RP LC Column Selection-Ricardo Martinez

RP LC Column Selection
Peru
2018
Ricardo Martínez
©2018 Waters Corporation COMPANY CONFIDENTIAL 1

Agenda
 LC Introduction
 Columns quality
 Particles & Ligands
 pH in LC
 New Tech Columns
 Solid-Cord Particles
 Conclusions

Caffeine
 Buffer- Transfer 6.8 g of monobasic potassium phosphate to 1L volumetric flask.

Dissolve the contents in 900 ml of water. Adjust with phosphoric acid to pH of 2.0. Dilute
to water to volume, add 0.2 ml of triethylamine, and mix well.
 Mobile phase- Prepare a mixture of Buffer an acetonitrile(84:16) and degas. Make
adjustements if necessary.
 Column 4.0 x 250 mm, 10 um, L1. Tailing Factor NMT 2.0

Common Practices in Selecting a Column
 Select the column that has worked before?
 Select a column from a “similar” application?
 Select a C18 Column?
 Try a Google search and hope?

Not all C18’s are the same!
 “There is a wide variety of reversed-phase liquid chromatography

columns available on the market today and this is certainly one of
the important reasons for the great popularity of this mode of
chromatographic separations.”1
1Kele and Guiochon, J. Chromatogr. A 830, 41 (1999)

Mikhail Tswett:
Inventor of Chromatography
 Russian Botanist
– May 14th 1872 – June 26th 1919
 Invented “liquid-adsorption column

chromatography” in 1903 as a technique to
separate chlorophylls and carotenoids from
plants

Tswett's Experiment (1903)
 Tall glass open column filled with sand-

like particles (alumina, or chalk)
 Ground-up plant extract
 Poured into the column and saw colored
“bands” develop as the extract
percolated down thru the column Different
Colored
 Different compounds had separated “Bands”
Greek
Chroma -- color
Graphy -- writing/study of
Note: Tswett in Russian means Color
COMPANY CONFIDENTIAL
8 8
©2018 Waters Corporation
Chromatography
Applications
pharmaceutical proteomics clinical food safety environmental

Chromatogram Characteristics
 Resolution
 Sensitivity
 Precision
 Time Analysis
 Method Simplicity
 Cost

Why is chromatography used?
Separating mixtures
What is it? (Identification)
How much is there? (Quantification)

HPLC System for Purification

Identification and Quantitation
Compound Identification Based on Retention Time

Acrylamide at 2.85 minutes
Data from PDA Detector
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
250.0 300.0 350.0

nm
m/z = 316.1
LC/MS/MS Instrument

The power of mass detection…. Identify co-elutions
Detect non-chromophoric
analytes

Identification and Quantitation
10X Area Count
Sample “A” has 10X the concentration of Acrylamide

Normal vs Reversed-Phase Chromatography
Normal Phase Reversed-Phase
Stationary Phase
Un-bonded Non-Polar Ligand (C18)

Silica (Polar) Surface Bonded to Silica Surface
Packing Polarity Polar Non-Polar
Mobile Phase Polarity Non-Polar Polar
Elution Order Most Non-Polar First Most Polar First
Effect of Increasing Mobile Phase Reduces Decreases
Polarity Retention Time Retention Time

Waters Column Product History
ACQUITY UPLC® BEH Amide
ACQUITY UPLC® BEH Glycan
XBridge Amide
XSelect HSS HPLC Columns ACQUITY UPLC® HSS
µBondapak™ ACQUITY UPLC® BEH Cyano & PFP columns
SunFire™ Columns ACQUITY UPLC®
Styragel® HSS C18 and HSS C18 SB
Spherisorb® SymmetryShield® XSelectTM HSS Cyano &
XTerra®
DeltaPak® PFP columns
PrepPak® Symmetry® XTerraPrep® XBridge™
1958
XP 2.5 µm Columns
1984 1992 1998 2002 2006 2008 2010 2012 2013
1964 1973 1976 1979 1986 1994 1999 2003 2004 2005 2007 2009 2011
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
Quality Systems
Minimize Risk with Dependable Column Performance
 Method Validation kits provide three

batches of chromatographic media
[derived from different base particles] to
judge the quality, reliability and
consistency of an analytical method
 Waters uniquely positioned as an

industry partner to minimize risk

Ligand Density Range Versus Resolution
0
5.37
X
4.70 0.81
Y
4.14 2.86
A

Quality Control Reference Materials
 Requirements for a QCRM Material
– Reproducible Lot to Lot
– Accurate
– Appropriate to analysis type (LCMS, LC-UV)
 The quality of the reference material is critical to the evaluation of analytical data
and instrument performance.
 Waters has an extensive portfolio of QCRMs designed to allow scientists to
determine if their system is in working order
Acenaphthene
Neutrals QCRM: 186006360
Naphthalene
0.050
0.040
Compound Type
Acetone
0.030
AU
Acetone (V0) Void Marker 0.020
Naphthalene Neutral 0.010
Acenaphthene Neutral 0.000
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

Min utes

Using a Reference Standard to Assess System Health
 A reference standard is any standard that is used independently of an assay method

to assess system health.
– Can be made by the analyst or purchased from an outside vendor
 There are three ways to use reference standards:

– Benchmarking
o Use standard to Benchmark performance when the system is in a “known good state”.
Known good states include:
• After installation, or performance maintenance and testing by a service engineer
– Qualification
o Use the standard to do routine Qualification checks to confirm continued good system
performance
o A failed Qualification check would trigger troubleshooting
– Troubleshooting
o Use standard to Troubleshoot system problems and check if repairs made have fixed the
problem
Before Starting Method Development...
Is My System Ready???
 Confirm Proper Instrument & Column Operation First!

– Run Built-in Instrument Diagnostics
o E.g. Pump Leak Test
– Run Well Behaved Standards & Compare to Reference Chromatograms
o Waters Quality Control Reference Materials (QCRMs) are ideal
Neutrals QCRM  QCRMs Can Help Monitor:
Acenaphthene
– Leaks
Naphthalene
0.020 Failed B Pump – Worn fitting

Acetone
AU
0.010
Check Valve
– Older column
0.000
0.06
– Carryover
0.04 Good B Pump – Dirty flow cell
AU
0.02 Check Valve – Mobile phase error

0.00
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 – Air bubbles
Minutes
– & more!
•
Trend Plots
USP Plate Count USP Tailing

1.40
150000
USP Plate Count
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

Neutrals QCRM used for Troubleshooting

What is a Silanol Group?
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

Silica Gel Pore Structure
Si--OH = Silanol
Si

Making a Bonded Phase Material: Monofunctional Synthesis
H3C C8 Silane “Ligand”

O OH C C C C
Si Si C C C CH
O 3
Cl
O + CH 3
Silica Gel Surface H3C

C C C C
Si C C C CH 3
O O
Si CH
O 3
O Siloxane Bond + HCl
C18 Bonded Silica Gel Pore
CH 3
H3C CCCCCCCCCCCCCCCCCC
Si
O
25 Å
“Steric Hindrance”
Physical hindrance due

to SIZE of reacting
Silane, limiting access to
un-reacted silanol groups

C18 Bonded and
“Fully Endcapped” Silica Gel Pore
Endcapping:a second bonding using a very small
(short) Silane (C1) to bond some of the silanols
which the Primary ligand (C18) could not reach
CH 3
H 3 C CCCCCCCCCCCCCCCCCC
Si
O
25 Å
“Steric Hindrance”
CH 3
H3C
Si CH 3
O
Endcap (2nd Bonding Step)
What do you still see? Silanols!

C18 Bonded and “Fully End-Capped”
High Purity Silica Gel Pore
C18  ~ 50% of surface
silanols remain due to
steric hindrance
End-Cap
Note: Difficulty bonding silanols in

micro-pores
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.
This is what causes tailing and excessive retention of bases.

Why Do You See Poor Peak Shape?
Answer: Ionization of Silanols
 Surface silanol charge changes with mobile phase pH
OH O
Si Si H+
Behaves as a Cation Exchanger
(pH 2) (pH 7)
Result: Strong interaction (cation exchange) between ionized surface

silanols (negative charge) and ionized basic analytes (positive charge)
Causes tailing and longer retention times.
©2018 Waters Corporation COMPANY CONFIDENTIAL Silanols are weak acids 34
Unbonded Silica Gel Particle:
Pore Surface - Silanol Groups
Si – OH
Si-OH  Si-O- + H+
Silanol Groups
(pH 2) (pH 7)
No charge Negatively charged
 Strong interaction between ionized

surface silanols (Si-O-) and basic
analytes (+)
 Creates potential for cation

exchange retention mechanism in
the column
At low mobile phase pH

Mixed Mode Retention on LC Column
Reversed-Phase Interaction with Ligands
No Ion Exchange
O- Si
OH
OH
OH
O- Si
Mobile Phase OH +
HN (CH 3 )
pH < 3 OH 2
Si - OH OH
O- Si
Substrate Protonated OH
(No Charge) O- Si
OH
OH
O- Si
Base

Mixed Mode Retention on LC Column
Reversed-Phase Interaction with Ligands Weak Cation Exchange & Reversed-Phase
No Ion Exchange High Silanol Activity
O- Si O- Si
OH O-
OH O-
OH O-
O- Si O- Si
Mobile Phase OH +
HN (CH 3 ) Mobile Phase
O- (CH ) HN
+
pH < 3 OH 2
pH > 5
OH O- 32
Si - OH O-
O- Si Si – O- O- Si
Substrate Protonated OH Substrate O-
(No Charge) O- Si O- Si
OH De-protonated
OH (Negative Charge) O-
O-
O- Si O- Si
Where base peak should be
N in pure reversed-phase
separation
Base has
Base increased
Base retention and
RP Cation X poor peak
shape
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

Different Silica Gel Manufacturing Processes
 Precipitation of sodium silicate: (irregular

particles)-µBondapak™
– Sodium silicate polymerized to silicic acid (silica gel) then
ground to fine particles
 “SolGel”1 Process: (spherical particles)-

