102
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

Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

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Page 1: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

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

Page 2: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

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

Page 3: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

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).

Page 4: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

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

Page 5: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

RTILs as GC Stationary Phases

RequirementsHigh thermal stability (250°C and above)High viscosityHigh wetability on fused silica capillary columnsProduces symmetrical, efficient peaks

Page 6: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

Anal. Chem., 71 (1999) 3873-3876.

Page 7: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

Volatilization of RTILs

220120 140 180160 200 260240

Column Temperature (Celsius)

FID

Det

ecto

r Res

pons

e

Page 8: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

Properties of High Stability Geminal Dicationic Ionic Liquids

Page 9: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

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.

Page 10: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

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

Page 11: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

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

Page 12: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

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

Page 13: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

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

Page 14: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

Comparison of trigonal ILs with a polar commercial column

100% cyanopropylpolisiloxane

Page 15: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

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

Page 16: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

GCXGC with Dicationic IL Columns in the 2nd Dimension

(Min.)

(Min

.)(M

in.)

Page 17: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

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.

Page 18: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

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

Page 19: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

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

Page 20: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

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

Page 21: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

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

Page 22: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

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

Page 23: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

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?

Page 24: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

24

Note that IL Stationary Phases of the Same Polarity as Current

Molecular Phases - STILL HAS DIFFERENT

SELECTIVITIES for MANY TYPES of COMPOUNDS, INCLUDING

ISOMERS-

Page 25: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

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.

Page 26: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

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

Page 27: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers
Page 28: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

Dissolution of Cellulose with Ionic LiquidsR.P. Swatloski, R.D. Rogers, et al. J.A.C.S. 124 (2002) 4974.

Page 29: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers
Page 30: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers
Page 31: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers
Page 32: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

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).

Page 33: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers
Page 34: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

• Towards a Second Generation of Ionic Liquid Matrices (ILM’s) for MALDI-MS of Peptides, Proteins, and Carbohydrates

Page 35: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

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

Page 36: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

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

Page 37: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

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

Page 38: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

Effective MS Analysis of Biodegradable Polymers with Second Generation Ionic

Liquid MALDI Matrices

Page 39: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

Characterization of Polycaprolactone

OH

O

n

Mn found by GPC Mn=10,000 Mn obtained from the manufacturer

Page 40: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

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

Page 41: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

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

ROOR

OR

OH

O

n

R=

Mn values are estimated by the manufacturer

Page 42: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

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

Page 43: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

• 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

Page 44: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

Quantitative Analysis of Anions

Anion quantification is important for:Environmental monitoringBiological analysisSemiconductor Industry

Current Methods of DeterminationIon chromatographyFlow-injection analysisIon-selective electrodes

Page 45: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

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.

Page 46: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

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+

Page 47: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

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+

Page 48: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

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.

Page 49: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

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.

Page 50: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

ESI-MS Analysis

Sample Sol’n

[Dication]2+

[Anion]-

[Dicat+Anion]+

MS

LC PumpH20/MeOH

LC Pump40 uM Additive in H20

LC ColumnFinnigan LXQ

Page 51: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

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

Page 52: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

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

Page 53: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

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

Page 54: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

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

Page 55: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

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

Page 56: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

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

Page 57: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

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

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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

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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-

Page 58: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

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.

Page 59: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

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

Page 60: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

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

Page 61: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

S/N=5

500pg

N+

N

N+

NN

+

N

N+

N

Page 62: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

Chiral Stationary Phases

Page 63: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

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.

Page 64: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

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).

Page 65: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

Bonded DerivatizedCyclodextrins

Summary of Derivatives of CYCOBOND 1 2000

Page 66: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

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.

Page 67: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

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

Page 68: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

Anal. Chem., 68, 2501 (1996).

Page 69: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers
Page 70: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

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

Page 71: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

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).

Page 72: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

Anal. Chem. 66 (1994) 1473

Page 73: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

J. Chromatogr. A 731 (1996) 123-137

Page 74: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers
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Principle of Complementary Separations

Page 76: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

P-CAP™

New Normal Phase CSP

Page 77: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

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

Page 78: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

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

Page 79: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

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.

Page 80: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

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

Page 82: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

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

Page 84: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

N

NH3C

OH2CC

OPh

HO

Conditions: 15 mM SCF6, 20 mM AmAc,10mMPhosphate, pH=4.7, 25 kv, 30 cm/40 cm capillary

Page 85: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

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

Page 86: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

Hydrophobic FaceHydrophilic Face

Side View

Cyclofructan Crystal Structure

Page 87: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

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

Page 89: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

3D Optimized Cyclofructan Isopropyl Derivativ

Hydrophobic Face Hydrophilic Face

Side View

Page 90: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

Side View

Cyclofructan Crystal Structure

Page 91: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

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

Page 93: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

RN 6 Side View

RN 12 Side View

RN 18 Side View

D Optimized Cyclofructan RN Derivative (6, 12, 1

Page 94: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

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.

Page 95: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

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

Page 96: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

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

Page 97: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

Separation of Chiral Alcohols

15 30Min

CF73,5-dimethylphenyl carbamateheptane/ethanol/TFA99/1/0.1

CF6R-naphthylethyl carbamateHeptane/isopropanol/TFA98/2/0.1

Page 98: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

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

Page 99: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

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

Page 100: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

SFC Separations of Derivatized Amino Acids

CF73,5-dimethylphenyl carbamateCO2/MeOH/FA 80/20/0.14 ml/min

Page 101: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers

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”.

Page 102: Ionic Liquids in Separations & Mass Spectrometry · Ionic Liquids in Separations & Mass Spectrometry ... 1,3,5-trichlorobenzene ... Determination of Stationary Phases Polarity Numbers