UHP & UFFLC Chromatography

1. UHP & UFFLC Chromatography High Performance Separations to 18,000 p.s.i. Saving You Time and Solvent

2. Detector 1-254nm Detector 1-254nm 160 160 Thermo150x4.6mm3um Hypersil Gold 2_1 x 50 140 140 120 120 100 100 mAU mAU 80 80 60 60 40 40 20 20 0 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Minutes UHP Runs: 3-micron; 4.6 X150 vs. 1.8-micron; 2.1X 50

3. Ultra Fast Flow Liquid ChromatographyUFFLC A New Era of UHP

4. UFFLC Scaling Factors for Column Diameters Pressure and column stability limit how fast you go

5. UHP, UFFLC

6. 18,000 p.s.i. PumpAllows UHP UFFLC and HPLC in one System

7. 15,000 p.s.i. Optimized Recompression

8. Specifications • Flow 0.001 – 5.000 ml/min • Pressure 18,000 p.s.i. • Pulsation >3000psi 1.5% • Accuracy < + 1% • 1ml/min 10,000psi Methanol • Precision + 0.5% • 30 sec average flow

9. Pump Design Innovations • True 18,000 p.s.i. pressure capability • 0.001 – 5.000 ml/min flow range • EZChrom & Xcalibur driver available • Low volume UHP mixer • Maintaining HPLC capability

10. Completing the system • Detectors • Autosampler • Data • Degasser

11. Detectors • UV, Diode Array, Fluorescence • 20-80 Hz data acquisition rate for fast chromatography • Standard and micro volume flow cells • Full digital control and data acquisition

12. UHP Autosampler • Allows injections to 18,000 p.s.i. • Fully programmable • Full or partial loop mode • Available cooling • Available sample prep

13. UHP Autosampler • Sample capacity :2*96 well or 2*384 or 2*48 standard vials or 2* 12 10ml prep vials • Injection cycle time: <15 sec • Injection: full loop, partial loop and µl pickup • RSD<=0,3% at full loop • Carry-over <0,05 %

14. Use Only What You Inject With µL Pick-up Zero sample lossFlush with transport solvent inject UHP: Transport liquid is in wash port available

15. How µL Pick-up Works 1 Pick-up sample load Inj.vol: (loop vol – 3x needle volume) / 2

16. How µL Pick-up Works 2 Transport sample to the center of the loop load

17. How µL Pick-up Works 3 Switch valve to Inject inject

18. Data and Control • EZ Chrom & EZ Start Drivers • Xcalibur drivers • Developing Analyst driver

19. Data and Control • EZ Start or EZ Chrom Elite • Full control and data analysis • From single instrument to enterprise based system • CFR 21 11 is available • Based on the Agilent/Scientific Software industry standard platform

20. TRUE Analysis Time Analysis time = Injector cycle to load and inject (10 seconds to 1 min) + Run time + Data analysis time (15 seconds to 1 min) + equilibration time (seconds to 30 min)

21. UFFLC UHP Reproducibility .3508min 4 Benzoate Esters Column: 1.5micron particle size 2.1mm X 10 cm C18 100 angstrom Mobile phase: A Pump Water B Bump Acetonitrile 35%A 65%B Flow: 1ml/min Pressure: 12,500psi Detector: UV at 254nm

22. Isocratic UFFLC UHP Reproducibility

23. 6X Faster=Real Savings • 1 run/min vs 1 run every 6 min on normal column = 6X the capacity of conventional HPLC • 1ml/sample for UHP 6ml/sample for conventional HPLC 1/6th the cost/analysis for solvents and solvent disposal

24. Gradient Reproducibility UFFLC UHP of Flavors Lemon Oils Column: 1.5micron particle size 2.1mm X 10 cm C18 100 angstrom Mobile phase: A Pump Water B Bump Acetonitrile 30%B to 90%B in 3min Flow: 1ml/min Pressure: 9,000-12,500psi Detector: UV at 214nm 3.424min

25. Gradient Reproducibility UFFLC UHP Flavors

26. Savings in Gradient UFFLC UHP • 3.5 min per run UHP vs. 30min per run Conventional HPLC = 10 X the throughput of a conventional system • 3.5ml per assay vs. 30ml/assay in conventional HPLC reduces the cost per assay substantially

27. Substantial Savings

28. UFFLC Savings in Method Development • 6-10 Times faster scouting runs • System equilibration time is reduced • Solvent changes are faster • You can do in a morning what normally takes days

