Column choices feb2008

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Column choices feb2008

  1. 1. A Look at Column Choices Does one-size-fit-all? LC Columns and Consumables Ed Kim Application Engineer February 13, 2008
  2. 2. Seminar Outline• Silica Surface Chemistry & Physical Characteristics• Silica Bonding Procedures – Special Column Chemistries• Column Choices Group/Presentation Title Agilent Restricted Month ##, 200X
  3. 3. Silica Column CharacteristicsSuface Chemistry Surface AreaSilanols Bonding Pore Particle Size Distribution Size Distribution NOTE: SILICA IS STABLE AT 1.0 > pH < 11.0* * range depends on manufacturer’s bonding Group/Presentation Title Agilent Restricted Month ##, 200X
  4. 4. ZORBAX Porous Silica Particles Particle Size Silica Sol 1.8 um + Δ - or -Urea (pH-2) 3.5 µm + - or - O2 5 µm CH2O - or - 7 µm ZORBAX Rx Pore 80Å, 95Å, or 300Å Group/Presentation Title 5 Agilent Restricted Month ##, 200X
  5. 5. History of HPLC Particle DevelopmentYear(s) of Particle Size Most Popular Plates / 15cmAcceptance Nominal Size1950’s 100µm 2001967 57µm (pellicular) 1,0001972 10µm 6,0001985 5µm 12,0001992 3.5µm 22,0002003 ☻ <2µm* >30,000 *Zorbax "Rapid Resolution HT”, product launch 5/1/03 Group/Presentation Title Agilent Restricted Month ##, 200X
  6. 6. Columns Packed with Smaller Particles Provide Higher Efficiency Sub 2 micron 3 micron N 5 micron 10 micron FLOW Group/Presentation Title Agilent Restricted Month ##, 200X
  7. 7. Pore Size RecommendationsUse 60 - 80Å pore size column packings to separate smallmolecules equal to or less than 4000MW to maximizeloading capacity and retention.Use 95 or 300Å pore size columns for larger molecules likepolypeptides ad proteins (from 4000 to 500,000 MW) tomaintain high efficiency.Increase column diameter to increase loading capacity. Group/Presentation Title Agilent Restricted Month ##, 200X
  8. 8. Chemistry Variations Are All C-18s the Same?1. Type of bonding2. Completeness of bonding (Carbon Load)3. Silica Chemistry4. Bonding Chemistries • End-capping NO! Group/Presentation Title Agilent Restricted Month ##, 200X
  9. 9. Monomeric vs. Polymeric Bonding Highly reproducibleMonomeric bondingTypical ZORBAX Bonding•Eclipse Plus•Eclipse XDB•StableBond•Bonus-RP•Agilent HC/TCPolymeric BondingEclipse PAH Trifunctional silane figure courtesy of Vydac Group/Presentation Title Agilent Restricted Month ##, 200X
  10. 10. 2. Carbon Load Carbon Load refers to the % carbon content of the silica bonded stationary phase. Generally speaking, a high carbon load (example 18-25%) results in a more hydrophobic surface. The surface is also more resistant to high pH. A high carbon load does not necessarily provide the best resolution. Group/Presentation Title Agilent Restricted Month ##, 200X
  11. 11. 3. The Surface of Silica Supports OH HO OH OH OH Si Si Si Si Free Geminol Associated Silanols Silanols Silanols decreasing acidity OH + + M M Si Surface Metal Internal Metal (activated silanol) (most acidic) Group/Presentation Title Agilent Restricted Month ##, 200X
