Convergence Chromatography:
Solving Complex Chromatographic Challenges
John Van Antwerp
©2012 Waters Corporation
1
What is Convergence Chromatography:
Why The Name?
Giddings, J.C. (1965) A critical evaluation of the theory of gas
chromatography. In Gas Chromatography. 1964, edited by A. Goldup, p.
3-24. Elsevier, Amsterdam
In this article Dr. Giddings stated “One of the most interesting features
of ultra high pressure gas chromatography would be convergence with
classical liquid chromatography.”
Prof. Calvin Giddings (1930-1996)
©2013 Waters Corporation
2
Evolution of Separation Technology
Gas Chromatography
©2012 Waters Corporation
Liquid Chromatography
Convergence Chromatography
GC
HPLC
SFC
Capillary GC
UPLC
UPC2
3
UPLC and UPC2
SPEED
SENSITIVITY
RESOLUTION
©2013 Waters Corporation
SIMPLICITY
SIMILARITY
ORTHOGONALITY
4
UPC² Adoption
 UPC² SIMPLIFIES the workflow
– Combines multiple techniques into ONE
o LC and GC
– Combines multiple methods into ONE
o NP and RP
– Reduces sample prep and analysis times
o Direct injection of organic solvents/extracts
 UPC² separates compounds with SIMILARITY
– Chiral, positional isomers, structural analogs, conjugates (biomarkers)
 UPC² provides ORTHOGONALITY
– More confidence in identifying impurities/degradants
– Full sample characterization
– Separation of analytes from matrix interferences (i.e., hydrophobic drugs
in bioanalysis)
©2012 Waters Corporation
5
How is SSO possible?
Rs =
N
4
α −1 k
α k +1
System efficiency [N] impacted by:


System dispersion
Reduction in particle size
Selectivity [α] and retentivity [k] impacted by:



Stationary phase (column selectivity)
Organic solvent (eluotropic series)
Mobile phase additives (pH and ionic strength)
Solvent
Stationary Phase
Pentane, Hexane, Heptane
Silica / BEH
Xylene
Impact on Rs
% Improvement
Double N
Double k
20-40%
15-20%
Double α > 400%
Toluene
2-ethylpyridine
Diethyl ether
Dichloromethane
Chloroform
Acetone
Dioxane
THF
MTBE
Ethyl acetate
DMSO
Cyano
Convergence
Chromatography
Selectivity Space
Unlimited solvent
and stationary
phase selectivity
Aminopropyl
Diol
Amide
PFP
Acetonitrile
Isopropanol
Ethanol
Methanol
©2012 Waters Corporation
Phenyl
C18 < C8
6
UPC2 Adoption
SIMPLICITY
SIMILARITY
ORTHOGONALITY
©2012 Waters Corporation
7
Combining Multiple Techniques
for Lipid Analysis
Gas Chromatography
Free fatty acids are
typically derivatized to
form the methyl esters
(FAMEs)
Analysis time 30 min
Liquid Chromatography
Analyzed by both HILIC
and RP
HILIC separates lipid
classes by polar head
group
RP separates based on
acyl chain length and
number of double bonds
©2012 Waters Corporation
Convergence Chromatography
Single methodology to
separate complex lipids
by class
Faster baseline
separation of lipids
based on chain length
and number of double
bonds
8
UPC2 Analysis of a Mouse Heart Extract
ACQUITY UPC2 BEH column
5-50% B
PC
TAG
PE
SM
LPC
TAG: Triacylglycerides
SM: Sphynogomyelin
©2012 Waters Corporation
PE: Phosphotidylethanolamine
LPC: Lysophosphotidylcholine
PC: Phosphotidylcholine
9
Separation of Neutral Lipids
