in
Separations & Mass Spectrometry
Daniel W. Armstrong
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
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
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
100% cyanopropyl
polisiloxane
Ionic Liquids in Tandem GC (GCXGC)
J.V. Seeley et al., Anal. Bioanal. Chem. 390 (2008) 323-332.
(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
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
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
0 15 25 50 100
Range of Alternative Polarities possible from Ionic Liquid GC
1,9-di(3-vinyl-imidazolium) nonane
bis(trifluoromethyl) sulfonyl imidate
O CF3
S
N N + N N 2 -N
+
S
O
CF3
O
0 15 25 50 100
Range of Alternative Polarities possible from Ionic Liquid GC
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.
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
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]+
+
[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
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.]
1.5 O
OH
1.0
HO
0.5 O
00
x104
Intens. [a.u.]
-
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 ++
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
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
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
Anion quantification is important for:
Environmental monitoring
Biological analysis
Semiconductor Industry
Current Methods of Determination
Ion chromatography
Flow-injection analysis
Ion-selective electrodes
A New Approach for Ultra-Sensitive Anion Analysis
2+ 1+
+ ClO4 -
ClO4-
[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
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
+
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
Trications 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
II 500 pg R R
S/N: 3
R
R=
tripropylphosphonium
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).
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
Si OH Si O Si O
NH
NH
Poly-DPEDA CSP
O CO2H
O2 N
N
H
N2O
4.17
8.94
5.42
25.74
OH
HO
12.63
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
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
H3 C H3C
CH3 CH3
3D Optimized Cyclofructan Isopropyl Derivativ
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
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
Br
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.
NH2
B. Polar organic mode
OH
Ph
O2 N
N R CO2 H
H
NO2
0 5 10 15
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
N N
N
2+
N
N
N
[Ru(dppz)3](Cl2) 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”.