11:00 a.m. EST Telephone Number: Chair Person: Tim Spaeder

Transcription

1 Stephen W. Coates LC Columns Marketing Specialist Consumables and Accessories Business Unit May 17, 2 High-Throughput Separations for QA/QC, LC/MS, LC/MS/MS, Combinatorial Chemistry, and Rapid Method Development 11: a.m. EST Telephone Number: Chair Person: Tim Spaeder

2 High-Throughput Separations for QA/QC, LC/MS, LC/MS/MS, Combinatorial Chemistry, and Rapid Method Development Slide 4

3 Why Shorter, Faster Columns? Need high-throughput/time LC/MS, LC/MS/MS Combinatorial chemistry QA/QC Rapid method development Save solvents Lower overall labor cost Need to use current equipment 129P1.PPT

4 Getting the Same Resolution With a Shorter Column and Smaller Particles 5 µm 25cm length = 3.5 µm 15cm length Resolution 1291P1.PPT

5 ZORBAX Rapid Resolution Columns Rapid Resolution columns use 3.5µm particles Use of smaller particle size allows use of shorter columns without loss of efficiency Use 2 micron (standard) frit to minimize clogging Column Length may be shortened to 15mm, 75mm, 5mm, 3mm, 15mm Time and solvent savings of from 4 to 9% Available in both 8Å and 3Å pore size Slide 7

6 Increasing Analysis Speed in HPLC While Maintaining Resolution 5µm 3.5µm 5µm 3.5µm Dimension 25 x 4.6 mm 15 x 4.6 mm 15 x 4.6 mm 75 x 4.6 mm Analysis Time (min) 3 min. 4% reduction 18 min. 18 min. 5% reduction 9 min. Solvent Waste, ml 3 ml 4% reduction 18 ml 18 ml 5% reduction 9 ml N Resolution α N 1/2 2, Unchanged 2, 12, 9% Difference 1, 788S1.PPT

7 3.5µm Rapid Resolution with Protein/Peptide Sample ZORBAX 3SB-C8 4.6 x 25 mm, 5 µm Rapid Resolution 4.6 x 15 mm, 3.5 µm Mobile Phase: A: 95:5, Water : ACN with.1% TFA B: 5:95, 28 Water 3: ACN with.85% TFA % B in 5 min. ÃPLQ 1-6% B in 3 min. ÃPLQ 1. Met-enkephalin 2. Leu-enkephalin 3. Angiotensin II 4. Neurotensin 5. RNase 6. Insulin (Bov) 7. Lysozyme 8. Calmodulin 9. Myoglobin 1. Carbonic Anhydrase Conditions: Rapid Resolution 4.6 x 5 mm, 3.5 µm 1-6% B in 1 min. Flow: 1. ml/min Detection: 215nm Sample: 1-1µg protein (1µL inj.) in 6M Guanidine HCL, ph7. ÃPLQ P1.PPT Slide 9

8 Maintaining Resolution and Shortening Run Time: Separation of Aspartame and Metabolites With Columns of 5 µm and 3.5 µm Particles µm Detector Response 1 A µm Mobile Phase: 15% ACN / 85%.1% trifluoroacetic acid Flow rate: 1. ml/min.; Temp: Ambient A. 4.6 x 15 cm Zorbax SB-C18, 5 µm particles B. 4.6 x 7.5 cm Zorbax SB-C18, 3.5 µm particles 1. Phenylalanine 2. 5-Benzyl-3,6-dioxo-2- piperazine-acetic acid 3. Asp-phe 4. Aspartame Retention Time, (min) Slide 1 122P1.PPT

9 Improved Throughput of deoxynucleosides Using New Column Configurations Zorbax SB-CN mau 3 2 C G T A 5 µm 4.6 x 15 mm LC: HP19 Flow: 1. ml / min. A:.1% TFA B: 97.5% MeOH : 2.5% H2O (.1% TFA) Temp.: 3 C Detec.: 254 nm 1 mau min 3.5 µm 4.6 x 5 mm 1 2 min min 1575S1.PPT

10 Increasing Analysis Speed in HPLC with Minimal Loss of Resolution 5µm 3.5µm 5µm 3.5µm Dimension 15 x 4.6 mm 75 x 4.6 mm 15 x 4.6 mm 15 x 4.6 mm Analysis Time (min) 18 min. 5% reduction 9 min. 18 min. 9% reduction 1.8 min. Solvent Waste, ml 18 ml 5% reduction 9 ml 18 ml 9% reduction 1.8 ml N 12, 12, 2, { Resolution α N 1/2 9% Difference 59% Difference {1, 1582S1.PPT

