Hydrophobic Neutral
Compounds
OAc
O
Me
HO
Me
OH
OH
H
H
OAc
OAc
OAc
AcO
H
O
2.
1. Prednisolone
D-Glucose
pentaacetate
Column size: 150 x 4.6 mm
Flow rate:
1.0 ml/min
Detection:
ELSD
Hydrophobic Zwitterions
HOOC
O
NH2
COOMe
N
H
T1 = 3.1min
T2 = 3.8min
Rs = 3.9
Primesep
P
O
MeCN – 35%
TFA 0.1%
MeCN – 35%
NH4Ac 30 mM pH 3.5
Column
T1 = 3.5min
T2 = 5.5min
Rs = 9.5
T1 = 4.5min
T2 = 4.5min
Rs = 0
Primesep
200
T1 = 3.2min
T2 = 4.2min
Rs = 5.5
T1 = 4.8min
T2 = 5.3min
Rs = 1.6
T1 = 4.6min
T2 = 5.3min
Rs = 1.1
Primesep
C
T1 = 4.3min
T2 = 4.5min
Rs = 0.7
T1 = 4.5min
T2 = 5.2min
Rs = 2.3
T1 = 4.6min
T2 = 52min
Rs = 2.5
Primesep
D
T1 = 4.4min
T2 = 4.2min
Rs = 0.7
T1 = 4.9min
T2 = 4.4min
Rs = 1.2
T1 = 4.5min
T2 = 4.9min
Rs = 1.56
5
2
NH2
4
6
Zwitterionic
Basic
Weak
base
Strong
base
8 mi
Hydrophobic Strong Bases
Column:
Primesep D
Column size: 150 x 4.6 mm
Mobile Phase:
MeCN/TFA — 20/0.15
Flow rate:
1.0 ml/min
Detector:
ELSD
Bu
Bu N Bu
Bu
5
1
1
1. DOPA
2. Tyrosine
3. Phenylalanine
NH
2
3
4
One
basic
group
Several
basic
groups
GLU
Na
NH2
NH4
H2N
NH3
Primesep columns. If you are dealing with complex mixtures that contain
several different compounds types, please check out additional method development resources at http://www.HplcMethodDevelopment.com
Weak base examples
Column:
Primesep D
Column size:
150 x 4.6 mm
Flow rate:
1.0 ml/min.
Mobile phase: MeCN 10-30 in 15
min with 0.1 % TFA
Detection:
UV - 250 nm
MET CYS
ILE
0
N
Alk
COOH
H
N
Ph
COOH
N
HNO2 O
O
Me
N
Me
NH2 HO NH2
Weak
acid
Strong
acid
Amitriptyline
HN
N
Ph
NO2
R P OH
H2SO4 HCl
H3PO4 HNO3
OH
Basic
5
CH COOH CH PO3H2
COO
SOH
………………….
Weak
acid
R
R
OH
5
10
15
Hydrophobic Compounds with
Several Strong Basic Groups
Hydrophobic Strong Acid
O
Column:
Primesep 500
Column size: 150 x 4.6 mm
Mobile phase: MeCN –7%,
NH4Acetate reverse gradient
from 50 to 10 mM in 10 min
Flow:
1.0 ml/min
Detection:
ELSD
Me
Me
O
N
Me
H
N
H
HO
H
Column:
Primesep C
Column size:
50 x 3.0 mm
Mobile phase: MeCN gradient
10-60% in 10 min,
AmFm 20 mM, pH 3.0
Flow: 1.0 ml/min
Rocuronium
Detection: ELSD
2
1
1.
2.
3.
4.
Hydrophobic Weak Acids
Column:
Primesep D
Column size: 50 x 3.0 mm
Mobile phase: MeCN – 80%
1. Gemfibrozil
AcOH-0.2%
2. Ibuprofen
Flow rate: 0.5 ml/min
Detection: UV 270 nm
Sodium
Hexanesulfonic Acid
Heptanesulfonic Acid
Octanesulfonic Acid
Asp-Arg-Val-Tyr-Val-His-Pro-Ile
Me
0
COOH
Me
Me
O
1
Me
Me
4
COOH
1.
2.
1
2
4
6
Column:
Primesep 100
Column size: 250 x 4.6 mm
Mobile Phase: Water/
1
MeCN/H2SO4–75/25/0.03
Flow rate:
0.3 ml/min.
