Column Selection Reverse phase
Reversed Phase Chromatography
Introduction to chemically bonded phases.Fighting tailing Introduction to reversed phase selectivityIsocratic reversed phase separation development.
Some general guidelines for selecting RP-bonded phases are summarized below:• C18—Very hydrophobic, retentive, and stable phase. First choice for most separations.
• C8—Preferred for lower organic mobile phase applications for more polar solutes. Note that C8 has similar selectivity as C18 but is much less retentive.
• CN—Less retentive and different selectivity than C8. Note that many CN phases are much less stable than C18.
• Phenyl—For medium-polarity components. Unique selectivity for aromatics
Ligand (Bonded-Phase)
Reversed Phase Chromatography
Introduction to chemically bonded phases.Fighting tailingIntroduction to reversed phase selectivityIsocratic reversed phase separation development.
Minutes0 5 10 15 20 25
Peak Tailing
Integration errorsReduced resolution Reduced sensitivity
AU
0,00
0,02
0,04
0,06
0,08
0,10
0,12
0,14
0,16
Minutes0,00 0,50 1,00 1,50 2,00 2,50 3,00 3,50 4,00 4,50 5,00 5,50
Peak tailing
What Causes Tailing?
Mixed-Mode RetentionHydrophobic interaction with bonded phaseIon exchange interaction with charged sites
Na
RNO
N1
H
2 R3 Si O Si-
H ++
- +R R R1 2 3R
Cl-
+Na+Cl- +
pH 7Cation Exchanger
Why Do You See Poor Peak Shape? Answer: Ionization of Silanols
Surface silanol charge changes with mobile phase pH
OH O
H+Si Si
Behaves as a Cation Exchanger(pH 2) (pH 7)
Result: Strong interaction between ionized surface silanols and
basic analytes (positive charge)
Mixed-Mode Retention:Hydrophobic Interaction
with Bonded PhaseIon Exchange Interaction
with Charged Sites
O-SiO-SiO-O-SiO-SiO-O-SiO-O-SiO-SiO-SiO-
O--SiO-Si
O-SiO-SiOHO-SiO-SiOHO-SiOHO-SiO-SiO-SiOH
O-SiO-Si
(CH3)2HN+(CH3
)2
HN+
Mobile Phase pH < 3
Si - OH
-Mobile Phase pH > 3
Si – OBase
Base
(Strong Base -- Amitriptyline pKa 9.4)
2 3 4 5 6 7 8Buffer pH
1
2
3
4
Taili
ng F
acto
r
Ideal Behavior Pure Polymer – No Silanols
High ligand density and high purity silica gel: Modern C18
Low ligand density and/or high metal content silica gel: Conventional C18
Peak Shape Over Wide pH Range
Si CH3
H3CCH3
O
Endcap
C18 Bonded and “Fully Endcapped” Silica Gel Pore
SiCCCCCCCCCCCCCCCCCCH3C
CH3
O
25 Å“Steric Hindrance”
What do you still see? Silanols!
Fighting tailing
Ion suppressionIon Pairing
Ultra pure silica (Symmetry 1994,Sunfire 2004)Reduced tailing
Embedded Polar groups (Symmetry Shield 1997)Reduced tailingLower tendency to dewet/hydrophobic collapse
Hybrid materials: High pH Ion suppression (XTerra 2000)
Expanded pH rangeReduced tailingHigh efficiency and stability
Why Not Just Run at pH’s Less Than 3 –Ion suppression?
pH 7.0
pH 2.0
Selectivity !!
amitriptyline
nortriptyline
Retention as a Function of pHAcidic Compound
pH
Cap
acity
Fac
tor (
k)
Non-ionized Acid
Ionized Acid
Retention Map : Capacity Factor (k) versus pH
N
COOH
CH3
O
Non-ionized
N
COO
CH3
O
-
Ionized
pKa = 5.8
50%
50%
pKa = pH where 50% of the molecules are
non-ionized
Retention as a Function of pHBasic Compound
pH
Cap
acity
Fac
tor (
k)
Ionized Base
Non-ionized Base
NHCH3
CH3
NCH3
CH3
Non-ionized
Ionized
Reversed-Phase Retention Behavior of Basic Compounds Relative to
Changes in pH
pH
Cap
acity
Fac
tor (
k)
Ionized Base
Non-ionized Base
pKa
± 2± 1
> ± 2 pH units provides stable retention (better reproducibil-ity at flat portions of curve)
Reversed-Phase Retention Behavior of Acidic, Basic and Neutral Compounds Relative
to Changes in pH
0
5
10
15
20
25
30
35
40
0 2 4 6 8 10 12
pH
Cap
acity
Fac
tor (
k) Acid
Base
Neutral
Note: Retention of neutral analytes not affected by pH
To increase retentionin these pH areas Ion-pair chromatographycan be used
Reversed Phase Chromatography
Introduction to chemically bonded phases.Fighting tailing Introduction to reversed phase selectivityIsocratic reversed phase separation development.
