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HPLC Method Development.pdf

The document discusses rapid HPLC method development. It outlines four critical aspects of rapid development: sample preparation, column selection, column configuration, and instrument optimization. Choosing the right bonded phase is key to achieving resolution. Common phases like C18 and C8 are recommended initially, with specialized phases for difficult samples. The document provides examples of developing methods for various sample types using different columns and conditions based on a rapid development scheme. Developing at low pH first and using temperature, then moving to mid pH if needed, can reduce analysis time while maintaining resolution and reproducibility.

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The Secrets of Rapid HPLC Method Development Choosing Columns for Rapid Method Development and Short Analysis Times
Slide 2 Dial 1- 904-779-4740 for e-Seminar Audio Rapid Analysis Is More Than Run Time • It is developing a method to meet a goal and developing and validating it quickly. • The final method should minimize analysis time for the greatest sample throughput. • It must be reproducible and robust.
Slide 3 Dial 1- 904-779-4740 for e-Seminar Audio Four Critical Aspects of Rapid Method Development and Analysis • Rapid Sample Preparation – minimum steps for maximum effectiveness, use updated tools (combination filters) and multi- sample preparation equipment (SPE 96-well plates) • Choose best bonded phases for high resolution – selecting from typical C18 and C8 bonded phases or those targeted to special sample types • Choose the best column configuration for minimum analysis time with high efficiency and resolution – best column length, internal diameter, particle size • Using your HPLC instrument to further reduce analysis time and increase sample throughput – optimizing the HPLC and using new features effectively
Slide 4 Dial 1- 904-779-4740 for e-Seminar Audio Bonded Phase Choice Drives Resolution • Changing selectivity (α) influences resolution the most • Bonded phase is the column choice that controls selectivity N= plates – increase by using longer column or reducing particle size α= selectivity – increase by changing bonded phase and mobile phase k= retention – increase by changing bonded phase and mobile phase does not improve Rs above k ≈10 Rs= N ( - 1) α k α 1+k ¼ ½
Slide 5 Dial 1- 904-779-4740 for e-Seminar Audio Selecting a Bonded Phase • Choose columns known to have long lifetimes at operating mobile phase pH. • Choose bonded phases on high purity, low acidity silica for best peak shape. • Select a C18 or C8 bonded phase first for good retention and resolution with typical acidic, basic and neutral samples. • If sample is expected to be difficult (i.e. very polar, difficult to retain, very basic) then select targeted bonded phases available for these types of samples.
Slide 6 Dial 1- 904-779-4740 for e-Seminar Audio Rapid Method Development Scheme – Low pH • Start at low pH for best peak shape, retention and long-term reproducibility • Select starting conditions • StableBond C18 or C8 for maximum lifetime – Rapid Resolution columns • pH 1 – 3 with 20 – 50 mM buffer for best peak shape • Acetonitrile or methanol – start high to scout • Adjust organic for maximum resolution and retention • Change organic if resolution not achieved – MeOH or ACN • Change bonded phase if resolution not achieved – SB-CN, SB- Phenyl, SB-C3 • Use elevated temperature to reduce analysis time further
Slide 7 Dial 1- 904-779-4740 for e-Seminar Audio Rapid Method Development Scouting Chromatograms on StableBond-C18 Rapid Resolution Columns 0.0 0.5 1.0 1.5 2.0 2.5 Time (min) 0.0 0.5 1.0 1.5 2.0 2.5 Time (min) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 Time (min) 5 4 3 2 1 A : B 20 : 80 40 : 60 60 : 40 Columns: Zorbax Rapid Resolution StableBond SB-C18 4.6 x 75 mm, 3.5 µ µ µ µm Mobile Phase: A: 25 mM NaH2PO4, pH 3.0 B: ACN Flow Rate: 2.0 mL/min. Temperature: 35°C UV Detection: 254 nm Sample: Cardiac Drugs
Slide 8 Dial 1- 904-779-4740 for e-Seminar Audio Rapid Method Development Scouting Chromatograms Change Organic Modifier Column: Zorbax Rapid Resolution SB-C18, 4.6 x 75 mm, 3.5 µ µ µ µm Mobile Phase: A: 25 mM NaH2PO4 , pH 3.0 B: MeOH Flow Rate: 2.0 mL/min Temperature: 35°C Detection: UV 254 nm Sample: Cardiac Drugs 1. Diltiazem 2. Dipyridamole 3. Nifedipine 4. Lidoflazine 5. Flunarizine Time(min) 1 2 3 4 5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Time(min) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Time(min) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 1 2 3 4 5 20 : 80 30 : 70 40 : 60 • Changing organic modifier can alter selectivity and improve peak shape. A : B
Slide 9 Dial 1- 904-779-4740 for e-Seminar Audio Rapid Method Development Process Optimizes Separation on StableBond-C18 at Low pH • A Rapid Resolution SB-C18 column at low pH was used to develop this thorough and rapid analysis of steroids and impurities following the rapid method development process. Column: Zorbax Rapid Resolution SB-C18, 4.6 x 75 mm, 3.5 µ µ µ µm Mobile Phase: 50% ACN 50% 20 mM NaH2PO4, pH 2.8 Flow Rate: 1.0 mL/min Temperature: RT Detection: UV 254 nm Sample: 1. Estradiol 2. Ethynylestradiol 3. Dienestrol 4. Norethindrone 1 2 3 4 0 2 4 8 10 6 12 Time(min)
Slide 10 Dial 1- 904-779-4740 for e-Seminar Audio Column: Zorbax SB-C18, 4.6 x 75 mm, 5 µ µ µ µm Mobile Phase: 74% 7.4 mM hexane sulfonate and 0.07% phosphoric acid 26% MeOH Flow Rate: 1 mL/min Detection: UV 280 nm Sample: B Vitamins min. 001599S1.PPT Method Optimization High Temperature Low Temperature Increase Temperature to Reduce Analysis Time • Agilent instruments provide reliable temperature control to 80 - 90°C allowing method development flexibility.
