Welcome to our E-Seminar: Choosing HPLC Columns for Faster Analysis Smaller and Faster

Transcription

1 Welcome to our E-Sear: Choosing HPLC Columns for Faster Analysis Smaller and Faster

2 High Throughput/Fast LC Requires. Short columns 0 mm or shorter Small particle sizes. µm Rapid Resolution or new.8 µm Rapid Resolution HT for maximum resolution rather than traditional µm or 0 µm particle size columns Optimized HPLC equipment to maintain resolution and eliate band broadening due to extra column volume And can be used for Faster method development Faster analysis Solvent savings Slide

3 Zorbax High Throughput and High Resolution HPLC with Rapid Resolution and Rapid Resolution HT Columns Rapid Resolution Columns. µm particle size 0 mm, 00 mm, 7 mm, 0 mm, 0 mm, mm column lengths Lower efficiency than Rapid Resolution HT Lower pressure than Rapid Resolution HT Rapid Resolution HT Columns.8 µm particle size 0 mm, 0 mm and mm column lengths 7 00% greater efficiency than Rapid Resolution Higher pressure than Rapid Resolution Slide

4 Rapid Resolution Chromatography Increases Sample Throughput for Isocratic or Gradient Elution Decrease Column Length proportional decrease in analysis time (time~length, L) proportional decrease in solvent usage (~L) decreases) N, plates~ L and thus N Decrease in Particle Size = maintain resolution, R increases N ~ /d p.8 and thus N increases R ~ N / P, pressure ~ / d p ; P Slide

5 ZORBAX Rapid Resolution and Rapid Resolution HT Column Lengths and Typical Efficiencies High Resolution High Throughput Resolution a N / Column Resolving Power Resolving Power Length N (. µm )* N (.8 µm) { 0,000 N.A. 00,000 N.A. { 7 0,00 N.A , Efficiency (N) Pressure Analysis Time Peak Volume * µm HPLC columns of the same length have 0% fewer plates (N-value);.6-mm i.d. N.A. Not currently available Slide

6 ZORBAX Rapid Resolution and Rapid Resolution HT Column Lengths Column Length (mm) Resolving Power N(. µm) Resolving Power N(.8 µm) Analysis Time* High Resolution 0 00,000,000 N.A. N.A. Efficiency (N) Analysis Time - -% 7 0 0,00 7,000 N.A.,00 Pressure Peak Volume -0% -67% High Throughput 0,00,00 6,00,00 Solvent Usage -80% -90% * Reduction in analysis time compared to 0mm column Slide 6

7 Reduce Analysis Time With Rapid Resolution Columns (.- µm) N= 0,.6 x 0 mm, µm N= 0, N=,.6 x 0 mm, µm N=, Conditions: Columns: ZORBAX SB-C8 Mobile Phase: A: 0% 0 mm NaH PO, ph.8 B: 0% ACN Flow Rate: ml/ Temperature: C Sample:. Estradiol. Ethynylestradiol. Dienestrol. Norethindrone N=,68.6 x 00 mm,. µm N=, N= 0,8 N= 0,8.6 x 7 mm,. µm N= 6,07.6 x 0 mm,. µm N= 6, Time () Slide 7

8 Reduce Analysis Time Even Further by using Rapid Resolution HT (.8- µm) Columns Rs(,) =.8 N=88.6 x 0 mm, µm 9.6 N= Rs(,) =..6 x 00 mm,. µm.7 N= N=668. Rs(,) =. N=60.6 x 0 mm,.8 µm ml/ N=66.09 N=660 Rs(,) = x 0 mm,.8 µm ml/ Columns: ZORBAX SB-C8 Mobile Phase: 0% 0 mm NaH PO, ph.8: 0% ACN Flow Rate: ml/ Temperature: RT Detection: UV 0 nm Sample:. Estradiol. Ethynylestradiol. Dienestrol. Norethindrone Slide 8

