Acetonitrile versus Propylene Carbonate Listed as VOC Not listed as - - PowerPoint PPT Presentation

Acetonitrile versus Propylene Carbonate Listed as VOC Toxic Flammable Acetonitrile shortage (economic crisis) Not listed as VOC Readily biodegradable Not flammable Available as highly purified solvent

Characteristics ACN PC MeOH EtOH CAS no. 75-05-8 108-32-7 67-56-1 64-17-5 Type of solvent Polar aprotic Polar aprotic Polar protic Polar protic Mw 41.04 102.09 32.04 46.07 Molecular dipole moment (D) 3.92 4.90 1.70 1.69 Dielectric constant 35.94 64.90 32.66 24.55 Hansen solubility parameters – Dispersive - D 15.3 20.0 15.1 15.8 Polar - P 18.0 18.0 12.3 8.8 Hydrogen bonding - H 6.1 4.1 22.3 19.4 Hildebrand solubility parameter T 24.3 27.3 29.6 26.5 Boiling point (oC) 81.6 241.7 64.7 78.0 Melting point (oC)

• 45.0
• 49.2
• 97.8
• 113.9

Density g/cm3 0.781 1.205 0.7918 0.789 Experimental Log Kow

• 0.34
• 0.41
• 0.77
• 0.31

Water solubility Fully miscible 240 g/L Fully miscible Fully miscible Viscosity / 25 oC (cps) 0.36 2.4 0.59 1.1 Vapor pressure kPa/20 oC 9.7 0.017 12.8 5.83 Flash point oC 2 132 11 13 Auto ignition temperature (oC) 524 455 385 365 Lower flammable limit (LFL) - % 4 1.7 6 3.5 Upper flammable limit (UFL) - % 16 32.5 36.5 19 Oral LD50 rat mg/kg 2460 29100 5600 15010 Dermal LD50 rabbit mg/kg 980 20001 15800 19999 Inhalation LC50 rat ppm 4 h 17100 >50000 64000 124700 Acute LC50 Daphnia 48 hrs mg/mL 3600 > 10000 3289 6386 HMIS Health 2 1 2 2 HMIS Flammability 3 1 3 3 HMIS Physical hazards Exempt VOC (acc. 40 CFR 51.100) No Yes No No Biodegradability ~ 40% / 10 days > 80% / 10 days readily biodegradable readily biodegradable

Comparative properties

Miscibility

• Solv. A = EtOH:H2O
• Solv. A / PC

1:9 1:4 3:7 2:3 1:1 3:2 7:3 4:1 9:1 EtOH 0% 10% 20% 30% fully miscible immiscible

• Solv. B = EtOH:PC

H2O / Solv. B 1:9 1:4 3:7 2:3 1:1 3:2 7:3 4:1 9:1 EtOH 0% 10% 20% 30% fully miscible immiscible

a) H2O / EtOH = 7/3 (v/v) and PC b) PC / EtOH = 7/3 (v/v) and H2O

Pressure Drop

Stationary Phase ECLIPSE XDB C18 Dimensions 15 cm L x 4.6 mm i.d. x 5 m d.p. 10 cm L x 2.1 mm i.d. x 3.5 m d.p. 5 cm L x 4.6 mm i.d. x 1.7 m d.p. Temperature 25 oC 25 oC 60 oC Flow Rate 0.8 mL/min 0.25 mL/min 1.0 mL/min

Pressure drop ratio (Palternative organic modifier/PACN)

% Org. Mod.

ACN/(PC+EtOH) ACN/PC ACN/(PC+EtOH) ACN/PC ACN/(PC+EtOH) ACN/PC

10 1.1 (82.9*) 2.0 1.2 (95.0) 2.1 1.1 (111.5) 1.7 20 1.3 (84.5) 2.0 1.3 (97.0) 2.0 1.3 (114.5) 1.7 30 1.6 (82.4) 2.1 1.6 (94.0) 2.1 1.5 (113.5) 1.8 40 2.0 (78.0) 2.3 2.1 (89.0) 2.3 1.7 (109.5) 2.0 50 2.4 (71.8) 2.6 2.4 (82.0) 2.6 2.0 (103.0) 2.2 60 2.8 (64.0) 2.9 2.8 (73.0) 3.0 2.3 (95.0) 2.5 70 3.2 (55.0) 3.4 3.3 (62.6) 3.5 2.7 (85.0) 2.9 80 3.8 (45.0) 4.1 3.9 (51.8) 4.2 3.0 (74.5) 3.4 90 4.3 (37.2) 5.1 4.5 (41.5) 5.3 3.5 (63.7) 4.3 100 5.3 (26.0) 9.2 4.7 (34.4) 8.0 3.8 (56.8) 6.3

* Pressure drop (bar) measured on using ACN/water mobile phases in the given conditions.