Nova-Pak®
– Colloidal silica spheres fused into chromatographic particle
 “SilGel”1 Process: (spherical particles)-

Symmetry®
– Pure silanes polymerized to form chromatographic particle

Particle Technologies
 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
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

Acidic Compounds:Two Different C18’s (Different
Brands – Same Ligand, cont.)
Ketoprofen and Tolmetin Note: Different

selectivity due to
different silica
Suprofen particle.
Naproxen
Col Brand A ODS
Suprofen Ketoprofen Naproxen Tolmetin

Col Brand B ODS
4 5 6 7 8 9 10
©2018 Waters Corporation COMPANY CONFIDENTIAL
Minutes 42
Aluminum in the Silica Gel Lattice
3D top view of silica particle

Bronsted surface with silanols pointing
upward
Acid
O O
O O
Si
Si Si Si
HO O Si
HO OH
Al
O O
Metal available for chelation

Correlation Between Base Tailing and Aluminum Content of Silica
Gel
Analyte: Chlorpheniramine
Mobile Phase: Acetonitrile/KH2PO4 pH 3.0 (20:80)
4 "High Purity Silica Gel"

Tailing 3
Region
Factor
2
1
0 100 200 300 400
Aluminum Content, ppm
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
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
0 50 Minutes 100 150

Test Compounds for the Measure of Residual Silanol
Activity
+
N
Amitriptyline Acenaphthene
pKa = 9.4 Neutral Compound
Basic Compound

Chromatographic Test Conditions
 Methanol /20 mM KH2PO4-K2HPO4 pH 7.0 (65/35)
 Temperature controlled 23 °C
 (Equilibration time controlled)
 New Columns

Impact of Choosing Two Different C18 Brands
on Peak Shape for a Base
Note: Different “brands”
(different silica particle) have
different silanol behaviors.
Waters SunFire™ C18
Amitriptyline Waters Spherisorb® ODS2
0 10 20 30 40 50 60 70 80 90
Minutes
Note: pH plays a role in peak shape

Pore Size
 Most silica gel packings are porous

– >99% of the surface area is contained within the particle (not on the surface)-
”Where the chromatography happens.”
 Rules of Thumb
 “The smaller the pore size, the greater the surface area.”
o (100 Å approx. 300 m2/gram)
o (300 Å approx. 100 m2/gram)
– “The greater the surface area, the greater the retention.”

 A typical 15 cm column holds a surface area of ~100-300 square meters

Silica Gel Pore Structure
Analyte MW Pore Size Recommendation
< 3,000 60 -130 Å

(6 -13 nm)
3,000 – 10,000 125-200 Å

(12.5-20 nm)
>10,000 300 – 1,000 Å (30 -100 nm)
Analyte molecule
Very Large Non-porous
Analyte needs to “fit” inside a pore to interact

with chromatographic surface
Si-OH = Silanol
Note: Pore Size is a distribution

Reversed-Phase Column Selectivity Chart
3.6 Waters Spherisorb S5 P
3.3
amitriptyline/acenaphthene)
3 ACQUITY UPLC BEH Phenyl

ACQUITY UPLC HSS C18 SB
XSelect HSS C18 SB
2.7 Waters Spherisorb S5CN XBridge Phenyl ACQUITY UPLC HSS PFP
Nova-Pak CN HP Inertsil Ph-3 XSelect HSS PFP Waters Spherisorb ODS1
Selectivity
2.4 Hypersil Phenyl

Resolve C18
ACQUITY UPLC BEH C18
2.1 ACQUITY UPLC CSH Fluoro-Phenyl
XSelect CSH Fluoro-Phenyl XBridgeWaters
C18 Spherisorb ODS2
ln [α]
1.8 ACQUITY UPLC HSS CN µBondapak C18 ACQUITY UPLC HSS T3

XSelect HSS CN YMC-Pack YMC J'sphere
XSelect
1.5 Phenyl ODS–L80 Nucleosil C18 HSS T3
Hypersil CPS Cyano Inertsil CN-3 Nova-Pak Phenyl CORTECS C18+
YMC J'sphere ODS–M80
1.2 Hypersil BDS Phenyl
Chromolith Nova-Pak YMC J'sphere ODS–H80
YMCbasic XTerra YMC-Pack™ ODS–AQ
RP-18 C18
0.9 YMC-Pack CN YMC-Pack Pro C4 PhenylNova-Pak Luna Atlantis dC18 Zorbax XDB C18
YMC-Pack Pro C8 C8 Phenyl Hexyl Atlantis T3
0.6 ACQUITY UPLC BEH C8 ®
XTerra MS C8 ACT Ace C18 Symmetry C8 YMC-Pack ODS-A
XBridge C8 Luna Luna C18 (2)
YMC-Pack Inertsil ODS-3
C8 (2)
0.3 ACQUITY UPLC CSH Phenyl-Hexyl SunFire C8 XTerra Pro C18
MS C18
SunFire ™ C18
Symmetry C18
XSelect CSH Phenyl-Hexyl SymmetryShield RP8
0 XTerra RP18
Zorbax SB C18
ACQUITY UPLC BEH Shield RP18 SymmetryShield RP18
-0.3 XBridge Shield RP18 XTerra RP8ACQUITY UPLC CSH ACQUITY UPLC HSS C18
XSelect CSH C18 XSelect HSS C18
-0.6 YMC-Pack PolymerC18
-1.5 -0.5 0.5 1.5 2.5 3.5
Hydrophobicity CORTECS C18
©2018 Waters Corporation COMPANY CONFIDENTIAL (ln [k] acenaphthene) 52
“Relative” Ranking of C18 Columns
Using a Standardized Test
– There are no bad C18 columns.
– There are only different C18 columns.

Creating a New Particle
Advantages Disadvantages
• Mechanically strong • Limited pH range
Inorganic • High efficiency • Tailing peaks for bases
(Silicon) • Predictable retention • Chemically unstable
• Wide pH range • Mechanically ‘soft’

Polymer • No ionic interactions • Low efficiency
(Carbon) • Chemically stable • Unpredictable retention
Hybrid (Silicon-Carbon) Particle Technology

Polymer versus Silica Gel Particle
Propranolol
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
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
MethylPolyethoxysilane Tetraethoxysilane Methyltriethoxysilane

(MPEOS) (TEOS) (MTEOS)
Ethylene Bridged Hybrid [BEH] Particle
U.S. Patent No. 6,686,035 B2 Bridged Ethanes within a silica matrix
Anal. Chem. 2003, 75, 6781-6788

The Resolution Equation
N α 1 k
Rs 
4 α k1
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

Column Length and Mechanical Separating Power*
Columns contain the same packing material, same particle size and same mobile phase,
only one is twice as long
Additional column length does provide a better separation.

However, several “costs” are incurred: more time (2X) for the analysis, use more solvent, increased back
pressure and the longer column costs more to buy.
A better approach, would be to try a different particle chemistry/mobile phase combination or a smaller particle size
that can create the separation in less time.
* This is also called “Efficiency”

Particle Size and Mechanical
Separating Power*
Columns contain the same packing material chemistry, are the same length with the
same mobile phase. One column has particles which are a third the size.
Smaller particle sizes provide for a better separation with the same
run time. However, back pressure will increase.
* This is also called “Efficiency”
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)
Efficiency vs. Flow Rate
Acenaphthene, 2.1 x 50 mm columns
70/30 MeCN/H2O, 30 °C, 254 nm
1.7 µm ACQUITY UPLC® BEH C18

16000
Plate Count (4 sigma)
2.5 µm XBridge™ C18

14000 2.6 µm Superficially Porous C18
12000
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)
1.7 µm ACQUITY UPLC® BEH C18

2.5 µm XBridge™ C18
20
2.6 µm Superficially Porous C18
15
10
0
0.0 0.5 1.0 1.5 2.0 2.5 3.0
Linear Velocity, u (cm/sec)
UPLC® Technology & The Fundamental Resolution
Equation
Rs 
N
4
( α 1
α
)( k
k 1
)
Physical Chemical
•In UPLC® systems, increasing N (efficiency) is the primary focus

•Selectivity and retentivity are the same as in HPLC
•Resolution, Rs, is proportional to the square root of N
Rs  N
If N ↑ 3x, Rs ↑ 1.7x
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
0.040 4.8 µm, 0.2 mL/min, 354 psi Theory

0.030
1.7X Resolution
AU
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
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
1.0 2.0 3.0

Flow Rate
{mL/min}
Optimal flow rate is inversely proportional to dp

1
F opt 
dp
Isocratic analysis time is inversely proportional to F
dp ↓ 3X, N ↑ 3X, Rs ↑ 1.7X, T ↓ 3X

(e.g., 5 µm to 1.7 µm)
(Rs α √N) 67
Speed Increases
Constant L/dp
Efficiency, N, is directly proportional to column length, L, and inversely
proportional to particle size, dp:
L
N 
dp
For same N and, therefore, same Rs
dp ↓ 3X, L ↓ 3X, N = 1X, Rs = 1X,

(e.g., 5 µm to 1.7 µm) (e.g., 150 mm to 50 mm)
F ↑ 3X, T ↓ 9X Efficiency & Resolution

(i.e., F increases 3X,
L decreases 3X) Remain Unchanged 68
Sensitivity Increases
Constant L/dp
Assuming same efficiency, Peak Height is inversely
proportional to column length, L:
1
Height 
L
For same efficiency, column length, L is decreased
proportionally to particle size, dp (constant L/dp)
dp ↓ 3X, L ↓ 3X, N = 1X, Rs = 1X,

(e.g., 5 µm to 1.7 µm)(e.g., 150 mm to 50 mm)
Efficiency & Resolution

T ↓ 9X,
(Increased optimal flow
Sensitivity ↑ 3X Remain Unchanged
rate & shorter column) (Peak height increases as peak width
©2018 Waters Corporation COMPANY CONFIDENTIAL and column length decreases) 69
Length Proportional to Particle Size
Similar L/dp
0.06
1.7 µm, 30 mm, 0.6 mL/min Reality
0.04 Same Resolution
8X Faster
AU
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
2.1 mm ID columns Backpressure

