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Ionic Liquids

in
Separations & Mass Spectrometry

Daniel W. Armstrong

Robert A. Welch Professor

University of Texas at Arlington


Department of Chemistry and Biochemistry
Arlington, TX 76019
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 temperature
™Properties:
™ Negligible vapor pressure
™ High thermal stabilities (~250-400°C)
™ Highly variable viscosities
™ Hydrophobic or hydrophilic
™ Capable of undergoing multiple solvation
interactions
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).


Uses of RTILs
™Novel solvents in organic synthesis and
liquid-liquid extraction
™Mobile phase additives in HPLC
™Run buffer additives in CE
™Additive for ESI-MS analysis of anions
™Matrixes in Matrix-Assisted Laser
Desorption Ionization (MALDI) mass
spectrometry
™Stationary phases in gas-liquid
chromatography
™Sensors
RTILs as GC Stationary Phases

™Requirements
™ High thermal stability (250°C and above)
™ High viscosity
™ High wetability on fused silica capillary
columns
™ Produces symmetrical, efficient peaks
Anal. Chem., 71
(1999) 3873-3876.
Volatilization of RTILs
FID Detector Response

120 140 160 180 200 220 240 260


Column Temperature (Celsius)
Properties of High Stability
Geminal Dicationic Ionic Liquids
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.
Incremental Max Temperature Studies,
Supelcowax 10 vs SLB-IL59
Bleed (pA) in incremental Temp Run

1200.0
1000.0
800.0
Bleed

bleed SLB-IL59
IL-36
600.0
bleed wax 10
400.0
200.0
0.0
200 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)
TCEP = (1,2,3-Tris(2-CyanoEthoxy)Propane

• H2C-O-CH2CH2CN * A Highly Polar, Fluid Stationary Phase


* *Oxygen and Moisture Sensitive
HC-O-CH2CH2CN * Maximum Operating Temp. = 140oC

H2C-O-CH2CH2CN
TCEP Mix on TCEP Column at 110 °C

1. n-Tridecane
2. Toluene
3. Ethylbenzene
2 4. p-Xylene
5. Isopropylbenzene (Cumene)
4 5
6. 1,2,4-Trimethylbenzene
7. 1,2,4,5-Tetramethylbenzene
6 (Durene)
1 8. Cyclohexanone
3

0 10 20
Time (min)
TCEP Mix on SLB-IL100
1. n-Tridecane
2. Ethylbenzene
3. p-Xylene
110 °C 80 °C 4. Isopropylbenzene
(Cumene)
2 5. 1,2,4-Trimethylbenzene
2 4 6. 1,2,4,5-
4
6 3 Tetramethylbenzene
5 (Durene)
1 3 5
1 7. Toluene
6
8
8. Cyclohexanone
7
8
7

1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0


1.0 2.0 3.0
Time (min)
Time (min)

•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
Comparison of trigonal ILs with a polar commercial column

100% cyanopropyl
polisiloxane
Ionic Liquids in Tandem GC (GCXGC)
J.V. Seeley et al., Anal. Bioanal. Chem. 390 (2008) 323-332.

• Traditional polar columns have a max temperature of ~280oC


• IL columns are stable to greater than 350oC

• 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
GCXGC with Dicationic IL Columns in the 2nd Dimension

(Min.)
(Min.)

(Min.)
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.
2D-GC Isolation of P-O Containing Compounds
Using a Triflate Ionic Liquid Column
3 3

Column 2 Time (sec), Triflate


Column 2 Time (sec), DB-WAX

DEMP DIMP (diisopropyl methylphosphonate)


DMMP 1,3,5-trichlorobenzene DMMP (dimethyl methylphosphonate)
2 2
DIMP Naphthalene
Br-benzene Naphthalene
DEMP TEP (triethyl phosphate)
1 1-Br-octane 1
30 Compounds
TEP that do not
contain P-O

0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7
Column 1 Time (min), DB-5
Column 1 Time (min), DB-5

Primary column = DB-5


Primary column = DB-5
Secondary column = SLB-IL100
Secondary column = DB-WAX H 2C
N N
+
N
+
N
CH2

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
Recent RTIL Polarity
Measurements
™Solvatochromic Dyes
™ Reichardt dye
™ Large shift in charge transfer absorption
band
™ Omax= 810 nm in diphenyl ether
™ Omax= 453 nm in water

