Introduction to the ACQUITY UPLC H-Class Amino Acid ... · PDF fileIntroduction to the ACQUITY UPLC H-Class Amino Acid Analysis System AMQ NH3 ... – Surplus amino acids are used

2012 Waters Corporation 1
Introduction to theACQUITY UPLC H-Class
Amino Acid Analysis System
AM
Q
NH
3 His
Ser Arg
Gly
Asp G
lu Thr Ala P
ro
Der
iv P
eak
Cys
Lys
Tyr
Met V
al IleLe
u
Phe
AU
0 . 0 0
0 .0 2
0 .0 4
0 .0 6
0 .0 8
0 .1 0
0 .1 2
0 .1 4
0 .1 6
0 .1 8
0 .2 0
0 .2 2
M in u te s1 .5 0 2 .0 0 2 .5 0 3 .0 0 3 .5 0 4 .0 0 4 .5 0 5 .0 0 5 .5 0 6 .0 0 6 .5 0 7 .0 0 7 .5 0 8 .0 0

Amino Acid Analysis
Required in many disciplines and applications Components of proteins Metabolic intermediates Bound vs. Free Nutritional value of foods and feeds
Difficult analytical problem Separation
o Wide range of propertieso Slight differences between pairs
Detectiono No chromophoreo Wide concentration range

Separation of Amino Acids
Cation-exchange Original method for amino acid analysis
Large historical data base of uncommon amino acids
Realistic run times near 30-60min
Reversed-phase HPLC of derivatized amino acids Best separation selectivity
Best sensitivity
Shortest run times

Detection of Separated Amino Acids
Post-column Reaction Ninhydrin or OPA with ion-exchange most common
ESI-MS with reversed phase HPLC and no derivatization
Pulsed Amperometric Detection
Pre-column derivatization Many reagents used successfully; some commercialized
Add UV or fluorescence detectability
Improve chromatography
Usually faster and more sensitive
Successful use requires care

Design Considerations
Base new method on AccQ-Tag Derivatization Established chemistry Well understood control of reaction Simple manual derivatization of batches
Base new method on ACQUITY UPLC H-Class System Highest resolution Well-controlled column chemistry Quality-tested eluents Established and available instrumentation
Base new method on Empower Software Fully-compliance ready Method templates Custom calculations

Chemistry of AQC Derivatization
N
N
NH
O
O
O
O N
NH
O
NHOH
O
N
NH2
NH2
O
OH
NH
O
OH
N
N
NH
O OHO
NN
NH
NH
O
AQC Reagent
Derivatized Amino Acids
6-Aminoquinolone (AMQ)
1o Amino Acid;t1/2

Ultra Performance LC Technology
A class of separation science Based on chromatography columns with very small particles
Based on instruments designed to take advantage of the small particles
Improves Resolution, Sensitivity, and Speed with no compromises
Suitable for chromatographic applications in general

UPLC Amino Acid Analysis Application Solution

Amino Acids Analysis Solution ACQUITY and H-Class
ACQUITYN
H3
HyP
roH
is
Asn
Tau
Ser
Gln
Arg
Gly As
p Glu Th
r
Ala
GA
BA
Pro
HyL
ys1
HyL
ys2 AA
BAO
rn
Cys
Lys
Tyr
Met
Val
NV
a Ile Leu
Phe
Trp
Minutes1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00
H-Class
AMQ
NH
3H
yPro
His
Asn
Tau Se
rG
lnAr
gG
ly
Asp
Glu
Thr
Ala
GAB
A Pro
HyL
ys1
HyL
ys2
AABA O
rnD
eriv
Pea
kC
ys Lys
Tyr
Met Va
lN
Va Ile Leu
Phe
Trp
Minutes1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00

ACQUITY UPLC H-Class and H-Class Bio Amino Acid Analysis System

Detector Comparison
AM
Q
NH
3H
yPro
His
Asn T
au Ser
Gln A
rgG
ly
Asp
Glu
Th
r Ala
GA
BA
Pro
HyL
ys1
HyL
ys2
AA
BA
Orn
Der
iv P
eak
Cys
Lys
Tyr
Met
Val NV
a
Ile
Leu
Phe Trp
AU
0.00
0.05
0.10
Minutes0.00 2.50 5.00 7.50
PDACell Culture,
PDA
AM
Q
NH
3
HyP
ro
His
Asn
Tau
Ser
Gln
Arg
Gly
Asp
Glu
Thr
Ala
GA
BA
Pro
HyL
ys1
HyL
ys2
AA
BA
Orn
Der
iv P
eak
Cys
Lys
Tyr
Met
Val
NV
a
Ile
Leu
Phe
Trp
EU
0.00
800.00
1600.00
Minutes0.00 2.50 5.00 7.50
Cell Culture, TUV
Cell Culture, FLRCell Culture, FLR
AM
Q
NH
3H
yPro
His
Asn T
au Ser G
lnArg G
ly Asp
Glu
Thr
Ala GABA Pro
HyL
ys1
HyL
ys2
AABA
Orn
Der
iv P
eak
Cys
Lys
Tyr
Met Val
NVa
Ile
Leu
Phe T
rp
AU
0.00
0.05
0.10
Minutes0.00 2.50 5.00 7.50