  12. 12. Chromatographic ImprovementCH2 -OH. Using Highly Purified Zorbax Rx-Sil Original ZORBAX, 1973 and other type A silicas (basic compound can tail) Conditions: Flow Rate: 2.0 mL / min. Mobile Phase: 5% 2-Propanol in Heptane CH2 -OH ZORBAX Rx-Sil, 1987 and other Type B silicas (basic compounds have less tailing; lower effective silanol pKa) Group/Presentation Title Agilent Restricted 9 Month ##, 200X
  13. 13. Potential Ion Exchange and Hydrogen BondingSecondary Interactions Ion-exchange _ _ SiO Na+ + R3 NH+ SiO N+R3 + Na+ 1. 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 -SiO . . . H + . . . OOCR 2. 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. Group/Presentation Title Agilent Restricted Month ##, 200X
  14. 14. Zorbax StableBond with Rx-SILImproves Peak Shape Mobile Phase: 75% 50 mM KH2PO4, pH 4.4 : 25% ACN Flow Rate: 1.5 mL/min Silica Type – More Acidic Silica Type – High Purity, Rx-Sil Column: ODS, 4.6 x 250 mm, 5 μm Column: SB-C18, 4.6 x 150 mm, 5 Plates: 92 μm USP Tf (5%): 2.90 Plates: 6371 USP Tf (5%): 1.09 Propranolol OH pKa 9.5 OCH CHCH NHCH(CH ) 2 2 3 2 0 5 10 15 0 5 10 Time (min) Time (min)• The high purity Rx-SIL improves the peak shape dramatically on a C18 column Group/Presentation Title Agilent Restricted Month ##, 200X
  15. 15. So What Bonded-Phase Do I Choose?*C18 offers the most retention, maybe too much for some samplesC8 less retentive than C18, but with similar selectivity in most casesPhenyl significantly less retentive for non-polar compounds but retains polar compounds, so reduces analysis time for mixture if polar/non polarCN least retentive, changes in selectivity, good for reducing analysis time with late eluters and avoiding gradient separations * Greater than 20% Organic Group/Presentation Title Agilent Restricted Month ##, 200X 001761S1.PPT
  16. 16. So What Bonded-Phases Do I Choose? High Aqueous* Mobile Phases C18 rarely offers retention advantages, and selectivity may not be optimum with little retention C8 often provides a little more retention than C18 with similar selectivity Phenyl most retentive with changes in selectivity C3 similar to Phenyl in retention but some changes in selectivity from C18, C8 and Phenyl CN least retentive, definite changes in selectivity, good resolution * Below 20% Organic Group/Presentation Title Agilent Restricted Month ##, 200X 001762S1.PPT
  17. 17. 4. ZORBAX Bonding TechnologyAll Purpose Rx-Silica ApplicationPhases Support Targeted Phases • StableBond • Bonus-RP • Eclipse XDB • Extend • Eclipse Plus • Ultra-pure • Spherical, consistent pore size • Fully-hydroxylated • Sol gel maximizes strength and lifetime Group/Presentation Title Agilent Restricted Month ##, 200X
  18. 18. Match Method pH and Column Choice Choose the Best Bonded-Phase for Each pH RangeStableBond, pH 1-6 Eclipse Plus & XDB, pH 2-9 Bonus-RP, pH 2-8 Extend-C18, pH 2-11.51. Uses bulky silanes 1. Proprietary double-endcapping 1. polar alkyl phase 1. unique bidentate structure2. Non-endcapped 2. triple endcapped 2. double endcapped 3. uses bulky silanes * R CH3 R1 O Si *R1 O Si C18 C18 O Si PG R CH3 Si Si R CH3 R1 O O OH O Si CH3 CH3 R CH3 Silica Support Si CH3 CH3 CH3 O Si O Si R1 R1 CH3 R CH3 O Si PG R O Si CH3 OH R1 CH3 R CH3 CH3 O Si Si CH3 O Si R1 CH3 CH3 R R1 O Si PG R R1 Group/Presentation Title Agilent Restricted 001241P2.PPT200X Month ##,