Pressure Drop (example)

Eclipse XDB C18 150*4.6 mm 5 um Flow rate 0.8 mL/min Temperature 25 oC

20 40 60 80 100 120 140 160 180 200 220 240 260 10 20 30 40 50 60 70 80 90 100

% Organic Solvent Pressure drop (bar)

ACN PC+EtOH PC

Experimental parameters / Retention data

Chromatographic Column: Purosphere Star C18e

Column dimensions: 7.5 cm L x 4 mm i.d. x 3 m d.p. Column temperature: 25 oC Mobile Phase Solvent A: aqueous 0.1% HCOOH (for acidic compounds) Solvent A’: aqueous 50 mM boric acid at pH=9 with NaOH (for alkaline compounds) Solvent A’’: water (for neutral compounds) Solvent B: acetonitrile Solvent A (or A’ or A’’) / Solvent B = 7/3 Solvent a: water/ethanol = 7/3 (v/v) with 0.1% HCOOH (for acidic compounds) Solvent a’: water/ethanol = 7/3 (v/v) with 50 mM boric acid at pH=9 with NaOH (for alkaline compounds) Solvent a’’: water/ethanol = 7/3 (v/v) (for neutral compounds) Solvent b: propylene carbonate Solvent a (or a’ or a’’) / Solvent b = different volumetric ratios Elution: isocratic Flow rate: 0.5 mL/min Injection volume: 5 L

Retention data

min 2.5 5 7.5 10 12.5 15 17.5 20 Toluene Fluorene Fluoranthene  = 254 nm 60% ACN 40% PC mAU min 2 4 6 8 10 12 14 Nicergoline Diltiazem Carbamazepine  = 220 nm 50% ACN 35% PC mAU

min 2 4 6 8 10 12 14 16 Simvastatic Acid Fenofibric Acid Lovastatic Acid  = 238 nm 50% ACN 35% PC mAU

Retention data

Toluene

y = -2.7975x + 5.7034 R2 = 0.9989 y = -3.1801x + 6.4259 R2 = 0.9891

0.000 0.200 0.400 0.600 0.800 1.000 1.200 1.400 1.600 1.800 2.000 0.000 0.500 1.000 1.500 2.000 2.500

log (%org) log (tR) ACN PC + EtOH

30 % 7 . % ; ) log(% ) log( ) log(% ) log(           PC Org t t a Org b t A ACN B t

Org R ACN R Org R ACN R

Retention data

y = 1.3685x - 36.701 R2 = 0.9999

y = 1.4345x - 41.759 R2 = 1 y = 1.357x - 34.713 R2 = 0.9998 y = 1.3139x - 33.631 R2 = 0.9997

0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0 10 20 30 40 50 60 70 80 90

% ACN %PC Averages Toluene Fenofibric Acid Diltiazem

) 7 . / 30 ( ] ) (% 10 ) 7 . / 1 [( %

) (

   

 b B b a A

ACN PC

Buffer concentration

2 4 6 8 10 12 14 50 100 150 200

C buffer (mM) Retention Time (min)

Carbamazepine Diltiazem Nicergoline 100 50 100 / 0 Acetonitrile Propylene carbonate

Salting-out effects (especially for last eluting peaks)!

Buffer concentration

4000 5000 6000 7000 8000 9000 10000 50 100 150 200

C buffer (mM) N

Carbamazepine Diltiazem Nicergoline 100 / 0 50 100 Acetonitrile Propylene carbonate

Loss of efficiency when using PC?

Buffer concentration

No significant loss in peak symmetry when using PC!

0.5 0.55 0.6 0.65 0.7 0.75 0.8 0.85 0.9 0.95 1 50 100 150 200

C buffer (mM) Peak Symmetry

Carbamazepine Diltiazem Nicerdoline 100 / 0 50 100 Acetonitrile Propylene carbonate

Van Deemter curves

0.0 5.0 10.0 15.0 20.0 25.0 30.0 0.00 5.00 10.00 15.00 20.00 25.00

u (cm/min) HETP (um)

Simvastatic acid (ACN) Fluoranthene (ACN) Simvastatic acid (PC) Fluoranthene (PC)

Low optimal flow rates when using PC!