Increase
Ratio of Column Length-to-Particle Size:
Maintaining Separation Power
Typical HPLC Column 4.6 x 150 mm, 5 µm
Column length (L) = 150 mm = 150,000 µm

dp = 5 µm
L = 150,000 = 30,000
dp 5
Separation Efficiency
Application Example L/dp
Index (N)
Easy Content Uniformity 5,000 15,000
Moderately
Related compound assay 12,000 30,000
Challenging
Difficult Impurity profiling 20,000 50,000
Extremely
Metabolite identification 35,000 85,000
Difficult

Ratio of Column Length to Particle Size
Resolution Capability
•50 mm, 1.7 µm
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
particle size (µm)

Proper Method Transfer
ACQUITY CSH Phenyl-Hexyl 4
50 mm, 1.7 µm 6
2
1
UPLC
α3,5= 1.07 5
3
0.00 1.00 2.00 3.00 4.00 5.00
XSelect CSH Phenyl-Hexyl 4

100 mm, 3.5 µm 6
2
1 HPLC
α3,5= 1.06 3 5
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00
XSelect CSH Phenyl-Hexyl 4

150 mm, 5 µm 6
2
1 HPLC/Prep
α3,5= 1.07 5
3
0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00
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
0.05 Rs (2,3) = 2.25 0.05

0.00 0.00
0.00 0.10 0.20 0.30 0.40 0.50 0.60
Minutes
0.70 0.80 0.90 1.00
1.10 0.00 0.10 0.20 0.30 0.40 0.50 0.60
Minutes
0.70 0.80 0.90 1.00
1.10
Note increase in sensitivity as particle size is decreased

INCREASED Resolution, SAME Speed
Constant Analytical Run Time
Independent Y- axis Set to same scale
0.10
0.02 10 µm – 50 mm
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
Defining the LC Categories by their System Dispersion
How are these categories differentiated?
Chromatographic Resolution Increases
Overall Run Time Decreases
Method Sensitivity Increases

What is at the Root of the Performance Differences across
the LC Categories?
 Dispersion – n. Broadening of an analyte band due to

both on-column effects (diffusion and mass transfer
kinetics which are both dependent on particle size and
linear velocity) and system effects (tubing internal
diameter (I.D.) and length, connections, detector flow cell
volumes, etc.)
 True separation performance is governed by the system

dispersion paired with a flow rate range that yields the
highest possible efficiency for a given analytical column

Interaction of System Dispersion
and Peak Volume
Peak Volume Decreases
Column i.d.
Impact of System Dispersion Increases

System Bandspread Influence Across Different Column i.d.s
Alliance HPLC: Bandspread 36 µL
0.06
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

Matching System Bandspread With Column I.D.
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

Column Internal Diameter and Instrument Dispersion
HPLC Instruments
Peak Volume = Peak Width x Flow Rate
Example: Pw = 0.1 minute, Pk volume = 30 µL @ 300 µL/minute
0.30
Waters Alliance HPLC 2.1 x 50 mm
0.30 mL/min
0.20
Dispersion >30 µL
AU
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
CORTECS 2.7 µm Particle Columns

UHPLC Instruments
0.20
Waters ACQUITY Arc UHPLC
2.1 x 50 mm
0.15
Dispersion 12 – 30 µL 0.30 mL/min
AU
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
CORTECS 2.7 µm Particle Columns

UPLC Instruments
0.15 Waters ACQUITY UPLC
2.1 x 50 mm
0.10 Dispersion < 12 µL 0.30 mL/min
AU
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
No Advantage to run a 4.6 mm i.d. Column on an ACQUITY UPLC System

What Causes Poor Peak
Shape with Basic Compounds?
Silanol Interactions with Basic Compounds

Impact of poor peak shape in methods
development
 Integration errors
 Reduced resolution
 Reduced sensitivity
0 5 10 15 20 25
Minutes

Influence of Poor Peak Shape on Integration
Tailing Factor = 1.00 Tailing Factor = 1.58

Recovered Peak Areas Recovered Peak Areas
99.9 % 97.8 %
99.8 % 95.3 %
99.6 % 92.3 %

Influence of Poor Peak Shape on Resolution
Rs = (tr Peak 2 – trPeak 1)/ 0.5 (w4.4% Peak 1 + w4.4% Peak2)

Rs = (11.5-10.6)/ 0.5 (2.0 + 0.5)
Rs = 0.72
Note: Baseline resolution = 1.5
0 5 10 15 20 25
Minutes

Tamoxifen: Influence of Poor Peak Shape on Sensitivity
0.0004 C18 Brand A Signal/Noise Ratio

AU A 11.0
0.0000 B 6.5
5 10
Minutes
0.0004 Note:
C18 Brand B Signal/Noise
AU Requirements
0.0000 LOQ>10
5 10 LOD>3
Minutes
MS Scans Certified Septa
TruView LCMS
LCMS Certified
LC/GC Certified

TruView LCMS Certified Vials vs. Other Vendor
Adsorption Results
 Followed conditions from ASMS paper for this analysis

 At 5 ng/mL, B’s vial adsorbed all the analyte; Gentamicin was barely detectable at the 12-hours
assay
* A. Shen, L. Morgan, M Barroso, X Zhang, T. Nguyen, “Method Development of LC-MS/MS Analysis of

Aminoglycoside Drugs: Challenges and Solutions” 2008 ASMS Denver, Co.
TruView LCMS Certified pH Test
product
 pH test – DI water in vial for set time

 Some glass will raise pH 2+ (pH) units
 Customers ask about degradation – their analyte is stable in our product but not
consistently stable in some vials.
– Analyte is pH labile – can degrade at higher pH
 The pH of water in the TruView Vial does not change
Polypropylene Vial
Contaminant from Vial
 Customer found mass in their assay – unexpected.

– Traced the mass to vial and questioned the vendor
– Vendor’s response “…the resin used in the manufacture of these vials was recently changed. The
previous resin contained whitening agents that gave the finished product a bright white
appearance. The new resin is better!”
– The new resin has an impurity the vendor did not detect.
 Customer is now a Waters vial user. The second scan is our PP vial – no mass at 17.61 min.
Chromatography was supplied by the customer.

Choosing the Right Vial for Your Application
 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
1 µg/mL or higher UV, RI (non-MS) LCGC Certified Vials

100’s ng/mL Single Quadropole or older Triple LCMS Certified Vials
Quadropole
1 ng/mL or lower Triple Quadropole or ToF (Time of Flight) TruView LCMS Certified Vials

Factors Affecting the Separation (Ligand Density)
 Stationary Phase Factors

 Type of bonded ligand
 Ligand Density
 Concentration of the ligand on the surface of the particle
“Ligand”
High Low
Density Density
 Mobile Phase Factors
 Organic strength adjusted to create appropriate elution times

Neutral Compounds: Two Different C18’s
(Same Brand – Different Ligand Density)
Naphthalene
Note: Similar selectivity due to same
silica particle. Different hydrophobicities.
YMC™ J’Sphere™
ODS-L80 (low)
Acenaphthene
YMC™ J’Sphere™ ODS-H80 (high)
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
CH2 CH3 CH2 CH2 CH2 CH3

CH3 HH3C Si CH3H
H3C Si H3C Si 3C CH3 H3C Si
CH3 H3C Si CH3H H Si CH3
H H O H H
O O O O O O O O O O O O
Si
O Si O Si O Si O Si O Si O Si O Si O Si O Si O Si Si
O
C8 alkyl chains H3C
H3C
Polar analytes easily CH2 Low Coverage
CH2
H2C interact with surface H2C Low Ligand
CH2 CH2
and ligands H2C Density
H2C Residual silanols CH2
CH2 H2C
H2C Endcap
CH2 CH CH 2
H3C Si CH3 H3C CH CH3H3C Si CH3
3 3 H3C Si CH3 H3C CH3
H H H Si CH
Si H H 3
H H H O
O O O O O O O O O O O
Si Si Si Si Si Si Si O
Si Si Si O O O O O O O O Si Si
O O
O O O O O O O O O O O O O
Low
Alkyl chains Coverage
O H2C Low Ligand
Non-polar H2C
portion of CH2 Density
CH2 Polar portion of
analyte interacts
NH analyte interacts H2C ~1.65
with H2C with silanols
bonded phase CH2
N O (hydrogen bonding CH2
(hydrophobic) H and/or ion exchange) H2C
H2C
NH 2
CH2
Residual silanols CH2
H2C Endcap
H2C N
CH2
CH2 CH3 CH3 CH3
H3C
H3C
CH3 H3C CH3
H3C
CH3 O H N Si CH3 H3C
CH3
Si Si Si H H H Si
H H H H
O O O O O O O O H
O O O O
O
Si Si Si Si Si Si Si
Si Si Si Si
O O O O O O Si
O O O O O O O
O O O O O O O
O O O
De-Wetting of Bonded
Chromatographic Surface
The particles are very porous,

like the pores of a sponge—
Mobile Phase 99% of chromatographic surface
is inside the pores
Analyte
If the pores are dry, the analyte

cannot get into the pores and it
De-Wetted Pore will not be retained by the
chromatographic surface.
This will greatly influence the chromatography

HSS T3 Versus HSS C18:
100% Aqueous Conditions
0.06 1 2 XSelect HSS T3
3 5
0.04 4
AU
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
Compatibility: Aqueous Mobile Phase
 Larger pore diameters and lower hydrophobic ligand density reduce dewetting
Increase pore diameter Lower C18 Coverage
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
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
acid (HVA) 25 Instrument: ACQUITY UPLCTM System with

1 5
ACQUITY UPLC™ 2996 PDA
3 4 7 ACQUITY UPLC™ BEH C18

2
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00
Minutes
NEW ACQUITY UPLC™ HSS T3
1 6
3 5
4 Increased
2 Retention
7
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00
Minutes
How Does T3 Bonding Work for both Nonpolar and Polar
molecules?
 Non-Polar
– Retention mechanism is classic reversed-phase, hydrophobic interaction with the C18 stationary phase
 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