11 Match the Column to Desired Resolution Rapid Separation of Analgesics SB-C x 15 mm all 7 components resolved in less than 1 min 4.6 x 3 mm Conditions: LC: Agilent 11 Columns: ZORBAX SB-C18, 3.5 µm Mobile Phase: 1 mm octane sulfonic acid, Na salt, ph 2.5 : ACN (8:2) UV: 275 nm; Flow: 2. ml / min.; 7 C Inj Vol: 1 µl 4.6 x 5 mm Absorbance Sample: 1. Acetaminophen (4-acetamidophenol) 2. Caffeine 3. 2-Acetamidophenol 4. Acetanilide 5. Aspirin (acetosalicyclic acid) 6. Salicylic acid 7. Phenacetin (acetophenetidin) 4.6 x 75 mm 4.6 x 15 mm min Slide P1.PPT

12 Obtaining Full Potential of Ultra-Fast HPLC Using a Shorter Column ANALGESICS 1. Acetaminophen (4-acetamidophenol) 2. Caffeine 3. 2-Acetamidophenol 4. Acetanilide 5. Aspirin (acetosalicyclic acid) 6. Salicylic acid 7. Phenacetin (acetophenetidin) mau mau sec 4.6 x 3 mm 4.6 x 15 mm min 7 ZORBAX SB-C18, 3.5 µm, F = 2 ml/min Slide 14

13 Calculated Effect of Particle Size on Chromatographic Parameters dp µm N = 13, k(last peak) = 5 Flow ml/min L cm Vm ml P psi tr min Vp (k=1) µl column i.d. = 4.6mm mobile phase = 1% H 2 O 783P1.PPT

14 Practical Particle Diameter is 3µm Good efficiency up to 2, + plates with 15mm column Reasonable pressure drop less than 3 psi with 4.6mm i.d. Generates peak volumes that allow use of standard HPLC equipment If particle size is increased slightly to 3.5 µm, then a 2 µm frit can be used instead of the prone to plug.5 µm frit 784P1.PPT

15 Particle Size Distribution for ZORBAX Rx-SIL 82P1.PPT

16 Comparison of Ultra-Fast Ambient Separations Using Totally Porous 3.5 mm and Non-Porous 1.5 mm Silicas 2 1 ZORBAX SB-C18, 2 ml/min Sample: 1. Methylparaben 2. Ethylparaben 3. Propylparaben 4. Butylparaben mau ml/min 4 Conditions: LC: Agilent 11 Columns: Zorbax SB-C18, 3.5 µm, 4.6 x 3 mm Mobile Phase:.1% H 3 PO 4 : ACN (5:5) UV: 254 nm; Flow: 2. ml / min.; ambient Inj Vol: 1 µl NPS ODS-1, 1 ml/min LC: Hewlett-Packard HP15 Columns: Micra NPS ODS-1, 1.5 µm, 4.6 x 33 mm Mobile Phase:.1% H 3 PO 4 : CH 3 CN (8:2) UV: 254 nm; Flow: 1. ml / min. Temp: ambient 1 2 min 1654S1.PPT Slide 19

17 Use 3.5 µm Totally Porous Silica to Full Potential mau non-porous silica, 4.6 x 33 mm, 1.5 µm ZORBAX SB-C18, 4.6 x 3 mm, 3.5 µm mau 25 2 ACN: 1% H3PO4 (28:72), F= 1mL/min 8 6 ACN: 1% H3PO4 (28:72), F= 1mL/min P= 247 bar 4 2 P= 6 bar min min mau ZORBAX SB-C18, 4.6 x 3 mm, 3.5 µm ACN: 1% H3PO4 (5:5), F= 2mL/min P= 16 bar 3.5 µm totally porous silica lower back pressure higher flow = reduced run times 1.5 µm non-porous silica pressure reduces practical flow rate small-pore frit is prone to blockage min Slide 2

18 Instrument Contributes to Gradient Performance Mobile Phase { Dwell Volume Extra Column Volume Data Device ECV = sample volume + connecting tube volume + fitting volume + detector cell volume Dwell Volume = volume from formation of gradient to top of column 786P1.PPT

19 Effect of Extra-Column Volume Extra- Column Volume 1 µl 2.1 x 15 mm F =.2 ml / min. Loss of Resolution 2 µl Time, min. 115P1.PPT

20 Approximate Plate Number (N) and Void Volume (Vm) for 5-µm and 3.5-µm Columns Column Size Particle Size Plate Number (N) Void Volume (V m ) 4.6 x 25 mm 5-mm 2, 2.5 ml 4.6 x 15 mm 5- m m 12, 1.5 ml 4.6 x 15 mm 3.5- m m Rapid Resolution 2, 1.5 ml 4.6 x 75 mm 3.5- m m Rapid Resolution 1,.75 ml 4.6 x 5 mm 3.5- m m Rapid Resolution 6,.5 ml 4.6 x 3 mm 3.5- m m Rapid Resolution 3,6.3 ml 4.6 x 15 mm 3.5- m m Rapid Resolution 2,.15 ml Slide 23

