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HPLC Peak Shape
HPLC Peak Shape
Efficiency
N = 5.54
( )
tR Wh
W f
0 . 0 5
0.05 h
Peak Maximum
Tf = W0.05/2f
Asymmetry
Peak Front Peak Tail h
h/2
W0 . 1 a b 0.1 h
Peak Maximum
As = b/a
Slide 4
Slide 5
Silica Type - Fully Hydroxylated and Metal Free Silica Reduces Acidity
OH Si Free Silanols HO Si Geminol Silanols decreasing acidity OH + M Surface Metal + M Si OH OH Si OH Si
Associated Silanols
Standard Silica
CH CH
2
NH
CH CH
2
CH -OH
2
Improve peak shape for basic compounds with high purity, fully hydroxylated silica such as Rx-SIL
Slide 8
Silica Type More Acidic Column: ODS, 4.6 x 250 mm, 5 m Plates: 92 USP Tf (5%): 2.90
Silica Type High Purity, Rx-Sil Column: SB-C18, 4.6 x 150 mm, 5 m Plates: 6371 USP Tf (5%):1.09
5 Time (min)
10
15
5 Time (min)
10
The high purity Rx-SIL improves the peak shape dramatically on a C18 column
Slide 9
Ionized silanols (SiO-) will ion-exchange with protonated bases (R3NH+) which can cause tailing and method variability. This occurs most often at mid pH where silanols are ionized.
Hydrogen Bonding
-SiOH + RCOO
2.
_
-SiO . . . H + . . . OOCR
Unprotonated acids can compete for H+ with protonated silanols. This can occur at low pH.
Some mobile phase additives can be added to the mobile phase to reduce these interactions and this will be discussed in the mobile phase section.
Slide 10
Slide 12
3 3
4 Time (min)
4 Time (min)
Double Endcapping Provides Superior Peak Shape Eclipse XDB-C18 vs. Competitive C18
Columns: 4.6 x 150 mm, 5 m Mobile Phase: 50% 25 mM Na2HPO4, pH 7.12: 50% MeOH Detection: UV 254 nm Flow Rate: 1.0 mL/min Temperature: 25C Sample: 1. Uracil 2. Amlodipine 3. Benazepril
Competitive L C18
2
Eclipse XDB-C18
2
6 8 Time (min)
10
12
14
0 2 4 6 8 Time (min) 10 12 14
Eclipse XDB shows superior peak shape for the basic compound, benazepril.
Slide 14
Good peak shape for basic compounds Polar alkyl-amide bonded phase Unique selectivity
Slide 15
Polar Alkyl Amide Bonded Phase Provides Better Peak Shape than Traditional Alkyl Phase
Columns: 4.6 x 150 mm Mobile phase: 25 mM K2HPO4, pH 7.2 / (MeOH: ACN, 50:50), 45/5 Flow rate: 1 mL/min. Detection: UV 254 nm Injection vol: 5 L Sample: Anorectics (Fen-phen) 1. Phentermine pKa10.1 2. Fenfluramine pKa 9.1 3. Impurity
1
mAU 15 10 5 0 mAU 15 10 5 0 0 1 2 3 4
2 3
Bonus-RP
Alkyl C8
2 3
min
Slide 16
Superior high pH stability up to pH 11.5 with silica particles Excellent reproducibility Patented bidentate, C18 bonding
Si
C18
Si
C18
Si
C18
Double endcapping
O Si C18
Slide 17
Extend-C18 pH 7
4 2,3 1 7
Extend-C18 pH 11
4
tR = 8.5
1 5 6 2
3 5
tR = 11.4
5 Time (min)
Time (min)
10
Slide 18
ZORBAX SB-Aq for Good Retention and Peak Shape in High Aqueous Mobile Phases
Bonded phases that are more wettable in high aqueous mobile phases improve chromatography both peak shape and retention reproducibility. A wettable bonded phase is one that does not fold over or collapse in a high aqueous mobile phase. These types of bonded phases can have polar endcapping or polar groups in the bonded phase, or other modifications to increase polarity. The SB-Aq column is an alkyl chain with more polar character that a typical C18 column.
Slide 19
Inconsistent Retention in High Aqueous Mobile Phase with Typical C18/C8 Columns
Example: Procainamides on Hydrophobic-C8 Column
1
Phase Collapse 50% loss in Resolution After equilibrating with 100% buffer Increase in peak tailing
3
5 4 5 4
5 Time (min)
10
5 Time (min)
10
Slide 20
No Phase Collapse No loss in Resolution After equilibrating with 100% buffer Good peak shape on all peaks
1
3 4 5
10
15 min
10
15 min
The SB-Aq column provides both good and consistent retention of the procainamides.
Slide 21
ZORBAX SB-Aq Provides Good Retention and Peaks Shape of Polar Pharmaceutical Compounds
1
Zorbax SB-Aq, 4.6 x 150 mm, 5 m 90% 0.2% TFA 10% ACN 25C 1.5 mL/min. UV 254 nm 1. Maleate 2. Phenylephrine 3. Phenylpropanolamine 4. Pyrilamine 5. Chlorpheniramine
8 Time (min)
10
12
14
16
Break Number 1
Slide 23
Large Molecules Need Large Pore Sizes for Good Peak Shape
Wide-pore 300 totally porous columns can be selected for efficient peaks when separating proteins and peptides. Select wide-pore columns for lower molecular weight molecules with large hydrodynamic volumes. Select Poroshell columns for more rapid mass transfer and improved efficiency of large peptides and proteins at higher flow rates.