  19. 19. ZORBAX StableBond BondingSuperior low pH stability – down to pH 1 R O SiNon-endcapped for selectivity and Rlifetime OH RPatented sterically protecting bonding O Si R OH5 different selectivities - C18, C8, CN, RPhenyl, C3 O Si RIdeal for most sample types at low pH Group/Presentation Title Agilent Restricted Month ##, 200X
  20. 20. StableBond Reaction to Make aSterically-Protected Surface H3C H CH 3 H 3C H CH 3 C CSi OH + X Si R Si O Si R C C H 3C H CH 3 H 3C H CH 3 X = Cl, OEt , etc . R = CN, C8, etc . • Diisopropyl silanes or diisobutyl silanes (C18) Group/Presentation Title Agilent Restricted Month ##, 200X
  21. 21. Bonded Phase Selectivity Differences in 30% ACNmAU RRHT SB-CN 5 Different bonded phases100 4.6 x 50 mm, 1.8 μm compared 0 1 Analysis time of each run ismAU RRHT SB-Phenyl only 2 minutes100 4.6 x 50 mm, 1.8 μm Comparison done in 0 optimum % organic 1 2mAU RRHT SB-AQ The fast runs mean a100 4.6 x 50 mm, 1.8 μm comparison can be done even 0 if you have a good separationmAU 1 2 on the C18 RRHT Eclipse Plus C18 More chances to100 4.6 x 50 mm, 1.8 μm optimize! 0 1 2mAU RRHT SB-C18100 4.6 x 50 mm, 1.8 μm 0 1 2 Group/Presentation Title Agilent Restricted Month ##, 200X
  22. 22. Traditional Stationary Phase Bonding andEndcapping Reaction CH 3 CH 3 O Si R OH O Si R CH 3 CH 3 CH 3 OH OH OH CH 3 CH 3 + Cl Si R OH CH 3 + Cl Si CH 3 O Si CH 3 OH CH 3 CH 3 CH 3 OH O Si R CH 3 R = C8, C18, etc . (TMS) CH 3 O Si R • Dimethyl silanes • Endcapped with TMS Group/Presentation Title Agilent Restricted Month ##, 200X
  23. 23. ZORBAX Eclipse Plus & XDB ColumnsImproved peak shape for basic CH3compounds – especially at Si Ointermediate pH CH3 ca CH3 Improved e Sili O Si CH 3 doubleGood for all sample types – Bas roved CH 3 endcappingacid, base, neutral I mp CH3 O Si CH3Wide useable pH range, pH 2-9 CH3 O Si CH3Double end-capped CH 3 CH3Pore size: O Si CH3XDB 80Å, Plus 95Å Group/Presentation Title Agilent Restricted Month ##, 200X
  24. 24. Selectivity of Polar Phases Provides OptimumSeparation of Steroids Versus Non-Polar C18/C8 Column: ZORBAX Eclipse XDB-CN 1 2 Column Dimensions: 4.6 x 150 mm, 5 μm Mobile Phase: 35:65 ACN:Water Flow Rate: 2.000 ml/min 4 5 Injection Volume: 2.00 μl 3 6 7 Column Temperature: 25 °C Detector: UV, 210 nm0 5 10 15 20 min 4,5 Column: ZORBAX Eclipse XDB-Phenyl 1 Mobile Phase: 48:52 ACN:Water Sample: 2 1. Estriol (0.00130 μg/μl), 3 6 7 2. β-Estradiol (0.00130 μg/μl), 3. Ethinyl Estradiol (0.00147 μg/μl),0 5 10 15 20 4. Dienestrol (0.00123 μg/μl), 5. Diethylstilbestrol (0.00128 μg/μl) 1 2 5 Column: ZORBAX Eclipse XDB-C8 6. Ethynylestradiol 3-methyl ether (0.00103 4 Mobile Phase: 54:46 ACN:Water 3 μg/μl) 6 7 7. Ethynodiol Diacetate (0.00139 μg/μl)0 5 10 15 20 Eclipse XDB-CN is the 4 3 5 optimum bonded phase – 12 Column: ZORBAX Eclipse XDB-C18 Mobile Phase: 60:40 ACN:Water the only one that can 6 7 resolve all the components0 5 10 15 20 in under 20 minutes. Group/Presentation Title Agilent Restricted Month ##, 200X
