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2013 Waters Corporation 1
Avances en cromatografa lquida
Teora y prctica
para el anlisis farmacutico
Oscar Cortes Ledezma, Qumico
oscar.cortes@scancotec.com
Adriana Murillo Ramrez, Qumica.
amurillo@scancotec.com
2013 Waters Corporation 2
AGRADECIMIENTOS
Dr Jorge Lpez Mora (UCIMED)
Dr Gustavo Senz Garca (UCIMED)
Dra Marisol Flores Campos (UCIMED)
Msc. Jens Voldum (Scanco Tecnologa)
Q.F. Ricardo Martinez (Waters Corporation)
Ing. Jeffry Aparicio (Scanco Tecnologa)
Licda. Vanessa Vagnarello
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Contenido
1. Regulaciones y requerimientos de validacin de mtodos
cromatogrficos.
1. Parmetros cromatogrficos y su optimizacin.
2. Seleccin de columnas.
3. Efecto de la fase mvil en el cromatograma.
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Regulaciones y requerimientos
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"System suitability tests are an integral part of gas and liquid chromatographic methods. They are used to verify that the resolution and reproducibility of the chromatographic system are adequate for the analysis to be done.
The tests are based on the concept that the equipment, electronics, analytical operations and samples to be analyzed constitute an integral system that can be evaluated as such.
Un SST debe contener:
Para un ensayo:
precisin + otro parmetro mas
Para ensayos de impurezas:
resolucin + precisin + otro parmetro ms
System suitability
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Categoras USP
Categora 1: Cuantificacin de componentes
mayoritarios o ingredients activos.
Categora 2: Determinacin de impurezas o
productos de degradacin.
Categora 3: Determinacin de caractersticas de
desempeo.
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USP Data Elements Required For Assay Validation
Analytical
Performance
Parameter
Assay Category 1
Assay Category 2Assay
Category 3Quantitative Limit Tests
Accuracy Yes Yes * *
Precision Yes Yes No Yes
Specificity Yes Yes Yes *
LOD No No Yes *
LOQ No Yes No *
Linearity Yes Yes No *
Range Yes Yes * *
Ruggedness Yes Yes Yes Yes
* May be required, depending on the nature of the specific test.
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Specificity-Analytical Parameter
Lack of specifity of an analytical proceduremaybe compensated for by other analyticalprocedure
For example, by MS
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Parmetros cromatogrficos
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Valores lmite segn la USP
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K = Capacity FactorA Measure of Retention
1.5
.5 - 1 Vfor k'
1
5.5
.5 - 3 Vfor k' 2
V0
V1
V2V3
TIME .5 1 3 4 6
kn = (tn-t0) / t0
kn = (Vn-V0) / V0
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= SelectivityA Measure of Peak Separation
V0
V1
V2V3
TIME .5 1 3 4 6
0V
1V
0V
2V
or
1 Vof k'
2 Vof k'
1.4.5 - 3
.5 - 4 Vfor 3
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RS = Resolution
2,2/11,2/1
RR
21
RR
S
12
12
18.1
)(2
1
hh WW
tt
WW
ttR
tR1 tR2
W1 W2
W1/2h,1
W1/2h,2
h1/2))((
1k
k1
4
NRs
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Theoretical Plate Number, N
W
W1/2H1/2
H
2
.21
R
R
/
R
W
t
W
2
2
2
545
16
Area
Ht
tN
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Impact of Instrument Band Spreading on Small Particle Column Performance
Minimize band spreading from the instrument and the column to improve chromatographic performance
1.7 m on an HPLC 1.7 m on a UPLC
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0.020"0.003" 0.040"
