CHROMATOGRAPHY FORUM OF THE DELAWARE VALLEY January 12, 2006 Media, PA
AQUEOUS NORMAL PHASE CHROMATOGRAPHY HOW IS IT DIFFERENT FROM HILIC? Joseph J. Pesek, Maria T. Matyska Department of Chemistry San Jose State University, San Jose, CA
ORDINARY SILICA
O O Si O O Si O O Si O
HYDRIDE SILICA
O OH OH OH
O Si O O Si O O Si O
O O O O
Si O Si O Si O
H H H
Bidentate Alkyl Phases O
O
H
Si
Si
C
Si
Si
C
O
O
R
O CH CH2
Structure I
R
O
Structure II
Ordinary Silica
Hydride Silica
Nonendcapped Normal C18 Bonded Phase
FTIR Spectra of Stored Hydride Silica Sample
SiH KROMASIL HYDRIDE YEAR 1996
% T R A N S M I T T A N C E
SiH
KROMASIL HYDRIDE YEAR 2003
SiH
KROMASIL HYDRIDE YEAR 2000
4000
3500
3000
2500
Wavenumbers
2000
1500
Biphenyl Phenol Tol uene
LONG TERM STABILITY TEST ON ENDCAPPED SILICA HYDRIDE
A
Phenol
Tol uene Biphenyl
Mobil Phase: 50:50 acetonitrile/water (0.05% H3PO4)
B
Biphenyl
A: Day 1 B: After six months
Phenol Tol uene
C Time (min.)
Name /t R
Day 1 Day 2 Day 5 6 mos. 8 mos. %RSD*
Phenol 1.34 1.32 1.32 1.34 1.38 0.87 Tol uene 2.52 2.49 2.48 2.49 2.49 0.69 Biphenol 3.57 3.55 3.53 3.52 3.55 0.63 * Data for 8 months obtai ned in a di fferent lab and on different equi pment are excluded from the calculations
C: After eight months
HYDRIDE COLUMN AFTER MORE THAN 2 YEARS OF USE IN 0.05% PHOSPHORIC ACID MOBILE PHASE
Solutes: 1 = pyridine; 2 = uracil; 3= phenol Mobile phase: 90:10 0.05% Phosphoric Acid in DI water/acetonitrile
Bidentate C18 after 1000 column volumes at pH = 10 Mobile Phase: 20 mM NH4OH/NH4COOH/Acetonitrile 1:1
Si-H
C-H
Equilibration of Hydride Silica Stationary Phases Reversed Phase Mode - Gradient Elution
Gradient program: 0-3 min ACN/water (50:50) 3-18 min to 100% ACN 18-23 min 100% ACN Equilibration to 50:50 ACN/water
Retention Time (min) Solute/Equilibration Time Benzene Naphthalene Phenanthrene Anthracene Pyrene
25 min 7.30 11.10 14.39 14.81 16.52
10 min 7.35 11.07 14.37 14.80 16.51
1 min 7.25 11.01 14.37 14.80 16.56
Equilibration of Hydride Silica Stationary Phases Reversed Phase Mode - Gradient Elution Gradient program: 0-3 min ACN/water (50:50) 3-18 min to 100% ACN 18-23 min 100% ACN Equilibration to 50:50 ACN/water
Solute/Equilibration Time Benzene Naphthalene Phenanthrene Anthracene Pyrene
25 min 5.49 9.00 12.70 13.03 15.08
Retention Time (min) 10 min 5.45 8.97 12.65 13.00 15.03
1 min 5.43 8.99 12.68 13.02 15.00
THREE MODES OF SEPARATION ONE COLUMN: REVERSED PHASE AQUEOUS NORMAL PHASE ORGANIC NORMAL PHASE
Neutral Compounds Hydride C18 or Cholesterol
C18 from Organosilanization
Flow Rate: 3ml/min Wavelength: 254nm Solvents A: Buffer B: 100%ACN
Typical Reversed Phase
Total Ion Chromatogram of Steroid Mixture on 4.2 x 150 mm Cholesterol Column
15 % Carbon bonded phase
5.00
10.00 15.00
20.00 25.00 30.00 35.00 40.00 45.00 50.00 Time (min)
Solutes: 1 = adrenosterone; 2 = corticosterone; 3 = 4-androstene-3,17-dione; 4= 11α-acetoxyprogesterone; 5 = estrone; 6 = estradiol.