  25. 25. ZORBAX Bonus-RP R1Good peak shape of basic O Si PG Rcompounds R1 CH3 Si CH3Polar alkyl-amide bonded phase CH3 R1Unique selectivity O Si PG R R1 CH3Enhanced low pH stability with Si CH3sterically protecting bonding CH3 R1 O Si PG RTriple endcapped R1 Group/Presentation Title Agilent Restricted Month ##, 200X
  26. 26. Improved Peak Shape of Basic Compounds Separation of Small Molecule Anorectics on ZORBAX Bonus-RP and Traditional Alkyl Phase Columns: 4.6 x 150 mm, 5 μm 1 2 Mobile Phase: 45% 25 mM K2HPO4, pH 7.2 : 55% (MeOH:ACN, 50:50) Flow Rate: 1.0 mL/min. Bonus-RP Detection: 254 nm Injection vol: 5 µL Sample: Anorectics (“Fen-phen”) 3 1. Phentermine pKa10.1 2. Fenfluramine pKa 9.1 3. Impurity 1 2 Alkyl C8 3 0 1 2 3 4 5 6 7 Time (min) Group/Presentation Title Agilent Restricted Month ##, 200X
  27. 27. ZORBAX Extend-C18Superior high pH stability – up to O Si C18pH 11.5 with silica particles O Si C18Excellent reproducibilityPatented bidentate, C18 bonding O Si C18Double endcapping O Si C18 Group/Presentation Title Agilent Restricted Month ##, 200X
  28. 28. Improved Retention of Basic Antihistamineson ZORBAX Extend-C18 pH 11 pH 7 30% 20 mM TEA 30% 20 mM Na2HPO4 70% MeOH 70% MeOH 7 7 4 4 Column: 4.6 x 150 mm, 5 μm Mobile Phase: See Above 2,3 Flow Rate: 1.0 mL/min 3 Temperature: RT 1 Detection: UV 254 nm 5 Sample: 1. Maleate 1 2. Pseudoephedrine 3. Scopolamine 5 4. Doxylamine 2 5. Chlorpheniramine 6 6 6. Triprolidine 7. Diphenhydramine0 5 10 0 5 Time (min) Time (min) Group/Presentation Title Agilent Restricted Month ##, 200X
  29. 29. HPLC ColumnsWithin the Column is where separation occurs.Proper choice of column is critical for success in HPLCColumn dimensions in HPLC:• Analytical [internal diameter (i.d.) 1.0 - 4.6-mm; lengths 15 – 250 mm]• Preparative (i.d. > 4.6 mm; lengths 50 – 250 mm)• Capillary (i.d. 0.1 – 0.5 mm; various lengths) LC Columns - analytical• Nano (i.d. < 0.1 mm, or sometimes stated as < 100 µm)Column Particle Sizes:• 7, 5, 3.5 (RR), & 1.8 um (RRHT)Materials of construction for the tubing• Stainless Steel (the most popular; gives high pressure capabilities)• Glass (mostly for biomolecules)• PEEK polymer (biocompatible and chemically inert to most solvents) Group/Presentation Title Agilent Restricted Month ##, 200X
  30. 30. Choose Column Configuration for Application Particle Column I.D. Lengths Flow Rate Sensitivity Sizes Applications Type (mm) (mm) Ranges Increase** (um) Proteomics Nano 0.1, 0.075 150* 3.5 100 – 600 nL/min 2000 LC/MS Peptide Mapping Capillary 0.3, 0.5 35 – 250 3.5, 5 1 – 10 μL/min 100 LC/MS High sensitivityMicroBore 1.0 30 – 150 3.5, 5 30 – 60 μL/min 20 LC/MS Sample limited,Narrow Bore 2.1 15 – 150 1.8, 3.5, 5 0.1 – 0.3 mL/min 5 LC/MS Solvent 3.0 150, 250 1.8, 3.5, 5 0.3 – 1.0 mL/min Analytical 2 Saver Analytical 4.6 15 – 250 1.8, 3.5, 5 1 – 4 mL/min Analytical 1 Small scale Semi-prep 9.4 50 – 250 5 4 – 10 mL/min -- Protein purification CombiChemPreparative 21.2 50 – 250 5, 7 20 – 60 mL/min -- purification ** Based on 4.6 mm id columns Group/Presentation Title Agilent Restricted Month ##, 200X
