Documenti di Didattica
Documenti di Professioni
Documenti di Cultura
Peru
2018
Ricardo Martínez
LC Introduction
Columns quality
Particles & Ligands
pH in LC
New Tech Columns
Solid-Cord Particles
Conclusions
Russian Botanist
– May 14th 1872 – June 26th 1919
Greek
Chroma -- color
Graphy -- writing/study of
Note: Tswett in Russian means Color
COMPANY CONFIDENTIAL
8 8
©2018 Waters Corporation
Chromatography
Applications
Resolution
Sensitivity
Precision
Time Analysis
Method Simplicity
Cost
Separating mixtures
What is it? (Identification)
How much is there? (Quantification)
Chromatogram
3D
Absorbance
l1
l2
Absorbanc
Spectrum
Time
Benefit: Peak
e
Identification
Wavelength
PDA detectors acquire 3D data,
2D data can be extracted
– Chromatograms
– Spectra
14
Highly similar UV, differentiating mass….
212.9 Metoclopramide
272.8272.8
309.1309.1
m/z = 300.1
Metoclopramide Impurity G
m/z = 316.1
©2018 Waters Corporation COMPANY CONFIDENTIAL 15
LC/MS/MS Instrument
Detect non-chromophoric
analytes
Stationary Phase
Nova-Pak® Atlantis®
XBridge™ HILIC ACQUITY UPLC®
ProteinPakTM AccQTagTM BEH125 SEC
Pico-TagTM Atlantis® T3 ACQUITY UPC2™
ACQUITY UPLC® HSS T3 Columns
Symmetry® 300 AccQTagTM Ultra
Atlantis® HILIC Silica BEH130 Columns CORTECS™ Columns
Prep OBD™ BEH300 Columns ACQUITY UPLC®
Intelligent SpeedTM ACQUITY UPLC® BEH200 SEC BEH450 SEC
BioSuite™ XSelect CSH HPLC columns ACQUITY APC™ Columns
NanoEase™ ACQUITY CSH Columns CSH130 Columns
Viridis SFC Columns
ProteinPak High Rs IEX
©2018 Waters Corporation COMPANY CONFIDENTIAL 20
Quality Systems
Minimize Risk with Dependable Column Performance
0
5.37
X
4.70 0.81
Y
4.14 2.86
A
Acenaphthene
Neutrals QCRM: 186006360
Naphthalene
0.050
0.040
Compound Type
Acetone
0.030
AU
Acenaphthene
– Leaks
Naphthalene
0.010
Check Valve
– Older column
0.000
0.06
– Carryover
0.04 Good B Pump – Dirty flow cell
AU
1.20
USP Tailing
1.00
100000
0.80
0.60
50000 0.40
0.20
0 0.00
0 200 400 600 800 1000 0 200 400 600 800 1000
System Pressure Injection
Injection
9000
Pressure (psi) at 0.4
8000
7000
6000
min
5000
4000
0 500 1000
Injection
Si – O – H
H H H
O O O
Si Si Si
O O O O
O O O
Comes from the silica gel particle (substrate) used to
make reversed-
reversed-phase packing materials
Si--OH = Silanol
Si
CH 3
H3C CCCCCCCCCCCCCCCCCC
Si
O
25 Å
“Steric Hindrance”
25 Å
“Steric Hindrance”
CH 3
H3C
Si CH 3
O
Depending on how a packing material is made, even a fully bonded and end-capped
particle can generate a significant negative charge (-) as you approach pH 7.
OH O
Si Si H+
Behaves as a Cation Exchanger
(pH 2) (pH 7)
Si – OH
Si-OH Si-O- + H+
Silanol Groups
(pH 2) (pH 7)
No charge Negatively charged
Base
Base has
Base increased
Base retention and
RP Cation X poor peak
shape
©2018 Waters Corporation COMPANY CONFIDENTIAL 37
Why not just run at pH’s less than 3?
Selectivity
0.04
pH 2.0
AU
0.02
0.00 A amitriptyline = A
0.00 2.00 4.00 6.00 8.00 10.00
M inutes
nortriptyline
pH 7.0
A A
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00
Non-porous
– Increased efficiency compared to fully porous
– Poor retention and mass loading
Fully porous
– Most common LC particle
– Encompass a wide range of pore diameters and pore
volumes
Superficially porous
– Provide increases efficiency over fully porous particles of
similar size
– Have lower backpressure when compared to fully porous
particles of similar size
©2018 Waters Corporation COMPANY CONFIDENTIAL 40
Types of Surface Silanols Found on Silica Gel
H
H
O O
O O O
Vicinal (Bridged) Si Si
O O
HO OH
OH O
O O O O
Geminal (Silanediol) Si Si Si
O O
OH
O O O
Lone (most active) Si Si
4 5 6 7 8 9 10
©2018 Waters Corporation COMPANY CONFIDENTIAL
Minutes 42
Aluminum in the Silica Gel Lattice
Analyte: Chlorpheniramine
Mobile Phase: Acetonitrile/KH2PO4 pH 3.0 (20:80)
1
0 100 200 300 400
Aluminum Content, ppm
©2018 Waters Corporation COMPANY CONFIDENTIAL 44
Peak Shapes of Chelating Agent (Hinokitiol)
M = metal
Low metals
O
Si
O M n+ O
Si O
High metals
2 4 Minutes 6 8
©2018 Waters Corporation COMPANY CONFIDENTIAL 45
Internal Metals:
A Much Bigger Problem
O
Electron withdrawing influence
CH3
Si O Si
Greater acidity for silanols
(C H 2 )17C H 3
O CH3 Exaggerated retention for bases,
M+ Si OH Major loss of peak shape for bases
O
Si O
CH3 Not a major problem for modern
Si CH
O CH3
3 particles
Waters Spherisorb® ODS2
Propranolol
Amitriptyline
Cation
Exchange
+
N
Amitriptyline Acenaphthene
pKa = 9.4 Neutral Compound
Basic Compound
Temperature controlled 23 °C
New Columns
0 10 20 30 40 50 60 70 80 90
Minutes
Note: pH plays a role in peak shape
Analyte molecule
Very Large Non-porous
3.3
amitriptyline/acenaphthene)
Advantages Disadvantages
• Mechanically strong • Limited pH range
Inorganic • High efficiency • Tailing peaks for bases
(Silicon) • Predictable retention • Chemically unstable
Note: Increased
Butylparaben
Naphthalene
Note:Reduced silanols
Acenaphthene
retention of improve peak shape for bases
Amitriptyline
neutral (loss of cation-exchange
compounds retention mechanism).
However, low efficiency
results in broader peaks.