Acidic Compounds 7
TYPE-B PHASE pH 8
Retention time (min.)
6 5 4 3
4-Hydroxybenzoic acid Acetylsalicylic acid
2
2-Hydroxybenzoic acid 1 0 0
10
20
30
40
50
60
70
80
90
100
% Acetonitrile
7
SILICA HYDRIDE PHASE pH 8
Retention time (min.)
6
Aqueous Normal Phase
5 4 Acetylsalicylic acid
3
4 Hydroxybenzoic acid 2 Hydroxybenzoic acid
2 1 0 0
10
20
30
40
50
60
70
80
90
100
% Acetonitrile
Solvents A: Buffer B: 100%ACN
Flow Rate: 3ml/min Wavelength: 254nm
Basic Compounds 8
Retention time (min.)
Retention tim e (m in.)
10
12
TYPE-B C18 Type-PHASE B phase pH pH88
Nortriptiline Imipramine Amiptriptyline
6 4 2
TYPE-C PHASE Hydride C18 phase pH pH88
10 8
Nortriptyline Imipramine Amitriptyline
6 4 2 0
0 20
40 60 %Acetonitrile
Retenion tim e (m in.) 8
0
100
20
40
60
80
100
% Acetonitrile
TYPE-C PHASE Hydride C18 phase pH 2.18 pH 2.18
14
TYPE-B PHASE C18 TypeB phase pHpH2.18 2.18
Nortriptyline Imipramine Amitriptyline
10
80
Retention time (min.)
0
6 4 2 0
12 10 8 6
Nortriptyline Imipramine Amitriptyline
4 2 0
0
20
40 60 % Acetonitrile
Reversed Phase Only
Flow Rate: 3ml/min Wavelength: 254nm
80
100
0
20
Reversed Phase
40 60 % Acetonitrile
80
100
Aqueous Normal Phase Solvents A: Buffer B: 100%ACN
Example of Separation Capabilities of Hydride Based Stationary Phase METFORMIN Aqueous Normal Phase
GLYBURIDE Reversed Phase
Reversed Phase
Aqueous Normal Phase
BD C18 on Hydride SILICA I
II Metformin - log P = -2.64 Glyburide - log P = +4.79
By changing solvent composition, the elution order can be reversed: I AQUEOUS NORMAL PHASE: NON POLAR FIRST II AQUEOUS REVERSE PHASE: POLAR FIRST
Hydride based BD C18, 4.6x75 mm, METFORMIN & GLYBURIDE 25
Comparison of Hydride Based C18 and Commercial C18 Columns in the Aqueous Normal Phase Mode
Retention time (min.)
20
Metformin - Acetonitrile
15
Glyburide - Acetonitrile 10
5
0 40
50
60
70
80
90
100
% organic in DI w ater, + 0.5% FA
LUNA C18, 4.6x150 mm
Agilent Zorbax C18, 4.6x150 mm
30
20 18
25
metformin glyburide
20
14 Retention time (min.)
Retention time (min.)
16
metformin glyburide
15
10
12 10 8 6 4
5
2 0
0 40
50
60
70 % Acetonitrile in DI w ater + 0.5% FA
80
90
100
40
50
60
70 % Acetonitrile in DI w ater + 0.5% FA
80
90
100
Aqueous Normal Phase Retention of the Basic Drug Tobramycin
Comparison of Peak Shapes on Hydride and Commercial Columns Endcapped Commercial C18 Column
Basic Drug Tobramycin
Hydride Cholesterol Column
AQUEOUS NORMAL PHASE RETENTION OF CARBOHYDRATES ON A HYDRIDE-BASED COLUMN
Carbohydrate Retention on Bidentate C18 50 45
Glucose (monosaccharide) Lactose(disaccharide) Raffinose (trisaccharide)
40
Retention Time (min.)