™ Nile Red
™ Experiences large bathochromic shift
™Results: RTILs have average
polarity similar to propanol
GC Column Polarity Scale
propanol
360°C 310°C 280°C 275°C 250°C 140°C
-1 -5 -20 -1701 -35 -50 Wax (PEG) -2331 -2560 TCEP

Non-Polar Intermediate Polar Polar Highly Polar

0 15 25 50 100
Range of Alternative Polarities possible from Ionic Liquid GC

• GC column polarity scale


•0 = squalane (considered the least polar GC stationary phase)
•100 = TCEP (considered the most polar GC stationary phase)
20
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: Experimental Conditions:

• SLB-IL111 Column Oven Temperature:


• SP-2340 120°C
• SLB-IL76
• SP-2330 Carrier gas Helium at constant linear velocity:
40 cm/sec
• SPB-225
• PAG Injection mode:
• SPB-50 7 Pm PDMS SPME Fiber
• SPB-35
• SPB-20
• SPB-Octyl
SLB-IL100

1,9-di(3-vinyl-imidazolium) nonane
bis(trifluoromethyl) sulfonyl imidate

O CF3
S

N N + N N 2 -N
+
S
O
CF3
O

FIRST COMMERCIAL IONIC LIQUID STATIONARY


PHASE 22
GC Polarity Scale
Where do current ILs fit on this scale?

360°C 310°C 280°C 275°C 250°C 140°C


-1 -5 -20 -1701 -35 -50 Wax (PEG) -2331 -2560 TCEP

Non-Polar Intermediate Polar Polar Highly Polar

0 15 25 50 100
Range of Alternative Polarities possible from Ionic Liquid GC

• GC column polarity scale


•0 = squalane (considered the least polar GC stationary phase)
•100 = TCEP (considered the most polar GC stationary phase)
23
Note that IL Stationary Phases of
the Same Polarity as Current
Molecular Phases
- STILL HAS DIFFERENT
SELECTIVITIES for MANY TYPES
of COMPOUNDS, INCLUDING
ISOMERS-

24
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 unique
selectivities 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.
IONIC LIQUIDS FOR MALDI-MS

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.
Dissolution of Cellulose with Ionic Liquids
R.P. Swatloski, R.D. Rogers, et al. J.A.C.S. 124 (2002) 4974.
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).
• Towards a Second Generation of Ionic
Liquid Matrices (ILM’s) for MALDI-MS of
Peptides, Proteins, and Carbohydrates
SA IMTBA CHCA IMTBA CHCA
x104
CHCA x104 x104
Lower laser intensity

Intens. [a.u.]

Intens. [a.u.]

Intens. [a.u.]

Intens. [a.u.]
[M + H]+
2.5 2.5 2.5 2500

2000

[M + H]+
2.0 2.0 2.0

[M + 2H]2+ [M + H] +
Bradykinin 1.5 1.5
[M + H]+ 1.5
1500

MW=1,060 Da 1.0 1.0 1.0


1000

500
0.5 0.5 0.5

0
0.0 0.0 0.0
1056 1057 1058 1059 1060 1061 1062 1063 1064 1065
600
600 800
800 1000
1000 1200
1200 1400
1400 1600
1600 1800
1800 600
600 800
800 1000
1000 1200
1200 1400
1400 1600
1600 1800
1800 600
600 800
800 1000
1000 1200
1200 1400
1400 1600
1600 1800
1800 1056 1057 1058 1059 1060 1061 1062 1063 1064 1065 1066
m/z
x104 x104 x104
Intens. [a.u.]

Intens. [a.u.]

Intens. [a.u.]
m/z m/z m/z
1.5 1.5 1.5

[M + 2H]2+ [M + H]+

Cytochrome C 1.0 1.0 1.0

MW=12,000 Da 0.5 0.5


[M + H]+
0.5

+
[M + H]
0.0 0.0 0.0

400
4000 600
6000 800
8000 1000
10000 1200
12000 1400
14000 1600
16000 1800
18000 400
4000 600
6000 800
8000 1000
10000 1200
12000 1400
14000 1600
16000 1800
18000 400
4000 600
6000 800
8000 1000
10000 1200
12000 1400
14000 1600
16000 1800
18000
x104 x104 x104 x104
Intens. [a.u.]

Intens. [a.u.]