Different C18 Bonding Levels:
Carbon Load
 Measured as weight of bonded phase per weight of silica (w/w %)
 Typical range for C18 ligand : 6 to 22%
Carbon Load Retention

Different Bonding Levels
Packing Column Silica Mass of Carbon Load Carbon in

Volume Density Packing (g) (%) Column (g)
(mL) (g/mL)
Nova-Pak® C18 1.8 0.8 1.44 7.3 0.11
µBondapak™ C18 1.8 0.40 0.72 9.8 0.07

Column Selectivity Comparison Chart (Methanol)
2 .8
Decreasing Silanol Activity

2 .3
µBondapak™ C18 Waters Spherisorb® ODS2

1 .8
9.8% with 1.42 11.5% with 2.84

1 .3 µmoles/m2 µmoles/m2
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

Column Selectivity Comparison Chart
2 .8
2 .3
Decreasing Silanol Activity
µBondapak™ C18
1 .8
1 .3
Sph1ODS-Low 14%
Sph2ODS-Med
9% Sph3ODS-High
0 .8
Nova-Pak® C18 22%

(ln [a]
0 .3
Symmetry® C18
Increasing Hydrophobicity
-0 .2
1 1 .5 2 2 .5 3 3.5

Selectivity Chart
2.7
NOTE: Do you see two C18’s
Resolve® C18
amitriptyline/acenaphthen
columns that look like they Waters Spherisorb® ODS1

2.2 are out of place? –Hint:
upper left quadrant
µBondapak™ C18 Waters Spherisorb® ODS2
1.7
YMC J'sphere™ ODS–L80 Nucleosil® C18
1.2 YMC J'sphere™ ODS–M80
YMCbasic™ YMC J'sphere™ ODS–H80
Hypersil® ODS
YMC-Pack™ Pro C4™Inertsil® C8 Nova-Pak® C18
0.7 Nova-Pak® C8 YMC-Pack™ ODS–AQ™
Kromasil® C8
YMC-Pack™ Pro C8™ YMC-Pack™ ODS-A™
(ln [a]
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)
“Base-Deactivated Packings”
 What are they?

– A class of reversed-phase packing materials that exhibit improved
peak shape for basic compounds---especially at pH’s 4-7
 How are they made?
– Various techniques in silica gel synthesis and/ or in the bonding
process that yields a particle surface with less silanol interaction with
basic analytes

“Base-Deactivated Packings”
a. Metal Impurities (silica gel)

a. Lower concentration of Al3+ and Fe3+
b. Ligand Density
c. Mixed C18 and Amino Ligands
d. Embedded polar ligands (advanced ligands)

Acid, Base, Neutral Chromatographic Evaluation Test at pH 3.0
H CH 3 H
OH
H 3C N
+ +H
N
H CH 3 O
HOOC
H O NH 2
N + Cl
- OOC
H
Chlorpheniramine Propranolol Maleic Acid Toluamide

pKa = 3.6, 9.2 pKa = 9.5 pKa = 1.9, 6.3
Base: Positive Charge Acid: Negative Charge
Base: Positive Charge Neutral: No Charge
Mobile Phase: 50 mM H3PO4-KH2PO4 pH 3.0/Acetonitrile (80/20)
Waters Spherisorb® ODS2 Standard C18

Maleic Acid
Toluamide
Propranolol
Chlorpheniramine
0 4 8 12 16 20 24 28 32
Minutes
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
Technique C: Pros and Cons (Better for Bases – Poor for
Acids)
Waters Spherisorb® ODSB

? “Base Deactivated”
Toluamide
Maleic Acid

Propranolol
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

Characteristics of Two-Step Synthesis Process
 Inexpensive process: aminopropyl silane and acyl chlorides

readily available
 Incomplete secondary reaction due to steric hinderance
resulting in the presence of an aminopropyl group
– Packing materials show anion exchange properties that are
undesirable (unusual retention of acidic compounds)
 Batch to batch reproducibility issues

Embedded Polar Ligand: Two Step Synthesis
Note: Similar results to Technique C: Mixed C18 and Amino Ligands
Chlorpheniramine
Toluamide
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
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
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)
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.
Shield RP18 Bonding
Alternative Selectivity Embedded Polar chains
CH3 CH3 CH3
(CH2)n (CH2)n (CH2)n Embedded Polar

group attracts
water to the
H2C H2C H2C surface, shielding
Embedded Polar unwanted
CH2 CH2 CH2
group acts as a interactions with
hydrogen bond HN HN HN residual silanols
acceptor which
can provide C O C O C O
alternative O
O O
selectivity when
compared to CH2 CH2 CH2
typical C18 phases H2C H2C Residual H2C Endcap
Silanol CH2
CH2 CH3 CH2 CH3
H3C
H3C H3C CH3 H3C
Si CH3 H3C CH3 Si CH3 H Si CH3
Si H H Si
H H H H
O O O O O O O O H
O O O O
O
Si Si Si Si
O O O O O O Si
O O O O O O O
O O O O O O O
O O O
Shield RP18
Selectivity Difference between C18 for Phenolic Compounds
CORTECS C18 2 1. Quercetin
2.1 x 50 mm
1 2. Kaempferol
3
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

Acidic Compounds:Embedded Polar versus Alkyl Linear
Ligands
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
 Reduced retention and decreased tailing of basic

compounds
 Unique selectivity
 Improved water wettability of the chromatographic bed
(hydrogen bonding of water to the particle surface) and
reduced risk of “hydrophobic collapse”

Ionization of Acids and Bases
Dissociation of the Molecule
Acid (PROTON DONOR)
+ -
HA H + A
(Un-Ionized) (Ionized)
Base (PROTON ACCEPTOR)
+ -
B BH + OH

Keep in MIND for Ionizable
Compounds in a Solution
Reversed-Phase : Non-polars are retained, Polars elute quickly
Proton DONOR H+ Proton ACCEPTOR H+

ACID BASE
UNCHARGED Un-Ionized {NON-POLAR} UNCHARGED Un-Ionized

{NON-POLAR}
Retained Retained
NEGATIVE - Charged {POLAR} POSITIVE + Charged {POLAR}
Little Retention Little Retention

Ionization of Acids:
Importance in Ion-Exchange Retention
Acid (PROTON DONOR)
+ -
HA H + A
50% @ pKa 50%

pH is 2 units
100% Low pH 0% away from pKa
0% High pH 100%
pKa = pH at which 50% of the analyte molecules in solution are charged
(ionized) and 50% are un-ionized
pKa of an ACID
0 pH 14
pKa <2 pKa 4 pKa 10

Strong Acid Moderate Acid Very Weak Acid
(It donates protons
N
EVEN when a lot
of protons present)
N
H
Imidazole (Acid) pka 7.6

Gallic Acid
H3PO4 pka 4.5 Acid Group
pKa’s 2.15, 7.09, 12.32 pKa 10 phenols pKa + pKb = 14
Reversed-Phase Retention Behavior of Acidic Compounds
Relative to Changes ± 1 pH Unit from pKa
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
Reversed-Phase Retention Behavior of Acidic Compounds
Relative to Changes ± 2 pH Units from pKa More Robust
Methods
35
Un-ionized Acid - 2 pH unit = 99% un-ionized

30
25
> ± 2 pH units provides stable
Capacity Factor (k)
20 retention (better reproducibility at

flat portions of curve)
15
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
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
If you adjust the pH

+ 2 pH units from the pKa,
you will make ~100%
of the molecules
either ionized or un-ionized

Ionization of Bases:
Importance in Ion-Exchange Retention
Base (PROTON ACCEPTOR)
+ -
B BH + OH
50% @ pKb* 50%

0% Low pH 100% pH is 2 units
away from pKa
100% High pH 0%
pKa = pH at which 50% of the analyte molecules in solution are charged * pK of conjugate acid
a
(ionized) and 50% are un-ionized
Reversed-Phase Retention Behavior of Basic Compounds
Relative to Changes in pH
35
30 > ± 2 pH units provides stable Un-ionized Base

retention (better reproducibility
25
Capacity Factor (k)
at flat portions of curve)

20
15
pKa
10
5
Ionized Base
0
0 1 2 3 4 5 6 7 8 9 10 11 12
±1 pH
±2
pKa of a BASE
0 pH 14
pKa 2 pKa 7 pKa 10

Weak Base Moderate Base Stronger Base
(It can pick-up a proton
EVEN when there are
only a few available)
N
N
Pyridine Chlorhexidine
Diphenylamine (Base) (Base) (Base)
pka 0.79
Amitriptyline
pka 5.2 pka 2.2; 10.3 pKa 9.4
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
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
Hybrid Particle pH Range

Neue et. al. American Laboratory 1999 (22) 36-39.
•
Relating Retention Maps to Chromatography
Reversed-Phase Retention Map

B A N
14
13 Neutral pH 2
12
11
Capacity Factor (k)
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


B A N
14
13 Neutral pH 2
12
11
Capacity Factor (k)
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


B A N
14
13 Neutral pH 2
12
11
Capacity Factor (k)
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
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
By varying pH, bases are now separated – pH is a powerful tool

©2018 Waters Corporation COMPANY CONFIDENTIALin methods development for selectivity 141
Impact of pH on the Retention of a Zwitterionic Compound
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 -
Peak Shape Over Wide pH Range
(Strong Base -- Amitriptyline pKa 9.4)
Low ligand density and

4 high metal content
Tailing Factor
silica gel: Conventional

C18
3 High ligand density and
high purity silica gel:
Modern C18
2
1
2 3 4 5 6 7 8
Buffer pH
Ideal Behavior Pure Polymer – No Silanols
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
1.00 are largely

0.80
0.60 1 unaffected by mobile
0.40
0.20 phase pH
0.00
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 Minutes
4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00
General Retention and Mobile Phase pH Rules
 To achieve stable chromatographic retention times

– Stay outside the ± 2 pH units from the analytes pKa value
– Always measure pH before adding the organic solvent

Retention Time Variability Influence of pH
Non-Column Influences:
pH
 Neutrals: No Influence
 Acids: Reduced Retention with Increasing pH
 Bases: Increased Retention with Increasing pH
 Up to 10% Change in Retention per 0.1 pH Unit

 (largest shift within +/- 1 pH unit of pKa)

Poor Method Reproducibility Near pKa’s
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
Range of poor retention reproducibility due to steep slopes near pKa

Small pH change – Large retention change

Poor Method Reproducibility Near pKa’s
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
B2 A, B1
B1 A B2
pH 5.5 pH 6.0
pH is a powerful tool in methods development for selectivity.