Slide 11 Dial 1- 904-779-4740 for e-Seminar Audio Rapid Method Development Scheme – Mid pH • Try pH 7 (pH 6 – 9) with Eclipse XDB-C18 or C8 and follow same process, selecting Eclipse XDB- Phenyl as alternate bonded phase • Use temperature to reduce analysis time further
Slide 12 Dial 1- 904-779-4740 for e-Seminar Audio Eclipse XDB-C18 is Bonded Phase Choice for High Resolution at Mid pH SB-C18, 4.6 x 75 mm, 3.5 µ µ µ µm Eclipse XDB-C18, 4.6 x 75 mm, 3.5 µ µ µ µm Mobile Phase: 20% Methanol: 80% 20 mM phosphate buffer + (10 mM TEA @ pH 7) Flow Rate: 1.0 mL/min Temperature: RT Detection: UV 254 nm Sample: 1. Nizatidine 2. Famotidine 3. Cimetidine 4. Pirenzipine • This sample is only resolved at mid pH on Eclipse XDB-C18 following rapid method development process. pH 3 pH 7 Time(min) 0 2 2 1 3 4 4 6 8 0 2 4 6 8 10 12 14 Time(min) 2 1 3 4
Slide 13 Dial 1- 904-779-4740 for e-Seminar Audio Break Number 1 For Questions and Answers Press *1 on Your Phone to Ask a Question
Slide 14 Dial 1- 904-779-4740 for e-Seminar Audio Choose Special Bonded Phases for Difficult Samples • Sample 1 – Highly basic compounds • May exhibit poor peak shape • May be difficult to retain • Column choice 1 – Bonus-RP for better peak shape • Column choice 2 – Extend-C18 at high pH for better retention and peak shape • Sample 2 – Highly polar compounds • May be difficult to retain • May require high aqueous mobile phases • Column choice 1 – SB-Aq for better retention with high aqueous mobile phases • Column choice 2 – Bonus-RP for use with high aqueous mobile phases
Slide 15 Dial 1- 904-779-4740 for e-Seminar Audio 0 1 2 3 4 5 6 7 8 9 10 Time (min) 0 1 2 3 4 5 6 Time (min) 2 3 1 1 2 3 Sample 1: Highly Basic Compounds Select Bonus-RP for Improved Peak Shape B. Bonus-RP A. Alkyl C8 TF 1. 1.1 2. 1.1 3. 1.1 TF 1. 1.8 2. 1.7 3. 2.1 Column: 4.6 x 150 mm, 5 µm Mobile Phase: A: 75% 25 mM NH4Ac, pH 5.5 : 25% ACN B: 80% 25 mM NH4Ac, pH 5.5 : 20% ACN Flow Rate: 1.5 mL/min Detection: UV 254 nm Sample: 1. Doxylamine 2. Chlorpheniramine 3. Triprolidine
Slide 16 Dial 1- 904-779-4740 for e-Seminar Audio Plates 1. 4903 2. 7621 3. 7567 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 2 1 3 Time (min) Sample 1: Highly Basic Compounds Extend-C18 Improves Retention and Efficiency of Procainamides at High pH Column: Zorbax Extend-C18, 4.6 x 150 mm, 5 µ µ µ µm Mobile Phase: See Below Flow Rate: 1.0 mL/min Detection: UV 254 nm Temperature: RT Sample: 1. Procainamide pKa 9.2 2. N-acetylprocainamide 3. N-propionylprocainamide Plates 1. 9475 2. 8823 3. 8794 50% 20 mM TEA, pH 11: 50% MeOH 50% 25 mM Na2HPO4, pH 7.0 : 50% MeOH 0.0 2.5 5.0 7.5 2 1 3 Time (min) • High efficiency improves resolution and column lifetime and the increase in retention results in a more rugged method.
Slide 17 Dial 1- 904-779-4740 for e-Seminar Audio Sample 2: Highly Polar Compounds Select New ZORBAX SB-Aq Columns for Method Development in High Aqueous Mobile Phases • Good retention for polar compounds in high aqueous mobile phases • Reproducible retention without “phase collapse” • Different selectivity vs. conventional C18 columns • Highly, stable at low pH and high temperature (up to 80°C)
Slide 18 Dial 1- 904-779-4740 for e-Seminar Audio Sample 2: Highly Polar Compounds ZORBAX SB-Aq Provides Good Retention of Polar Compounds 16 0 2 4 6 8 Time (min) 10 12 14 2 1 3 4 5 Columns: Zorbax SB-Aq, 4.6 x 150 mm, 5 µ µ µ µm Mobile Phase: 90% 0.2% TFA 10% ACN Temperature: 25°C Flow Rate: 1.5 mL/min. Detection: UV 254 nm Sample: 1. Maleate 2. Phenylephrine 3. Phenylpropanolamine 4. Pyrilamine 5. Chlorpheniramine • These small polar compounds are difficult to retain on most columns. • The SB-Aq provides excellent retention with a 90% aqueous mobile phase.
Slide 19 Dial 1- 904-779-4740 for e-Seminar Audio Sample 2: Highly Polar Compounds ZORBAX SB-Aq Provides Good Retention of Water Soluble Vitamins without Ion Pairing Time (min) 1 2 3 4 0.0 0.5 1.0 1.5 2.0 Columns: Zorbax SB-Aq, 4.6 x 150 mm, 5 µ µ µ µm Mobile Phase: 95% 0.1% TFA 5% MeOH Temperature: 35°C Flow Rate: 2.0 mL/min. Detection: UV 254 nm Sample: 1. Thiamine – B1 2. Nicotinic Acid 3. Pyridoxine – B6 4. Niacinamide – B3 • The SB-Aq column provides good retention of these polar compounds without ion pairing. • The result is a simpler method without the reproducibility problems associated with ion pairing.
Slide 20 Dial 1- 904-779-4740 for e-Seminar Audio Sample 2: Highly Polar Compounds ZORBAX SB-Aq Provides Good Retention of NADH/NAD+ with LC/MS Compatible Mobile Phase 1 2 3 Time (min) 0 2 4 6 8 Columns: Zorbax SB-Aq, 4.6 x 150 mm, 5 µ µ µ µm Mobile Phase: 94% 10 mM ammonium acetate, pH 5.5 6% MeOH Temperature: 25°C Flow Rate: 1.0 mL/min. Detection: 254 nm Sample: 1. Uracil 2. NADH 3. NAD+ • These coenzymes are difficult to retain and analyze by LC/MS. • The SB-Aq provides baseline resolution without ion pairing.