Intens. [a.u.]
Intens. [a.u.]
m/z m/z m/z
+
[M + H] + [M + H] [M + H]+
[M + H]+
2.5 2.5 2.5 2.5

2.0

+
2.0 2.0
[2M + H]+ 2.0

[2M + H] [2M + H]+


BSA 1.5 1.5 [2M + H]+ 1.5 1.5

MW=66,000 Da [3M + H]+


1.0 1.0
[3M + H] + 1.0 1.0
[3M + H]+
+
[3M + H]
0.5 0.5 0.5
[4M + H]+ 0.5

0.0 0.0 0.0 0.0


50000 100000 5 150000
1X10 200000 5 250000
2X10 300000 5 350000
3X10 00000 5 450000
4X10
4 500000 5
5X10 50000 100000 5 150000
1X10 200000 5 250000
2X10 300000 5 350000
3X10 00000 5 450000
44X10 5000005 5
5X10
5X10 50000 100000 5 150000
1X10 200000 5 250000
2X10 300000 5 350000
3X10 00000 5 450000
44X10 5000005 5
5X10
5X10 50000 100000 5 150000
1X10 200000 5 250000
2X10 300000 5 350000
3X10 00000 5 450000
4X10
4 00000 5
5X10
5
x104 x104 x104
Intens. [a.u.]

Intens. [a.u.]

Intens. [a.u.]
m/z m/z m/z m/z
O
2.5 2.5 2.5
[M + H]+
2.0 2.0
[M + 2H]2+ 2.0
O
-

[2M + H]+
Urease 1.5 1.5 1.5

N
HO
[M + H]+ [3M + H]+
MW=90,000 Da 1.0 1.0 1.0

+
H N
0.5 0.5 0.5

00 00 00
50000 100000 5 150000
1X10 200000 5 250000 3X10
2X10 3000005 350000 400000 5 450000
4X10 5X105 50000 100000 5 150000
1X10 200000 5 250000 3X10
2X10 3000005 350000 400000 5 450000
4X10 5X105 50000 100000 5 150000
1X10 200000 5 250000
2X10 300000 5 350000
3X10 00000 5 450000
44X10 5X105
Detection of Catalase (Monomer=60,000 Da)
x104
Intens. [a.u.]

[M + H]+
Į-cyano-4-hydroxycinnamic acid: CHCA
2.0 O

1.5
OH

1.0
HO N
0.5

00
x104
Intens. [a.u.]

2.0 [M + H]+ Sinapinic acid


O

1.5 O
OH

1.0
HO

0.5 O

00
x104
Intens. [a.u.]

[2M + H ]+ [4M + H]+


2.0
[M + H]+
[3M + H] +
IMTBA CHCA
1.5
[5M + 2H]2+ O

-
1.0 O +
H N

0.5
HO N

00
50000 100000 150000 200000 250000 300000
Cation Properties vs. Performance
PA GB Performance vs.
Amine name pKa
(kJ/mole) (kJ/mole) Solid Matrix
triethanolamine 7.8 941 NA X
triisobutylamine 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


Effective MS Analysis of Biodegradable
Polymers with Second Generation Ionic
Liquid MALDI Matrices
Characterization of Polycaprolactone
O

O
H
n
Mn obtained from the manufacturer
Mn found by GPC Mn=10,000
4

Intens. [a.u.]
Intens. [a.u.]
x10 4
x10
DEA CHCA Mn=4250 Da 1.0
1.0
HABA Mn=2447 Da Mn=4162 Da
1200
1200

Mw=4732 Da Mw=4059 Da Mw=4758 Da


1000
1000
pd=1.11 0.8
0.8
pd=1.66 pd=1.14
800
800

Intensity
0.6
0.6
Intensity

600
600

0.4
0.4

400
400

0.2
0.2

200
200

00 0
0.0
1000 2000 3000 4000 5000 6000 7000 8000 1000 2000 3000 4000 5000 6000 7000 8000
1000 2000 3000 4000 5000 6000 7000 8000 m/z
1000 2000 3000 4000 5000 6000 7000 8000 m/z

m/z m/z
Intens. [a.u.]

Intens. [a.u.]
DHB Mn=1345 Da Mn=1764 Da DCTB Mn=784 Da
5000
5000
Mw=1570 Da Mw=1959 Da 6000
6000
Mw=815 Da
4000
4000
pd=1.17 pd=1.11 pd=1.04
Intensity
Intensity

4000
4000

3000
3000

2000
2000

2000
2000

1000
1000

00 00
1000 2000 3000 4000 5000 6000 7000 8000
1000 2000 3000 4000 5000 6000 7000 8000 m/z
1000
1000 2000
2000 3000
3000 4000
4000 5000
5000 6000
6000 7000
7000 8000
8000 m/z
m/z m/z
Characterization of Polycaprolactone Triol
(Mn= 300 and 900 Da)
RO
OR O

O
R=
H
n
OR

Mn values are estimated by the manufacturer


Characterization of Polycaprolactone Triol
(Mn= 300 and 900 Da)
x1044

Intens. [a.u.]
x10
Intens. [a.u.]

