J-L. Veuthey , D. Guillarme SFTA, Paris, 22 Janvier 2016 · 2016-05-15 · Peptide mapping of...
39
FACULTÉ DES SCIENCES SECTION DES SCIENCES PHARMACEUTIQUES QUOI DE NEUF EN CHROMATOGRAPHIE AU XXI ÈME SIÈCLE? J-L. Veuthey, D. Guillarme SFTA, Paris, 22 Janvier 2016
Transcript of J-L. Veuthey , D. Guillarme SFTA, Paris, 22 Janvier 2016 · 2016-05-15 · Peptide mapping of...
FACULTÉ DES SCIENCES SECTION DES SCIENCES PHARMACEUTIQUES
QUOI DE NEUF EN CHROMATOGRAPHIE AU XXI ÈME SIÈCLE? J-L. Veuthey, D. Guillarme
SFTA, Paris, 22 Janvier 2016
Pression atmosphérique
Vide : 1 Torr ≤ 10-2 Torr ≤ 10-5 Torr
Gaz de nébulisation
Ecumoires
Gaz de séchage
Quadrupole Lentilles
Zone de fragmentation
Octopole
Détecteur
Flux LC
Capillaire
LC-MS | the gold standard
Analysis
Automation Rapidity
Simplicity Selectivity
Data handling Separation Detection Sample
preparation MS Detection
2D -
separation
Evolution of liquid chromatography
1903 2000 2004 2007 1965 2010
First HPLC systems
Discovery of chromatography Monoliths Core-shell
technology
Green LC and SFC
Biomolecules
2008
SiOSi
Si
SiSi
Si
O
O
O OOSiSi
O
O
O
SiO-
Si
SiO
Si
O
Si
O
O
Si
O
Si
O
Si
O H
O H
O
HO -
O-
SiSiOSi
Si
SiSi
Si
O
O
O OOSiSi
O
O
O
SiO-
SiSi
SiSiO
Si
O
Si
O
Si
O
Si
O
O
Si
O
SiSi
O
Si
O
SiSi
O
Si
O
SiSi
O H
O H
O
H
O
HO -
O-
OH
HOH
H
O
H
HO
H
H
ACN
H2O
NH
N
NH3+
O
NHN
NH3+
O
NH
NN
O
H
H
SiOSi
Si
SiSi
Si
O
O
O OOSiSi
O
O
O
SiO-
Si
SiO
Si
O
Si
O
O
Si
O
Si
O
Si
O H
O H
O
HO -
O-
SiSiOSi
Si
SiSi
Si
O
O
O OOSiSi
O
O
O
SiO-
SiSi
SiSiO
Si
O
Si
O
Si
O
Si
O
O
Si
O
SiSi
O
Si
O
SiSi
O
Si
O
SiSi
O H
O H
O
H
O
HO -
O-
OH
HOH
H
O
H
HO
H
H
ACN
H2O
NH
N
NH3+
O
NHN
NH3+
O
NH
NN
O
H
H
NH
NN
O
H
H
HILIC
First UHPLC systems and
sub-2µm columns
Pressure
High pressure required
Fast and ultra-fast analysis in UHPLC
Tr÷9
5 µm
N ≈cst
High Throughput
150 mm
1.7 µm
50 mm
N ≈ 10’000 plates tana< 2 min (k=10)
Reduced extra column volume
High Resolution N ≈ 90’000 plates tana< 20 min (k=10)
Tr ≈ cst
150 mm 450mm
N ×9
5 µm 1.7 µm
Method transfer | HPLC towards UHPLC
1
2
1
2
1
2
LL
dpdp
tt
ana
ana ×=
32
31
1
2
1
2
dpdp
LL
PP
×=Δ
Δ
Ø Analysis time : proportional to column dead time
Ø Backpressure : inversely proportional to dp3 (at uopt) and column length
Ø Solvent consumption : proportional to internal diameter and column length
PRESSUREPRESSURE
÷ 14
÷ 9
x 9
1
221
22
1
2
LL
dcdc
VV
×=
5µm
4.6 x 150 mm
1.7µm
2.1 x 50 mm W
ater
s co
rpor
atio
n
Wat
ers
corp
orat
ion
http://www.unige.ch/sciences/pharm/
Separation of drugs and impurities
0.0 5.0 10.0 15.0 20.0 25.0 30.0
0.0
0.1
0.2
