Does one-size-fit-all?

A Look at Column Choices: Does one-size-fit-all? Bill Champion Agilent Technologies, Inc. 800-227-9770 opt 3/opt3/opt 2 william_champion@agilent.com Page 1

Separation Techniques Hydro - philic Volatile Carboxylic acids Sulfonamides Aldehydes Ketones Glyphosate Glycols Amino acids Synthetic food dyes PG, OG, DG phenols Enzymes Aflatoxins Sugars alcohols Inorganic ions Sugars Polarity Nitrosamine TMS derivatives of sugar Epoxides Nitriles Organophosphorus pesticides BHT, BHA, THBQ antioxidents PCBs Alcohol Aromatic amines Fatty acids Antibiotics Flavonoids PAHs Anabolica Natural food dyes Fat soluble vitamins i Essential Oils Polymer monomers Triglycerides Phospholipids Hydrophobic C2-C6 hydrocarbons Fatty acid methylesters Volatile Gas Phase Aromatic esters Nonvolatile - Liquid Phase Volatile Volatility Nonvolatile Page 2

A Look at Column Choices: Rationale for column selection Page 3

Presentation Outline Common terms & definitions Overview of Major HPLC Modes Reversed-Phase Chromatography Column Dimensions - Length, Particle Size Page 4

Presentation Objectives Understand effect of roles of Selectivity and Column Efficiency i in improving i Resolution Column Selection Effect of Small Particles - Cost and Benefit Page 5

Mechanism of Chromatographic Separation Analyte partitions between stationary phase and mobile phase, Selectivity is obtained by difference in physical properties of the analytes Page 6

Some Basic Chromatography Parameters Retention Factor (k), Capacity Factor (k ) Selectivity or Separation Factor (α) Column Efficiency as Theoretical Plates (N) Resolution (R s ) Page 7

Definition of Resolution t = R-2 -t R R-1 = t R s (w 2 + w )/2 1 w Resolution is a measure of the ability to separate two components Page 8

Resolution Determined by 3 Key Parameters Efficiency, Selectivity and Retention The Fundamental Resolution Equation N = Column Efficiency Column length and particle size α = Selectivity Mobile phase and stationary phase, α = k 2/ k 1 k = Retention Factor Mobile phase and stationary phase, k = (t R t 0 )/ t 0 Page 9

Chromatographic Terms Resolution The distance between two neighboring peaks R = 1.5 is baseline resolution R = 2 is highly desirable during method development R s = (t Resolution is improved by increasing R2 t R1 ) difference in t R increasing α Resolution is improved by reducing peak (w 1 + w 2 )/2 width - increasing N Original t 0 > N value < k values > k values >α value Page 10

Presentation Outline Common terms & definitions Overview of Major HPLC Modes Reversed-Phase Chromatography Column Dimensions - Length, Particle Size Page 11

ZORBAX Porous Silica Particles Particle Size Silica Sol 1.8 µm + -or- Δ Urea (ph-2) 3.5 µm + -or- O CH 5 µm O 2 2 -or- 7 µm Pore 80Å, 95Å, or 300Å ZORBAX Rx Page 12

Reversed Phase Bonding Monomeric Si-OH + Cl-Si-(CH 3 3) 2 -R Si-O-Si-(CH 3 3) 2 -R Si-OH + Cl 3 -Si-R Si-OH Si-O Si-O Si Cl R Polymeric Si-OH Si-O O-CH 2 -CH 3 + (CH 3 -CH 2 -O) 3 -Si-R Si Si-OH Si-O R Page 13

Major Separation Modes of HPLC There are four major separation modes that are used dto separate most compounds: Reversed-phase (RPLC) chromatography (most popular) Normal-phase (NPLC) and adsorption chromatography Ion exchange chromatography (IEX) Size exclusion chromatography (SEC, GPC).Let s look briefly at each mode Page 14

Normal Phase or Adsorption Chromatography Key Points Column packing is polar silica (strongest)>amino>diol>cyano (weakest) Mobile phase is non-polar hexane, iso-octane, methylene chloride, ethyl acetate, etc. Polar compounds more retained Retention decreases as polarity of mobile phase increases Reasons to choose normal phase Analyze very hydrophobic (or lipophilic) compounds. Isomer separation. Sample injection solvent is non-polar. Recovery in non-polar solvents is desirable. Separation of Nitroanilines on HPLC Column packed with silica gel using hexane (mobile phase component A) mixed with methylene chloride (mobile phase component B) Page 15