Symmetry® C18
PolymerC18™
0 10 20 30 40
©2018 Waters Corporation COMPANY CONFIDENTIAL
Minutes 55
1st Generation Hybrid
(MethylSiloxane/Silica) Particles
Methyl Groups
on Hybrid Surface
(Better Peak Shape)
and
in Hybrid Particle
(High pH Life-time)
Waters Patented technology
US Patent: 6,686,035 B2
Date of Patent: Feb. 3, 2004
N α 1 k
Rs
4 α k1
Mechanical Contributions Chemical/Physical Contributions
Ultra-low dispersion system
Operate at optimal linear velocity Complementary bonded phases
Particle morphology Multiple particle substrates
Small particles Ability to utilize high pH
Well-packed columns Increase retentivity
Smaller particle sizes provide for a better separation with the same
run time. However, back pressure will increase.
* This is also called “Efficiency”
©2018 Waters Corporation COMPANY CONFIDENTIAL 61
Waters Particle Technology
60 µm Human Hair (very fine hair)
5 µm 1.7 µm
Analytical Particles ACQUITY UPLC™ Particles
(can fit 12 across hair) Optimal Particle Size Distribution
Images are on For Max Efficiency
same scale at a given Pressure
(Bar = 10 µm)
©2018 Waters Corporation COMPANY CONFIDENTIAL 62
Efficiency vs. Flow Rate
Acenaphthene, 2.1 x 50 mm columns
70/30 MeCN/H2O, 30 °C, 254 nm
10000
8000
6000
4000
2000
0
0.0 0.5 1.0 1.5 2.0 2.5
©2018 Waters Corporation COMPANY CONFIDENTIAL Flow Rate (mL/min) 63
van Deemter Curves
Plate Height (Non-reduced, H)
Acenaphthene, 2.1 x 50 mm columns
70/30 MeCN/H2O, 30 °C, 254 nm
25
Plate Height (H, 4 sigma)
15
10
0
0.0 0.5 1.0 1.5 2.0 2.5 3.0
Linear Velocity, u (cm/sec)
©2018 Waters Corporation COMPANY CONFIDENTIAL 64
UPLC® Technology & The Fundamental Resolution
Equation
Rs
N
4
( α 1
α
)( k
k 1
)
Physical Chemical
Rs N
If N ↑ 3x, Rs ↑ 1.7x
©2018 Waters Corporation COMPANY CONFIDENTIAL 65
Constant Column Length
Flow Rate Proportional to Particle Size
0.050
0.040
1.7 µm, 0.6 mL/min, 7656 psi Reality
0.030 1.5X Resolution
AU
0.020
2.6X Faster
0.010
0.000
1.4X Sensitivity
0.00 1.00 2.00 3.00
Minutes
4.00 5.00
6.00 22X Pressure
0.050
0.020
0.010
3X Faster
0.000
1.7X Sensitivity
0.00 2.00 4.00 6.00 8.00
Minutes
10.00 12.00
15.00 25X Pressure
2.1 x 50 mm columns Very High
System Pressure
©2018 Waters Corporation COMPANY CONFIDENTIAL 66
Resolution (and Speed)
Constant Column Length
Plates, Flow Rate and Particle Size
12000
11000
10000
9000 Smaller Particle Optimal Smaller Particle Size
8000 *Increased N,
7000
Rs N 6000
5000
4000
*Higher, optimal u
*Increased pressure
3000
Larger Particle
2000
1000
L
N
dp
For same N and, therefore, same Rs
0.02
2.5X Sensitivity
0.00 11X Back Pressure
0.00 1.00 2.00 3.00
0.06 4.00
Minutes Theory
0.04 Same Resolution
4.8 µm, 100 mm, 0.2 mL/min 9X Faster
AU
0.02
3X Sensitivity
0.00
9X Back Pressure
0.00 5.00 10.00 15.00 20.00 25.00
30.00 Manageable
Minutes
Moderately
Related compound assay 12,000 30,000
Challenging
Extremely
Metabolite identification 35,000 85,000
Difficult
150 mm
100000
90000
•75 mm, 2.5 µm L/dp ~ 30,000
•100 mm, 3.5 µm Extremely
250 mm
80000
•150 mm, 5 µm Difficult
150 mm
100 mm
70000
250 mm
60000
L/dp Ratio
150 mm
75 mm
100 mm
50000
Difficult
Impurity
150 mm
100 mm
40000
75 mm
50 mm
Moderately
100 mm
75 mm
30000
50 mm
30 mm
75 mm
Challenging
50 mm
30 mm
50 mm
20000
30 mm
30 mm
10000
Easy
0
5.0
0.005 3.5
0.0035 2.5
0.0025 1.7
0.0017
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00
0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00
©2018 Waters Corporation COMPANY CONFIDENTIAL 73
SAME Resolution, INCREASED Speed
Constant L/dp
Independent Y- axis Set to same scale
0.15
0.020
0.10
AU
AU
0.010 10 µm – 250 mm 0.05
Rs (2,3) = 1.54
0.000 0.00
0.00 5.00 10.00 15.00 20.00 25.00 30.00
35.00
0.00 5.00 10.00 15.00 20.00
Minutes
25.00 30.00 35.00
0.15
0.06
0.10
5 µm – 150 mm
AU
AU
0.04
0.05
0.02 Rs (2,3) = 2.69
0.00
0.00
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00
9.50
0.00 1.00 2.00 3.00 4.00 5.00
Minutes
6.00 7.00 8.00 9.50
0.08
0.15
0.06
3.5 µm – 100 mm 0.10
AU
AU
0.04
0.02 Rs (2,3) = 2.29 0.05
0.00 0.00
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00
4.50 0.00 0.50 1.00 1.50 2.00 2.50
Minutes
3.00 3.50 4.00 4.50
0.15 0.15
0.10 1.7 µm – 50 mm 0.10
AU
AU
Rs (2,3) = 0.8
AU
0.05
0.00
0.00
0.10
5 µm – 50 mm
0.02
Rs (2,3) = 1.2
AU
AU
0.05
0.00
0.00
0.10
0.05
3.5 µm – 50 mm
AU
AU
Rs (2,3) = 2.0 0.05
0.00 0.00
0.10 0.10
0.05 1.7 µm – 50 mm
AU
AU
0.05
Rs (2,3) = 2.7
0.00 0.00
0.00 0.50 1.00 1.50 2.00 2.50
3.00 0.00 0.50 1.00 1.50 2.00 2.50
3.00
Minutes Minutes
Note increase in sensitivity as particle size is decreased
©2018 Waters Corporation COMPANY CONFIDENTIAL 75
Defining the LC Categories by their System Dispersion
Column i.d.
