tips & tricks to achieve fast, sensitive, and reproducible separation of … · 2017-09-07 ·...

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


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


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


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


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




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)

Fluorenylmethoxy

chloroformate

(FMOC)

8


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


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


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


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


Fast and Rugged Amino Acids Separation

13

DAD = 338 nm DAD = 262 nm


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


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.


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%


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


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)


4.6 × 100 mm4.5


3 × 100 mm4.6

Protein hydrolysate

sample


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


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


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


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


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


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,



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,



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


Traditional Lifetime Testing


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


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


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


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


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


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


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


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


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


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


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

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Tips & Tricks - Troubleshooting


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


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

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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!



34

23 + 23 = 46 minutes for 2 samples





Time (min) %B

0 2

0.35 2

13.4 57

13.5 100

15.7 100

15.8 2

18 stop



35





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


How-To Guide – Step by Step Instructions & Method Details

All the information you need

Document number 5991-7694EN

36


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.

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tips & tricks to achieve fast, sensitive, and reproducible separation of … · 2017-09-07 ·...

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