1500 1500
Estimated Mn=593 Da Estimated Mn=1930 Da
300 Da Mw=629 Da 1.0
1.0
900 Da Mw=2081 Da
1250
1250

pd=1.06 pd=1.08
0.8
0.8

1000
1000

Intensity
Intensity

0.6
0.6
750
750

0.4
0.4
500
500

250
250 0.2
0.2

00 0
0.0
400 600 800 1000 1200 500 1000 1500 2000 2500 3000 3500
400 600 800 1000 1200 m/z
500 1000 1500 2000 2500 3000 3500 m/z

m/z m/z

Precision
Mn=593 ± 3 Da Mn=1930 ± 16 Da
Conclusions
• 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
Quantitative Analysis of Anions
ƒ Anion quantification is important for:
ƒ Environmental monitoring
ƒ Biological analysis
ƒ Semiconductor Industry
ƒ Current Methods of Determination
ƒ Ion chromatography
ƒ Flow-injection analysis
ƒ Ion-selective electrodes
A New Approach for Ultra-Sensitive Anion Analysis

Take a Di-cationic Reagent

• Anion exchange to fluoride form


• Fluoride complex not detected in the MS

REFERENCES: Anal. Chem., 2005, 77, 4829.


Anal.Chem., 2007, 79, 7346.
J. Am. Soc. Mass Spec., 2008, 19, 261.
Anal. Chem., 2008, 80, 2612.
Gas-Phase Ion Association in
ESI-MS
ƒ Using ionic additives as ion-pairing agents
for MS
ƒ First application was published in Anal. Chem.
77 (2005) 4829-4835.
ƒ We are expanding to additional ions.

2+ 1+
+ ClO4 -
ClO4-

m/z +145 m/z - 99 m/z +389


(MW 290)
Detection of Anions
• Three different ways to detect ions
1) Anion SIM
2) Cationic complex SIM
3) 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+ 1+
ClO4-
Why Positive Ion Mode?
ƒ MeOH/Water based solvent systems not
ideal to provide stable signal in negative
mode
ƒ Low gas-phase proton affinities lead to
protonation of analyte
ƒ Corona discharge more prevalent in
negative mode
ƒ Leads to unstable signal
ƒ Higher background noise
REFERENCES: Anal. Chem., 2005, 77, 4829.
Anal. Chem., 2007, 79, 7346.
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
35 - 34 -
same M/Z ( ClO4 vs. H SO4 )

REFERENCES: Anal. Chem., 2005, 77, 4829.


Anal.Chem., 2007, 79, 7346.
ESI-MS Analysis

[Dication]2+
LC Pump
40 uM Additive in H20
[Dicat+Anion]+
MS
Finnigan LXQ
LC Column

Sample Sol’n
[Anion]-

LC Pump
H20/MeOH
Anions
Inorganic Organic
- - - -
• ClO4 , BrO3 , IO3 , IO4 • Perfluoronated octanoic Acid
(PFOA)
• Cl-, Br-, I-
- - • 2-Bromooctanoic acid
• NO3 , NO2 • Halogenated and acetic acids
• SCN-, OCN-, CN- (TFA, TCA, BrClA, Cl2A, MBrA,
- - MClAA)
• BF4, PF6
- • Acetic, Formic, Benzoic acids
• MnO4 • Benzensulfonate
-
• H2AsO4 • Trifluoromethanesulfanate
(TFO)
• Trifluoromethanesulfonimide
(NTF2)
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
Positive and Negative LC-ESI-MS
(A) TFO (B) TFO
RT: 0.00 - 17.99 SM: 15G Area: 8.9E5 RT: 0.00 - 18.00 SM: 15G

S/N: 88
NL:
4.62E3
Area: 4.9E5 NL:
0

NTF2
m/z=
347.50-348.50 S/N: 67 m/z=
57.50-58.50
MS Genesi s NTF2 MS

SCN BZSN Area: 1.4E5 AnionmixtureM


S_040307c
Area: 2.5E5
AnionmixtureM
S_040207d

Area: 7.3 E5 Area: 2.9E6 S/N: 30 NL:


4.60E3
S/N: 32
NL:
2.46E3
m/z= m/z=
S/N: 138 S/N: 204 438.50-439.50
MS Genesi s BZSN PFOA 148.50-149.50
MS Genesis