9 Reduce Analysis Time by 80% Using Rapid Resolution HT Columns.6 x 0 mm, µm 6 7 A B C D E Rapid Resolution.6 x 7 mm,.µm Rapid Resolution.6 x 0 mm,.µm Rapid Resolution HT.6 x 0 mm,.8µm Rapid Resolution.6 x 0 mm,.µm Columns: ZORBAX Eclipse XDB-C8 Mobile Phase: 7% MeOH, 7% 0mM Phosphate Buffer, ph 7.0 Part Numbers: A) Flow Rate: ml/ B) Temperature: Ambient C) Detection: UV nm D) Sample: ) uracil, ) naproxen, ) mefanamic acid, ) butyl paraben, E) ) propranolol, 6) napthalene, 7) dipropyl phthalate Slide 9

10 High Speed Separation of Analgesics Impact of Particle Size on Resolution mau mau mau um. um um -Acetamidophenol Caffeine -Acetamidophenol Acetanilide Acetylsalicylic Acid 6 Phenacetin 7 Salicylic Acid LC Conditions Column: SB-C8,.6 x 0 mm Detector: nm Injector: ul, Mobile Phase: % Formic Acid Acetonitrile (8:8) Flow:.0 ml/ Slide 0

11 Chromatographic Comparisons on Columns with Different Particle Sizes Separation of Analgesics Particle Size Chromatographic Parameter um. um.8 um Peak width, µl Acetylsalicylic acid Peak width, µl Salicylic acid Resolution Acetylsalicylic acid/ Phenacetin Resolution Phenacetin/Salicylic acid Efficiency (plates) Acetylsalicylic acid Efficiency (plates) Salicylic acid The Rapid Resolution HT column with.8 um particles provides the best results, measured by any parameter. Slide

12 Column Particle Size Comparison - Van Deemter Curve for Acetonitrile/Water 0.0 Plate Height (mm) um SB-C8. um SB-C8.8 um SB-C8 ml/ ml/ ml/ 0 6 Velocity (mm/sec) High flow rates can be used with acetonitrile/water mobile phases. Slide

13 Column Particle Size Comparison - Van Deemter Curve for Methanol/Water Plate Height (mm) um SB-C8. um SB-C8.8 um SB-C8 ml/ ml/ 0 Velocity (mm/sec) Slide

14 Columns with Smaller Particle Sizes are Reliable Long Lifetime with Zorbax Rapid Resolution HT Columns.. RT TF 0. 0 Inj# Injection Number Both retention time and tailing factor are consistent for many injections indicating long column lifetime Slide

15 Break Number Slide

16 High Resolution HPLC Applications with Rapid Resolution and Rapid Resolution HT Save time by using ZORBAX Rapid Resolution and Rapid Resolution HT HPLC columns to: Use more efficient method development techniques Develop rapid separations Upgrade methods Slide 6

17 Run Method Development Scouting Chromatograms on ZORBAX Rapid Resolution Columns Nucleotides-Isocratic Conditions A:B 70:0 80:0 90:0 6 Equilibration Volume: 7. ml 6 0 Time () 0 Time () Time () Column: Rapid Resolution Eclipse XDB-C8,.6 x 7 mm,. µm Mobile Phase: A: 0 mm Na HPO / 0 mm TBA (ph 7) B: Acetonitrile / 0 mm TBA Flow Rate:.0 ml/ Temperature: C Detection: UV nm Sample:. NAD. GMP. AMP. NADH. ADP 6. ATP Slide 7

18 Develop More Rapid Isocratic Separations using Rapid Resolution.-mm Columns Resolution of Peaks and Detere Method Analysis Time Isocratic HPLC - Separation of Test Mix Eclipse XDB-C8.6 x 0 mm, µm N =,000 Analysis Time Solvent Waste 7. 7 ml Rapid Resolution Eclipse XDB-C8.6 x 7 mm,. µm N = 0,00 Analysis Time Solvent Waste. ml Time () 0 0 Time () Mobile Phase: % 0 mm NaH PO, ph 7: 6% Methanol Temperature: C Sample:. Uracil. Butyl Paraben. Propranolol. Naphthalene. Dipropyl Phthalate 6. Acenaphthene 7. Amitriptyline Slide 8