Based on Chain Length and Number of Double Bonds
ACQUITY UPC2 HSS C18 SB column
1-10% B
22:0
100
20:0
18:0
Peak
24:0
16:0
1
ESI negative mode
Free Fatty Acids (FFA)
containing 8-24 acyl chain
%
14:0
2
3
12:0
10:0
8:0
0
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
2.30
2.40
2.60
2.70
2.80
2.90
3.00
3.10
10
100
ESI positive mode
Triacylglycerols (TG) and
Cholesterol esters (CE)
9
%
1
2
4
0.70
0.80
0.90
1.00
©2012 Waters Corporation
1.10
1.20
1.30
1.40
1.50
1.60
1.70
1.80
12
5
4
5
18:1(∆9Tr)/18:1(∆9Tr)/18:1(∆9Tr) TG
6
17:0/17:0/17:0 TG
7
18:1(∆9Tr)/18:1(∆9Tr)/18:1(∆9Tr) TG
8
18:3 CE
9
18:2 CE
7 8
6
1.90
2.00
2.10
11
13
11
2.20
2.30
2.40
2.50
2.60
2.70
2.80
2.90
3.00
15:0/15:0/15:0 TG
18:3(∆9,12,15Cis)/18:3(∆9,12,15Cis)/18:
3(∆9,12,15Cis) TG
16:0/16:0/16:0 TG
18:2(∆9,12Cis)/18:2(∆9,12Cis)/18:2(∆9,1
2Cis) TG
10
3
0
0.60
2.50
Lipid Species
17:0 CE
18:1 CE
18:0/18:0/18:0 TG
12
18:0 CE
13
19:0 CE
Time
3.10
10
Metabolomics/Lipidomics
Shulaev Metabolomics Lab
Metabolic Signaling Pathway Research
University of Northern Texas Collaboration
Cottonseed Extracts
©2012 Waters Corporation
11
Global Profiling Workflow
Interclass
Intraclass
•UPC2 BEH
Stationary Phase
•Neutral and Polars
Lipids from Cottonseed extracts
•UPC2
HSS C18 SB
Stationary Phase
•Neutral Lipids
UNT Goals: Global Profile and Targeted Analysis
ANALYZE
Qualitative
INTERPRET
•Synapt G2 - S
Quantitative •Xevo TQ-S
©2012 Waters Corporation
12
How does UPC2 Compare to Current
Lipidomic Approaches?
Historically
UPC2 Trends
BEH: Effect of FA chain length
# FA chain
50
40
30
20
ACQUITY UPC2 BEH
10
0
7.2
7.25
7.3
7.35
7.4
Rt (min)
 Separation based on adsorption of
the head group to the NP material
for lipid class separation.
 Separation based on hydrophobic
interaction of the FA chain and RP
material for lipid molecular species
separation
©2012 Waters Corporation
PC:
PC:
PC:
PC:
PC:
14:0/14:0
16:0/16:0
17:0/17:0
18:0/18:0
23:0/23:0
Trends Similar based
on stationary phase
C18: Effect of FA chain length
50
# FA chain
1.
2.
3.
4.
5.
40
30
20
ACQUITY UPC2 HSS C18 SB
10
0
7
8
9
10
11
12
Rt (min)
13
UPC2 Neutral Lipid Method Mix
UPC2 HSS C18 SB 1.7 µm (3.0x100mm)
ACQUITY UPC2 HSS C18 SB column
1-10% B
22:0
100
20:0
18:0
24:0
16:0
ESI negative mode
Free Fatty Acids (FFA)
containing 8-24 acyl chain
%
14:0
Peak
1
12:0
2
10:0
3
8:0
0
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
2.30
2.40
100
ESI positive mode
Triacylglycerols (TG) and
Cholesterol esters (CE)
2.60
2.70
2.80
2.90
3.00
3.10
9
%
1
2
4
0.70
0.80
0.90
1.00
©2012 Waters Corporation
1.10
1.20
1.30
1.40
1.50
1.60
1.70
1.80
12
5
3
0
0.60
2.50
10
7 8
2.00
2.10
11
2.20
2.30
2.40
2.50
2.60
4
5
18:1(∆9Tr)/18:1(∆9Tr)/18:1(∆9Tr) TG
6
17:0/17:0/17:0 TG
7
18:1(∆9Tr)/18:1(∆9Tr)/18:1(∆9Tr) TG
8
18:3 CE
9
18:2 CE
11
2.70
2.80
2.90
3.00
Time
3.10
15:0/15:0/15:0 TG
18:3(∆9,12,15Cis)/18:3(∆9,12,15Cis)/18:
3(∆9,12,15Cis) TG
16:0/16:0/16:0 TG
18:2(∆9,12Cis)/18:2(∆9,12Cis)/18:2(∆9,1
2Cis) TG
10
6
1.90
13
Lipid Species
17:0 CE
18:1 CE
18:0/18:0/18:0 TG
12
18:0 CE
13
19:0 CE
14
UNT Cottonseed Extract