Hydrophilic Interaction Chromatography (HILIC) Page 16

Why HILIC Is Used Retention of polar components Similar to NPLC but with aq mobile phase (General reversal of elution from RPLC) Variety of different stationary phases available MS compatible May simplify sample prepp Page 17

Typical Mobile Phase Water (at least 2-3%, ~ 25%)/ACN Buffer (e.g., AmOAc) ph control, if necessary Page 18

Typical Stationary Phases - Polar Silica Amine Diol Amide Page 19

Typical Elution Order for Test Compounds on HILIC Column RPLC Column Test Mixture mau 4-chloronitrobenzene 40 Column 4.6 x 150 mm Mobile phase 75% ACN: 25% H2O 35 Flow Rate 0.5 ml/ml Phenol Col Temp 25 C 30 Injection Volume 5 µl 25 20 15 10 Naphthalene Uracil 1. Notice Uracil normally unretained in RPLC retained ed in HILIC 5 0 1 2 3 4 5 6 7 8 9 min 2. Notice completely different retention order than in RPLC 20

Ion Exchange Chromatography In ion exchange: Column packing contains ionic groups, (e.g. sulfonate, tetraalkylammonium) t l Mobile phase is an aqueous buffer (e.g. phosphate, formate, etc.) Used infrequently Similarities to ion-pair chromatography Well suited to the separation of inorganic and organic anions and cations in aqueous solution. Basic proteins on strong cation exchanger (-SO 3- ) 1. RNA polymerase 2. Chymotrypsinogen 3. Lysozyme Page 21

Ion-pair Chromatography Allows Retention of Ionized Compounds under RPLC Conditions polar non-polar - COO COO -+ IP---------- Amphiphilic compound is added to mobile phase - Quaternary amines for anions - Sulfonates for cations Electrostatic interaction with oppositely charged analyte to increase retention of analyte in RPLC Page 22

Advantages Separation of very polar compounds in RPLC Some variables can be varied continuously (ph, [buffer], [IP], %B, Temp) Allows mixed mode retention - Separate ionic compounds (IEX) - Separate non-ionized compounds (RPLC) Page 23

Disadvantages Higher level of complexity Larger region of sub-optimal performance Many IP reagents are non-volatile, need volatile IP reagents for MS Slow equilibrium (too slow for gradients) Difficult to wash from column Need to have high purity IP reagents Poor reproducibility Page 24

Comparison Separation of Catecholamines: Reversed-Phase and Ion-Pair HPLC amine V 0 acid amine V 0 amine acid Note that t amines (cations) are more retained and acids (anions) are less retained with anionic ion-pairing agent. Page 25

Size Exclusion Chromatography (SEC) There are two modes: non-aqueous SEC [sometimes termed Gel Permeation Chromatography (GPC)] aqueous SEC [sometimes referred to as Gel Filtration Chromatography (GFC)] No interaction ti between the sample compounds and packing material Molecules diffuse into pores of a porous medium Molecules are separated depending on their size relative to the pore size molecules larger than the pore opening do not diffuse into the particles while molecules smaller than the pore opening enter the particle and are separated large molecule elute first, smaller molecules elute later The mobile phase is chosen mainly to dissolve the analyte Used mainly for polymer characterization ti and for proteins. Gel Permeation Chromatogram of Polybutadiene polymer on non- aqueous SEC (GPC) column; The monomers elute after the polymer. Column: PLgel mixed-d gel Mobile phase: Tetrahydrofuran (THF) Page 26

Mechanism of SEC A Molecules (A,B) Enter Pores B Full entry Partial entry -SiOH + RCOO _ Molecules must freely enter and exit pores to be separated. Largest molecules elute first, followed by intermediate size molecules and finally the smallest molecules elute last. C -SiO _... H +... _ OOCR Packing Pore Molecule (C) Will be Excluded from Pores B C A Signal Time Page 27

Presentation Outline Common terms & definitions Overview of Major HPLC Modes Reversed-Phase Chromatography Column Dimensions - Length, Particle Size Page 28

Reversed-Phase Chromatography (RPLC) Principle: Partition of analytes between polar mobile phase and non-polar stationary phase Nonpolar (nonspecific) interactions of analyte with hydrophobic (or lipophilic) stationary phase: C18, C8, Phenyl, C3, etc. Different sorption affinities between analytes results in their separation More polar analytes are less retained Analytes with larger hydrophobic part are retained longer Mobile phase: water (buffer) + water-miscible organic solvent e.g. MeOH, ACN Can be used for non-polar, polar, ionizable and ionic molecules Gradient elution is often used - re-equilibration times are acceptable Page 29