H-ClassUPLC
System CD Users Guide
Empower projects
AccQTag Ultra Chemistry Package AccQTag Ultra Eluent A Concentrate AccQTag Ultra Eluent B AccQTag Ultra Column AccQTag Ultra Derivatization Kit Amino Acid Hydrolysate Standard
Low flow inlet tubing
Column in-line filter kit
Comparison of Application Kit
System CD Users Guide
Empower projects
AccQTag Ultra Chemistry Package AccQTag Ultra Eluent A Concentrate AccQTag Ultra Eluent B AccQTag Ultra Column AccQTag Ultra Derivatization Kit Amino Acid Hydrolysate Standard
Low flow inlet tubing
Column in-line filter kit
150mm column stabilizer
2L sample loop

ACQUITY UPLC H-Class and H-Class BioAmino Acid Analysis System
Turn-key UPLC Amino Acid Analysis application Optimized for the ACQUITY UPLC H-
Class Systems Dedicated QC-tested column and
reagents Application-specific Performance
Installation Test and Training Same result day-to-day,
instrument-to-instrument, lab-to-lab, around the world
Target Applications Protein and peptide identification
and quantitation Monitoring cell culture media Analysis of Foods and Feeds

H-Class Amino Acid Analysis SystemCell Culture Amino Acids
AM
Q
NH
3H
yPro
His
Asn T
au Ser G
lnArg G
ly Asp
Glu
Thr
Ala
GABA Pro
HyL
ys1
HyL
ys2
AABA
Orn
Der
iv P
eak
Cys
Lys
Tyr
Met Val
NVa
Ile
Leu
Phe T
rp
AU
0.00
0.05
0.10
Minutes0.00 2.50 5.00 7.50

Retention Time Reproducibility

Area Reproducibility

Quantitative LinearityDerivatized Amino Acids
0.05 0.075 1.0 2.5 25 50AMQ 6033932 6020972 6101437 6056268 5069175 3648294NH3 4064 4810 9094 14396 86830 155693His 167 304 4064 9618 104287 204119Ser 334 330 4052 9459 102749 199653Arg 230 320 3994 9516 102770 201123Gly 591 682 4260 9608 100540 196412Asp 211 272 4053 9704 101645 190396Glu 238 303 3988 9330 100969 190784Thr 238 329 4115 9617 104228 201799
CM Cys 197 310 4110 9726 105289 211792Ala 269 336 4029 9447 102180 195455
PE Cys 240 339 4281 10014 109782 224055Pro 219 308 3807 8916 96538 187201
D-Peak 94086 84767 94312 89565 62635 46575Cys 172 262 3566 8315 91527 179095Lys 334 499 6810 15852 173792 329877Tyr 217 320 4177 9874 106770 209182Met 176 268 4049 9354 104998 204492Val 274 374 4127 9765 105725 204246NVa 219 331 3893 8911 99315 198500Ile 373 435 4323 9899 106378 205866
Leu 225 300 4084 9521 103758 201006Phe 237 348 4312 9970 106180 208031
Peak Name
Conc(pmol/uL),AvePeakAreas
y=4098.3x+54.943R=0.9999
0
50000
100000
150000
200000
250000
0 5 10 15 20 25 30 35 40 45 50
His,Areavs.Conc

Analysis of Protein Hydrolysates
Purified Proteins or Simple Pharmaceutical Formulations
Confirmation of Protein Identity
Determination of Protein Amount
Requirements Unequivocal peak identification
Two order-of-magnitude linear dynamic range
Limitations Destruction of Tryptophan, Asparagine, and Glutamine
Variable, but poor recovery of sulfur-containing amino acids
Other variables in hydrolysis procedure
Matrix interference
Compositional accuracy on the order of 5%

Analysis of Protein HydrolysateBovine Serum Albumin
NH
3
His
Ser
Arg
Gly
Asp
Glu
Thr
Ala
Pro
Der
iv P
eak
Cys
Lys
Tyr
Met
Val
Ile
Leu
Phe
AU
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0

Documents

Introduction to the ACQUITY UPLC H-Class Amino Acid ... · PDF fileIntroduction to the ACQUITY UPLC H-Class Amino Acid Analysis System AMQ NH3 ... – Surplus amino acids are used