29. 80 60 mAU 40 20 0 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 Minutes UFFLC 2-Minute Clinical Hemoglobin Rapid Screen SAMPLE: Hemoglobin FASC standard COLUMN: PolyCAT A, 100x2.1-mm; 3-µm, 1500-Å FLOW: 1.2 ml/min A415 Pressure: 6938 psi GRADIENT: 0-0.7’: 18-55% B; 0.7-1.1’: 55-85% B; 1.1-1.2’: 85-100% B; 1.2-1.3’: 100% B; 1.4’: Initial A) 40 mM Bis-Tris + 2 mM KCN, pH 6.5 B) 40 mM Bis-Tris + 2 mM KCN + 200 mM NaCl, pH 6.8 A S C F A1c A3 A2

30. Top Down ProteomicsUFFLC of yeast lysate: COLUMN: PolyCAT A + PolyWAX LP, 200x4.6-mm; 5 µm, 1000-Å GRADIENT: 0 – 0.8 M NaCl in 20 mM HEPES, pH 7.0 A280 4 ml/min 60 1 ml/min 40 mAU 20 0 0 5 10 15 20 25 30 35 40 45 Minutes

31. Top Down ProteomicsUFFLC of yeast lysate: COLUMN: PolyCAT A + PolyWAX A280 4 ml/min (4709 psi) (X-axis expanded 5x) 2 4 6 8 Minutes 1 ml/min (1051 psi) 0 10 20 30 40 Minutes

32. 25 20 15 mAU 10 5 0 0 1 2 3 4 5 6 7 Minutes Top Down Proteomics UFFLC of E. coli lysate: COLUMN: PolyCAT A + PolyWAX LP, 200x4.6-mm; 5 µm, 1000-Å A280 4 ml/min 4709 psi GRADIENT: 0-2’: 0-8 %B; 2-5.5’: 8-95 %B; 5.5-6’: 95-100 %B A) 20 mM HEPES, pH 7.0 B) 20 mM HEPES + 0.8 M NaCl, pH 7.0

33. PROTEOMICS TODAY BY 2-DIMENSIONAL LC/ MS/MS MudPIT: Multidimensional Protein Identification Technology Typically identifies 2.5-3 peptides per protein; rugged method. Good where sample size is limited ADVANTAGES OVER 2-D ELECTROPHOPHORESIS 1) Greater dynamic range: Most abundant protein detected Least abundant protein detected 2) Works better for hydrophobic and very basic proteins 3) Handling and automation easier - from Link et al., Nat. Biotechnol. 17 (1999) 676-682 -

34. 1200 1000 800 600 # of proteins 400 200 0 4 5 6 7 8 9 10 11 12 13 pI value Complexity of the Proteome Complicate Bottom UP Analysis Acidic proteins; Basic proteins; Use anion-exchange Use cation-exchange From: H. Wang et al., J. Proteome Res. 5 (2006) 361 pI Distribution of the Predicted Mouse Proteome

35. 5000 4356 4500 4000 3500 3000 2500 Number of Proteins 2000 1500 1115 1000 300 500 103 34 28 20 31 16 0 1 2 3 4 5 6 7 8 >8 Number of Unique Peptides PROTEINS IDENTIFIED IN LOW-MOL.-WT. MOUSE SERUM Method: SCX (96 fractions)-RPC Shows Bottom Up Limitation Ref: B.L. Hood et al., J. Am. Soc. Mass Spectrom. 16 (2005) 1221 NOT ACCEPTABLE One Hit Wonders. ACCEPTABLE Identifying proteins via 2 peptides or more

36. UFFLC of Intact Proteins Solves the Problem & Increases Detection of Low Abundance Proteins Low-abundance Protein X is 0.1% of total protein 1 2 3 4 5 6 7 8 9 10 Now Protein X is 1.0 % of total protein in Fraction #6. After digestion, its peptides will be 10x higher a percentage of the total in that fraction than would have been true in a digest of the unfractionated mixture. That greatly increases the chances of identifying Protein X through 2-3 of its fragments rather than just one.

37. The Future Of Proteomics Top-Down UFFLC

38. See The Whole Picture Histone H4 Acetylation & Methylation Variants 0 Acetyl The most minor variants can be the most critical VOLTAGE METHYLATION H2A 1 Acetyl INTERPHASE 2 Acetyl 27.0 32.0 37.0 42.0 47.0 52.0 57.0 62.0 0-Acetyl 1-Acetyl H2A VOLTAGE MITOSIS METHYLATION 2-Acetyl 3-Acetyl 4-Acetyl 0 20 30 40 50 60 70 TIME (Min) (courtesy James Pesavento - U. of Ill.)

39. Advantages • See lower abundance proteins • Compare MS of intact proteins to proteins found by bottom up method • Allows further analysis of fractions of interest • High speed allows multiple runs to be set up giving higher utilization of the MS • Reduces number of One Hit Wonders

40. UHP UFFLC • Higher throughput than any other system • Allows use of standard columns and UHP columns • Allows normal chromatography and small scale prep as well as UHP &UFFLC • Highest pressure and flow limits on the market today. Why limit yourself

41. HPLC, UHP, UFFLC Exclusively from LabAlliance