  31. 31. Resolving Power Column Resolving Resolving Resolving Typical Analysis Length Power Power Power Pressure Time* (mm) N(5 µm) N(3.5 µm) N(1.8 µm) Bar (1.8 µm) 150 12,500 21,000 32,500 N.A. Analysis 100 8,500 14,000 24,000 420 Time -33% 75 6000 10,500 17,000 320 -50% Peak 50 4,200 7,000 12,000 210 Volume -67% 30 N.A. 4,200 6,500 126 -80% Solvent Usage -90% 15 N.A. 2,100 2,500 55* Reduction in analysis time compared to 150 mm column• pressure determined with 60:40 MeOH/water, 1ml/min, 4.6mm ID Group/Presentation Title Agilent Restricted Month ##, 200X
  32. 32. Pick the Column andParticle Size to Meet Your Needs 1 2 Rs(1,2) = 4.8 3 4.6 x 250 mm, 5 μm 29.65 N=21848 4 N=22680 0 1 5 10 15 20 25 30 min 2 3 Rs(1,2) = 3.5 4.6 x 100 mm, 3.5 μm 12.71 4 N=11691 . 0 1 5 10 15 20 25 30 min 2 3 N=6568 4.6 x 30 mm, 1.8 μm 4.15 Rs(1,2) = 3.3 1 mL/min 4 N=6104 0 5 10 15 20 25 30 min 12 N=6463 3 Rs(1,2) = 3.1 4.6 x 30 mm, 1.8 μm 4 2.09 2 mL/min N=6460 0.5 1 1.5 2 2.5 0 5 10 15 20 25 30 minColumns: ZORBAX SB-C18 Mobile Phase: 50% 20 mM NaH2PO4, pH 2.8: 50% ACN Flow Rate: 1 mL/min Temperature: RT Detection: UV 230 nm Sample: 1. Estradiol 2. Ethynylestradiol 3. Dienestrol 4. Norethindrone Group/Presentation Title Agilent Restricted Month ##, 200X
  33. 33. Smaller Particles Maintain Efficiency Over Wider Flow Rate Ranges H = A + B/u + Cu 0.0030 Column: ZORBAX Eclipse XDB-C18 Dimensions: 4.6 x 50/30mm Eluent: 85:15 ACN:Water 0.0025 Flow Rates: 0.05 – 5.0 mL/min Temp: 20°C HETP (cm) 0.0020 Sample: 1.0μL Octanophenone in Eluent 5.0μm 0.0015 3.5μm 1.8μm 0.0010 0.0005 0.0000 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 Volumetric Flow Rate (mL/min)Smaller particle sizes yield flatter curves, minima shift to higher flow rates Group/Presentation Title Agilent Restricted Month ##, 200X
  34. 34. Different C18 Bonded Phases for Max Selectivity 1st choice 1 2 4 Eclipse Plus C18 Best Resolution & Peak Shape 3 Mobile phase: (69:31) ACN: water Flow 1.5 mL/min. 1 2 3 4 5 Temp: 30 °C2nd choice Detector: Single Quad ESIGood alternate selectivity due 1 2 4 StableBond positive mode scan 3 Columns: RRHTto non-endcapped SB-C18 4.6 x 50 mm 1.8 um 1 2 3 4 5 min Sample: 1. anandamide (AEA) 3rd choice 1 2 4 Eclipse 2. Palmitoylethanolamide (PEA) Good efficiency & peak shape 3 XDB-C18 3. 2-arachinoylglycerol (2-AG) Resolution could be achieved 4. Oleoylethanolamide (OEA)4th choice 1 2 3 4 5 Multiple bonded phasesResolution not likely, 1 4 Extend-C18 for most effective methodOther choices better, for this 2,3 development.separation. Match to one you are currently using. 1 2 3 4 5 Group/Presentation Title Agilent Restricted Month ##, 200X
  35. 35. # 1 – Analytical 4.6x250mm, 5.0µm PAH Column –Separation of 16 PAH’s in EPA 610mAU 2 1 1 = Toluene1400 Conditions: 2 = Naphthalene 3 = Acenaphthylene Det. 220,4nm No Ref.; 4 = Acenaphthene Flow 2.00 ml/min 5 = Fluorene Mobile Phase A = Water; B = Acetonitrile1200 6 = Phenanthrene Initial %B = 40 7 = Anthracene Gradient: Time (Min) %B 8 = Fluoranthene 0.00 45 9 = Pyrene 17.5 1001000 10 = Benzo(a)anthracene 24.0 100 11 = Chrysene 25.5 40 12 = Benzo(b)fluoranthene 27.5 40 800 13 = Benzo(k)fluoeanthene Stop Time = 25.0 14 = Benzo(a)pyrene Temp. = 25° C 15 = Dibenzo(a,h)anthracene 500 nanogm on Col for each Component Rs = 2.2 16 = Benzo(g,h,i)perylene 600 17 = indeno(1,2,3-c,d)pyrene 4 400 3 8 10 12 6 11 200 5 15 13 16 7 9 14 17 0 5 10 15 20 25 min Group/Presentation Title Agilent Restricted Month ##, 200X