19 Update Existing Methods with Rapid Resolution HT Columns USP Analysis of Guaifenesin Sample:. Guaifenesin 0.0 µg/µl,. Benzoic Acid 0.0 µg/µl USP L Column: ZORBAX Eclipse XDB-C8.6x0mm, µm Eluent: 0% Methanol:60% Water:.% Glacial Acetic Acid Flow:.0 ml/, Injection: 8µL, Temp: C Peak T R N Rs 6.6, , Rapid Resolution HT Column (L) ZORBAX Eclipse XDB-C8,.6x0mm,.8µm Eluent: 0% Methanol:60% Water:.% Glacial Acetic Acid Flow:.0 ml/, Injection: µl, Temp: C Peak T R N Rs.0, 0., Rapid Resolution HT Column (L) ZORBAX Eclipse XDB-C8,.6x0mm,.8µm Eluent: 0% Methanol:60% Water:.% Glacial Acetic Acid Flow:.0 ml/, Injection: µl, Temp: C Peak T R N Rs 0.8,8 0. 7, Slide 9

20 Updating Existing Methods with Rapid Resolution HT Columns USP Analysis of Triamcinolone Sample:. Triamcinolone - 0. µg/µl,. Hydrocortisone - 0. µg/µlminimum Resolution Required =.0 USP L Column: mbondapak C8,.9x00mm, 0µm Eluent: 7% Methanol:% Water, Flow:. ml/ Injection: µl, Temp: C Peak T R N Rs Rapid Resolution HT Column (L) ZORBAX Eclipse XDB-C8:.6x0mm,.8µm P/N Eluent: 7% Methanol:% Water, Flow:. ml/ Injection: µl, Temp: C Peak T R N Rs Rapid Resolution HT Column (L) ZORBAX Eclipse XDB-C8:.6x0mm,.8µm P/N Eluent: 60% Methanol:0% Water, Flow:. ml/ Injection: µl, Temp: C Peak T R N Rs Slide 0

21 High Throughput HPLC Applications High Speed Separations Isocratic Gradient Analytical (.6-mm i.d.) Narrow-Bore (.-mm i.d.) Slide

22 Improved Efficiency and Resolution on Rapid Resolution HT- Particle Size Comparison Conditions: Column: Eclipse XDB-C8,.6 x 0mm Mobile Phase: 7% Methanol, % 0mM Phosphate Buffer, ph 7 Flow Rate: ml/, Temperature: Ambient Injection Volume:.0 µl Inj., Detection: UV nm Sample: ) Uracil, ) Naproxen, ) Mefanamic Acid, ) Butyl Paraben, ) Propranolol, 6) Dipropyl Phthalate, 7) Naphthalene. Rapid Resolution Eclipse XDB-C8,. µm N 7 = 6,88 Rs, =. 6 7 Rapid Resolution HT Eclipse XDB-C8,.8 µm N 7 =,98 Rs, = Resolution improves and efficiency nearly doubles using the Rapid Resolution HT column. Slide

23 Rapid Resolution Columns Provide Very High Efficiency and Very Short Analysis Times Columns: ZORBAX SB-C8,.6 x 0 mm Mobile Phase: % Water: 7% MeOH Flow Rate:. ml/ Temperature: RT Detection: UV nm Sample: QC:. Uracil. Phenol. -Cl-Nitrobenzene. Toluene Rapid Resolution.6 x 0 mm,. µm Plates (N) Rapid Resolution HT.6 x 0 mm,.8 µm Plates (N) Analysis time of < ute for high throughput analysis with very high efficiency on Rapid Resolution HT. Slide

24 Isocratic High-Throughput HPLC Increase Flow Rate to Reduce Analysis Time Column: Rapid Resolution StableBond SB-C8,. µm,.6 x 0 mm Sample:. Acetaophen (-acetamidophenol). Caffeine. -Acetamidophenol. Acetanilide. Acetylsalicylic acid 6. Salicylic acid 7. Acetophenetidin Pressure 90 bar Rs, =. 6 Rs 7,6 =. 7 ml/ bar 6 7 ml/ 8 bar 60 psi Rs, =. 6 Rs 7,6 = Time () ml/ Slide

