ionic liquids in separations & mass spectrometry · ionic liquids in separations & mass...
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Ionic Liquids in
Separations & Mass Spectrometry
Daniel W. Armstrong
Robert A. Welch Professor
University of Texas at ArlingtonDepartment of Chemistry and Biochemistry
Arlington, TX 76019
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What is a Room Temperature Ionic Liquid?
Liquid salt consisting of at least one organic component (cation or anion)Room temperature ionic liquid (RTIL) if melting point is below room temperatureProperties:
Negligible vapor pressureHigh thermal stabilities (~250-400°C)Highly variable viscositiesHydrophobic or hydrophilicCapable of undergoing multiple solvation interactions
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Ethyl ammonium nitrate (EtNH+3)(NO-
3), which has a melting point of 12°C, was first described in 1914.
P. Walden, Bull. Acad. Imper. Sci. (St. Petersburg) 1800 (1914).
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Uses of RTILsNovel solvents in organic synthesis and liquid-liquid extractionMobile phase additives in HPLCRun buffer additives in CEAdditive for ESI-MS analysis of anionsMatrixes in Matrix-Assisted Laser Desorption Ionization (MALDI) mass spectrometryStationary phases in gas-liquid chromatographySensors
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RTILs as GC Stationary Phases
RequirementsHigh thermal stability (250°C and above)High viscosityHigh wetability on fused silica capillary columnsProduces symmetrical, efficient peaks
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Anal. Chem., 71 (1999) 3873-3876.
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Volatilization of RTILs
220120 140 180160 200 260240
Column Temperature (Celsius)
FID
Det
ecto
r Res
pons
e
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Properties of High Stability Geminal Dicationic Ionic Liquids
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Figure 2-Thermal stability diagram for four of the ILs tested in this analysis. The plot shows the bleeding temperatures for the IL stationary phases, which corresponds to their decomposition or volatilization temperatures. The thermal stability test was done with 1 ml/min He flow, a temperature ramp of 3°C/min, and FID detection. Compounds A1, A4, A3, and D3 represent unique ILs.
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Incremental Max Temperature Studies, Supelcowax 10 vs SLB-IL59
Bleed (pA) in incremental Temp Run
0.0
200.0
400.0
600.0
800.0
1000.0
1200.020
0 oC
210
oC
220
oC
230
oC
240
oC
250
oC
260
oC
270
oC
280
oC
290
oC
300
oC
310
oC
320
oC
330
oC
340
oC
350
oC
Temp (4hrs)
Ble
ed bleed IL-36bleed wax 10
SLB-IL59
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TCEP = (1,2,3-Tris(2-CyanoEthoxy)Propane
• H2C-O-CH2CH2CN * A Highly Polar, Fluid Stationary Phase
* *Oxygen and Moisture Sensitive
* Maximum Operating Temp. = 140oC
H2C-O-CH2CH2CN
HC-O-CH2CH2CN
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TCEP Mix on TCEP Column at 110 °C
0 10 20Time (min)
1
2
3
4 5
6
7
8
1. n-Tridecane2. Toluene3. Ethylbenzene4. p-Xylene 5. Isopropylbenzene (Cumene) 6. 1,2,4-Trimethylbenzene7. 1,2,4,5-Tetramethylbenzene
(Durene)8. Cyclohexanone
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1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0Time (min)
TCEP Mix on SLB-IL100
1.0 2.0 3.0Time (min)
1 3
42
5
6
78
110 °C
1
34
2
5
6
7 8
80 °C
1. n-Tridecane2. Ethylbenzene3. p-Xylene 4. Isopropylbenzene
(Cumene) 5. 1,2,4-Trimethylbenzene6. 1,2,4,5-
Tetramethylbenzene(Durene)
7. Toluene8. Cyclohexanone
•A 1,9-di(3-vinyl-imidazolium)nonane bis(trifluoromethyl) sulfonyl imidate (SLB-IL100) Ionic liquid phase has a polarity and selectivity similar to TCEP.
•SLB-IL100 has an approximate maximum temperature of at least 230 °C which is a significant improvement over the 140 °C maximum temperature of TCEP
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Comparison of trigonal ILs with a polar commercial column
100% cyanopropylpolisiloxane
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Ionic Liquids in Tandem GC (GCXGC)J.V. Seeley et al., Anal. Bioanal. Chem. 390 (2008) 323-332.
• DB-1=100% Dimethylpolysiloxane, non-polar, general purpose, bonded and crosslinked• DB-Wax= PEG columns, high polarity, good for resolving low BP compounds• DB-210= (50%-Trifluoropropyl)-methylpolysiloxane high polarity, bonded and crosslinked • Perkin-Elmer Autosystem XL GC, modulation period = 1.5 s
• Traditional polar columns have a max temperature of ~280oC• IL columns are stable to greater than 350oC
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GCXGC with Dicationic IL Columns in the 2nd Dimension
(Min.)
(Min
.)(M
in.)
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Figure 6. GC x GC separation of diesel fuel on the (a) IL x HP-5 column combination, (b) the DB-Wax x HP-5 column combination, and (c) the HP-50+ x HP-5 column combination. Both the IL x HP-5 and DB-Wax x HP-5 configurations generated distinct chromatographic regions for the saturated hydrocarbons, monoaromatics, and diaromatics. The HP-50+ x HP-5 configuration had nearly complete separation of the saturated hydrocarbons from the aromatics, but no clear separation of the aromatics into monoaromatic and diaromatic regions.
Anal. Bioanal. Chem. 390 (2008) 323-332.