0.3
0.4
0.5
0.6 0.7
AU
1
2 3 4
5
6
7
8 9
10 11
12 27 min
Active compound
HPLC 4.6 x 150 mm, 5 µm
F = 1000 µL/min ΔP = 80 bars
ORIGINAL METHOD
AU
0.00
0.20
0.40
0.60
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8
0.00
0.20
0.40
0.60
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8
1 2
3
4
5
6 7
8 9
10 11
12
3 min
TRANSFERRED METHOD
Impurity profiling
Guillarme et al., Eur. J. Pharm. Biopharm., 68 (2008) 430-440
UHPLC 2.1 x 50 mm, 1.7 µm
F = 600 µL/min ΔP = 520 bars
AU
0.00
0.20
0.40
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.40 2.60 2.80 3.00
0.00
0.20
0.40
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.40 2.60 2.80 3.00
1 2
3
4
5
6 7
8 9
10 11 12
1.8 min
UHPLC 2.1 x 50 mm, 1.7 µm
F = 1000 µL/min ΔP = 980 bars
Ø Important to minimize extra-column band broadening
Ø Need to increase acquisition rate
Ø Short injection cycle time
Column C18 2.1 x 50mm, 1.7µm, Flow rate 1.7 mL/min, Oven temperature 90 C, UV Detection 25 ms, 80 Hz
90 C
0.00
0.20
0.40
0.60
0.00 0.40
1
2 3
4
5
6
7
8
9
Time (min) 0.20 0.60 0.80
45 s
1.Acetazolamide, 2.Chlortalidone, 3.Clopamide, 4.Dexamethasone, 5.Furosemide, 6.Indapamide, 7.Bumetanide, 8.Probenecide, 9.Ethacrinic acid
High throughput analysis in HT-UHPLC
Time (s) 0.0
0.00
0.20
0.40
2.0 4.0 6.0 8.0 10.0 12.0
0.60
1
2 3
4
5
10 s
1.Uracil, 2.methylparaben, 3.ethylparaben, 4. Propylparaben, 5. Butylparaben
D.T.T. Nguyen et al., J. Chromatogr. A, 1167 (2007) 76–84
High resolution analysis in HPLC
High Resolution N ≈ 90’000 plates tana< 20 min (k=10)
Tr ≈ cst
150 mm 450mm
N ×9
5 µm 1.7 µm
High pressure required
Tr÷9
5 µm
N ≈cst
High Throughput
150 mm
1.7 µm
50 mm
N ≈ 10’000 plates tana< 2 min (k=10)
Reduced extra column volume
Metabolite profiling of complex plant extract
Analysis of a standardized extract of Ginkgo Biloba
Column: 300x2.1mm, 1.7µm
T=30C, F= 200 µL/min
Gradient length: 120 minutes
ΔP= 950 bar
E. Grata et al., J. Chromatogr. A, 1216 (2009) 5660–5668 Time (minutes)
10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00 110.00 120.00
%
0
20
Peak capacity > 300
0.00
Peptide mapping of therapeutic mAb Tryptic digest (peptide mapping) of panitumumab. Columns: Acquity BEH C18 300A - 150 mm x 2.1 mm, 1.7 µm (1 and 3 columns in series). Mobile phase A: 0.1% TFA in water, mobile phase B: 0.1% TFA in acetonitrile. Gradient: 10-55 %B, flow: 0.30 and 0.10 mL/min for the 150 and 450 mm long columns, respectively. Temperature: 50C, Detection FL: 280-360 nm. Injected volume: 5 and 15 µL for the 150 and 450 mm long columns, respectively.
