SilicaBased HyperCrosslinked Strong Cation Exchanger SO3HCC8: Separation of Catecholamines

1
Silica-Based Hyper-Crosslinked Strong Cation
Exchanger -SO3-HC-C8 Separation of Catecholamines
_

• Hao Luo, Lianjia Ma, Peter W. Carr
• University of Minnesota
• Nov. 14th, 2005

2
Significance and Challenges of Catecholamine
Separation

• Catecholamines biogenic amines containing a
  catechol function (3,4-dihydroxyphenyl group) and
  an amino function on a side chain.

• Play an important role in nervous system
• Cause and treatment of Parkinsons disease

• Due to the extremely hydrophilic structures of
  catecholamines, typical separation using RPLC is
  quite difficult
• Long ODS column, longer than 15cm
• Minute amount of organic modifier, less than 10
• Strong ion-pairing reagent, e.g. alkyl sulfonate

3
Acid
Base
Amino Acid
4
Catecholamine Separations
1
Commercial ODS 10/90 MeCN/H2O 0.1 TFA 10mM
C6H13SO3Na
5
3
6
7
2
9
4
8
Commercial silica-based CEX 10/90 MeCN/H2O 0.02
TFA
1
3
8
2
5
7
6
4
9
-SO3-HC-C8 24/76 MeCN/H2O 0.02 TFA
1
2
Column 50.46cm Temperature 40 ?C for 2
commercial columns 65 ?C for -SO3-HC-C8 Flow
rate 1mL/min Other m.p. additives 0.2mM EDTA
3
5
6
7
4
8
9
5
Efficiency Comparison (N/meter)
-SO3-HC-C8 provides excellent efficiency in
catecholamine separation
6
Synthesis of -SO3-HC-C8 from HC-C8
Reversed Phase
HC-C8
Development of a Hyper-Crosslinked, Acid Stable
RPLC Phase for the Separation of Organic Bases
Lianjia Ma, Hao Luo, Jun Dai, Peter W. Carr
(submitted to J. Chrom. A.)
7
Sulfonation Degree and Hydrophobicity
Synthesis conditions sulfonated with 0.11M
ClSO3H dichloromethane solution at -61?C for
0.5hour value estimated from the slope of k
vs. 1/C in cation separation.
-SO3-HC-C8 exhibits both cation exchange sites
(SO3-) and hydrophobic interaction sites
8
0.02 TFA
Effect of Trifluoroacetic Acid (TFA) Concentration
1
2
3
5
6
7
4
8
9
0.06 TFA
1
3,5
2
6,4
7
8
9
1
0.1 TFA
5,3
6
2
4
7
9
24/76 MeCN/H2O containing TFA and 0.2mM oxalic
acid, 40?C, 1mL/min, 50.46cm
8
9
Effect of TFA on Retention Factors of
Catecholamines
Dominant Mechanism
Acids (protonated) reversed phase
Retention range (k) and selectivity (a) can be
tuned by changing TFA concentration.
10
24/76 MeCN/H2O
Effect of Acetonitrile Fraction
1
2
3
5
6
7
4
8
9
50/50 MeCN/H2O
1
2
4,5
3
7
6
8
9
1,2
80/20 MeCN/H2O
7,8
6
MeCN/H2O containing 0.02 TFA and 0.2mM oxalic
acid, 40?C, 1mL/min, 50.46cm
3,4
5
9
11
Effect of Acetonitrile on the Retention Factors
of Catecholamines
Retention range (k) and selectivity (?) can be
tuned by changing MeCN fraction.
12
Gradient Elution of Catecholamines by Changing
TFA Concentration or Acetonitrile Composition
280nm
24/76 MeCN/H2O containing TFA and 0.2mM oxalic
acid A 0.02 TFA B 0.1 TFA 0-6-6.01-8min
at 0-100-0-0 B 40?C, 1mL/min
1
2
7
5
6
3
9
4
280nm
1
MeCN/H2O containing 0.02 TFA and 0.2mM oxalic
acid A 24/76 MeCN/H2O B 80/20
MeCN/H2O 0-4-12-12.01-14min at 0-0-100-0-0 B
40?C, 1mL/min
2
3
5
6
7
9
4
13
40 ?C
1
Effect of Temperature
2
3
5
6
7
4
8
9
1
50 ?C
2
3
5
6
4
7
8
9
1
65 ?C
2
3
5
7
6
4
8
9
1
80 ?C
2
3
5
6
4
7
8
9
1
100 ?C
2
24/76 MeCN/H2O containing 0.02 TFA and 0.2mM
EDTA, 1mL/min, 50.46cm
3
5
7
4
6
8
9
14
Conclusions