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Col
umn
2 Ti
me
(sec
), D
B-W
AX
N+
NCH 2
N+
NC H 2
TfO-TfO-
V. R. Reid, J.R. Crank, D. W. Armstrong, R. E. Synovec, Submitted Journal of Separation Science, 2008. Cooperation between the University of Washington and University of Texas at Arlington
Secondary column = DB-WAXSecondary column = SLB-IL100Primary column = DB-5
Primary column = DB-5Column 1 Time (min), DB-5
1 2 3 4 5 60
3
1
7
DMMP
TEP
DIMP
DEMP
Naphthalene
1,3,5-trichlorobenzene
Br-benzene
1-Br-octane
2
Col
umn
2 Ti
me
(sec
), Tr
iflat
e
Column 1 Time (min), DB-5
1 2 3 4 5 60
3
2
1
7
DMMP (dimethyl methylphosphonate)
TEP (triethyl phosphate)DEMP
DIMP (diisopropyl methylphosphonate)
Naphthalene
30 Compounds that do not
contain P-O
2D-GC Isolation of P-O Containing Compounds Using a Triflate Ionic Liquid Column
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Recent RTIL Polarity Measurements
Solvatochromic DyesReichardt dye
Large shift in charge transfer absorption band
max= 810 nm in diphenyl ethermax= 453 nm in water
Nile RedExperiences large bathochromic shift
Results: RTILs have averagepolarity similar to propanol
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20
GC Column Polarity Scale
•GC column polarity scale•0 = squalane (considered the least polar GC stationary phase)•100 = TCEP (considered the most polar GC stationary phase)
0 50 100
280°CWax (PEG)
310°C-20 -1701 -35 -50
360°C-1 -5
275°C 250°C 140°C-2331 -2560 TCEP
Non-Polar Intermediate Polar Polar Highly Polar
2515Range of Alternative Polarities possible from Ionic Liquid GC
propanol
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Determination of Stationary Phases Polarity Numbers according to McReynolds-Rorhschneider constants determination and comparison with those obtained on the most widely used polar and nonpolar columns
Evaluated Columns:
• SLB-IL111 • SP-2340• SLB-IL76 • SP-2330• SPB-225• PAG• SPB-50• SPB-35• SPB-20• SPB-Octyl
Experimental Conditions:
Column Oven Temperature: 120°C
Carrier gas Helium at constant linear velocity: 40 cm/sec
Injection mode: 7 m PDMS SPME Fiber
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22
SLB-IL100
1,9-di(3-vinyl-imidazolium) nonanebis(trifluoromethyl) sulfonyl imidate
N N NN N
S
S
O
CF3
O
O
O CF3
-2+ +
FIRST COMMERCIAL IONIC LIQUID STATIONARY PHASE
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23
GC Polarity Scale
•GC column polarity scale•0 = squalane (considered the least polar GC stationary phase)•100 = TCEP (considered the most polar GC stationary phase)
0 50 100
280°CWax (PEG)
310°C-20 -1701 -35 -50
360°C-1 -5
275°C 250°C 140°C-2331 -2560 TCEP
Non-Polar Intermediate Polar Polar Highly Polar
2515Range of Alternative Polarities possible from Ionic Liquid GC
Where do current ILs fit on this scale?
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Note that IL Stationary Phases of the Same Polarity as Current
Molecular Phases - STILL HAS DIFFERENT
SELECTIVITIES for MANY TYPES of COMPOUNDS, INCLUDING
ISOMERS-
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How Will Ionic Liquid Stationary Phases Fit into the Pantheon of GC Columns?
I) New IL stationary phases will be introduced that are engineered to produce identical separations to current, often flawed commercial stationary phases.
Example: The polar stationary phase TCEP does some unique separations,but it has an upper temperature of 140oC.
II) New IL stationary phases will be introduced that will have completely uniqueselectivities compared to any/all commercial columns.
III) New IL stationary phases of ultra-high thermal stability are at hand.Caveat: Some multifunctional Ils have greater thermal stability than the
outer polyimide coating of the fused silica capillaries
IV) IL stationary phases should play a significant role in multidimensional separations because of their unique group selectivity and their natural or
engineered orthogonality to existing stationary phases.
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GENERAL PROPERTIES of MALDI MATRICIES
a) They must dissolve (liquid matrix) or co-crystallize (solid matrix) with the sample.
b) They must strongly absorb the laser light (e.g., 337 nm).
c) They must remain in the condensed phase under high vacuum conditions.
d) They must stifle both chemical and thermal degradation of the sample.
e) They must promote the ionization of the sample via any number of mechanisms.
IONIC LIQUIDS FOR MALDI-MS
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Dissolution of Cellulose with Ionic LiquidsR.P. Swatloski, R.D. Rogers, et al. J.A.C.S. 124 (2002) 4974.
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MALDI mass spectra of the three oligonucleotides (d(pT)10, d(pC)11, and d(pC)12) in different matrixes: (a) 3-HPA + 10% ammonium citrate, (b) ionic solid 21, and (c) ionic solid 26. Spectra obtained cumulating 100 UV 237 nm laser shots. For the three experiments, the oligonucleotide-to-matrix molar ratio was 1:500000 and the laser fluence was the same (attenuation 10). The signal strength is expressed in arbitrary units corresponding to the accumulation of 100 shots on a good spot. The 3-HPA scale (top spectrum) differs 8 times from that for the two salts (bottom spectra).
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• Towards a Second Generation of Ionic Liquid Matrices (ILM’s) for MALDI-MS of Peptides, Proteins, and Carbohydrates
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0
500
1000
1500
2000
2500
Inte
ns. [
a.u.
]
1056 1057 1058 1059 1060 1061 1062 1063 1064 1065m/z
0.0
0.5
1.0
1.5
2.0
2.5
4x10
Inte
ns. [
a.u.