Biological samples targeted to Neutral Lipids
TAG
UPC2/Synapt G2 with MSE:
DAG
Low collision
energy
50%
PC
NAPE
High collision
energy
2%
©2012 Waters Corporation
ACQUITY UPC2 BEH
15
Sample Analysis
Goals:
 Discriminate between Wild type and treated cottonseeds
 Search for homo- and hetero- geneity between treatments
©2012 Waters Corporation
16
Combining Multiple LC Methods
Fat-soluble vitamins
Vitamin A
Normal phase
12 minutes
0.40
0.38
0.34
Vitamin E
Normal phase
30 minutes
0.26
E
0.32
A Acetate
0.30
0.28
E Acetate
0.20
0.18
0.16
0.14
Lycopene
D2
0.22
A Palmitate
0.24
0.12
Lycopene
Normal phase
10 minutes
0.10
Vitamin K1
Reversed-phase
12 minutes
0.02
K2
AU
β-carotene
Normal phase
10 minutes
K1
Vitamin D3
Normal phase
20 minutes
Beta carotene
0.36
0.08
0.06
0.04
0.00
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
2.00 2.20
Minutes
2.40
2.60
2.80
3.00
3.20
3.40
3.60
3.80
4.00
Simultaneous Analysis of Fat-soluble Vitamins
and Carotenoids in 5 minutes
©2013 Waters Corporation
17
Reducing Sample Prep and Analysis Time
β-carotene Analysis
Official
AOAC Method
Modified
AOAC Method
UPC2 Method
3.0e-1
0.91
β-carotene standard
Dissolve/Hydrolyze
Dissolve/Extract
Dissolve/Extract
AU
2.5e-1
2.0e-1
1.5e-1
1.0e-1
5.0e-2
0.0
0.00
30 min
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
1.00
1.10
1.20
1.30
1.40
1.50
1.40
1.50
0.91
β-carotene capsule
Filter
3.0e-1
Filter
AU
Extract
2.0e-1
1.0e-1
120 min
0.0
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0.90
0.91
Dilute
UPC² Analysis
3.0e-1
AU
LC Analysis
30 min
2 min
Carotenoids Mix
2.0e-1
1.31
0.77
1.0e-1
0.0
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0.90
Filter
LC Analysis
6 Replicates
Peak Area
Average
RSD%
10326
0.34
1.00
1.10
1.20
1.30
Retention
Time (min)
0.91
0
Label Claim: 15 mg/capsule
30 min
20 samples
~12.5 hrs
©2013 Waters Corporation
20 samples
~10 hrs
20 samples
~ 40 minutes
Assay
#1
Assay
#2
Assay
#3
Average
RSD%
15.13
15.39
15.24
15.25
0.84%
18
Directly injecting organic solvent extracts
Eliminating Potential Sources of Error
100
Sample
Pretreatment/
Homogenization
%
LLE
Protein Precipitation
SPE
0.1 ng/mL Desipramine
D3 in Human Urine
0
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
2.30
2.40
2.50
2.60
2.70
2.80
2.90
1.20
1.30
1.40
1.50
1.60
1.70
1.80
1.90
2.00
2.10
2.20
2.30
2.40
2.50
2.60
2.70
2.80
2.90
100
%
Extraction
Samples directly injected
from mixed-mode SPE
Centrifugation/
Filtration
Desired
injection
point
Blank Human Urine
0
Time
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0.90
1.00
1.10
Standard Curve results for amitriptyline
Evaporation
Reconstitution
©2013 Waters Corporation
Concentration
0.1 ng/mL
0.2 ng/mL
0.5 ng/mL
1 ng/mL
5 ng/mL
10 ng/mL
RT (min)
1.48
1.48
1.48
1.48
1.48
1.48
Area
Counts
16161
27061
60531
103149
467997
999886
% Deviation
-3.3
2.7
7.9
-3.6
-7.9
-0.9
%
Accuracy
96.7
102.7
107.9
96.4
92.1
99.1
19
Analysis of Non-Ionic Surfactants
©2013 Waters Corporation
20
Background
•Non-ionic surfactants are used in cosmetics, industrial materials, and
many other products.