• Hydrophobic hyper-crosslinked strong cation
  exchanger -SO3-HC-C8 is very useful in the
  separation of highly hydrophilic cations, such as
  catecholamines, morphine, methamphetamine, etc.
• Flexible optimization
• Tuning TFA concentration pH acetonitrile
  fraction temperature
• TFA or acetonitrile gradient
• Excellent efficiency (gt 100,000/m)
• Silica-based hyper-crosslinked polymer networks
  serves as a novel and stable platform for the
  synthesis of a family of reversed and ion
  exchange phases.
• Hydrophobic hyper-crosslinked strong cation
  exchanger -SO3-HC-C8 can be synthesized by
  controlled sulfonation of reversed
  hyper-crosslinked phase HC-C8.

15
Acknowledgement

• University of Minnesota
• NIH
• Dr. Bill Barber at Agilent
• Carr group members
• Adam Schellinger, Dwight Stoll, Xiaoli Wang,
  Yu Zhang, and Chang Yub Paek

16
Gradient Elution of Catecholamines by Changing
TFA Concentration or Acetonitrile Composition
260nm
24/76 MeCN/H2O containing TFA and 0.2mM oxalic
acid A 0.02 TFA B 0.1 TFA 0-6-6.01-8min
at 0-100-0-0 B 40?C, 1mL/min
1
2
7
5
6
4
3
9
8
260nm
1
MeCN/H2O containing 0.02 TFA and 0.2mM oxalic
acid A 24/76 MeCN/H2O B 80/20
MeCN/H2O 0-4-12-12.01-14min at 0-0-100-0-0 B
40?C, 1mL/min
2
3
4
5
9
6
7
8
17
Catecholamine Separation on SB C18
3,7
0mM C6H13SO3Na
6
5
1
9
4
2
8
5mM C6H13SO3Na
7,1
3
5
6
2
9
4
8
1
10mM C6H13SO3Na
5
3
6
7
2
10/90 MeCN/H2O containing 0.1 TFA, 0.2mM EDTA
and C6H13SO3Na 40 ?C, 1mL/min, 50.46cm
9
4
8
18
Effect of Hexanesulfonate Concentration on the
Retention Factors of Catecholamines in RPLC
SB C18 (50.46cm) 10/90 MeCN/H2O containing 0.1
TFA, 0.2mM EDTA and C6H13SO3Na , 40 ?C, 1mL/min,
19
Gradient Elution of Catecholaminesby Changing
the TFA Concentration
260nm
1
2
7
5
6
4
3
9
8
280nm
1
24/76 MeCN/H2O containing TFA and 0.2mM oxalic
acid A 0.02 TFA B 0.1 TFA 0-6-6.01-8min
at 0-100-0-0 B 40?C, 1mL/min
2
7
5
6
3
9
4
20
Gradient Elution of Catecholamines by Changing
Acetonitrile Composition
1
260nm
2
3
4
9
5
7
6
8
280nm
1
MeCN/H2O containing 0.02 TFA and 0.2mM oxalic
acid A 24/76 MeCN/H2O B 80/20
MeCN/H2O 0-4-12-12.01-14min at 0-0-100-0-0 B
40?C, 1mL/min
2
3
5
6
7
9
4
21
Effect of Temperature on the Retention Factors of
Catecholamines
Retention range and selectivity can also be tuned
by Temperature
22
Reproducibility of TFA Gradient
280nm
1
2
24/76 MeCN/H2O containing x TFA and 0.2mM oxalic
acid A 0.02 TFA B 0.1 TFA 0-6-6.01-8min
at 0-100-0-0 B 40?C, 1mL/min
7
5
6
3
9
4
23
Reproducibility of MeCN Gradient
280nm
1
2
MeCN/H2O containing 0.02 TFA and 0.2mM oxalic
acid A 24/76 MeCN/H2O B 80/20 MeCN/H2O 1-3 run
0-4-12-12.01-14min at 0-0-100-0-0
B 4-6 run 0-4-12-12.01-15min at
0-0-100-0-0 B 7-9 run 0-4-12-12.01-14min at
0-0-100-0-0 B 40?C, 1mL/min
3
5
7
6
9
4
24
Hyper-Crosslinked Platform Prepared by Orthogonal
Friedel-Crafts Chemistry
DM-CMPES
Amplified View of A Porous Silica Particle
Surface
Friedel-Crafts reaction catalyst SnCl4
25
Synthesis of HC-C8 and -SO3-HC-C8
Secondarily Crosslinked DM-CMPES
A novel platform for a family of new phases with
different separation modes
26
Acid Stability of HC-C8
Superb acid stability is achieved by the
formation of hyper-Crosslinked polymer networks