2
Detection:
UV 210 nm
0
2
4
6
8
10 mi
5
7.5
10 min
0
1
2
3
4
5
10
12
1
0
1.
2.
3.
4.
-3
Column: Primesep D
Column size: 50 x 3.0 mm
Mobile phase: MeCN –10%
NH4Acetate gradient from 20
to 200 mM in 10 min
Flow rate: 0.5 ml/min
Detection: ELSD
min
4
2
2.5
3
8
Me
0
Hydrophilic Strong Acid
SO4 - 2
2
Maleic acid
Malic acid
Succinic acid
Fumaric acid
2
4
6
8
min
0
2
4
6
0
Weak
base
Strong
base
8
min
1.
2.
N
4
6
8
min
Mobile phase:
MeCN –50%,
HCOOH – 0.1%
N
N
5
10
Column:
Column size:
Flow rate:
Detection:
Several
basic
groups
One
basic
group
Column:
Primesep 100
Column size: 150 x 4.6 mm
Mobile Phase:
H2O/MeCN/TFA - 95/5/0.1
Flow rate: 1.0 ml/min
Detector:
ELSD
1.
2.
3.
Strong
acid
H
N
0
NH2
H
N
NH2
H2 N
2
4 min
5
10
1
2
Column:
Primesep C
Column size: 150 x 3.2 mm
Mobile phase:
Water/MeCN–75/25 with
NH4 Acetate 20 mM pH 4.1
Flow rate:
0.5 ml/min
Detection:
ELSD
5
min
DOPA
Tyrosine
Phenylalanine
Norepinephrine
Epinephrine
Dopamine
3
Column :
Primesep 200
Column size: 150 x 4.6 mm
Mobile phase: MeCN – 10%,
AmFm 10 mM pH 2.9
Flow rate:
1.0 ml/min
Detection:
UV 205 nm
2
15
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min
0
5
6
5
3
Benzenesulfonic acid
p-Toluenesulfonic acid
min
Acetylcholine
Methylacetylcholine
4
NH2
1
10
N
1
2
2
3
5
1.
2.
0
1.
2.
3.
4.
5.
6.
1
N
Na
1
H 2N
min
Monoethanolamine
Diethanolamine
Triethanolamine
O
H
N
15
Primesep 100
150 x 4.6 mm
1.0 ml/min
UV 270 nm
Hydrophilic Strong Bases
Column:
Primesep C
Column size:
50 x 4.6 mm
Mobile phase: gradient from A to B in 10
min + 10 min hold of 100% B
A: MeCN – 20% AmAc 25mM pH 4.0
B: MeCN – 20% AmFm 25 mM pH 3.0
Flow rate: 1.0 ml/min
0
2
O
0
3
3
1
NH2
HO
2
2
2
H
H
OH OH
H2N
1
min
20
0
N
Hydrophilic Compounds with
Several Strong Basic Groups
min
Column:
Primesep D
Column size:
4.6 x 50
Mobile phase: MeCN –60%
NH4Acetate 25 mM
Flow rate: 1.0 ml/min
Detection: UV 270 nm
3
6
ARG
NH2
Mobile phase:
MeCN –50%,
AmFm 20 mM
pH 3.0
O
Ascorbic Acid
Methylmalonic
Acid
Succinic Acid
3.
LYS
SO2NH2
O
PO4
Me
3
(Zemuron)
Column: Primesep N
Column size: 150 x 4.6 mm
Mobile phase: MeCN -75%
NH4Acetate 15 mM
1
Flow rate: 1.0 ml/min
Detection: ELSD
15
2
Hydrophilic Weak Acids
mi
10
SO2NH2
O
0
HIS
PHE
Column:
Primesep 100
Column size:
150 x 4.6 mm
Mobile phase: MeCN – 10%
HCOOH – 0.1%
Flow rate:
1.0 ml/min
Detection:
UV 230 nm
Zwitterionic
NH2
NH2
N(Me)3
H2C
min
Hydrophilic Weak Bases
Zwitterion examples
Weak acid examples
10
Hydrophilic
Hydrophilic compounds - compounds with a better solubility in water than in organic solvents, or completely nonsoluble in organic solvents (et. acetonitrile, ethanol). Examples of such compounds are amino acids, sugars, peptides,
Acidic
nucleotides, protonated amines and any other molecules with one or several polar groups (Fig. 1). The presence of
Acidic
some functional groups does not make a molecule significantly polar in terms of LC hydrophobic interaction (Fig. 2).
