Automatic Combination of Orthogonal Separation Modes for Multiple Components in Mixtures

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1 Automatic Combination of Orthogonal Separation Modes for Multiple Components in Mixtures Thomas E. Wheat, Amanda B. Dlugasch, and Patricia R. McConville Waters Corporation IFPAC January 26, Waters Corporation 1

2 Value of LC for PAT The workhorse tool for determining product purity Specificity for multiple components Dynamic range for quantitative analysis of all components Methods can be used in process and cross over to QC no method transfer/re-validation LC often has simple linear calibration models the technique upon which spectroscopic quantitative models are built Chiral and achiral information possible with LC 2016 Waters Corporation 2

3 Challenges Facing LC in PAT Off-line analytical laboratories are the most common deployment Viewed as too slow for process analysis Too complicated for routine in-process monitoring Samples often require preparation prior to analysis Liquid chromatographic systems are not robust enough Complete Resolution May Require Method Development 2016 Waters Corporation 3

4 When is 2D Chromatography Necessary? Increased information and sample characterization Convert non-volatile mobile phases (i.e., phosphate buffers) to acquire MS information Improved selectivity needed Complex samples Peak co-elution Additional sensitivity needed Sensitivity may not be adequate for assay requirements Improved assay robustness needed Co-elution of matrix and analyte(s) of interest may provide inconsistent MS ionization 2016 Waters Corporation 4

5 Waters Multidimensional Capabilities: Functional Examples of 2D UPLC Trap and back-flush elution Improve assay robustness and increase sensitivity Trap and forward-flush elution Improve assay robustness Trap and back-flush elution with at-column dilution Reduce manual sample preparation, increase sensitivity and improve assay robustness Heart cutting Increase resolution and improve assay robustness Comprehensive 2D Increased sample characterization Parallel column regeneration Increase sample throughput 2016 Waters Corporation 5

6 Standard Two-Dimensional System 2016 Waters Corporation 6

7 Conventional Chromatography Sample in High Organic 2016 Waters Corporation 7

8 At-Column-Dilution: Sample Loading Sample in high organic carried to T by elution solvent or mobile phase T Mix with initial highly aqueous mobile phase 2016 Waters Corporation 8

9 At-Column-Dilution Sample Loading Completion of Sample Loading Concentration of sample at head of column Strong Solvent Gradient Elution Elution of Compounds with Gradient Elution of Strong Solvent 2016 Waters Corporation 9

10 At Column Dilution Permits injection of large volumes of strong solvents Improves both mass capacity and resolution Increases system ruggedness Extends column life 2016 Waters Corporation 10

11 Classic Configuration Chromatography in Dimension Waters Corporation 11

12 Classic Configuration Trapping in Dimension Waters Corporation 12

13 Classic Configuration Rinsing Trap 2016 Waters Corporation 13

14 Classic Configuration Elution in Dimension Waters Corporation 14

15 Multi-Dimensional System Selectable At-column Dilution On the right valve (VR) port 1, 5 and 6 are closed off with Pin Plugs Waters Corporation 15

16 Instrument Set-Up Columns: First-Dimension: BEH C x 50 mm, 1.7 um, p/n Second Dimension: BEH C x 50 mm, 1.7 um, p/n Trapping column: XBridge C 18 Direct Connect 2.1 x 30 mm, p/n Mobile phases: QSM: Solvent A: Water Solvent B: Acetonitrile Solvent C: 1% Formic Acid in Water BSM: Solvent A: 0.1% Ammonium Hydroxide in Water Solvent B: 0.1% Ammonium Hydroxide in Acetonitrile ISM: Water Needle Wash: 80/20 ACN/water Seal wash: 10% ACN Injection volume: 3.0 µl and 0.5 µl PDA: Scan: 210 to 400 nm Channel: 254 nm QDa: Scan: 100 to 650 Da Temperatures: Column 1: 40 o C Column 2: 40 o C Trap Column: Room Temperature 2016 Waters Corporation 16

