System Design and Performance Overview

1. System Design and Performance Overview nano LC nano MDLC ht LC

2. nano LC • Nanoscale binary gradient chromatography • No flow splitting and dynamic flow control • Additional loading pump for high speed sample loading and de-salting

3. Nano-Flow HPLC • Active flow control of each mobile phase • Measurement of flows with active feedback • Accounts for changes in viscosity and back pressure in the separation column • No flow splitting • Direct measurement of column flow and back pressure • Reductions in mobile phase preparation and disposal • Rapid system response • Prompt step changes in flow or composition • High flow loading/washing and “peak parking” integral to system

4. Traditional Analytical vs. Capillary HPLC

5. Repeated Injections of BSA digest: chromatograms and column pressures • BSA tryptic digest • 150 x 0.075 mm 5um ProteoPep2 C18 column • water/acetonitrile gradient with 0.1% formic acid • 250 nL/min gradient • column back pressure shows run to run conditions of column

6. Dynamic Flow Control for Peak Parking • Rapid flow rate reduction (500-50 nL/min) • Gradient profile resumed after parking • Parking flow can be set at any rate • No additional hardware or plumbing • Triggered within software or by external trigger

7. Gradient separation of Cytochrome c tryptic digest Col: 150 x 0.100 mm m 3 m C18 Inj: 100 nL Flow rate: 300 nL/min Park rate: 50 nL/min Peak parking example PARK 214 nm Absorbance 0 5 10 15 20 25 Time (min)

8. XIC of +EPI: Exp 3, 784.0 to 784.9 amu Max. 3.8e6 cps. NO Peak Parking Intensity, cps < 30 sec 0.0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Time, min XIC of +EPI: Exp 3, 784.1 to 785.1 amu Max. 9.5e6 cps. Peak Parking Activated > 1.5 min Intensity, cps 0.0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Time, min Phosphopeptide Identification with Peak Parking • Mixture of a and b casein with BSA tryptic digests • Q-Trap triple quadrupole with linear trap • Neutral loss (49 amu) survey scan to identify phosphopeptides • Peak parking from 500 nL/min to 50 nL/min activated by MS software when NL detected

9. Increased Phosphopeptide ID with Peak Parking a-casein and its phosphopeptides identified with peak parking * Neutral Loss of 49 was used to target doubly charged phosphopeptides specifically

10. nano MDLC • Dual binary gradient chromatography (4 pumps) • 20 – 1000 nL/min • 1 – 20 microL/min • No flow splitting and dynamic flow control

11. Rapid Sample loading on parallel traps to increase throughput of dilute samples

12. Rapid Sample Loading Data 1st trap: rapid sample load 2nd trap: loaded while 1st trap elutes • 5 mL of BSA tryptic digest loaded at 5 mL/min • 0.5mm x 2 mm CapTrap (Michrom) • eluted at 500 nL/min onto 150mm x 0.1mm C18 analytical column • 5 mL of BSA tryptic digest loaded at 500 nL/min • 0.3mm x 5 mm PepMap C18 trap (LC Packings) • eluted at 500 nL/min onto 150mm x 0.1mm C18 analytical column

13. Microfluidic Flow Control Technology • Flow meters in each mobile phase measure the actual flow rate • Active Flow Control uses feedback to control a rapid, electronically adjusted variable pressure source • Flow rate is maintained regardless of system back pressure or viscosity • Binary system includes dual controllers

14. 1 nL/min step 2 nL/min step Accurate Flow Control

15. ExpressLC • Fast gradient chromatography • 2-30 microL/min • Optimized for 300 micoL columns • Direct real-time flow control • Very fast, highly reproducible gradients

16. ht LC System Layout CTC autosampler- various versions integrated with system Temperature controlled column holder Integrated injection system- software selectable, delivers precise injection volumes from 10 nl to 1 ul Microfabricated flow cell with array-based UV absorbance from 200 – 380 nm

17. ht LC Instrument Design • Direct pumping system with precision flow control  • Reduce instrumental variance to achieve highest performance allowed by column • Instrumental variance includes • Injection (dispersion of “plug”) • Connection tubing • Dispersion from fittings/connectors • Detection cell s2 = scol2 + sinj2 + stube2 + sfittings2 + sdet2

18. 15,000 13,500 12,000 10,500 9,000 7,500 s2=150 s2=1000 s2=500 s2=2000 • Plates Assumptions 15,000 plates s2col = 3700 nL2 s2=3700 K’ Loss in Efficiency Due to System Dispersion for 150 x 0.3 mm Column

19. 60nl from variable injector Time-Sliced Injections Provide Low Variance 60nl fixed internal loop

20. Variation of Injection Time / Volume 540 nL/min

21. Linear Variation of Injection Volumes using Timed Injections Flow rate: 4.0 mL/min Duplicate injections for each volume

22. Conversion of Flow Rates and Injection Volumes

23. Low Dispersion / High Sensitivity Detection Cell • Chip-based flow cell • 200-380 nm array detection • Fiber optic coupling • 4 mm pathlength, 45 nL detection cell • Each separation column will have independent detection cell and detection array

24. Dual Beam Spectrometer Performance • Temperature-controlled detector arrays and light source • Methods developed for stable fiber illumination and transmission • Typical drift £ 0.4 mAU/hr • Typical short-term noise £ 0.04 mAU rms