Shortest length is best
Smallest
ID is best
Flow Direction
Tubing with a large ID increases the overall system volume and contributes to band spreading
Excessive tubing lengths contribute to band spreading
Decreasing tubing ID and increasing length increases system pressure
System Band Spreading:Effect of Tubing ID and Length
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Small Particles Necessitate Low Band Spread Instruments
Band spread is a measure of system dispersionLow band spread ensures separated peaks stay separated
System Band Spread (L) 5 sigma
Shimadzu UFLC 41
Alliance HPLC 29
1260 Infinity 6 L/5 mm bore FC 28
1290 Infinity 1 L/10 mm MaxLight FC (dual column) 23
Original Accela 21
1290 Infinity 1 L/10 mm MaxLight FC (single column) 20
Dionex Ultimate 3000 2.5 L/7 mm FC 17
1260 Infinity 2 L/3 mm bore FC 16
ACQUITY UPLC H-Class with Column Manager 12
ACQUITY UPLC H-Class with Column Heater 9
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UPLC Technology & The Fundamental Resolution Equation
))((1k
k1
4
NRs
NRs
Physical Chemical
dp 3X, If N 3x, Rs 1.7x
Sensitivity 1.7X
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Constant Column LengthFlow Rate Proportional to Particle Size
AU
0.000
0.010
0.020
0.030
0.040
0.050
Minutes
0.00 2.00 4.00 6.00 8.00 10.00 12.00 15.00
4.8 m, 0.2 mL/min, 354 psi
AU
0.000
0.010
0.020
0.030
0.040
0.050
Minutes
0.00 1.00 2.00 3.00 4.00 5.00 6.00
Theory1.7X Resolution3X Faster1.7X Sensitivity25X Pressure
Reality1.5X Resolution2.6X Faster1.4X Sensitivity22X Pressure
1.7 m, 0.6 mL/min, 7656 psi
2.1 x 50 mm columns Very High System Pressure
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Ultra Performance LCTM
Velocidad incrementa volumen de muestras
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
1.
Th
iou
rea -
0.4
30
2.
tolu
en
e -
1.0
34
3.
pro
pylb
en
zen
e -
1.7
42
4.
bu
tylb
en
zen
e -
2.4
13
5.
hexylb
en
zen
e -
5.0
58
No. de componentes: 5
Completa separacin: 6.00 min0.18
0.20
0.22
0.24
1.
Thio
ure
a -
0.0
46
2.
tolu
ene -
0.0
88
3.
pro
pylb
enzene -
0.1
37
4.
buty
lbenzene -
0.1
82
5.
hexylb
enzene -
0.3
60 UPLCTM HPLC
AU
0.00
0.10
0.20
Minutes0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60
UPLC
No. de componentes: 5
Completa separacin: : 0.60 min
UPLCTM incremento de velocidad por arriba de 9X
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USP Chromatographydefines Allowable adjustments
Adjustments to a USP method may be made to meet system suitability
requirements
Verification tests must be performed after changes
Full re-validation not required
Must use the same L-designation of column
Isocratic hold or dwell volume adjustments are allowed
USP 37 NF 32 S1
- to be official Aug. 1, 2014
- significant changes to Chapter Chromatography
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Changes to Effective August 1, 2014
Current (USP36-NF31)
isocratic & gradient isocratic gradient
Particle Size -50%
Column Length 70%
Flow Rate 50% *based on particle size and 50% no changes allowed
Column ID Any allowed Any allowed no changes allowed
Injection Volume Any reduction Any allowed Any allowed
Column Temperature 10% 10% 10%
Mobile Phase pH 0.2 unit 0.2 unit 0.2 unit
VariableAfter Aug 1, 2014 (USP37-NF32 S1)
Allowable Adjustments in USP Chromatography
per constant L/dp or N: -25% to +50% no changes allowed
Flow rate (isocratic): F2 = F1 x [(dc22 x dp1)/(dc1
2 x dp2)]
L/dp = column length (L) to particle size (dp) ratioN = theoretical plate count (for solid-core particles)