100 100
PFOA
AnionmixtureM
S_040307c 100 100
Area: 3.6E5 Area: 4.0E4 AnionmixtureM
S_040307e

95
NL:
1.15E4 95
S/N: 31 S/N: 26 NL:
1.11E4

90
Area: 1.0E5 m/z=
446.50-447.50 90
m/z=
156.50-157.50

85
S/N: 162 MS Genesi s
AnionmixtureM 85
MS Genesis
AnionmixtureM
S_040307c S_040307e

80 80
NL:
9.83E3
m/z=
80 80
NL:
1.51E4
m/z=
75 569.50-570.50 75 279.50-280.50
MS Genesi s MS Genesis
Relative Abundance

Relative Abundance
70 AnionmixtureM 70 AnionmixtureM
S_040307c S_040307e
NL: NL:
65 65
7.46E3 2.01E3
m/z= m/z=

60 60 702.50-703.50
60
60 412.50-413.50

Relative Abundance
Relative Abundance

MS Genesi s MS Genesis
55 AnionmixtureM 55 AnionmixtureM
S_040307c S_040307e
50 50

45 45
SCN
40
40 40

35
40 35

30 30

25 25

20 20
20 15 20 15

10 10

5 5

0 0

0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
0 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

0 3 6 9Tim e (min)
12 15 3 6 9Tim e (min)
12 15
Time (min) Time (min)
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 ȝL/min. In (A) the dicationic salt solution (40
ȝM in MeOH) was added post-column at 100 ȝL/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.
Detection of Related Species
Arsmix_032707o #56-60 RT: 0.85-0.91 AV: 5 NL: 1.59E3
T: ITMS + p ESI u Z ms [ 413.50-433.50]
Arsmix_032707o #43-50 RT: 0.65-0.76 AV: 8 NL: 2.00E3
T: ITMS + p ESI u Z ms [ 413.50-433.50]
O 429.24

1550

1500
68
66
428.22
HO As CH3
64
1450

1400
62
60
O
58
1350 56
54
MMAV
1300 52
414.42
50
1250
48

1200
HO 46

OH
1150
44
42 As 418.31

40 432.22
1100 38
O
Intensity

36
1050
34

1000 32
30
Arsenite
950 420.88
28
415.40
26
900 24 421.28
22
850
Intensity

20 416.92

800 18
16 418.91
750 14
422.88

700
12
10
40X 419.41
422.25 424.83
424.94 432.46
O
650
8
6
416.39
419.94 423.18

423.40
O 433.26

600 4
2
426.35
HO As OH
550 0
414 416 418 420 422 424 426 428
H3C As CH3
430 432
500 m/z
O
450 O
400
Arsenate
350
DMAV
300 431.23
427.26 430.24
250

200

150
100 417.30
428.22 432.23
50 414.42 426.88
415.40 416.92 418.31 419.42 420.88 422.27 422.87 424.84 433.23
426.39
0
414 416 418 420 422 424 426 428 430 432
m/z
Recommended Dications

 + (CH 2)3 N+
+
(CH2) 3 P+ N N N N N
+
( CH2) 5 N+ N N
+
P
HO

 + (CH 2)5 + + (CH2 )5 +


N N N N N N

 +
N
+ (CH2) 5 P+ + (CH 2) 5 +
P  + ( CH) 9 N+ N N
N N N

 + +
 +
N
+
N
N (CH2) 5 N +
N N
N
+
P
+ (CH2) 9 P+ N
 N N
+ CH2 CH2 (C F2 )4CH 2CH2 N
+
N + +
N (CH2) 12 N

HO OH
 N N
+
(CH2)5 N+ N OH
CH3
+
N CH3 O OMe
+ +
+ +
P P N (CH2)5 N N


HO
N N
+
O O O N
+
N OH O
+
MeO OH N
H3C H

 
+
P
+ + (CH 2)5 N+ + +
P O O O N N N N (CH2)5 P
ƒTrications A6 and B1 performed the best overall
Trications Core Charged Groups

A1 A B  +
1) R= N N
A2 
A5 R R R R +
A6 5) R= N

 +
B1 R R 2) R= N N
B2
B4 C P
+

R R
6) R=
B6
N

 +
C1 3) R= N N OH
R
C2 HN
+

C3 7) R= N
D
C4
C5 R N
N
N
R 
C6 5 5
4) R=
N N

O O
C7
N
R
D2 5
O
D6
LC-MS Positive vs. Negative Ion
Mode
Positive 
RT: 0 .0 0 - 5 .01 SM : 7G
Negative
AA: 38 7 N L:
RT: 0.00 - 5. 02 SM: 7G
AA: 7969 NL: 100 1 00
S N: 6 1 .02 E 2
TIC MS