Slide 21 Dial 1- 904-779-4740 for e-Seminar Audio Rapid Resolution Columns Reduce Method Development and Analysis Time • Rapid Resolution columns (3.5 µm) are available in many configurations • Available in analytical, narrow bore, microbore, and capillary internal diameters for compatibility with any sample size. • Semi-preparative and preparative columns use 5 µm particles. • Rapid Resolution columns reduce isocratic and gradient run times with: • Shorter column lengths • Higher flow rates • Optimized HPLC instrument
Slide 22 Dial 1- 904-779-4740 for e-Seminar Audio Choose Column Configuration for Application Large scale prep Small scale prep (mg) Analytical Analytical High sensitivity LC/MS Higher sensitivity LC/MS Max sensitivity LC/MS Applications 20 – 60 mL/min 5, 7 50 – 250 21.2 Preparative 4 – 10 mL/min 5 50 – 250 9.4 Semi-prep 1 – 4 mL/min 3.5, 5 15 – 250 4.6 Analytical 0.3 – 1.0 mL/min 3.5, 5 150, 250 3.0 Solvent Saver 0.1 – 0.3 mL/min 3.5, 5 15 – 150 2.1 Narrow Bore 30 – 60 µL/min 3.5, 5 30 – 150 1.0 MicroBore 1 – 10 µL/min 3.5, 5 35 – 250 0.3, 0.5 Capillary Flow Rate Ranges Particle Sizes (µ µ µ µm) Lengths (mm) I.D. (mm) Column Type
Slide 23 Dial 1- 904-779-4740 for e-Seminar Audio High Resolution with Rapid Resolution Columns Isocratic Separation of Aspartame Column: Zorbax StableBond SB-C18 Mobile Phase: 85% Water with 0.1% TFA : 15% Acetonitrile Flow Rate: 1.0 mL/min. Temperature: 35°C Sample: 1. Phenylalanine 2. 5-Benzyl-3,6-dioxo-2-piperazine acetic acid 3. Asp-phe 4. Aspartame 4.6 x 150 mm, 5 µ µ µ µm 4.6 x 75 mm, 3.5 µ µ µ µm 4.6 x 50 mm, 3.5 µ µ µ µm 4.6 x 30 mm, 3.5 µ µ µ µm 4.6 x 15 mm, 3.5 µ µ µ µm Time (min) 0 1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4
Slide 24 Dial 1- 904-779-4740 for e-Seminar Audio 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Time (min) 5 4 3 2 1 0 1 2 3 4 5 6 Time (min) 5 4 3 2 1 Rapid Resolution Columns Reduce Isocratic Analysis Time by 50% or More Mobile Phase: 45% 25 mM NaH2PO4, pH 3.0 55% MeOH Flow Rate: 2.0 mL/min. Temperature: 35°C Detection: UV 254 nm Sample: Cardiac Drugs 1. Diltiazem 2. Dipyridamole 3. Nifedipine 4. Lidoflazine 5. Flunarizine Rapid Resolution StableBond SB-C18 4.6 x 75 mm, 3.5 µm StableBond SB-C18 4.6 x 150 mm, 5 µm Analysis Time: 5.2 min Analysis Time: 11.7 min
Slide 25 Dial 1- 904-779-4740 for e-Seminar Audio Higher Flow Rates Reduce Analysis Time with Rapid Resolution Columns Column: Zorbax Rapid Resolution StableBond SB-C18, 4.6 x 30 mm, 3.5 µ µ µ µm Sample: 1. Acetaminophen (4-acetamidophenol) 2. Caffeine 3. 2-Acetamidophenol 4. Acetanilide 5. Acetylsalicylic acid 6. Salicylic acid 7. Acetophenetidin 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Time (min) 1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7 2 mL/min 3 mL/min 4 mL/min Pressure 90 bar 133 bar 181 bar 2650 psi Rs2,1 = 2.1 Rs2,1 = 2.2 Rs7,6 = 2.2 Rs7,6 = 2.2
Slide 26 Dial 1- 904-779-4740 for e-Seminar Audio at 0.2 mL/min at 1.0 mL/min Gradient Re-equilibration Times are Minimal on Short Rapid Resolution Columns Column Volume (Vm) and Equilibration Time Gradient Analysis Time = Run Time + Equilibration Time ( single step return) 4.6 x 50 0.5 mL 5 min 4.6 x 30 0.3 mL 3 min 4.6 x 15 0.15 mL 1.5 min 4.6 x 150 1.54 mL 15 min 2.1 x 50 0.10 mL 5 min 60 sec 2.1 x 30 0.06 mL 3 min 36 sec 2.1 x 15 0.03 mL 1.5 min 18 sec Column Internal Equilibration Time Dimension (mm) Volume (Vm) at 1.0 mL/min (Vm x 10 x F)
Slide 27 Dial 1- 904-779-4740 for e-Seminar Audio Length = 50 mm, 3.5 µ µ µ µm tG = 6 min Short, Rapid Resolution Columns Reduce Gradient Analysis Time Gradient Time α α α α Column Length Column: Zorbax Eclipse XDB-C8, 4.6 mm i.d. Gradient: 45-90% B in tG minutes Mobile Phase: A: 25 mM Na2HPO4, pH 3 B: Methanol Temperature:35°C Flow Rate: 1.0 mL/min Sample: Cardiac Drugs: 1. Diltiazam 2. Dipyridamole 3. Nifedipine 4. Lidoflazine 5. Flunarizine Length = 75 mm, 3.5 µ µ µ µm tG = 9 min 0.0 2.5 5.0 7.5 Time (min) 0.0 2.5 5.0 Time (min) 1 2 3 4 5 1 2 3 4 5
Slide 28 Dial 1- 904-779-4740 for e-Seminar Audio Rapid Gradient CombiChem Analysis 1.5 min Injection-to-Injection mAU min 0 0.2 0.4 0.6 0.8 1 Column: Zorbax SB-C18, 2.1 x 30 mm, 3.5µm Flow Rate: 0.832mL/min Temperature: 20°C Mobile Phase Gradient A=0.1% Formic Acid, H2O; B=0.1%Formic Acid, ACN; 30-98 %B in 0.6 min P1-A-02 P1-A-03 P1-A-04 P1-A-05 P1-A-07 P1-A-08 P1-A-09 P1-A-10 P1-B-01 P1-B-02 Well-Plate Position 1 min
Slide 29 Dial 1- 904-779-4740 for e-Seminar Audio 1 2 3 4 5 1 2 3 4 5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Time (min) 0.0 0.5 1.0 1.5 2.0 Time (min) F = 3.0 mL/min tG = 2 min F = 2.0 mL/min tG = 3 min Increasing Flow Rate Reduces Gradient Analysis Time Further Column: Zorbax Eclipse XDB-C8 4.6 x 50 mm, 3.5 µ µ µ µm Gradient: 45-90% B in tG minutes Mobile Phase: A: 25 mM Na2HPO4, pH 3 B: MeOH Flow Rate: 1.0 mL/min Temperature: 35°C Sample: Cardiac Drugs 1. Diltiazam 2. Dipyradamole 3. Nifedipine 4. Lidoflazine 5. Flunarizine If tG x F = constant then the elution pattern is unchanged
Slide 30 Dial 1- 904-779-4740 for e-Seminar Audio Break Time For Questions and Answers Press *1 on Your Phone to Ask a Question
Slide 31 Dial 1- 904-779-4740 for e-Seminar Audio HPLC Instrument Optimization Increases Efficiency and Decreases Method Development and Analysis Time • Choose the best column configuration for your instrument type and application (i.e. Capillary LC for highest sensitivity or compatibility with LC/MS) • Instrument optimization can improve efficiency for isocratic and gradient separations • New instrument features can help you reduce analysis time