17 Sample Mixture of the following standards: Waters UPC2 Standard Mix, p/n Waters Analgesic Mix Standard, p/n Waters ACQUITY/Quattro MS Start Up Solution Kit, p/n Final Sample Solution: 0.45 mg/ml Sulfadimethoxine 0.45 mg/ml Terfenadine 0.45 mg/ml Reserpine 0.45 mg/ml Acetaminophen 0.45 mg/ml Caffeine 90ug/mL Acetamidophenol 90ug/mL Acetanilde 90ug/mL Acetylsalicylic Acid 90ug/mL Phenacetin 0.20 mg/ml Salicylic Acid 0.20 mg/ml 3-benzoylpyridine 0.20 mg/ml Cortisone 0.20 mg/ml 4-nitroaniline 0.20 mg/ml 4,4 -biphenol 2016 Waters Corporation 17

18 Method Details Dimension 1-QSM Dimension 2-BSM ISM Valves Time Flow %A %B %C Time Flow %A %B Time Flow Time VL VR Waters Corporation 18

19 Dimension µl Injection Volume PDA: 254 nm with cut peak 2016 Waters Corporation 19

20 Dimension 1 Standard Separation 2016 Waters Corporation 20

21 Dimension µl Injection Volume PDA: 254 nm 2016 Waters Corporation 21

22 Dimension 1 Trapping Target Region 2016 Waters Corporation 22

23 Dimension 1 Cut Peak Cut Peak 2016 Waters Corporation 23

24 Dimension 1 Rinsing Trapped Analytes 2016 Waters Corporation 24

25 Elution From Trap with ACD Separation in Dimension 2 PDA VL ACD T ISM- Dilution Pump VR Waste Trap cartridge BSM Second dimension pump Dimension 1 FTN QSM First dimension pump Dimension 2 QDa 2016 Waters Corporation 25

26 Dimension 2 Separation of Trapped Analytes QDa: TIC Plot 2016 Waters Corporation 26

27 Replicated Dimension 2 Separation Extracted Ion Chromatograms Black= Sulfadimethoxine Blue= Phenacetin Green= 3-Benzoylpyridine 2016 Waters Corporation 27

28 Replicate Injections Sulfadimethoxine Area Average Std. Dev. Replicate 1 2.1E+08 Replicate 2 1.4E+08 Replicate 3 1.8E E E+07 Replicate 4 2.0E+08 Replicate 5 1.6E+08 Replicate 6 1.9E+08 Phenacetin Replicate 1 7.6E+07 Replicate 2 4.5E+07 Replicate 3 4.9E E E+07 Replicate 4 7.5E+07 Replicate 5 4.3E+07 Replicate 6 5.1E+07 3 Benzoylpyridine Replicate 1 1.1E+08 Replicate 2 6.3E+07 Replicate 3 7.6E E E+07 Replicate 4 1.1E+08 Replicate 5 6.6E+07 Replicate 6 8.2E Waters Corporation 28

29 Linearity: PDA with 2 nd Peak Cut Injection 1: 0.5µL Injection 2: 1.5µL Injection 3: 2.5µL Injection 4: 3.5µL Injection 5: 4.5µL Injection 6: 5.0µL 2016 Waters Corporation 29

30 Linearity: QDa XIC 195 Da: 2 nd Peak Injection 1: 0.5µL Injection 2: 1.5µL Injection 3: 2.5µL Injection 4: 3.5µL Injection 5: 4.5µL Injection 6 : 5.0µL 2016 Waters Corporation 30

31 Conclusions Complete Chromatographic Resolution Can Be Approached by Combining Orthogonal Modes Using a Multi-dimensional System At-column Dilution Can Be Used to Ensure Compatibility Between Chromatographic Dimensions System Fluidics and Control Can Be Configured such that Atcolumn Dilution Is Applied to Any or All Dimensions At-column Dilution Expands the Usable Dynamic Range of a Chromatographic System or Method 2016 Waters Corporation 31

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