Reverse Phase Chromatography

Reversed Phase HPLC
Chromatographic Process
Reversed Phase Advanced Features

Dr. Shulamit Levin
Analytical Department
B+A
Hydrophobic Stationary Phase
Mobile phase Water, Buffers MeOH, Acetonitril, IPA
Medtechnica
Email: levins @medtechnica .co.il shulal@zahav .net.il
A B Distribution: K = Cs /Cm B (More Hydrophobic)
Tel: 03-9254040 Cell: 052-448632 Fax: 03-9249977 Home page: http://www.forumsci.co.il/HPLC
A Elution through the Column Chromatogram
Reversed-Phase Chromatography
Non-polar stationary phase
O-S O-Si i OH O- Si O- Si OH O-Si OH O- Si O-Si O- Si OH O- Si O- Si
RP Method Development Tools
Polar mobile phase Analyte Y Non-Polar Analyte X Polar

Flow
low C18
Phenyl C4 Columns C8 CN
MeOH
pH Additives
Solvents MeCN THF
Well retained (like attracts like)
Poorly retained
high
Dr. Shulamit Levin, Medtechnica

levins@medtechnica.co.il
Reversed Phase HPLC

ELUTION ORDER IN REVERSED PHASE
1 R E S P O N S E 2 3
LIPOPHYLIC
3
0.30
Reversed Phase Elution Order

OH
2
CH 3
3
CH3
0.25
OH
0.20
CH3
O
AU 0.15
1
OH
2
OH
O
0.10
H3 C
H3C
VOID 1
OH
0.05
Methylparabene 2 3
OH
0.00
0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00
OH
Propylparabene
OH
TIME (MIN.)
Minutes
PAH Analysis with Alliance System and PDA Using a Binary Gradient
UV@254nm Column- HibarRT 125-4 LiChrosphere PAH Eluent A: Water Eluent B: Acetonitrile Gradient: Linear A to B 11 minutes, Hold 10 minutes Back to initial conditions Flow Rate: 1.0 ml/min Injection: 20ul
11 5 2
0.06 0.04 0.02 0.00 4.00 6.00 8.00 10.00 12.00 Minutes 14.00 16.00 18.00 20.00
Hydrophobicity
3.0
6
0.16 0.14 0.12 0.10 AU 0.08
10 4 7 3 8 9 12 13 14 15
16
1- Naphthalene - 20 ppm 2- Acenaphthylene- 40 ppm 3- Acenaphthene- 20 ppm 4- Fluorene- 4 ppm 5- Phenanthrene- 2 ppm 6- Anthracene- 2 ppm 7- Fluoranthene- 4 ppm 8- Pyrene- 2 ppm 9- Benzo(a)anthracene- 2 ppm 10- Chrysene- 2 ppm 11- Benzo(b) fluoranthene- 4 ppm 12- Benzo(k) fluoranthene- 2 ppm 13- Benzo(a) pyrene- 2 ppm 14- Dibenzo(a,h) anthracene- 4 ppm 15- Benzo(g,h,I) perylene-4 ppm 16- Indeno(1,2,3-cd)pyrene-2 ppm
7 e
log k'
4
2.0
b a 3 1 c 2 d
1.0 1.0 2.0 3.0
log P

Reversed Phase HPLC
ISOCRATIC vs GRADIENT
9
IONIZABLE
11
R4 R 1 N R3 R2
AMINES - 1,2,3,4
R E S P 0 O N S E
2 3 4 5
10
O C
CARBOXYLIC ACIDS
OH
VOID
NH R OH
9 1 2 3 4 5 67 8 10 11
OH R O S OH O
SULPHATES
ALCOHOLS
OH R O P OH O
PHOSPHATES
OH R P OH O
PHOSPHONATES
OH R S OH O
SULPHONATES
0 VOID
R SH
THIOLS
TIME (MIN.)
MOBILE PHASE
* * * * * * TYPE OF MODIFIER (MeOH, ACN) SOLVENT STRENGTH (% modifier) pH TYPE OF BUFFER (phosphate, acetate) IONIC STRENGTH (Salts, buffer concentration) ION-PAIRING REAGENTS (alkyl-amines, -sulfonates) * * * * * *
MOBILE PHASE
TYPE OF MODIFIER (MeOH, ACN)
SOLVENT STRENGTH (% modifier) pH TYPE OF BUFFER (phosphate, acetate) IONIC STRENGTH (Salts, buffer concentration) ION-PAIRING REAGENTS (alkyl-amines, -sulfonates)

Reversed Phase HPLC

OPTIMIZATION: CHOICE OF SOLVENTS
MOBILE PHASE
* * TYPE OF MODIFIER (MeOH, ACN)
20% MeOH
SOLVENT STRENGTH (% modifier)

pH TYPE OF BUFFER (phosphate, acetate) IONIC STRENGTH (Salts, buffer concentration) ION-PAIRING REAGENTS (alkyl-amines, -sulfonates)
20% ACN
* *
20% EtOH
* *
20% THF
main solvent: H2 O
SOLVENT STRENGTH Analyte Retention as a Function of % Modifier
OPTIMIZATION: % SOLVENTS
20% MODIFIER
k (retention) for each analyte changes independently as % Modifier changes. Thus, the resolution between peaks changes.
ln k peak 8 peak 9 peak 10 peak 11
40% MODIFIER
60% MODIFIER
% Acetonitrile
80% MODIFIER

Reversed Phase HPLC
MOBILE PHASE
* * * * * * TYPE OF MODIFIER (MeOH, ACN) SOLVENT STRENGTH (% modifier)
Ionization of Acids and Bases

Dissociation of Molecule
Acid HA
(Un-ionized) 50% 100% 0% @ pKa Low pH High pH
pH
TYPE OF BUFFER (phosphate, acetate) IONIC STRENGTH (Salts, buffer concentration) ION-PAIRING REAGENTS (alkyl-amines, -sulfonates)
+ A
(Ionized) 50% 0% 100%
Ionization of Acids and Bases