]
600 800 1000 1200 1400 1600 1800m/z
0.0
0.5
1.0
1.5
2.0
2.5
4x10
Inte
ns. [
a.u.
]
600 800 1000 1200 1400 1600 1800m/z
0.0
0.5
1.0
1.5
2.0
2.5
4x10
Inte
ns. [
a.u.
]
600 800 1000 1200 1400 1600 1800m/z
0.0
0.5
1.0
1.5
4x10
Inte
ns. [
a.u.
]
4000 6000 8000 10000 12000 14000 16000 18000m/z
0.0
0.5
1.0
1.5
4x10
Inte
ns. [
a.u.
]4000 6000 8000 10000 12000 14000 16000 18000
m/z
0.0
0.5
1.0
1.5
4x10
Inte
ns. [
a.u.
]
4000 6000 8000 10000 12000 14000 16000 18000m/z
CHCA SA IMTBA CHCA
0.0
0.5
1.0
1.5
2.0
2.5
4x10
Inte
ns. [
a.u.
]
50000 100000 150000 200000 250000 300000 350000 400000 450000 500000m/z
0.0
0.5
1.0
1.5
2.0
2.5
4x10In
tens
. [a.
u.]
50000 100000 150000 200000 250000 300000 350000 400000 450000 500000m/z
0.0
0.5
1.0
1.5
2.0
2.5
4x10
Inte
ns. [
a.u.
]
50000 100000 150000 200000 250000 300000 350000 400000 450000 500000m/z
0.0
0.5
1.0
1.5
2.0
2.5
4x10
Inte
ns. [
a.u.
]
50000 100000 150000 200000 250000 300000 350000 400000 450000 500000m/z
00
0.5
1.0
1.5
2.0
2.5
4x10
Inte
ns. [
a.u.
]
50000 100000 150000 200000 250000 300000 350000 400000 45000000
0.5
1.0
1.5
2.0
2.5
4x10
Inte
ns. [
a.u.
]
50000 100000 150000 200000 250000 300000 350000 400000 45000000
0.5
1.0
1.5
2.0
2.5
4x10
Inte
ns. [
a.u.
]
50000 100000 150000 200000 250000 300000 350000 400000 450000
BradykininMW=1,060 Da
Cytochrome CMW=12,000 Da
BSAMW=66,000 Da
UreaseMW=90,000 Da
Lower laser intensity
600 800 1000400 1200 1400 1600 1800 600 800 1000400 1200 1400 1600 1800 600 800 1000400 1200 1400 1600 1800
600 800 1000 1200 1400 1600 1800 600 800 1000 1200 1400 1600 1800 600 800 1000 1200 1400 1600 1800
1X105 2X105 3X105 4X105 5X105 5X105
1X105 2X105 3X105 4X105 5X105
IMTBA CHCA
1X105 2X105 3X105 4X105 5X105 1X105 2X105 3X105 4X105 5X105
1X105 2X105 3X105 4X105 5X105 5X1051X105 2X105 3X105 4X105 5X105 1X105 2X105 3X105 4X105 5X105
[M + H]+
[M + H]+[M + H]+
[M + H]+
[M + H]+
[M + 2H]2+ [M + H]+
[M + H]+
[M + 2H]2+[M + H]+
[2M + H]+
[3M + H]+
[M + H]+ [M + H]+
[2M + H]+
[3M + H]+
[M + H]+
[2M + H]+
[3M + H]+
[M + H]+
[2M + H]+
[3M + H]+
[M + 2H]2+
[M + H]+
1056 10581057 1059 1060 10621061 1063 1064 10661065
[2M + H]+
[3M + H]+[4M + H]+
H N+
NOH
O-
O
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Detection of Catalase (Monomer=60,000 Da)
0 0
0.5
1.0
1.5
2.0
4x10
Inte
ns. [
a.u.
]
0 0
0.5
1.0
1.5
2.0
4x10
Inte
ns. [
a.u.
]
0 0
0.5
1.0
1.5
2.0
4x10
Inte
ns. [
a.u.
]
50000 100000 150000 200000 250000 300000
[M + H]+
[M + H]+
[M + H]+[2M + H ]+
[3M + H]+[4M + H]+
[5M + 2H]2+
NOH
O-
O
IMTBA CHCA
H N+
NOH
OH
O
-cyano-4-hydroxycinnamic acid: CHCA
Sinapinic acidO
OH
O
OH
O
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Cation Properties vs. PerformanceAmine name pKa
PA (kJ/mole)
GB (kJ/mole)
Performance vs. Solid Matrix
triethanolamine 7.8 941 NA Xtriisobutylamine 9.5 967.6 998.5 X
tributylamine 9.9 998.5 967.6 -butylamine 10.6 921.5 886.6 -
2-amino butane 10.7 929.9 895.7 +N-isopropyl-N-methyl-t-butylamine 10.9 NA NA ++
N,N-diisopropylethylamine 11.4 994.3 963.5 ++
•Cation pKa should be 11•Cation PA should be 930 kJ/mole
•Anion pKa and PA were examined but no correlation was found
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Effective MS Analysis of Biodegradable Polymers with Second Generation Ionic
Liquid MALDI Matrices
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Characterization of Polycaprolactone
OH
O
n
Mn found by GPC Mn=10,000 Mn obtained from the manufacturer
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0
200
400
600
800
1000
1200Inte
ns. [
a.u.
]
1000 2000 3000 4000 5000 6000 7000 8000m/z
0
1000
2000
3000
4000
5000
Inte
ns. [
a.u.
]
1000 2000 3000 4000 5000 6000 7000 8000m/z
0
2000
4000
6000
Inte
ns. [
a.u.