100 100
S N: 35 1. 45E3
TI C MS
95 5 ng G en e s i s
N eg H e xC l

Hexachloroplatinate
Gen esi s P d_ 1 02 60
95
500 pg TricatL_HC
P _102607 90 S/N: 6 7b

90
S/N: 35 85

85
80 80

PtCl62-
80 80
75

75
ec 70

70
na
ce
65

na
65
dn 60 60

dn 60 bu
60

R el ati v e Ab u nd a nc e
55

R elative Ab undanc e
ub 55
A 50

50 ev
A it 45

(Trication A6) e
vi
ta 40
le
R
45

40

35
lae 40
R
40

35

30
30
25
25
20 20

20 20
15
15

10
10
5
5
0 0

0 0
0. 0 0. 5 1.0 1. 5 2.0 2.5 3. 0 3.5 4.0 4 .5 5.0
0
0.0 0 .5

1
1.0 1 .5

2
2.0

3
2.5
Ti m e (m i n )
3.0 3.5

4
4 .0 4.5

5
5.0

Tim e (m in)
0 1 2 3 4 5 Time (min)
Time (min)
RT: 0.00 - 5. 01 SM: 7G
RT: 0 .00 - 5 .0 2 SM : 7 G AA: 13 2 NL:

100 AA: 20 0 04 N L:
100
Benzenedisulfonate
SN: 3 2. 71E1
S N: 5 6 2 .7 4 E3 100 TI C MS
1 00
TIC M S Gen es i s

95 500 pg G e n es i s
Tri C atB _ 1
2 9 07
95
5 ng Neg OB DS
A_102 607c

90 S/N: 56 90
S/N: 3
85
85

80 80 80 80

SO 3- 75
75

70

ec 70
ec 65

an na
65

d 60 60 60

nd
60

nu

R elative Ab undanc e
ub
Re l a ti v e Ab u n da nc e

55
55

b 50
A 50
A
SO3- ev e 45

ivt
45

it 40
lae 40
40
al 40

e 35
35

(Trication B1) 20
R 30

25

20 20
R 30

25

20

15
15

10
10

5
5

0 0
0.0 0 .5 1.0 1 .5 2.0 2.5 3.0 3 .5 4 .0 4 .5 5 .0
0 0
0. 0 0. 5 1.0 1. 5 2.0 2.5 3. 0 3.5 4.0 4 .5 5.0

0 1 2 Ti m e (m i n )
3 4 5 0 1 2
Tim e (m in)
3 4 5
Time (min) Time (min)
Trications with More Flexibility?
• Rigid trications not performing quite as well as the
more flexible dications
• Synthesized: CH3

N N
+
+
CH3

P
+
H3C P
H3C
CH3

H3C

• 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 From:
hexachloroplatinate.
D. W. Armstrong & co-workers, J. Am. Soc. Mass Spec.
2008, in press.
H3C CH3
n=3
I 500 pg P
+
CH2 + CH2 P
+
CH3
H3C N N
n n
S/N: 21
CH3 H3C

II 500 pg R R

S/N: 3

R
R=
tripropylphosphonium

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.
O
OH
-
O RT 4.21 HO
O
O-
O RT 6.58
O
RT 8.45
100

95

90

85

80

75

70

65
+ + +
60 P 10 N N 10 P
Relative Abundance

55

50

45

40
Tricationic pairing
35 reagent
30

25

20 m/z 865.8
15

10 m/z 859.8
5

0
m/z 953.8
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Tim e (m in )

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 ȕ-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.
+
+ N
N N
N

+
N
N
N
+
N

500pg

S/N=5
Chiral Stationary Phases
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)
W-cyclodextrin (or cyclooctaamylose). (B) E-cyclodextrin (or cycloheptaamylose), and (C) D-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.
Simplified schematics illustrating two different enantioselective retention
mechnisms for the native E-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).
Summary of Derivatives of CYCOBOND 1 2000

Bonded
Derivatized
Cyclodextrins
The separation of three racemates using an (S)-NEC-E-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.
OR 1
CH 2 O
CH
2 OR
OR 2 O O 1
2O