Column Selection Strategy Purpose of Analysis Chemistry of Analytes Sample Matrix Detection Page 30

So What Bonded-Phase Do I Choose? Lipophilic Compound (e.g., > 20% Organic in Mobile Phase) C18 C8 Phenyl CN offers the most retention, maybe too much for some samples less retentive than C18, but with similar selectivity in most cases significantly less retentive for non-polar compounds but retains polar compounds, so reduces analysis time for mixture if polar/non polar least retentive, changes in selectivity, good for reducing analysis time with late eluters and avoiding gradient separations Page 31

So What Bonded-Phase Do I Choose? Hydrophilic Compound (e.g., < 20% Organic in Mobile Phase) C18 C8 Phenyl C3 CN may not have sufficient retention, lower limit to %B in mobile phase often provides a little more retention than C18 with similar selectivity most retentive with changes in selectivity similar to Phenyl in retention but some changes in selectivity from C18, C8 and Phenyl least retentive, definite changes in selectivity, good resolution Page 32

Match Method ph and Column Choice Choose the Best Bonded-Phase for Each ph Range StableBond, ph 1-6 1. Uses bulky silanes 2. Non-endcapped Eclipse Plus & XDB, ph 2-9 1. Proprietary double-endcapping Bonus-RP, ph 2-8 1. polar alkyl phase 2. triple endcapped 3. uses bulky silanes Extend-C18, ph 2-11.5 1. unique bidentate structure 2. double endcapped * R O Si R OH R O Si R OH R O Si * R1 R1 R1 O O O O O CH3 Si CH3 CH3 Si CH3 CH3 Si CH3 CH3 Si CH3 CH3 Si CH3 CH3 CH3 O CH3 Si CH3 O CH3 Si CH3 R 1 Si R 1 CH3 R 1 Si R 1 CH3 PG PG R R C18 Si O C18 Si O Silica Support R R 1 O Si PG R R 1 Page 33

1 st choice Best Resolution & Peak Shape 2 nd choice Different ZORBAX C18 Bonded Phases for Different Selectivity/Applications Good alternate selectivity due to non-endcapped 1 2 1 2 3 4 5 3 1 2 3 4 4 Eclipse Plus C18 StableBond SB-C18 Mobile phase: (69:31) ACN: water Flow 1.5 ml/min. Temp: 30 C Detector: Single Quad ESI positive mode scan Columns: RRHT 4.6 x 50 mm 1.8 um 1 2 3 4 5 3 rd choice 1 2 4 Eclipse XDB-C18 Good efficiency & peak shape Resolution could be achieved 4 th choice Resolution not likely, Other choices better, for this separation. 1 2 3 4 5 1 3 2,3 4 min Extend-C18 1 2 3 4 5 Sample: 1. anandamide (AEA) 2. Palmitoylethanolamide l th l id (PEA) 3. 2-arachinoylglycerol (2-AG) 4. Oleoylethanolamide (OEA) Multiple bonded phases for most effective method development. Match to one you re currently using. Page 34

ZORBAX StableBond Bonding Superior low ph stability down to ph 1 Non-endcapped for selectivity and lifetime Patented sterically protected bonding 5 different selectivities - C18, C8, CN, Phenyl, C3 Ideal for most sample types at low ph O OH O OH O R Si R R Si R R Si R Page 35

300Å Pore size Page 36

Traditional Stationary Phase Bonding and Endcapping Reaction OH OH OH OH CH 3 + Cl O Si R CH 3 CH 3 OH CH 3 Si R OH CH 3 + Cl Si CH 3 CH 3 CH 3 O Si R R = C8, C18, C8,etc. CH 3 (TMS) CH 3 O OH Si CH 3 CH 3 R O Si CH 3 CH 3 CH 3 O Si R Dimethyl silanes Endcapped with TMS Page 37

ZORBAX Eclipse Plus & XDB Columns Improved peak shape for basic compounds especially at intermediate ph Good for all sample types acid, base, neutral Wide useable ph range, ph 2-9 Double end-capped Pore size: O XDB 80Å, Plus 95Å O O O O CH Si 3 CH3 CH Si CH CH Si 3 CH 3 CH Si CH CH Si CH Improved 3 CH3 double 3 endcapping 3 3 3 3 CH 3 Page 38