35 30 25 20 15 10 5 0 70
75
80
85
90
95
100
% Acetonitrile in DI Water + 0.5% FA
Detection by MS in the APCI+ mode
AQUEOUS NORMAL PHASE RETENTION OF NUCLEOSIDES ON A HYDRIDE-BASED COLUMN
Nucleoside Retention
Nucleoside Isomer Drug Separation 18
6 Nucleoside 1 Nucleoside 2
Cyt Dev 1 Cyt Dev 2
16
5
Retention Time (min.)
Retention Time (min.)
14 4
3
2
12 10 8 6 4
1
2 0
0 50
55
60
65
70
75
80
85
90
95
100
50
55
60
65
70
75
80
85
90
95
% Acetonitrile in 0.5% FA
% Acetonitrile in DI water + 0.5% FA
Column: Cogent UDC Cholesterol 4.6 x 150 mm
Column: Cogent UDC Cholesterol 4.6 x 150 mm Nucleoside Analog
MW
BpKa
LogP
Nucleoside Analog
MW
BpKa
LogP
Compound 1
494.5
3.73
3.184
Cytidine Derivative 1
358.4
3.73
-0.163
Compound 2
494.5
3.73
3.184
Cytidine Derivative 2
356.4
3.73
-0.952
RETENTION OF BASIC SOLUTES ON A CARBOXYLIC ACID PHASE BONDED TO A HYDRIDE SURFACE 1 Uracil 2 Tobramycin 20% Acetonitrile
35
Retention Time (min.)
30
10-Undecynoic Acid Column
2
1
Choline Tobramycin
25
20
15
10
5
0 0
10
20
30
40
50
60
70
80
90
% Acetonitrile
O O Si O CH O Si O
(CH2)8
CH2COOH
Structure I
O O Si O O Si O
C C H
(CH2)7
CH2COOH
Structure II
COMPARISON OF TWO CARBOXYLIC ACID COLUMNS
Retention Time (Min.)
Choline Retention on UDA Columns 35
Vydac (150 x 4.6 mm)
30
AstroSil (100 x 4.6 mm)
25
20
15
10
5
0
10
20
30
40
50
60
% Acetonitrile in ) 0.5% FA
70
80
90
SHORT COLUMN FORMAT (Qx2) FOR DIRECT INJECTION INTO THE MASS SPECTROMETER
Metformin/Glyburide on Bidentate C18 Qx2 60:40 ACN/Water
50:50 ACN/Water
40:60 ACN/Water
Aqueous Phase Contains 0.5% formic acid
4.52 c , ~, , , I' , , , i' , , I" , .1.'~~ .I' ., , I' .,
Separation of Choline/Acetylcholine on Standard Cholesterol Column
~x291 AcAcetylcholine400C 4.52
On Qx2 with 91% ACN
Acet = 3.50 min, Chol = 4.53 min
On Qx2 with 92% ACN
Acet = 4.11 min, Chol = 5.23 min
Reproducibility on Bidentate C18 Qx2
Mobile Phase 80:20 ACN/Water + 0.5% Formic Acid
Bidentate C18
Comparison of Stationary Phases in Qx2 Format
Cholesterol Metformin m/z = 130 Glyburide m/z = 369 Mobile Phase: 50:50 ACN/Water + 0.5% Formic Acid
30 Acetylcholine Choline
Retention time (min.)