21 Obtaining Full Potential in Ultra-Fast HPLC Role of Response Time Rs = 2.77 N = 23 mau Response Time HP11 DAD.1 sec mau sec mau.5 sec Rs =.86 N = 26 mau mau 1 sec 2 sec mau sec min ZORBAX SB-C18, 3.5 µm, 4.6 x 3 mm, F=2 ml/min Slide 24

22 Obtain Full Potential in Ultra-Fast Analyses Using Proper Data-Collection Rate At least 9 Data Points Necessary for Properly Defined Peaks mau 4 2 mau pts collected 5 pts collected 1 pts collected 12 pts collected min Data Rate 2 Hz Res. Time.1 sec PW <.1 min Data Rate 2.5 Hz Res. Time 2 sec PW >.1 min ZORBAX XDB-C8, 4.6 x 3 mm, 3.5 µm, F= 2 ml/min Slide 25

23 Sensitivity: Effect of Column Configuration on Peak Height Ciprofloxacin, k' = 1.7; ZORBAX SB-C8; 6 C Mobile Phase, 2/8 acetonitrile/.1% TFA; 2 µl 8P1.PPT

24 Effect of Particle Size on Plate Number and Flow Rate ZORBAX SB-C8 Columns; Column i.d., 4.6mm; Ciprofloxacin; 22 C Mobile Phase, 2% ACN / 8% Aqueous.1% TFA 81P1.PPT

25 Gaining Full Potential by Increasing Flow Rate Pressure 9 bar mau Rs 2,1 = 2.2 Rs 7,6 = 2.2 Flow Rate 2 ml/min 133 bar mau ml/min 181 bar mau Rs 2,1 = 2.1 Rs 7,6 = sec min 4 ml/min ZORBAX SB-C18, 3.5 µm, 4.6 x 3 mm Slide 28

26 Ion-Pairing Chromatography at Elevated Temperature for Significantly Reduced Run Times Low Temperature Method Optimization Isocratic Ion-Pairing ZORBAX SB-C18 (4.6 x 75 mm) mixture containing 7.4 mm hexane sulfonate and.7% phosphoric acid Sample: B Vitamins High Temperature min. Slide S1.PPT

27 Improved Throughput of deoxynucleosides Using Optimal Bonded Phase (Selectivity) LC: HP19 Columns: 4.6 x 5, 3.5 µm Flow: 1. ml / min. A:.1% TFA B: 9% MeOH : 1% H2O (.1% TFA) Temp.: 3 C Detec.: 254 nm mau mau G C C T A G T 2 min A ZORBAX SB-C18 92% A : 8% B 6 min ZORBAX SB-CN 97.5% A : 2.5% B min 1574S1.PPT

28 LC for Combinatorial Chemistry 4-1, compounds/month Desire consensus method capable of separating a group of molecules of varying properties - nonideal method development May require follow-up methods capable of high resolution separation Goal is rapid resolution in 1-4 minutes Preparative separations usually 15-5 mg

29 Optimized Column Configuration and Operating Parameters for High-Throughput Analyses* Vendor A-C x 3 mm Flow: 4 ml / min. Gradient: -1% B / 2 min. Detect.: 22 nm A: 1/9, MeOH / H 2 O +.2%H 3 PO 4 B: 9/1, MeOH / H 2 O +.2% H 3 PO 4 2 mm each compound, 5µL inj. Relative Absorbance 1.2 min <1 min 3 min Eclipse XDB-C x 3 mm Eclipse XDB-C x 3 mm LC: 11 Flow: 4 ml / min. Gradient: -1% B / 2 min. Detect.: 22 nm LC: 11 Flow: 4 ml / min. Gradient: -1% B / 1 min. Detect.: 268 nm min * Note improved baseline for HP 11 DAD 1577S1.PPT

30 CombiHT Columns Used for 15-5 mg Semipreparative Separations A B C Collected Peak A. Analytical Separation of Crude Sample Column: (4.6 x 5mm), P/N: Det. (21nm) Mobile Phase: A=.1% TFA/H2O, B=.1%TFA/ACN Gradient: 1-5%B/1 min, F= 3mL/min B. Preparative Separation of Crude Sample (~35mg) Column: (21.2 x 5mm) Prep, P/N: Det. (21nm) Mobile Phase: A=.1% TFA/H2O, B=.1%TFA/ACN Gradient: 1-4%B/2 min, F= 3mL/min C. Fraction from Zorbax Combi HT Conditions: as in Panel A Slide 33

