Choosing Columns and Conditions for the Best Peak Shape

Choosing Columns and Conditions for the Best Peak Shape

What is Good Peak Shape and Why is it Important? Good peak shape can be defined as a symmetrical or gaussian peak and poor peak shape can include both peak fronting and tailing. Good peak shape can be defined by. Tailing factor of.0 High efficiency Narrow peak width Good peak shape is important for. Improved resolution (Rs) More accurate quantitation Longer usable column lifetime (based on system suitability criteria) Page

Measures USP Tailing Factor at 5% of peak height* Asymmetry at 0% of peak height Skew rd moment measure column testing* Indicators Efficiency plates* Peak Width peak width at ½ height* * Available in ChemStation reports Page

USP Tailing Factor Efficiency peak front peak tail h N = 5.54 t R W ½ h = f W 0. 0 5 0.05 h peak maximum T = W / f 0. 0 5 Asymmetry Peak Width at ½ Height peak front peak tail h h W h / h/ W 0. = a = b 0. h W t peak maximum T f = b /a Page 4

Column packing factors Silica type Bonded phase and endcapping Mobile phase factors Sample factors Page 5

Silica type/acidity Fully hydroxylated silica and silica metal content Silica silanol ionization* Hydrogen bonding with silica silanols* Column bonding Endcapping Type of bonded phase Packing Pore Size/Structure * Controlled by mobile phase Page 6

H H H H H Si Si Si Si Free Silanols Geminol Silanols Associated Silanols decreasing acidity M + M + Surface Metal H Si Internal Metal (activated silanol) (most acidic) Page 7

Mobile Phase: 5% -Propanol in Heptane Flow Rate:.0 ml/min. Temperature: 5 C Standard Silica High Purity, Low Acidity ZRBAX Rx-SIL CH -H NH CH CH NH CH CH CH -H Improve peak shape for basic compounds with high purity, fully hydroxylated silica such as Rx-SIL Page 8

ZRBAX StableBond with Rx-SIL Improves Peak Shape Mobile Phase: 75% 50 mm KH P 4, ph 4.4 : 5% ACN Flow Rate:.5 ml/min Silica Type More Acidic Column: DS, 4.6 x 50 mm, 5 mm Plates: 9 USP T f (5%):.90 Silica Type High Purity, Rx-Sil Column: SB-C8, 4.6 x 50 mm, 5 mm Plates: 67 USP T f (5%):.09 Propranolol pka 9.5 H CH CHCH NHCH(CH ) 0 5 0 5 0 5 0 The high purity Rx-SIL improves the peak shape dramatically on a C8 column Page 9

Potential Ion Exchange and Hydrogen Bonding Secondary Interactions Ion-exchange Si _ Na+ + R NH + Si _ N + R + Na +. Ionized silanols (Si - ) will ion-exchange with protonated bases (R NH + ) which can cause tailing and method variability. This occurs most often at mid ph where silanols are ionized. Hydrogen Bonding -SiH + RC _ -Si _... H +... _ CR. 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. Page 0

Bonded Phase Types for Better Peak Shape Bonded phases with endcapping Eclipse XDB columns are double endcapped This minimizes the number of unreacted silanols and potential peak tailing interactions Bonded phases with embedded polar groups Bonus-RP provides unique silanol shielding reducing peak tailing for basic compounds Bonded phases for high ph Extend-C8 is stable at high ph Basic compounds can not ion-exchange with the silica thereby reducing peak tailing Bonded phases for high aqueous SB-Aq is more wettable improving analyte interactions and peak shape 00764S.PPT Page

Eclipse XDB is Double Endcapped for Best Peak Shape Improved peak shape for basic compounds especially at intermediate ph Double endcapped with two different reagents for more complete silanol coverage Good for all sample types acid, base, neutral Wide useable ph range ph -9 CH Si CH CH Si CH CH Si CH CH Si CH CH Si CH CH CH Page

