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New Systems Based on the VersaGrad

New Systems Based on the VersaGrad. Current Binary Configurations Available. Detectors Available. 1200 UV/Vis 1 channel programmable 1200 MWD 8 channel programmable 1200 Diode Array Full wavelength spectral capability 1200 Refractive Index 1200 ELSD 1200 Fluorescence.

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New Systems Based on the VersaGrad

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  1. New Systems Based on the VersaGrad

  2. Current Binary Configurations Available

  3. Detectors Available • 1200 UV/Vis 1 channel programmable • 1200 MWD 8 channel programmable • 1200 Diode Array Full wavelength spectral capability • 1200 Refractive Index • 1200 ELSD • 1200 Fluorescence

  4. Automation and PC Reactors • 1200 Autosampler • Alias Autosampler • 1200 instant pilot • Post Column Reaction system • Fract R1 and R2 Fraction collectors • Column Oven

  5. Versatility to Meet Any Application • Custom build a system by line items: • Choose a pump • Add a detector (s) • Add an injector or Autosampler • Add the degasser if desired • Add a system accessory kit • Add the solvent tray • Add EZChrom with control options required • Add Instant pilot, PCR or Fraction collector if desired • Add a column oven • Quote as a package price not by line item

  6. Pre Configured Systems • Standard HPLC Systems • Series III Manual with EZChrom • Series 1500 with Autosampler and EZChrom • QGrad with Autosampler PDA and EZChrom • Ultra High Pressure Systems • Series III UHP with Autosampler and EZChrom • Series UHP with Autosampler and EZChrom • Application Based Systems • Advanced Protein Liquid Chromatography System APLC • Proteome UFFLC System • PCR system for Carbamates

  7. Binary & Quaternary HPLC Systems Features Single and dual piston models available Pressures up to 6,000psi SS 5,000psi PEEK Manual or automated injection Match the performance to the application flows from 0.001ml/min to 100ml/min Full range of detectors EZChrom control and data Reduction Elegance at an affordable price

  8. VersaGrad Series III Manual • Entry level system with EZChrom control & data includes: • VersaGrad Series III • Degasser & solvent tray • EZChrom on preloaded computer • Manual injector • Standard UV/Vis detector Shown with optional Instant pilot

  9. VersaGrad Series III System with Automation Option • Shown with optional autosampler • Add other options at any time to upgrade a system • Modularity allows reconfiguration for many applications

  10. VersaGrad 1500 Binary System • High end automated Binary system containing: • VersaGrad Model 1500 • 1200 Autosampler • 1200 UV detector • 1200 Degasser • Solvent tray • PC with EZChrom pre loaded

  11. QGrad Quaternary Gradient System • Quaternary high end DAD based system with: • QGrad Pump • 1200 Diode Array Detector • 1200 Autosampler • EZChrome Elite Workstation with DAD Pre installed • 1200 Degasser & Solvent tray

  12. Ultra High Pressure (UHP) Systems Features Single and dual piston models available Pressures up to 12,000psi Single piston 18,000psi dual piston  Automated injection Full range of detectors EZChrom control and data Ultimate Performance at an affordable price

  13. Series III HP Binary with EZChrom • 12,000psi entry level UHP system with: • VersaGrad Series III UHP, 12,000psi pump • Alias Autosampler • 1200 UV detector • 1200 Degasser • Solvent tray • PC with EZChrom pre loaded

  14. UHP Binary System • 18,000 psi ultimate UHP system including: • VersaGrad UHP dual piston pump • Alias Autosampler • 1200 UV detector • 1200 Degasser • Solvent tray • PC with EZChrom pre loaded

  15. Substantial Savings

  16. The Ultra High Pressure Binary Systems 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

  17. UHP Reproducibility 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

  18. UHP Reproducibility – Previous Data

  19. Gradient Reproducibility 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

  20. Gradient Reproducibility UHP of Flavors –Previous Data

  21. UHP Binary Pump Retention Time Study at HPLC Pressure

  22. New UHP Binary Pump Retention Time Study

  23. Application Specific Systems Features Tailored to a given application 18,000psi pumps advantages are exploited in the UFFLC EZChrom control and data