Dissociation of Molecule
Retention Factor versus pH for Acids, Basesand Neutrals
Retention Factor -Linear
Acids (HA)
( pKa =4.8 ) Un-Ionized
k =
k HA + k
a +
K [H ]
a +
B
Un-Ionized
Base B
(Un-ionized) 50% 0% 100% @ pKa Low pH High pH
K 1+ [H
Neutral
BH
+ OH
(Ionized) 50% 100% 0%
ABases (BH )
( pKa = 9.0 ) - Ionized +
Ionized
10 11 12 13 14
pH
Apparent pKa

Reversed Phase HPLC

Resolution of Two Acidic Compounds at Different Mobile Phase pHs
Enhanced Resolution of Basic Compounds at High pH
Silica gel high pH limit
k versus pH
CH3
Amitriptyline
Capacity Factor (k)
pH 2
0 1 Minutes 2 3
Best retention
Decreasing retention Critical pH control necessary
Indoprofen
Ketoprofen
Silanols ionized
CH3
Amitriptyline
10
Nortriptyline
Amitriptyline
N H CH3
pH 8
Nortriptyline
pH control critical 2 Amitriptyline 3
Silanols Un-ionized
8 pH 2
Nortriptyline
Minutes
pH 2
0 1 Minutes 2 0
pH 8
1 Minutes 2
Nortriptyline
pH 10
Phoebe, Tran
pH
Minutes
Dependence of Selectivity on pH
23
0.115
Impact of pH on the Retention of a Zwitterionic Compound
5 4
pH 2.5
AU
6
0.115
12
3 4 5
Conditions: Column:
pH 5.0
AU
0.075
0.075
XTerra RP 18, 3.9 X 150 mm, 5 m Mobile Phase: 65% 20 mM Buffers, 35% ACN Column Temp.: 30 C Flow Rate: Detector: 1.0 mL/min 210nm for pH 2.5, pH 5.0, and pH 7.0; 230nm for pH 10.6
Aqueous pKa = 4.3
Aqueous pKa = 9.5

Negative Charge
0.035
0.035
6
4 8 12
-0.005 0
12
16
20
24
28
-0.005 0
Time (min)
0.135 0.121 0.107 0.093 0.079 0.025
Time (min)
16
0.015
Positive Charge
1 6 2
pH 8.0
AU
5 3 4
4 8 12 16 20
pH 10.6
2 53 4
AU
0.065 0.051 0.037 0.023 0.009 -0.005 0
1:acetaminophen 2:lidocaine 3:doxepin 4:imipramine 5:nortriptyline 6:ibuprofen
Dual Charge
pH
H N OH
0.005
-0.005 0
10
20
30
40
50
Time (min) Lu, Cheng; Waters
Time (min)
pH the most powerful selectivity tool
Fexofenadine (Antihistamine)
C H3 C H3 C OO -
+
HO

Reversed Phase HPLC

UV/Vis Spectral Change Between Ionized and Non-ionized Forms
Typical Chromatograms for pH Failure of an Ordinary C18-Silica Column
1.00 1.20 1.00 0.80 AU 0.60 0.40 0.20 0.00 220 240 260 280 300 320 340 360 380 nm
252 277 286 216
2 4
Astemizole
254 nm pH 2.0 pH 10.0
0.80 0.60 0.40 0.20 0.00
Ketoconazole
3
Initial
5
254 nm pH 2.0 pH 10.0

293
1. Uracil 2. Propranolol 3. Naphthalene 4. Acenaphthene 5. Amitriptyline
1 2 4 5
After failure
3
220 240 260 280 300 320 340 360 380 nm
Phoebe, Tran
40 minutes
MOBILE PHASE
* * * * TYPE OF MODIFIER (MeOH, ACN) SOLVENT STRENGTH (% modifier) pH
Recommended Buffers for pHs 2-7

Additive or Buffer TFA Acetic Acid Acetate pK a pH range ( 1 pH unit) Volatile or NonVolatile Volatile Volatile Volatile Volatile/No n-volatile Volatile/No n-volatile Nonvolatile Nonvolatile Recommended for use with Extended pH Packings Yes (0.02 -0.1%) Yes (0.1 -1.0%) Yes (0.1 -1.0%) Yes (1-10mM) NH 4, Na, K Yes (1-10mM) NH 4, Na, K Yes No for pHs >7.0 (lower the
temperature the longer the column lifetime)
0.3 4.76 4.76 3.76 5.76 3.75 2.75 4.75 2.15 1.15 3.15 7.20 6.20 8.20
Formic Acid 3.75
TYPE OF BUFFER (phosphate, acetate) IONIC STRENGTH (Salts, buffer concentration)

ION-PAIRING REAGENTS (alkyl-amines, -sulfonates)
Formate Phosphate
*
*

Reversed Phase HPLC

Types of Buffers and Ionic Strength
MOBILE PHASE
* * * TYPE OF MODIFIER (MeOH, ACN) SOLVENT STRENGTH (% modifier) pH TYPE OF BUFFER (phosphate, acetate) IONIC STRENGTH (Salts, buffer concentration)
pH 10: Borate
20 mM H 3 BO 3
pH 7: Phosphate
20 mM K 2HPO
4
pH 4-5: Acetate
10 mM CH
3
COONH
* * *
100 mM CH 3COOH
pH 2-3.5: Phosphate
20 mM H 3 PO4 - KH 2PO 4
ION-PAIRING REAGENTS (alkyl-amines, -sulfonates)
Ion Pair Reagent

Alkylamines
CH3CH2 Triethylamine (TEA) CH3CH2 N+ OHCH3CH2
Concentration of Ion-Pair Reagent in the Mobile Phase
Alkylsulfonates
Pentanesulfonate - Na+ SO 3 SO3 - Na+
The larger the alkyl, the longer are retention times

The larger alkyls saturate the stationary phase at lower concentrations
C8 C7 C6
CH2CH2 CH3 CH2 CH3CH2 CH2 CH2
Hexanesulfonate
OH-
N+ CH2CH2CH2CH3 CH2CH2CH2 CH3
Heptanesulfonate
SO3 Na+ SO 3- Na+ SO 3 Na+
k'
C5
Tetrabutylamine (TBA)
Octanesulfonate
H3C CH3CH2 CH2 CH2 OHN+ CH2CH2CH2CH3 CH3 Dibutyl -dimethylamine
Dodecylsulfonate
5 mM Conc. of Ion Pair Reagent in the Mobile Phase