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30
mm
30
mm
30
mm 30
mm
50
mm
50
mm 50
mm
50
mm
75
mm 75
mm
75
mm
75
mm
10
0 m
m 10
0 m
m
10
0 m
m
10
0 m
m
15
0 m
m
15
0 m
m
15
0 m
m
15
0 m
m
25
0 m
m
25
0 m
m
0
10000
20000
30000
40000
50000
60000
70000
80000
90000
100000
0.005 0.0035 0.0025 0.0017
L/d
p R
atio
particle size (m)
1.7 2.5 3.5 5.0
Moderately Challenging
Easy
ExtremelyDifficult
Difficult
Ratio of Column Length to Particle SizeResolution Capability
50 mm, 1.7 m75 mm, 2.5 m100 mm, 3.5 m150 mm, 5 m
L/dp ~ 30,000
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Improve HPLC Productivity Today:Directly Scalable to UPLC Technology
XP 2.5 m
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Introducing CORTECS 2.7 m Columns
CORTECS 2.7 m solid-core particles demonstrate an efficiency equivalent to that of 2.2 m fully porous particles and a backpressure equivalent to that of 3.1 m fully porous particles:
CORTECS Particle Size
Equivalent Porous Particles
Efficiency Backpressure
1.6 m 1.3 m 1.8 m
2.7 m 2.2 m 3.1 m
2.22
m 3.1 m2.7 m
Same Efficiency
Same back pressure
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High Efficiency at HPLC Backpressures
AU
0.00
0.02
0.04
0.06
0.08
0.10
AU
0.00
0.02
0.04
0.06
0.08
0.10
AU
0.00
0.02
0.04
0.06
0.08
0.10
Minutes
1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50
1
2 34
Rs 4.9
Rs 6.3
Rs 8.2
Fully Porous C18, 5 mPsi: 1900N peak 4: 10,400
Fully Porous C18, 3.5 mPsi: 2900N peak 4: 17,600
CORTECS C18, 2.7 mPsi: 3200N peak 4: 23,600
1. Estradiol2. Ethinyl estradiol3. Estrone4. Levonorgestrel
Configuration: 4.6 x 150 mm
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Seleccin de columnas
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Stationary Phase Selectivity:Bonded-Phase [Ligand] and Particle Substrate
Silanol activity and surface charge
Secondary interactions
Affects peak shape and sample loadability
Hydrophobicity
Longer alkyl chain lengths will provide increased retention
Shorter, ionizable ligands will increase polarity
Ligand density
Influences retention and sample loadability
Hydrolytic stability
the number of attachment points to the particle surface can impact
column lifetime
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Surface of a Silica Gel Bonded-Phase Packing Material
High Coverage
High LigandDensity
Low Coverage
Low LigandDensity
H
SiO
O
O
SiO
O
O
SiO
O
O
SiO
O
O
SiO
O
O
SiO
O
O
SiO
O
O
SiO
O
OSi
O
O
O
SiO
OO
SiO
O
O
Si
CH2H3C CH3
H2CCH2
H2C
CH2
H2C
CH2
H3C
Si
CH3H3C CH3 Si
CH2H3C CH3
H2CCH2
H2C
CH2
H2C
CH2
H3C
SiO
O
O
Si
CH3H3C CH3 Si
CH3H3C CH3
HHHHHH H
C8 alkyl chains
Residual silanolsEndcap
Polar analytes are not able to energetically fit between
ligands cant interact with surface or ligands
Si
SiO
O
OSi
O
O
O
SiO
O
O
SiO
O
O
SiO
O
O
SiO
O
O
SiO
O
O
SiO
O
OSi
O
O
O
SiO
OO
SiO
O
O
Si
CH2H3C CH3
H2CCH2
H2C
CH2
H2C
CH2
H3C
Si
CH2H3C CH3
H2CCH2
H2C
CH2
H2C
CH2
H3C
CH2H3C CH3
H2CCH2
H2C
CH2
H2C
CH2
H3C
Si
CH2H3C CH3
H2CCH2
H2C
CH2
H2C
CH2
H3C
SiO
O
O
Si
CH3H3C CH3 i
CH3H3C CH3
HHHHHH H
Residual silanol
Endcap
Polar analytes easily interact with surface
and ligands
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SiC
CC
CC
CC
CH3
CH3
H3C
OSi
O
O
O
Hydrolysis of a Bonded Phase Material: Monofunctional Ligand
+ HCl
OHSi
O
O
O+
SiC
CC
CC
CC
CH3
CH3
H3C
Cl
OHSi
O
O
O+ Si
CC
CC
CC
CCH3
CH3
H3C
HO
Low pH (hydrolysis of ligand)
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Making a Bonded Phase Material: Multifunctional Synthesis
+
+ HCl
C8 Trichlorosilane Ligand
Multi-Point Attachment
ClSi
Cl
ClCH3
OHOH
OHSi
SiSi
O
O
