H-Class & H-Class Bio AutoBlend and AutoBlend plus. Jean-Michel Plankeele May Waters Corporation 1

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1 H-Class & H-Class Bio AutoBlend and AutoBlend plus Jean-Michel Plankeele May Waters Corporation 1

2 UPLC H-Class Introduction in 2010 What Were The Requests? More flexibility? More solvent capability o Some applications require more than 2 solvents o Easier & faster method development Variable volume auto-sampler o Ease of use Technology enablers: Quaternary Solvent Manager: o Low pressure mixing system with UPLC pressure capability Sample Manager FTN, Flow-Through-Needle concept o Needle remains a part of the fluidic path 2012 Waters Corporation 2

3 Auto Blend For Easy Method Development Water Acetonitrile Lines A, B, C are configured with pure solvents Line D is configured a concentrated buffer/modifier Only D has to be changed when another buffer/modifier is required Alcohol Concentrated Modifier 2012 Waters Corporation 3

4 AU AU AU Peptide Map Development Varying TFA Concentration D : 1% TFA % TFA 2.5% D % TFA 5% D % TFA 10% D Minutes 6 different mobile phase preparations required with BSM 3 mobile phase preparations required with QSM Time savings (solvent preparation, switching from one experiment to other experiment) Less solvent waste 2012 Waters Corporation 4

5 ACQUITY UPLC H-Class Bio Product Definition Based on ACQUITY UPLC H-Class System Same specifications, same features Engineered with a bio-inert flow path made of non-stainless-steel materials Inert system for the analysis of biological molecules Eliminate system corrosion o Buffers specific to ion exchange or SEC Best sample recovery o Limit sample adsorption o Limit damage to molecules, especially oxidation Eliminate adduct formation in MS detection Same options as the H-Class Column ovens, solvent select valve True UPLC performance 2012 Waters Corporation 5

6 Wetted Materials Component Modified Part Ti 6AL4V Ti Grade 2 Ti Grade 4 Ti 6AL4V with DLC MP35N PEEK Inconel 600 PPS SS QSM sinkers X X primary pump X X X X X accumulator pump X X X X mixer manifold X I 2 Valve X X X X transducer X vent valve X X mixer X X solvent select valve X X tubing assemblies X ferules X SM stationary assembly needle assembly X X inject valve X X ferrules X Column Heater APH Tubing assembly X APH Ferrules X post column tubing X* TUV/PDA Flow cells unions X bodies X PDA (only) tubing X 2012 Waters Corporation 6

7 Design Considerations Tubing (MP35N) SS MP35N 50x 100x 50x 100x Cleaner draw (ID has good form) Smoother less burring Better than existing tubing by 2.5x More consistent finish 2012 Waters Corporation 7

8 Auto Blend Plus Technology Auto Blend allows the automatic blending of up to four solvents in accurate proportions Auto Blend Plus allows the formation of independent salt and ph gradients in the same method The system is configured with salts (acidic and basic) No ph adjustment required Auto Blend Plus is unique to Waters! Details, videos on the website ph gradient Salt stock solutions Salt gradient 2012 Waters Corporation 8

9 Auto Blend for Proteins 2012 Waters Corporation 9

10 Auto Blend Plus Technology Program methods directly in units of ph and molarity Calculation of required proportions from physical constants pk, using Henderson-Hasselbalch equation Corrected pk with salt compensation Empirical calibration table covering operating range Proportions calculated at each pump stroke for best fidelity Independent gradients for ph and salt concentration True linear ph gradients as required Available with ACQUITY UPLC H-Class Bio Systems ACQUITY UPLC H-Class Systems Patented 2012 Waters Corporation 10

11 Conventional Method Editor Program Percentage Flow 2012 Waters Corporation 11

12 Auto Blend Plus Method Editor Program ph and Salt Concentration 2012 Waters Corporation 12

13 Auto Blend Plus Method Editor Select Buffer System 2012 Waters Corporation 13

14 Auto Blend Plus Method Editor Program Required Gradient 2012 Waters Corporation 14

15 AU AU AU AU AU Auto Blend Plus Method Development ph 6.1 A B C ph 6.9 A B C ph 7.0 A+B C ph 7.1 B A C ph 7.6 B A C Minutes Method development for proteins Cation exchange chromatography No pre-mixing of mobile phases required 2012 Waters Corporation 15

16 Auto Blend Plus Creating an Empirical Table Measure the ph of a set of mixtures of the buffers and salts to be used in the separation Composition of the mixtures 3x3 table High, medium and low ratios of the acidic and basic buffer stocks High, medium and low salt concentrations 2012 Waters Corporation 16

17 Auto Blend Plus Method Editor Recording Empirical Table 2012 Waters Corporation 17

18 Summary Auto Blend Technology and Auto Blend Plus Technology use the four reservoir capability to simplify methods development and routine operation Auto Blend Technology allows composition programming as a percentage of the four reservoirs Auto Blend Plus Technology adds Program methods directly in units of ph and molarity Proportions calculated at each pump stroke for best fidelity True linear ph gradients as required Patent Pending Available with ACQUITY UPLC H-Class Systems ACQUITY UPLC H-Class Bio Systems 2012 Waters Corporation 18

19 Need To Learn More? Waters Corporation 19

20 Back Up Slides 2012 Waters Corporation 20

21 Auto Blend for Proteins 2012 Waters Corporation 21

22 Waters Auto-Blend Table Sodium Phosphate Buffer Eluent A = 100mM sodium phosphate monobasic Eluent B = 100mM sodium phosphate dibasic 2012 Waters Corporation 22

23 Automated Column Management Automated Column Management for Protein Ion Exchange. The method specifies the column to be used and the bypass (B) permits matching the buffer to the column Waters Corporation 23

24 Buffer Management for Protein Ion Exchange Fluid Path of ACQUITY UPLC H-Class Bio System configured for multi-buffer use The optional solvent select valve provides 6 selectable choices that are associated with particular method names. The four physical reservoirs, labeled A, B, C, and D1-D6 can be assigned as needed as the Acid, Base, Salt, and Aqueous components Waters Corporation 24

25 Solutions for AutoBlend Plus Technology Methods Development 2012 Waters Corporation 25