S. Fekete et al, J. Pharm. Biomed. Anal. 83 (2013) 273
Lcol = 150 mm
tgrad = 30 min
Flow = 0.3 mL/min
nc = 326
Lcol = 450 mm
tgrad = 270 min
Flow = 0.1 mL/min
nc = 704
0 5 10 15 20 0 20 40 60 80
EU
retention time (min) 0 50 100 150 0
100
200
EU
retention time (min)
Evolution of liquid chromatography
1903 2000 2004 2007 1965 2010
First HPLC systems
Discovery of chromatography Monoliths Core-shell
technology
Green LC and SFC
Biomolecules
2008
SiOSi
Si
SiSi
Si
O
O
O OOSiSi
O
O
O
SiO-
Si
SiO
Si
O
Si
O
O
Si
O
Si
O
Si
O H
O H
O
HO -
O-
SiSiOSi
Si
SiSi
Si
O
O
O OOSiSi
O
O
O
SiO-
SiSi
SiSiO
Si
O
Si
O
Si
O
Si
O
O
Si
O
SiSi
O
Si
O
SiSi
O
Si
O
SiSi
O H
O H
O
H
O
HO -
O-
OH
HOH
H
O
H
HO
H
H
ACN
H2O
NH
N
NH3+
O
NHN
NH3+
O
NH
NN
O
H
H
SiOSi
Si
SiSi
Si
O
O
O OOSiSi
O
O
O
SiO-
Si
SiO
Si
O
Si
O
O
Si
O
Si
O
Si
O H
O H
O
HO -
O-
SiSiOSi
Si
SiSi
Si
O
O
O OOSiSi
O
O
O
SiO-
SiSi
SiSiO
Si
O
Si
O
Si
O
Si
O
O
Si
O
SiSi
O
Si
O
SiSi
O
Si
O
SiSi
O H
O H
O
H
O
HO -
O-
OH
HOH
H
O
H
HO
H
H
ACN
H2O
NH
N
NH3+
O
NHN
NH3+
O
NH
NN
O
H
H
NH
NN
O
H
H
HILIC
First UHPLC systems and
sub-2µm columns
Pressure
Superficially porous particles : SPP
2nd generation: Technology developed by J.J. Kirkland and commercialized by Agilent in the 90’s under the trademark Poroshell (particle size of 5 µm, shell thickness of 0.25 µm).
Analysis of peptides/proteins by RPLC
3rd generation: Technology updated by J.J. Kirkland and commercialized by various companies since 2007 (particle size of 2.6 - 2.7 µm, shell thickness of 0.35 - 0.5 µm).
Analysis of small and large molecules by RPLC
1st generation: Pellicular particles originally imagined by Horvath et al. in the 60’s (particle size of 50 – 100 µm, shell thickness of 1 µm).
Analysis of proteins by ion exchange chromatography
SPP = superficially porous particles = core-shell = fused-core
2.5-2.7 µm SPP phases on the market Advanced Material Technologies HALO 2.7 µm (core 1.7 µm)
Supelco ASCENTIS 2.7 µm (core 1.7 µm) 2007
2009 2010
2011
Agilent POROSHELL 2.7 µm (core 1.7 µm)
Phenomenex KINETEX 2.6 µm (core 1.9 µm) et 1.7 µm (1.25 µm)
Thermo ACCUCORE 2.6 µm (core 1.6 µm)
Macherey-Nagel NUCLEOSHELL 2.6 µm (core 1.6 µm)
ChromaNik SUNSHELL 2.6 µm (core 1.6 µm)
Perkin-Elmer BROWNLEE SPP 2.7 µm (core 1.7 µm)
Fortis SPEEDCORE 2.6 µm (core 1.8 µm)
Nacalai COSMOCORE 2.6 µm (core 1.6 µm)
Restex RAPTOR 2.7 µm (core 1.7 µm) 2012