31 LC/MS Considerations and Rapid Resolution Chromatography Slide 35

32 Switching to an LC/MS-Compatible Mobile Phase Volatile Mobile Phase (LC/MS) Conditions: LC: Agilent 11 Columns: ZORBAX SB-C18, 3.5 µm, 4.6 x 3 mm Mobile Phase: H 2 O, ph 2.5 : ACN (86:14); 1% formic acid overall UV: 275 nm; Flow: 2. ml / min.; 7 C Inj Vol: 1 µl Absorbance 1min Non-Volatile Mobile Phase Conditions: LC: Agilent 11 Columns: ZORBAX SB-C18, 3.5 µm, 4.6 x 3 mm Mobile Phase: 1 mm octane sulfonic acid, Na salt, ph 2.5 : ACN (8:2) UV: 275 nm; Flow: 2. ml / min.; 7 C Inj Vol: 1 µl.5 1 min ANALGESICS 1. Acetaminophen (4-acetamidophenol) 2. Caffeine 3. 2-Acetamidophenol 4. Acetanilide 5. Aspirin (acetosalicyclic acid) 6. Salicylic acid 7. Phenacetin (acetophenetidin) Slide P1.PPT

33 Column Selection for LC-MS Applications Ideal Particle Size and Column Length Short Column Increased Throughput Single - Ion Monitoring (SIM) Mode Multiple - Ion Mode Simple Sample Easily Distinguishable M.W mm 3.5 µm Particle Increased Sensitivity (> peak heights) Increased Resolution (< peak widths) Increased Throughput (scale down) May need Single - Ion Monitoring (SIM) Mode Complex Sample (<Quenching) Isobaric Components 1578S1.PPT

34 Column Selection for LC-MS Applications Determine Ideal Column Diameter On the HP11 LC/MSD, most separations can be performed using a 4.6 mm i.d. column Use 3. and 4.6 mm i.d. columns for higher flow rates Use 1., 2.1, and 3. mm i.d. columns for increased sensitivity and lower flow rates Use smaller i.d. columns for sample-limited conditions 1581S1.PPT

35 Rapid Separation of Complex Anthocyanins Using LC/MS Anthocyanins are complex mixtures of pigments found in fruits and other plant parts Heterogeneity is generated as a result of the 6 base molecules and large number of possible substitutions The chromatograms are used as fingerprints to characterize plants extracts, for food products, and nutraceuticals MS is a powerful tool for investigating structural differences R OH HO A + O B R O - Glyc. OH Slide 39

36 Heterogeneity of Anthocyanins R OH HO A + O B R O - Glyc. OH Anthocyanidin R R Cyanidin OH H Delphinidin OH OH Malvidin OCH 3 OCH 3 Pelargonidin H H Peonidin OCH 3 H Petunidin H OCH P1.PPT

37 Comparison of Vaccinium Anthocyanin Profiles.8.7 Lowbush Cumberland 2X Highbush Coville V. Angustifolium V. Corymbosum Absorbance (52 nm) Lowbush Fundy 3127 V. Angustifolium 9DFLQLXPÃ \UWLOXV Bilberry 2634 V. Myrtillus (.1) Retention Time, min. 1423P1.PPT

38 Initial Anthocyanin Separation -- Zorbax SB-C18 Mobile Phase A=3% H 3 PO 4, B=1% MeOH 4.6 x 25 mm, 5µm Time Percent B 23% 26% 6% 4.6 x 15 mm, 3.5µm Time Percent B 23% 26% 6% 4.6 x 75 mm, 3.5µm Time Percent B 23% 26% 6% HWKRGÃGHYHORSPHQWÃXVHGÃ89ÃGHWHFWLRQ 852P1.PPT

39 Comparison of Column Configuration in Rapid LC/MS Analysis of Anthocyanins Relative Abundance Base-Peak Chromatograms x 15mm ZORBAX SB-C18 min 4.6 x 5mm ZORBAX SB-C min Multisegment Gradients of A: 5% formic acid, B: 1% MeOH

40 EICs (extracted ion chromatograms) Based on Anthocyanin Fragments Myrtillus extract on 5 mm column Malvidin m/z 331 Peonidin m/z 31 Petunidin m/z 317 Cyanidin m/z Delphinidin m/z 33 BPC min The MS detector allows component analysis without the same high resolution necessary for UV detection Slide 44

41 Detection of Minor Components Using DAD and MS Detection EIC m/z 31 P$8 UV λ max 519 nm PLQ QP 1RUP EIC m/z 31 m/z 55 ** MS à peonidin à peonidin 3-Acgal/glu PLQ P] MS detection reveals a minor sample component; while, the diode array further confirms its identity Slide 45

42 Summary Save time and money by choosing the shortest possible column with smallest particle size to achieve the desired separation 15- vs. 15-mm length 3.5µm vs. 5µm particles Because of its sensitivity and specificity, LC/MS benefits from short fast columns Adjust system parameters to obtain optimal separation response time data collection rate extra-column volume dwell volume flow rate vs. particle size column selectivity 1295P1.PPT