  36. 36. # 4 – Rapid Resolution – 4.6x150mm, 3.5µm PAHColumn – Separation of 16 PAHs in EPA 610mAU 1 2 1 = Toluene Conditions: 2 = Naphthalene Det. 220,4nm No Ref.; Stop time = 20.0min 3 = Acenaphthylene Flow 2.00 ml/min1400 4 = Acenaphthene Mobile Phase A = Water; B = Acetonitrile 5 = Fluorene Gradient: Time (Min) %B 6 = Phenanthrene 0.00 401200 7 = Anthracene 1.13 40 8 = Fluoranthene 15.00 100 9 = Pyrene 18.5 100 10 = Benzo(a)anthracene 20.0 401000 11 = Chrysene 21.5 40 12 = Benzo(b)fluoranthene Stop Time = 23.5 13 = Benzo(k)fluoeanthene Temp. = 25° C800 Rs = 2.6 14 = Benzo(a)pyrene 500 nanogm on Col for each Component 15 = Dibenzo(a,h)anthracene 16 = Benzo(g,h,i)perylene 4 17 = indeno(1,2,3-c,d)pyrene600 3400 8 10 11 12 6 15 5 13 16200 7 9 14 17 0 2 4 6 8 10 12 14 16 18 min Group/Presentation Title Agilent Restricted Month ##, 200X
  37. 37. #6- Rapid Resolution Eclipse PAH 4.6x50mm,3.5µm Column – Separation of 16 PAHs in EPA 610mAU 1 1 = Toluene1750 2 2 = Naphthalene Conditions: 3 = Acenaphthylene Det. 220,4nm No Ref. 4 = Acenaphthene Flow 2.00 ml/min1500 5 = Fluorene Mobile Phase A = Water; B = Acetonitrile 6 = Phenanthrene Gradient: Time (Min) %B 7 = Anthracene 0.00 42 8 = Fluoranthene 0.33 421250 9 = Pyrene 10.00 100 10 = Benzo(a)anthracene 12.5 100 11 = Chrysene 13.5 42 12 = Benzo(b)fluoranthene 15 421000 13 = Benzo(k)fluoeanthene Stop Time = 15.1 14 = Benzo(a)pyrene Temp. = 25° C Rs = 2.0 15 = Dibenzo(a,h)anthracene 500 nanogm on Col for each Component 16 = Benzo(g,h,i)perylene 750 17 = indeno(1,2,3-c,d)pyrene 4 500 3 8 10 15 6 11 12 16 250 5 13 17 7 9 14 0 2 4 6 8 10 min Group/Presentation Title Agilent Restricted Month ##, 200X
  38. 38. #7- Rapid Resolution HT Eclipse PAH 4.6x100mm,1.8 µm Column - Separation of 16 PAHs in EPA 610 1 = ToluenemAU 2 = Naphthalene Conditions:100 3 = Acenaphthylene Agilent 1200SL 4 = Acenaphthene DAD 220,4nm No Ref. DAD 5 = Fluorene Flow 2.0 ml/min 6 = Phenanthrene Mobile Phase A = Water; B = Acetonitrile 7 = Anthracene Gradient: Time (Min) %B80 8 = Fluoranthene 0.00 40 9 = Pyrene 0.9 40 10 = Benzo(a)anthracene 12 100 11 = Chrysene 14.5 100 12 = Benzo(b)fluoranthene Rs = 3.63 15 4060 13 = Benzo(k)fluoeanthene Stop Time = 16 14 = Benzo(a)pyrene Temp. = 25° C 15 = Dibenzo(a,h)anthracene 50 nanogm on Col for each Component 16 = Benzo(g,h,i)perylene no Mixer & no Pulse Dampener 17 = indeno(1,2,3-c,d)pyrene4020 0 2 4 6 8 10 12 14 min Group/Presentation Title Agilent Restricted Month ##, 200X