]
1000 2000 3000 4000 5000 6000 7000 8000m/z
0.0
0.2
0.4
0.6
0.8
1.0
4x10
Inte
ns. [
a.u.
]
1000 2000 3000 4000 5000 6000 7000 8000m/z1000 2000 3000 4000 5000 6000 7000 8000
1000 2000 3000 4000 5000 6000 7000 8000 1000 2000 3000 4000 5000 6000 7000 8000
1000 2000 3000 4000 5000 6000 7000 80000
200
400
600
800
1000
1200
0
0.2
0.4
0.6
0.8
1.0
0
2000
4000
6000
x104
0
1000
2000
3000
4000
5000
m/z m/z
m/z m/z
Inte
nsity
Inte
nsity
Inte
nsity
Inte
nsity
DEA CHCA HABA
DHB DCTB
Mn=4250 DaMw=4732 Dapd=1.11
Mn=4162 DaMw=4758 Dapd=1.14
Mn=1764 DaMw=1959 Dapd=1.11
Mn=784 DaMw=815 Dapd=1.04
Mn=2447 DaMw=4059 Dapd=1.66
Mn=1345 DaMw=1570 Dapd=1.17
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Characterization of Polycaprolactone Triol(Mn= 300 and 900 Da)
ROOR
OR
OH
O
n
R=
Mn values are estimated by the manufacturer
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0
250
500
750
1000
1250
1500
Inte
ns. [
a.u.
]
400 600 800 1000 1200m/z
0.0
0.2
0.4
0.6
0.8
1.0
4x10
Inte
ns. [
a.u.
]
500 1000 1500 2000 2500 3000 3500m/z400 600 800 1000 1200
0
250
500
750
1000
1250
1500
500 1000 1500 2000 2500 3000 35000
0.2
0.4
0.6
0.8
1.0
x104
m/z
Inte
nsity
m/z
Inte
nsity
Mn=593 DaMw=629 Dapd=1.06
Mn=1930 DaMw=2081 Dapd=1.08
Estimated 300 Da
Estimated 900 Da
Characterization of Polycaprolactone Triol(Mn= 300 and 900 Da)
PrecisionMn=593 ± 3 Da Mn=1930 ± 16 Da
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• 16 biodegradable polymers were characterized– DEA CHCA typically produced almost
Gaussian analyte peak distributions– DEA CHCA typically produced larger Mn’s and
Mw’s with the least degradation• When DEA CHCA was used as a matrix,
Mn’s and Mw’s were shown to be both precise and accurate
Conclusions
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Quantitative Analysis of Anions
Anion quantification is important for:Environmental monitoringBiological analysisSemiconductor Industry
Current Methods of DeterminationIon chromatographyFlow-injection analysisIon-selective electrodes
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Take a Di-cationic Reagent
• Anion exchange to fluoride form• Fluoride complex not detected in the MS
A New Approach for Ultra-Sensitive Anion Analysis
REFERENCES: Anal. Chem., 2005, 77, 4829.Anal.Chem., 2007, 79, 7346.
J. Am. Soc. Mass Spec., 2008, 19, 261.Anal. Chem., 2008, 80, 2612.
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Gas-Phase Ion Association in ESI-MS
Using ionic additives as ion-pairing agents for MS
First application was published in Anal. Chem. 77 (2005) 4829-4835.We are expanding to additional ions.
2++ ClO4
-
m/z +145(MW 290)
m/z +389m/z - 99
ClO4-
2+ 1+
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Detection of Anions• Three different ways to detect ions
1) Anion SIM2) Cationic complex SIM3) Use MS/MS
- Trap m/z of complex- Excite this m/z to break complex- Monitor m/z of deprotonated cation (m/z
289)1+
ClO4-
1+
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Why Positive Ion Mode?
MeOH/Water based solvent systems not ideal to provide stable signal in negative mode
Low gas-phase proton affinities lead to protonation of analyte
Corona discharge more prevalent in negative mode
Leads to unstable signalHigher background noise
REFERENCES: Anal. Chem., 2005, 77, 4829.Anal. Chem., 2007, 79, 7346.
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Other Advantages
• Can select cation to place complex in a low noise M/Z (shift to higher mass region)
• Can bring smaller ions out of low mass cutoff (LMCO=50 for LXQ)
• May help distinguish between ions of same M/Z (35ClO4
- vs. H34SO4-)
REFERENCES: Anal. Chem., 2005, 77, 4829.Anal.Chem., 2007, 79, 7346.