Slide 32 Dial 1- 904-779-4740 for e-Seminar Audio Choose HPLC Instrument for Application 1. Purificatio n 1. Analytical 2. MS compatible 1. Sample limited 2. Most MS compatible Applications Preparative and CombiChem prep 1 - 100 mL/min Semi-prep, Prep Prep LC Standard analytical more semi-prep than low volume Work at 3- 4mL/min 0.05 – 5 mL/min* binary 0.2 – 10 mL/min* isocratic/quaternar y Narrow-bore, Solvent Saver, Analytical, Semi-prep LC 5 µL dwell volume, best low volume gradient reproducibility 1 µL/min – 2.5 mL/min Capillary, MicroBore, Narrow-bore, Solvent Saver, Analytical Capillary LC Why? Flow Rate Range Compatible Columns Agilent 1100 Type * Optimum range
Slide 33 Dial 1- 904-779-4740 for e-Seminar Audio Optimizing Results with Rapid Resolution Columns Isocratic Separations • Minimize extra column volume to minimize band broadening • Keep the injection volume small (< 5 µL) • Use semi-micro or micro detector cells ( 5 µL or less) • Use 0.12 mm i.d. tubing (0.005”) • Prepare the sample in an injection solvent with the same or weaker solvent strength than the mobile phase • Overlap injections • Select the Agilent 1100 Capillary HPLC for capillary and microbore columns • Select correct detector response time for rapidly eluting peaks
Slide 34 Dial 1- 904-779-4740 for e-Seminar Audio Time (min) 0 2.5 5 2 1 3 4 Time (min) 0 2.5 5 2 1 3 4 Effect of Detector Cell Volume on Peak Width 4.6 x 75 mm, 3.5 µ µ µ µm Column: Zorbax StableBond SB-C18 Mobile Phase: 85% H20 with 0.1% TFA : 15% ACN Flow Rate: 1.0 mL/min Temperature: 35°C Sample: 1. Phenylalanine 2. 5-benzyl-3,6-dioxo-2-piperazine acetic acid 3. Asp-phe 4. Aspartame N 1. 8700 2. 8700 3. 8300 4.10,000 Microflow Cell - 2.5 µ µ µ µL Standard Flow Cell - 8 µ µ µ µL N 1. 6200 2. 6700 3. 7000 4. 9250 1. -29% 2. -23% 3. -16% 4. -7%
Slide 35 Dial 1- 904-779-4740 for e-Seminar Audio Optimizing Results with Rapid Resolution Columns Gradient Separations • Minimize dwell volume – includes mixing volume and injector volume • Use high pressure mixing and reduce mixer volume • Run autosampler in bypass – reduce dwell volume by 300 µL • Minimize extra column volume • Overlap injections • Select correct detector response time for rapidly eluting peaks • Select Agilent 1100 Capillary HPLC with 5 µL dwell volume for capillary and microbore columns
Slide 36 Dial 1- 904-779-4740 for e-Seminar Audio Compound rsd RT for 10 runs rsd Area for 10 runs Dimethylphthalate 0.09 0.98 Diethylphthalate 0.10 0.87 Biphenyl 0.08 1.83 o-Terphenyl 0.07 0.90 Compound rsd RT for 10 runs rsd Area for 10 runs Dimethylphthalate 1.09 5.91 Diethylphthalate 0.55 5.64 Biphenyl 0.52 5.30 o-Terphenyl 0.43 6.87 min 0 0.2 0.4 0.6 0.8 1 1.2 1.4 Norm. -20 0 20 40 60 80 100 min 0 0.2 0.4 0.6 0.8 1 1.2 1.4 Norm. 0 0.02 0.04 0.06 0.08 0.1 0.12 Low pressure mixing, Brand X Reproducible, Fast Gradient Analysis with High Pressure Mixing Column: Zorbax StableBond C18, 2.1 x 50 mm Agilent 1100, binary pump high pressure mixing
Slide 37 Dial 1- 904-779-4740 for e-Seminar Audio Reduce Gradient Analysis Time and Dwell Volume with Bypass of the Sample Loop mAU 0 100 200 mAU 0 100 200 min 0 0.2 0.4 0.6 0.8 1 1.2 Agilent 1100 WPS Auto Bypass By-Pass On Column: Zorbax SB-C18, 4.6 x 30 mm, 3.5µm Flow Rate: 0.832 mL/min Temperature: 20°C Mobile Phase Gradient: A= 0.1% Formic Acid, H2O; B = 0.1% Formic Acid, ACN; 30-98 %B in 0.6 min Agilent 1100 WPS Auto Bypass By-Pass Off Time Savings
Slide 38 Dial 1- 904-779-4740 for e-Seminar Audio min 0.2 0.4 0.6 0.8 1 1.2 1.4 mAU 0 250 500 750 1000 1250 1500 1750 2000 Overlapped Injection Cycle time is ~ 2min 12sec Total time for 20 runs: 44 min 22 sec Standard Injection Cycle time is ~ 2min 57sec Total time for 20 runs: 58 min 20 sec Switch of injection valve Run time: 1.5 min / Post time: 0.5 min for both runs Overlapped Injection Reduces Analysis Time • Overlapped injections – sample is drawn up during previous injection – reduce gradient and isocratic run times.
Slide 39 Dial 1- 904-779-4740 for e-Seminar Audio Effect of Detector Response Time on Ultra-Fast Gradient Analyses 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 Time(min) 1.0 Agilent 1100 DAD Agilent 1100 WPS with ADVR Column: Poroshell 300SB-C18 2.1 x 75 mm, 5 µm Mobile Phase: A: 95% H2O, 5% ACN with 0.1% TFA B: 5% H2O, 5% ACN with 0.1% TFA Flow Rate: 2 mL/min Temperature: 70°C Detector: UV 215 nm Piston stroke: 20 Sample: 1. Neurotensin 3. Lysozyme 2. RNaseA 4. Myoglobin 0.1 sec 0.2 sec 0.5 sec 1.0 sec 1st peak = 1.2 sec At 20 pts/sec = 24 pts/sec Response Time 2.0 sec 1st peak = 1.2 sec At 5 pts/sec = 6 pts/sec • You may have to adjust the response rate of your detector for rapid peak detection.
Slide 40 Dial 1- 904-779-4740 for e-Seminar Audio Summary • C18 and C8 bonded phases are the best for initial rapid method development with typical sample types • Choosing the most sample appropriate bonded phase and using special, targeted bonded phases, such as SB-Aq for polar, difficult to retain compounds can decrease method development time • Rapid Resolution columns are needed to reduce method development time • Rapid Resolution columns reduce both gradient and isocratic analysis time and permit high throughput rapid analysis • Rapid Resolution columns can work effectively with your HPLC • HPLC instruments may have additional capabilities to speed up method development and reduce analysis time
Slide 41 Dial 1- 904-779-4740 for e-Seminar Audio Break Time For Questions and Answers Press *1 on Your Phone to Ask a Question
Slide 42 Dial 1- 904-779-4740 for e-Seminar Audio HPLC Column Technical Support 800-227-9770 (phone: US & Canada)* 302-993-5304 (phone)* * Select option 4, then option 2. 916-608-1964 (fax) www.agilent.com/chem

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HPLC Method Development.pdf

  • 1.