Reversed Phase HPLC

Stationary Phase Characterization
The Evolution of the Silica Gel Particle Platform
1960s 1970s 1980s 1990s 2000s
Pellicular native silica Irregular 10 m native silica Spherical 5 m native silica Spherical 3-5 m high purity silica Hybride Silica-Gel (co-polymer organic/Inorganic) high purity silica
Improvement in Peak Shape for Bases
neutral
Conventional C 18 base 1970's base Modern C18

Early 1990's
Not all C18s are the same!
neutral
base neutral
Embedded Polar Technology -- Today
neutral
0
base
Hybride Silica Technology -- Today

50
Time (min)

Reversed Phase HPLC

Different Columns Different Chromatograms
7
V0 = 0.86 min
Different Columns Different Chromatograms

7
V0 = 1.05 min
3 4 5 6 8
Symmetry C 18
9 10
3 4 5
9 6 8
YMC Hydrosphere
10
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00 9.00 Minutes
10.00
11.00 12.00
13.00
14.00 15.00
1.00
2.00
3.00
4.00
5.00
6.00
V0 = 0.92 min
2 3 4 5
7 6 8
9 10
SymmetryShield RP18
V0 = 0.93 min
7.00 8.00 Minutes
9.00
10.00
11.00
12.00
13.00
14.00
15.00
Phenomenex Luna
8 9 10
7 2 3 4 5 6
1
1.00 2.00 3.00
4.00
5.00
6.00
7.00 8.00 Minutes
9.00
10.00
11.00
12.00
13.00
14.00
15.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 Minutes 9.00 10.00 11.00 12.00 13.00 14.00 15.00
YMC ODS AQ
8 9 10
7
V0 = 1.00 min
V0 = 0.98 min
3 4 5
3 4 5 6
9 8
Phenomenex Aqua
10
1
7.00 8.00 Minutes 9.00 10.00 11.00 12.00 13.00 14.00 15.00
1.00
2.00
3.00
4.00
5.00
6.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00 8.00 Minutes
9.00
10.00
11.00
12.00
13.00
14.00
15.00
Relative Ranking of C18 Columns Using a Standardized Test
Making a Bonded Phase Material: Monofunctional Synthesis
O n
OH Si O
H3 C Cl Si
C8 Silane Ligand
C C CH 3 C C C C C CH 3
There are no bad C18 columns. There are only different C18 columns.
Silica Gel Surface

O Si O O
H3 C Si O
C C CH 3
C C
C C
C CH 3
+ HCl

10
Reversed Phase HPLC

Surface of a Silica Gel Bonded-Phase Packing Material
C8 alkyl chains
CH2 H 2C CH2 H 2C CH2 H 2C H3 C H O Si O O O H2 C residual silanol CH 2 Si O Si O CH 3 H 3C H O Si O CH 3 Si O CH 2 HC CH 3 3 H O Si O O O Si O Si O O CH 3 H O Si O O H2C CH 2 H2C CH 2 H2 C CH 2 H 2C CH 2 H 3C Si O Si O O
Stationary Phase Properties

H3C
H 3C
H3C CH 2
H3 C CH 2 H2 C CH 2
CH 2 H2C CH 2 H2C CH 2 H 2C CH 2 H 3C H O Si O O
CHEMISTRY: * BONDED HYDROCARBON: C-18, C-8, C-4, C-1, CN, phenyl * % COVERAGE * TYPE OF SILICA GEL
endcap
CH 3
CH 3
Si O Si
CH 3 H3C H O O O Si O H Si O Si O O
CH 3
GEOMETRY * SPHERE- IRREGULAR * PARTICLE DIAMETER * POROSITY
Si O O
Note: ~50% of the surface silanols remain even with high bonding densities
Stationary Phase Ligands

Stationary phase
C
18 8 2
Neutral Compounds: C18 versus C4 (Same Brand Different Ligands)
Functionality
Si(CH ) C H
3 2 18 37
Butylparaben
Note: Similar selectivity due to same silica particle.

Acenaphthene
Naphthalene
Si(CH ) C H
3 2 5 2 3 3 2 2 8
17
tC
SiC H
2
Aminopropyl Cyanopropyl Diol
Si(CH ) NH
Si(CH ) (CH ) CN
2 3
Si (CH 2 )3 O CH 2CH(OH)CH Retention time
OH
YMC-Pack Pro C18 YMC-Pack Pro C4 0 4 8 12 Minutes 16 20 24
Chain length CN Phenyl NH2 C4 C8 C18

11
Reversed Phase HPLC
Type of Ligands
Reversed-Phase Packing with an Embedded Polar Ligand

Polar Group
O Si CH3 CH3
Embedded Polar Group Ligand
O Si CH3 CH3
Traditional, Straight Chain Alkyl Ligand
Commercial Phases with Embedded Polar Group
Embedded Polar Ligand: Possible Mechanism

Polar group increases water concentration in surface layer
Me Si Me O
O C N H R
SymmetryShield RP (Waters)
O H H
O C Si O Si O C H3 C C O C N H (CH 2)7 C H3
O Si N H C R Discovery RP Amide16 (Supelco)
O Si O O
O
OH3 C
Si
Me
- reduces
N H
Me Si Me
O C
N H
Prism, Spectrum (Keystone)
interaction with silanols selectivity water wettability

Me N H
O C R
- modifies - improves
Shields Negative Silanols

Bonus RP (Hewlett Packard)
No additional hydrophobic retention Reduced retention of bases Reduced peak tailing

12
Reversed Phase HPLC
Embedded Polar Goups

Embedded Polar Wetted Surface
Water Water "Shield" "Shield" Layer Layer
O O
Mixed-Mode Retention:
Hydrophobic Interaction with Bonded Phase
O-Si O-Si OH O- Si O- Si OH O- Si OH O- Si O- Si O-Si OH O- Si O-Si
Ion exchange Interaction with Charged Sites