  24. UFFLC of the Proteome • Comes standard with • VersaGrad UHP 18,000psi pump • Alias Autosampler • 1200 UV detector • 1200 Degasser • Solvent tray • PC with EZChrom pre loaded • Fract R1 Fraction collector

  25. 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 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

  26. Mixed-bed ion-exchange HPLC of yeast lysate: Effect of flow rate 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

  27. Mixed-bed ion-exchange HPLC of yeast lysate: Effect of flow rate COLUMN: PolyCAT A + PolyWAX LP, 200x4.6-mm; 5 µm, 1000-Å A280 GRADIENT: (1 ml/min) 0-30’: 0-50 %B; 30-40’: 50-100 %B; (4 ml/min) 0-2’: 0-8 %B; 2-7’: 8-100 %B A) 20 mM HEPES, pH 7.0; B) 20 mM HEPES + 0.8 M NaCl, pH 7.0 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

  28. 25 20 15 mAU 10 5 0 0 1 2 3 4 5 6 7 Minutes Mixed-bed ion-exchange HPLC of E. coli lysate: Fast flow 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

  29. PROTEOMICS BY 2-DIMENSIONAL LC/ MS/MS MudPIT: Multidimensional Protein Identification Technology 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 -

  30. 1200 1000 800 600 # of proteins 400 200 0 4 5 6 7 8 9 10 11 12 13 pI value 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

  31. 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 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

  32. Fractionating Intact Proteins Increases Detection of Peptides from Proteins of Low Abundance 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.

  33. Top-Down UHP Proteomics

  34. Histone H4 Acetylation & Methylation Variants COLUMN: PolyCAT A (104CT0315; HILIC mode) 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.)

  35. 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 • FPLC , is now obsolete with the speed and resolution of UFFLC • Top down proteomics is now much easier

  36. PCR System • Comes standard with • VersaGrad Series III • 1200 Fluorescence detector or UV • 1200 Degasser • Solvent tray • PC with EZChrom pre loaded • Sensivate post column reaction system • Manual injector

  37. SensivatePost-Column Reactor Applications Carbamates by OPA Glyphosates by OPA Aflatoxin by Iodine Amino Acids by OPA Amino Acids by Ninhydrin

  38. Application Reactor 1 Temp. Reactor 2 Carbamate NaOH 100o C OPA Glyphosate NaOCl 40o C OPA Aflatoxins Iodine 40o C (not used) Carbohydrates NaOH 40o C (not used) Lipids Phenacyl 100o C (not used) Amino Acids -Ninhydrin Ninhydrin 140o C (not used) Amino Acids - OPA NaOCl 40o C OPA Amino Acids - Primary OPA 30o C (not used) PCR System Applications

  39. State of the Art Separations Aflatoxin Glyphosate Carbamate

  40. Amino Acids

  41. APLC System • Going beyond FPLC • VersaGrad Series III • 1200 UV Detector • 1200 Degasser • Solvent tray • PC with EZChrom pre loaded • Fract R1 Fraction collector

  42. Mixed-bed ion-exchange HPLC of yeast lysate: COLUMN: PolyCAT A + PolyWAX LP, 200x4.6-mm; 5 µm, 1000-Å A280 GRADIENT: (1 ml/min) 0-30’: 0-50 %B; 30-40’: 50-100 %B; A) 20 mM HEPES, pH 7.0; B) 20 mM HEPES + 0.8 M NaCl, pH 7.0 A280 1 ml/min (1051 psi) 0 10 20 30 40 Minutes