Double Endcapping Improves Peak Shape ph 7 Columns: 4.6 x50 mm, 5 mm Mobile Phase: 60% ACN : 40% 0 mm phosphate buffer ph 7.0 Flow Rate:.5 ml/min. Temperature: 40 C Sample:. Nortriptyline pka 9.7. Doxepin pka 9.0. Amitriptyline pka 9.4 4. Trimipramine Single Endcapped Waters Symmetry C8 Double Endcapped Eclipse XDB-C8 Tf..95..90..54 4.. 4 Tf..79..4..4 4..0 4 0 4 5 6 7 8 0 4 5 6 7 8 Each column is made from high purity silica Fewer silanol interactions on the double endcapped column reduce tailing and retention Page

Double Endcapping Provides Superior Peak Shape Eclipse XDB-C8 vs. Luna C8 Columns: 4.6 x 50 mm, 5 mm Mobile Phase: 50% 5 mm Na HP 4, ph 7.: 50% MeH Detection: UV 54 nm Flow Rate:.0 ml/min Temperature: 5 C Sample:. Uracil. Amlodipine. Benazepril Phenomenex Luna C8 Eclipse XDB-C8 Tf(5%)..07..50 Tf(5%)..0..08 0 4 6 8 0 4 0 4 6 8 0 4 Eclipse XDB shows superior peak shape for the basic compound, benazepril. Page 4

Bonus-RP with Embedded Polar Groups Good peak shape for basic compounds R Si PG R R Polar alkyl-amide bonded phase CH Si CH CH R Unique selectivity Si PG R Enhanced low ph stability with sterically protecting bonding R CH Si CH CH Triple endcapped R Si PG R R Page 5

Polar Alkyl Amide Bonded Phase Provides Better Peak Shape than Traditional Alkyl Phase Columns: 4.6 x 50 mm Mobile phase: 5 mm K HP 4, ph 7. / (MeH: ACN, 50:50), 45/5 Flow rate: ml/min. Detection: UV 54 nm Injection vol: 5 µl Sample: Anorectics ( Fen-phen ). Phentermine pka0.. Fenfluramine pka 9.. Impurity mau 5 0 Bonus-RP 5 0 mau 5 0 5 Alkyl C8 0 0 4 5 6 7 min Good peak shape of highly basic compounds is readily achieved on Bonus-RP. 00767S.PPT Page 6

ZRBAX Extend-C8 for High ph Superior high ph stability up to ph.5 with silica particles Si C8 Excellent reproducibility Patented bidentate, C8 bonding Double endcapping Si C8 Si C8 Si C8 Page 7

Extend-C8 Improves Retention and Peak Shape of Basic Compounds at High ph Column: ZRBAX Extend-C8, 4.6 x 50 mm, 5 mm Mobile Phase: 0% buffer: 70% MeH ph 7 buffer=0 mm Na HP 4 ph buffer = 0 mm TEA Flow Rate:.0 ml/min Temperature: RT Detection: UV 54 nm Sample:. Maleate. Scopolamine pka 7.6. Pseudoephedrine pka 9.8 4. Doxylamine pka 9. 5. Chlorpheniramine pka 9. 6. Triprolidine pka 6.5 7. Diphenhydramine pka 9.0 Extend-C8 ph 7 4, 7 Extend-C8 ph t R = 8.5 t R =.4 4 7 5 5 6 6 0 5 0 5 0 The retention of this sample of basic compounds increases at high ph on Extend-C8. Page 8

Large Molecules Need Large Pore Sizes for Good Peak Shape Wide-pore 00Å 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. Page 9

Choose 00Å Columns for Good Peak Shape with Peptides and Proteins Effect of Pore Size and Molecular Size on Peak Width, Gradient Separations 0. 0.8 0.6 00SB-C8 (00Å) SB-C8 (80Å) 0.4 PW / 0. 0. 0.08 0.06 0.04 0.0 0 Leu Enkephalin M.W. = 556 Angiotensin II M.W. = 046 Insulin B M.W. =,496 Cyt C M.W. =,7 RNase M.W. =,684 Lysozyme M.W. =,900 Proper pore size selection results in sharper peaks for large molecules Page 0