O- Si O- Si OO- Si O-Si + O- (CH ) HN 32 O- Si OO- Si O- Si O- Si OO-- Si O- Si
H N
C
H N
O C O
Water layer repels Carbon chain -- keeping ligand extended -- maintaining retention
Mobile Phase pH < 3
+ HN (CH 3 )
Embedded Polar Ligand versus Linear Alkyl Ligand on Silica Gel
Silica Surface
Si - OH
Me Si
Me O Si Me O C
Me
O
Me Si O
Me
Me Si O
Me O C O
H N
Mobile Phase pH > 3
Si O -
H N
Base
Base
Impact on Selectivity - Retention

Mobile Phase: 20 % CH 80 % pH 3 KH PO
2 3 4
CN Buffer
Amitriptyline SymmetryShield RP18 TF USP = 1.13

SymmetryShield RP 8
3 4 1 2 Sample: 1. Benzyl Alcohol 2. Phenol 3. Benzoic Acid 4. Benzylamine
3 4 1 2
Symmetry C18 TF USP = 1.95
Symmetry C
10
Time (minutes)
15
10 Minutes
20
30
B. A. Alden
Reduced Retention of Amines

(Shielding of Silanols )

13
Reversed Phase HPLC

Selectivity Difference: Furazolidone Impurities
SymmetryShield RP8
0.01 0.01

CHEMISTRY: * BONDED HYDROCARBON: C-18, C-8, C-4, C-1, CN, phenyl * % COVERAGE * TYPE OF SILICA GEL GEOMETRY * SPHERE- IRREGULAR * PARTICLE DIAMETER * POROSITY
Symmetry C8
0.008
0.008
0.006
0.006
0.004
0.004
0.002
0.002
-0.002 0 10 20 30
-0.002 0 10 20 30
El Fallah
minutes
minutes
CARBON LOAD
H3C

H3C CH 2 H2C CH 2 H 2C CH2 H 2C H 2C CH2 CH 2 CH2 H 2C CH 2 H 2C H 3C CH 2 H 2C CH 2 H 3C CH 2
Increasing carbon load on a similar geometrical shaped particles increases retention.

O Si O
H 2C H3C H
CH 2 Endcap H2C H2 C Residual silanol H2 C CH2 CH2 CH 2 CH 3 CH 2 CH 3 3C H 3C H H 3 C H 3 C CH 3 H3 C CH 3 CH3 CH3 CH3 H Si CH 3 Si Si Si Si Si H H H H O H O O O O O O O O O O O Si Si Si Si Si Si Si Si Si Si O Si O O O O O O O O O O O O O O O O O O O O O CH 2
H2C
High Coverage High Ligand Density
Retention time
H3 C CH 2 H2 C CH 2
C8 alkyl chains
Polar analytes easily interact with surface
H 3C CH 2 H 2C CH 2 H 2C
Low Coverage Low Ligand Density
Carbon load 5% 7% 9% 12% 15% 17%

O Si O
H2 C H 2C H3C H CH 2
Residual silanols
CH 2 CH 3 CH 3 CH 3 H3 C Si CH 3 H CH3 H 3C Si Si H H O O O O O O Si Si Si Si Si Si O O O O O O O O O O O O
H O O Si O
CH 2 H2C Endcap CH 2 CH 3 H3 C CH 3 H 3C Si CH 3 Si H H O H O O O O Si Si Si O O O O Si O O O O

14
Reversed Phase HPLC

Silica based "bonded phases"
H 3C
C8 alkyl chains
CH2
H3C CH 2 H2C
H3 C CH 2 H2 C CH 2 CH 2 H2 C CH 2 CH 2 H 2C CH 2 CH 3 H 3C H O Si O O O Si O Si O O
H3C CH 2 H2C CH 2 H2C CH 2 H 2C CH 2 CH 3 H 3C H O Si O O Si O Si O CH 3 H3C H O O Si O H Si O Si O O O
H 2C CH2 H 2C CH2 H 2C H3 C H
H2C H2 C residual silanol
endcap
CH 3 CH 3
CH 2 Si O Si O O O O CH 3 H 3C H O Si O
CH 3 Si O
CH 2 HC CH 3 3 H O Si O O O Si O Si O
Bulky alkylsilane ligands can not react with all available silanols due to the steric hindrance.
O Si
Si O O
Note: ~50% of the surface silanols remain even with high bonding densities
Ligand Density (Surface Coverage)

=
2
Better Way to Compare: Ligand Density (Surface Coverage)

%C
Ligand Density (Surface Coverage)

moles/m Silica Silanols : Highest Bonding Reported : 6 -8 4.2
100
SA
nC
12
1 - %C 100
MW - 1 nC 12
moles/m
SA - Specific Surface Area MW - Molecular Weight of Ligand
%C % Carbon Load nC - # of Carbon Atoms in Ligand
Residual Silanols (Best Case)
2.0
[ ~ 30% ]
Ligand Density
Retention
Silanols
Residual Silanols (Typical)
> 3.5
[ > 50% ]
Surface Area
%C
Ligand Density

15
Reversed Phase HPLC

Acidic Compounds:C18 versus C8 (Same Brand Different Ligand)
Relative Hydrophobicities of General Purpose Analytical Packings
Suprofe n Naproxen Fenoprofen
Diclofenac
Note: Similar selectivity due to same silica particle.
Inertsil ODS-2
Symmetry C18
Puresil C18 Zorbax Rx C18
Nova-Pak C18
Inertsil C8
Symmetry C8 YMC-Pack Pro C18 YMC-Pack Pro C8 Bondapak C18 0 4 8 Minutes 12 16 20 0 5 10 15

Zorbax Rx C8
Nova-Pak C8
k' acenapthene

CHEMISTRY: * BONDED HYDROCARBON: C-18, C-8, C-4, C-1, CN, phenyl * % COVERAGE * TYPE OF SILICA GEL Native/Synthetic-Pure GEOMETRY * SPHERE- IRREGULAR * PARTICLE DIAMETER * POROSITY
Types of Silica
Silanols pH stability Metal content Temperature stability

16
Reversed Phase HPLC
Structure of Silica Gel
What are Silanols?

Silanol (Si-O-H)
=H =O = Si
Silicon Oxygen
Amorphous, porous matrix of silicon atoms joined together with oxygen atoms to form siloxane bonds = (Si O Si)
Residual unreacted surface hydroxyl groups left over from polymerization Reactive sites for use in bonding ligands (C18) to the silica gel surface
Surface Silanols Found on Silica Gel