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ESI-MS Analysis
Sample Sol’n
[Dication]2+
[Anion]-
[Dicat+Anion]+
MS
LC PumpH20/MeOH
LC Pump40 uM Additive in H20
LC ColumnFinnigan LXQ
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Anions
• ClO4-, BrO3
-, IO3-, IO4
-
• Cl-, Br-, I-
• NO3-, NO2
-
• SCN-, OCN-, CN-
• BF4, - PF6
-
• MnO4-
• H2AsO4-
• Perfluoronated octanoic Acid (PFOA)
• 2-Bromooctanoic acid• Halogenated and acetic acids
(TFA, TCA, BrClA, Cl2A, MBrA, MClAA)
• Acetic, Formic, Benzoic acids• Benzensulfonate• Trifluoromethanesulfanate
(TFO)• Trifluoromethanesulfonimide
(NTF2)
Inorganic Organic
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Positive Ion Limits of Detection for Anions Using Dicationic Reagent
Anion SIM Mass SIM LOD (ng) SRM Mass SRM LOD (ng)
Perfluorooctanoic acid (PFOA) 703 1.22 x10-4 289 7.32 x10-5
Nitrate (NO3-) 352 1.84 x10-3 289 1.38x10-3
Tetrafluoroborate (BF4-) 376 1.96 x10-3 289 3.90x10-1
Thiocyanate (SCN-) 348 2.00 x10-3 289 2.00x10-3
Benzenesuflonate (BZSN) 447 2.06 x10-3 289 4.12x10-4
Trifluoromethanesulfonimide (NTF2-) 570 2.26 x10-3 289 2.26x10-3
Hexafluorophosphate (PF6-) 435 4.28 x10-3 289 2.14x10-3
Iodide (I-) 417 6.00x 10-3 289 2.00x10-1
Perchlorate(ClO4-) 389 1.02 x10-2 289 1.02x10-2
Dichloroacetic acid (DCA) 417, 419 1.50 x10-2 289 2.00x10-2
Monochloroacetic acid (MCA) 383, 385 1.50 x10-2 289 1.90x10-0
Bromochloroacetic acid (BCA) 461, 463 1.54 x10-2 289 1.54x10-02
Periodate (IO4-) 481 4.48 x10-2 289 1.12x10-0
Bromate (BrO3-) 417, 419 5.00 x10-2 289 5.00x10-02
Iodate (IO3-) 465 6.00 x10-2 289 1.39x10-02
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PFOAArea: 4.0E4S/N: 26
RT: 0.00 - 18.00 SM: 15G
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e Ab
unda
nce
NL:0m/z= 57.50-58.50 MS AnionmixtureMS_040207dNL:2.46E3m/z= 148.50-149.50 MS Genesis AnionmixtureMS_040307eNL:1.11E4m/z= 156.50-157.50 MS Genesis AnionmixtureMS_040307eNL:1.51E4m/z= 279.50-280.50 MS Genesis AnionmixtureMS_040307eNL:2.01E3m/z= 412.50-413.50 MS Genesis AnionmixtureMS_040307e
RT: 0.00 - 17.99 SM: 15G
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
NL:4.62E3m/z= 347.50-348.50 MS Genesis AnionmixtureMS_040307cNL:4.60E3m/z= 438.50-439.50 MS Genesis AnionmixtureMS_040307cNL:1.15E4m/z= 446.50-447.50 MS Genesis AnionmixtureMS_040307cNL:9.83E3m/z= 569.50-570.50 MS Genesis AnionmixtureMS_040307cNL:7.46E3m/z= 702.50-703.50 MS Genesis AnionmixtureMS_040307c
0 3 6 9 12 15Time (min)
100
80
60
40
20
00 3 6 9 12 15Time (min)
100
80
60
40
20
0
Rel
ativ
e A
bund
ance
Rel
ativ
e A
bund
ance
SCNArea: 7.3 E5S/N: 138
TFOArea: 8.9E5S/N: 88
BZSNArea: 2.9E6S/N: 204
NTF2Area: 1.4E5S/N: 30
PFOAArea: 1.0E5S/N: 162
TFOArea: 4.9E5S/N: 67
BZSNArea: 3.6E5S/N: 31
NTF2Area: 2.5E5S/N: 32
SCN
(A) (B)
A comparison of the chromatographic separation and sensitivity of 5 anions on a Cyclobond I column detected in the (A) positive and (B) negative SIM modes. The mass injected in (B) is 10x that of (A) for SCN, TFO, and BZSN, 5x for PFOA, and the same for NTF2. The mass injected in (A) is :1.43 ng SCN, 9.92 ng TFO, 1.16ng BZSN, 0.68 ng NTF2, and 1.30 ng PFOA. The column was equilibrated with 100% Water with a linear gradient to 100 % MeOH beginning at 3 minutes and complete at 9 minutes. Flow rate was 300 L/min. In (A) the dicationic salt solution (40
M in MeOH) was added post-column at 100 L/min where as in (B) it is methanol only. SCN: thiocyanate; TFO: triflate; BZSN: benzenesulfonate; PFOA: perfluorooctanoic acid; NTF2: trifluoromethanesulfonimide. From: D. W. Armstrong et al., Anal. Chem. 2007, 79, 7346.