2013 2014
Ace ACEULTRACORE 2.5 µm (core 1.6 µm)
Waters CORTECS 2.7 µm (core 1.9 µm)
Acquity
0.00
0.05
0.10
0.15
0.20
0.25
Minutes0.00 0.40 0.80 1.20 1.60 2.00 2.40 2.80 3.20 3.60
2
0.00
0.10
0.20
0.30
0.40
Minutes0.00 0.40 0.80 1.20 1.60 2.00 2.40 2.80 3.20 3.60
Poroshell
21
6
3
11
129+13
54
8
10
Minutes
0.00
0.10
0.20
0.30
0.40
0.00 0.40 0.80 1.20 1.60 2.00 2.40 2.80 3.20 3.60
Kinetex
0.00
0.10
0.20
0.30
0.40
Minutes0.00 0.40 0.80 1.20 1.60 2.00 2.40 2.80 3.20 3.60
Halo
1
3+6
11
9+12+13
54
7
8
10
2 111
54
7
8
10
3+6
9+12+13
2 16
3
11
129+13
54
7
8
10
Acquity
0.00
0.05
0.10
0.15
0.20
0.25
Minutes0.00 0.40 0.80 1.20 1.60 2.00 2.40 2.80 3.20 3.60
2
0.00
0.10
0.20
0.30
0.40
Minutes0.00 0.40 0.80 1.20 1.60 2.00 2.40 2.80 3.20 3.60
Poroshell
21
6
3
11
129+13
54
8
10
Minutes
0.00
0.10
0.20
0.30
0.40
0.00 0.40 0.80 1.20 1.60 2.00 2.40 2.80 3.20 3.60
Kinetex
0.00
0.10
0.20
0.30
0.40
Minutes0.00 0.40 0.80 1.20 1.60 2.00 2.40 2.80 3.20 3.60
Halo
1
3+6
11
9+12+13
54
7
8
10
2 111
54
7
8
10
3+6
9+12+13
2 16
3
11
129+13
54
7
8
10
Acquity
0.00
0.05
0.10
0.15
0.20
0.25
Minutes0.00 0.40 0.80 1.20 1.60 2.00 2.40 2.80 3.20 3.60
2
0.00
0.10
0.20
0.30
0.40
Minutes0.00 0.40 0.80 1.20 1.60 2.00 2.40 2.80 3.20 3.60
Poroshell
21
6
3
11
129+13
54
8
10
Minutes
0.00
0.10
0.20
0.30
0.40
0.00 0.40 0.80 1.20 1.60 2.00 2.40 2.80 3.20 3.60
Kinetex
0.00
0.10
0.20
0.30
0.40
Minutes0.00 0.40 0.80 1.20 1.60 2.00 2.40 2.80 3.20 3.60
Halo
1
3+6
11
9+12+13
54
7
8
10
2 111
54
7
8
10
3+6
9+12+13
2 16
3
11
129+13
54
7
8
10
Acquity
0.00
0.05
0.10
0.15
0.20
0.25
Minutes0.00 0.40 0.80 1.20 1.60 2.00 2.40 2.80 3.20 3.60
2
0.00
0.10
0.20
0.30
0.40
Minutes0.00 0.40 0.80 1.20 1.60 2.00 2.40 2.80 3.20 3.60
Poroshell
21
6
3
11
129+13
54
8
10
Minutes
0.00
0.10
0.20
0.30
0.40
0.00 0.40 0.80 1.20 1.60 2.00 2.40 2.80 3.20 3.60
Kinetex
0.00
0.10
0.20
0.30
0.40
Minutes0.00 0.40 0.80 1.20 1.60 2.00 2.40 2.80 3.20 3.60
Halo
1
3+6
11
9+12+13
54
7
8
10
2 111
54
7
8
10
3+6
9+12+13
2 16
3
11
129+13
54
7
8
10
Analysis of drugs: core-shell vs porous particles
Similar performance, no major change in retention/selectivity Pressure is strongly reduced
Waters Acquity
FPP 1.7 µm
Agilent Poroshell
SPP 2.7 µm
Phenomenex Kinetex
SPP 2.6 µm AMT Halo
SPP 2.7 µm
7 7
7 7
Columns of 50x2.1mm; gradient: 1 min at 5%ACN, then 5-95%ACN in 3 min @ 500 µL/min; 0.1%FA in both ACN and water.