ZORBAX Bonus-RP Good peak shape of basic compounds O R 1 Si R 1 PG R CH 3 Polar alkyl-amide bonded phase Si CH 3 CH 3 R 1 Unique selectivity O Si R 1 PG R Enhanced low ph stability with sterically protecting bonding CH 3 Si CH 3 CH 3 R 1 Triple endcapped O Si R 1 PG R Page 39

Improved Peak Shape of Basic Compounds Separation of Small Molecule Anorectics on ZORBAX Bonus-RP and Traditional Alkyl Phase Bonus-RP 1 2 Columns: 4.6 x 150 mm, 5 μm Mobile Phase: 45% 25 mm K 2 HPO 4, ph 7.2 : 55% (MeOH:ACN, 50:50) Flow Rate: 1.0 ml/min. Detection: 254 nm Injection vol: 5 µl Sample: Anorectics ( Fen-phen ) 3 1. Phentermine pka10.1 2. Fenfluramine pka 9.1 3. Impurity 1 Alkyl C8 2 3 0 1 2 3 4 5 6 7 Time (min) Page 40

ZORBAX Extend-C18 Superior high ph stability up to ph 11.5 with silica particles O Si C18 Excellent reproducibility O Si C18 Patented bidentate, C18 bonding O Si C18 Double endcapping O Si C18 Page 41

Improved Retention of Basic Antihistamines on ZORBAX Extend-C18 ph 11 30% 20 mm TEA 70% MeOH ph 7 30% 20 mm Na 2 HPO 4 70% MeOH 1 2 3 4 5 6 7 Column: 4.6 x 150 mm, 5 μm Mobile Phase: See Above Flow Rate: 1.0 ml/min Temperature: RT Detection: UV 254 nm Sample: 1. Maleate 2. Pseudoephedrine 3. Scopolamine 4. Doxylamine 5. Chlorpheniramine 6. Triprolidine 7. Diphenhydramine 1 2,3 4 5 6 7 0 5 10 Time (min) 0 5 Time (min) Page 42

Column Summary Increase retention CN > C8 > C18 Phenyl, C3, Bonus RP, SB-Aq can handle high water level SB not end-capped, ph 1-8 XDB, Eclipse Plus end-capped, ph 2-9 Extend end-capped, ph 2-11.5 Silanols can interact with amines Silanols are less active in higher purity silica End capping blocks interaction with silanols Page 43

Presentation Outline Common terms & definitions Overview of Major HPLC Modes Reversed-Phase Chromatography Column Dimensions - Length, Particle Size Page 44

Resolution as a Function of Selectivity, Column Efficiency, or Retention 7.00 5.00 Selectivity Affects Resolution Most 6.00 Change bonded phase Change mobile phase α Resolution 4.00 3.00 R = ½ s N /4 (α-1)/α k/(k+1) 2.00 1.00 Increase N Increase Alpha Increase k' k N 0.00 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Plates: 5000 10000 15000 20000 25000 Alpha: 1.10 1.35 1.60 1.85 2.1 k: 2.0 4.5 7.0 9.5 12.0 Page 45

If α Has the Most Effect, Why Focus on N? It s Easy to Predict High plate number (N) provides: Sharp and narrow peaks Better detection Peak capacity to resolve complex samples But Resolution increases only with the square root of the plate number. Plate number increase is limited by experimental conditions (analysis time,..pressure) Selectivity (α) helps best but Is difficult to predict, so method development is slower (experience helps, model retention) Note: Software supported, optimization for separation of multi-component mixtures can reduce method development time (ChromSword, DryLab) Page 46

Peak Width Decreases with Increasing N Column Efficiency 5um N = 5000 theoretical plates 3.5um N = 10,000 theoretical plates 18um 1.8um N = 20,000 theoretical plates 1 3 5 7 9 11 13 15 Time (min) Page 47

Decreasing Particle Size 5 μm 8 min, 170 bar 35μm 3.5 6 min, 204 bar 1.8 μm 3.4 min, 300 bar Page 48

Putting it Together The van Deemter Equation Plate Height H H min H = L/N H = A + B/u + C u Longitudinal diffusion Large Particle Small Particle Sum Curve: van-deemter Resistance to Mass Transfer Eddy Diffusion u opt Linear Velocity u The smaller the plate height, the higher the plate number and the greater the chromatographic resolution Page 49

Columns Packed with Smaller Particles Provide Higher Efficiency sub 2 micron N α 1/(d 3.5 micron p ) N 5 micron P α 1/(d p ) 2 10 micron Velocity Page 50