•Their composition has to be monitored because the differences in
ethoxy chain length affect the viscosity, solubility, polarity, and other
characteristics of the mixture
©2013 Waters Corporation
21
Current separation methods for nonionic surfactants
•Normal phase HPLC
•Hexane:Methylene chloride:Methanol gradient
•24 min to elute all components
•Approx. 16 oligomers separated and detected
•(Sigma-Aldrich)
•High temperature GC
•19 min to elute all components
•Approx. 18 oligomers separated and detected (Atas GL)
©2013 Waters Corporation
22
Purpose/Competitive Technology
•Typically analyzed by HPLC, SFC, GC
•Analysis by GC and HPLC very time-consuming
•SFC uses high column temperatures which can limit analysis of
thermally labile compounds
•HPLC might require derivatization for non-UV absorbing
surfactants
•Incomplete baseline separation for oligomers in some cases
©2013 Waters Corporation
23
Peak20 - 1.248
0.10
Peak19 - 1.217
0.20
Peak17 - 1.158
Peak1 - 0.271
0.30
Peak18 - 1.188
Peak2 - 0.455
0.40
Peak15 - 1.093
Peak3 - 0.574
0.50
Peak16 - 1.119
0.60
Peak14 - 1.067
Peak4 - 0.662
AU
0.70
Peak12 - 1.013
0.80
Peak13 - 1.041
Peak5 - 0.733
0.90
Peak11 - 0.985
1.00
Peak10 - 0.954
Peak6 - 0.792
1.10
Peak9 - 0.921
Triton X (10 mg/mL in IPA)
Peak7 - 0.841
1.20
Peak8 - 0.884
1.30
0.00
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
0.70
0.75
0.80
Minutes
0.85
0.90
0.95
1.00
1.05
1.10
1.15
1.20
1.25
1.30
1.35
1.40
1.45
1.50
Sample Set Id: 1462 SampleName: Triton X-100 Date Acquired: 6/21/2012 8:54:56 AM EDT Injection Id: 1522
•UPC2
•CO2 and Methanol gradient @ 40°C
•1.4 min to elute all components
•Approx. 20 oligomers separated and detected
©2013 Waters Corporation
24
Improving Workflow with
Convergence Chromatography
 Samples Incompatible with Water
– Tree extracts analyzing for phenolics
– Resins crash out when in contact with water
– Impossible to run by Reversed Phase
– Needed to be Compatible with MS so NP was out
©2013 Waters Corporation
25
Simplifying the Workflow with UPC2
Application
Example
UPC2 Advantages
Other application areas this
might apply
Combining multiple
techniques (LC and
GC into CC) Lipids
1) Single technique for neutral and polar
lipids
2) No derivatization for neutral lipids
(needed for GC)
3) Faster baseline separation than LC
1) Glyceride analysis in food and fuels
2) Lipid profiling in drug discovery
studies
Combining multiple
methods (NPLC
and RPLC into CC)
Fat-soluble
Vitamins and
Carotenoids
1) Directly inject organic extracts
2) One technique to replace RPLC and
NPLC
1) Pre-mixes and formulated samples
2) Raw materials testing
3) Samples prepared by LLE
Reducing sample
prep and analysis
times
β-carotene
Analysis
Directly injecting
organic solvent
extracts
Tricyclic
anti-depressants
(TCAs)
©2013 Waters Corporation
1) Reduced sample prep steps
2) Faster run times
3) Higher overall throughput
1) Direct injection of SPE extract
2) No evaporation and reconstitution =
less experiment error
3) No solvent exchange needed prior to
analysis
1) Samples prepared by SPE, LLE, or
protein precipitation that need
evaporation and reconstitution
2) Bioanalysis/DMPK
26
UPC2 Adoption
SIMPLICITY
SIMILARITY
ORTHOGONALITY
©2013 Waters Corporation
27
Structural Similarity
 Isomers and structural analogs can be challenging to separate
due to small differences in structure, or due to their chirality.
