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New Systems Based on the VersaGrad Slide 2 Current Binary Configurations Available Slide 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 Slide 4 Automation and PC Reactors 1200 Autosampler Alias Autosampler 1200 instant pilot Post Column Reaction system Fract R1 and R2 Fraction collectors Column Oven Slide 5 Versatility to Meet Any Application Custom build a system by line items: 1. Choose a pump 2. Add a detector (s) 3. Add an injector or Autosampler 4. Add the degasser if desired 5. Add a system accessory kit 6. Add the solvent tray 7. Add EZChrom with control options required 8. Add Instant pilot, PCR or Fraction collector if desired 9. Add a column oven 10. Quote as a package price not by line item Slide 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 Slide 7 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 Binary & Quaternary HPLC Systems Slide 8 VersaGrad Series III Manual Entry level system with EZChrom control & data includes: 1. VersaGrad Series III 2. Degasser & solvent tray 3. EZChrom on preloaded computer 4. Manual injector 5. Standard UV/Vis detector Shown with optional Instant pilot Slide 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 Slide 10 VersaGrad 1500 Binary System High end automated Binary system containing: 1. VersaGrad Model 1500 2. 1200 Autosampler 3. 1200 UV detector 4. 1200 Degasser 5. Solvent tray 6. PC with EZChrom pre loaded Slide 11 QGrad Quaternary Gradient System Quaternary high end DAD based system with: 1. QGrad Pump 2. 1200 Diode Array Detector 3. 1200 Autosampler 4. EZChrome Elite Workstation with DAD Pre installed 5. 1200 Degasser & Solvent tray Slide 12 Features Single and dual piston models available Pressures up to 12,000psi Single piston 18,000psi dual piston A utomated injection Full range of detectors EZChrom control and data Ultimate Performance at an affordable price Ultra High Pressure (UHP) Systems Slide 13 Series III HP Binary with EZChrom 12,000psi entry level UHP system with: 1. VersaGrad Series III UHP, 12,000psi pump 2. Alias Autosampler 3. 1200 UV detector 4. 1200 Degasser 5. Solvent tray 6. PC with EZChrom pre loaded Slide 14 UHP Binary System 18,000 psi ultimate UHP system including: 1. VersaGrad UHP dual piston pump 2. Alias Autosampler 3. 1200 UV detector 4. 1200 Degasser 5. Solvent tray 6. PC with EZChrom pre loaded Slide 15 Substantial Savings AssayAnalysis Time Flow ml/min Test per Year Cost per test Cost per 1000 Samples Solvent cost per 1000 Savings per 1000 samples Conventional Isocratic 6119200$9.03$9,025.63$1,005 UHP Isocratic11115200$1.50$1,504.27$167$7,521.36 Conventional Gradient 3013840$45.13$45,128.17$5,024 UHP Gradient3.5132914$5.26$5,264.95$586$39,863.21 Slide 16 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 The Ultra High Pressure Binary Systems Slide 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 Slide 18 UHP Reproducibility Previous Data Slide 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 Slide 20 Gradient Reproducibility UHP of Flavors Previous Data Slide 21 UHP Binary Pump Retention Time Study at HPLC Pressure Methyl Benzoate Ethyl Benzoate 1.1891.353 1.1891.353 1.1871.35 1.1871.35 1.1881.351 1.1881.351 1.1881.352 1.1881.352 1.1881.352 1.1881.352 ColumnZorbax C18 5um 4.6 X 15 SolventH20 35% CH3CN 65% Flow1.5ml/mi DetectionUV254nm Pressure1268psi Slide 22 Methyl Benzoate Ethyl Benzoate Mean1.188Mean1.3516 Standard Error0.00021082Standard Error0.00034 Median1.188Median1.352 Mode1.188Mode1.352 Standard Deviation0.00066667Standard Deviation0.001075 Sample Variance4.4444E-07Sample Variance1.16E-06 Kurtosis0.08035714Kurtosis-0.88203 Skewness-6.2444E-13Skewness-0.32201 Range0.002Range0.003 Minimum1.187Minimum1.35 Maximum1.189Maximum1.353 Sum11.88Sum13.516 Count10Count10 Relative Std Dev0.06% 0.08% New UHP Binary Pump Retention Time Study Slide 23 Features Tailored to a given application 18,000psi pumps advantages are exploited in the UFFLC EZChrom control and data Application Specific Systems Slide 24 UFFLC of the Proteome Comes standard with 1. VersaGrad UHP 18,000psi pump 2. Alias Autosampler 3. 1200 UV detector 4. 