Resolution Efficiency
0.04 4.6 x 50 mm
AU
3.6 7200
0.02
0.00
0.06
0.04
4800 3.0 x 50 mm
AU
0.02 2.5
0.00
0.06
0.04
2.1 x 50 mm
AU
0.02
1.4 2900
0.00
0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50
Minutes
0.06 Alliance
0.04
Resolution Efficiency 4.6 x 50 mm
AU
3.6 7200
0.02
0.00
0.06
ACQUITY Arc 3.0 x 50 mm
0.04
7600
3.8
AU
0.02
0.00
0.06
ACQUITY UPLC H-Class
0.04
2.1 x 50 mm
7300
AU
0.02
3.7
0.00
0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50
Minutes
0.10
0.00
X
0.20 3.0 x 50 mm
0.15
0.61 mL/min
AU
0.10
0.05
0.00
X
0.14
0.12
0.10
4.8 Times the 4.6 x 50 mm
1.44 mL/min
Peak Volume
AU
0.08
0.06
0.04
0.02
0.00
0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80 1.90 2.00 2.10 2.20
Minutes
0.10
0.05
0.00
X
0.14
0.12 3.0 x 50 mm
0.10 0.61 mL/min
AU
0.08
0.06
0.04
0.02
0.00
0.14
0.12 4.6 x 50 mm
0.10
1.44 mL/min
AU
0.08
0.06
0.04
0.02
0.00
0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80 1.90 2.00
Minute s
0.05
0.00
0.15 3.0 x 50 mm
0.10
0.61 mL/min
AU
0.05
0.00
0.10
0.08 4.6 x 50 mm
1.44 mL/min
AU
0.06
0.04
0.02
0.00
X
0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80 1.90 2.00
Minute s
Integration errors
Reduced resolution
Reduced sensitivity
0 5 10 15 20 25
Minutes
0 5 10 15 20 25
Minutes
TruView LCMS
LCMS Certified
LC/GC Certified
product
The vial chosen should be based on the application of the work and the method
of detection.
Analyte Concentration Method of Detection Recommended Waters Vial
High Low
Density Density
Mobile Phase Factors
Organic strength adjusted to create appropriate elution times
Naphthalene
Note: Similar selectivity due to same
silica particle. Different hydrophobicities.
YMC™ J’Sphere™
ODS-L80 (low)
Acenaphthene
0 10 20 30 40
Minutes High ligand density provides
©2018 Waters Corporation COMPANY CONFIDENTIAL more retention 95
Surface of a Silica Gel
Bonded-Phase Packing Material
Polar analytes are not able to “energetically fit” between ligands
High
– can’t interact with surface or ligands
H3C H3C
Coverage
H3C H3C
CH2 CH2 CH2 CH2 High
H2C H2C H2C H2C
CH2 CH2 CH2 CH2 Ligand
H2C H2C H2C H2C
Endcap Density
H2C CH H2C CH
2
H2C CH Residual silanolH2C CH
2 2 2
0.02
Initial
0.00
0.06
AU
0.04
0.02
After dewetting
0.00
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00
5
1
0.06
2 4 XSelect HSS C18
3
AU
0.04
0.02
Initial
0.00
0.15
Pks 1 - 5
AU
0.10
0.05 After dewetting
0.00
0.00 0.50 1.00 1.50 2.00
Minutes 2.50 3.00 3.50 4.00
Peak i.d.: 1) thiourea 2) 5-Fluorocytosine 3) adenine 4) thymidine-5’-monophosphate 5) thymine
Conditions: 10mM Ammonium Formate pH 3; 0.2mL/min; 30ºC; 2.1 x 50 mm
©2018 Waters Corporation COMPANY CONFIDENTIAL 99
Compatibility: Aqueous Mobile Phase
Larger pore diameters and lower hydrophobic ligand density reduce dewetting
T. Walter, P. Iraneta M. Capparella, Mechanism of retention loss when C8 and C18 HPLC columns are used with highly aqueous mobile phases; J. Chrom. A 1075 (2005), 177
©2018 Waters Corporation COMPANY CONFIDENTIAL 100
Excellent Peak Shape and Retention with Simple MS
Compatible Mobile Phases
The popular void marker Uracil is
well retained and is actually peak 3
Conditions
Compounds:
Column: AtlantisTM dC18 4.6 x 150 mm, 5 µm
Mobile Phase A: H2O
1. Cytosine Note excellent peak
2. 5-Fluorocytosine
Mobile Phase B: ACN shape for strong polar
Mobile Phase C: 100 mM CH3COONH4, pH 5.0 3. Uracil
4. 5-Fluorouracil base Adenine
Flow Rate: 1.0 mL/min
Gradient: Time Profile 5. Guanine at pH 5.0
(min) %A %B %C 6. Thymine 7
0.0 90 0 4 7. Adenine
10
10.0 84 6 1
10
V0 = 1.83 min
5 6
Injection Volume: 10 µL
Temperature: 30 oC
2 3
Detection: UV @ 254 nm
Instrument:: AllianceTM 2695, 2996 PDA
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
Minutes
Grumbach, Diehl
At pH 5.0, the strong polar base
Adenine still exhibits excellent peak shape
©2018 Waters Corporation COMPANY CONFIDENTIAL (reason: fully endcapped) 101
Increased Polar Compound Retention with ACQUITY
UPLC™ HSS T3 Columns
Compounds: Conc (mg/mL) Conditions:
1. Norepinephrine 25 Columns: As Indicated
2. Epinephrine 25 Mobile Phase A: 10mM CH3COONH4, pH 5.0
3. Dopamine 10 Mobile Phase B: ACN
Flow Rate: 0.438 mL/min
4. 3,4- Dihydroxyphenylacetic acid Isocratic Mobile
25 Phase Composition: 2% B
5. Serotonin (5-HT) 30 Injection Volume: 0.7 µL
6.
7.