 In this section we will look at applications that are of interest
due to their structural similarity using UPC2, including:
1. Chiral Separations (Enantiomers & diastereomers)
2. Positional isomers (differ in location of functional groups)
3. Structural analogs

Biomarkers (conjugated/unconjugated)

Drugs (metabolites, impurities, degradants)
©2013 Waters Corporation
28
Chiral Separations
UPC²
0.3 min
Key advantages of moving to UPC2
– Results that are equal to or better than NPLC
– Drastic reduction in analysis time (up to 30X)
– Nearly 75X reduction in solvent
– Drastic reduction in cost of analysis (up to 100X)
o
Waste generation and disposal
AU
NPLC
11 min
0.30
0.00
0.00
2.00
4.00
©2013 Waters Corporation
6.00
8.00
Minutes
10.00
12.00
14.00
29
Cyfluthrin Chiral Separation
F
0.50
Cl
UPC2
(A)
IC + OJ-H
*
O
Cl
0.40
AU
*
O
0.30
*
O
CN
cyf luthrin
0.20
0.10
0.00
0.00
2.75e-1
2.5e-1
2.25e-1
1.00
2.00
3.00
4.00
Minutes
5.00
6.00
7.00
8.00
(B) Traditional SFC
2 x IC + 2 x OJ-H
2.0e-1
AU
1.75e-1
1.5e-1
1.25e-1
1.0e-1
7.5e-2
5.0e-2
2.5e-2
0.0
0.00
Time
2.00
4.00
©2012 Waters Corporation
6.00
8.00
10.00
12.00
14.00
16.00
18.00
20.00
22.00
24.00
26.00
28.00
30.00
32.00
34.00
30
Methamphetamine
UPC2/TQD In house Control Urine
sample
11000.0
10000.0
9000.0
8000.0
l_Methamphet - 5.570
d_Methamphet - 4.784
12000.0
Intensity
7000.0
6000.0
5000.0
4000.0
3000.0
2000.0
1000.0
0.0
1.50
©2013 Waters Corporation
2.00
2.50
3.00
3.50
4.00
4.50
Minutes
5.00
5.50
6.00
6.50
7.00
7.50
31
Chiral Separations
 Pesticides
 Drugs of Abuse
 Beta-blockers
Fast Chiral Separations
 Binol
 Warfarin
 Benzyl Mandelate (enantiomeric excess)
 Chiral screening
 Carprofen (chiral method development)
 Chiral method development
 Pantoprazole and Oxfendazole (chiral
method development with MS)
 Chiral inversion studies
 Clenbuterol
 Enantiomeric excess
– MS and UV detection
 Phenylalanine methyl esters
 Flurbiprofen
 Cyclometalated Iridium (III) Complexes
www.waters.com/upc2
©2013 Waters Corporation
32
Positional isomers
DMBA
0.90
2,4
2.552
0.80
0.70
0.60
3,4
2,6
3.430
0.30
2.245
2.342
2.134
AU
0.40
2,3
3,5
2.682
2,5
0.50
0.20
0.10
0.00
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80 2.00
Minutes
2.20
2.40
2.60
2.80
3.00
3.20
3.40
3.60
3.80
Mixture of 6 positional isomers of DMBA
Each at 0.2 mg/mL in isopropanol (IPA)
3.0 x 100 mm, 1.7 µm ACQUITY UPLC BEH125 (custom configuration)
CO2 / MeOH with 0.2% formic acid
©2013 Waters Corporation
33
Structural Analogs
Steroids
Androstenedione
17α-Hydroxyprogesterone
Estradiol
©2013 Waters Corporation
Testosterone
Corticosterone
11-Deoxycortisol
Estrone
Aldosterone
Cortisol
34
Steroids by UPC2
0.14
0.12
0.10
0.08
0.06
0.04
0.02
0.00
4
1
3
5
AU
6
9
8
ACQUITY
UPC2 BEH
2
0.00
0.50
1.00
Minutes
3
1
0.00
AU
0.14
0.12
0.10
0.08
0.06
0.04
0.02
0.00
0.00