COMPANY CONFIDENTIAL 148
However, if near pKa’s, pH control is critical
Example:
Significant Retention Shifts with pH Change
Compounds 0.40
1
1. p-Toluamide -neutral
0.30
pH 5.0
0.20
3
2. Lidocaine – base (pKa = 7.9) 0.10 2
0.00
3. Ibuprofen- acid (pKa = 4.4) 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
0.40
Method developed at pH = 5.0

0.30
1 pH 5.2
0.20
High reproducibility risk — 3
0.10 2
Requires tight pH control 0.00
to maintain separation 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 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

Good Reproducibility
2 Units from Analyte pKa Values
100
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
Improved retention reproducibility due to flattening of curves

Large pH change – Small retention change
Separation robustness
in pH ranges 2 pH units
from analyte pKa values
pH Test for Robustness
Good Results

Impact of Retention Factor (k)
on Prep LC
In Pure REVERSED-PHASE Mode
pH Limit of Silica gel
100
Acid (Un-Ionized)
Base (Un-Ionized)
log (k)
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
Note: Column Particle, Temperature and % Organic Held Constant

Basic Compounds
(ln [a] amitriptyline/acenaphthene)
2 .8
W aters Spherisorb® O DS1

Resolve® C18
2 .3
W aters Sp herisorb® O DS2

µBondapak™ C18
1 .8
YM C J' sphere™ O DS–L80 N ucleosil® C18
1 .3 Amine additives YM C J' sphere™ O DS–M 80
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
Buffers required Symmetry® C18
-0 .2
1 1 .5 2 2 .5 3 3 .5
(ln [k] acenaphthene)

Impact of Organic Solvent Composition
on Retention Factor (k), pH, and pKa
 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
Organic Solvent Composition c

Note: k, pKa and pH are a function of the
Retention Factor k organic solvent composition
Slope of relationship between
Log k and % Organic B

Impact of Organic Concentration on the pKa of the Analyte
 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

Amitriptyline
+ CH 3 CH 3
NH N
CH 3 CH 3
Ionized Un-ionized
pKa (H20) 9.4
apparentpKa (20% ACN) 8.5
Note: you cannot determine the degree or the direction of the
pKa shift with out experimentally running each organic
concentration
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)
10 Apparent pKa = 8.0

5 Compound
ionized
0
0 1 2 3 4 5 6 7 8 9 10 11 12
Aqueous pH 157
Naproxen
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
Note: you cannot determine the degree or the direction

of the pKa shift with out experimentally running each
organic concentration
What is the practical application of this information?
 For new methods development: aqueous buffers at pH 2 (for acidic

analytes) and pH 10 (for basic analytes) when mixed with organic
solvents may provide stable chromatographic regions where most
analytes will be un-ionized
 Caution: Must consider
– Un-ionized analyte hydrophobicity
– Organic solvent strength (Methanol versus THF)

pH Limitations of Silica Based
Packing Materials
Hydrolysis of Bonded Ligand Dissolution of silica particle

10
1
0 2 4 6 pH 8 10 12 14

Hydrolysis of a Bonded Phase Material:
Monofunctional Ligand
O H 3 C
OH C C C C
Si Si C C C CH
+
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”
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)
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
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
Peak Shape: Monofunctional Silane versus
Trifunctional Silane
Risk: trifunctional silane can increase silanols
Silica gel particle Monofunctional

C18
amitriptyline TF = 1.8
0
1 2 3
Silica gel particle Trifunctional C18

amitriptyline TF = 2.6
1 2 3
Minutes

Trifunctional Synthesis: Higher Ligand Densities,
Greater Hydrolytic Stability
OH
HO CH
Si 3
OH
O
Si CH 3
“Bonded” but not actually
HO O reacted to substrate
Si CH 3
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
Mobile Phase pH and Column Life-time
Even when bonded with ligands – it will still dissolve

240- Silica Solubility Curve
Solubility of Silica in Water (ppm)
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
Effects of High pH Mobile Phases
Surface Modified Silica Particles Hybrid Particles
•Slow rate of surface dissolution

• Complete dissolution of Silica • Incorporated methyl groups uncovered
• Catastrophic column failure slows rate of dissolution
• Short lifetimes • Longer column lifetimes
Mechanical -- Column
Packing Material Flush
Well packed column

Good Plate Count Results
(Instrument OK , Column OK, Connections OK)
Well packed column

Triethylamine….temperature….pH

Column Bed Collapse – Mechanical
Poor Plate Count
Instrument OK, Connections OK
Voided column Packing Material Settled
- Channeling or dissolved

Column Collapse (voiding)
(due to shock / high pH {dissolution of particle})
All Peaks Distorted
Voids - high back pressure,

©2018 Waters Corporation double peaks
COMPANY CONFIDENTIAL 173
Extended pH Range of XBridge/BEH Columns Simplifies Methods
Development
(One Column Instead of Three)
Low pH High pH polymeric

silica gel column column
Intermediate pH
silica gel column
Broad pH range Xbridge/BEH columns
1 2 3 4 5 6 7 8 9 10 11 12

pH 174
Initial Phases Chosen for Selectivity

Symmetry
C8 C18 Shield RP8 Shield RP18
Symmetry300 C4 Symmetry300 C4

SunFire
C8 C18 Silica

Two Fully-Scalable LC Column Platforms
Family designed and optimized Family designed and optimized for

for pH stability selectivity
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
XBridge/BEH Family
 Excellent general-purpose HPLC

and UPLC columns for small
molecule separations
Family designed and optimized for
– Broadest range of compound
pH stability classes
– Widest pH range (1 – 12)
– Widest temperature range
– Ultra-low MS bleed
 Direct scalability between UPLC

Technology, analytical HPLC and
preparative HPLC
1.7 [UPLC], 2.5 XP, 3.5, 5 and 10 µm
Ethylene Bridged Hybrid [BEH] Chemistries
 Rugged, reproducible, fully-scalable column chemistries for reversed-phase and

HILIC separations
– BEH C18: First column choice, widest pH range, LC/MS
– BEH C8: For hydrophobic compounds, widest pH range, LC/MS
– BEH Shield RP18: Embedded carbamate group, alternate selectivity
– BEH Phenyl: Most stable phenyl column, wide pH range
– BEH HILIC: Unbonded, rugged BEH particle for HILIC separation of very polar bases
– BEH Amide: General-purpose HILIC column for very polar compounds such as
sugars, saccharides, carbohydrates, etc.

Industry Leading pH Stability
Column lifetimes in acidic mobile phases
Shorter bar
equals longer
lifetime under
low pH
conditions
Test conditions: 1% TFA in water (pH 1.0) at 80oC.

©2018 Waters Corporation COMPANY CONFIDENTIAL Comparative separations may not be representative of all applications. 182
Industry Leading pH stability
Column lifetimes in alkaline mobile phases
 High pH mobile phases (>pH 10)

rapidly dissolve silica-based
stationary phases
 Only modern hybrid-based sorbents,

[BEH] and [CSH], extend the usable
mobile phase pH range from 1-12
©2018 Waters Corporation COMPANY CONFIDENTIAL Comparative separations may not be representative of all applications. 183
XSelect HSS Family
 Highest pressure tolerance; first

silica-based particle (1.8 µm)
designed for UPLC applications
 Alternate selectivities (vs.
BEH & CSH particle columns)
 Increased retention
1.8 [UPLC], 2.5 XP, 3.5 and 5 µm HSS

HSS Chemistries
 For general-purpose C18 columns for low to neutral pH reversed-phase

separations
– HSS C18: Superior peak shape & acid stability
– HSS C18 SB: Alternate Selectivity for Basic (SB) compounds
– HSS T3: Enhanced reversed-phase retention of polar compounds
– HSS CN: Stable, normal phase-compatible, reproducible CN chemistry
– HSS PFP: Exceptional selectivity and retention for positional isomers, halogenated
compounds and polar basic compounds

Charged Surface Hybrid [CSH] Particles
Step 1 Step 2 Step 3
CHARGED SURFACE HYBRID PROCESS

Surface Complexity
C18 C18 C18 C18
I I
O O
TMS Si Si OH TMS Si Si OH X+
OH OH
O O O O O O
H O H O Si
H O -
O B+ H O
O
O O
Silica or Hybrid Silica or Hybrid
 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, Phenyl, Fluoro-Phenyl
C18+
©2018 Waters Corporation K. Wyndham,

COMPANY LC-GC 2012 (Suppl.) 15
CONFIDENTIAL Patents Pending 189
Selectivity Comparison
2 1. 1-pyrenesulfonic acid
2. Flavone
6 3. Imipramine
ACQUITY UPLC BEH C18 4 7 4. Fenoprofen
1 5. Amitriptyline
3 5 6. Diclofenac
7. Octanophenone
3 2
5 6
ACQUITY UPLC 7
CSH C18 1 4
3 2
5
ACQUITY UPLC 6 7
CSH Phenyl-Hexyl 1
4
3 2
5
ACQUITY UPLC 6 7
1
CSH Fluoro-Phenyl 4
0.00 1.00 2.00 3.00 4.00 5.00

All columns 2.1 x 50 mm, 1.7 µm Minutes
NOT All Peaks are Created Equally!
Most common peak shapes