O
O
O O
O
O
Si
O
OHSi
SiSi
CH3
O
O
O
O
O O
O
OH
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Effects of High pH Mobile Phases
Surface Modified Silica Particles Hybrid Particles
Complete dissolution of Silica Catastrophic column failure Short lifetimes
Slow rate of surface dissolution Incorporated methyl groups uncovered slows rate of dissolution Longer column lifetimes
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Reversed-PhaseColumn Selectivity Chart
(ln [k] acenaphthene)
SunFire C18
YMC-Pack PolymerC18
Hypersil CPS Cyano
YMC-Pack CN
Waters Spherisorb S5 P
Hypersil BDS Phenyl
Nova-Pak Phenyl
YMC-Pack
Phenyl
Hypersil Phenyl
Inertsil Ph-3
YMC-Pack Pro C4
YMCbasic
Symmetry C8
YMC-Pack Pro C8
Nova-Pak
C8
XTerra MS C18Symmetry C18
YMC-Pack Pro C18 Inertsil ODS-3
YMC-Pack ODS-A
Nova-Pak
C18
YMC J'sphere
ODSL80 Nucleosil C18
Waters Spherisorb ODS2
Waters Spherisorb ODS1
Resolve C18
Bondapak C18
YMC-Pack ODSAQ
YMC J'sphere ODSH80
YMC J'sphere ODSM80
Inertsil CN-3
Waters Spherisorb S5CN
Nova-Pak CN HP
SymmetryShield RP8
SymmetryShield RP18
XTerra RP8XTerra RP18
-0.6
-0.3
0
0.3
0.6
0.9
1.2
1.5
1.8
2.1
2.4
2.7
3
3.3
3.6
-1.5 -0.5 0.5 1.5 2.5 3.5
(ln
[
] am
itri
pty
lin
e/a
cen
ap
hth
en
e)
XTerra MS C8
Luna C18
(2)ACQUITY
UPLC BEH C18
XTerra Phenyl Luna
Phenyl Hexyl
ChromolithTM
RP-18
Atlantis dC18
Zorbax XDB C18ACT Ace
C18
Zorbax SB C18
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Waters column Selectivity Chart
http://www.waters.com/waters/promotionDetail.htm?id=10048475&locale=en_CA
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Efecto de la fase mvil en
el cromatograma
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Selectivity Differences:Methanol versus Acetonitrile
Selectivity shifts observed.Acetonitrile is a stronger elution solvent.Acetonitrile and methanol can be blended to fine tune separations.
Jenkins, Diehl
XBridge Shield RP18
100% MeOH
100% ACN
Low pH
1:1 ACN:MeOH
1
2
43
56
2
8
79
6
10
12
11
8
9
10 11
10,11
12
128,91 6
7
2
3 4
43
15,7
5
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Mobile Phase pH Selectivity:Basic and Neutral Compounds
IA
P
Fl
Fe
D
O
Minutes
0.00 1.20 2.40 3.60 4.80
I
A
P
Fl
Fe
DO
Minutes
0.00 1.20 2.40 3.60 4.80
0.1% formic acid pH 3
Test Probes:I: Imipramine [B]A: Amitriptyline [B]Fl: Flavone [N]O: Octanophenone [N]
ACQUITY UPLC CSH C18
Acetonitrile
0.1% NH4OH pH 10
Observations:
At high pH, bases are in their neutral
(unionized) form, resulting in greater
retention
Neutral compounds are unaffected by pH
2013 Waters Corporation 38
Mobile Phase pH Selectivity:Acidic Compounds
IA
P
Fl
Fe
D
O
Minutes
0.00 1.20 2.40 3.60 4.80
I
A
P
Fl
Fe
DO
Minutes
0.00 1.20 2.40 3.60 4.80
Test Probes:P: 1-pyrenesulfonic acid [A]Fe: fenoprofen [A]D: diclofenac [A]Fl: Flavone [N]O: Octanophenone [N]
0.1% formic acid pH 3
0.1% NH4OH pH 10
ACQUITY UPLC CSH C18,
Acetonitrile
Observations:
At low pH, acids are in their neutral
(unionized) form, resulting in greater
retention
Elution order change for acidic compounds
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Ion Pair Mechanism
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Reference
Exerpts from USP37-NF32 1S (Published)
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Allowable adjustments
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Changes to Particle Size
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Changes to Flow Rate
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Changes to Other factors
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Example provided