Improved Peak Shape for Large Molecules in Solution with 00Å Columns Columns: 4.6 x 50 mm, 5 mm Mobile Phase: 60% MeH: 40% 0.% TFA Flow Rate: 0.75 ml/min Temperature: RT Detection: UV 8 nm Sample: Tylosin (MW 96) SB-C (80Å) H CH CH H C CH CH CH CH H C CH CH CH H H C H N CH CH 00SB-C (00Å) H CH H CH Pw / = 0.44 Pw / =0.5 0 5 0 0 5 0 The size of a molecule in solution determines which pore size column is best. The narrower peak width indicates unrestricted access to the pores. Page

Poroshell Particles Provide More Efficient Peaks for Peptides and Proteins Porous Shell SLID CRE 5 µ m Poroshell 00SB-C8 is for the separation of large peptides and proteins Poroshell particles have a solid silica core with a superficially porous surface with a 00Å pore size This results in more efficient mass transfer and sharper peaks with large proteins and peptides Page

Fast, High Resolution Separation of Peptides and Proteins With Poroshell 00SB-C8... In Seconds Columns: Poroshell 00SB-C8. x 75 mm, 5 mm Mobile Phase: A: 0.% TFA B: 0.07% TFA in ACN Gradient: 5 00% B in.0 min. Flow Rate:.0 ml/min. Temperature: 70 C Pressure: 50 bar Detection: UV 5 nm 4 5 6 7 8 Sample:. Angiotensin II. Neurotensin. Rnase 4. Insulin 5. Lysozyme 6. Myoglobin 7.Carbonic Anhydrase 8.valbumin 0 0.5.0 nly Poroshell can provide high efficiency at higher flow rates for extremely rapid separations of proteins and peptides. This is due to more rapid mass transfer of the superficially porous particle Page

Effect of Increasing Flow Rate in Protein Analysis with use of Totally Porous Silica Agilent 00 WPS with AutoBypass; Piston Stroke: 0 µl, Temperature: 70 C Detection: UV 5 nm Mobile Phase: A= 95% H, 5% ACN with 0.%TFA; B= 5% H, 95% ACN, with 0.07%TFA 0.5 ml/min 0-00%B in 4 min 0 0.5.5.5.5 4 ml/min 0-00%B in min 0 0.5.5.5.5 4 ml/min 0-00%B in min Totally Porous.x75mm, 5µm. Neurotensin. Rnase A. Lysozyme 4. Myoglobin 0 0.5.5.5.5 4 ml/min 0-00%B in 0.67 min 0 0.5.5.5.5 4 Efficiency and resolution are lost at high flow rates on a totally porous 00Å RP column Page 4

High Efficiency at High Flow Rates for Ultra-Fast Protein Analyses with Poroshell 0.5 ml/min 5 00%B in 4 min 0 ml/min 5 00%B in min ml/min 5 00%B in min ml/min 5 00%B in 0.67 min 4 ml/min 5 00%B in 0.5 min Agilent 00 DAD Agilent 00 WPS with ADVR Column: Poroshell 00SB-C8. x 75 mm, 5 mm Mobile Phase: A: 95% H, 5% ACN with 0.% TFA B: 5% H, 5% ACN with 0.% TFA Temperature:70 C Detector: UV 5 nm Sample:. Neurotensin. Lysozyme. RNaseA 4. Myoglobin 0 0. 0.4 0.6 min Resolution is maintained at high flow rates with Poroshell, allowing for efficient peaks and rapid analysis times. Page 5

Mobile Phase Factors for Improved Peak Shape ph Buffers rganic modifiers Additional mobile phase modifiers (TEA, TFA) Page 6