J. Ruta et al., J. Chromatogr. A, 2012, 1228, 221
High resolution separation of a tryptic digest
Evolution of liquid chromatography
1903 2000 2004 2007 1965 2010
First HPLC systems
Discovery of chromatography Monoliths Core-shell
technology
Green LC and SFC
Biomolecules
2008
SiOSi
Si
SiSi
Si
O
O
O OOSiSi
O
O
O
SiO-
Si
SiO
Si
O
Si
O
O
Si
O
Si
O
Si
O H
O H
O
HO -
O-
SiSiOSi
Si
SiSi
Si
O
O
O OOSiSi
O
O
O
SiO-
SiSi
SiSiO
Si
O
Si
O
Si
O
Si
O
O
Si
O
SiSi
O
Si
O
SiSi
O
Si
O
SiSi
O H
O H
O
H
O
HO -
O-
OH
HOH
H
O
H
HO
H
H
ACN
H2O
NH
N
NH3+
O
NHN
NH3+
O
NH
NN
O
H
H
SiOSi
Si
SiSi
Si
O
O
O OOSiSi
O
O
O
SiO-
Si
SiO
Si
O
Si
O
O
Si
O
Si
O
Si
O H
O H
O
HO -
O-
SiSiOSi
Si
SiSi
Si
O
O
O OOSiSi
O
O
O
SiO-
SiSi
SiSiO
Si
O
Si
O
Si
O
Si
O
O
Si
O
SiSi
O
Si
O
SiSi
O
Si
O
SiSi
O H
O H
O
H
O
HO -
O-
OH
HOH
H
O
H
HO
H
H
ACN
H2O
NH
N
NH3+
O
NHN
NH3+
O
NH
NN
O
H
H
NH
NN
O
H
H
HILIC
First UHPLC systems and
sub-2µm columns
Pressure
0
50
100
150
200
250
300
350
1991
1992
1994
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
2011
2012
2013
# pa
pers
per
yea
rHILIC: an alternative to RPLC
1990: Alpert coined the term « HILIC » to describe the hydrophilic partitioning between a water enriched layer at the surface of the stationary phase and the mobile phase.
RPLC
NPLC
HILIC
IEX
eluent
Charged compounds
Type of stationary phase
Type of mobile phase
B. Buszewski et al., Anal. Bioanal. Chem., 402 (2012) 231-247
Keyword: HILIC
Keyword: HILIC + MS
Research in April 2013 SciFinder Scholar
HILIC for drugs and metabolites
Gradient90-75%[email protected]/min,pH6,T=45C,
λ=220nm
0.00
0.02
0.04
0.06
0.08
Minutes0.00 0.20 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00
ColumnAmide2.1x100mm,1.7µm
TMD
VLX
N-TMD N-VLX
N,N-TMD
N,O-VLX
O-VLX
O-TMD
AU
0.00
0.02
0.04
0.06
0.08
0.10
Minutes0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00
VLX
TMD
N-VLX
O-TMD
N,O-VLX
N,N-TMD
O-VLX
N-TMD
ColumnBEHC182.1x50mm,1.7µm
ColumnCSHC182.1x50mm,1.7µm
+Gradient10-80%[email protected]/min,pH9,T=45C,
λ=220nm
238bar
827bar
0.40 0.60 0.80
AU
J. Ruta et al., J. Pharm. Biomed. Anal. 63 (2012) 95
HILIC
RPLC
Evolution of liquid chromatography
1903 2000 2004 2007 1965 2010
First HPLC systems
Discovery of chromatography Monoliths SPP
technology
Green LC and SFC
Biomolecules
2008
SiOSi
Si
SiSi
Si
O
O
O OOSiSi
O
O
O
SiO-
Si
SiO
Si
O
Si
O
O
Si
O
Si
O
Si
O H
O H
O
HO -
O-
SiSiOSi
Si
SiSi
Si
O
O
O OOSiSi
O
O
O
SiO-
SiSi
SiSiO
Si
O
Si
O
Si
O
Si
O
O
Si
O
SiSi
O
Si
O
SiSi
O
Si
O
SiSi
O H
O H
O
H
O
HO -
O-
OH
HOH
H
O
H
HO
H
H
ACN
H2O
NH
N
NH3+
O
NHN
NH3+
O
NH
NN
O
H
H
SiOSi
Si
SiSi
Si
O
O
O OOSiSi
O
O
O
SiO-
Si
SiO
Si
O
Si
O
O
Si
O
Si
O
Si
O H
O H
O
HO -
O-
SiSiOSi
Si
SiSi
Si
O
O
O OOSiSi
O
O
O
SiO-
SiSi
SiSiO
Si
O
Si
O
Si
O
Si
O
O
Si
O
SiSi
O
Si
O
SiSi
O
Si
O
SiSi
O H
O H
O
H
O
HO -
O-
OH
HOH
H
O
H
HO
H
H
ACN
H2O
NH
N
NH3+
O
NHN
NH3+
O
NH
NN
O
H
H
NH
NN
O
H
H
HILIC
First UHPLC systems and
sub-2µm columns
Pressure
Modern SFC: a viable alternative to RPLC?