25 Increase Temperature to Reduce Analysis Time on Rapid Resolution and RR HT Columns Temp. RT ºC 0ºC Pressure bar bar 00 bar Column: Rapid Resolution HT SB-C8.6 x 0 mm,.8 µm Mobile Phase: 0% water: 60% methanol Flow Rate: ml/ Detection: UV nm Temperature:As noted Sample:. Triamcinolone. Hydrocortisone T = 0 C F =.0 ml/ T = 0 C F =. ml/ ºC 89 bar Can increase flow rate to decrease analysis time even further Increasing temperature reduces analysis time by 0% or more and pressure by %. Slide

26 Break Number Slide 6

27 How To Use Small Volume Columns Effectively for Isocratic and Gradient Separations Critical instrument parameters include: Isocratic and Gradient Gradient only Data rate Flow cell size Tubing Mixing volume (part of gradient delay volume) Gradient delay volume (dwell volume) Slide 7

28 Optimization of Results with Rapid Resolution HT Columns - Data Acquisition Rate Comparison Column: ZORBAX Rapid Resolution HT SB-C8.6 x 0 mm,.8 µm Mobile Phase: 60% Methanol: 0 Water Flow Rate: ml/ Temperature: RT Detection: UV nm Sample: QC Test. Uracil. Phenol. -Cl-Nitrobenzene. Toluene Data Acquisition Rate = sec Plates # Plates # Data Acquisition Rate = 0. sec Plates # Plates # Slide 8

29 HPLC Instrumentation Contribution to Isocratic and Gradient Performance Mobile Phase Dwell Volume Extra Column Volume (ECV) Data Device ECV = sample volume connecting tube volume fitting volume detector cell volume Dwell Volume = volume from formation of gradient to top of column Slide 9

30 How to Use Rapid Resolution and Rapid Resolution HT Columns Effectively LC A = standard LC, plumbed with 0.7 mm (green) tubing, semi-micro flow cell ( 8 ul) and 80 ul mixer for gradient runs. LC B = standard LC, plumbed with 0. mm (red) tubing, semi-micro (8 ul) or micro high pressure flow cell (.7 ul) and 80 ul or no mixer. You can further optimize with the 00 nl flow cell for the. x mm columns and no mixer for gradient separations. Flow cells listed are for DAD Slide 0

31 How to Use Short Rapid Resolution HT Columns Effectively for Isocratic and Gradient Separations Column Length 0 mm 0 mm mm Internal Diameter Typical k k = k = k = 0 k = k = k = 0 k = k = k = LC A..6.*.6 LC B LC A = standard LC, with 0.7 mm (green) tubing, semi-micro flow cell (8 ul, DAD) and 80 ul mixer (0-6) for gradients. LC B = standard LC, with 0. mm (red) tubing, semi-micro (8 ul) or micro high pressure (.7 ul) flow cell, and 80 ul (0-6) or no mixer for gradient analysis. *the 00nL flow cell can be used with the. x mm to improve results * *.* Slide

32 Effect of Flow Cell Volume on Resolution. x mm Rapid Resolution HT Column Column: Rapid Resolution HT SB-C8,. x mm,.8 µm Mobile Phase: 0% Methanol:0% water Flow Rate 0. ml/ Detection: UV nm Sample: QC test. Uracil. Phenol. -Chloronitrobenzene. Toluene mau Rs (,) =.8.7 ul Flow Cell Rs (,) = mau 7 00 nl Flow Cell Rs (,) =. Rs (,) = On the smallest volume column, resolution can be improved with the smallest flow cell. Slide

33 Benefits of Using Gradient Elution for High Throughput HPLC Provides more efficiency because of gradient compression effects Preferred over isocratic elution when separating a number of unknown compounds Easily separates compounds that differ widely in polarity or have high molecular weight (i.e. peptides and proteins) Eluted peaks have essentially the same peak width, so sensitivity is similar throughout chromatogram Exposes a column continuously to solvents of increasing strength, imizing sample and impurity build-up Equilibration time is not a limitation (small column volumes allows faster regeneration after gradient elution) Slide

34 Gradient High-Throughput HPLC on Rapid Resolution.-µm Columns Reducing Column Length Reduces Run Time Gradient Time (t G ) ~ Column Length Column: Eclipse XDB-C8,.6 mm i.d. Gradient: -90% B in t G utes Mobile Phase: A: mm Na HPO, ph B: Methanol Temperature: C Flow Rate:.0 ml/ Sample: Cardiac Drugs:. Diltiazam. Dipyridamole. Nifedipine. Lidoflazine. Flunarizine Length = 7 mm,. µm t G = 9 Length = 0 mm,. µm t G = Time () Time () Slide