25
20
15
10
5
0 40
50
60
70
80
90
100
90
100
% Acetonitrile in DI w ater + 0.5% FA
Hydride Based BD C18: Choline/Acetocholine 25
20 Choline Retention time (min.)
Comparison of Choline and Acetylcholine Elution on Cholesterol and BD C18 Columns under Aqueous Normal Phase Conditions
Hydride Based Cholesterol: Choline/Acetylcholine
Acetylcholine 15
10
5
0 40
50
60
70
% Acetonitrile in DI w ater + 0.5% FA
80
Tobramycin/Glyburide on Bidentate C18 Qx2 Column
Mobile Phase: 40:60 ACN/Water + 0.5% Formic Acid
EXAMPLES OF TYPICAL HILIC PHASES SILICA
Si-OH
AMINO
Si-R-NH2
CYANO
Si-R-CN
DIOL
Si-R-CH-CH2 OH OH
DIFFERENCES BETWEEN AQUEOUS NORMAL PHASE AND HILIC z
z
z
z
Aqueous Normal Phase Retains nonpolar compounds by reversed phase mechanism Retains polar compounds by normal phase mechanism Both reversed phase and normal phase mechanisms can operate simultaneously Can separate samples with both polar and nonpolar compounds
z
z z
HILIC Retains polar compounds by a normal phase mechanism Does not retain nonpolar compounds Cannot usually separate samples having both polar and nonpolar compounds
Normal Phase Operation on Hydride-Based C18
25
Hydride C18 phase Sample: CARVONE Flow rate: 1 mL/min TYPE-C PHASE 255 nm Sample:CARVONE
Retention time (min.)
20
Flow rate: 1mL/min.
% DI water/ THF
15
% DI water/acetonitrile % DI water/methanol
10
% hexane/isopropanol
5
0 0
20
40
60
Mobile phase - %B
80
100
NORMAL PHASE SEPARATION OF SUBSTITUTED PHENOLS
Atmospheric Pressure Chemical Ionization in positive mode - APCI+ Column: Bidentate C18, mobile phase: 95:5 Hexane/Ethyl Acetate Flow rate: 1.0 mL/min. Samples: 1 – phenol with aldehyde, 2 – parent phenol, 3 – phenol with ketone, 4 – phenol with acid
USE OF HYDRIDE COLUMN IN NORMAL PHASE MODE
A: HYDRIDE COLUMN B: COMMERCIAL SILICA COLUMN
USING 100 % AQUEOUS MOBILE PHASE ON BIDENTATE C18 HYDRIDE SILICA
After 3 hours of 100% aqueous exposure, results are the same as with the first injection
1- OXALIC ACID 2- FORMIC ACID 3- ACETIC ACID 4- SODIUM AZIDE 5- URACIL 6- FUMARIC ACID 7- PROPIONIC ACID 100% 0.05% v/v H3PO4
Flow Rate:1ml/mn Column: 75 x 4.6mm id UV: 2155
Proprietary Vitamin Tablet – column: C18 Bidentate Multi- Vitamin Standards
1- Niacidamide 2. Thiamine
3
3. Pyriadoxine 2 4. Folic Acid 5. Riboflavin
Dimensions: 250 mm x 4.6 mm, Detection: 270 nm, Mobile phase: 0-4 min: 5% MeCN/95% (0.1% v/v TFA DI Water), 4-10 min: 15% MeCN/ 85% (0.1% v/v TFA DI Water), Flow Rate; 1 mL/min, Standard C18 columns are not able to retain these very polar compounds well. Bidentate C18 provides separation of these analytes. In addition the column quickly equilibrates between mobile phase compositions making the analysis more efficient.
