Modernizing HPLC Methods II
Upgrading Current Methods
www.interface.co.kr
Why Modernize a Method?
• Improved technology available
• Save time
• Save solvent
• Improve results
• Peak shape
• Resolution
• Reproducibility or accuracy
• Accommodate new applications or
formulations
• Difficulty in duplicating results
Page 2
1
When to Modernize a Method
• Method will be used many times so
changes are cost effective
• Other parameters
• Reference method does not meet
needs
• Before any new method becomes
final
Page 3
Common Sources of Reference
Methods
• USP – Pharmaceutical compounds
• NIOSH – Industrial pollutants
• AOAC – Food components
• EPA – Environmental methods
Page 4
2
USP Provides Many Reference
Methods
• Important criteria are specified for each
method
• HPLC column type – “L” designation
• Resolution
• Tailing factor
• Relative retention times
Page 5
USP Methods – Common Liquid
Chromatography Column (L)
Designations
• L1 – Octadecyl silane chemically bonded to porous silica or
ceramic microparticles, 3 – 10 ? m in diameter (C18 or ODS)
• L3 – Porous silica microparticles, 3 – 10 ? m in diameter Sil)
• L7 – Octyl silane chemically bonded to totally porous microsilica
particles, 3 – 10 ? m in diameter (C8)
• L10 – Nitrile groups chemically bonded to porous silica
microparticles, 3 – 10 ? m in diameter (CN)
• L11 – Phenyl groups chemically bonded to porous silica
particles, 5 – 10 ? m in diameter (Phenyl)
• There are more than 40 HPLC column types designated by the
USP.
Page 6
3
ZORBAX L1 (C18) Columns
Column Carbon
pH
Type
Load Range
Pore
Size
SB-C18
80Å
10%
0.8 – 8.0
Eclipse
XDB10% 2.0 – 9.0
C18
Extend2.0 –
12.5%
C18
11.5
Silica Surface Surface Upper
Type
Area Coverage Temp
2.0
B
180 m 2/g
90°
C
? mole/m 2
80Å
B
180 m 2/g
80Å
B
180 m 2/g
Rx-C18
12%
2.0 – 9.0
80Å
B
ODS
20%
2.0 – 8.0
70Å
A
3.6
60°
C
? mole/m 2
3.0
40/60 °
C
? mole/m 2
3.3
180 m 2/g
60°
C
? mole/m 2
2.9
300 m 2/g
60°
C
? mole/m 2
Page 7
ZORBAX L7 (C8) Columns
Column Carbon
pH
Type
Load Range
Pore
Size
Silica
Type
Surface Surface Upper
Area Coverage Temp
SB-C8
Rx-C8
5.5%
0.8 – 8.0
80Å
B
180 m 2/g
2.0
90°
C
? mole/m 2
Eclipse
XDB-C8
7.6%
2.0 – 9.0
80Å
B
180 m 2/g
3.6
60°
C
? mole/m 2
C8
12%
2.0 – 8.0
70Å
A
300 m 2/g
2.9
60°
C
? mole/m 2
SB-C8 is identical to Rx-C8
Page 8
4
USP Analysis of Diazepam Capsules
• Requirements:
CH
• L1 (C18) column
3
O
N
• 3.9 x 300 mm
recommended
• Resolution between
ethylparaben and
diazepam is > 4.5
N
Cl
CH
6
5
•USP requirements permit changes in column configurations up to 50%.
Page 9
USP Analysis of Diazepam Capsules
Column Comparison
Mobile Phase: 35% Water: 65% MeOH
Flow Rate: 1.2 mL/min
Rx-C18
4.6 x 250 mm, 5 ? m
1
Sample: 1. Ethylparaben 2. Diazepam
? Bondapak C18
3.9 x 300 mm, 10 ? m
Retention
Peak Time Plates
Rs
2
1
3.47 11836 0.00
2 7.01 8885 14.75
1
Retention
Peak Time Plates Rs
2
1 4.64 5947 0.00
2 8.95 5216 11.24
Analysis Solvent
Time
Used
8 min
9.6 mL
0
5
10
Time (min)
15
0
Analysis Solvent
Time
Used
10 min 12 mL
5
10
15
Time (min)
• The Rx-C18 column provides higher resolution and greater efficiency in a
shorter analysis.