5-Hydroxy-3-indoleacetic acid
4-Hydroxy-3-methoxyphenylacetic
25 6 Temperature:
Detection: UV @ 280 nm
30oC
Polar
– Dominant retention mechanism is still reversed-phase
o Retention maximized using 100% aqueous mobile phases (enabled by having the wider pore
diameter)
– Retention maximized by using reduced C18 coverage
o Polar analytes can “fit” between C18 ligands and interact with surface silanols and alkyl chains
– Secondary interactions due to residual silanols that are more accessible due to reduced C18 coverage
– Cation-exchange interactions
– Hydrogen bonding interactions
2 .8
amitriptyline/acenaphthene)
Nova-Pak® C18
0 .8
7.3% with 2.80 µmoles/m2
(ln [a]
0 .3
Symmetry® C18
Increasing Hydrophobicity
-0 .2 20% with 3.20 µmoles/m2
1 1.5 2 2.5 3 3.5
2 .8
amitriptyline/acenaphthene)
2 .3
Decreasing Silanol Activity
Waters Spherisorb® ODS2
µBondapak™ C18
1 .8
1 .3
Sph1ODS-Low 14%
Sph2ODS-Med
9% Sph3ODS-High
0 .8
0 .3
Symmetry® C18
Increasing Hydrophobicity
-0 .2
1 1 .5 2 2 .5 3 3.5
2.7
NOTE: Do you see two C18’s
Resolve® C18
amitriptyline/acenaphthen
Symmetry® C8
Hypersil® BDS C8 YMC-Pack™ Pro C18™ Kromasil®Inertsil®
C18
ODS-3
Hypersil® BDS C18 Inertsil® ODS-2
0.2
e)
Symmetry® C18
-0.3
1 1.5 2 2.5 3 3.5 4
(ln [k]
acenaphthene)
©2018 Waters Corporation COMPANY CONFIDENTIAL 108
“Base-Deactivated Packings”
H CH 3 H
OH
H 3C N
+ +H
N
H CH 3 O
HOOC
H O NH 2
N + Cl
- OOC
H
Propranolol
Chlorpheniramine
0 4 8 12 16 20 24 28 32
Minutes
©2018 Waters Corporation COMPANY CONFIDENTIAL 112
Repulsion of Cations with Technique C
O O (CH 2 ) 11 CH 3
O Si Si
O O O
Si
O - Reduced silanol effect
O
O
Si O
Si O +
NH 3 on basic analyte
O O
Si O resulting in better peak
O Si shape
H CH 3
H 3C N
+
H
Repulsion of cationic N + Cl
“base” resulting in
shorter retention times
©2018 Waters Corporation COMPANY CONFIDENTIAL 113
Technique C: Pros and Cons (Better for Bases – Poor for
Acids)
Chlorpheniramine
0 4 8 12 16 20 24 28 32
©2018 Waters Corporation COMPANY CONFIDENTIAL Minutes 114
Making an Embedded Polar Bonded Phase Material: Two-Step
Synthesis
OH
Cl
OH Si NH 2
Amination Step
OH Cl
+ Cl with trifunctional
silane
OH O
OH NH
Si 2
O
O
+ Cl
C (CH 2)n
CH 3
O Acylation
OH
OH
Si N
C (CH 2 )n
CH 3
Step
O O OH H
O
O Si
Note: Residual amine on particle
OH Amide surface due to steric hindrance
NH (incomplete acylation).
2
Propranolol
Supelcosil™ LC-ABZ+Plus
Technique D: Two-Step
Maleic Acid
Waters Spherisorb® ODSB
Technique C
0 2 4 6 8 10 12 14 16 18 20
Minutes
©2018 Waters Corporation COMPANY CONFIDENTIAL 117
Making an Embedded Polar Bonded Phase Material: One-
Step Synthesis
Monofunctional silane
O
O OH H 3C
Si C (CH 2 )7
O Si O N CH 3
O
+ Cl
CH 3 H
Carbamate
Silica Gel Surface group built into
O starting silane
H 3C
C (CH 2 )7
Si O N CH 3
O O
CH H
Si 3
O
O
+ HCl
Note: No residual amines on silica gel surface
©2018 Waters Corporation COMPANY CONFIDENTIAL 118
Embedded Polar Ligand: Possible Mechanism
Reduced retention of bases
Water Layer
Reduced peak tailing
Reason 1
Water layer
increases
dielectric
constant,
reducing ionic
interactions Particle
Reason 2
The carbamate
group may
Both reasons given hydrogen bond with
describe why the Shield RP18
silanol groups,
boning provides excellent peak
shape observed for bases shielding analytes
from interacting
©2018 Waters Corporation COMPANY CONFIDENTIAL with them 119
SymmetryShield™ RP18
Chlorpheniramine
Propranolol
Maleic Acid
Toluamide
0 2 4 6 8 10 12 14
Minutes
Technique D: One-step process (great peak shape for acids and bases)
©2018 Waters Corporation COMPANY CONFIDENTIAL 120
Embedded Polar Ligand versus Linear Alkyl
Ligand on Silica Gel
Amitriptyline
SymmetryShield™ RP18
TF USP = 1.1
Symmetry® C18
TF USP = 1.9
0 10 15 20 25 30
Minutes Note: Reduced retention, 25 –> 15 min.
and improved peak shape for the base.
©2018 Waters Corporation COMPANY CONFIDENTIAL 121
Shield RP18 Bonding
Alternative Selectivity Embedded Polar chains
CH3 CH3 CH3
3. Isorhamnetin
2
CORTECS Shield RP18 1
2.1 x 50 mm
3 Increased Retention
Change in Selectivity
0.00 0.55 1.10 1.65 2.20 2.75 3.30 3.85 4.40 4.95 5.50
Minutes
S
N Symmetry® C18
D In
K T Suprofen = S
F Ketoprofen = K
Naproxen = N
Ib Tolmetin = T
Fenoprofen = F
S In Diclofenac = D
T N Indomethacin = In
K D
Ibuprofen = Ib
F
Ib SymmetryShield™ RP18
0 4 8 12 16 20
©2018 Waters Corporation COMPANY CONFIDENTIAL Minutes Note: No change in mobile phase. 124
Chromatographic Impact of Embedded Polar Ligands
+ -
HA H + A
(Un-Ionized) (Ionized)
+ -
B BH + OH
(Un-Ionized) (Ionized)
Retained Retained
+ -
HA H + A
(Un-Ionized) (Ionized)
0 pH 14
35
Un-ionized Acid
30
- 1 pH unit = 91% un-ionized
25
Capacity Factor (k)
Small Change
20
in pH = Large
change in k
pKa
15
(potential
reproducibility
10 + 1 pH unit = 91% ionized
problems)
5
Ionized Acid
0
0 1 2 3 4 5 6 7 8 9 10 11 12
±1 pH
©2018 Waters Corporation COMPANY CONFIDENTIAL 130
Reversed-Phase Retention Behavior of Acidic Compounds
Relative to Changes ± 2 pH Units from pKa More Robust
Methods
35
25
> ± 2 pH units provides stable
Capacity Factor (k)
10
pKa + 2 pH unit = 99% ionized
5
Ionized Acid
0
0 1 2 3 4 5 6 7 8 9 10 11 12
±2 pH