 Gaussian peak shape due to single
interaction with the surface
– Linear isotherm
 Exponential tail due to secondary

interactions
– Acidic silanols
 Shark fin peak shape due to mass

overload
– Convex isotherm

Benefits of CSH Technology:
Loading Comparison
3.0 ACQUITY UPLC® CSH C
18 Quetiapine Propiophenone
2.0 2.1 x 50 mm, 1.7 µm (base) (neutral)
AU
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
0.04 1.0 % Amitriptyline

0.5 %
0.02
CSH Technology
0.1 %
0.00
enables superior peak
shape and efficiency
0.00 0.20 0.40 0.60 0.80 1.00
Minutes
1.20 1.40 1.60 1.80 2.00
in low ionic strength
0.10
mobile phases
Superficially Porous C18 1.7 µm
0.08
0.1 % formic acid Improved sensitivity
Imipra
for trace level

mine
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
CSH Technology:
Imipramine
0.015 ACQUITY CSH C18
A 0.1%
impurity 0.1% formic acid
AU
0.000
0.015 Imipramine 0.1% Superficially porous C18

B impurity
AU
0.1% formic acid
0.000
Imipramine
0.015
0.1% Superficially porous C18
C
AU
impurity 0.05% TFA

0.000
0.00 0.60 1.20 1.80 2.40 3.00

Minutes

CSH Technology:
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
8x108 Superficially porous C18
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

Why Haven’t I Seen This Before?
 Remember: when using low pH buffers as part of your method

development protocols (e.g., ammonium formate, sodium phosphate,
etc.) these ‘pH switching’ effects do not occur
 ‘pH switching’ effects only occur when using acidic, low ionic strength
additives
 ALL high pH-capable columns EXCEPT ACQUITY CSH and XSelect

Columns exhibit this behavior

XSelect CSH Family
Family designed and optimized for

selectivity
 Designed for LC/MS
 Improved peak shape for basic analytes Multiple particle substrates to
under low-ionic strength acidic mobile solve multiple chromatographic
phases problems
 Reduces overload effect
 Rapid equilibration; prevents
retention time drift with changes in
pH
1.7 [UPLC], 2.5 XP, 3.5, 5 and 10 µm CSH

What Column(s) Should I Choose?
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

CSH Chemistries
 For general-purpose C18 columns for low to neutral pH reversed-phase

separations
– CSH C18: Rapid pH switching for method development
– CSH Phenyl-Hexyl: High performance phenyl column, best in additive-based mobile
phases, different selectivity vs BEH Phenyl
– CSH Fluoro-Phenyl: Truly different selectivity, optimized for low pH separations,
enhanced retention of acidic compounds

Widest Selection of Unique Particle Offering
BEH Technology HSS Technology CSH Technology
Maximum retention Exceptional loading

Unparalleled pH stability capacity
Mobile phase and Particle and ligand Superior peak shape for
temperature versatility selectivity basic analytes
Seamless scalability Seamless scalability Seamless scalability

UPLC to HPLC UPLC to HPLC UPLC to HPLC
Waters is the ONLY company that has

fully porous and solid-
solid-core sub-
sub-2-µm particle columns
Selectivity Differences:
Different Base Particles
Low pH, ACN
Ziprasidone and related compounds
0.20 1
2 BEH C18
AU
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

eXtended Performance Columns

2.5 mm 202
Addressing the Challenges:
2.5 µm eXtended Performance Columns
Addressing the challenges
Utilize XP 2.5 µm columns to

improve productivity or achieve
higher resolution
Compatible with bandspread

volumes of UHPLC systems
Implement XP 2.5 µm columns to

decrease backpressure or increase
flow rate

Flexible Options to Improve LC Productivity

Allowable Adjustments in Chromatography <621>
Variable Allowable Changes

Particle Size -50%
Column Length ±70%
Flow Rate ±50%
Column ID Any allowed
Injection Volume Any reduction
Column Temperature ±10%
Mobile Phase pH ±0.2 unit
System Suitability. USP 37. Pag 306

306--308

What is L/dp?
 L/dp is directly related to the resolving power

– Higher the L/dp, higher the efficiency (N), higher resolving power
Relative Values
Column Length Particle Size L/dp N Run Time
(L, mm) (dp, mm)
250 10 25,000 0.8 3.3

150 5 30,000 1.0 1.0
100 3.5 28,600 1.0 0.5
75 2.5 30,000 1.0 0.3
50 1.7 29,400 1.0 0.1

Columns Options

Solid-Core Particles

Solid-Core Particles
 Solid-Core particles have been around since the 1970’s

– Waters Corasil I 30-80 µm particles
– Problems with early solid-core columns, poor efficiency and poor loading capacity
 Today's modern core-shell particles are primarily prepared by the multi-layering of

silica sols around a solid silica core.
– Thicker shell, allows for better Loading capacity
– Smaller particles, better particle design, and improvements in packing provide higher
efficiency
Particle
FIB SEM Images Solid-
Core
Core
Laboratory Impact
 The higher efficiency provides increased resolution.

– More peaks are baseline resolved, easier to identify and quantify
N α 1 k Resolution equation:
Rs 
4 α k1 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

Lower Backpressure? How?
Kozeny-Carman Equation
F  L 180  (1   e ) 2
P  2
r 2 dp  e3
 Particle size, (dp) and the external porosity (ee

) have a significant impact on column
backpressure
– As the particle size in reduced, pressure
increases by the difference squared
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
2000  CORTECS 1.6 µm particles:

– Slightly smaller than fully porous 1.7 µm
1000
o Would expect higher backpressures
0 – Pack less densely
o Lower backpressure
– Net result is similar backpressures
Conditions: 20% Acetonitrile in water; 600 µL/min; 40 oC 1A. Daneyko, Anal. Chem. 2011, 83, 3903–3910
Understanding Mass Transfer [Diffusion]:
Influence of Particle Size
Diffusion distance is shorter with decreasing particle size resulting in

a narrower, more efficient, chromatographic band

Understanding Mass Transfer [Diffusion]:
Superficially Porous Particles
Fused – Core Particle
Diffusion distance is short because the analyte band can only diffuse
into the porous layer of material

However, as of today…
19,700 39% higher efficiency

20,000
Plates (4 sigma)
16,000
14,150
or up to 3x faster!
12,000
8,000 CORTECS UPLC 1.6 µm C18+

ACQUITY UPLC 1.7 µm BEH C18
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
 Any place where the analytical band “moves”

adds to the instruments dispersion
– Injector
– Tubing
o Pre-column
o Post-column
– Oven design
– Flow cell volume
Impact of System Dispersion on Observed Efficiency of
CORTECS Columns
ACQUITY UPLC I-Class
5.5 µL USP N: 18,000
ACQUITY UPLC H-Class USP N: 11,700

12 µL
53% Increase in Observed CORTECS Column

Efficiency on ACQUITY UPLC I-Class System
Acetonitrile/ Water (70/30 v/v), 0.4 mL/ min, 30oC, 0.5 µL injection. Peak i.d.: Acetone, Naphthalene, Acenaphthene
2.1 x 50 mm CORTECS C18 Column

CORTECS 1.6 µm and 2.7 µm Particles
Fully Scalable
FIB SEM Images
Rho (ρ) = 0 → fully porous 1.6 µm 2.7 µm
particle
Particle
Particle
ρ = 1 → nonporous particle Solid- Solid-
Core Core
Core Core
r = core diameter / particle diameter
Attribute CORTECS
r 0.7
Particle Size 1.6 µm, 2.7 µm

Maintaining the rho value, Pore Volume 0.26 cm³/g
Allows for scalability Pore Size 90 Å*
between the particle sizes Surface Area 100 m²/g
©2018 Waters Corporation COMPANY CONFIDENTIAL *T3 chemistry is 120 Å 218

Transferability
 Method Transfer
– Scaled synthetic process
o Rho value scaled
– UPLC HPLC
o Seamless method transfer
o Future proofing
1.6 µm UPLC 2.7 µm HPLC/UHPLC

Compatible with Existing Methods
USP Method Transfer
1. Loratidine Related Compound A
3 2. Loratidine Related Compound B
0.08 USP Method 1 2 3. Loratidine
0.06 Conditions 4.6 x 150 mm
AU
0.04 System Suitability XBridge BEH C8 5 µm

Rs 2,1 NLT 1.5: 2.7 1.00 mL/min
0.02 tR3 %RSD NMT 2.0%: 0.7%
0.00
HPLC
3
0.10 12 Meets all regulatory accepted guidelines
3.0 x 100 mm
AU
System Suitability CORTECS C8 2.7 µm

0.05 Rs 2,1 NLT 1.5: 3.5
tR3 %RSD NMT 2.0%: 0.5%
0.80 mL/min
0.00
UHPLC
0.06 3
Meets all regulatory accepted guidelines
0.04
2.1 x 50 mm
AU
12 System Suitability CORTECS UPLC C8 1.6 µm

0.02 Rs 2,1 NLT 1.5: 2.5 10x Faster 0.60 mL/min
0.00
tR3 %RSD NMT 2.0%: 0.5% 10x Solvent Savings UPLC
0.00 2.00 4.00 6.00 8.00 10.00
Minutes
Method Transfer Across LC Platforms
0.80 1 CORTECS C18, 2.7 µm
4.6 x 150 mm Compatible with
AU
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 UHPLC for Maximum

0.31 mL/min
0.00 Productivity
0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0
0.80 CORTECS C18 1.6 µm
2.1 x 50 mm
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
Porous Particle Equivalence
Same Efficiency Same back pressure
2.22 µm
2.7 µm 3.1 µm
CORTECS Equivalent Porous Particles

Particle Size Efficiency Backpressure
1.6 µm 1.3 µm 1.8 µm
2.7 µm 2.2 µm 3.1 µm

CORTECS 2.7 µm
High Efficiency Separation at HPLC Backpressures
0.10 1. Estradiol Configuration: 4.6 x 150 mm
0.08 2. Ethinyl estradiol
3. Estrone 1 Fully Porous C18, 5 µm
0.06 2 3
AU
4. Levonorgestrel 4 Psi: 1900