©2013 Waters Corporation
1.50
2.00
2,5
4
7 8
AU
0.14
0.12
0.10
0.08
0.06
0.04
0.02
0.00
7
Steroids
1. Androstenedione
2. Estrone
3. 17α-Hydroxyprogesterone
4. Testosterone
5. 11-Deoxycortisol
6. Estradiol
7. Corticosterone
8. Aldosterone
9. Cortisol
9
6
ACQUITY
UPC2 BEH 2-EP
0.50
1.00
Minutes
4
1
3
2
0.50
6
5
7
8
9
1.00
Minutes
1.50
2.00
ACQUITY
UPC2 CSH Fluoro-Phenyl
1.50
2.00
35
Structural Analogs
Sulfated Estrogens
C H3 O
C H3 O
C H3 O
C H3 O
H
H
H
H
O
O
H
H
O
O
S
O
O
S
OH
O
O
O
O
O
S
OH
OH
Equilin (2)
MW = 348.41
Estrone (1)
MW = 350.43
S
CH 3 O H
O
OH
∆-8,9-Dehydroestrone (3)
MW = 348.41
Equilenin (4)
MW = 346.41
C H3 O H
C H3 O H
C H3 O H
H
H
H
H
O
O
O
S
O
O
17α-Estradiol (5)
MW = 352.45
S
O
O
OH
OH
S
O
O
Molecular weights are
color coded to denote
isobaric compounds
17α-Dihydroequilin (7)
MW = 350.43
S
O
17β-Dihydroequilin (8)
MW = 350.43
C H3 OH
H
H
O
O
O
17α-Dihydroequilenin (9)
MW = 348.41
©2013 Waters Corporation
H
OH
C H3 O H
OH
H
O
OH
17β-Estradiol (6)
MW = 352.45
O S
H
O
O
S
O
OH
17β-Dihydroequilenin (10)
MW = 348.41
36
USP 35-NF 30 GC method for
Conjugated Estrogens
CH3 OH
CH3 OH
H
H
H
O
O
O
S
O
O S
17α-Estradiol (5)
MW = 352.45
1
O
OH
OH
17β-Estradiol (6)
MW = 352.45
5
2
7
6
©2013 Waters Corporation
10
9
8
3
4
37
Sulfated Estrogens by UPC2
Sulfated Estrogens
1. Estrone
2. Equilin
3. ∆-8,9-Dehydroestrone
4. Equilenin
5. 17α-Estradiol
6. 17β-Estradiol
7. 17α-Dihydroequilin
8. 17β-Dihydroequilin
9. 17α-Dihydroequilenin
10. 17β-Dihydroequilenin
3
0.24
0.22
0.20
0.18
5
0.16
6
0.14
AU
1
0.12
0.10
10
4
9
2
7
8
0.08
0.06
0.04
0.02
0.00
5.00
5.50
©2013 Waters Corporation
6.00
6.50
7.00
7.50
8.00
8.50
9.00
9.50
Minutes
10.00
10.50
11.00
11.50
12.00
12.50
13.00
38
Structural Similarity with UPC2
Application
Example
Enantiomers and
diastereomers
(Chiral separations)
UPC2 Advantages
1) Faster and cheaper than NPLC
2) 10X faster, >85% solvent savings
3) Meets “green” initiatives
Other potential
applications
1) Chiral screening
2) Chiral method development
(MS and UV detection)
3) Chiral inversion studies
4) Enantiomeric excess
Positional isomers
DMBA
1) Faster separation than NPLC
2) Low solvent usage and waste production
3) Compatibility with NP diluents and
extraction solvents
4) Separation of both geometric and
enantiomeric isomers
1) Starting materials analysis
2) Reaction monitoring
3) Asymmetric catalysis (Chemical
Materials)
Structural Analogs
Steroids/
Estrogens
1)
2)
3)
4)
1) Non-polar biomarkers
2) Natural product API and
formulation analysis
©2013 Waters Corporation
No derivatization required (takes 2.5 hrs)
Faster and better separation than LC or GC
Resolution of conjugated steroids
Directly compatible with MS
39
UPC2 Adoption
SIMPLICITY
SIMILARITY
ORTHOGONALITY
©2013 Waters Corporation
40
Orthogonal Separations
 Why do I need an orthogonal separation mode?
