determination of amino acid composition using … · pre-column derivitization and superficially...
TRANSCRIPT
Determination of Amino Acid Composition using Automated Pre-Column Derivitization and Superficially Porous Columns
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
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 Poroshellparticle 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 orOPA
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
DerivatizationDetector
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
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)
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)
Fluorenylmethoxychloroformate (FMOC)
8
For Research Use Only. Not for use in diagnostic procedures
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
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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
Robust Columns for AAA
10
• 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 proprietarytechnology for particle synthesis
For Research Use Only. Not for use in diagnostic procedures
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
11
Time (min) %B
0 2
0.35 2
13.4 57
13.5 100
15.7 100
15.8 2
18 stop
Fast and Rugged Amino Acids Separation
12
DAD = 338 nm DAD = 262 nm
For Research Use Only. Not for use in diagnostic procedures
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
13
Primary AA
Secondary AA
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
ara
gin
e
Se
rin
e
Glu
tam
ine
His
tid
ine
Gly
cin
e
Arg
inin
e
Ty
rosi
ne
Ala
nin
e
Cy
stin
e
Va
lin
e
Me
thio
nin
e
No
rva
lin
e
Try
pto
ph
an
Ph
en
yla
lan
ine
Iso
leu
cin
e
Leu
cin
e
Lysi
ne
Asp
art
ic a
cid
Th
reo
nin
e
Elution Profile with and without Sodium Azide
14
• 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
For Research Use Only. Not for use in diagnostic procedures
Reproducible Separations
15
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
ara
gin
e
Se
rin
e Glu
tam
ine
His
tid
ine Gly
cin
e
Arg
inin
e
Ty
rosi
ne
Ala
nin
e
Cy
stin
e
Va
lin
e
Me
thio
nin
e
No
rva
lin
e Try
pto
ph
an
Ph
en
yla
lan
ine
Iso
leu
cin
e
Leu
cin
e
Asp
art
ic a
cid
Th
reo
nin
e
Lysi
ne
Hy
dro
xy
pro
lin
e
Sa
rco
sin
e
Pro
lin
e
DAD = 338 nm DAD = 262 nm
1 nmol amino acid standards4.6 x 100 mm column
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
• Retention time %RSD mostly under 1%• Peak area %RSD mostly under 3%
For Research Use Only. Not for use in diagnostic procedures
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
16
LeucineIsoleucine
≥ 1.5
Ref: Ph.Eur.9.0 (2.2.56) Amino Acid Analysis
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,
Asp
art
ic a
cid
Glu
tam
ic a
cid
Se
rine
Try
osi
ne
His
tidin
e Gly
cin
eT
hre
on
ine
Ala
nin
e
Cys
tein
e
Va
line
Me
thio
nin
e
Ph
en
yla
lan
ine
Iso
leu
cin
e
Leu
cin
eL
ysin
e
. P
rolin
e
Rs = 4.35
Arg
inin
e
17
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
18
min4 6 8
mAU
0
20
40
60
80
100
Asp
ara
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 + 2.6479
R² = 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 + 0.3716
R² = 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 + 6.0767
R² = 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
19
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 = LODS/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
ara
gin
e
Se
rin
e
Glu
tam
ine
His
tid
ine
Gly
cin
e
Th
reo
nin
e
Arg
inin
e
Ty
rosi
ne
Ala
nin
e
Cy
stin
e
Va
lin
e
Me
thio
nin
e
No
rva
lin
e
Ph
en
yla
lan
ine
Iso
leu
cin
e
Leu
cin
e
Lysi
ne
Glu
tam
ic a
cid
Glu
tam
ine
His
tid
ine
Th
reo
nin
e
Arg
inin
e
Ty
rosi
ne
Ala
nin
e Cy
stin
e
Va
lin
e
Me
thio
nin
e
Ph
en
yla
lan
ine
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
20
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
21
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
ara
gin
e
Se
rin
e
Glu
tam
ine
His
tid
ine
Gly
cin
e
Th
reo
nin
e
Arg
inin
e
Ty
rosi
ne
Ala
nin
e
Cy
stin
e
Va
lin
e
Me
thio
nin
e
No
rva
lin
e
Ph
en
yla
lan
ine
Iso
leu
cin
e
Leu
cin
e
Lysi
ne
Hy
dro
xy
pro
lin
e
Sa
rco
sin
e
Pro
lin
e
Asp
art
ic a
cid
A
spa
rtic
aci
d
Ala
nin
eAsp
ara
gin
e
Glu
tam
ic a
cid
Gly
cin
e
Pro
lin
eSe
rin
e
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
22
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
200G
luta
mic
aci
d
Asp
ara
gin
e
Se
rin
e
Glu
tam
ine
His
tid
ine
Gly
cin
e
Th
reo
nin
e
Arg
inin
e
Ty
rosi
ne
Ala
nin
e
Cy
stin
e
Va
lin
e
Me
thio
nin
e
No
rva
lin
e
Try
pto
ph
an
Ph
en
yla
lan
ine
Iso
leu
cin
e
Leu
cin
e
Lysi
ne
Hy
dro
xy
pro
lin
e
Sa
rco
sin
e
Pro
lin
e
Asp
art
ic a
cid
Pro
lin
e
Arg
inin
eAsp
ara
gin
e
Cy
stin
e
Gly
cin
e
His
tid
ine
Hy
dro
xy
pro
lin
e
Iso
leu
cin
e
Leu
cin
e
Lysi
ne
Me
thio
nin
e
Ph
en
yla
lan
ineS
eri
ne
Th
reo
nin
e
Try
pto
ph
an
Ty
rosi
ne
Va
lin
eGlu
tam
ic a
cid
Asp
art
ic a
cid
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
% I
nit
ial
Eff
icie
ncy
mL Stress Buffer
Non-HPH silica column
Poroshell HPH-C18
Poroshell HPH C18 last longer in phosphate buffer
23
For Research Use Only. Not for use in diagnostic procedures
For Research Use Only. Not for use in diagnostic procedures
Traditional Lifetime TestingUsing 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.
24
For Research Use Only. Not for use in diagnostic procedures
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
25
Test like a customer runs: 100 injections (3 days), Store (4 days)
min0 2 4 6 8 10 12
mAU
020406080
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
.10
4
11
.15
3
min0 2 4 6 8 10 12
mAU
020406080
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
.07
0
11
.12
0
min0 2 4 6 8 10 12
mAU
020406080
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
.04
1
11
.08
3
min0 2 4 6 8 10 12
mAU
020406080
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
.50
9
7.7
71
7.9
42
8.8
87
9.0
39
9.5
33
9.9
71
11
.02
3
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
26
For Research Use Only. Not for use in diagnostic procedures
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
27
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
.846
6.0
71
Quantity and diversity of amino acids is evident, can be used to monitor reactions and compare batches
28
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.
29
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
30
Tips & Tricks - Troubleshooting
For Research Use Only. Not for use in diagnostic procedures
Damaged Column
31
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
32
Re-equilibration
Separation
High Organic Wash
Sample Derivatization
Time (min) %B
0 2
0.35 2
13.4 57
13.5 100
15.7 100
15.8 2
18 stop
23 minutes
Initial mobile phase conditions are
running through the column!
Tips & Tricks – Saving Time
33
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
34
20.8 + 20.8 = 41.6 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
How-To Guide – Step by Step Instructions & Method Details
All the information you need
Document number 5991-7694EN
35
For Research Use Only. Not for use in diagnostic procedures
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.
36