O 1

OR 3
2 OR

OR
OR

3
OR
2
CH

O
3
OR
2O
OR 3

OR
3
R

1
CH 2OO 1

OR

OR
2 O
OR 2 O

OR
3

CH
O OR3
OR2
OR
2
O
CH
2O

O
O R1

CH2OR1
DNP-O-E-CD Family
Anal. Chem., 68, 2501 (1996).
CE EVALUATION OF VANCOMYCIN
CHIRALITY 6:496-509 (1994)

Capillary electropherograms showing the resolution of (A) Capillary electropherograms showing the resolution of
three racemic AQC amino acids and (B) racemic dansyl- nonsteroicantiinflammatones: (A) naproxen, (B) carprofen, and (C)
valine. The separations were done with a 50 Pm x 30.5 flurbiprofen. The separations of carprofen and flurbiprofen were done
cm (25 cm to detector) containing 0.1 M phosphate buffer with a 50 PM x 30 5 cm (25 cm detector) containing pH 7. 0.1 M
and 5 mM vancomycin. The voltage was + 5 kV, and the phosphate buffer and 5 mM vancomycin. The voltage was 5 kV, and
analytes were detected via absorbance at 254 nm. The the analytes were detected via absorbance at 254 nm. The
pH of the run buffer was 7.0 for the AQC amino acids, separation of naproxen was done under the same conditions except
and 4.9 for the dansyl amino acids. that the vancomycin concentration was 2 mM and pH was 6.0
JOURNAL OF LIQUID CHROMATOGRAPHY, 17(3), 1695-1707 (1994)

TLC chromatogram showing the separation of all four TLC chromatogram showing the separation of (A)
isomers (2 pairs of enantiomers) of AQC-leucyl-leucine. indoprofen, and (B) coumachlor. The mobile
The stereochemistry of the compounds represented by phase consisted of 0.05 M vancomycin in 4:6 (by
each spot is indicated. This was determined by volume) acetonitrile: 0.6 M NaCl(aq). Diphenyl-F
developing pure standards in a separated experiment. TLC plates (5 x 20 cm) were used. Spots were
The mobilie phase consisted of 0.02 M vancomycin in detected using a 365 nm UV hand lamp (see
1:3 (by volume) acetonitrile: 0.6 M Experimental).
Anal. Chem. 66 (1994) 1473
J. Chromatogr. A 731 (1996) 123-137
Principle of Complementary Separations
P-CAP™
New Normal Phase CSP
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
Free Radical Polymerization on
Functionalized Silica Gel O
O
CN
Si O Si NH NH
Silica
Gel N CHCl 3, 60oC
+ O
N
Si O Si NH
NH
CN
O

NH

O NH
O

Si O Si NH
CN NH
NH
O
O
Silica O
Gel O
NH NH
CN

O Si NH
Si
O
O NH

NH
Elemental Analysis: %C 10.36; %H 1.68; %N 2.19
FT-IR (KBr): 3078, 2941, 2860, 2237, 1646, 1542, 1451 cm-1 O

Weight Increment: 16.55%


O
H3CO O
H3CO Si O
Si OH H3CO Si O Si O
O

Si OH Si O Si O

NH

NH

Poly-DPEDA CSP

Synthesis of the poly-DPEDA chiral stationary


phase.
Comparison of separation in two mobile
phase modes

O CO2H
O2 N
N
H

N2O

4.17
8.94
5.42

25.74

D =3.93 , Rs=5.1 D =2.31 , Rs=2.6


Mobile phase: EtOH/Heptane/TFA=40/60/0.1 Mobile phase: ACN/MeOH/TFA=100/1/0.1
Comparison of separation in three synthetic
polymeric CSPs

OH
HO

23.65 30.88 32.82


10.13 28.48

12.63

P-CAP CSP P-CAP-DP CSP New polymeric CSP


Mobile phase: Heptane/EtOH/TFA=90/10/0.1 Mobile phase: Heptane/EtOH=80/20 Mobile phase: Heptane/EtOH=50/50
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 etal.CarbohydrateResearch313(1998)91Ͳ105
Sulfated Cyclofructan (SCF6) 0
(1)PyridineͼSO in Pyridine80Ͳ85 C,6h
3
C36H42O12(OH)18 C36H42O14(OH)18Ͳn(OSO2Na) n
(2)CH3OHͼCH3COONa

13
14

12

15
11

Q.Sunetal.Di’er Junyi Daxue Xuebao,27,453


Ph O N
HO C OH2C
N
H3C

Conditions:15mMSCF6, 20mMAmAc,10mMPhosphate,pH=4.7,25kv,30cm/40cmcapillary
Poor Separations on Native
CF6
Native CF6
Acetonitrile/methanol/AA/TEA
90/10/0.3/0.2