Thermodynamic Aspects

Van’t Hoff plots with ACN as organic modifier!

ACN

y = 1090.5x - 2.9537 R2 = 0.9943 y = 980.91x - 2.2741 R2 = 0.9955 y = 506.74x - 0.4084 R2 = 0.943 y = 1310.7x - 2.4373 R2 = 0.9966 y = 1690.8x - 3.1192 R2 = 0.9986 0.000 0.500 1.000 1.500 2.000 2.500 3.000 0.00310 0.00315 0.00320 0.00325 0.00330 0.00335 0.00340 0.00345 0.00350

1/T ln (k)

Fenofibric Acid Toluene Simvastatic Acid Fluorene Fluoranthene

Thermodynamic Aspects

Van’t Hoff plots with PC/EtOH as organic modifier!

PC

y = 1191.1x - 2.8826 R2 = 0.9967 y = 1037.6x - 2.1934 R2 = 0.9983 y = 366.76x + 0.6339 R2 = 0.7229 y = 1224.8x - 2.0723 R2 = 0.9962 y = 1447.5x - 2.4518 R2 = 0.9965 0.000 0.500 1.000 1.500 2.000 2.500 3.000 0.00310 0.00315 0.00320 0.00325 0.00330 0.00335 0.00340 0.00345 0.00350

1/T ln (k)

Fenofibric Acid Toluene Simvastatic Acid Fluorene Fluoranthene

Thermodynamic Aspects

Parameter

Solvent

Fenofibric Acid Toluene Simvastatic Acid Fluorene Fluoranthene ACN 1090.5 980.9 506.7 1310.7 1690.8 B (slope of the van’t Hoff plot) PC 1191.1 1037.6 366.8 1224.8 1447.5 ACN

• 2.9537
• 2.2741
• 0.4084
• 2.4373
• 3.1192

A (intercept of the van’t Hoff plot) PC

• 2.8826
• 2.1934

0.6339

• 2.0723
• 2.4518

ACN 0.9971 0.9977 0.9711 0.9983 0.9993 rxy (correlation coeff. of the van’t Hoff plot) PC 0.9983 0.9991 0.8502 0.9981 0.9982 ACN

• 9067
• 8156
• 4213
• 10898
• 14058

 H0 (J)

PC

• 9903
• 8627
• 3049
• 10184
• 12035

ACN

• 16.9
• 11.3

4.2

• 12.6
• 18.3

 S0 (J)

PC

• 17.8
• 12.1

11.4

• 11.1
• 14.2

ACN

• 4022
• 4796
• 5478
• 7133
• 8603

 G0 (25 oC) (J)

PC

• 4589
• 5021
• 6452
• 6878
• 7788

No essential changes in the thermodynamics of the partition process arises when PC+EtOH are used instead ACN as organic modifier of the mobile phase!

Sensitivity (UV)

min 10.2 10.4 10.6 10.8 11 11.2 11.4 mAU 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5

= 254 nm

S/N = 8.61 ACN Fluorene

min 10.8 11 11.2 11.4 11.6 11.8 12 mAU 0.5 0.55 0.6 0.65 0.7 0.75 0.8 0.85 0.9

= 254 nm

S/N = 3.51 PC Fluorene

No addition to the mobile phase!

Sensitivity (UV)

min 9.2 9.4 9.6 9.8 10 mAU

• 0.05

0.05 0.1 0.15 0.2

Lovastatic Acid ACN S/N = 5.52

= 238 nm

min 8.8 9 9.2 9.4 9.6 9.8 10 10.2 mAU

• 0.8
• 0.7
• 0.6
• 0.5
• 0.4
• 0.3
• 0.2
• 0.1

S/N = 7.44 Lovastatic Acid PC

= 238 nm

Acid addition (HCOOH) to the mobile phase!