H H
Mixed-Mode Retention:
O O O Si O
Mobile Phase pH < 3
Vicinal (Bridged)
O O Si O
O
Hydrophobic Interaction with Bonded Phase

O-Si O-Si OH O- Si O- Si OH O- Si OH O- Si O- Si O-Si OH O- Si O-Si
Ion exchange Interaction with Charged Sites

O- Si O- Si OO- Si O-Si + O- (CH ) HN 32 O- Si OO- Si O- Si O- Si OO-- Si O- Si
HO OH O Si O Si O
OH O
O Si O
+ HN (CH 3 )
Si - OH
Geminal (Silanediol)
Mobile Phase pH > 3
OH
Si O -
Lone (most active)
O Si
O Si
Base
Base

17
Reversed Phase HPLC

Bonded Phase on Particles Bonded Hybrid versus Bonded Silica Gel Surfaces
H3 C
C 18 H 37 C 18 H 37 CH 3 CH 3 H 3 C CH 3 H3 C CH 3 Si Si Si OH OH OH
C18-Bonded Silica Gel 1/2 -OSi(CH3)2R 1/2 -OH
CH3
C18 H37 CH3 CH3 H3 C CH3 H3 C Si Si OH CH3 OH
C18-Bonded Hybrid 1/3 -OSi(CH3)2R 1/3 -CH3 1/3 -OH
Silica Gel C18 Materials 1/2 free silanols
XTerra C18 Materials 1/3 free silanols
Hybrid versus Silica Gel Particle
PERFORMANCE BY ONE PEAK

ASYMMETRY FACTOR
Af =
RETENTION FACTOR or CAPACITY RATIO
B (10% h)
(10%
h)
Amitriptyline
k' =
tR - t 0 t0 t
k =
Cs Cm
TAILING FACTOR
Tf =
(5% h)
A+B 2A
Symmetry C18 TF USP = 1.95 XTerra MS C18 TF USP = 1.35 0 10 20 Minutes 30 40
NUMBER OF THEORETICAL PLATES
t0 w
N = 16
tR w

18
Reversed Phase HPLC

Amitriptyline Peak Tailing Over Extended pH Range 1-12 Integration Errors Caused by Tailing
T = 1.00
T = 1.58 Recovered Peak Areas 97.8 % 95.3 % 92.3 %
Tailing Factor USP
Conventional C18
Recovered Peak Areas 99.9 % 99.8 %
Pure Silica C18

2
99.6 %
Ideal Tailing Factor

0 1 2 3 4 5 6 7 8
Hybrid C18
Buffer pH
10
11
12
Types of Silica
pH Limitations of Silica Based Packing Materials
Hydrolysis of Bonded Ligand
pH

19
Reversed Phase HPLC

Hydrolysis of a Bonded Phase Material: Monofunctional Ligand
Typical Chromatograms for Reference C18-Silica Column
O O Si O
OH
H3C Cl Si
C C C H3
C C
C C
C C H3
2 4
+
H3C Si Si O
Initial
3 5
C C CH 3
C C
C C
C CH 3
O O O
+ HCl
1 2 4 5
1. Uracil 2. Propranolol 3. Naphthalene 4. Acenaphthene 5. Amitriptyline
Low pH (hydrolysis of ligand)

OH
After failure
3
O O Si O
H3C Si HO
C C CH 3
C C
C C
C CH3
40 minutes
Types of Silica
Metal Content in Silica Aluminum in the Silica Gel Lattice

Bronsted Acid
O Si Si HO O Si
3D top view of silica particle surface with silanols pointing upward

O O Si O Si OH O
H O
Al
Metal available for chelation

20
Reversed Phase HPLC

Correlation Between Base Tailing and Aluminum Content of Silica Gel Correlation between Metal Content of Silica Gel and Peak Retention and Shape
Propranolo l Methanol Acenaphthene Naphthalene
Waters Symmetry C18

Amitriptylin e
Analyte: Chlorpheniramine Mobile Phase: Acetonitrile/KH2PO 4 pH 3.0 (20:80)

4
Butylparab en
Al conc.< 10 ppm TF USP = 1.95
Propranolol and Butylparaben
Tailing 3 Factor
2 1
"High Purity Silica Gel" Region
10
20 Minutes
30
40
Naphthalene
Waters Nova -Pak C18

Acenaphthene
Methanol
Al conc.= ~375 ppm TF USP = 6.5

Amitriptyline
100 200 300 Aluminum Content, ppm
400
5 15 Minutes 25
35
45
Peak Shapes of Chelating Agent (Hinokitiol)
Types of Silica
Low metals
Mobile phase:20 mM MPhosphate = metal Buffer pH 3.6 with 0.05% EDTA: Acetonitrile (50:50)
O O Si O Mn+ O
Si
High metals
2 4 Minutes 6 8

21
Reversed Phase HPLC

Temperature Effects on Resolution Effect of Temperature
(Isocratic separations)
Resolution can be temperature dependent
N=2250
60 0 C 50 0 C
1160 psi
Higher Temperature: Shorter Run Time Sharper Peaks Better Sensitivity Lower Back Pressure
Temperature can be a critical parameter to control in order to achieve reproducible separations.