Page 10
5
USP Analysis of Diazepam on C18
Columns
Column: 4.6 x 250 mm Mobile Phase: 35% Water: 65% MeOH Flow Rate: 1.2 mL/min Sample: 1. Ethylparaben 2. Diazepam
ODS
SB-C18
1
1
2
2
Retention
Peak Time Plates Rs
1 3.81 8034 0.00
2 7.97 6020 13.45
Retention
Peak Time Plates Rs
1
4.17 13850 0.00
2
9.76 12154 0.28
0
5
10
15
0
Time (min)
5
10
15
Time (min)
•Several C18 columns give acceptable results.
•Newer C18 provides higher efficiency.
Page 11
Rapid Resolution HPLC Columns
Reduce
Analysis Time and Solvent Waste
5 ?m
3.5 ? m
5 ?m
3.5 ? m
Dimension 4.6 x 250 mm
4.6 x 150 mm 4.6 x 150 mm 4.6 x 75 mm
40%
50%
Analysis
30 min.
18 min.
18 min. reduction 9 min.
reduction
Time (min)
Solvent
Waste (mL)
20,000
unchanged
Resolution ?
50%
18 mL reduction 9 mL
12,00
0
10,000
}
20,000
}
N
40%
18 mL
reduction
30 mL
9% difference
N 1/2
Page 12
6
Update Method to Save Time and
Solvent
Mobile Phase: 35% water: 65% methanol
Sample: 1. Ethylparaben 2. Diazepam
Rapid Resolution, RxC18 4.6 x 75 mm, 5 ? m
Flow Rate: 1.2 mL/min
Solvent Saver, Rx-C18
3.0 x 250 mm, 5 ? m
Flow Rate: 0.5 mL/min
1
1
2
2
Analysis Solvent Solvent
Time
Used
Saved
2.5 min 3.0 mL 6.6mL
Analysis Solvent Solvent
Time
Used
Saved
8 min
4.0 mL 5.6 mL
Rs(1,2) = 12.05
0
Rs(1,2) = 8.62
2.5
5
Time (min)
7.5
0
2.5
5
7.5
Time (min)
•Consider all column configuration options for saving time or solvent.
Page 13
USP Analysis of Triamcinolone
• Requirements
CH 2OH
• L1 (C18) column
• (3.9 x 300 mm)
• Resolution between
triamcinolone and
hydrocortisone is >
3.0
CO
H
CH3
HO
H
H
CH 3
F
OH
OH
H
O
Page 14
7
USP Analysis of Triamcinolone
Column Comparison
Mobile Phase: 40% water: 60% methanol
Sample: 1. Triamcinolone 2. Hydrocortisone
? Bondapak C18
3.9 x 300 mm, 10 ? m
Rx-C18
4.6 x 250 mm, 5 ? m
2
1
1
2
Retention
Peak Time Plates Rs
1 2.28 8426 0.00
2 3.23 6892 4.79
Retention
Peak Time Plates Rs
1
3.39 4078 0.00
2
5.22 3769 6.16
Analysis Solvent
Time
Used
4 min
6 mL
0
1
2
3
4
5
6
7
8
9
Analysis Solvent
Time
Used
6 min
9 mL
10
0
1
2
3
Time (min)
4
5
6
7
8
9
10
Time (min)
Page 15
Update Method to Save Time and
Solvent
Mobile Phase: 40% water: 60% methanol
Solvent Saver, Rx-C18
3.0 x 250 mm, 5 ? m
Flow Rate: 0.6 mL/min
1
Sample: 1. Triamcinolone 2. Hydrocortisone
Rapid Resolution, Rx-C18
4.6 x 75 mm, 5 ?m
Flow 1 Rate: 1.5 mL/min
2
2
Analysis Solvent
Solvent
Time Used
Saved
1.5 min 1.5 mL 4.5mL
Analysis Solvent olvent
Time
Used Saved
4 min
2.4 mL 3.6mL
Retention
Peak Time
Rs
1
2.40 0.00
2
3.58 2.27
0
1
Retention
Peak Time
Rs
1
0.61 0.00
2
0.98 2.27
2
Time (min)
3
4
5
0
1
2
Time (min)
Page 16
8
Benefits of High Purity Silica for the
Analysis of Basic Compounds
• Improved peak shape
• More accurate quantitation
• Simplified mobile phases (no amine
modifiers)
• Note - Different silicas – Type A and
Type B – can still be the same “L”
designation with same kind of bonded
phase
Page 17
Chromatographic Benefits of Base
Deactivated Silica
Mobile Phase: 5% 2-Propanol in Heptane
Flow Rate: 2.0 mL/min. Temperature: 35 °C
High Purity, Low
Acidity
ZORBAX Rx-SIL
Standard Silica
NH
2
CHCH
2
3
CH-OH
2
NH
2
CHCH
2
3
CH-OH
2
• Improve peak shape for basic compounds with high purity, fully
hydroxylated silica such as Rx-SIL
Page 18
9
USP Analysis of Propranolol
Mobile Phase: 75% 50 mM KH2PO4 , pH 4.4 : 25% ACN
Flow Rate: 1.5 mL/min
• USP Requirements
Column:ODS, 4.6 x 250mm, 5? m
Plates: 92
USP Tf (5%): 2.90
• L1(C18) column
• 1000 plates
Propranolol
pKa 9.5
• Tf < 3
OH
• ODS column has “Type A”
silica
OCHCHCHNHCH(CH)
2
2
3
2
• L1 columns available in 198485 all had Type A silica
• This requires a high tailing
factor specification
0
5
10
15
Time (min)
•Higher tailing factors are typical of basic compounds on older silica.