25. Detection Cell Variance Time [s] 0 5 10 15 20 25 30 Eksigent : ~300 nL2 LC Packings : ~6500 nL2 Eksigent 45 nL Flow Cell Absorbance LC Packings 45 nL Flow Cell 0 500 1000 1500 Volume [nL]

26. Eksigent Microfabricated UV Flow Cell Provides Excellent Linearity

27. Full UV Data Displayed in Eksigent Control Software

28. High Resolution: Isocratic Separation Column: Zorbax 300XDB-C18 3.5 mm 150 x 0.3 mm Mobile Phase: A: H2O B: ACN Gradient: Isocratic 30% A : 70% B Flow Rate: 4 mL/min Injection: 40 nL Sample: 1. Uracil 2. Acetophenone (19000) 3. Propiophenone (20300) 4. Butyrophenone (19700) 5. Valerophenone (19600) 6. Hexanophenone (19400) 7. Heptanophenone (19500)

29. Near Theoretical Efficiencies Small C-term allows fast analysis

30. High Resolution Gradient Separation Column: Zorbax 300XDB-C18 3.5 mm 150 x 0.3 mm Mobile Phase: A: H2O B: ACN Gradient: Gradient 20  80% in 15 min Flow Rate: 6 mL/min Injection: 40 nL Sample: 1. Uracil 2. Acetophenone 3. Propiophenone 4. Butyrophenone 5. Valerophenone 6. Hexanophenone 7. Heptanophenone

31. Method 2 Time (min)A (mL/min)B (mL/min) 0 16 0.8 0.25 0.08 24 0.7 0.08 24 Rapid Gradient Separations

32. Chromatographic Hydrophobicity Assay Acetophenoe 5-phenyl-1H-tetrazole Butyrophenone Colchicine Valerophenone Propiohenonee Indole Theophylline

33. 2400 1900 1400 Absorbance Mixing Gradient Reset and Re-equilibration 900 500 ul 30 sec Conventional 6 min 3 min 2.5 min 400 300 nl 2 sec ExpressLC -100 0 1 2 3 4 5 6 7 1.5 min 28 sec 1 min Time (min) Reduction of Analysis Time

34. Ten Assays in 15 Minutes Retention Time 2%  98% B in 45 sec. , hold 15 seconds, pre-run flush 30 sec. A : 10 mM Ammonium Acetate pH 7.4 B : 10 mM AA/ACN 14 microL/min; 40 nL inject

35. Simple Method Transfer with Increased Gradient Speed Gradient is both reproducible and linear

36. A stir diffuse t-dispersive B tb td Anatomy of a Fast Gradient g(t) tb = 2 td’ • The delivered gradient is a function of delay volume and system blur • Re-equilibration also requires a similar transition • Therefore the delay occurs 2 times per analysis tb = 0 td=td’+tb stir & diffuse  mix mobile phase t td’ tb

37. System Comparisons • Vdelay divided by Vcolumn provides a relative comparison of delay times between different systems- data is normalized for column diameter • Delay volumes based on manufacturer specifications • Data below are for a 5 cm column Compared to Eksigent, next best system is 9X worse in relative delay.

38. Analysis of Buspirone Metabolites LC/MS/MSExpressLC / API 2000 • Column: Zorbax SB C18, 3.5 mm, 0.30 x 50 mm • Flow rate: 10 mL/min. • Gradient: Multi-step • A: Water / 0.1 % Formic Acid • B: ACN / 0.06% Formic Acid Time (min)%B 0 5 0.1 5 0.6 25 1 60 1.1 98 1.4 98

39. MS Method • Parameters • CUR: 10 • GS1: 10 • GS2: 20 • IS: 5000 • TEM: 150 • ihe: ON • CAD: 8 • DP: 80 • EP: 10 • CE: 50 • CXP: 5 • MS: AB/MDS Sciex API2000 • Ionization mode: Turbo Spray with 150 mm OD / 30 mm ID capillary inserted through spray tip. • Polarity: Positive

40. RLM Metabolites

41. ExpressLC / API 2000 using Zorbax 1.8 micron C18 Time (min)%B 0 20 0.1 35 1.1 50 1.3 98 1.9 98 Zorbax 1.8mm C18 50 x 0.3 mm

42. Long Term Repeatability Gradient Separations • Column: Aquasil C18, 5 mm, 0.320 x 50 mm • Column temperature: 30 C • Flow rate: 12 mL/min • Gradient: 30-95% in 1 minute • A: Water (0.1% formic acid) • B: Acetonitrile (0.1% formic acid) • Injection: 80 nL • Samples diluted in 70/30 water/acetonitrile • Detection: 300 nm @ ~ 5 Hz data collection rate • Sample: compound in TPG5/Water • Each sample was injected 250 times

43. System Repeatability: 250 Injections 200 100 Injection # 10

44. Retention time RSD = 0.19 % Retention Time: 250 Injections

45. Area RSD = 0.72 % Peak Area: 250 Injections

46. Peak Width: 250 Injections

47. Reserpine Dioctyl Phthalate Aspartame Cortisone 200 µg/mL Test System Linearity on Pfizer Test Mix Column: Zorbax C18 300SB 3.5 mm 50 x 0.3 mm Mobile Phase: A: H2O (0.05% TFA) B: ACN (0.04% TFA) Gradient: Gradient 2%  98% B in 3 min Flow Rate: 6 mL/min Injection: 40 nL

48. Serial Dilutions

49. Linearity Plots

50. Serial Dilutions