The new Agilent 1290 Infinity Quaternary LC How binary pump-like performance can enhance your lab efficiency Sebastian Krahe Produktspezialist HPLC Agilent Technologies Page 1

The 1290 Infinity Quaternary LC System Leveraging the Performance of the 1290 Infinity Binary LC System 1290 Infinity DAD with 60mm cell (& HDR optional) 1290 Infinity TCC with broadest valve selectivity 1290 Infinity Autosampler (with Flexible Cube for lowest carry-over) 1290 Infinity Quaternary Pump Page 2

Innovation: 1290 Infinity Quaternary Pump Quaternary Pump with Binary Pump-like Performance 1290 Infinity Quaternary Pump • Boost Performance Highest accuracy and precision for composition and flow, with exceptionally low delay volume • Save Time Accelerates transfer of existing HPLC methods to UHPLC • Reduce Costs Outstanding UHPLC performance at HPLC-like costs of ownership Page 3

1290 Infinity Quaternary Pump Various tools enabling the high performance Inlet-Weaver Switchable Jet Weaver Mixer Multi-PurposeValve 4-channel Degasser MCGV (MultiChannel-GradientValve) In-line Filter Pump Head Page 4

Multipurpose valve functions for highest comfort Standard Application for standard Delay Volume Page 5

Agilent 1290 Infinity Quaternary Pump Specifications & Benefits Power Range For any kind of analysis Composition Accuracy and Precision < 0.15 % RSD or 0.02 min SD ± 0.4 % (1-99 % Composition B) High RT precision in gradient runs Flow Accuracy and Precision < 0.07 % RSD or 0.01 min SD ± 1.0 % or 10 µL High RT precision in isocratic runs Composition Range 1-99 % Wide analytical range Delay Volume < 350 µL For fast quaternary gradients Page 6

INFINITELY BETTER Different mixtures (H20, MeOH, ACN) at -> different flow rates Programmed Composition Measured Composition Time [mini - Agilent Technologies

Flow and Composition Accuracy and Precision Isocratic analysis of Glucocorticoids 1: Hydrocortisone 2: Beclomethasonedi-proprionate 3: Hydrocortisone acetate Column: Agilent ZORBAX Eclipse Plus C18, 4.6 x 100 mm, 5 µm (p/n 959996-902) Mobile phase: Water/ Acetonitrile = 30/70 Flow rate: 1 mL/min Stop time: 5 min Injection vol.: 3 µL (needle wash for 3 sec) Column Temp.: 30 °C DAD: 254/10 nm, (ref 400/ 80 nm), 20 Hz, slit 4 nm Page 8

Flow and Composition Accuracy and Precision Isocratic analysis of Glucocorticoids mAU 1 RT of compounds very sensitive regarding changes in mobile phase composition! 30 25 2 20 15 10 3 Water/ACN 29:71 5 Water/ACN 31:69 0 Water/ACN 30:70 1 1.5 2 2.5 3 3.5 min Page 9

Precision of Retention Time Peak number Agilent Technologies

Accuracy and Precision Dynamically vs. pre-mixed solvents Isocratic composition by dynamic mixing: A/B = 70:30 Which method is better? Isocratic composition by premixed solvent: RSD RT(%) A/B = 70:30 Page 11

Accuracy and Precision Dynamically vs. pre-mixed solvents Retention time precision Single operator, single bottle Dynamic mixing For illustration only, data can be found in Agilent Application Note 5991-0098EN Page 12

Accuracy and Precision Dynamically vs. pre-mixed solvents Retention time precision Single operator, single bottle Dynamic mixing Single operator, multiple bottles For illustration only, data can be found in Agilent Application Note 5991-0098EN Page 13

Accuracy and Precision Dynamically vs. pre-mixed solvents Retention time precision Single operator, single bottle Dynamic mixing Single operator, multiple bottles Multiple operators, multiple bottles For illustration only, data can be found in Agilent Application Note 5991-0098EN Page 14

Accuracy and Precision Gradient Analysis of Clopidole Clopidole is used as prophylactics and/or therapeutics in the prevention or treatment of coccidiosis in the poultry and rabbit industries. It is added to finished feed in a concentration range of 100 to 125mg/kg. Clopidole Column: Agilent ZORBAX SB-C18, 4.6 x 250 mm, 3.5 µm (p/n 884950-567) Mobile phase: A = Water/ACN (95/5) + 0.1 % TFA B = Water/Acetonitrile(40/60) + 0.1 % TFA Gradient: 0 – 100 % B in 15min Flow rate: 1 mL/min Stop time: 15 min Injection vol.: 1 µL Column Temp.: 30 °C DAD: 263/10 nm, (ref. 400/ 80 nm), 10 Hz Page 15

Accuracy and Precision Dynamically vs. pre-mixed solvents Gradient from 0 to 100 % with premixed solvent: H2O +0.1% TFA 0 100 % B ACN +0.1% TFA Gradient from 5 to 60 % B by dynamic mixing: H2O +0.1% TFA 5 60 % B Which method is better? RSD RT(%) ACN +0.1% TFA Page 16

Accuracy and Precision Dynamically vs. pre-mixed solvents Retention time precision 0.5 RSD RT (%) 0.4 0.3 0.2 Specification (< 0.15 %) 0 100 % B 0.1 5 60 % B 0 Dynamic mixing Premixed Page 18

Power Range Composition Accuracy and Precision Flow Accuracy and Precision Composition Range Delay Volume Agilent Technologies

Ternary/Quaternary Gradients Blend Assist You need different concentrations of modifiers in your analysis, would like to have just one stocksolution and do online dilution to profite from the quaternary mixing capability of your pump? Here is a simple tool – BlendAssist! Water Water 1% TFA Desired method conditions - example: 1. 5 to 95% gradient of ACN with 0.1% TFA in Water and 0.08% TFA in ACN 2. 20 – 80% gradient of ACN with 0.5% TFA in Water and 0.4% TFA in ACN Without BlendAssist you need to either pre-mix the required solvents or by using stock-solutions of TFA in Water and ACN to program...

Ternary/Quaternary Gradients Blend Assist, different Phosphate buffer concentrations H3C CH3 H3C H2C O N N O CH3 NH2 OH H3C N N Disopyramide Quinidine Column: Agilent ZORBAX Eclipse Plus C18, 4.6 x 100 mm, 1.8 μm Mobile phase: A = Water B = Water/Acetonirile 20:80 + 0.05 M NaH2PO4, pH=4.6 Gradient: var. Flow rate: 1.5 mL/min Stop time: 8.5 min Injection vol.: 1 µL Column Temp.: 40 °C DAD: 270/10 nm, (ref. 360/ 100 nm), 20 Hz Page 21

Ternary/Quaternary Gradients Blend Assist, different Phosphate buffer concentrations Quinidine mAU 400 Procainamide AcetylProcainamide 300 0.05 M buffer 200 0.025 M buffer 100 Disopyramide 0.01 M buffer 0 0 1 2 3 4 5 6 7 8 min Page 22