Good Peak Shape at Low ph ph 7.0 ph.0 USP TF( 5%) 4..5 USP TF (5%) 4.. Column: ZRBAX Eclipse XDB-C8 4.6 x 50 mm, 5 mm 4 5 4 5 Mobile Phase:85% 5 mm Na HP 4 5% ACN Flow Rate:.0 ml/min. Temperature: 5 C Sample: Amphetamines. Phenylpropanolamine pka 9.4. Ephedrine pka 9.6. Amphetamine pka 9.9 4. Methamphetamine pka 0. 5. Phentermine pka 0. 0.0.5 5.0 0.5 5.0 These basic compounds have good peak shape when the ph is lowered and the silica silanols are protonated. Page 7

Effect of ph on Peak Shape at or Near the Sample pka Column: ZRBAX SB-C8 4.6 x 50 mm, 5 mm Mobile Phase: 40% 5 mm KH P 4 : 60% ACN Flow Rate:.0 ml/min. Temperature: RT ph 4.4 ph.0 CH CHCH CH CH(CH ) Ibuprofen pk a = 4.4 0 4 5 6 7 8 9 0 0 4 5 6 7 8 9 0 Inconsistent and tailing peaks may occur when operating close to an analyte pka and should be avoided. Page 8

Use Buffered Mobile Phases for Best Peak Shape and Retention Column: ZRBAX Rapid Resolution Eclipse XDB-C8, 4.6 x 75 mm,.5 µm Mobile Phase: 44% A : 56% methanol Flow Rate:.0 ml/min Temperature: 5 C Detection: UV 50 nm Sample:. ketoprofen. ethyl paraben. hydrocortisone pka 5. 4. Fenoprofen pka 4.5 5. propyl paraben 6. Propranolol pka 9.5 7. Ibuprofen pka 4.4 A = ph 7.0 water A = ph 7.0, 5 mm phosphate buffer, 4, 6, 7 4 5 5 6 7 0 4 0 4 5 Buffered mobile phases enhance retention, resolution, and peak shape. Page 9

Increasing Buffer Concentration Decreases Tf 0 mm Phosphate ph 7.0 5 mm Phosphate ph 7.0 USP Tf..6..65..6 4..77 5..8 6.. USP Tf..4..50.. 4..9 5..6 6..00 Columns: ZRBAX Eclipse XDB-C8 4.6 x 50 mm, 5 µm Mobile Phase: 40% phoshpate buffer 60% ACN Flow Rate:.5 ml/min. 4 5 6 4 5 Temperature: 40 C Sample: Tricyclic Antidepressants. Desipramine. Nortriptyline 6. Doxepin 4. Imipramine 5. Amitriptyline 6. Trimipramine 0.5 5.0 7.5 0.5 5.0 Higher ionic strength at mid ph is very effective at masking silanol interactions and reducing peak tailing. Page 0

Mobile Phase rganic Modifier Acetonitrile vs. Methanol 40% 5 mm Na HP 4 ph 7.0 60% ACN 40% 5 mm Na HP 4 ph 7.0 60% MeH 4 5 USP TF 5%....9..66 4..94 5..95 6.. 6 USP TF 5%. 0.7..45..4 4..5 5..09 6..0 4 5 Columns: ZRBAX Eclipse XDB-C8 4.6 x 50 mm, 5 µm Flow Rate:.5 ml/min. Temperature: 40 C Sample: Tricyclic Antidepressants. Desipramine. Nortriptyline. Doxepin 4. Imipramine 5. Amitriptyline 6. Trimipramine 6 0.5 5.0 7.5 0 5 50 Changing the organic modifier may improve peak shape due to secondary interactions. Page

Mobile Phase Modifiers Effect of TEA on Peak Shape of Basic Compounds Column: ZRBAX Eclipse XDB-C8, 4.6 x 50 mm, 5 µm Mobile Phase: 85% 5 mm Na HP 4 : 5% ACN Flow Rate:.0 ml/min. Temperature: 5 C Sample: Amphetamines. Phenylpropanolamine. Ephedrine. Amphetamine 4. Methamphetamine 5. Phenteramine ph 7 No TEA 0 mm TEA 4 5 USP TF 5%..9....6 4..5 5..57 4 5 USP TF 5%..9..8..0 4..6 5..4 0.0.5 5.0 0.5 5.0 Page