  39. 39. # 8 – Rapid Resolution HT Eclipse PAH 4.6x50mm,1.8µm Column – Separation of 16 PAHs in EPA 610mAU 2 Conditions: 1 = Toluene Agilent 1200SL160 2 = Naphthalene DAD 220,4nm No Ref. DAD 1 3 = Acenaphthylene Stop Time = 5.60min 4 = Acenaphthene Flow 2.00 ml/min140 5 = Fluorene Mobile Phase A = Water; B = Acetonitrile 6 = Phenanthrene Gradient: Time (Min) %B 7 = Anthracene 0.00 45120 8 = Fluoranthene 3.5 100 9 = Pyrene 4.9 100 10 = Benzo(a)anthracene 5.2 45 Rs = 2.0 11 = Chrysene100 Stop Time = 5.6 12 = Benzo(b)fluoranthene Temp. = 25° C 13 = Benzo(k)fluoeanthene 50 nanogm on Col for each Component 4 14 = Benzo(a)pyrene no Mixer & no Pulse Dampener 80 15 = Dibenzo(a,h)anthracene 16 = Benzo(g,h,i)perylene 17 = indeno(1,2,3-c,d)pyrene 60 1 3 10 12 40 11 8 13 15 6 16 5 14 17 20 7 9 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 min Group/Presentation Title Agilent Restricted Month ##, 200X
  40. 40. #10 Solvent Saver Eclipse PAH 3.0x250 mm, 5 µmColumn - Separation of 16 PAHs in EPA 610 1 = Toluene 2 = Naphthalene Conditions:mAU 3 = Acenaphthylene Agilent 1200SL140 4 = Acenaphthene DAD 220,4nm No Ref. DAD 5 = Fluorene 6 = Phenanthrene Flow 0.85 ml/min120 7 = Anthracene Mobile Phase A = Water; B = Acetonitrile 8 = Fluoranthene Gradient: Time (Min) %B 9 = Pyrene 0.00 40 10 = Benzo(a)anthracene 2 40100 11 = Chrysene 17 100 12 = Benzo(b)fluoranthene 24 100 13 = Benzo(k)fluoeanthene 24.1 4080 14 = Benzo(a)pyrene Stop Time = 27 15 = Dibenzo(a,h)anthracene Temp. = 25° C 16 = Benzo(g,h,i)perylene 50 nanogm on Col for each Component 17 = indeno(1,2,3-c,d)pyrene no Mixer & no Pulse Dampener60 Rs = 2.154020 0 5 10 15 20 25 min
  41. 41. #11 Narrow Bore Eclipse PAH 2.1x250 mm, 5 µmColumn - Separation of 16 PAHs in EPA 610 1 = Toluene 2 = Naphthalene Conditions:mAU 3 = Acenaphthylene Agilent 1200SL 4 = Acenaphthene DAD 220,4nm No Ref. DAD 5 = Fluorene Flow 0.417 ml/min 6 = Phenanthrene Mobile Phase A = Water; B = Acetonitrile 7 = Anthracene Gradient: Time (Min) %B200 8 = Fluoranthene 0.00 40 9 = Pyrene 2 40 10 = Benzo(a)anthracene 17 100 11 = Chrysene 24 100 12 = Benzo(b)fluoranthene 24.1 40150 13 = Benzo(k)fluoeanthene Stop Time = 27 14 = Benzo(a)pyrene Temp. = 25° C 15 = Dibenzo(a,h)anthracene 50 nanogm on Col for each Component 16 = Benzo(g,h,i)perylene no Mixer & no Pulse Dampener 17 = indeno(1,2,3-c,d)pyrene100 Rs = 2.0050 0 5 10 15 20 25 min
  42. 42. #13 Narrow Bore Rapid Resolution Eclipse PAH2.1x100mm, 3.5 µm Column - Separation of 16 PAHs in EPA 610 1 = ToluenemAU 2 = Naphthalene Conditions: 3 = Acenaphthylene Agilent 1200SL300 4 = Acenaphthene DAD 220,4nm No Ref. DAD 5 = Fluorene Flow 0.417 ml/min 6 = Phenanthrene Mobile Phase A = Water; B = Acetonitrile 7 = Anthracene Gradient: Time (Min) %B250 8 = Fluoranthene 0.00 40 9 = Pyrene 0.9 40 10 = Benzo(a)anthracene 12 100 11 = Chrysene 14.5 100200 12 = Benzo(b)fluoranthene 15 40 13 = Benzo(k)fluoeanthene Stop Time = 16 14 = Benzo(a)pyrene Temp. = 25° C 15 = Dibenzo(a,h)anthracene 50 nanogm on Col for each Component150 16 = Benzo(g,h,i)perylene no Mixer & no Pulse Dampener 17 = indeno(1,2,3-c,d)pyrene100 Rs = 2.5250 0 2 4 6 8 10 12 14 min Group/Presentation Title Agilent Restricted Month ##, 200X