1. A common concern in many applications is that an impurity,
degradation peak, or similar compounds may be overlooked
(isobaric, co-elution)
2. Orthogonal methods that provide different relative retention of
peaks are needed to ensure full characterization
3. Ability to see more information about the sample
4. Separation of desired compounds from matrix interferences
©2013 Waters Corporation
41
Orthogonal to RPLC
Metoclopramide Related Substances
Metoclopramide
AU
0.026
2
1
0.013
3
9
4
Reversed-Phase
6
5
8
0.000
0.00
1.20
2.40
3.60
4.80
6.00
Minutes
7.20
8.40
9.60
12 minutes
10.80
Metoclopramide
0.009
ACQUITY UPC2
0.006
AU
2
0.003
0.000
-0.003
0.00
1.10
2.20
3.30
4.40
5.50
Minutes
©2013 Waters Corporation
6.60
7.70
8.80
9.90
12 minutes
42
Separation of Compounds from Matrix
Interferences (Bioanalysis)
Clopidogrel
(RPLC)
Interfering Phospholipids
(RPLC)
Interfering Phospholipids
(UPC2)
Clopidogrel
(UPC2)
©2013 Waters Corporation
43
Separation of Compounds from Matrix
Interferences (Bioanalysis)
Clopidogrel
(RPLC)
Interfering Phospholipids
(RPLC)
Interfering Phospholipids
(UPC2)
Clopidogrel
(UPC2)
©2013 Waters Corporation
44
Separation of Compounds from Matrix
Interferences (Bioanalysis)
Clopidogrel
(RPLC)
Interfering Phospholipids
(RPLC)
Interfering Phospholipids
(UPC2)
Clopidogrel
(UPC2)
©2013 Waters Corporation
45
Separation of Compounds from Matrix
Interferences (Bioanalysis)
Clopidogrel
(RPLC)
Interfering Phospholipids
(RPLC)
Interfering Phospholipids
(UPC2)
Clopidogrel
(UPC2)
©2013 Waters Corporation
46
Orthogonal Separations with UPC2
Application
Example
UPC2 Advantages
Other potential applications
1)
2)
3)
4)
Impurity/degradant analysis
Agrochemical APIs and formulations
Stability indicating methods (OLEDs)
Non-polar compounds that elute late
in RPLC
Full characterization
Metaclopramide
1) Different elution order than RPLC
2) Resolves peaks not resolved by LC
3) Compatible with MS for
identification of unknowns
Orthogonal to LC
Chamomile
1) Direct injection of organic extracts
(i.e., microwave extraction)
2) MS compatibility
1) Complex mixture analysis
Ability to see more
(isobaric)
Chamomile
1) Separation of isobaric species
1) Isobaric separations
Separation from Matrix
Interferences
Bioanalysis
(phospholipids)
1) Analytes of interest are eluted
away from the matrix
2) Less matrix interferences = less
potential suppression
3) More precise and accurate
quantitation
1) Bioanalysis, DMPK (hydrophobic
compounds)
2) Other matrices containing lipids (food,
fuels, tissues)
©2013 Waters Corporation
47
National Center for Natural Products Research
School of Pharmacy, The University of Mississippi
University of Mississippi Collaboration
©2012 Waters Corporation
48
Global Profiling Workflow
Chamomile Extracts
Ole Miss Goals: To explore new entities via different extraction procedures
EXTRACT
Solvent Solvent
Microwave
SFE
©2012 Waters Corporation
•MeOH
•Hexane
ANALYZE
UPC2 2-EP
Stationary
Phase
INTERPRET
TransOmics •TOIML
•IPA
•Hexane
•IPA:Hexane
•Different Percentages
•Different Modifiers
Xevo Q-Tof
G2 S
49
Chamomile Profiling
 Compare to MeOH extracts and
Hexane extracts of two species of
chamomile
Anthemis nobilis
(Roman Chamomile)
– UPC2 method development
required
 Compare to previous studies
– HPTLC v. UPLC/MS v. GC-MS
Matricaria recutita
(German Chamomile)
 Identify benefits
– New discoveries?
– Examination of extracts on both
systems?
©2012 Waters Corporation
50
Roman Chamomile
0.88
A.
UPLC Reversed Phase
UV 350 nm
2
1. apigenin-7-O-glucoside
2. chamaemeloside
3. apigenin
3
AU
0.66
0.44
1
0.22
0.00
0.00
0.20
3.20
6.40
9.60
12.80
19.20
22.40
11.00
Minutes
13.20
15.40
25.60
28.80
32.00
17.60
19.80
22.00
3
B.