OTHER PHASES UNDER INVESTIGATION BIDENTATE C8 C30
1-octyne column A
Reversed-phase separation on C8 column made from 1-octyne. A; polypeptides; mobile phase: solvent A; 0.05% TFA in acetonitrle, solvent B; 0.05% TFA in water. Gradient: 20-35% A in 15 minutes. Flow rate; 1.3 mL /min. Detection at 214 nm. Solutes: 1 = angiotensin III, 2 = bradykinin, 3 = angiotensin I. B; Tricyclic antidepressants; mobile phase: acetonitrile/ Methanol/10mM phosphate buffer (35:25:40). Flow rate 1.0 mL/min. Detection at 254 nm. Solutes: 1 = desipramine, 2 = imipramine, 3 = clomipramine. C; Barbiturates; mobile phase: methanol/water (37:63). Flow rate 1.5 mL/min. Detection at 220 nm. Solutes: 1 = butabarbital, 2 = amobarbital, 3 = secobarbital, 4 = phenobarbital, 5 = hexobarbital.
C30 bonded phase Separation of tricyclic antidepressant mixture on C30 bonded phase. Moblie phase: 45:45:10 methanol/acetonitrile/12mM phosphate buffer (pH = 6.7) Solutes: 1 = doxepin, 2 = propranolol, 3 = clomipramine, 4 = nortriptiline and 5 = desipramine.
SPECIAL FEATURES OF CHOLESTEROL PHASE SHAPE SELECTIVITY TEMPERATURE EFFECTS
CHOLESTEROL MOIETY BONDED TO SILICA HYDRIDE UDC Cholesterol O O Si CH2 (CH2)9 O O Si H O O Si H O O Si H O CH2 (CH2)9 O Si O O Si H O
CH3 CH3
O C
H
O
H
H
H CH3 CH3
O C
O H
H
H
H
1.Prednisolone 2.Cortic osterone 3.Estradiol 4.Ethiny l Estradiol 5.Estrone 6.Norgestere l 7.Progesterone
Chromatogram A: Near baseline resolution was rapidly achieved for these steroids. Chromatogram B: A near optimal resolution is shown. The selectivity shown here employs shape recognition as the predominant separation mechanism. Selectivity based on hydrophobicity exhibitied in Chromatogram A is similar to other standard C18 polar embedded columns when used with similar conditions. Many variations of the relative elution orders of peaks 3,4,5 & 6 were possible by controlling temperature and or the relative percent concentrations of MeOH and Acetonitrile with the UDCCholesterol™ column shown in Chromatogram B.
Cholesterol column 55:45 methanol/DI Water
50:50 acetronitrile/DI water
1 150
Bidentate C18 column 55:45 methanol/DI water
1 2
1
2
150
2 1
1
200
2
200
2
10
12
14
16 min.
Solutes:
2
450
1+2
1
450
1.
Nogestrel
2.
Ethynylestradiol
Elution behavior of Bidentate C18 as a function of temperature is independent of solvent 2 6
8
10
12
14 min.
3
min.
1
22 1
6
5
6
5
43
3
0.00
10.00
1 1
4
A 20.00
30.00 40.00 Time (min)
4
5 4
3
20.00
70.00
66
B 10.00
60.00
22
3
0.00
50.00
30.00 40.00 Time (min)
50.00
Solutes 1 = andrenosterone; 2 = corticosterone; 3 = estrone; 4 = 4-androstene-3,17-dione;
5
60.00
70.00
SEPARATION OF STEROIDS ON THE CHOLESTEROL HYDRIDE COLUMN
5 = estradiol; 6 = 11-α-acetoxyprogesterone
A: Qx2 format, 10.5% carbon B: Standard 4.6 x 75 mm column, 15.1% carbon
IMPORTANT FEATURES OF HYDRIDE BASED STATIONARY PHASES 1.
Hydride phases operate in the normal phase, aqueous normal phase and reversed phased modes
2.
Hydride phases can be used in 100% aqueous mobile phases with no stationary phase collapse
3.
Bases are retained at low pH so high pH mobile phases may not be necessary for many applications
4.
Surface absorbs very little water so there is reproducible retention in the normal phase and rapid equilibration for gradient separations
5.
Bidentate phases can be used over a broad range of pH and can be used at high temperatures
Funding Provided by: National Science Foundation National Institutes of Health Camille and Henry Dreyfus Foundation
Special Thanks to MicroSolv Technology Corp.