Page 19
Improved Analysis of Propranolol
SB-C18 Rapid Resolution
4.6 x 75 mm, 3.5 ?m
SB-C18
4.6 x 150 mm, 5 ? m
Plates: 6371
USP Tf (5%): 1.09
Retention
Time: 6.50 min
Solvent
Used: 12 mL
0
5
Time (min)
Mobile Phase: 75% 50 mM KH2 PO4 , pH
4.4
25% ACN
Flow Rate:
1.5 mL/min
Sample:
1. Propranolol
10
0
Plates: 6370
USP Tf (5%): 1.14
Retention
Time: 3.11 min
Solvent
Used: 6 mL
5
10
Time (min)
• Excellent peak shape on SB-C18 columns, easily meets requirements with
very low tailing.
• Rapid Resolution SB-C18 reduced analysis time with equivalent results.
Page 20
10
Separation of ? -Blockers on
High Purity Silica
1
•Use the same ultra-pure
silica based column for
best peak shape when
separating related
compounds.
Column:
ZORBAX Rapid Resolution SB -C18
4.6 x 75 mm, 3.5 mm
Mobile Phase: 75% 50 mM KH2 PO4 , pH 4.4
25% ACN
Flow Rate:
1.5 mL/min
Sample:
1. Pindolol
2. Metoprolol
3. Oxprenolol
4. Propranolol
5. Alprenolol
4
•Rapid Resolution columns
reduce analysis time.
2
5
3
0
1
2
3
4
5
Time (min)
Page 21
ZORBAX Eclipse XDB Technology
• Features
• Benefits
• Dense dimethyl bonding
• Long lifetime
• Double endcapping
• Excellent peak shape
• All accessible silanols
reacted
• Wide usable pH range
• Ultrapure fullyhydroxylated silica
• Excellent all-purpose
column
• Applied to C18, C8 and
Phenyl bonded phases
• Different selectivities for
optimum resolution
(2 – 9)
Page 22
11
Analysis of Dexamethasone
• Requirements:
CHOH
2
• L7 (4.0 x 250 mm)
CO
• Recommendations:
OH
3
HO
H
CH
• 70:30 Water:ACN,
2 mL/min
• Retention time = 7
min.
CH
H
H
3
CH
3
H
F
O
Page 23
Analysis of Dexamethasone
Mobile Phase: 70% water: 30% ACN
Eclipse XDB-C8
4.6 x 75 mm, 3.5
? mFlow Rate: 1
mL/min
Eclipse XDB-C8
4.6 x 250 mm, 5 ? m
Flow Rate: 2 mL/min
Retention
USP
Peak Time Plates Tf (5%)
1
6.59 13540 1.00
Retention
USP
Peak Time Plates Tf (5%)
1 4.38
9069 1.00
Analysis Solvent
Time
Used
7 min
14 mL
0
2.5
Analysis Solvent
Time
Used
5 min
5 mL
5.0
Time (min)
7.5
0
1
2
3
4
5
6
7
8
9
10
Time (min)
• Updating this method saves substantial solvent and reduces cost.
Page 24
12
Analysis of Diphenhydramine
Children’s Chewable Tablet
1
USP Method
• L10 (CN) Column
• Mobile Phase:
50% ACN: 50% water
0.5% TEA
pH 6.5 with
glacial acetic acid
Column:
ZORBAX SB-CN
4.6 x 150 mm, 5 ? m
Mobile Phase:
A: 70% 25 m M
Na2 HPO4 , pH 3.0
B: 30% Methanol
Flow Rate:
1.0 mL/min
Temperature: 35°
C
UV Detection:
254 nm
Sample:
1. Unknown
2. Unknown
3. Diphenhydramine
2
Modified Method
• Mobile Phase:
Phosphate buffer, pH 3
No TEA necessary
3
0
5
10
Time (min)
•Update this method for greater simplicity and reliability.
