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Agilent & SSI/ LabAlliance Working Together to Bring High Value

Agilent & SSI/ LabAlliance Working Together to Bring High Value Systems to the LabAlliance Network SSI Quality Pumps & Components + Agilent Field Proven Detectors + EZChrom Data and Control = Unmatched Performance for HPLC, UHP, and Preparative Chromatography

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Agilent & SSI/ LabAlliance Working Together to Bring High Value

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  1. Agilent & SSI/LabAlliance Working Together to Bring High Value Systems to the LabAlliance Network • SSI Quality Pumps & Components • + Agilent Field Proven Detectors • + EZChrom Data and Control • = Unmatched Performance for HPLC, • UHP, and Preparative Chromatography • Increased Capability and Unmatched • Value

  2. NEW THIS YEAR • VersaGrad Pump line • Prep 250 Pump • Agilent Detectors & Autosamplers • Fract R1 & R2 Fraction Collectors • New Systems • Single Piston UHP Pump • UFFLC • Vista Compatible EZChrom with Simplified Chinese & Japanese Let us show You More

  3. Versatile HPLC systems VersaGrad Series III Manual Binary System VersaGrad 1500 Automated Binary Gradient System QGrad Quaternary System with Diode Array

  4. 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

  5. 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

  6. QGrad Quaternary Gradient System • Quaternay 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

  7. Ultra High Pressure LC 12,000 psi binary gradient system 18,000 psi binary gradient system

  8. 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

  9. 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

  10. Substantial Savings

  11. 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

  12. 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

  13. UHP Reproducibility – Previous Data

  14. UHP Binary Pump Retention Time Study at HPLC Pressure

  15. New UHP Binary Pump Retention Time Study

  16. Application Based systems Post Column Reaction UFFLC (Ultra Fast Flow Chromatography) APLC (Advanced Protein Liquid Chromatography

  17. 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

  18. 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 mMBis-Tris + 2 mM KCN, pH 6.5 B) 40 mMBis-Tris + 2 mM KCN + 200 mMNaCl, pH 6.8 A S C F A1c A3 A2

  19. 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

  20. 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.

  21. Top-Down UHP Proteomics

  22. 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 MITOSIS VOLTAGE METHYLATION 2-Acetyl 3-Acetyl 4-Acetyl 0 20 30 40 50 60 70 TIME (Min) (courtesy James Pesavento - U. of Ill.)

  23. 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

  24. 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

  25. State of the Art Separations Aflatoxin Glyphosate Carbamate

  26. Amino Acids

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

  28. 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

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

  30. 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 mMHCl = 2:1)

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

  32. ________________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]?

  33. SPE OF PEPTIDES: Unexpectedly Selective Extraction by Carbon and C18 NuTips Urea (1 M) = Transferrintryptic 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 "

  34. 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)

  35. 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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