Mobile Phase Modifiers Effects of Competing Acids on the Peak Shape of Acidic Compounds A: ph 5 mm NaH P 4 ph 5 mm NaH P 4 % acetic acid ph.5 0.% TFA Column: ZRBAX StableBond SB-C8 4.6 x 50 mm, 5 µm Mobile Phase: 40% A: 60% ACN Flow Rate:.0 ml/min. Temperature: Room Temperature Sample: Ibuprofen pka 4.4 T f =.8 T f =.0 T f =.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. Page

Sample and Additional Considerations for Good Peak Shape Sample overload (mass/volume) Sample solvent/injection solvent Metal complexation Page 4

Sample verload May Cause Peak Fronting and Tailing Peak fronting from sample mass overload more sample than can effectively partition results in some sample preceding the rest of the peak Peak tailing from overload of silanols with basic compounds may be seen as peak tailing Reduce sample load to eliminate 00 mau for compounds with average absorptivity Page 5

Strong Injection Solvent May Cause Poor Peak Shape Column: ZRBAX StableBond SB-C8, 4.6 x 50 mm, 5 µm Mobile Phase: 8% H : 8% ACN Injection Volume: 0 µl Sample:. Caffeine. Salicylamide A. Injection Solvent 00% Acetonitrile B. Injection Solvent Mobile Phase 0 0 0 0 Injecting in a solvent stronger than the mobile phase can cause peak shape problems, such as peak splitting or broadening. Page 6

Metal Complexation May Cause Poor Peak Shape Analytes that may complex with metals may show poor peak shape Both tailing and fronting may result from metal complexation Metals are present in LC systems from solvents, tubing, and stainless steel frits High purity silica eliminates silica as a source of metals Page 7

Metal Sensitive Compounds Can Chelate Hint: Look for Lone Pair of Electrons on :: or N Which Can Form 5 or 6 Membered Ring with Metal : C :H : H C M + H + M + : : H Salicylaldehyde 6-membered ring complex N: M + H : : C C : : N : H M + 8-hydroxyquinoline 5-membered ring complex α-benzoinoxomine 5-membered ring complex Page 8

Acid Wash Can Improve Peak Shape Before Acid Wash After Acid Wash 50 00 mls % H P 4 H H H H H.... H H H Columns: ZRBAX SB-Phenyl 4.6 x 50 mm Mobile Phase: 75% 5 mm ammonium phosphate buffer 5% ACN Flow Rate:.0 ml/min. Temperature: RT Sample Size: 5 µl Tf:.7 Tf:. A % H P 4 solution is used on SB columns, 0.5 % can be used on endcapped columns. Page 9

Purpurin Column: Zorbax SB-C8, 4.6 x 50 mm, 5 mm Mobile Phase: 0% 0.0% TFA in water: 80% MeH Flow Rate: ml/min Detection: UV 54 nm Temperature: 4 C A. Before Acid Wash B. After Acid Wash H % phosphoric acid, 0 min. Purpurin H H Tf=.6 Tf=.8 0 4 5 6 0 4 5 6 Both fronting and tailing are evident on purpurin before the acid wash Page 40

Guidelines for Improved Peak Shape Select columns based on high purity fully hydroxylated silica Zorbax Rx-Sil based columns, such as StableBond, Eclipse XDB, Bonus-RP and Extend Select double endcapped columns for mid ph or difficult basic compounds, such as Eclipse XDB Select special bonded phases (Bonus-RP, Extend-C8) for better peak shape at mid and high ph Select wide-pore columns for high molecular weight analytes Use buffered low ph mobile phases to reduce secondary interactions Use 5 50 mm buffered mobile phases at every ph Use additional additives only when needed Check sample solvents Page 4

HPLC Column Technical Support 800-7-9770 (phone: US & Canada) * 0-99-504 (phone) * * Select option 4, then option. 96-608-964 (fax) www.agilent.com/chem Page 4