©2018 Waters Corporation COMPANY CONFIDENTIAL 131
pKa Value
Ionizable Compounds
pKa
– pH at which the molecules of the analyte in solution are 50% ionized (charged) and 50%
are un-ionized
+ 2 Rule
+ -
B BH + OH
(Un-Ionized) (Ionized)
15
pKa
10
5
Ionized Base
0
0 1 2 3 4 5 6 7 8 9 10 11 12
±1 pH
±2
©2018 Waters Corporation COMPANY CONFIDENTIAL 134
pKa of a BASE
0 pH 14
N
N
Pyridine Chlorhexidine
Diphenylamine (Base) (Base) (Base)
pka 0.79
Amitriptyline
pka 5.2 pka 2.2; 10.3 pKa 9.4
©2018 Waters Corporation COMPANY CONFIDENTIAL 135
Ionization
pH 14 % Ionized
pH Molecules
Cation
12.6 Un-ionized
Exchanger
(-) Cation
pKa 10.6 50%
BASE (+)
pH 7 8.6 ~100%
6.7 ~100%
ACID (-)
Anion
Exchanger pKa 4.7 50%
(+) Anion
2.7 Un-ionized
pH 1
©2018 Waters Corporation COMPANY CONFIDENTIAL 136
Factors:
pH Considerations – pH vs. Retention
40 Retention of neutral analyte not affected by pH
35
Neutral Analyte
30
25
pH pH
Retention 20
Factor (k) Increases Increases
15 Retention of Retention of
10
Acidic Analyte Basic Analyte
5 Basic Analyte Acidic Analyte
0
0 2 4 6 8 10 12
pH
Silica pH Range
0 1 2 3
10
9
Acid
8
7
6
5
4
3
2
Base
1
0
0 1 2 3 4 5 6 7 8 9 10 11 12
pH
0 1 2 3
10
Acid A,B
9
N pH 5
8
7
6
5
4 0 1 2 3
3
2
Base
1
0
0 1 2 3 4 5 6 7 8 9 10 11 12
pH
0 1 2 3
10
Acid A,B
9
N pH 5
8
7
6
0 1 2 3
5 B N
4 A
3 pH 10
2
Base
1
0 1 2 3
0 Minutes
0 1 2 3 4 5 6 7 8 9 10 11 12
pH Note: pH is a powerful
selectivity tool
©2018 Waters Corporation COMPANY CONFIDENTIAL 140
Using pH to Create Separations
100
Acid [HA]
Base 2 [B2]
10
Base 1 [B1]
( Log k)
Neutral
1
Acid [A-]
Bases 1+2
[BH1+, BH2+]
0.1
0 2 4 6 pH 8 10 12 14
N
N
B1, B2 A
B1
A B2
pH 2 pH 10
120
100
Aqueous pKa = 4.3 Aqueous pKa = 9.5
Capacity Factor
80
Negative
Charge
60 Positive -
BA
Charge Dual
40
+ Charge
20
B A
(k)
+
0
0 1 2 3 4 5
B A-
6 7 8 9 10 11 12
pH
H Fexofenadine
N (Antihistamine)
OH + CH 3
CH 3
HO COO -
©2018 Waters Corporation COMPANY CONFIDENTIAL 142
Peak Shape Over Wide pH Range
1
2 3 4 5 6 7 8
Buffer pH
Ideal Behavior Pure Polymer – No Silanols
©2018 Waters Corporation COMPANY CONFIDENTIAL 143
The Importance of Mobile Phase pH: Rapid Method
Development
1.70 3
1.60
1.50
1.40
2 4 5
6 pH 2 Using a wide mobile
1.30
1.20
1.10 phase pH range is an
AU
1.00
0.90
0.80
0.70
1 effective approach to
0.60
0.50
0.40
0.30 change compound
0.20
0.10
0.00 selectivity
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00
Minutes
1.70 3 5
1.60 4 Increase selectivity
1.50
1.40
2 6 pH 7
1.30 for:
1.20 1
1.10
1.00
AU
0.90 Acids
0.80
0.70
0.60
(Green
Green//Brown
Brown))
0.50
0.40
0.30
0.20 Bases
0.10
0.00 (Red
Red/
/Yellow
Yellow)
)
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00
1.80 3 2 Minutes
1.60
6 5 pH 12
1.40 4 Neutrals (Peaks 2 & 5)
1.20
AU
Non-Column Influences:
pH
Neutrals: No Influence
Neutral
1
Acid [A-]
Bases 1+2
[BH1+, BH2+]
0.1
0 2 4 6 pH 8 10 12 14
Acid [HA]
Base 2 [B2]
10
Base 1 [B1]
( Log k)
Neutral
1
Acid [A-]
Bases 1+2
[BH1+, BH2+]
0.1
0 2 4 6 pH 8 10 12 14
N N
B2 A, B1
B1 A B2
pH 5.5 pH 6.0
0.30 1
0.20 pH 5.4
0.10 3 2
0.00
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
0.40
0.30 1
0.20
3 pH 5.6
0.10 2
0.00
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
Grumbach, Diehl
Acid [HA]
Base 2 [B2]
10
( Log k)
Base 1 [B1]
Neutral
-2 +2
1 -2 +2
Acid [A-]
Bases 1+2
[BH1+, BH2+]
0.1
0 2 4 6 pH 8 10 12 14
10
Neutral
1
Base + (Ionized) -
Acid (Ionized)
0.1
0 2 4 6 8 10 12 14
pH
Maximum Loading for Prep is when analyte is in the UN-IONIZED Form
ACID at Low pH BASE at High pH
2 .8
1 .8
YM C J' sphere™ O DS–L80 N ucleosil® C18
Hypersil® O DS
required with buffers N ova-Pak® C18
YM C J' sphere™ O DS–H80
0 .8
YM C-Pack™ O DS–AQ ™
Kromasil® C1 8
YM C-Pa ck™ Pro C18™ Inertsil® ODS-3
0 .3 Hypersil® BDS C18 Inertsil® O DS-2
-0 .2
1 1 .5 2 2 .5 3 3 .5
pK c pH c
k HA , 0 e B HA c k A , 0 e B HA c 10 a
k pK c pH c
1 10 a
In general:
– Basic Compounds: pKa will decrease with the addition of an organic solvent
– Acidic Compounds: pKa will increase with the addition of an organic solvent
The specific change in pKa will be compound dependent
+ CH 3 CH 3
NH N
CH 3 CH 3
Ionized Un-ionized
pKa (H20) 9.4
apparentpKa (20% ACN) 8.5
apparentpKa (30% ACN) 8.3
apparentpKa (40% ACN) 8.0
Note: you cannot determine the degree or the direction of the
pKa shift with out experimentally running each organic
concentration
©2018 Waters Corporation COMPANY CONFIDENTIAL 156
Shift of pKa With Organic Solvent Composition: Amitriptyline
(Basic Compound)
40
Compound
35 un-ionized
30
Capacity Factor
Aqueous
25
pKa = 9.4
20
15 40% Acetonitrile
(k)
CH 3 CH 3
COOH COO
-
H 3 CO H 3 CO
Un-ionized Ionized
pKa (H20) 4.2
apparent pKa (20% ACN) 4.6
apparent pKa (30% ACN) 4.8
1
0 2 4 6 pH 8 10 12 14
+
3
O Cl
O CH 3
H 3 C
C C C C
O
Si
O
Si
CH 3
C C C CH 3
+ HCl
O
O
Low pH Mobile Phase
(hydrolysis of ligand)
O OH H3C
C C C C
Si
O Si C C C CH 3
+
O HO
CH 3
“Silane Bleed”
©2018 Waters Corporation COMPANY CONFIDENTIAL 161
Making a Bonded Phase Material:
dC18 Difunctional Synthesis
O OH C8 Dichlorosilane Ligand
O Si
O OH CH3
Si OH Si
O
O
Si + Cl
Cl
CH3
O O
Synthesis
O OH CH3
O Si Si CH3
O O
Si HCl
O O Si
O Two siloxane bonds