    The Secrets ofRapid HPLC Method Development Choosing Columns for Rapid Method Development and Short Analysis Times
  • 2.
    Slide 2 Dial 1-904-779-4740 for e-Seminar Audio Rapid Analysis Is More Than Run Time • It is developing a method to meet a goal and developing and validating it quickly. • The final method should minimize analysis time for the greatest sample throughput. • It must be reproducible and robust.
  • 3.
    Slide 3 Dial 1-904-779-4740 for e-Seminar Audio Four Critical Aspects of Rapid Method Development and Analysis • Rapid Sample Preparation – minimum steps for maximum effectiveness, use updated tools (combination filters) and multi- sample preparation equipment (SPE 96-well plates) • Choose best bonded phases for high resolution – selecting from typical C18 and C8 bonded phases or those targeted to special sample types • Choose the best column configuration for minimum analysis time with high efficiency and resolution – best column length, internal diameter, particle size • Using your HPLC instrument to further reduce analysis time and increase sample throughput – optimizing the HPLC and using new features effectively
  • 4.
    Slide 4 Dial 1-904-779-4740 for e-Seminar Audio Bonded Phase Choice Drives Resolution • Changing selectivity (α) influences resolution the most • Bonded phase is the column choice that controls selectivity N= plates – increase by using longer column or reducing particle size α= selectivity – increase by changing bonded phase and mobile phase k= retention – increase by changing bonded phase and mobile phase does not improve Rs above k ≈10 Rs= N ( - 1) α k α 1+k ¼ ½
  • 5.
    Slide 5 Dial 1-904-779-4740 for e-Seminar Audio Selecting a Bonded Phase • Choose columns known to have long lifetimes at operating mobile phase pH. • Choose bonded phases on high purity, low acidity silica for best peak shape. • Select a C18 or C8 bonded phase first for good retention and resolution with typical acidic, basic and neutral samples. • If sample is expected to be difficult (i.e. very polar, difficult to retain, very basic) then select targeted bonded phases available for these types of samples.
  • 6.
    Slide 6 Dial 1-904-779-4740 for e-Seminar Audio Rapid Method Development Scheme – Low pH • Start at low pH for best peak shape, retention and long-term reproducibility • Select starting conditions • StableBond C18 or C8 for maximum lifetime – Rapid Resolution columns • pH 1 – 3 with 20 – 50 mM buffer for best peak shape • Acetonitrile or methanol – start high to scout • Adjust organic for maximum resolution and retention • Change organic if resolution not achieved – MeOH or ACN • Change bonded phase if resolution not achieved – SB-CN, SB- Phenyl, SB-C3 • Use elevated temperature to reduce analysis time further
  • 7.
    Slide 7 Dial 1-904-779-4740 for e-Seminar Audio Rapid Method Development Scouting Chromatograms on StableBond-C18 Rapid Resolution Columns 0.0 0.5 1.0 1.5 2.0 2.5 Time (min) 0.0 0.5 1.0 1.5 2.0 2.5 Time (min) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 Time (min) 5 4 3 2 1 A : B 20 : 80 40 : 60 60 : 40 Columns: Zorbax Rapid Resolution StableBond SB-C18 4.6 x 75 mm, 3.5 µ µ µ µm Mobile Phase: A: 25 mM NaH2PO4, pH 3.0 B: ACN Flow Rate: 2.0 mL/min. Temperature: 35°C UV Detection: 254 nm Sample: Cardiac Drugs
  • 8.
    Slide 8 Dial 1-904-779-4740 for e-Seminar Audio Rapid Method Development Scouting Chromatograms Change Organic Modifier Column: Zorbax Rapid Resolution SB-C18, 4.6 x 75 mm, 3.5 µ µ µ µm Mobile Phase: A: 25 mM NaH2PO4 , pH 3.0 B: MeOH Flow Rate: 2.0 mL/min Temperature: 35°C Detection: UV 254 nm Sample: Cardiac Drugs 1. Diltiazem 2. Dipyridamole 3. Nifedipine 4. Lidoflazine 5. Flunarizine Time(min) 1 2 3 4 5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Time(min) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Time(min) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 1 2 3 4 5 20 : 80 30 : 70 40 : 60 • Changing organic modifier can alter selectivity and improve peak shape. A : B
  • 9.
    Slide 9 Dial 1-904-779-4740 for e-Seminar Audio Rapid Method Development Process Optimizes Separation on StableBond-C18 at Low pH • A Rapid Resolution SB-C18 column at low pH was used to develop this thorough and rapid analysis of steroids and impurities following the rapid method development process. Column: Zorbax Rapid Resolution SB-C18, 4.6 x 75 mm, 3.5 µ µ µ µm Mobile Phase: 50% ACN 50% 20 mM NaH2PO4, pH 2.8 Flow Rate: 1.0 mL/min Temperature: RT Detection: UV 254 nm Sample: 1. Estradiol 2. Ethynylestradiol 3. Dienestrol 4. Norethindrone 1 2 3 4 0 2 4 8 10 6 12 Time(min)
  • 10.
    Slide 10 Dial 1-904-779-4740 for e-Seminar Audio Column: Zorbax SB-C18, 4.6 x 75 mm, 5 µ µ µ µm Mobile Phase: 74% 7.4 mM hexane sulfonate and 0.07% phosphoric acid 26% MeOH Flow Rate: 1 mL/min Detection: UV 280 nm Sample: B Vitamins min. 001599S1.PPT Method Optimization High Temperature Low Temperature Increase Temperature to Reduce Analysis Time • Agilent instruments provide reliable temperature control to 80 - 90°C allowing method development flexibility.
  • 11.
    Slide 11 Dial 1-904-779-4740 for e-Seminar Audio Rapid Method Development Scheme – Mid pH • Try pH 7 (pH 6 – 9) with Eclipse XDB-C18 or C8 and follow same process, selecting Eclipse XDB- Phenyl as alternate bonded phase • Use temperature to reduce analysis time further
  • 12.
    Slide 12 Dial 1-904-779-4740 for e-Seminar Audio Eclipse XDB-C18 is Bonded Phase Choice for High Resolution at Mid pH SB-C18, 4.6 x 75 mm, 3.5 µ µ µ µm Eclipse XDB-C18, 4.6 x 75 mm, 3.5 µ µ µ µm Mobile Phase: 20% Methanol: 80% 20 mM phosphate buffer + (10 mM TEA @ pH 7) Flow Rate: 1.0 mL/min Temperature: RT Detection: UV 254 nm Sample: 1. Nizatidine 2. Famotidine 3. Cimetidine 4. Pirenzipine • This sample is only resolved at mid pH on Eclipse XDB-C18 following rapid method development process. pH 3 pH 7 Time(min) 0 2 2 1 3 4 4 6 8 0 2 4 6 8 10 12 14 Time(min) 2 1 3 4
  • 13.