N= 1680
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
40 0 C 30 0 C
Minutes
1920 psi
Effect of Temperature on Column Efficiency
Dependence of Retention on Temperature

0.024 0.022 0.020 0.018 0.016 0.014
0.024 0.022
1 2
300 C
AU
0.020 0.018 0.016 0.014 0.012 0.010
1 2
400 C
3
Conditions: Column:
Plate height, H [m]
35 30 5C 25 20 15 10 0 1 2 3 4 5 6 7 8 9 60C 25C
0.012 AU 0.010 0.008 0.006 0.004 0.002
0.008 0.006 0.004 0.002
XTerra MSC18, 2.1 X 50 mm, 2.6 m Mobile Phase: 25% ACN/75% buffer (10 mM, pH5, NH4AC) Flow Rate: 0.6 mL/min Injection Vol.3 L
0.000
0.000
-0.002 0.00 1.00 2.00 3.00 4.00 5.00 6.00 Minutes 7.00 8.00 9.00 10.00 11.00 12.00
-0.002 0.00
1.00
2.00
3.00
4.00
5.00
6.00 Minutes
7.00
8.00
9.00
10.00
11.00
12.00
Detector:
210 nm
0.024 0.022 0.020 0.018 0.016 0.014 0.012 0.010 0.008 0.006 0.004 0.002 0.000 -0.002 0.00
0.024
1 2
500 C
3
AU
0.022 0.020 0.018 0.016 0.014 0.012 0.010 0.008 0.006 0.004 0.002 0.000
1 2
600 C
3
Analyte Conc. (g/ml) 1: doxepin 0.5 2: imipramine 1.0 3: amitriptyline 3.0
AU
1.00
2.00
3.00
4.00
5.00
6.00 Minutes
7.00
8.00
9.00
10.00
11.00
12.00
-0.002 0.00
1.00
2.00
3.00
4.00
5.00
6.00 Minutes
7.00
8.00
9.00
10.00
11.00
12.00
Linear velocity, u [mm/s]
Lu, Cheng
Higher Temp. Shorter Run Time Higher Signal

22
Reversed Phase HPLC

Temperature Effects on Resolution - Gradient
High Temperature Phosphate Buffer Test

0.10
1 4
0.10
0.300 0.200 AU 0.100 0.000 25.00
5, 6
Rs (5,6) = 0 Rs (8,9) = 2.39

7
30.00
10 9 8
35.00
30 C
Conditions - Column: Symmetry300,,C 5 m, 3.9x150mm

18 AU
Day 21
XTerra RP
Peak Number 1. 2. 3. 4.
USP Tailing Factor 1.2 1.1 1.1 1.0
18
AU
11
Minutes
40.00
- Sample: Tryptic digests of bovine cytochrome c - Injection: 20 L

- 0.005
3
- 0.005 4.00 5.00 Minutes 6.00 7.00 8.00 9.00 10.00 1.00 2.00 3.00
Doxepin Nortriptyline Amitriptyline Trimipramine
0.300 0.200 AU 0.100 0.000 25.00
5, 6
Rs (5,6) = 0 Rs (8,9) = 2.07

7
30.00
3
4.00 5.00 6.00 7.00 8.00 9.00 10.00
10 9 8 11
35 C
- Mobile Phase: Solvent A: 0.1% TFA in water Solvent B: 0.1% TFA in acetonitrile - Gradient: 0-45 min., 0-3 0 % B
40.00
1.00
2.00
3.00
0.48
0.26
Minutes
C 18 - silica particle
AU
Day 2
4
Minutes
35.00
0.300 0.200 AU 0.100 0.000 25.00
Rs (5,6) = 0.84 Rs (8,9) = 1.63

5 6 7
30.00
10 9 8 11
40 C
- Flow rate: 0.75 mL/min. - Detection: 214 nm
4 2
- 0.02
AU
3
0.00 5.00 Minutes 6.00 7.00 8.00 9.00 10.00 1.00 2.00 3.00
2
4.00 5.00 Minutes 6.00
3
7.00 8.00 9.00 10.00
1.00
2.00
3.00
4.00
Minutes
35.00
40.00
Tricyclic Antidepressant Separation

Spherical and Irregular particles
Electron microphotograph of spherical and irregular silica particles. [W.R.Melander, C.Horvath, Reversed-Phase Chromatography, in HPLC Advances and Perspectives, V2, Academic Press, 1980]

23
Reversed Phase HPLC

Resolution (Arbitrary Units)
Resolution - Time Diagram

90' s 80' s
12 10 8
70' s
Length / dp 30 cm / 10 m 15 cm / 5 m 9 cm / 3 m
0 1 10 100 1000
Analysis Time [min]
Comparison of the van Deemter Plots for 5 m and 2.5 m XTerra MS C 18 Particles
(50/50, acetonitrile / water mobile phase)
30 28 26 24
Challenge of producing smaller particles

Contains a proportion of 2 m particles
Both are commercial 2m packings
22 20 18 16 14 12 10 8 6 4 2 0
Alden
5 m XTerra Particle
Smaller Particles Higher Flow Rate provide Shorter Run Time Higher Efficiency 2.5 m XTerra Particle
H (m)
Centered at 2.4 m Narrower distribution

4
0.5
Linear Velocity (mm/sec)
1.5
2.5
3.5
(Waters proprietary technology)

24
Reversed Phase HPLC

Fast Gradient Application
Columns: XTerra MS C18 2.1 x 20 mm, 2.5 m; XTerra MS C18 2.1 x 50 mm, 5 m Mobile Phase: A = 0.1% TFA in water, B = 0.08% TFA in MeCN Gradient: 5 - 95% B in 45 seconds and 120 seconds Column Temperature: 60 C Flow Rate: 1.5 mL/min. Detector: 254 nm Injection Volume: 1 L 0.20 Column: XTerra MS C18
Fast Gradient of 12 Standards
2.1 x 20 mm, 2.5 m

Gradient 0 - 100% B in 2.5 min, A: 0.1% TFA in water B: 0.08% TFA in MeCN Flow rate: 1.5 mL/min Temperature: 60 C
0.50 0.40 0.30 AU 0.20
2.5 m 2.1 x 20 mm
AU 0.10
0.10 0.00 0.20 0.80 0.60 AU 0.40 0.20 0.00 0.20 0.40 0.60 0.80 1.00 Minutes 1.20 1.40 1.60 1.80 2.00 0.40 0.60
Minutes
0.80
1.00
1.20
1.40 0.00
5 m 2.1 x 50 mm
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
2.00
2.20
2.40
Minutes
Carmody
Fast LC/MS Applications

2.1 mm 5 m and 2.5 m columns length: 5 cm and 3 cm flow rates 0.2 and 0.6 mL/min Conditions:
n n n n n n n n 100
Fast LC-MS Analysis