Ionizable compounds – molecules which have at least one charged functional group in a solution within a pH range
from 2 to 7. Under these conditions, basic compounds are positively charged and acidic compounds are negatively
charged.
Fig. 1 Polar functional groups Weak basic compounds - compounds that go through a
Fig. 2 Non-polar functional groups
O
transition state from positively charged to neutral within a
O
F
C O C
C C
HO
N
N
pH range from 2 to 7 (pKa ≤ 6).
S O
C
N
Cl
C C C
O
Weak acids - compounds that go through the transition
C C
O
from a negatively charged state to a neutral state within a
HN N
O
C N
Br C Si
pH range from 2 to 7 (pKa ≥ 3). Strong acids - compounds
O
C O C
OH
S
C NO2
I
which are significantly ionized in solutions at a pH of 3
P O
O
(pKa<3).
NH3
OH
C O
Strong bases - compounds which are significantly ionized in solutions
Strong acid examples
at a pH of 6 (pKa >6).
Zwitterionic compounds - compounds that bear an equal number of positively charged and
O
O
negatively charged functional groups in solutions at a pH within the range of 2 to 7.
Ph S OH F3C C OH
Recommended buffers: Low UV methods: sulfuric acid, phosphoric acid,
Strong base examples
O
perchloric acid, phosphate buffers, TFA. MS/ELSD methods: formic acid,
HOOC
COOH
Bu
NH2
acetic acid, ammonium formate, ammonium acetate, TFA.
O
Complex mixtures: each compound type can be analyzed on different
Bu N Bu HN C NH R
Alk
3
VAL
ASP
Ionizable
Bu
2
Column: Primesep 100
Column size: 250x4.6 mm
Mobile phase:
MeCN/H2O–30/70
TFA gradient from 0.05 to
0.3% in 25 min
Flow rate: 1.0 ml/min
Detector: ELSD
Neutral
Your compound is…?
1
5
ALA
min
15 min
10
0
O
allows you to find conditions for different detection techniques (UV, MS, ELSD, IR), and
K
0
O
Before you start… become familiar with the terms used in this guide:
3
2
0
Ionizable
min
H 2N
0
small molecules with different properties. The flexibility of mixed mode chromatography
Hydrophobic
2
3
2
2
Column:
Primesep 200
Column size: 150 x 4.6 mm
Mobile phase:
MeCN /H2O/HCOOH – 20/80/0.1
Flow:
1.0 ml/min
Detection:
UV 250 nm
O
OH
N
H
Neutral
Column:
Primesep D
Column size: 150 x 4.6 mm
Mobile phase: MeCN –20%
HCOOH –0.1%
1
Flow rate: 1.0 ml/min
Detection: UV 250 nm
NH2
OH
during the same run. This guide will help in developing methods for the separation of
works well with difficult sample matrices and a variety of sample diluents.
Hydrophobic Weak Bases
1
N
O
zwitterions and neutral molecules can be separated with high selectivity and efficiency
Column size: 150 x 4.6 mm
Flow rate:
1.0 ml/min
Detection:
UV 270 nm
15
HO
Column:
Primesep N
Column size: 250 x 4.6 mm
Detection:
UV 250 nm
Sample:
solution in
MeCN/H2O-90/10
Flow:
1.0 ml/min
Mobile phase:
MeCN/ H2O-90-10
ion-exclusion or pi-pi interactions. Polar and hydrophobic, acidic and basic compounds,
Hydrophilic Zwitterionic Compounds
NH
1
pounds. These separations are based on a combination of reverse phase, ion-exchange,
provides easy scale-up and high throughput capabilities. This mixed mode technology
10
O
Hydrophilic Neutral
Compounds
Mixed mode chromatography is a powerful technique for the separation of various com-
Aspartame
Column: Primesep D
Mobile phase:
MeCN – 5%,
NH4Acetate
Column: Primesep 100
25mM pH 3.0
Mobile phase:
MeCN – 20%
NH4Formate
25mM pH 3.0
0
A Guide for HPLC Separation of
Small Molecules
MeCN – 35%
NH4Fm 30 mM pH 3.0
10
15
min