UHPSFC 1.7 µm
UHPLC 1.7 µm
UHPSFC 1.7 µm
UHPLC 1.7 µm
Kine%cperformance Pressuredrop
10 -10 -5 0 5
High versatility of SFC vs other techniques
An%bio%csPolysaccharides
MetabolitesNeurotransmi:ersAminoacids/pep%desNucleo%des/Nucleosides
MostofusualdrugsLipids(triglycerides)LiposolublevitaminsFewplantcomponents
IEX
HILIC
RPLC
Requires a dedicated LC system. Not MS compatible
Requires a dedicated LC system. Not MS compatible.
Suitable till a certain polarity limit.
Polarity limits of SFC have still to be defined.
SFC
Sameinstrumentwithoutdras%cchangesinanaly%calcondi%ons
NPLC
Limited retention of polar and ionisable compounds.
Ultra-fast analysis of steroids AU
0.00
0.01
0.02
0.03
0.04
Minutes
0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00
Mixtureof5structurallyrelatedsteroids.ColumnUPC²BEH(3.0x100mm,1.7µm).Gradientfrom10%to12%MeOHin0.6minfollowedbyanisocra]cstepof0.4min.Backpressureregulatorandoventemperatureweresetat145barand40oC,respec]vely.
For10successiveinjec]ons,RSDontrwerebetween0.35and0.59%
Injec]oncycle]me~50s
Silica
OH
Maximalpressure(414bar)
High resolution analysis of benzodiazepines AU
0.00
0.02
0.04
0.06
0.08
Minutes
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00
P = 143 P = 143
AU
0.00
0.02
0.04
0.06
0.08
Minutes
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00
P = 143 P = 143
AU
0.00
0.02
0.04
0.06
0.08
Minutes
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00
P = 186P = 186
AU
0.00
0.02
0.04
0.06
0.08
Minutes
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00
P = 186P = 186
AU
0.00
0.02
0.04
0.06
0.08
Minutes
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
P = 196 P = 196
AU
0.00
0.02
0.04
0.06
0.08
Minutes
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
P = 196 P = 196
AU
0.00
0.02
0.04
0.06
0.08
Minutes
0.00 4.00 8.00 12.00 16.00 20.00 24.00 28.00 32.00 36.00 40.00
P = 258P = 258
AU
0.00
0.02
0.04
0.06
0.08
Minutes
0.00 4.00 8.00 12.00 16.00 20.00 24.00 28.00 32.00 36.00 40.00
P = 258P = 258
Mixture of 11 structurally related benzodiazepines (elution order: prazepam, flunitrazepam, clorazepate, desmethylflunitrazepam, nitrazepam,
clonazepam, midazolam, brotizolam, 7-aminoflunitrazepam, alprazolam and triazolam).
100 mm column Gradient run time: 5 min Flow rate: 2.5 mL/min.
100 mm column Gradient run time: 10 min
Flow rate: 2.5 mL/min.
200 mm column Gradient run time: 20 min Flow rate: 1.25 mL/min.
200 mm column Gradient run time: 40 min Flow rate: 1.25 mL/min.
Silica
OH
( )∑+= n
grad
wn
tP
1%4.13/1
1
1.7 µm
Selectivity on structurally related compounds
Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00
AU
0.00
0.02
0.04
0.06
0.08
1 2 3 4 5 6 7
BoldenoneNandrolone
Methytestosterone
Testosterone
Stanozolol Methandienone
Androstendione
Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00
AU
0.00
0.10
0.20
0.30
0.40UHPLC5
42 7
6 1 3UHPSFC 5
4
2
7 6
13
Evolution of liquid chromatography
1903 2000 2004 2007 1965 2010
First HPLC systems
Discovery of chromatography Monoliths Core-shell
technology
Green LC and SFC
Biomolecules
2008
SiOSi
Si
SiSi
Si
O
O
O OOSiSi
O
O
O
SiO-
Si
SiO
Si
O
Si
O
O
Si
O
Si
O
Si
O H
O H
O
HO -
O-
SiSiOSi
Si
SiSi
Si
O
O
O OOSiSi
O
O
O
SiO-
SiSi
SiSiO
Si
O
Si
O
Si
O
Si
O
O
Si
O
SiSi
O
Si
O
SiSi
O
Si
O
SiSi
O H
O H
O
H
O
HO -
O-
OH
HOH
H
O
H
HO
H
H
ACN
H2O
NH
N
NH3+
O
NHN
NH3+
O
NH
NN
O
H
H
SiOSi
Si
SiSi
Si
O
O
O OOSiSi
O
O
O
SiO-
Si
SiO
Si
O
Si
O
O
Si
O
Si
O
Si
O H
O H
O
HO -
O-
SiSiOSi
Si
SiSi
Si
O
O
O OOSiSi
O
O
O
SiO-
SiSi
SiSiO
Si
O
Si
O
Si
O
Si
O
O
Si
O
SiSi
O
Si
O
SiSi
O
Si
O
SiSi
O H
O H
O
H
O
HO -
O-
OH
HOH
H
O
H
HO
H
H
ACN
H2O
NH
N
NH3+
O
NHN
NH3+
O
NH
NN
O
H
H
NH
NN
O
H
H
HILIC
First UHPLC systems and
sub-2µm columns
Pressure
Analytical characterization of molecules
Rituximab MW ~150,000 Da
mAbs
salicylic acid MW 138 Da
Small molecules
triptorelin MW 1,311 Da
Peptides Proteins
human growth hormone
MW 22,125 Da
Analytical complexity
Important micro-heterogeneity during the production of biologics
ADC
Brenduximab-vedotin
MW ~150,000 Da
Analytical challenge !