CF6
3,5-dimethylphenyl carbamate
Acetonitrile/methanol/AA/TEA
75/25/0.3/0.2

0 10 20 30 40
Time (min)
Cyclofructan Crystal Structure

Hydrophobic Face
Hydrophilic Face

Side View
Native vs. Derivatized CF6

Native CF6
Acetonitrile/methanol/AA/TEA
90/10/0.3/0.2

CF6
3,5-dimethylphenyl carbamate
Acetonitrile/methanol/AA/TEA
75/25/0.3/0.2

0 10 20 30 40
Time (min)
Structure of Chemically-Bonded CF CSPs
Diverse derivatization groups
4 R •Aliphatic derivatization CH3
O CH3
RO
H2
O
OR CH3 C CH3 CH C CH3

1 O
RO OR

O
OR
O •Aromatic derivatization CH3
O OR
CH3
Suppot O O
2
RO

OR
Cl Cl Cl
O O
OR O
OR
O
O CH3 Cl CH3
RO
RO OR
RO
O H3C CH3
1. silica gel support OR
2. covalent linker between cyclofructans and solid support
CH3 Cl NO2 CF3
3. cyclofructan 6~8
4. derivatization groups or hydrogen OR

1~3

CH3 Cl NO2 CF3

H3 C H3C

CH3 CH3
3D Optimized Cyclofructan Isopropyl Derivativ

Hydrophobic Face Hydrophilic Face

Side View
Cyclofructan Crystal Structure

Side View
Separation of Primary Amines

NH2


OH
H3C
1S,2R/1R,2S

NH2

CH
CH3

6 10 14 18 min

Column: IPCF; 60ACN/40MEOH/0.3AA/0.2TEA


D Optimized Cyclofructan RN Derivative (6, 12, 1

RN 12 Hydrophilic Face
RN 6 Hydrophobic Face

RN 18 Hydrophilic Face
D Optimized Cyclofructan RN Derivative (6, 12, 1

RN 6 Side
View

RN 12 Side
View

RN 18 Side
View
Comparison Between Different Modes

Normal phase mode


70 heptane/30ethanol

Br

Reversed phase mode


OH

HO 50 water/50 acetonitrile
Br

4 6 8 10 12 min

Generally, normal phase mode is better than reversed phase mode for
separating neutral analytes, due to higher selectivity and efficiency.
CF-based CSPs can be used alternately in polar organic,
reversed-phase and normal phase solvents without damage.

A. Normal phase mode


NH 2
H2 N

NH2
B. Polar organic mode
OH

Ph

C. Reversed phase mode O Ph

O2 N
N R CO2 H
H

NO2

0 5 10 15

Mobile phase: (A) 70% heptane/30% ethanol: (B) 60% acetonitrile/40%


methanol/0.3% acetic acid/0.2% triethylamine. CSP: CF6-RN
Separation of Chiral Acids

CF6
bis(trifluoromethylphenly carbamate
Acetonitrile/methanol/AA/TEA
75/25/0.3/0.2

CF7
3,5-dimethylphenyl carbamate
heptane/ethanol/TFA
80/20/0.1

0 Min 20
Separation of Chiral Alcohols

CF6
R-naphthylethyl carbamate
Heptane/isopropanol/TFA
98/2/0.1

CF7
3,5-dimethylphenyl carbamate
heptane/ethanol/TFA
99/1/0.1

15 30
Min
Separation of Chiral Pharmaceutical Compounds

CF6
3,5-dimethylphenyl carbamate
Heptane/isopropanol/TFA
Thalidomide 98/2/0.1

CF7
R-naphthylethyl carbamate
heptane/ethanol/TFA
99/1/0.1
Bendroflumethiazide

20 Min 55
Separation of Ru tris(diimine) Complexes

2+
N
N

k1=0.883, D=1.51, Rs=4.4


N
[Ru(phen)3](Cl2) Ru

N N

N
2+
N

N
N

[Ru(dppz)3](Cl2) N N

k1=1.27, D=2.81, Rs=12.1


Ru
N N N
N

N
N

0 4 8 12 16 min

RNCF6 column
60methanol/40acetonitrile/25 mM NH4NO3
SFC Separations of Derivatized Amino Acids

CF7
3,5-dimethylphenyl carbamate
CO2/MeOH/FA 80/20/0.1
4 ml/min
Conclusions
1. 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”.