    Slide 13 Dial 1-904-779-4740 for e-Seminar Audio Break Number 1 For Questions and Answers Press *1 on Your Phone to Ask a Question
  • 14.
    Slide 14 Dial 1-904-779-4740 for e-Seminar Audio Choose Special Bonded Phases for Difficult Samples • Sample 1 – Highly basic compounds • May exhibit poor peak shape • May be difficult to retain • Column choice 1 – Bonus-RP for better peak shape • Column choice 2 – Extend-C18 at high pH for better retention and peak shape • Sample 2 – Highly polar compounds • May be difficult to retain • May require high aqueous mobile phases • Column choice 1 – SB-Aq for better retention with high aqueous mobile phases • Column choice 2 – Bonus-RP for use with high aqueous mobile phases
  • 15.
    Slide 15 Dial 1-904-779-4740 for e-Seminar Audio 0 1 2 3 4 5 6 7 8 9 10 Time (min) 0 1 2 3 4 5 6 Time (min) 2 3 1 1 2 3 Sample 1: Highly Basic Compounds Select Bonus-RP for Improved Peak Shape B. Bonus-RP A. Alkyl C8 TF 1. 1.1 2. 1.1 3. 1.1 TF 1. 1.8 2. 1.7 3. 2.1 Column: 4.6 x 150 mm, 5 µm Mobile Phase: A: 75% 25 mM NH4Ac, pH 5.5 : 25% ACN B: 80% 25 mM NH4Ac, pH 5.5 : 20% ACN Flow Rate: 1.5 mL/min Detection: UV 254 nm Sample: 1. Doxylamine 2. Chlorpheniramine 3. Triprolidine
  • 16.
    Slide 16 Dial 1-904-779-4740 for e-Seminar Audio Plates 1. 4903 2. 7621 3. 7567 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 2 1 3 Time (min) Sample 1: Highly Basic Compounds Extend-C18 Improves Retention and Efficiency of Procainamides at High pH Column: Zorbax Extend-C18, 4.6 x 150 mm, 5 µ µ µ µm Mobile Phase: See Below Flow Rate: 1.0 mL/min Detection: UV 254 nm Temperature: RT Sample: 1. Procainamide pKa 9.2 2. N-acetylprocainamide 3. N-propionylprocainamide Plates 1. 9475 2. 8823 3. 8794 50% 20 mM TEA, pH 11: 50% MeOH 50% 25 mM Na2HPO4, pH 7.0 : 50% MeOH 0.0 2.5 5.0 7.5 2 1 3 Time (min) • High efficiency improves resolution and column lifetime and the increase in retention results in a more rugged method.
  • 17.
    Slide 17 Dial 1-904-779-4740 for e-Seminar Audio Sample 2: Highly Polar Compounds Select New ZORBAX SB-Aq Columns for Method Development in High Aqueous Mobile Phases • Good retention for polar compounds in high aqueous mobile phases • Reproducible retention without “phase collapse” • Different selectivity vs. conventional C18 columns • Highly, stable at low pH and high temperature (up to 80°C)
  • 18.
    Slide 18 Dial 1-904-779-4740 for e-Seminar Audio Sample 2: Highly Polar Compounds ZORBAX SB-Aq Provides Good Retention of Polar Compounds 16 0 2 4 6 8 Time (min) 10 12 14 2 1 3 4 5 Columns: Zorbax SB-Aq, 4.6 x 150 mm, 5 µ µ µ µm Mobile Phase: 90% 0.2% TFA 10% ACN Temperature: 25°C Flow Rate: 1.5 mL/min. Detection: UV 254 nm Sample: 1. Maleate 2. Phenylephrine 3. Phenylpropanolamine 4. Pyrilamine 5. Chlorpheniramine • These small polar compounds are difficult to retain on most columns. • The SB-Aq provides excellent retention with a 90% aqueous mobile phase.
  • 19.
    Slide 19 Dial 1-904-779-4740 for e-Seminar Audio Sample 2: Highly Polar Compounds ZORBAX SB-Aq Provides Good Retention of Water Soluble Vitamins without Ion Pairing Time (min) 1 2 3 4 0.0 0.5 1.0 1.5 2.0 Columns: Zorbax SB-Aq, 4.6 x 150 mm, 5 µ µ µ µm Mobile Phase: 95% 0.1% TFA 5% MeOH Temperature: 35°C Flow Rate: 2.0 mL/min. Detection: UV 254 nm Sample: 1. Thiamine – B1 2. Nicotinic Acid 3. Pyridoxine – B6 4. Niacinamide – B3 • The SB-Aq column provides good retention of these polar compounds without ion pairing. • The result is a simpler method without the reproducibility problems associated with ion pairing.
  • 20.
    Slide 20 Dial 1-904-779-4740 for e-Seminar Audio Sample 2: Highly Polar Compounds ZORBAX SB-Aq Provides Good Retention of NADH/NAD+ with LC/MS Compatible Mobile Phase 1 2 3 Time (min) 0 2 4 6 8 Columns: Zorbax SB-Aq, 4.6 x 150 mm, 5 µ µ µ µm Mobile Phase: 94% 10 mM ammonium acetate, pH 5.5 6% MeOH Temperature: 25°C Flow Rate: 1.0 mL/min. Detection: 254 nm Sample: 1. Uracil 2. NADH 3. NAD+ • These coenzymes are difficult to retain and analyze by LC/MS. • The SB-Aq provides baseline resolution without ion pairing.
  • 21.
    Slide 21 Dial 1-904-779-4740 for e-Seminar Audio Rapid Resolution Columns Reduce Method Development and Analysis Time • Rapid Resolution columns (3.5 µm) are available in many configurations • Available in analytical, narrow bore, microbore, and capillary internal diameters for compatibility with any sample size. • Semi-preparative and preparative columns use 5 µm particles. • Rapid Resolution columns reduce isocratic and gradient run times with: • Shorter column lengths • Higher flow rates • Optimized HPLC instrument
  • 22.
    Slide 22 Dial 1-904-779-4740 for e-Seminar Audio Choose Column Configuration for Application Large scale prep Small scale prep (mg) Analytical Analytical High sensitivity LC/MS Higher sensitivity LC/MS Max sensitivity LC/MS Applications 20 – 60 mL/min 5, 7 50 – 250 21.2 Preparative 4 – 10 mL/min 5 50 – 250 9.4 Semi-prep 1 – 4 mL/min 3.5, 5 15 – 250 4.6 Analytical 0.3 – 1.0 mL/min 3.5, 5 150, 250 3.0 Solvent Saver 0.1 – 0.3 mL/min 3.5, 5 15 – 150 2.1 Narrow Bore 30 – 60 µL/min 3.5, 5 30 – 150 1.0 MicroBore 1 – 10 µL/min 3.5, 5 35 – 250 0.3, 0.5 Capillary Flow Rate Ranges Particle Sizes (µ µ µ µm) Lengths (mm) I.D. (mm) Column Type
  • 23.
