
51
4.7
93
4.8
74
5.1
98
5.3
35
5.4
74
5.6
56
5.7
44
5.8
47
min0 1 2 3 4 5 6 7
mAU
-50
0
50
100
150
200
DAD1 A, Sig=338,10 Ref=390,20, TT (WJL_HPH_AA\WJL_AA2 2015-03-29 13-52-07\AA_HPH0000004.D)
0.6
92
1.1
26
2.0
84
2.2
37
2.5
82 2.7
75
2.9
07 3.1
92
3.3
86
3.5
24
4.0
54
4.7
91
4.8
84
5.2
18
5.5
48
5.6
60
5.7
43
5.8
60
min0 1 2 3 4 5 6 7
mAU
-50
0
50
100
150
200
DAD1 A, Sig=338,10 Ref=390,20, TT (WJL_HPH_AA\WJL_AA2 2015-03-29 13-52-07\AA_HPH0000006.D)
0.6
94
1.1
24
2.0
85
2.2
33
2.5
78 2
.766
3.1
87 3.3
79
3.5
15
4.0
47
4.7
82
4.8
81
4.9
78
5.2
18 5
.535
5.6
48
5.7
47 5
.84
6
6.0
71
Quantity and diversity of amino acids is evident, can be used to monitor reactions and compare batches
29
For Research Use Only. Not for use in diagnostic procedures.
Tips & Tricks - Maintenance
• Replace derivatization reagent, borate buffer, amino acid standard daily
• Recalibrate for retention times and response factors daily
• Check column and guard column performance by following specs (Rs for 2 pairs
of AA)
• Replace mobile phase A and B with fresh ones every other day
• Exchange guard column if high back pressure develops
• Avoid using MAX mixing speed during sample derivatization
• The max speed on newer LCs is much faster than older LCs (1100s, 1200s), and can cause
excessive wear on the autosampler.
30
For Research Use Only. Not for use in diagnostic procedures.
Poor chromatographic resolution?
• Cell culture media does not require any sample preparation, however appropriate dilutions have to be made to suit detector response
• In all cases, use the low-volume heat exchanger with short red tubing to minimize extra column volume
• Ensure proper connections
• Damaged guard or analytical column
Low intensity chromatogram?
• OPA/FMOC reagent deteriorated
• Air bubble in vial insert
Column storage?
• Never leave the column in mobile phase A even if it’s just overnight
• For short term always store the column in mobile phase B
• For long term, store column in 50/50 acetonitrile/H2O
31
Tips & Tricks - Troubleshooting
For Research Use Only. Not for use in diagnostic procedures.
Damaged Column
32
min2 4 6 8 10 12 14
mAU
0
50
100
150
200
250
300
350
DAD1 A, Sig=338,10 Ref=390,20, TT (AAA FINAL\AAA 46 X 100 COLUMN DAMAGE 2 (2)\1DF-0101.D)
After three days in mobile phase A
For Research Use Only. Not for use in diagnostic procedures.
Tips & Tricks – Saving Time
• After Injection 1, during the sample derivatization for Injection 2, the initial mobile phase condition is
flowing through the LC and the column
• Save time by shortening the length of the re-equilibration time at the end of the method by the
amount of time consumed by sample derivatization
33
Time (min) %B
0 2
0.35 2
13.4 57
13.5 100
15.7 100
15.8 2
18 stopRe-equilibration
Separation
High Organic Wash
Sample
Derivatization
23 minutes
Initial mobile phase
conditions are
running through the
column!
For Research Use Only. Not for use in diagnostic procedures.
Tips & Tricks – Saving Time
34
23 + 23 = 46 minutes for 2 samples
• After Injection 1, during the sample derivatization for Injection 2, the initial mobile phase condition is
flowing through the LC and the column
• Save time by shortening the length of the re-equilibration time at the end of the method by the
amount of time consumed by sample derivatization
Time (min) %B
0 2
0.35 2
13.4 57
13.5 100
15.7 100
15.8 2
18 stop
For Research Use Only. Not for use in diagnostic procedures.
Tips & Tricks – Saving Time
35
• After Injection 1, during the sample derivatization for Injection 2, the initial mobile phase condition is
flowing through the LC and the column
• Save time by shortening the length of the re-equilibration time at the end of the method by the
amount of time consumed by sample derivatization
Time (min) %B
0 2
0.35 2
13.4 57
13.5 100
15.7 100
15.8 2
18 stop
20.8 + 20.8 = 41.6 minutes for 2 samples
For Research Use Only. Not for use in diagnostic procedures.
How-To Guide – Step by Step Instructions & Method Details
All the information you need
Document number 5991-7694EN
36
For Research Use Only. Not for use in diagnostic procedures.
Summary
• We used the Agilent AdvanceBio AAA solution for the automated online derivatization and separation of amino acids.
• Area and RT precision of the method were excellent, and Leu/Ile resolution met the system suitability requirement.
• Linearity curves with ten standard concentrations of three amino acids, ranging from 0.9pmol to 1nmol, had excellent coefficient of linearity values, indicating that the method was quantitative and accurate.
• The LOD and LOQ for the amino acids were 0.9pmol and 3.8 pmol respectively, indicating that the method was sensitive.
• This method was able to separate and detect, amino acids from a variety of samples, including cell culture media, protein hydrolysate, and fermentation reactions.
37
For Research Use Only. Not for use in diagnostic procedures.