IEX
Analytical methods for macromolecules
Electrophoresis
SEC
RPLC
• Charge heterogeneity
• PEGylations
• MW determination
• Aggregation
• MW determination
• Charge heterogeneity
• Quality control
• Characterization of biosimilars
A. Staub et al., JPBA, 2011, 55, 810-822
+ _+ _+ _+ _+ _
Need for high resolution because of sample complexity Importance to combine methods with MS detection
Bevacizumab (IgG1) Cancer
Analysis of intact mAbs
Rituximab (IgG1) Leukemia
Panitumumab (IgG2) Cancer
C18
- 1.
7 µm
C
18 -
3.6 µm
SPP
Impressive peak shapes for RPLC of an intact 150 kDa antibody
Charge variants
1.7 µm
3.6 µm
Columns of 150x2.1mm, T = 80°C (Waters Acquity C18, 1.7µm, 300A) and 90°C (Phenomenex Aeris C18, 3.6 µm SPP, widepore)
Evolution of liquid chromatography
1903 2000 2004 2007 2030
Discovery of chromatography
SPP
2015
• Instrumentation – robustness
• Miniaturization (3D printed micro devices)
• New stationary phases: monoliths, pillar array,
Sphere-on sphere particles, silica-colloidal particles, PLOT, parallel-
segmented flow chromatography
• 2D or 3D chromatography
• New MS or detection systems Monoliths
UHPLC and sub-2µm columns
Pressure
Acknowledgments
Dao Nguyen Josephine Ruta Aline Staub Szabolcs Fekete Alexandre Grand-Guillaume Perrenoud Aurélie Periat Serge Rudaz Jean-Luc Wolfender Alain Beck
Thank you for your attention !
Réponses aux questions
1. Lachromatographieenphasesupercri]que
A:estréservéeauxcomposésapolaires NON
B:permetdessépara8onsrapides OUI
C:estadaptéeauxbiomoléculesdegrandetaille NON
D:u8liseunephasemobilecons8tuéed’unmélangedeCO2etdeméthanol OUI
E:peut-êtrecoupléeàlaspectrométriedemasse OUI
Instrumental constraints
S. Fekete et al. J. Pharm. Biomed. Anal., 87, 2014, 105-119
( )( )col
20
col
2r2
col Nk1V
NV +⋅
==σ
σ²ext = 20 µL²
k=5
4.6 mm
3 mm
2.1 mm
1 mm
N=13’500
N=11’200
N=9’000
N=3’600
m
cccellcell
injinjext D
FlrFVKV
K×
××+++=
6.7²²
1212
4222 τσ
injector detector tubing
2012 2004 2000 1990
Anewgenera8onofUHPSFCsystems
SFC UHPLC UPC2
Dwellvolume(VD) 2270µL 90µL 440µL
Isocra%cstepforgenericcondi%ons
1.13min(F=2mL/min)
0.15min(F=0.6mL/min)
0.22min(F=2mL/min)
Extracolumnvolume(Vext)
118µL 13µL 60µL
Suitablecolumndimensions
150x4.6mm(volume~1750µL)
50x2.1mm(volume~120µL)
100x3mm(volume~490µL)
EXTRA-COLUMN VOLUME : Vext Injector
TubingDetector
Column
Programatcolumninlet
Time
Programatpumpoutlet%B
Dwell]me
DWELL VOLUME : VD
Superficially porous particles : SPP
v A small layer (0.5µm) of uniform porous particles is grown around a solid core of non porous silica material (1.7µm).