Page 25
Separation of Cold Capsule
Components
Retention
USP
Peak Time Plates Tf (5%)
1
2.67
8864 1.00
2
11.35
6113 1.36
•Chlorpheniramine
Extended Release- L1
10 mm, 900 Plates, T f < 2
Column:
•Phenylpropanolamine –
Ion-pair chromatograpy,
L1, T f < 2.5
1
•Phenylpropanolamine
and Chlorpheniramine
hard to analyze together
0
2
5
10
ZORBAX SB-CN
4.6 x 150 mm, 5 ? m
Mobile Phase:
A: 80% 25 mM
Na2 HPO4 , pH 3.0
B: 20% Methanol
Flow Rate:
1.0 mL/min
Temperature: 35°
C
UV Detection:
254 nm
Sample:
1. Phenylpropanolamine
2. Chlorpheniramine
15
Time (min)
•Update and combine methods to save time.
Page 26
13
Steps in Developing a Modern
Method
• Select best silica for basic compounds
• Select best bonded phase
• Select most time saving configuration
• Plan for LC/MS compatibility
• Evaluate method for robustness and
ruggedness
Page 27
Separation of Herbicides on Different
StableBond Bonded-Phases
2
5&6
1
8
3
SB-C8
4
9
7
10
40
0
1
2
6
8
4
3
SB-Phenyl
9
7
10
40
0
6&7
2
8
1
SB-CN
9
3 4
5
0
10
T ime (min)
40
Columns:
4.6 x 250 mm, 5 ? m
Mobile Phase:
65%
H2O
35% Acetonitrile
Flow Rate:
1.0 mL/min
Temperature: room temperature
UV Detection:
254
nm
Sample:
1. Bentazon
2. Tebuthiuron
3. Simazine
4. Atrazine
5. Prometron
6. Diuron
7. Propazine
8. Propanil
9. Prometryne
10. Metolachlor
• Short chain bonded phases can reduce analysis time for late eluting
compounds, offer selectivity advantages and eliminate the need for
gradient elution.
Page 28
14
ZORBAX SB-C8 and SB-C18
Comparison Separation
ZORBAX SB-C8
Rs USP
1. ---2. 5.60
3. 12.37
Rs USP
1. ---2. 4.81
3. 10.35
2
Column:
ZORBAX Rapid
Resolution 4.6 x 75 mm, 3.5 µ m
Mobile Phase:90% 25
mMNa2HPO4, pH 3.0
10% ACN
1
Flow Rate: 1.5 mL/min
Temperature:
40°
C
Sample:
1. Theobromine
2. Theophylline
3. Caffeine
0
ZORBAX SB-C8
2
TF (5%)
1.25
1.18
1.15
0.5
1.0
3
3
1
1.5
2.0
2.5
3.0
3.5
4.0
0
0.5
1.0
Time (min)
1.5
2.0
2.5
TF (5%)
1.44
1.45
1.31
Column:
ZORBAX Rapid
Resolution 4.6 x 75 mm, 3.5 µ m
Mobile Phase:
90%50 mM Na2HPO4, pH 3.0
10% ACN
Flow Rate: 1.5 mL/min
Temperature:
40°
C
Sample:
1. Theobromine
2. Theophylline
3. Caffeine
3.0
3.5
4.0
Time (min)
•More efficient interaction with the C8 bonded phase in a high aqueous
mobile phase results in lower tailing factors.
Page 29
Plan for LC/MS Compatibility
• Is HPLC compatible with narrow-bore
columns?
• Do you need a capillary LC?
• Review adjusting gradient and isocratic
conditions for different column dimensions.
• Will mobile phase be sufficiently volatile and
allow efficient sample ionization?
Page 30
15
Adapting Isocratic Method to
Different Sized Columns
tr – Vm/F
k=
Vm1
V
= m2 = 1.5 = 0.3
F1
F2
1.