XTerra MS C18, 2.1 x 50 mm ( 5 m)
N
2.56
295 8.11e4
100
295
N
4.59e4 296
(4)
0 100 1.57 280 2.21e5
0
N
100
O
(2)
280 1.13e5 281 264 233 265 260 9.25e4 7.67e4
HPLC: 65/35 0.1% formic acid / MeCN 1 L injection of 200 ng/mL of samples MS: ESI+; SIR 4 channels HV: 3.15 kV, Cone 25 V Drying Gas: 380 L/h Source Temp: 175oC
0 100 2.16 264 1.26e5
0 100
NH
(3)
0 100 1.29 260 1.53e5
0 100
O OH N H
(1)
0 100 125 150 175 200 225 250 Mass/Charge (m/z)
261 275 300
0 100 1.57 2.16 1.29 2.56 TIC 3.50e5
1.00
2.00
3.00
4.00
5.00
Ding
Time (min)
10 L injection of 200 ng/mL sample (in 40% MeOH),1= Propranolol, 2= Doxepin, 3= Nortriptyline, 4=Trimipramine, 65/35 0.1 % Formic Acid / MeCN 0.2 mL/min

25
Reversed Phase HPLC

Masschromatograms of Std.10ppm (APcI +/- )
std.,
std_ A
. .
Fast LC-MS Analysis

XTerra MS C18: 5 m vs.. 2.5 m
ppm
Tinuvin 770DF
: Scan AP+ . . e
2
100 0.51 3 0.74 0.91
std_ A
. .
Chmassorb944LD
. . . . . . . . . . . . . . .
: Scan AP+ . . e
1
0.38
2.5 m
TIC 8.06e5
Relative Abundance (%)
std_ A
Tinuvin 326 % std_ A
: Scan AP. . e
2.1 x 30 mm 0.6 mL/min split 0.2 mL/min to MS 0 100
HPLC: 65/35 0.1% formic acid / MeCN 1 L injection of 200 ng/mL of samples MS: ESI+ SIR 4 channels HV: 3.15 kV Cone 25 V Drying Gas: 380 L/h Source Temp: 175oC 1= 2= 3= 4= Propranolol Doxepin Nortriptyline Trimipramine
2 1
1.13
1.44
3
2.04
4
2.44
Irganox 1076 +
% std_ A
. . . . .
. . . . . . . .
: Scan AP+ . . e
5 m
2.1 x 50 mm 0.2 mL/min
TIC 1.15e6
Irganox 1076 % . . . . . . . . .
: Scan AP. . e
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
Time . . .
Ding
Time (min)

Pore size, shape and distribution
Macroporous spherical silica particle. [K.K.Unger, Porous silica, Elsevier , 1979]
Pore size defines an ability of the analyte molecules to penetrate inside the particle and interact with its inner surface. This is especially important because the ratio of the outer particle surface to its inner one is about 1:1000. The surface molecular interaction mainly occurs on the inner particle surface.

26
Reversed Phase HPLC
Pore Size
Most silica gel packings are porous
n
Silica Gel Pore Structure

100
* Silica is Porous * Pore Size, or nm -- distribution * Specific Pore Volume, mL/g Range: 0.3 -- 1.3 mL/g SV Particle Strength
Pore Size Recommendation 60 - 130 (6 - 13 nm) 100 (10 nm) 300 - 1,000 (30 - 100 nm) non- porous
>99% of the surface area is contained within the particle (not on the surface)-Where the chromatography happens.
The smaller the pore size, the greater the surface area. w (100 approx. 300 m2/gram) w (300 approx. 100 m2/gram)
Rules of Thumb
Analyte MW < 3,000 3,000 - 10,000 >10,000 Very Large
The greater the surface area, the greater the retention.
A typical 15 cm column holds a surface area of ~100-300 square meters
Exclusion Inclusion Effects

Ligand
R H
3
Pore Size Effects on Resolution

Conditions
Symmetry, C18, 5m, 4.6 X 150 mm, 100
0.30 10 56 23 1 4 7 9 8 11 12 13
R H
3 3
C Si
CH
C Si
CH
3
0.20 0.10 AU
Micropore 6
O
0.00
Si
Si
CH
3
H
3
-0.10 0.00
20.00 Minutes
40.00
- Sample: Tryptic digests of cytochrome c (bovine) - Injection: 20 L - Mobile Phase: Solvent A: 0.1% TFA in water Solvent B: 0.1% TFA in acetonitrile - Gradient: 0-50 min., 0-30%B - Temperature: 35 C - Flow Rate: 0.75 mL/min. - Detection: 214 nm
Si
CH
3
H
3
Si
O Si
O CH
3
H
3
Si
0.500 0.500
O
5,6 9 2 1 3
20.00
Symmetry300, C , 5 m, 4.6 X 150 mm, 300 10 13

18
O Si O
CH
3
CH
3
0.400 0.400 0.300 0.300 AU 0.200 0.200 0.100 0.100 0.000 0.000 0.00 Minutes
12 11
O Si Si
Si
4 7
- Different pore sizes change selectivity.
XTerra
Matrix
40.00
Carmody

27
Reversed Phase HPLC

Mechanism of Retention of Polar Compounds on Aqueous Columns
Polarity/Aqueous Columns:
Low ligand density High pore volumn
Polar analytes are not able to Fit between ligands cant interact with surface
H3C CH 2 H2C CH 2 H2C H 2C H 2C H3C H O Si O CH 2 H 2C CH2 H 2C CH2 CH 2 H3C CH2 H 2C CH 2 H 2C H 3C CH 2 H 2C CH 2 H 3C CH 2
High Coverage High Ligand Density
CH 2 Endcap H2C H2 C Residual silanol H2 C CH2 CH2 CH 2 CH 3 CH 2 CH 3 3C H 3C H H 3 C H 3 C CH 3 H3 C CH 3 CH3 CH3 CH3 H Si CH 3 Si Si Si Si Si H H H H O H O O O O O O O O O O O Si Si Si Si Si Si Si Si Si Si O Si O O O O O O O O O O O O O O O O O O O O O
H3 C CH 2 H2 C CH 2 H2 C H 2C CH 2
C8 alkyl chains Polar analytes easily interact with surface Residual silanols
H 3C CH 2 H 2C CH 2 H 2C
Low Coverage Low Ligand Density
CH 2 CH 3 CH 3 H3C CH 3 H3 C Si CH 3 H CH3 H 3C Si Si H H H O O O O O O O Si Si Si Si Si Si Si O O O O O O O O O O O O O
H O O Si O
CH 2 H2C Endcap CH 2 CH 3 H3 C CH 3 H 3C Si CH 3 Si H H O H O O O O Si Si Si O O O O Si O O O O
Polar Compounds - Aqua Columns