+
O O
Difunctional = Attached to silica at two points
Less susceptible to low pH hydrolysis (column bleed)
©2018 Waters Corporation COMPANY CONFIDENTIAL 162
Making a Bonded Phase Material: Multifunctional
Synthesis
O OH
O Si C8 Trichlorosilane “Ligand”
O OH Cl
Si OH Si CH
O
O
Si + Cl
Cl
3
O O
OH
O OH
O Si Si CH 3
O O
HCl
O
Si
O
Si
O
+
O O
Multi--Point Attachment
Multi -
usually not 3
©2018 Waters Corporation COMPANY CONFIDENTIAL 163
Making a Bonded-Phase Material: Trifunctional
Synthesis
Notice: need to break 3 bonds before
OH
Silane bleeds
Risks:
O
O
O Si Si CH Notice creation of
3
O O additional silanols from
Si HO the trifunctional
O Si CH 3 synthesis
O O
Si O Attachment of silanes
O
Si CH may not be attached to
O 3
O silica gel surface
Si O Potential for poor batch-
batch-
O
O to--batch reproducibility
to
Si
O OH
O Si
1 and 2-point attachments to silica gel surface
O O
More hydrolytically stable
©2018 Waters Corporation COMPANY CONFIDENTIAL 164
Peak Shape: Monofunctional Silane versus
Trifunctional Silane
Risk: trifunctional silane can increase silanols
1 2 3
1 2 3
Minutes
HO O
O
OH
O Si Si CH 3 Risks:
O OH
O
Si
O Si CH 3 Notice creation of additional silanols
O O
O
Si O from the trifunctional synthesis
Si CH 3
O
O Attachment of silanes may not be
Si O
O
O
attached to silica gel surface
Si
O
O
Potential for poor batch-to-batch
reproducibility
©2018 Waters Corporation COMPANY CONFIDENTIAL 166
Mobile Phase pH and Column Life-time
220- Elevated
200- Temperature causes
Silica pH 2 - 8 more rapid failure
180-
Polymer pH 2 -12
160-
140-
120-
At pH >8 silica dissolves pH > 8 causes
100- VOIDING
80- for traditional silica/
60- bonded particles
40-
20-
0- 1 2 3 4 5 6 7 8 9 10
pH
©2018 Waters Corporation COMPANY CONFIDENTIAL 167
Effects of High pH Mobile Phases
- Channeling or dissolved
Intermediate pH
silica gel column
1 2 3 4 5 6 7 8 9 10 11 12
Symmetry300 C4 Symmetry300 C4
C8 C18 Silica
1.7 [UPLC], 2.5 XP, 3.5, 5 and 10 µm 1.7 [UPLC], 2.5 XP, 3.5, 5 and 10 µm CSH
1.8 [UPLC], 2.5, 3.5 and 5 µm HSS
©2018 Waters Corporation COMPANY CONFIDENTIAL 178
©2018 Waters Corporation COMPANY CONFIDENTIAL 179
XBridge/BEH Family
Shorter bar
equals longer
lifetime under
low pH
conditions
©2018 Waters Corporation COMPANY CONFIDENTIAL Comparative separations may not be representative of all applications. 183
©2018 Waters Corporation COMPANY CONFIDENTIAL 184
XSelect HSS Family
Lack of surface charge control results in: Surface Charge Control Technology:
– Equilibration issues – Ultra-low level surface modification with
ionizable silanes
– Changes in retention times
– Improved basic analyte peak shape with low
– Poor peak shape for ionized analytes
pH, low ionic strength
High ionic strength mobile phases can mask these issues – MS Compatible – no bleed
C18+
3 2
5
ACQUITY UPLC 6 7
CSH Phenyl-Hexyl 1
4
3 2
5
ACQUITY UPLC 6 7
1
CSH Fluoro-Phenyl 4
1.0
0.0
3.0
Fully porous silica C18 Propiophenone
2.0 2.1 x 50 mm, 1.8 µm (neutral)
AU
Quetiapine
(base)
1.0
0.0
3.0
Competitive Core-Shell C18 Propiophenone
2.0 2.1 x 50 mm, 1.7 µm (neutral)
AU
Quetiapine
1.0 (base)
0.0
0.6 0.8 1.0 1.2 1.4 1.6
©2018 Waters Corporation COMPANY CONFIDENTIAL Minutes Comparative separations may not be representative of all applications
192
CSH Technology:
Influence of Sample Loading on Trace Impurity Detection
0.10 ACQUITY CSH C18 1.7 µm
0.1 % formic acid
0.08
Imipra
2.0 %
mine
0.06
Observations
AU
0.06
impurity analysis
AU
0.04
2.0 %
0.02
1.0 %
0.5 %
0.1 %
0.00
Imipramine concentration held
0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 constant at 0.5 mg/mL;
Minutes
0.1% formic acid mobile phase
©2018 Waters Corporation COMPANY CONFIDENTIAL 193
CSH Technology:
Influence of Sample Loading on Trace Impurity Detection
Imipramine
0.015 ACQUITY CSH C18
A 0.1%
impurity 0.1% formic acid
AU
0.000
0.000
Imipramine
0.015
0.1% Superficially porous C18
C
AU
Imipramine
8x108
ACQUITY UPLC® CSH C18
Total Ion
0.1% impurity 0.1% formic acid
Chromatogram
(m/z 278.3)
Intensity
4x108
0
0.60 1.20 1.80 2.40 3.00
Minutes
Imipramine
Total Ion
Chromatogram 0.05% TFA
UV Trace
Intensity
4x108
0.1% impurity
0
0.60 1.20 1.80 2.40 3.00
Minutes
‘pH switching’ effects only occur when using acidic, low ionic strength
additives
CSH Columns
Use additives AND buffers
Impurity Profile work
Isolation/purification
Prefer to work at low pH with occasional high pH work
Switch back/forth between low & high pH (additives)
LC/MS laboratory
Seeking additional ACQUITY UPLC column selectivities
Designed for Selectivity
Mobile phase and Particle and ligand Superior peak shape for
temperature versatility selectivity basic analytes
0.10 3
0.00
1
2
0.20
3 CSH™ C18
AU
0.00
1
0.20
2 HSS T3
AU
0.10 3
0.00
1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
Minutes
Relative Values
Column Length Particle Size L/dp N Run Time
(L, mm) (dp, mm)
Particle
FIB SEM Images Solid-
Core
Core
©2018 Waters Corporation COMPANY CONFIDENTIAL 209
Laboratory Impact
N α 1 k Resolution equation:
Rs
4 α k1 Increase in N provides greater Rs
The lower backpressure allows for faster analysis and/or use longer columns.