Ø Lower mass transfer resistance, longitudinal and eddy diffusion: better chromatographic performance
Ø Compatible with fast analysis and high resolution chromatography Ø Initially developed for high molecular weight compounds (MW>600g.mol-1) such
as peptides and proteins (low Dm) but today applied to all molecules
www.sigmaaldrich.com
www.sigmaaldrich.com
SPP: a good compromise…
Fully porous particles (FPP)
Superficially porous particles (SPP)
Diameter: 2.6-2.7 µm
Porous layer: 0.35-0.5 µm
Good kinetic performance
Good mechanical stability
Large specific surface area
• High loading capacity
• Good retention
Non-porous particles (NPP)
Low specific surface area
• Poor loading capacity
• Limited retention
High kinetic performance
Huge mechanical stability
Good loading capacity and retention Large choice of column dimensions and chemistries/providers
High backpressure with small particles
Frictional heating in UHPLC ?
100x3mm I.D., 1.7µm
150x3mm I.D., 1.7µm
50x3mm I.D., 1.7µm
F. Gritti, G. Guiochon, Anal. Chem., 2008
Theoretical approach Practical approach
Pressure
L. Novakova, J.L. Veuthey, D. Guillarme, J. Chromatogr. A, (2011), 1218, 7971-7981
Effect of high pressure on retention
Column: C18 (50 mm x 2.1 mm, 1.7µm), mobile phase: water (0.1% TFA) + acetonitrile (0.1% TFA) : 70 + 30 v/v, flow-rate: varied between 100, 500 and 900 µL/min, temperature: 30 °C, injected volume: 0.5 µL, detection: 210 nm. Peaks: lidocaine (1), salicylic acid (2), bupivacaine (3), propranolol (4), propylparaben (5) and testosterone (6).
Case of small analytes
Pure effect of pressure (experiments with restrictor
capillary)
Effect of pressure + frictional heating
Sensitivity in HILIC-MS vs. RPLC-MS ?
16%
43% 9% 18%
14%
bupropion
x 6600
acebutolol < 1 fold
1 to 5-fold 5 to 10-fold
10 to 30-fold
> 30 fold
pseudoephedrine
x 2.6
÷2.2
Gain in sensitivity in HILIC vs. RPLC @ 600µL/min
0.00 1.00 2.00 3.00 4.00 5.00 Time (min)
0.00 1.00 2.00 3.00 4.00 5.00 Time (min)
0.00 1.00 2.00 3.00 4.00 5.00 Time (min)
56 basic drugs of diverse polarity were tested on modern UHPLC-ESI-MS/MS instrument
Comparison of current LC technologies
+ Comparison made for butylparaben (MW=200 g.mol-1) with η=0.89 cp @ 30°C and 0.41 cp @ 90°C. Pressure was equal to 400 bar in all cases except
those cited at 1000 bar and UHPLC and SPP 1.3µm, monoliths at 200 bar and SPP 2.6 µm at 600 bar.
Thro
ughp
ut
(Req
uire
d tim
e fo
r N =
10’
000
plat
es)
_ + _
Efficiency (Highest possible N for t0 = 30 min)
HTLC 5 µm
t0 = 11 s, N = 208,000
+
HPLC 5 µm
t0 = 55 s, N = 79,000 5 µm, 1000 bar
t0 = 38 s, N = 127,000
+ Pressure
Sub-2µm particles
t0 = 11 s, N = 31,000
Monoliths t0 = 15 s, N = 151,000
Not available
UHPLC Pressure
+
t0 = 6 s, N = 73,000
(e.g. k=5, tana=3 h)
D. Guillarme et al., Anal. Bioanal. Chem., 2010, 397, 1069
SPP 2.6 µm
t0 = 6 s, N = 98,000
SPP 5 µm t0 = 14 s, N = 238,000
SPP 1.3 µm
t0 = 3 s, N = 63,000
Highest throughput
Highest resolution