  43. 43. #14 – Narrow Bore RRHT Eclipse PAH 2.1x100mm1.8 µm – Separation of 16 PAHs in EPA 610 1 = ToluenemAU Conditions: 2 = Naphthalene Agilent 1200SL 3 = Acenaphthylene DAD 220,4nm No Ref. DAD 4 = Acenaphthene Flow 0.417 ml/min175 5 = Fluorene Mobile Phase A = Water; B = Acetonitrile 6 = Phenanthrene Gradient: Time (Min) %B 7 = Anthracene 0.00 40150 8 = Fluoranthene 0.9 40 9 = Pyrene 12 100 10 = Benzo(a)anthracene 14.5 100 11 = Chrysene125 15 40 12 = Benzo(b)fluoranthene Rs = 3.61 Stop Time = 16 13 = Benzo(k)fluoeanthene Temp. = 25° C 14 = Benzo(a)pyrene100 50 nanogm on Col for each Component 15 = Dibenzo(a,h)anthracene no Mixer & no Pulse Dampener 16 = Benzo(g,h,i)perylene 17 = indeno(1,2,3-c,d)pyrene7550 toluene25 0 2 4 6 8 10 12 14 min
  44. 44. #15 – Narrow Bore RRHT Eclipse PAH 2.1x50mm,1.8µm PAH Column - Separation of 16 PAHs in EPA 610 1 = ToluenemAU 2 = Naphthalene Conditions:350 3 = Acenaphthylene Agilent 1200SL 4 = Acenaphthene DAD 220,4nm No Ref. DAD 5 = Fluorene Stop Time = 7.0min 6 = Phenanthrene Flow 0.417 ml/min300 7 = Anthracene Mobile Phase A = Water; B = Acetonitrile 8 = Fluoranthene Gradient: Time (Min) %B 9 = Pyrene 0.00 45 10 = Benzo(a)anthracene 3.5 100250 11 = Chrysene 4.9 100 12 = Benzo(b)fluoranthene 5.2 45 13 = Benzo(k)fluoeanthene Stop Time = 7200 14 = Benzo(a)pyrene Temp. = 25° C 15 = Dibenzo(a,h)anthracene 50 nanogm on Col for each Component 16 = Benzo(g,h,i)perylene no Mixer & no Pulse Dampener 17 = indeno(1,2,3-c,d)pyrene150 Rs = 2.16100 50 0 1 2 3 4 5 6 min Group/Presentation Title Agilent Restricted Month ##, 200X
  45. 45. How To “Match” a Column to a ZORBAX RRHTColumn General Phase Type Starting ZORBAX Choice Typical “endcapped” C18 or C8 Eclipse Plus C18 or C8 bonded phases, newer columns Endcapped C18 or C8 columns, Eclipse XDB-C18 or C8 older generation Non-endcapped columns StableBond C18 Older types of columns StableBond C18, C8 etc. Aqueous “type” columns SB-AQ CN or Phenyl SB-CN, SB-Phenyl Group/Presentation Title Agilent Restricted Month ##, 200X
  46. 46. Conclusion There is no one-size-fits all columnChoose the best for your application needs Group/Presentation Title Agilent Restricted Month ##, 200X
  47. 47. Agilent LC Columns and Agilent J&W GCColumns Scientific Technical Support • 800-227-9770 (phone: US & Canada)* • 302-993-5304 (phone) • For LC columns • Select option 4, then option 2 • For GC Columns • * Select option 4, then option 1. • www.agilent.com/chem
  48. 48. Looking for More Information on Agilent’s LC Systems andSoftware? Agilent offers a full range of LC training courses including hands-on courses with the latest 1200 series equipment including Rapid Resolution, and additional 1100 series courses. Each course includes a course manual for future reference and a certificate of completion. All courses are taught by industry experts. Call 800.227.9770, Option 5 or visit www.agilent.com/chem/education to register today!
  49. 49. New On Demand Webinar:Utilizing Sub-Two Micron Particles to Optimize HPLC Methods A Four Part Workshop on Managing Chemistry and Pressure for Faster and More Efficient HPLC Separations www.SeparationsNOW.com/agilentwebinar Be sure to register after today’s event.Our measure is your success.
  50. 50. Upcoming LC and GC e-Seminars Introduction to Capillary GC February 20, 2008 – 1:00 p.m. EST Selection of a Capillary GC Column March 13, 2008 – 2:00 p.m. EST Method Development March 18, 2008 – 2:00 p.m. EST

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