F2
0
Vm/F
1.5 F2 = 0.3
F2 = 0.2 mL/min
Column
1. 4.6 x 150 mm
3.0 x 150 mm
2. 2.1 x 150 mm
Column Volume
1.5 mL
0.6 mL
0.3 mL
Flow Rate
1.0 mL/min
0.4 mL/min
0.2 mL/min
Page 31
Isocratic Separation of Antibacterials
on Columns of Different Diameters
1
Column:
ZORBAX SB-C18
4.6 x 150 mm, 5 ? m
Flow Rate:
1.0 mL/min
Injected:
3?L
Detector Cell Volume: 8 ? L
Solvent Waste: 31 mL
4
6
2
5
3
1
Mobile Phase: 80% Acetonitrile
20% Citrate/phosphate pH
2.6
Temperature: Ambient
Sample:
Antibacterials
1. Sulfamerazine
2. Furazolidone
3. Oxolinic acid
4. Sulfadimethoxine
5. Sulfaquinoxaline
6. Nalidixic acid
0
Column:
Saver
4
2
5
3
1
6
3.0 x 150 mm, 5 ? m
Flow Rate:
0.5 mL/min
Injected:
2?L
Detector Cell Volume: 8 ? L
Solvent Waste: 15 mL
Column:
ZORBAX SB-C18
2.1 x 150 mm, 5 ? m
Flow Rate:
0.25 mL/min
Injected:
1?L
Detector Cell Volume: 2 ? L
Solvent Waste: 8 mL
4
2
3
5
Time (min)
ZORBAX SB-C18 Solvent
6
40
Page 32
16
Gradient Separation of Herbicides on
SB-C18 Columns with Different
Configurations
4.6 x 250 mm
tG= 30 min
F = 1.0 mL/min
• Each separation has the same profile;
b (gradient
steepness) is the same.
8
2
1
4 5
3
0
5
10
2.1 x 150 mm
tG= 18 min
F = 0.25
mL/min
15
6
7
20
25
30
8
2
1
6
4
3
0
5
7
5
10
15
20
Mobile Phase: A: 80% water + 0.1%
TFA
B: 20% ACN
Gradient:
20 – 60%B
Flow Rate:
0.25 mL/min
Sample:
Herbicides
1. Tebuthiuron
2. Prometryne
3. Simazine
4. Atrazine
5. Bentazon
6. Diuron
7. Propazine
8. Propanil
Time (min)
Page 33
Changing Gradient Retention (k*) to
Improve Gradient Resolution
8
2.1 x 150 mm
tG = 30 min
6
2
1
•To increase k*, decrease gradient
steepness, b
- use longer gradient time
•Changing k* or b by increasing tG
can improve resolution
4
5
7
3
0
5
10
15
Time (min)
20
25
30
Mobile Phase: A: 80% water + 0.1% TFA
B: 20% ACN
Gradient:
20 – 60%B
Flow Rate:
0.25 mL/min
Sample:
Herbicides
1. Tebuthiuron
2. Prometryne
3. Simazine
4. Atrazine
5. Bentazon
6. Diuron
7. Propazine
8. Propanil
Page 34
17
Adapting Gradient Methods to
Different Column Dimensions
To adjust gradient methods to different column
dimensions keep gradient steepness (b) the same.
1/k* ?
gradient
S ? ? ? ? Vm
=
b
=
steepness
tG ? F
S = constant
? ? = change in % organic
during the gradient run
Vm = void volume of column
F = flow rate
tG = gradient time
k* = k of solute at mid point
of column
If “b” is kept constant from run-to-run peaks will elute
in the same relative pattern.
Page 35
Adjusting a Gradient from a 4.6 x 250
mm Column to a 2.1 x 150 mm
Column
2.1 x 150 mm
4.6 x 250 mm
ΔФ= 40 (20% - 60%)
Vm = 2.5 mL
F = 1.0 mL/min
tG = 30 min
ΔФ • Vm
=
F • tG
Using b = 3.33
3.33 =
40 • 0.30
0.2 • tG
ΔФ= 40 (20% - 60%)
Vm = 0.3 mL
F = 0.2 mL/min
tG = ? (18 min)
40 • 2.5
= 3.33
1.0 • 30
Using new dimensions
Solve for tG
tG =
40 • 0.3
= 18 min
0.2 • 3.33
Page 36
18
Conclusion
• Improved results and lower costs may justify
time spent updating methods
• Evaluate Rapid Resolution and Solvent Saver
configurations for increased speed and reduced
solvent usage
• Check for ways to save time and solvent with any
method
• Apply current technology when appropriate –
scale down column dimensions and particle size
to meet speed and sample requirements
• Plan for compatibility with LC/MS
Page 37
19