Experimental parameters / sensitivity MS/MS

Tube dimensions:

200 cm L x 0.12 mm i.d. Column temperature: 25 oC Carrier Solvent A: aqueous 0.1% HCOOH Solvent B: acetonitrile Solvent A / Solvent B = 1/1 (v/v) Solvent a: aqueous 0.1% HCOOH Solvent b: propylene carbonate / ethanol = 7/3 (v/v) Solvent a / Solvent b = 1/1 (v/v) Elution: isocratic Flow rate: 0.8 mL/min Injection volume: 1 L Sample concentration: 10 ppb

ESI parameters

Ion polarity: (+) or (-) Gas temperature: 350 oC Drying gas Flow (N2): 13 L/min Nebulizer pressure (N2): 60 psi Capillary voltage: 4000 V Dwell time: 200 ms  EMV: 600 V CID Collision gas: nitrogen Collision voltage: 15 V MS/MS transitions: specific to the analyte

Sensitivity (MS/MS +)

(+) Positive Ionization Acetonitrile Propylene carbonate # Analyte Peak Area

• St. Dev.

RSD% Peak Area

• St. Dev.

RSD% Yield (%) PC/ACN

1 Amiodarone 3251750 22705 0.7 487979 4853 1.0 15.0 2 Amlodipine 549565 34320 6.2 199353 13378 6.7 36.3 3 Atenolol 2561733 22491 0.9 881120 1942 0.2 34.4 4 Bromazepam 1492690 23508 1.6 414118 8945 2.2 27.7 5 Carbamazepine 5182413 160493 3.1 2078770 14758 0.7 40.1 6 Diltiazem 11604295 32081 0.3 2684578 30014 1.1 23.1 7 Enalapril 9092329 148271 1.6 1914998 33000 1.7 21.1 8 Enalaprilat 4801810 95705 2.0 940381 13463 1.4 19.6 9 Gliclazide 2469090 37301 1.5 630132 4523 0.7 25.5 10 Meloxicam 6588510 204680 3.1 1835548 26601 1.4 27.9 11 Metformin 6414215 67996 1.1 2729679 54359 2.0 42.6 12 Pentoxiphylline 2694353 57536 2.1 1056381 7838 0.7 39.2 13 Ranitidine 556601 4289 0.8 191422 5960 3.1 34.4 14 Simvastatin 91169 643 0.7 13130 177 1.3 14.4 15 Spironolactone 349875 19659 5.6 24886 1059 4.3 7.1 16 Tramadol 19349247 331553 1.7 4704267 138561 2.9 24.3 17 Trimetazidine 7174337 565960 7.9 3078189 55669 1.8 42.9

Signal suppression in (+) MS mode when using PC!

Sensitivity (MS/MS -)

Signal enhancement in (-) MS mode when using PC!

(-) Negative Ionization Acetonitrile Propylene carbonate # Analyte Peak Area

• St. Dev.

RSD% Peak Area

• St. Dev.

RSD% Yield (%) PC/ACN

1 Bromazepam

29729 377 1.3 26164 1183.5 4.5 88.0

2 Enalapril

41981 1085 2.6 71328 1818.5 2.5 169.9

3 Enalaprilat

43990 518 1.2 44188 1076.0 2.4 100.4

4 Furosemide

257727 6341 2.5 341865 14154.8 4.1 132.6

5 Gliclazide

750070 6155 0.8 1043481 7716.5 0.7 139.1

6 Indapamide

228600 7642 3.3 264781 9920.6 3.7 115.8

7 Meloxicam

1316722 34150 2.6 970591 7081.1 0.7 73.7

8 Nimesulide

180741 7664 4.2 241230 16389.5 6.8 133.5

9 Ranitidine

95294 2132 2.2 152655 2039.9 1.3 160.2

Other separation mechanisms (IP)

O O N H CH3 CH3 H OH O N H CH3 CH3 H OH O H O O H Betaxolol Betaxolol Impurity B Betaxolol Impurity D C H3 O N H CH3 CH3 H OH O O O H O O N H CH3 CH3 H OH C H3 Betaxolol Impurity A Betaxolol Impurity C Betaxolol Impurity E

O O N H CH3 CH3 OH Betaxolol Impurity F

Related impurities in betaxolol

Other separation mechanisms (IP)

Chromatographic column: Zorbax SB-C18, 5 cm L x 4.6 mm i.d. x 1.8 m d.p. Column temperature: 70 oC Alternative 1. Solvent A: ACN Solvent B: aqueous Sodium heptane sulphonate 15 mM and 0.1% H3PO4 Elution: Isocratic; Solvent A / Solvent B = 25 / 75 (v/v) Flow rate: 2 mL/min Detection: UV, 273 nm Alternative 2. Solvent A: Propylene carbonate / Ethanol = 7 / 3 (v/v) Solvent B: aqueous Sodium heptane sulphonate 15 mM and 0.1% H3PO4 Elution: Isocratic; Solvent A / Solvent B = 25 / 75 (v/v) Flow rate: 2 mL/min Detection: UV, 273 nm