0.04
Rs 4.9
0.02 N peak 4: 10,400
0.00
0.10
0.08
Fully Porous C18, 3.5 µm
0.06
AU
0.04 Psi: 2900

Rs 6.3
0.02 N peak 4: 17,600
0.00
0.10
0.08 CORTECS C18, 2.7 µm
0.06
AU
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
Note: 2.5 µm fully porous Psi ~6000 223
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
0.12 9. Omeprazole-n-oxide/ Sulphone (362.42)

10. Unknown Degradent
11. Unknown Degradent
0.08 6/7 11
8/9
0.04
10
5
0.00
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00
Minutes

Increased Speed and Resolution
Lipid Soluble Antioxidents
1. Propyl Gallate (0.05mg/mL)
2. 2,4,5-trihydroxybutyrophenone (0.05mg/mL)
0.15 1
2 3. Tert-butylhydroquinone (0.1mg/mL)
4 4. Hydroxyanisole (0.1mg/mL)
0.10
5. 2,6 di-tert-butyl-4-hydroxymethyphenol (0.1 mg/mL)
6. Octyl Gallate (0.05 mg/mL)
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

CORTECS HILIC
Higher Resolution of Local Anesthetics
1 5
0.20 1. Lidocaine
2. Butacaine 2 4
3. Tetracaine
ACQUITY BEH HILIC
0.15
4. Procaine 3 2.1 x 50 mm 1.7 µm
5. Procainamide
AU
0.10 USP Resolution2,3: 1.2
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
USP Resolution2,3: 2.2

0.05
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

The CORTECS Family
C18 General Purpose, balanced acidic,

basic, neutral retention
C18+ Basic analyte peak shape when using

low ionic strength mobile phases
HILIC Very polar compounds using

HILIC
General purpose, less hydrophobic
C8
than typical C18 chemistries
Different selectivity vs C18, particularly for
Phenyl aromatic analytes
Balanced retention of non-polar and
T3 polar compounds using RPLC
Different selectivity to C18. Improved peak

Shield RP18 shapes for basic analytes
One Way to Visualize Selectivity: pH 7 Selectivity Chart
3.6 Waters Spherisorb S5 P
3.3
3 ACQUITY UPLC HSS C18 SB
ACQUITY UPLC BEH Phenyl

XSelect HSS C18 SB
2.7 Waters Spherisorb S5CN XBridge Phenyl ACQUITY UPLC HSS PFP
Nova-Pak CN HP Inertsil Ph-3 XSelect HSS PFP Waters Spherisorb ODS1
Selectivity
2.4 Hypersil Phenyl

Resolve C18
ACQUITY UPLC BEH C18
2.1 ACQUITY UPLC CSH Fluoro-Phenyl CORTECS
XSelect CSH Fluoro-Phenyl XBridgeWaters
C18 Spherisorb ODS2
ACQUITY UPLC HSS CN Phenyl
ln [α]
1.8 µBondapak C18 ACQUITY UPLC HSS T3

XSelect HSS CN YMC-Pack YMC J'sphere
Phenyl ODS–L80 XSelect
Nucleosil C18 HSS T3
1.5 Hypersil CPS Cyano Inertsil CN-3 Nova-Pak Phenyl CORTECS C18+
YMC J'sphere ODS–M80
1.2 Hypersil BDS Phenyl
Chromolith Nova-Pak YMC J'sphere ODS–H80
YMCbasic XTerra YMC-Pack™ ODS–AQ
RP-18 C18
0.9 YMC-Pack CN YMC-Pack Pro C4 PhenylNova-Pak Luna Atlantis dC18 Zorbax XDB C18
YMC-Pack Pro C8 C8 Phenyl Hexyl Atlantis T3
0.6 ACQUITY UPLC BEH C8 ®
XTerra MS C8 ACT Ace C18 Symmetry C8 YMC-Pack ODS-A
XBridge C8 Luna Luna C18 (2)
CORTECS C8 YMC-Pack Inertsil ODS-3
C8 (2)
0.3 ACQUITY UPLC CSH Phenyl-Hexyl Pro C18 SunFire ™ C18
CORTECS T3 SunFire C8 XTerra MS C18 Symmetry C18
XSelect CSH Phenyl-Hexyl SymmetryShield RP8
0 Zorbax SB C18
XTerra RP18
ACQUITY UPLC BEH Shield RP18 SymmetryShield RP18
-0.3 XBridge Shield RP18 XTerra RP8ACQUITY UPLC CSH ACQUITY UPLC HSS C18
XSelect CSH C18 XSelect HSS C18
-0.6 YMC-Pack PolymerC18
CORTECS
-1.5 -0.5 0.5 1.5 Shield RP18 2.5 3.5
Hydrophobicity
CORTECS C18
Rugged Bonding Technology
Low pH Stability
XBridge C18
Shorter the
CORTECS Phenyl
bar the better
CORTECS C18
the stability!
CORTECS C8
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
Transfer to 2.7 µm 0.02 CORTECS 2.7 µm C18

4.6 x 75 mm
4X Faster method
AU
0.01 1.85 mL/min

2X Less solvent Alliance HPLC
0.00
3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0
0.02 CORTECS 2.7 µm C18

Compatible with 3.0 x 75 mm
UHPLC systems 0.79mL/min
AU
0.01
4X Less Solvent ACQUITY Arc UHPLC

0.00
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
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
Plates
5000
0
10000
15000
20000
25000

1013531716001
1013531716002
1013531716003
1013531716004
1013531716005
1013531716006
1013531716007
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
 Understanding column packing provides equivalent column

performance
6.00
across all column dimensions
Consistent packing quality
5.50 across all three diameters
This is difficult
Reduced Plate Height, h (USP)
provides for improved

5.00
scalability and smooth method to achieve!
4.50 transfer
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)

Batch-to-Batch Reproducibility
Controlling the manufacturing (hardware, particle synthesis, column
packing) reduces the variability in our columns!
CORTECS UPLC 1.6 µm C18 CORTECS 2.7 µm C18
0.08 2 0.08
3 4 Batch A
AU
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 Shield RP18 Configurations
 1.6 µm – 10 column configurations  1.6 µm – VanGuard Pre-Columns

 2.7 µm – 15 column configurations
 2.7 µm – VanGuard Cartridges
 1 packs, 3 packs, and Method Validation Kits – 2.1 & 3.9 mm i.d.
(MVKs)

Waters Particle Technologies
Hybrid Silica
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
Fully Scalable: UPLC UHPLC HPLC

Factors: Column Considerations
Waters Particles and Chemistries
Hybrid
FULLY POROUS FULLY POROUS
BEH Technology (XBridge

(XBridge)) CSH Technology (XSelect
(XSelect))
Unparalleled pH, mobile phase and Controlled Surface Charge
temperature versatility Unparalleled peak symmetry for bases in
formic acid

Factors: Column Considerations
Waters Particles and Chemistries (cont.)
Silica
FULLY POROUS SOLID-CORE
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
Summary
 CORTECS Solid-Core columns provide increased efficiency at lower

backpressures when compared to fully porous particle columns of equivalent size
 Seven Chemistries provide a wide selectivity space, which is helpful in methods

development
 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
 Developed using Waters quality column manufacturing process, CORTECS Columns

are highly reproducible; provide greater efficiency, resolution, and throughput

Reversed Phase Column Chemistries
Hybrid Silica
BEH Technology CSH Technology HSS Technology CORTECS
(XBridge) (XSelect) (XSelect) Solid--Core Technology
Solid
C18
+
+
+
+++
C18+ ++
C8
Phenyl
T3
Shield RP18

From
Column Selectivity Chart

 Search alternative to
your existing column

 Search column by
USP designation

 Search column by
compound class

Column Coach is Mobile Friendly

Caffeine
pKa 10.4 (at 40 °C)
 Buffer- Transfer 6.8 g of monobasic potassium phosphate to 1L volumetric flask.

Dissolve the contents in 900 ml of water. Adjust with phosphoric acid to pH of 2.0. Dilute
to water to volume, add 0.2 ml of triethylamine, and mix well.
 Mobile phase- Prepare a mixture of Buffer an acetonitrile(84:16) and degas. Make
adjustements if necessary.
 Column 4.0 x 250 mm, 10 um, L1. Tailing Factor NMT 2.0

Silica Ionization
Hydrophobic Interaction with Ion exchange Interaction

Bonded Phase with Charged Sites
High Silanol Activity
O-Si O-Si
O-Si O-Si
OH O-
O-Si O-Si
Mobile O-Si +(CH ) O-Si
-
OH HN 3 2 +
O (CH3)2HN
Phase pH < 3 O-Si O-Si
OH O-
O-Si O-Si Si – O
Si - OH O-Si
O-Si O-Si
OH O-Si Mobile
O-Si O- pH > 5
O--Si
N
O-Si
Substrate Protonated - no charge
O-Si
Phase
Substrate De-protonated -- Negative Charge
-
Base Increased
Base retention and
RP Cation X
POOR Peak
Shape
SAME COLUMN

Tailing Factor

C18
Modern C18
2
1
2 3 4 5 6 7 8
Buffer pH
Hybrid Silica
? Solid
C18
+
+
+
+++
C18+
?
++
? C8
Phenyl
T3 ?
Shield RP18 ?