– Run at higher flow rates, run more samples/day
– Use longer columns that provide increased resolution
Pressure
– As the interstitial porosity is reduced, pressure
increases by the difference cubed
(psi)
Fully porous columns tend to have higher ee
<0.37, higher pressure
CORTECS solid-core columns tend to have
ee 0.39, lower pressure External or Interstitial Porosity, ee
Flow Rate (F) = 0.5 mL/min
©2018 Waters Corporation COMPANY CONFIDENTIAL A. Daneyko, Anal. Chem. 2011, 83, 3903–3910 211
Backpressure Comparisons
Columns: 2.1 x 50 mm
7000
CORTECS 2.7 µm particles:
6000 – Larger particle than fully porous 2.5 µm
o Generate lower backpressure
Backpressure (psi)
5000
– Packed less densely
4000
o Even lower backpressures1
3000
Conditions: 20% Acetonitrile in water; 600 µL/min; 40 oC 1A. Daneyko, Anal. Chem. 2011, 83, 3903–3910
©2018 Waters Corporation COMPANY CONFIDENTIAL 212
Understanding Mass Transfer [Diffusion]:
Influence of Particle Size
Diffusion distance is short because the analyte band can only diffuse
into the porous layer of material
16,000
14,150
or up to 3x faster!
12,000
4,000
0.00 0.25 0.50 0.75 1.00 1.25
Flow Rate (mL/min)
2.1 x 50 mm column. A standard ACQUITY UPLC I-Class using 70% Acetonitrile in H2O at 30 °C
©2018 Waters Corporation COMPANY CONFIDENTIAL with 0.5 µL injections from a 1 µL FL injector 215
Instrument (System) Dispersion
What is it & Where is it
Instrument dispersion is the broadening of the
analytical band due to the instruments flow path
volume
– It’s part of all LC systems, and varies significantly
depending on the configuration
Particle
Particle
ρ = 1 → nonporous particle Solid- Solid-
Core Core
Core Core
r = core diameter / particle diameter
Attribute CORTECS
r 0.7
Method Transfer
– Scaled synthetic process
o Rho value scaled
– UPLC HPLC
o Seamless method transfer
o Future proofing
2 HPLC
0.40
3 4 HPLC and UHPLC
1.51 mL/min
0.00
0.0 5.0 10.0 15.0 20.0 25.0
0.80 CORTECS C18, 2.7 µm
4.6 x 150 mm
AU
0.40
UHPLC
0.00
1.51 mL/min
0.0 5.0 10.0 15.0 20.0 25.0
0.80 CORTECS C18, 2.7 µm Scalable From
3.0 x 75 mm UHPLC to UPLC
AU
0.40 UPLC
0.53 mL/min 1. Epigallocatechin
0.00 2. Catechin
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 3. Epicatechin
Minutes
4.50 5.00 5.50
4. Gallocatechin
©2018 Waters Corporation COMPANY CONFIDENTIAL 221
Porous Particle Equivalence
2.22 µm
2.7 µm 3.1 µm
0.04
Rs 8.2 Psi: 3200
0.02 N peak 4: 23,600
0.00
1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50
Minutes
COMPANY CONFIDENTIAL
Note: 2.5 µm fully porous Psi ~6000 223
©2018 Waters Corporation
High Resolution Separation Omeprazole Acid Degradation
Analysis
Alliance HPLC with PDA CORTECS C18+, 2.7 µm
1.2 mL/min 4.6 x 150 mm
Psi 2530 4
0.24 1. Unknown Degradent (298.14)
3 2. Omeprazole (346.40)
3. 5-methoxy-2-thiol (181.23)
0.20 4. Omeprazole Sulphide (330.42)
2
1 5. Omeprazole Desmethoxy (316.39)
0.16 6. Related Compound F/G (312.16)
7. Related Compound F/G (312.16)
8. Omeprazole-n-oxide/ Sulphone (362.42)
AU
3
6 Atlantis T3 5 µm
0.05
5 4.6 x 150 mm
0.00
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00
Minutes
1
0.15
4 CORTECS T3 2.7 µm
2
3.0 x 75 mm
0.10
AU
Increase resolution
3 6 amongst 3 unknown
0.05
5 peaks 50% Faster
5x Solvent Savings
0.00
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00
Minutes
0.05
0.00
1
5
0.20 2
4 CORTECS UPLC HILIC
0.15 3 2.1 x 50 mm 1.6 µm
0.10
AU
0.00
-0.05
0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00
Minutes
3.3
3 ACQUITY UPLC HSS C18 SB
amitriptyline/acenaphthene)
CORTECS T3
CORTECS C18+
CORTECS RP18
Kinetex C18
0 5 10 15 20 25 30
% Loss in Methyl Paraben Retention Factor using Accelerated Low pH
Stability Challenge Conditions (0.5% Aqueous TFA, 60 °C)
Accelerated low
COMPANY
©2018 Waters Corporation pH stability testing conditions: 0.5% aqueous TFA, 60 °C
CONFIDENTIAL 229
Method Transfer:
HPLC UHPLC UPLC
0.02 Fully Porous C18, 5 µm
2
1 4.6 x 150 mm
Initial Method 3
AU
0.01 1.00 mL/min
Alliance® HPLC 4 5
0.00
12.0 14.0 16.0 18.0 20.0 22.0 24.0 26.0 28.0 30.0
3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0
0.01
3.5 4.0 4.5 5.0 5.5 6.0 6.0 7.0 7.5 8.0
0.02
Transfer to 1.6 µm CORTECS 1.6 µm C18
2.1 x 50 mm
9X Faster method
AU
0.01
0.65 mL/min
15X Less solvent 0.00
H-Class
1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6
Minutes
©2018 Waters Corporation COMPANY CONFIDENTIAL 230
Monitoring the Process Over Time
Process Control Charting
Carbon
%
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 55 57 59 61 63 65 67 69 71 73
Batch Number
Carbon content ACQUITY UPLC BEH C18 sorbent
– Recent batches with control limits plotted for one of the 337 essential response factors
– 43 quality control tests
©2018 Waters Corporation COMPANY CONFIDENTIAL 231
Plates
5000
0
10000
15000
20000
25000
COMPANY CONFIDENTIAL
1013531716008
1013531716009
1013531716010
1013531716011
1013531716012
1013531716013
1013531716014
Serial#
1013531716015
n=27
1013531716016
1013531716017
Individuals Chart
1013531716018
1013531716019
1013531716020
Cortecs Phenyl 1.6um 2.1x50mm