Slide 24
Choose 300 Columns for Good Peak Shape with Peptides and Proteins
Effect of Pore Size and Molecular Size on Peak Width, Gradient Separations
0.2 0.18 0.16 0.14
PW 1/2
Le u M En .W k . = eph 55 ali 6 n
A n M g io .W te . = ns 1 0 in 46 II
Proper pore size selection results in sharper peaks for large molecules
Slide 25
.W RN . = as 13 e ,6 84 M Ly .W so . = zy 1 3 me ,9 00
M Ins .W u . = lin 3, B 49 6
.W Cy .= tC 12 ,3 27
Improved Peak Shape for Large Molecules in Solution with 300 Columns
Columns: 4.6 x 150 mm, 5 m Temperature: RT Mobile Phase: 60% MeOH: 40% 0.1% TFA Flow Rate: 0.75 mL/min Detection: UV 282 nm Sample: Tylosin (MW 916)
O CH3 CHO CH2 H3C O O CH3 OH HO O H3C O N CH3 O CH3 O
SB-C3 (80)
300SB-C3 (300)
HO CH3 CH3 OH
Pw1/2 = 0.442
Pw1/2 =0.125
5 Time (min)
10
5 Time (min)
10
The size of a molecule in solution determines which pore size column is best.
The narrower peak width indicates unrestricted access to the pores.
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Slide 27
pH 3.0
USP TF( 5%) 4. 2.35
2 3
Column:
4
4 5
Temperature: 35C Sample: pKa 9.4 pKa 9.6 pKa 9.9 pKa 10.1 pKa 10.1
0.0
5.0
2.5
Time (min)
5.0
These basic compounds have good peak shape when the pH is lowered and the
silica silanols are protonated.
Slide 28
pH 4.4
pH 3.0
CH2CH(CH3)2
4 5 6 Time (min)
10
4 5 6 Time (min)
10
Use Buffered Mobile Phases for Best Peak Shape and Retention
Column: ZORBAX Rapid Resolution Eclipse XDB-C8, 4.6 x 75 mm, 3.5 m Mobile Phase: 44% A : 56% methanol Flow Rate: 1.0 mL/min Temperature: 25C Detection: UV 250 nm Sample: 1. ketoprofen 2. ethyl paraben 3. hydrocortisone pKa 5.1 4. Fenoprofen pKa 4.5 5. propyl paraben 6. Propranolol pKa 9.5 7. Ibuprofen pKa 4.4
A = pH 7.0 water
1, 4, 6, 7
7
2 5
6 3
2 Time (min)
Time (min)
25 mM Phosphate pH 7.0
USP Tf 1. 1.41 2. 1.50 3. 1.33 4. 1.39 5. 1.36 6. 1.00
3 4 5 6
2 3
Columns:
Mobile Phase:40% phoshpate buffer 60% ACN Flow Rate: Sample: 1.5 mL/min. Tricyclic Antidepressants 1. Desipramine 2. Nortriptyline 3. Doxepin 4. Imipramine 5. Amitriptyline 6. Trimipramine Temperature: 40C
2.5
Higher ionic strength at mid pH is very effective at masking silanol interactions and
reducing peak tailing.
Slide 31
7.5
5.0
ZORBAX Eclipse XDB-C8 4.6 x 150 mm, 5 m 1.5 mL/min. Tricyclic Antidepressants 1. Desipramine 2. Nortriptyline 3. Doxepin 4. Imipramine 5. Amitriptyline 6. Trimipramine
Temperature: 40C
2.5
7.5
25 Time (min)
50
Changing the organic modifier may improve peak shape due to secondary interactions.
Slide 32
pH 7
No TEA
USP TF 5% 1. 1.29 2. 1.33 3. 1.63 4. 2.35 5. 1.57
5
10 mM TEA
3 2
4 5
0.0
5.0
5.0
Slide 33
Mobile Phase Modifiers Effects of Competing Acids on the Peak Shape of Acidic Compounds
A: pH 3 5 mM NaH2PO4 pH 3 5 mM NaH2PO4 1% acetic acid pH 2.5 0.1% TFA
Column: ZORBAX StableBond SB-C18 4.6 x 150 mm, 5 m Mobile Phase: 40% A: 60% ACN Flow Rate: 1.0 mL/min. Temperature: Room Temperature Sample: Ibuprofen pKa 4.4
Tf = 1.8
Tf = 1.0
Tf = 1.09
Both acetic acid and TFA (trifluoroacetic acid) act as competing acids.
TFA can be used at a lower concentration and is the preferred choice.
Slide 34
Break
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0 Time (min)
10
0 Time (min)
10
Injecting in a solvent stronger than the mobile phase can cause peak shape problems, such as peak splitting or broadening.
Slide 38
Slide 39
: : : :
H + M +2
O O H
M +2
OH
Salicylaldehyde
N: M +2
: : :
O N
M +2 OH
: :
OH
1.
2.
1.
2.
OH
Columns: ZORBAX SB-Phenyl 4.6 x 150 mm Mobile Phase: 75% 25 mM ammonium phosphate buffer 25% ACN Flow Rate: 1.0 mL/min. Temperature: RT Sample Size: 5 L
Tf: 3.7
Tf: 1.2
Purpurin
Column: Zorbax SB-C8, 4.6 x 250 mm, 5 m Mobile Phase: 20% 0.02% TFA in water: 80% MeOH Flow Rate: 1 mL/min Detection: UV 254 nm Temperature: 24C
Purpurin
O OH
Tf= 1.36
Tf= 1.18
3 Time (min)
3 Time (min)
Both fronting and tailing are evident on purpurin before the acid wash
Slide 42
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