Polar and Non -Polar Compounds Test Mix
Conditions Columns: 4.6 x 150 mm, 5 m Mobile Phase A: H2 O Mobile Phase B: ACN Mobile Phase C: 100 mM NH4 COOH, pH 3.0 Flow Rate: 2.0 mL/min Gradient: Time Profile (min) %A %B %C 0.0 80 10 10 10.0 0 95 5 15.0 0 95 5 Injection Volume: 5.0 L o Temperature: 30 C Detection: UV @ 254 nm
V0 = 0.98 min Compounds USP Tailing: 1. Uracil 1.04 2. Acetanilide 0.95 3. Triamcinolone 1.02 4. Hydrocortisone 1.03 5. 2-Amino-7-chloro -5-oxo -5H-[1]benzopyrano[2,3-b]pyridinecarbonitrile 1.01 6. 6a-Methyl -17a-hydroxyprogesterone 1.01 7. 3-Aminofluoranthene 0.97 8. 2-Bromofluorene 1.00 9. Perylene 0.99 10. Naphthol[2,3-a]pyrene 0.95
Chromolith Packing
By utilising an innovative new "Gel-Sol" technology, a silica gel polymer is formed, which after ageing, is dried into the required form of a straight rod of highly porous silica with a bimodal pore structure. Chromolith macroporestructure
Chromolith mesopore structure
7 9 8 6 10
3 4 5
Altantis dC 18
1.00
2.00
3.00
4.00
5.00
6.00
7.00 8.00 Minutes
9.00
10.00
11.00
12.00
13.00
14.00
15.00

28
Reversed Phase HPLC

Batch-to-Batch Reproducibility of Columns
Columns: Symmetry C 83.9 mm X 150 mm with Sentry Guard Column 3.9 mm X 20 mm Sample: Mobile Phase: Barbiturate Standard 100 mM potassium phosphate, pH 6.9/acetonitrile/water 20:30:50 3: Sulfathiazole 2: Sulfadiazine
Chromatogram of lifetime test

Conditions
1: Sulfanilamide
1 2
3 4 5
Column:
Symmetry C
8 3.9 mm X 150 mm with Sentry Guard Column 3.9 mm X 20 mm
Mobile Phase: water/methanol/glacial acetic acid 79:20:1
4: Sulfamerazine
Batch A
2
Batch B Batch C
0 2 4 6 8 10 12 5: Sulfamethazine
1 0 2 4 6 8 10
Injection 5020
Start
Minutes
6: Succinylsulfathiazole
Minutes

29

Reverse Phase Chromatography

Uploaded by

Document Information

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Reverse Phase Chromatography

Uploaded by

Copyright:

Available Formats

Reversed Phase HPLC

Reversed Phase Advanced Features

Mobile phase Water, Buffers MeOH, Acetonitril, IPA

A B Distribution: K = Cs /Cm B (More Hydrophobic)

Tel: 03-9254040 Cell: 052-448632 Fax: 03-9249977 Home page: http://www.forumsci.co.il/HPLC

A Elution through the Column Chromatogram

RP Method Development Tools

Polar mobile phase Analyte Y Non-Polar Analyte X Polar

Solvents MeCN THF

Well retained (like attracts like)

Dr. Shulamit Levin, Medtechnica

Reversed Phase HPLC

Reversed Phase Elution Order

1.0 1.0 2.0 3.0

Dr. Shulamit Levin, Medtechnica

Reversed Phase HPLC

Dr. Shulamit Levin, Medtechnica

Reversed Phase HPLC

SOLVENT STRENGTH (% modifier)

SOLVENT STRENGTH Analyte Retention as a Function of % Modifier

Dr. Shulamit Levin, Medtechnica

Reversed Phase HPLC

Ionization of Acids and Bases

(Ionized) 50% 0% 100%

Ionization of Acids and Bases

Retention Factor versus pH for Acids, Basesand Neutrals

Retention Factor -Linear

(Ionized) 50% 100% 0%

Dr. Shulamit Levin, Medtechnica

Reversed Phase HPLC

Enhanced Resolution of Basic Compounds at High pH

Silica gel high pH limit

Capacity Factor (k)

Decreasing retention Critical pH control necessary

pH control critical 2 Amitriptyline 3

Impact of pH on the Retention of a Zwitterionic Compound

Aqueous pKa = 4.3

Aqueous pKa = 9.5

0.065 0.051 0.037 0.023 0.009 -0.005 0

1:acetaminophen 2:lidocaine 3:doxepin 4:imipramine 5:nortriptyline 6:ibuprofen

Time (min) Lu, Cheng; Waters

pH the most powerful selectivity tool

Dr. Shulamit Levin, Medtechnica

Reversed Phase HPLC

0.80 0.60 0.40 0.20 0.00

254 nm pH 2.0 pH 10.0

1. Uracil 2. Propranolol 3. Naphthalene 4. Acenaphthene 5. Amitriptyline

220 240 260 280 300 320 340 360 380 nm

Recommended Buffers for pHs 2-7

Formic Acid 3.75

TYPE OF BUFFER (phosphate, acetate) IONIC STRENGTH (Salts, buffer concentration)

Dr. Shulamit Levin, Medtechnica

Reversed Phase HPLC

ION-PAIRING REAGENTS (alkyl-amines, -sulfonates)

Ion Pair Reagent

Concentration of Ion-Pair Reagent in the Mobile Phase

The larger the alkyl, the longer are retention times

CH2CH2 CH3 CH2 CH3CH2 CH2 CH2

N+ CH2CH2CH2CH3 CH2CH2CH2 CH3

SO3 Na+ SO 3- Na+ SO 3 Na+

5 mM Conc. of Ion Pair Reagent in the Mobile Phase

Dr. Shulamit Levin, Medtechnica

Reversed Phase HPLC

1960s 1970s 1980s 1990s 2000s