35 Gradient High-Throughput HPLC on Rapid Resolution.-µm Columns Increasing Flow Rate Reduces Gradient Run Time Even Further and Improves Sample Throughput Note: If t G x F = constant, then the elution pattern is unchanged F =.0 ml/ = t G Samples per day Time () Column: Gradient: Rapid Resolution Eclipse XDB-C8.6 x 0 mm,. µm -90% B in t G utes Mobile Phase: A: mm Na HPO, ph B: Methanol Flow Rate: Temperature: C Sample:.0 ml/ Cardiac Drugs. Diltiazam. Dipyradamole. Nifedipine. Lidoflazine. Flunarizine F =.0 ml/ = t G Samples per day Time () Slide

36 Gradient Narrow-Bore HPLC on Rapid Resolution.-µm Columns.-mm i.d. Column Reduces Solvent Waste and Improves Sensitivity Mobile Phase: A: Water with 0.% formic acid B: Methanol with 0.% formic acid Temperature: C Inj: µl Sample: Organic Acids:.Gallic. Protocatechuic. Hydrocaffeic. Gentisic. Vanillic 6. Syringic 7. Sinapinic 8. Salicylic Rapid Resolution SB-C8.6 x 0 mm,. µm MAU 0 Rapid Resolution SB-C8. x 0 mm,. µm Gradient: -% B in 6. Flow Rate:.0 ml/ 00 Gradient: -% B in 6. Flow Rate: 0. ml/ 8 Solvent Used:.6 ml MAU Solvent Used:. ml Time () Time () Instrument is optimized for lowest dwell volume and extra-column-volume Slide 6

37 Higher Resolution, Faster Analyses for Complex Samples. mm i.d. Rapid Resolution HT.x0mm, µm P/N gradient 0. ml/ 0 peaks 60 s.x0mm,.8µm P/N gradient 0.mL/ peaks 0 s!.x0mm,.8µm P/N gradient 0.mL/ peaks! s 0 0 Conditions: Mobile Phase A: Water w/ 0.% TFA, B: ACN w/0.% TFA, Gradient %B to 0%B, Temperature: 0 C Detection: UV nm Sample: HSA Tryptic Digest Slide 7

38 Conclusions Shorter column lengths offer reduced analysis time whether using a.-,.6- or.-mm i.d. HPLC column under isocratic or gradient conditions. Rapid Resolution.-µm particles provide good resolution for fast analysis. Smaller,.8-µm particles provide even more resolution for high throughput with the highest resolution available; for highest performance some instrument modification may be necessary. Gradient elution offers advantage over isocratic elution when using short, fast HPLC columns. Peaks elute with the same peak width and columns are protected against sample and mobile phase contaation as gradient displaces strongly retained materials. Gradient equilibration time is essentially a non-issue with very short, Rapid Resolution columns. Short, Rapid Resolution, narrow-bore columns offer very good resolving power in less than 60 seconds by using steep gradients and flow rates >.0 ml/. Slide 8

39 Rapid Resolution HT Application Notes. Increasing Sample Throughput Using ZORBAX Eclipse XDB-C8 Rapid Resolution HT Columns pub no EN. The Influence of Sub Two Micron Particles on HPLC Performance pub no EN. Fast Liquid Chromatography and Liquid Chromatography/ Mass Spectrometry Analysis of Antibiotics Using Rapid Resolution HT HPLC Columns with Sub Two-Micron (.8 mm) Particles pub no EN. Application Examples with Rapid Resolution HT HPLC columns pub no EN. High Efficiency, High Throughput LC and LC/MS Applications Using ZORBAX Rapid Resolution HT Columns pub no EN 6. ZORBAX Rapid Resolution HT Columns A Breakthrough in High- Throughput HPLC Column Technology pub no. 989-XXXXEN 7. Separation Times (Vol 6, No., 00 High Throughput HPLC with Short Columns and New Sub Two-Micron Particles available as pub #988-96EN, Vol 6 No., 00) Slide 9