Analytical conditions

Other separation mechanisms (IP)

m in

2 4 6 8 1 0 1 2

mAU

2 0 4 0 6 0 8 0

Betaxolol

Betaxolol Impurity B Betaxolol Impurity C Betaxolol Impurity D Betaxolol Impurity A Betaxolol Impurity F Betaxolol Impurity B

Degradation byproduct of Betaxolol Impurity C (C1) Degradation byproduct of Betaxolol Impurity C (C1)

2 0 4 0 6 0 8 0

• Solv. A = PC / EtOH = 7 / 3 (v/v)
• Solv. A = ACN

Other separation mechanisms (HILIC)

min 2 4 6 8 10 mAU 5 10 15 20 25 30 35 40 45 50 55 60  = 260 nm Nicotinamide Pyridoxine Thiamine

ACN / H2O / aq. 100 mM CH3COONH4 pH 5.8 = 95 / 5 / 5 (v/v/v) PC / EtOH / aq. 100 mM CH3COONH4 pH 5.8 = 89 / 4.1 / 6.9 (v/v/v) Chromatographic Column: Luna 3u HILIC, 150 mm L x 2 mm i.d. x 3 m d.p. Column temperature: 25 oC Elution: Isocratic Flow rate = 0.15 mL/min

N = 7516

• Sym. = 0.75

N = 5481

• Sym. = 0.55

N = 4960

• Sym. = 0.57

N = 2452

• Sym. = 0.56

N = 2621

• Sym. = 0.45

N = 3131

• Sym. = 0.43

Large Volume Injection of non-miscible diluents

Column = Zorbax Eclipse XDB C-18 Dimensions = 150 mm L x 4.6 mm i.d. x 5 m d.p. Temperature = 25 oC Flow rate = 1 mL/min Solvent A = Water Solvent B = Organic Solvent A / Solvent B = 4 / 1 (v/v) Elution = Isocratic Detection = UV (270 nm) Injection volumes = 1, 5, 10, 20, 50, 75, 100 L

Analytical conditions

Large Volume Injection of non-miscible diluents

Reduction of retention with the injection volume

y = -0.0042x + 2.2732 R2 = 0.9989 y = -0.0107x + 5.8775 R2 = 0.9996 y = -0.0036x + 1.183 R2 = 0.9565 y = -0.0116x + 7.0539 R2 = 1 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 20 40 60 80 100 120

• Vol. Inj. (uL)

k

PTX / ACN PTX / PC+EtOH MePar / ACN MePar / PC+EtOH

Large Volume Injection of non-miscible diluents

Variation of efficiency with the injection volume

4000 6000 8000 10000 12000 14000 16000 20 40 60 80 100 120

• Vol. Inj. (uL)

N

PTX / Hex / ACN MePar / Hex / ACN PTX / Hex / PC+EtOH MePar / Hex / PC+EtOH

Large Volume Injection of non-miscible diluents

Hexane as diluent

min 2 4 6 8 10 12 mAU

• 2

2 4 6 8 10 12  = 270 nm 20% ACN 20% (PC / EtOH = 7/3, v/v) Diluent = Hexane Injected volume = 75 L Pentoxiphylline (PTX) Methylparabene (MePar)

Large Volume Injection of non-miscible diluents

1-Octanol as diluent

min 2 4 6 8 10 12 mAU 2.5 5 7.5 10 12.5 15 17.5 20  = 270 nm Pentoxiphylline (PTX) Methylparabene (MePar) 20% ACN 20% (PC / EtOH = 7/3, v/v) Diluent = 1 - Octanol Injected volume = 75 L

Large Volume Injection of non-miscible diluents

min 2 4 6 8 10 12 mAU 2 4 6 8 10  = 270 nm Pentoxiphylline (PTX) Methylparabene (MePar) 20% ACN 20% (PC / EtOH = 7/3, v/v) Diluent = CCl4 Injected volume = 75 L

Carbon tetrachloride as diluent

Final problem (not an analytical one): PRICES ?

Final problem (not an analytical one): PRICES ?

Conclusions

Authors acknowledge the financial support given through the Romanian project PNII_ID_PCE_2011_3_0152.