Column Dimensions Trade-Off s Smaller particles, proportional increase in efficiency (N) R s increases as square root of N Smaller particles, pressure increase as (1/d p ) 2 Longer columns (L), proportion increase in: - Analysis time - Solvent usage - N - Pressure To decrease peak width Increase N, Decrease L Smaller particles come with price - Increase pressure - Less forgiving Page 51

Summary HPLC is a powerful analytical tool There are a variety of separation modes Most applications are RP Choice of stationary phase can affect separation by changing g selectivity (α) Efficiency (N) can be improved in predictable, brute force way: - Increase column length, - Decrease particle size Smaller particles come with price - Increase pressure - Less forgiving Page 52

THANK YOU! william_champion@agilent.com 1-800-227-9770, opt 3, opt 3, opt 2 Page 53

Chromatographic Profile Equations Describing Factors Controlling R S Retention Factor k = (t R-t 0 ) t 0 Selectivity α = k 2 /k 1 Theoretical Plates-Efficiency N = 16(t R / t W ) 2 = 5.54(t R / W 1/2 ) 2 Page 54

Resolution as a Function of Selectivity, Column Efficiency, or Retention 7.00 5.00 Selectivity Affects Resolution Most 6.00 Change bonded phase Change mobile phase α Resolution 4.00 3.00 R = ½ s N /4 (α-1)/α k/(k+1) 2.00 1.00 Increase N Increase Alpha Increase k' k N 0.00 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Plates: 5000 10000 15000 20000 25000 Alpha: 1.10 1.35 1.60 1.85 2.1 k: 2.0 4.5 7.0 9.5 12.0 Page 55

ZORBAX Porous Silica Particles Particle Size Silica Sol 1.8 μm + -or- Δ Urea (ph-2) 3.5 µm + -or- O CH 5 µm O 2 2 -or- 7 µm Pore 8nm nm, 95nm 9.5 nm, or30nm ZORBAX Rx Page 56

ZORBAX Porous Silica Particles Particle Size Silica Sol 1.8 μm + -or- Δ Urea (ph-2) 3.5 µm + -or- O CH 5 µm O 2 2 -or- 7 µm Pore 0.008008 μm, 0.00950095 μm, or 0.030030 μm ZORBAX Rx Page 57

SEC Calibration Page 58

Column Selection Strategy Purpose of Analysis Prep good loading Low Level sharp (tall) peaks Impurity Profile selectivity Assay Main Component selectivity Reaction Monitoring fast Chemistry of Analytes Sample Matrix Detection Page 59

Column Selection Strategy Purpose of Analysis Chemistry of Analytes Acid low ph Bases (amines) low ph, high ph Protein Non-polar - Lipophilic High Polarity - Hydrophilic Sample Matrix Detection Page 60

Column Selection Strategy Purpose of Analysis Chemistry of Analytes Sample Matrix Aqueous Water immiscible Biological Reaction Mixture Detection Page 61

Column Selection Strategy Purpose of Analysis Chemistry of Analytes Sample Matrix Detection UV-Vis, Fluorescence MS, MS/MS RI ELSD Page 62

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Selectivity of Polar Phases Provides Optimum Separation of Steroids Versus Non-Polar C18/C8 1 2 3 Column: ZORBAX Eclipse XDB-CN Mobile Phase: 35:65 ACN:Water 4 5 6 7 0 5 10 15 20 min 45 4,5 Column: ZORBAX Eclipse XDB-Phenyl 1 2 Mobile Phase: 48:52 ACN:Water 3 6 7 0 5 10 15 20 1 5 Column: ZORBAX Eclipse XDB-C8 2 3 4 Mobile Phase: 54:46 ACN:Water 6 7 Column Dimensions: 4.6 x 150 mm, 5 μm Flow Rate: 2.0 ml/min Injection Volume: 2.0 μl Column Temperature: 25 C Detector: UV, 210 nm Sample: 1. Estriol (0.00130 μg/μl), 2. β-estradiol (0.00130 μg/μl), 3. Ethinyl Estradiol (0.00147 μg/μl), 4. Dienestrol (0.00123 μg/μl), 5. Diethylstilbestrol (0.00128 μg/μl) 6. Ethynylestradiol 3-methyl ether (0.00103 μg/μl) 7. Ethynodiol Diacetate (0.00139 μg/μl) 0 5 10 15 20 4 3 5 1 Column: ZORBAX Eclipse XDB-C18 2 Mobile Phase: 60:40 ACN:Water 6 7 0 5 10 15 20 Page 64