Column Selectivity
Hydrophobicity - carbon load.Silanol Group Interaction - selectivity chart.Alternative Column Chemistries
Different bonded phase functionality.Polymer based stationary phase.Hybrid stationary phases.
Different Selectivity and Great Peak Shape on XTerra™ Particles (pH 7 Evaluation Test)
5 10 15 20 250
XTerra™ RP18
Embedded Polar Ligand
TF USP = 1.1
Amitriptyline
Minutes10 20 30 400
XTerra™ MS C18
Straight Chain Ligand
TF USP = 1.3
Peaks1 Gallic Acid2 Epigallocatechin3 Catechin4 Caffeine5 Epicatechin6 Epigallocatechin Gallate7 Gallocatechin Gallate8 Epicatechin Gallate9 Catechin Gallate
Minutes5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00
Minutes5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00
XTerra® RP865
4
32
1
7 89
XTerra® MS C8
6 54
32
17
89
XTerra® Phenyl65
432
1
Minutes5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00
78
9
Reversed-PhaseChromatographyon Different Columns
What chromatographic parameter has primarily changed ?
Bonding Chemistries
SpacerO S iCH3
CH3
O CO
NH
CH2CH2CH2 CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH3
O S iO
CH3
CH2 CHCH3
O S iCH3
CH3
O CO
NH
CH2CH2CH2 CH2CH2CH2CH2CH2CH2CH2CH3
C18
Spacer
C8
Ph
O S iO
CH2CH2CH2CH2CH2CH2CH2CH3
O
O S iO
CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH3
O
Chromatographic Characterization
Mobile Phase: 65/35 Methanol/ 20 mM KH2PO4 Buffer pH 7.05 µm materials in 4.6 x 150 mm columns, 1.0 mL/min, 23°C
Naphthalene
Acenaphthene
N H+
Amitriptyline pKa = 9.4
O NH2+
OH
Propranolol pKa = 9.6
Uracil
HN
N H
O
O
O
O H
O
Butyl Paraben
Testing column characteristics
Tailing factor of two basesamitryptyline and propranolol
Hydrophobic retentionretention of acenapthene
Hydrophobic selectivityRelative retention acenapthene/naphalene
Silanol activityRelative retention acenapthene/amitryptyline and acenapthene/propranolol
Polar interactionRelative retention acenapthene/butyl paraben and acenapthene/dipropylpthalate
Reversed-Phase Selectivity Chart
Hydrophobicity (Log Scale)
YMC-Basic
1
10Platinum ™ C18
Inertsil ® C8
Kromasil ® C8Zorbax ® XDB C 8
Symmetry ® C8
Hypersil ® BDS C 18
Inertsil ® ODS-2Prodigy ™ C18
Symmetry ® C18
Kromasil ® C18
YMC J'Sphere H80
Zorbax ® Rx C 18
Nova-Pak ® C18
Zorbax ® SB C 18
Hypersil ® ODS
Alltima ™ C18YMC J'Sphere M 80
Waters Spherisorb ® ODS 2µBondapak ® C18
Zorbax ® SB C 8
YMC J'Sphere L 80Lichrosorb ® Select B
Waters Spherisorb ® C8
Nova-Pak ® C8
Hypersil ® BDS C 8
Prodigy ™ C8
SymmetryShield ™ RP8
Zorbax ® Rx C 8
Alltima ™ C8
Lichrospher ® Select B
Purospher ® RP18
Nucleosil ® C18
Inertsil ® ODS-3
1 10
Sila
nol A
ctiv
ityWaters Spherisorb ® ODS 1
Resolve® C 18
Zorbax ® XDB C18
Hypersi Hypurity Elitel®
2 3 4 5 6 7 8 9
2
3
4
5
6
7
8
920
(Log
Sca
le)
Reversed-Phase Column Selectivity Chart:The “C18 Zone”
-0.6
-0.3
0
0.3
0.6
0.9
1.2
1.5
1.8
2.1
2.4
2.7
3
3.3
3.6
-1.5 -0.5 0.5 1.5 2.5 3.5
(ln [α
] am
itrip
tylin
e/ac
enap
hthe
ne)
(ln [k] acenaphthene)
The “C18 Zone”(~80% of all separations)
-0.6
-0.3
0
0.3
0.6
0.9
1.2
1.5
1.8
2.1
2.4
2.7
3
3.3
3.6
-1.5 -0.5 0.5 1.5 2.5 3.5
(ln [α
] am
itrip
tylin
e/ac
enap
hthe
ne)
(ln [k] acenaphthene)
The Modern“C18 Zone”
Reversed-Phase Column Selectivity Chart:
The Modern “C18 Zone”
(ln [k] acenaphthene)
XTerra® MS C18
Hypersil® Elite C18
Inertsil® ODS-3Kromasil® C18
Inertsil® ODS-2Hypersil® HyPurity Elite C18