1013531716021
1013531716022
Process Control Chart Example – column efficiency
1013531716023
1013531716024
1013531716025
Monitor: efficiency, USP tailing factor, back pressure, retention
1013531716026
1013531716027
LCL
UCL
Mean
Plates
232
Column Packing Consistency
4.00
3.50 2.1 x 50 mm
3.00 3.0 x 50 mm
4.6 x 50 mm
2.50
2.00
1.50
1.00
0.0 5.0 10.0 15.0 20.0 25.0 30.0
Linear Velocity (cm/min)
AU
0.04 1 5 Batch A 0.04
0.00 0.00
0.08 0.08
Batch B
AU
AU
0.04 0.04
Batch B
0.00 0.00
0.08 0.08
Batch C
AU
AU
0.04 0.04
Batch C
0.00 0.00
0.08 0.08
Batch D
AU
AU
0.04 0.04
Batch D
0.00 0.00
0 Minutes 12 0 Minutes 12
1) uracil, 2) promethazine, 3) amitriptyline, 4) butylparaben, 5) naphthalene
ACQUITY UPLC 2.1x50 mm, 30˚C
0.25 mL/min 35:65 acetonitrile/15.4mM ammonium formate, pH 3
CORTECS
BEH Technology CSH Technology HSS Technology Solid-Core
Solid-
FULLY POROUS FULLY POROUS FULLY POROUS Technology
SOLID-CORE
Unparalleled pH, Controlled Surface Mechanical Stability of
mobile phase and Charge Pure Silica Particle High efficiency and
temperature versatility resolution, high
Unparalleled peak Increased retention speed optimized for
symmetry for bases in backpressure
formic acid
CORTECS
HSS Technology (XSelect
(XSelect))
Solid-Core Technology
Solid-
Mechanical Stability of Pure Silica Particle High efficiency and resolution
Increased retention High speed optimized for backpressure
lower ligand density
C18
+
+ +
C18+ ++ +
+
+
non-endcapped C8
Phenyl
lower ligand density
T3
Shield RP18
©2018 Waters Corporation COMPANY CONFIDENTIAL 238
Summary
CORTECS Columns are fully scalable between particle sizes, and can be used
with any UPLC, UHPLC, and HPLC system in your laboratory.
Ultimate Efficiency = CORTECS 1.6 µm Columns
Ultimate Utility = CORTECS 2.7 µm Columns
C8
Phenyl
T3
Shield RP18
1
2 3 4 5 6 7 8
Buffer pH
Ideal Behavior Pure Polymer – No Silanols
©2018 Waters Corporation COMPANY CONFIDENTIAL 248
Reversed Phase Column Chemistries
Hybrid Silica
FULLY POROUS SOLID-CORE
BEH Technology CSH Technology HSS Technology CORTECS
(XBridge) (XSelect) (XSelect) Solid--Core Technology
? Solid
C18
+
+
+
+++
C18+
?
++
? C8
Phenyl
T3 ?
Shield RP18 ?
1
2 3 4 5 6 7 8
Buffer pH
Ideal Behavior Pure Polymer – No Silanols
©2018 Waters Corporation COMPANY CONFIDENTIAL 252
Impact On Retention Factor (k)
By Changing Mobile Phase pH -- HPLC
Note: Column Particle,Temperature
and % Organic Held Constant
In REVERSED-PHASE Mode
100 pH Limit of Silica gel
Acid(Un-Ionized)
Base (Un-Ionized)
10
log (k')
Neutral
1
Base
(Ionized) Acid
(Ionized)
0.1
0 2 4 6 8 10 12 14
pH
Range of poor reproducibility due to steep slopes - small pH change -- large retention change
C8
Phenyl
T3
Shield RP18
1
2 3 4 5 6 7 8
Buffer pH
Ideal Behavior Pure Polymer – No Silanols
©2018 Waters Corporation COMPANY CONFIDENTIAL 257
Reversed Phase Column Chemistries
Hybrid Silica
FULLY POROUS SOLID-CORE
BEH Technology CSH Technology HSS Technology CORTECS
(XBridge) (XSelect) (XSelect) Solid--Core Technology
Solid
C18
+
+
+
+++
C18+ ++
C8
Phenyl
T3
Shield RP18
Neutral
1
Base
(Ionized) Acid
(Ionized)
0.1
0 2 4 6 pH 8 10 12 14
Greatly improved reproducibility due to flattening of curves—Robustness
Note: Column Particle, Temperature and % Organic Held Constant
©2018 Waters Corporation COMPANY CONFIDENTIAL 260
Reversed Phase Column Chemistries
Hybrid Silica
FULLY POROUS SOLID-CORE
BEH Technology CSH Technology HSS Technology CORTECS
(XBridge) (XSelect) (XSelect) Solid--Core Technology
Solid
? C18
?
+
+
+
+++
C18+ ++
C8
Phenyl
T3
Shield RP18
Mobile Phase: (Note- Prepare the Mobile phase fresh daily.) Mix 400 ml
of acetonitrile, 400 ml of methanol, and 280 ml of water. Add 2 ml of
ammonium hydroxide and mix.
Column: A 4 mm x 10-cm pre-column that contains packing L4 (silica
gel), installed between the pump and the injector (replaced daily) and a
5-mm x 25-cm analytical column that contains packing L1.
Why is it there???
It is constantly dissolving (voiding) creating a saturated MP that reduces the amount of dissolution
of the C18 Column silica (extending its lifetime).
It is BEFORE the Injector to keep voiding from causing poor mechanical peak shapes for the
sample (Poor Plate Count)
1
2 3 4 5 6 7 8
Buffer pH
Ideal Behavior Pure Polymer – No Silanols
©2018 Waters Corporation COMPANY CONFIDENTIAL 266
Impact on Retention Factor (k) on Robustness By
Changing Mobile Phase pH Range - HPLC
In Pure REVERSED-PHASE Mode
100
pH Limit of Silica gel
Acid Base
(Un-Ionized)
10 (Un-Ionized)
log (k')
Neutral
1
Base
(Ionized) Acid
(Ionized)
0.1
0 2 4 6 pH 8 10 12 14
Greatly improved reproducibility due to flattening of curves—Robustness
Note: Column Particle, Temperature and % Organic Held Constant
©2018 Waters Corporation COMPANY CONFIDENTIAL 267
Reversed Phase Column Chemistries
Hybrid Silica
FULLY POROUS SOLID-CORE
BEH Technology CSH Technology HSS Technology CORTECS
(XBridge) (XSelect) (XSelect) Solid--Core Technology
Solid
C18
+
+
+
+++
C18+ ++
C8
Phenyl
T3
Shield RP18
C8
Phenyl
T3
Shield RP18
Thank you