1200 Degasser 5. Solvent tray 6. PC with EZChrom pre loaded 7. Fract R1 Fraction collector Slide 25 UFFLC 2-Minute Hemoglobin Rapid Screen SAMPLE: Hemoglobin FASC standard COLUMN: PolyCAT A, 100x2.1-mm; 3-m, 1500- 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 1c F A A2A2 S C A3A3 Slide 26 Minutes 051015202530354045 mAU 0 20 40 60 Mixed-bed ion-exchange HPLC of yeast lysate: Effect of flow rate COLUMN: PolyCAT A + PolyWAX LP, 200x4.6-mm; 5 m, 1000- GRADIENT: 0 0.8 M NaCl in 20 mM HEPES, pH 7.0 A 280 1 ml/min 4 ml/min Slide 27 Mixed-bed ion-exchange HPLC of yeast lysate: Effect of flow rate COLUMN: PolyCAT A + PolyWAX LP, 200x4.6-mm; 5 m, 1000- A 280 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 Minutes 2468 010203040 A 280 1 ml/min (1051 psi) 4 ml/min (4709 psi) (X-axis expanded 5x) Slide 28 Mixed-bed ion-exchange HPLC of E. coli lysate: Fast flow COLUMN: PolyCAT A + PolyWAX LP, 200x4.6-mm; 5 m, 1000- A 280 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 Slide 29 PROTEOMICS BY 2-DIMENSIONAL LC/ MS/MS 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 - MudPIT: Multidimensional Protein Identification Technology Slide 30 # of proteins pIvalue pI Distribution of the Predicted Mouse Proteome From: H. Wang et al., J. Proteome Res. 5 (2006) 361 Acidic proteins; Basic proteins; Use anion-exchange Use cation-exchange Slide 31 4356 1115 300 103 34 2820 16 31 0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 12345678>8 Number of Unique Peptides Number of Proteins 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 Slide 32 Fractionating Intact Proteins Increases Detection of Peptides from Proteins of Low Abundance Low-abundance Protein X is 0.1% of total protein 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. 12345678910 Slide 33 Top-Down UHP Proteomics Slide 34 Histone H4 Acetylation & Methylation Variants COLUMN: PolyCAT A (104CT0315; HILIC mode) 27.032.037.042.047.052.057.062.0 VOLTAGE 0 Acetyl 1 Acetyl 2 Acetyl H2A METHYLATION TIME (Min) 203040506070 VOLTAGE 0 4-Acetyl 3-Acetyl 2-Acetyl 1-Acetyl 0-Acetyl H2A METHYLATION (courtesy James Pesavento - U. of Ill.) MITOSIS INTERPHASE The most minor variants can be the most critical Slide 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 Slide 36 PCR System Comes standard with 1. VersaGrad Series III 2. 1200 Fluorescence detector or UV 3. 1200 Degasser 4. Solvent tray 5. PC with EZChrom pre loaded 6. Sensivate post column reaction system 7. Manual injector Slide 37 Sensivate Post-Column Reactor Applications Carbamates by OPA Glyphosates by OPA Aflatoxin by Iodine Amino Acids by OPA Amino Acids by Ninhydrin Slide 38 PCR System Applications ApplicationReactor 1Temp.Reactor 2 CarbamateNaOH100 o COPA GlyphosateNaOCl40 o COPA AflatoxinsIodine40 o C(not used) CarbohydratesNaOH40 o C(not used) LipidsPhenacyl100 o C(not used) Amino Acids - Ninhydrin Ninhydrin140 o C(not used) Amino Acids - OPANaOCl40 o COPA Amino Acids - PrimaryOPA30 o C(not used) Slide 39 State of the Art Separations AflatoxinGlyphosateCarbamate Slide 40 Amino Acids Slide 41 APLC System Going beyond FPLC 1. VersaGrad Series III 2. 1200 UV Detector 3. 1200 Degasser 4. Solvent tray 5. PC with EZChrom pre loaded 6. Fract R1 Fraction collector Slide 42 Mixed-bed ion-exchange HPLC of yeast lysate: COLUMN: PolyCAT A + PolyWAX LP, 200x4.6-mm; 5 m, 1000- A 280 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 Minutes 010203040 A 280 1 ml/min (1051 psi) Slide 43 TIME (Min) 0.05.010.015.020.0 A220 0 40000 80000 120000 160000 phosphorylation variants 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 Slide 44 Cation-Exchange HPLC of BMP-14 (Bone Morphogenetic Protein 14) COLUMN: PolyCAT A, 100x4.6-mm; 3-m, 1000- (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 g BMP-14 in 20 l of (Mobile Phase A:10 mM HCl = 2:1) Slide 45 BMP-14: Blowup to show deamidation variants 1 2 3 4 Slide 46 BMP-14: More gradual gradient to improve separation of variants; resembles preceding growth factors profile QUESTION: Do all recombinant growth factors contain ~ 8% Met[O] or mismatched -S-S- variants (@ cysteine knot motif)? New shoulder; deamidation variant? Another new shoulder? Slide 47 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 NH 4 -Formate, pH 2.2, with 70% ACN MP B: [TOP] 100 mM NH 4 -Formate, pH 2.2, w. 64% ACN; [BOTTOM] 20 mM NH 4 -Formate, pH 2.2, w. 10% ACN Which to choose: [salt] or [ACN]? Slide 48