Luna™ C18YMC-Pack™ Pro C18™ Zorbax® Eclipse® XDB C18Zorbax® Rx C18
Prodigy™ C18
Symmetry® C18
SymmetryShield™ RP18
Supelcosil™ LC-ABZ+Plus
Supelcosil™ LC DB-C18
Zorbax® Extend C18
XTerra® RP18
Luna™ C18(2)
Polaris™ C18-A
-0.2
0
0.2
0.4
0.6
0.8
1 1.5 2 2.5 3 3.5
(ln [α
] am
itrip
tylin
e/ac
enap
hthe
ne)
1.0
1.2
Atlantis™ dC18YMC-Pack™ ODS–AQ™
EXPANDED VIEW
ACQUITY UPLC™ C18
ACQUITY UPLC™Resolution through
Efficiency
ChromolithTM RP-18
Nucleosil® C181.5The Modern “C18 Zone”
Resolution through Efficiency
(ln [k] acenaphthene)
XTerra® MS C18
Hypersil® Elite C18
Inertsil® ODS-3Kromasil® C18
Inertsil® ODS-2Hypersil® HyPurity Elite C18
Luna™ C18YMC-Pack™ Pro C18™ Zorbax® Eclipse® XDB C18Zorbax® Rx C18
Prodigy™ C18
Symmetry® C18
SymmetryShield™ RP18
Supelcosil™ LC-ABZ+Plus
Supelcosil™ LC DB-C18
Zorbax® Extend C18
XTerra® RP18
Luna™ C18(2)
Polaris™ C18-A
-0.2
0
0.2
0.4
0.6
0.8
1 1.5 2 2.5 3 3.5
(ln [α
] am
itrip
tylin
e/ac
enap
hthe
ne)
1.0
1.2
Atlantis™ dC18YMC-Pack™ ODS–AQ™
EXPANDED VIEW
ACQUITY UPLC™ C18
ACQUITY UPLC™Resolution through
Efficiency
ChromolithTM RP-18
Nucleosil® C181.5The Modern “C18 Zone”
Resolution through Efficiency
Column Differences within the Same Trademark
C18/C8little selectivity difference
Phenylsome selectivity difference
CNlarge selectivity differenceneeds redevelopment of mobile phase stability problems!!!
Embedded polar grouplarge selectivity difference
Reversed Phase Chromatography
Introduction to chemically bonded phases.Fighting tailing Introduction to reversed phase selectivityIsocratic reversed phase separation development.
Separation Development
Select a columnC18, Embedded polar group- ScoutingTest 3 pH rages – ScoutingTest MeOH, AcN - ScoutingOptimizeShort columns (50 to 100mm)are good for fast screening of separation conditions.
Developing a Separation
Our first goal is to adjust the mobile phase strength to elute the sample components at a reasonable k’
Use a high flow rate - k’ is independent of flow rate.Use a short column - k’ is independent of column length. This allows a relatively fast experiment.
Developing a Separation
Select an initial mobile phase.Sample solubilityCompatible with the detectortypically a blend of a “strong” eluent and a “weak” eluent
Always choose a mobile phase that is too strong for the first try. Why?
Developing a Separation
Experimental conditions:Symmetry C18 column, 100 x 4.6 mmMethanol and phosphate/acetate buffer, 1.5 ml / min. (about 1,5 column volumes / min)Alliance 2690 Separation Module UV detection at 250 nm.
5 minutes for new mobile phase to wash through.10 minute chromatogram = k’ of almost 15
Developing a Separation
With optimized k’s, we can look at the selectivity of the separation.
If necessary we can change the mobile phase or the column to change the selectivity as was noted in the theory chapter.
Developing a Separation
A reversed phase exampleIsocratic method development techniqueExcedrin brand analgesic and salicylamide
CaffeineAcetaminophenAspirinSalicylamide
Sample and Acetone Overlay
80% MeOH
Spiked Excedrin Sample
80% MeOH
60%MeOH
Spiked Excedrin Sample
60% MeOH
50% MeOH
Spiked Excedrin Sample
50% MeOH
40% MeOH
Spiked Excedrin Sample
40% MeOH
30% MeOH
Spiked Excedrin Sample
30% MeOH
20% MeOH
Void Volume Overlay Vs 30% MeOH
Vo
Optimizing pHpH 2.8 vs pH 3.3
pH 2.8
pH 3.3