    Slide 23 Dial 1-904-779-4740 for e-Seminar Audio High Resolution with Rapid Resolution Columns Isocratic Separation of Aspartame Column: Zorbax StableBond SB-C18 Mobile Phase: 85% Water with 0.1% TFA : 15% Acetonitrile Flow Rate: 1.0 mL/min. Temperature: 35°C Sample: 1. Phenylalanine 2. 5-Benzyl-3,6-dioxo-2-piperazine acetic acid 3. Asp-phe 4. Aspartame 4.6 x 150 mm, 5 µ µ µ µm 4.6 x 75 mm, 3.5 µ µ µ µm 4.6 x 50 mm, 3.5 µ µ µ µm 4.6 x 30 mm, 3.5 µ µ µ µm 4.6 x 15 mm, 3.5 µ µ µ µm Time (min) 0 1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4
  • 24.
    Slide 24 Dial 1-904-779-4740 for e-Seminar Audio 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Time (min) 5 4 3 2 1 0 1 2 3 4 5 6 Time (min) 5 4 3 2 1 Rapid Resolution Columns Reduce Isocratic Analysis Time by 50% or More Mobile Phase: 45% 25 mM NaH2PO4, pH 3.0 55% MeOH Flow Rate: 2.0 mL/min. Temperature: 35°C Detection: UV 254 nm Sample: Cardiac Drugs 1. Diltiazem 2. Dipyridamole 3. Nifedipine 4. Lidoflazine 5. Flunarizine Rapid Resolution StableBond SB-C18 4.6 x 75 mm, 3.5 µm StableBond SB-C18 4.6 x 150 mm, 5 µm Analysis Time: 5.2 min Analysis Time: 11.7 min
  • 25.
    Slide 25 Dial 1-904-779-4740 for e-Seminar Audio Higher Flow Rates Reduce Analysis Time with Rapid Resolution Columns Column: Zorbax Rapid Resolution StableBond SB-C18, 4.6 x 30 mm, 3.5 µ µ µ µm Sample: 1. Acetaminophen (4-acetamidophenol) 2. Caffeine 3. 2-Acetamidophenol 4. Acetanilide 5. Acetylsalicylic acid 6. Salicylic acid 7. Acetophenetidin 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Time (min) 1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7 2 mL/min 3 mL/min 4 mL/min Pressure 90 bar 133 bar 181 bar 2650 psi Rs2,1 = 2.1 Rs2,1 = 2.2 Rs7,6 = 2.2 Rs7,6 = 2.2
  • 26.
    Slide 26 Dial 1-904-779-4740 for e-Seminar Audio at 0.2 mL/min at 1.0 mL/min Gradient Re-equilibration Times are Minimal on Short Rapid Resolution Columns Column Volume (Vm) and Equilibration Time Gradient Analysis Time = Run Time + Equilibration Time ( single step return) 4.6 x 50 0.5 mL 5 min 4.6 x 30 0.3 mL 3 min 4.6 x 15 0.15 mL 1.5 min 4.6 x 150 1.54 mL 15 min 2.1 x 50 0.10 mL 5 min 60 sec 2.1 x 30 0.06 mL 3 min 36 sec 2.1 x 15 0.03 mL 1.5 min 18 sec Column Internal Equilibration Time Dimension (mm) Volume (Vm) at 1.0 mL/min (Vm x 10 x F)
  • 27.
    Slide 27 Dial 1-904-779-4740 for e-Seminar Audio Length = 50 mm, 3.5 µ µ µ µm tG = 6 min Short, Rapid Resolution Columns Reduce Gradient Analysis Time Gradient Time α α α α Column Length Column: Zorbax Eclipse XDB-C8, 4.6 mm i.d. Gradient: 45-90% B in tG minutes Mobile Phase: A: 25 mM Na2HPO4, pH 3 B: Methanol Temperature:35°C Flow Rate: 1.0 mL/min Sample: Cardiac Drugs: 1. Diltiazam 2. Dipyridamole 3. Nifedipine 4. Lidoflazine 5. Flunarizine Length = 75 mm, 3.5 µ µ µ µm tG = 9 min 0.0 2.5 5.0 7.5 Time (min) 0.0 2.5 5.0 Time (min) 1 2 3 4 5 1 2 3 4 5
  • 28.
    Slide 28 Dial 1-904-779-4740 for e-Seminar Audio Rapid Gradient CombiChem Analysis 1.5 min Injection-to-Injection mAU min 0 0.2 0.4 0.6 0.8 1 Column: Zorbax SB-C18, 2.1 x 30 mm, 3.5µm Flow Rate: 0.832mL/min Temperature: 20°C Mobile Phase Gradient A=0.1% Formic Acid, H2O; B=0.1%Formic Acid, ACN; 30-98 %B in 0.6 min P1-A-02 P1-A-03 P1-A-04 P1-A-05 P1-A-07 P1-A-08 P1-A-09 P1-A-10 P1-B-01 P1-B-02 Well-Plate Position 1 min
  • 29.
    Slide 29 Dial 1-904-779-4740 for e-Seminar Audio 1 2 3 4 5 1 2 3 4 5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Time (min) 0.0 0.5 1.0 1.5 2.0 Time (min) F = 3.0 mL/min tG = 2 min F = 2.0 mL/min tG = 3 min Increasing Flow Rate Reduces Gradient Analysis Time Further Column: Zorbax Eclipse XDB-C8 4.6 x 50 mm, 3.5 µ µ µ µm Gradient: 45-90% B in tG minutes Mobile Phase: A: 25 mM Na2HPO4, pH 3 B: MeOH Flow Rate: 1.0 mL/min Temperature: 35°C Sample: Cardiac Drugs 1. Diltiazam 2. Dipyradamole 3. Nifedipine 4. Lidoflazine 5. Flunarizine If tG x F = constant then the elution pattern is unchanged
  • 30.
    Slide 30 Dial 1-904-779-4740 for e-Seminar Audio Break Time For Questions and Answers Press *1 on Your Phone to Ask a Question
  • 31.
    Slide 31 Dial 1-904-779-4740 for e-Seminar Audio HPLC Instrument Optimization Increases Efficiency and Decreases Method Development and Analysis Time • Choose the best column configuration for your instrument type and application (i.e. Capillary LC for highest sensitivity or compatibility with LC/MS) • Instrument optimization can improve efficiency for isocratic and gradient separations • New instrument features can help you reduce analysis time
  • 32.