Positive and Negative LC-ESI-MS
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Detection of Related SpeciesArsmix_032707o #56-60 RT: 0.85-0.91 AV: 5 NL: 1.59E3T: ITMS + p ESI u Z ms [ 413.50-433.50]
414 416 418 420 422 424 426 428 430 432m/z
0
50
100
150
200
250
300
350
400
450
500
550
600
650
700
750
800
850
900
950
1000
1050
1100
1150
1200
1250
1300
1350
1400
1450
1500
1550
Inte
nsity
429.24
431.23427.26 430.24
417.30428.22 432.23414.42 426.88418.31415.40 420.88 424.84416.92 419.42 422.87422.27 433.23426.39
DMAV Arsenate
Arsmix_032707o #43-50 RT: 0.65-0.76 AV: 8 NL: 2.00E3T: ITMS + p ESI u Z ms [ 413.50-433.50]
414 416 418 420 422 424 426 428 430 432m/z
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30
32
34
36
38
40
42
44
46
48
50
52
54
56
58
60
62
64
66
68
Inte
nsity
428.22
414.42
418.31
432.22
420.88415.40
421.28
416.92
418.91
422.88
424.83422.25432.46424.94
419.41433.26419.94 423.18
423.40416.39 426.35
40X
Arsenite
OH As OH
O
AsO
OOHOH
AsO
OCH3CH3
AsO
OCH3OH
MMAV
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Recommended Dications
N+
N+
OH
N+
N+
N+
N+
O
O
OH
OH
CH3CH3
CH3 H
OMe
MeON
+N
OH N+
N OHO O O
N+
N CH2CH2(C F2)4CH 2CH2 N+N
N+
N N+
N
P+
P+
P+
P+
O O O
N+
N(CH2)5N+
P+
(CH2)5N+
N+
N(CH) 9N+N
(CH2)3 P+
P+
(CH2)5 P+
P+
(CH2)9 P+
P+
N+
N(CH2)3N+N
N+
N(CH2)5N
+N N
+(CH2)5N+
N+(CH 2)5N
+
(CH2)5 N+
N+
(CH2)12 N+
N+
N+
N( CH2)5N+
N
N+
N(CH2)5N
+N
N+N(CH2)5 OHN
+N
OH
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RR
R
RR
R
NRR
R
A B
C
R= NN+
2)
R= NN+
1)
R= NN+
OH3)
R= P+
6)
R= N+5)
C1C2C3C4C5C6C7
A1A2A5A6
B1B2B4B6
Trications Core Charged Groups
NNN
R R
OO
N
R
O
5
5
5
D
D2D6
R=4) N N
NH+
NR=7)
Trications A6 and B1 performed the best overall
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LC-MS Positive vs. Negative Ion Mode
RT: 0.00 - 5 .01 SM : 7G
0.0 0.5 1.0 1 .5 2.0 2.5 3.0 3.5 4 .0 4.5 5.0
Tim e (m in)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e Ab
und
anc
e
AA: 387S N: 6
N L:1 .02E 2TIC MS G enes is
N egH exC lP d_102607b
Time (min)
100
80
60
40
20
0
RelativeAbundance
0 1 2 3 4 5
5 ngS/N: 6
RT: 0.00 - 5. 02 SM: 7G
0.0 0.5 1.0 1. 5 2.0 2.5 3.0 3.5 4.0 4 .5 5.0Tim e (m in)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e Ab
unda
nce
AA: 7969S N: 35
NL:1.45E3TIC MS Gen esi s TricatL_HCP_102607
0 1 2 3 4 5Time (min)
100
80
60
40
20
0
RelativeAbundance
500 pgS/N: 35
RT: 0.00 - 5.01 SM: 7G
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4 .5 5.0Tim e (m in)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e Ab
unda
nce
AA: 13 2SN: 3
NL:2.71E1TIC MS Gen esi s Neg OBDSA_102 607c
0 1 2 3 4 5Time (min)
100
80
60
40
20
0
RelativeAbundance
5 ngS/N: 3
RT: 0 .00 - 5 .02 SM : 7G
0.0 0.5 1.0 1 .5 2.0 2.5 3.0 3 .5 4 .0 4 .5 5 .0Tim e (m in)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rela
tive
Abu
nda
nce
AA: 20004S N: 56
N L:2 .74E3TIC M S G enes is TriC atB _12907
0 1 2 3 4 5Time (min)
100
80
60
40
20
0
RelativeAbundance
500 pgS/N: 56
HexachloroplatinatePtCl6
2-
(Trication A6)
Benzenedisulfonate
(Trication B1)
Positive Negative
SO3-
SO3-
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Trications with More Flexibility?• Rigid trications not performing quite as well as the
more flexible dications• Synthesized:
• Preliminary results show that this flexible linear trication performs better than any of the other more rigid trications
• Lowest LOD for SO4, S2O3, dibromosuccinate, and FPO3.
• Ranking near the top for detection of Cr2O7, nitroprusside, and hexachloroplatinate.
P+
CH3
CH3CH3
N+
N
P+
CH3
CH3
CH3
From: D. W. Armstrong & co-workers, J. Am. Soc. Mass Spec.2008, in press.
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Figure 2. Comparison of the detection of sulfate in the positive mode using tricationic ion-paring reagents D3 (I) and E2 (II). Note, Sulfate has a M/Z of 48 and is completely undetectable in the negative ion mode.
I
II
500 pgS/N: 21
500 pgS/N: 3
nCH2P
+
CH3
CH3
CH3
N N+ CH2 P
+
CH3
CH3
CH3
n
n=3
R
R
R
R= tripropylphosphonium
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An extracted ion chromatogram representing the LC separation of camphorate, phenylsuccinate, and naphthalene-1,5-disulfonate with the retention times (RT) listed. This separation was performed on a -cyclodextrin stationary phase (2.1mm x 25 cm) which was equilibrated with 100% methanol. A step gradient to 100% water was applied at 5 minutes. The flow rate was 300 uL/min and 40 uM LTC 1 was teed into the effluent at a flow rate of 100 uL/min. The three trication-dianion complex masses were monitored simultaneously in SIM mode.
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14Tim e (m in)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
RT 4.21
RT 6.58RT 8.45
m/z 865.8
m/z 859.8
m/z 953.8
HOOH
O
OO-
-O
O
O
Tricationic pairing reagent
P+
N+NP
+10 10
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S/N=5
500pg
N+
N
N+
NN
+
N
N+
N
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Chiral Stationary Phases
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D.W. Armstrong, Journal of Chromatographic Science, Vol. 22, September, 1984, pg. 412.
Schematic diagram showing the structure and relative size of the three most common cyclodextrin molecules. (A) -cyclodextrin (or cyclooctaamylose). (B) -cyclodextrin (or cycloheptaamylose), and (C) -cyclodextrin (or
cyclohexaamylose).
A schematic of cyclodextrin bonded to a silica gel support and reversibly forming an inclusion complex with a chiral molecule. Neither the linkage nor the cyclodextrin contain nitrogen (e.g., amines or amides) in any form.