  43. Anion-Exchange of Ovalbumin Phosphorylation Variants COLUMN: PolyWAX LP (#104WX0510) SAMPLE: Sigma Grade VI (99%) GRADIENT: 10 mM K-PO4, pH 7.0; 60-300 mM NaCl in 20’ 160000 phosphorylation variants 120000 A220 80000 40000 0 0.0 5.0 10.0 15.0 20.0 TIME (Min)

  44. Cation-Exchange HPLC of BMP-14 (Bone Morphogenetic Protein 14) COLUMN: PolyCAT A, 100x4.6-mm; 3-µm, 1000-Å (item# 104CT0310; ser# A2562C) MOBILE PHASE: A) 20 mM K-PO4, pH 6.0, with 20% ACN; B) Same + 0.6 M NaCl GRADIENT: 20’ linear, 0-100% B, then 5’ at 100% B 1.0 ml/min A220 SAMPLE: 25 µg BMP-14 in 20 µl of (Mobile Phase A:10 mM HCl = 2:1)

  45. BMP-14: Blowup to show deamidation variants 3 4 2 1

  46. BMP-14: More gradual gradient to improve separation of variants; resembles preceding growth factor’s profile QUESTION: Do all recombinant growth factors contain ~ 8% Met[O] or mismatched -S-S- variants (@ cysteine knot motif)? Another new shoulder? New shoulder; deamidation variant?

  47. ________________KEY_________________ A: SLYSSSPGGAYVTR (Vimentin(51-64)) B: SVNFSLTPNEIK (MAP 1B(1271-1282)) C: WWGSGPSGSGGSGGGK (#1644) A+P: SLYSSS(p)PGGAYVTR B+P: SVNFSLT(p)PNEIK C+P: WWGSGPSGSGGS(p)GGGK (#1645) D+P: GGAAGLGY(p)LGK (#1876) 200 A, B & C 150 D+P A+P 100 A280 B+P C+P 20  100 mM salt 260 nm 270 nm 50 70  10% ACN 0 0 5 10 15 20 25 30 35 40 45 50 Minutes ERLIC of Phosphopeptides with Volatile Solvents Column: PolyWAX LP (104WX0503) Flow rate: 1 ml/min. Detection: A270 (blue) or A260 (red) Gradient: 0-5’: 0%B; 5-45’: 0-100% B; 45-50’: 100%B MP A: 20 mM NH4-Formate, pH 2.2, with 70% ACN MP B: [TOP] 100 mM NH4-Formate, pH 2.2, w. 64% ACN; [BOTTOM] 20 mM NH4-Formate, pH 2.2, w. 10% ACN Which to choose:  [salt] or  [ACN]?

  48. SPE OF PEPTIDES: Unexpectedly Selective Extraction by Carbon and C18 NuTips Urea (1 M) = Transferrin tryptic digest (30 µg) = Peptides extracted/released by NT200C18 from 500 µg digest = " " NT200CAR " " = " " (second set of releasing washes) 75 50 25 Millivolts 0 -25 0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 Minutes Column: PolyHYDROXYETHYL A (item# 204HY0503), HILIC mode. Detection: A220 Gradient: 40’ linear, 0-70% B. A) 15 mM TEAP, pH 2.7, with 85% ACN B) " " 10 "

  49. I + M Comparison of ERLIC vs. HILIC of Amino Acids MOBILE PHASE: 10 mM TEAP, pH 4.0, w. 65% ACN; 1 ml/min pGLU + Q PolyWAX LP (basic) Cya H M N L A R T E P G K S I D V Cya pGLU T PolyHYDROXYETHYL A (neutral) N H A D P E K R V ABSORBANCE (215 nm) pGlu Cya Q + S D H T N E V A G TSKgel Amide-80 (acidic) P R K L 0 10 20 30 40 TIME (Min)

  50. Systems For Every Application • From HPLC to UHP in pressure • From UFFLC to Prep in flow • Versatile selection of detectors • Full automation • Data to meet your labs requirements • Simplicity and functionality at an affordable price LabAlliance has the system your customer needs

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