UPC2
UV 350 nm
2
0.15
AU
16.00
Minutes
0.10
1
0.05
0.00
0.00
2.20
4.40
©2012 Waters Corporation
6.60
8.80
51
Separation of Isobaric Species
XIC of m/z = 475 Da
1.6x10 6
A. UPC2/MS
Interrogation of the MS data for both LC and SFC
based techniques displayed some differences when
comparing isobaric separations indicated by the XIC
of selected m/z = 475
Intensity
1.2x10 6
8.0x10 5
XIC of m/z=475. An additional peak was
found in the UPC2 trace (A)…
4.0x10 5
0.0
4.62
320000
5.28
5.94
6.60
7.26
7.92
Minutes
8.58
9.24
9.90
10.56
…when compared to the UPLC-RP (B) trace.
B. UPLC/MS
Intensity
240000
160000
80000
0
11.60 11.80
©2012 Waters Corporation
12.00
12.20
12.40
12.60
12.80
13.00
13.20
13.40 13.60
Minutes
13.80
14.00
14.20
14.40
14.60
14.80
15.00
15.20
15.40
52
OPLS/OPLS-DA (9172 vs. 9254)
Within group variation
(from different extraction techniques)
German - 9172
Roman - 9254
GC
RC
Between group variation
©2012 Waters Corporation
53
S-Plot (9172 vs. 9254)
Compounds up
regulated in 9254
Compounds up
regulated in 9172
Exported to excel file for the list of up regulated features,
currently the collaborator have identified new features not
previously seen by other techniques (yet to be published)
©2012 Waters Corporation
54
Difficult Separations - Orthogonality
D-JSC-000007-857, MW: 404.2
UV Chromatogram - UPLC-MS
Column: BEH C18 1.7 mm, 2.1 x 50
mm
Gradient: 5-95% AcCN in 1 min
Solvent: 0.1% FA in H2O, 0.1% FA in
AcCN
100%
404.2
0.46
UV Detector: 220
1.2
AU
1.0
1.452
Range: 1.379
8.0e-1
6.0e-1
4.0e-1
2.0e-1
Tim e
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
0.70
0.75
0.80
0.85
UV Detector: 220
1.997
Range: 1.997
0.12
UV Chromatogram - UPC2-MS
Column: BEH 2-EP 1.7 um, 2.1 x 50
mm
Gradient: 5-35% MeOH in 1 min
Solvent: CO2, MeOH
1.5
AU
1.25
1.0
87%
404.2
0.58
7.5e-1
5.0e-1
13%
404.2
0.63
2.5e-1
0.0
0.10
©2012 Waters Corporation
Time
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
0.70
0.75
0.80
0.85
0.90
0.95
1.00
55
Similar Structures - Regioisomers
D-JSC-000006-0010
TIC Trace - UPLC-MS
0010A
0010B
TIC Trace- UPC2-MS
0010A
©2012 Waters Corporation
0010B
Column: BEH C18 1.7 mm, 2.1 x 50
mm
Gradient: 5-95% AcCN in 1 min
Solvent: 0.1% FA in H2O, 0.1% FA in
AcCN
Column: BEH 2-EP 1.7 um, 2.1 x 50
mm
Gradient: 5-35% MeOH in 1 min
Solvent: CO2, MeOH
56
Conclusion
 ACQUITY UPC2 Technology, using compressed carbon dioxide (CO2) as the
primary mobile phase, is a separation tool that solves both routine and complex
chromatographic problems, especially for samples possessing a wide range of
polarities
 UPC2 offers scientists unique workflow, application, and environmental impact
benefits compared to LC and GC platforms
 Because UPC2 is built utilizing UPLC Technology, customers are assured of its
optimized performance through holistic design of instrumentation, detectors,
software data systems and chemistries.
 UPC2 provides an exceptional increase in available selectivity, making this
technology widely applicable to a diverse range of compound types
– 80-85% overlap of compounds that can be analyzed by CC and RPLC
– Any compound soluble in an organic solvent
©2013 Waters Corporation
57
Thank You for Your Attention !
©2013 Waters Corporation
58