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Simplified schematics illustrating two different enantioselective retention mechnisms for the native -cyclodextrin/propanol system. Case “A” is the polar organic mode where acetonitrile occupies the hydrophobic cavity and the analyte is retained via a combination of hydrogen bonding and dipolar interactions at the mouth of the cyclodextrin. Steric interactions also can contribute to chiral recognition (8,9). In case “B”, (the reversed phase mode) retention is mainly due to hydrophobic inclusion compexation, while enantioselectivity also requires hydrogen bonding and steric interactions at the mouth of the cyclodextrin cavity (1-4).
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Bonded DerivatizedCyclodextrins
Summary of Derivatives of CYCOBOND 1 2000
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The separation of three racemates using an (S)-NEC- -cyclodextrin column, including the (a) normal phase separation of the 3,5-dinitrobenzoyl derivative of racemic 1-(1naphthyl)ethylamine, (b) reversed phase separation of racemic bendroflumethiazide, and (c) separation of the enantiomers of ciprofibrate. Column dimensions: 25 cm x 4.6 mm; mobile phase (a): 70:30 (v/v) hexane-isopropyl alcohol; (b): 30:70 (v/v) acetonitrile 1 vol % triethylammonium acetate in water; (c): 80:20:1 (v/v/v) acetonitrile – ethanol-acetic acid. Flow rate: 1.0 mL/min.
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DNP-O- -CD FamilyO
CH 2OR 1
O
OR 2
OR 3
OC
H2O
R1
OO
R2
OR
3
OC
H2 O
R1O
OR
2O
R3
OCH2OR1
OOR2
OR3
O CH2OR1
O OR2
OR3
OCH
2 OR1
OOR
2
OR3
OCH2OR1
OOR2OR3
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Anal. Chem., 68, 2501 (1996).
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Capillary electropherograms showing the resolution of (A) three racemic AQC amino acids and (B) racemic dansyl-valine. The separations were done with a 50 m x 30.5 cm (25 cm to detector) containing 0.1 M phosphate buffer and 5 mM vancomycin. The voltage was + 5 kV, and the analytes were detected via absorbance at 254 nm. The pH of the run buffer was 7.0 for the AQC amino acids, and 4.9 for the dansyl amino acids.
CE EVALUATION OF VANCOMYCIN
CHIRALITY 6:496-509 (1994)
Capillary electropherograms showing the resolution of nonsteroicantiinflammatones: (A) naproxen, (B) carprofen, and (C) flurbiprofen. The separations of carprofen and flurbiprofen were done with a 50 M x 30 5 cm (25 cm detector) containing pH 7. 0.1 M phosphate buffer and 5 mM vancomycin. The voltage was 5 kV, and the analytes were detected via absorbance at 254 nm. The separation of naproxen was done under the same conditions exceptthat the vancomycin concentration was 2 mM and pH was 6.0
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JOURNAL OF LIQUID CHROMATOGRAPHY, 17(3), 1695-1707 (1994)
TLC chromatogram showing the separation of all four isomers (2 pairs of enantiomers) of AQC-leucyl-leucine. The stereochemistry of the compounds represented by each spot is indicated. This was determined by developing pure standards in a separated experiment. The mobilie phase consisted of 0.02 M vancomycin in 1:3 (by volume) acetonitrile: 0.6 M
TLC chromatogram showing the separation of (A) indoprofen, and (B) coumachlor. The mobile phase consisted of 0.05 M vancomycin in 4:6 (by volume) acetonitrile: 0.6 M NaCl(aq). Diphenyl-F TLC plates (5 x 20 cm) were used. Spots were detected using a 365 nm UV hand lamp (see Experimental).
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Anal. Chem. 66 (1994) 1473
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J. Chromatogr. A 731 (1996) 123-137
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Principle of Complementary Separations
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P-CAP™
New Normal Phase CSP
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P-CAP
• New bonded polymeric chiral stationary phase
• No solvent limitations• Reversible elution order as R,R and S,S
configurations• High efficiency – thin, ordered layer bonded
to the silica surface gives fast kinetics • Derivatization has little effect on selectivity
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Elemental Analysis: %C 10.36; %H 1.68; %N 2.19FT-IR (KBr): 3078, 2941, 2860, 2237, 1646, 1542, 1451 cm-1
Weight Increment: 16.55%
NH
NH
O
O
SilicaGel
Si
Si
O
O
Si
Si
NH
NH
O
O
CN
N
CN
N
CHCl3, 60oC+
SilicaGel
Si O Si NH
O
NHCN
ONH
O
NHO
NH
O
Si O Si NH
O
NHCN
ONH
O
NHO
NH
O
Free Radical Polymerization on Functionalized Silica Gel
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Poly-DPEDA CSP
Si OH
Si OH
H3CO SiH3CO
H3COO
O
Si O
Si O
Si
Si
O
O
O
O
NH
NH
O
O
Synthesis of the poly-DPEDA chiral stationary phase.