    Slide 32 Dial 1-904-779-4740 for e-Seminar Audio Choose HPLC Instrument for Application 1. Purificatio n 1. Analytical 2. MS compatible 1. Sample limited 2. Most MS compatible Applications Preparative and CombiChem prep 1 - 100 mL/min Semi-prep, Prep Prep LC Standard analytical more semi-prep than low volume Work at 3- 4mL/min 0.05 – 5 mL/min* binary 0.2 – 10 mL/min* isocratic/quaternar y Narrow-bore, Solvent Saver, Analytical, Semi-prep LC 5 µL dwell volume, best low volume gradient reproducibility 1 µL/min – 2.5 mL/min Capillary, MicroBore, Narrow-bore, Solvent Saver, Analytical Capillary LC Why? Flow Rate Range Compatible Columns Agilent 1100 Type * Optimum range
  • 33.
    Slide 33 Dial 1-904-779-4740 for e-Seminar Audio Optimizing Results with Rapid Resolution Columns Isocratic Separations • Minimize extra column volume to minimize band broadening • Keep the injection volume small (< 5 µL) • Use semi-micro or micro detector cells ( 5 µL or less) • Use 0.12 mm i.d. tubing (0.005”) • Prepare the sample in an injection solvent with the same or weaker solvent strength than the mobile phase • Overlap injections • Select the Agilent 1100 Capillary HPLC for capillary and microbore columns • Select correct detector response time for rapidly eluting peaks
  • 34.
    Slide 34 Dial 1-904-779-4740 for e-Seminar Audio Time (min) 0 2.5 5 2 1 3 4 Time (min) 0 2.5 5 2 1 3 4 Effect of Detector Cell Volume on Peak Width 4.6 x 75 mm, 3.5 µ µ µ µm Column: Zorbax StableBond SB-C18 Mobile Phase: 85% H20 with 0.1% TFA : 15% ACN Flow Rate: 1.0 mL/min Temperature: 35°C Sample: 1. Phenylalanine 2. 5-benzyl-3,6-dioxo-2-piperazine acetic acid 3. Asp-phe 4. Aspartame N 1. 8700 2. 8700 3. 8300 4.10,000 Microflow Cell - 2.5 µ µ µ µL Standard Flow Cell - 8 µ µ µ µL N 1. 6200 2. 6700 3. 7000 4. 9250 1. -29% 2. -23% 3. -16% 4. -7%
  • 35.
    Slide 35 Dial 1-904-779-4740 for e-Seminar Audio Optimizing Results with Rapid Resolution Columns Gradient Separations • Minimize dwell volume – includes mixing volume and injector volume • Use high pressure mixing and reduce mixer volume • Run autosampler in bypass – reduce dwell volume by 300 µL • Minimize extra column volume • Overlap injections • Select correct detector response time for rapidly eluting peaks • Select Agilent 1100 Capillary HPLC with 5 µL dwell volume for capillary and microbore columns
  • 36.
    Slide 36 Dial 1-904-779-4740 for e-Seminar Audio Compound rsd RT for 10 runs rsd Area for 10 runs Dimethylphthalate 0.09 0.98 Diethylphthalate 0.10 0.87 Biphenyl 0.08 1.83 o-Terphenyl 0.07 0.90 Compound rsd RT for 10 runs rsd Area for 10 runs Dimethylphthalate 1.09 5.91 Diethylphthalate 0.55 5.64 Biphenyl 0.52 5.30 o-Terphenyl 0.43 6.87 min 0 0.2 0.4 0.6 0.8 1 1.2 1.4 Norm. -20 0 20 40 60 80 100 min 0 0.2 0.4 0.6 0.8 1 1.2 1.4 Norm. 0 0.02 0.04 0.06 0.08 0.1 0.12 Low pressure mixing, Brand X Reproducible, Fast Gradient Analysis with High Pressure Mixing Column: Zorbax StableBond C18, 2.1 x 50 mm Agilent 1100, binary pump high pressure mixing
  • 37.
    Slide 37 Dial 1-904-779-4740 for e-Seminar Audio Reduce Gradient Analysis Time and Dwell Volume with Bypass of the Sample Loop mAU 0 100 200 mAU 0 100 200 min 0 0.2 0.4 0.6 0.8 1 1.2 Agilent 1100 WPS Auto Bypass By-Pass On Column: Zorbax SB-C18, 4.6 x 30 mm, 3.5µm Flow Rate: 0.832 mL/min Temperature: 20°C Mobile Phase Gradient: A= 0.1% Formic Acid, H2O; B = 0.1% Formic Acid, ACN; 30-98 %B in 0.6 min Agilent 1100 WPS Auto Bypass By-Pass Off Time Savings
  • 38.
    Slide 38 Dial 1-904-779-4740 for e-Seminar Audio min 0.2 0.4 0.6 0.8 1 1.2 1.4 mAU 0 250 500 750 1000 1250 1500 1750 2000 Overlapped Injection Cycle time is ~ 2min 12sec Total time for 20 runs: 44 min 22 sec Standard Injection Cycle time is ~ 2min 57sec Total time for 20 runs: 58 min 20 sec Switch of injection valve Run time: 1.5 min / Post time: 0.5 min for both runs Overlapped Injection Reduces Analysis Time • Overlapped injections – sample is drawn up during previous injection – reduce gradient and isocratic run times.
  • 39.
    Slide 39 Dial 1-904-779-4740 for e-Seminar Audio Effect of Detector Response Time on Ultra-Fast Gradient Analyses 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 Time(min) 1.0 Agilent 1100 DAD Agilent 1100 WPS with ADVR Column: Poroshell 300SB-C18 2.1 x 75 mm, 5 µm Mobile Phase: A: 95% H2O, 5% ACN with 0.1% TFA B: 5% H2O, 5% ACN with 0.1% TFA Flow Rate: 2 mL/min Temperature: 70°C Detector: UV 215 nm Piston stroke: 20 Sample: 1. Neurotensin 3. Lysozyme 2. RNaseA 4. Myoglobin 0.1 sec 0.2 sec 0.5 sec 1.0 sec 1st peak = 1.2 sec At 20 pts/sec = 24 pts/sec Response Time 2.0 sec 1st peak = 1.2 sec At 5 pts/sec = 6 pts/sec • You may have to adjust the response rate of your detector for rapid peak detection.
  • 40.
    Slide 40 Dial 1-904-779-4740 for e-Seminar Audio Summary • C18 and C8 bonded phases are the best for initial rapid method development with typical sample types • Choosing the most sample appropriate bonded phase and using special, targeted bonded phases, such as SB-Aq for polar, difficult to retain compounds can decrease method development time • Rapid Resolution columns are needed to reduce method development time • Rapid Resolution columns reduce both gradient and isocratic analysis time and permit high throughput rapid analysis • Rapid Resolution columns can work effectively with your HPLC • HPLC instruments may have additional capabilities to speed up method development and reduce analysis time
  • 41.
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    Slide 42 Dial 1-904-779-4740 for e-Seminar Audio HPLC Column Technical Support 800-227-9770 (phone: US & Canada)* 302-993-5304 (phone)* * Select option 4, then option 2. 916-608-1964 (fax) www.agilent.com/chem