Dibucaine
Reported pKa = 8.9
 Eluent: 1.2 g sodium lauryl sulfate (sodium dodecyl sulfate), 0.2g

sodium acetate, 2.0 ml triethylamine in 300 ml water, adjust pH to 5.6
and add 700 ml methanol
 Column: 3.9 x 300 mm L1. Tailing Factor NMT 3.0

Silica Ionization

O-Si O-Si
O-Si O-Si
OH O-
O-Si O-Si
-
OH HN 3 2 +
O (CH3)2HN
OH O-
O-Si O-Si Si – O
Si - OH O-Si
O-Si O-Si
OH O-Si Mobile
O-Si O- pH > 5
O--Si
N
O-Si
O-Si
Phase
-
Base Increased
Base retention and
RP Cation X
POOR Peak
Shape
SAME COLUMN

Tailing Factor

C18
Modern C18
2
1
2 3 4 5 6 7 8
Buffer pH
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

Hybrid Silica
Solid
C18
+
+
+
+++
C18+ ++
C8
Phenyl
T3
Shield RP18

Quinidine
pKa 8.56 (at 25 °C)
 Solution A: Add 35 ml of methanesulfonic acid to 20 ml of glacial acetic acid,

and dilute with water to 500 ml
 Solution B: Dissolve 10 ml of Diethylamine in water to obtain 100 ml of solution
 Mobile Phase Acetonitrile, Solution A, Solution B, and Water (10:1:1:40)
 Column 4.6 x 250 mm, 5 um, L1.

Silica Ionization

O-Si O-Si
O-Si O-Si
OH O-
O-Si O-Si
-
OH HN 3 2 +
O (CH3)2HN
OH O-
O-Si O-Si Si – O
Si - OH O-Si
O-Si O-Si
OH O-Si Mobile
O-Si O- pH > 5
O--Si
N
O-Si
O-Si
Phase
-
Base Increased
Base retention and
RP Cation X
POOR Peak
Shape
SAME COLUMN

Tailing Factor

C18
Modern C18
2
1
2 3 4 5 6 7 8
Buffer pH
Hybrid Silica
Solid
C18
+
+
+
+++
C18+ ++
C8
Phenyl
T3
Shield RP18

Alprostadil
pKa 4.85 (at 25 °C)
 Eluent: Prepare a filtered and degassed mixture of methanol,

acetonitrile, and 0.02 M monobasic potassium phosphate (2:1:1) and
adjust with phosphoric acid to pH 3. Make adjustments if necessary.
 Column: 4.6 x 250 mm L1.

Impact on Retention Factor (k) on Robustness By
Changing Mobile Phase pH Range - HPLC
100
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
Hybrid Silica
Solid
? C18
?
+
+
+
+++
C18+ ++
C8
Phenyl
T3
Shield RP18

Tioconazole (Imidazole Antifungal – Basic Compound)
and Related Compounds A, B, and C
Predicted pKa = 6.19
Structures for Related

Compounds A-C
Unknown
 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.

USP Example of Using Silica Based C18 at High
pH Tioconazole (Imidazole Antifungal—Basic Compound) and
Related Compounds A, B, and C Cl
Predicted pKa = 6.19

Mix of Mix of ACN/MeOH
N
Structures for Related Cl Cl
for selectivity?
Compounds A-C Unknown O
N
Needed to use high pH
S
• USP 24 Chromatographic Related Compounds

• 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 (high pH)
• Column(s): 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(Silica C18).
Why is it there???
Silica Injector C18 Silica

MP Detector
Pump
USP Example of Using Silica Based C18 at High
pH Tioconazole (Imidazole Antifungal—Basic Compound) and
Related Compounds A, B, and C Cl
Mix of Mix of ACN/MeOH for selectivity?

Predicted pKa = 6.19 Needed to use high pH
Has to use daily saturator/sacrificial
N
Structures for Related Cl Cl silica column for lifetime
ACN/MeOH for selectivity?
Compounds A-C Unknown O
N
S Needed to use high pH
• USP 24 Chromatographic Related Compounds

• 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 (high pH)
• Column(s): 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(Silica C18).
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)
Silica Injector C18 Silica

MP Detector
Pump
Silica Ionization

O-Si O-Si
O-Si O-Si
OH O-
O-Si O-Si
-
OH HN 3 2 +
O (CH3)2HN
OH O-
O-Si O-Si Si – O
Si - OH O-Si
O-Si O-Si
OH O-Si Mobile
O-Si O- pH > 5
O--Si
N
O-Si
O-Si
Phase
-
Base Increased
Base retention and
RP Cation X
POOR Peak
Shape
SAME COLUMN

Tailing Factor

C18
Modern C18
2
1
2 3 4 5 6 7 8
Buffer pH
Impact on Retention Factor (k) on Robustness By
Changing Mobile Phase pH Range - HPLC
100
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
Hybrid Silica
Solid
C18
+
+
+
+++
C18+ ++
C8
Phenyl
T3
Shield RP18

Succinylcholine
Nicotinic acetilcholine receptor agonist
 Mobile Phase: Prepare a solution in water containing 3.85 g per L of

sodium 1-pentanesulfonate anhydrous, 2.9 g per L of sodium chloride,
and 1% (v/v) of 1 N sulfuric acid. Prepare a filtered and degassed
mixture of Buffer solution and acetonitrile (95:5)
 Column: A 4.6-mm x 25-cm, 5 um, analytical column that contains
packing L1.
Hybrid Silica
Solid
C18
+
+
+
+++
C18+ ++
C8
Phenyl
T3
Shield RP18

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 H 2C H 2C
Endcap Density
H2C CH H2C CH
CH 2 2
H2C CH
2 Residual silanol 2
H2C
CH2 CH3 CH2 CH2 CH2 CH3
CH3 HH3C Si CH3H
H3C Si H3C Si 3C CH3 H3C Si
CH3 H3C Si CH3H H Si CH3
H H O H H
Si Si Si Si O Si O Si O Si O Si O Si O Si Si Si
O O O O O O
C8 alkyl chains H3C
H3C
Polar analytes easily CH2 Low Coverage
CH2
H2C interact with surface H2C Low Ligand
CH2 and ligands CH2
H2C Density
H2C Residual silanols CH2
CH2 H2C Endcap
H2C
CH2 CH3 CH 2
H3C Si CH3 H3C CH3 H3C H3C Si CH3 H3C CH3
H H Si CH3H Si CH3 H H H H H Si CH
3
O O O O O O O O O O O O O
Si Si Si Si Si Si Si Si Si Si Si
O Si
O O O O O O O
O O O
O
Low
Alkyl chains Coverage
O H2C Low Ligand
Non-polar H2C
portion of CH2 Density
CH2 Polar portion of
analyte interacts
NH analyte interacts H2C ~1.65
with H2C with silanols
bonded phase CH2
N O (hydrogen bonding CH2
(hydrophobic) H and/or ion exchange) H2C
H2C
NH 2
CH2
Residual silanols CH2
H2C Endcap
H2C N
CH2
CH2 CH3 CH3 CH3
H3C
H3C
CH3 H3C CH3
H3C
CH3 O H N Si CH3 H3C
CH3
Si Si Si H H H Si
H H H H
O O O O O O O O H
O O O O
O
Si Si Si Si
O O O O O O Si
O O O O O O O
O O O O O O O
O O O
Questions
Thank you

RP LC Column Selection-Ricardo Martinez

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RP LC Column Selection-Ricardo Martinez

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RP LC Column Selection

©2018 Waters Corporation COMPANY CONFIDENTIAL 1

©2018 Waters Corporation COMPANY CONFIDENTIAL 2

 Buffer- Transfer 6.8 g of monobasic potassium phosphate to 1L volumetric flask.

©2018 Waters Corporation COMPANY CONFIDENTIAL 3

 Select the column that has worked before?

 Select a column from a “similar” application?

 Select a C18 Column?

 Try a Google search and hope?

©2018 Waters Corporation COMPANY CONFIDENTIAL 5

 “There is a wide variety of reversed-phase liquid chromatography

1Kele and Guiochon, J. Chromatogr. A 830, 41 (1999)

©2018 Waters Corporation COMPANY CONFIDENTIAL 6

 Invented “liquid-adsorption column

©2018 Waters Corporation COMPANY CONFIDENTIAL 7

 Tall glass open column filled with sand-

pharmaceutical proteomics clinical food safety environmental

©2018 Waters Corporation COMPANY CONFIDENTIAL 9

©2018 Waters Corporation COMPANY CONFIDENTIAL 10

©2018 Waters Corporation COMPANY CONFIDENTIAL 11

©2018 Waters Corporation COMPANY CONFIDENTIAL 12

Compound Identification Based on Retention Time

250.0 300.0 350.0

©2018 Waters Corporation COMPANY CONFIDENTIAL 16

©2018 Waters Corporation COMPANY CONFIDENTIAL 17

10X Area Count

Sample “A” has 10X the concentration of Acrylamide

Normal Phase Reversed-Phase

Un-bonded Non-Polar Ligand (C18)

©2018 Waters Corporation COMPANY CONFIDENTIAL 19

 Method Validation kits provide three

 Waters uniquely positioned as an

©2018 Waters Corporation COMPANY CONFIDENTIAL 21

©2018 Waters Corporation COMPANY CONFIDENTIAL 22

Acetone (V0) Void Marker 0.020

Naphthalene Neutral 0.010

Acenaphthene Neutral 0.000

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

©2018 Waters Corporation COMPANY CONFIDENTIAL 23

 A reference standard is any standard that is used independently of an assay method

 There are three ways to use reference standards:

 Confirm Proper Instrument & Column Operation First!

Neutrals QCRM  QCRMs Can Help Monitor:

0.020 Failed B Pump – Worn fitting

0.02 Check Valve – Mobile phase error

USP Plate Count USP Tailing

©2018 Waters Corporation COMPANY CONFIDENTIAL 26

©2018 Waters Corporation COMPANY CONFIDENTIAL 27

©2018 Waters Corporation COMPANY CONFIDENTIAL 28

©2018 Waters Corporation COMPANY CONFIDENTIAL 29

H3C C8 Silane “Ligand”

Silica Gel Surface H3C

Physical hindrance due

©2018 Waters Corporation COMPANY CONFIDENTIAL 31

Endcap (2nd Bonding Step)

What do you still see? Silanols!

Note: Difficulty bonding silanols in

This is what causes tailing and excessive retention of bases.

 Surface silanol charge changes with mobile phase pH

Result: Strong interaction (cation exchange) between ionized surface

 Strong interaction between ionized

 Creates potential for cation

©2018 Waters Corporation COMPANY CONFIDENTIAL 35

©2018 Waters Corporation COMPANY CONFIDENTIAL 36