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Comparison of separation in two mobile phase modes
O2N
N2O
O
N
CO2H
H
8.94
25.74
4.17
5.42
Mobile phase: EtOH/Heptane/TFA=40/60/0.1=3.93 Rs=5.1,
Mobile phase: ACN/MeOH/TFA=100/1/0.1=2.31 Rs=2.6,
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Comparison of separation in three synthetic polymeric CSPs
30.88 32.8223.65
28.4810.13
12.63
OHHO
P-CAP CSP P-CAP-DP CSP New polymeric CSPMobile phase: Heptane/EtOH/TFA=90/10/0.1 Mobile phase: Heptane/EtOH=50/50Mobile phase: Heptane/EtOH=80/20
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Cyclofructan (CF)• Cyclic oligosacchride• Inuline,fructosyltransferas
e• 6,7,8 fructofuranose units• Crown ether skeleton• Disk-shape with shallow
central indentation • UV transparent• Solubility: >1.2g/ml• No health hazardous
S. Immel et al. Carbohydrate Research 313 (1998) 91 105
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Sulfated Cyclofructan (SCF6)C36H42O12(OH)18 (2) CH3OH CH3COONa
(1)Pyridine SO3 in Pyridine 80 850C,6hC36H42O14(OH)18 n(OSO2Na) n
Q. Sun et al. Di’er Junyi Daxue Xuebao, 27, 453
13
12
11
14
15
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N
NH3C
OH2CC
OPh
HO
Conditions: 15 mM SCF6, 20 mM AmAc,10mMPhosphate, pH=4.7, 25 kv, 30 cm/40 cm capillary
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Poor Separations on Native CF6
0 10 20 30 40Time (min)
CF63,5-dimethylphenyl carbamateAcetonitrile/methanol/AA/TEA75/25/0.3/0.2
Native CF6Acetonitrile/methanol/AA/TEA90/10/0.3/0.2
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Hydrophobic FaceHydrophilic Face
Side View
Cyclofructan Crystal Structure
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Native vs. Derivatized CF6
0 10 20 30 40Time (min)
CF63,5-dimethylphenyl carbamateAcetonitrile/methanol/AA/TEA75/25/0.3/0.2
Native CF6Acetonitrile/methanol/AA/TEA90/10/0.3/0.2
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Structure of Chemically-Bonded CF CSPs
O
O
O
OO
O
O
O
O
O
O
O
OR
RO
RO
OR
OR
OR
O
OR
OR
OR
RO
O
RO
RORO
OR
OROR
1~3
Suppot
1
2
3
4 R
1. silica gel support2. covalent linker between cyclofructans and solid support3. cyclofructan 6~84. derivatization groups or hydrogen
Diverse derivatization groups
•Aliphatic derivatization
•Aromatic derivatizationCH3
H2C CH3 CH
CH3
CH3
C
CH3
CH3
CH3
CH3 Cl
CH3
CH3
Cl
Cl
NO2
NO2
CF3
CF3
CH3CH3
H3C H3C
Cl
CH3
Cl
CH3
Cl
H3C
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3D Optimized Cyclofructan Isopropyl Derivativ
Hydrophobic Face Hydrophilic Face
Side View
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Side View
Cyclofructan Crystal Structure
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Separation of Primary Amines
min6 10 14 18
CHCH3
NH2
H3COH
NH2
1S,2R/1R,2S
Column: IPCF; 60ACN/40MEOH/0.3AA/0.2TEA
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D Optimized Cyclofructan RN Derivative (6, 12, 1
RN 6 Hydrophobic Face RN 12 Hydrophilic Face
RN 18 Hydrophilic Face
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RN 6 Side View
RN 12 Side View
RN 18 Side View
D Optimized Cyclofructan RN Derivative (6, 12, 1
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Comparison Between Different Modes
min4 6 8 10 12
Normal phase mode
70 heptane/30ethanol
Reversed phase mode
50 water/50 acetonitrileOH
Br
HO
Br
Generally, normal phase mode is better than reversed phase mode for separating neutral analytes, due to higher selectivity and efficiency.
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CF-based CSPs can be used alternately in polar organic, reversed-phase and normal phase solvents without damage.
0 5 10 15
A. Normal phase mode
B. Polar organic mode
C. Reversed phase mode
H2NNH2
NH2
OH
Ph
Ph
CO2HO2N
NO2
NH
O
R
Mobile phase: (A) 70% heptane/30% ethanol: (B) 60% acetonitrile/40% methanol/0.3% acetic acid/0.2% triethylamine. CSP: CF6-RN
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Separation of Chiral Acids
0 Min 20
CF73,5-dimethylphenyl carbamateheptane/ethanol/TFA80/20/0.1
CF6bis(trifluoromethylphenly carbamateAcetonitrile/methanol/AA/TEA75/25/0.3/0.2
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Separation of Chiral Alcohols
15 30Min
CF73,5-dimethylphenyl carbamateheptane/ethanol/TFA99/1/0.1
CF6R-naphthylethyl carbamateHeptane/isopropanol/TFA98/2/0.1
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Separation of Chiral Pharmaceutical Compounds
20 Min 55
CF7R-naphthylethyl carbamateheptane/ethanol/TFA99/1/0.1
CF63,5-dimethylphenyl carbamateHeptane/isopropanol/TFA98/2/0.1Thalidomide
Bendroflumethiazide
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Separation of Ru tris(diimine) Complexes
min0 4 8 12 16
RNCF6 column
60methanol/40acetonitrile/25 mM NH4NO3
[Ru(phen)3](Cl2)
[Ru(dppz)3](Cl2)
N
N
RuN
N
N
N
2+
N
N
RuN
N
N
N
2+2+
N
N
Ru
NN
NN N
N
N
N
N
N
2+
N
N
Ru
NN
NN N
N
N
N
N
N
2+2+
k1=0.883, =1.51, Rs=4.4
k1=1.27, =2.81, Rs=12.1
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SFC Separations of Derivatized Amino Acids
CF73,5-dimethylphenyl carbamateCO2/MeOH/FA 80/20/0.14 ml/min
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Conclusions1. Native cyclofructans are poor chiral selectors.2. Derivatization relaxes the CF structure.3. Small aliphatic groups are best for exposing the 18-
crown-6 core.4. We have a nearly universal chiral selector for
separating primary amines and it works best in organic and SF solvents.
5. Aromatic derivatives of CF6 & CF7 behave quite differently and are broadly selective.
6. More extensive mechanistic studies are needed and are underway. It is likely that we have just “scratched the surface”.
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