Am I getting the very best value from my UHPLC analyses?

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Am I getting the very best value from my UHPLC analyses? Stephen Luke LC Columns Product Manager 1

Primary reasons for UHPLC use Very fast Very high resolution Column 2.1 x 50 mm Column 2.1 x 150 mm Run time 0.35-min Run time 20-min 2

Requirements for successful UHPLC The key requirement for UHPLC is very high efficiency separations This has consequences Peaks are very narrow and have a low volume Fast enough detector sampling rate Very low instrument dispersion volume Small particles are more efficient but generate high pressures High pressure rated columns and instruments 3

Minimize instrument dispersion volume Agilent 1290 Infinity II Ultra Low Dispersion Kit Part no G4267-87020 5500-1207 G7116-60021 5067-6602 5500-1208 Description High Pressure Seat Assembly 0.075 mm (PEEK) Capillary ST 0.075 mm x 500 mm - Multisampler to Heat exchanger Quick Connect Heat exchanger Ultra Low Dispersion Quick Connect / Quick Turn Assembly ST 0.075 mm x 105 mm - Heat exchanger to column Capillary ST 0.075 mm x 250 mm - Column outlet to Flow Cell Note: The small diameter capillaries in this kit will significantly increase the backpressure of the system at high flow rates - for example +165 bar with water at 1 ml/min flow rate 4

Impact of ultra-low dispersion In this example the use of the ultra-low dispersion kit increased efficiency by 26%, with an 8% increase in pressure 2.1 x 50 mm columns, 40% 20 mm Sodium Phosphate ph 7, 55 or 60% Acetonitrile, 0.5 ml/min, 0.5 ul, 25C, 254 nm, 80 Hz 5

Minimize instrument dispersion - connections Quick Connect and Quick Turn Fittings Most commonly used fittings in UHPLC are non-adjustable 2-piece or 3-piece metallic fittings. Since different manufacturers of column hardware have different design in column end fittings, as shown in Figure 1, a new set of tubing and fittings needs to be installed for every brand of column to guarantee that the stem length, namely the length between the bottom of the ferrule and the end of tubing, fits the column end fitting. Importance of the Spring Loaded Feature The spring-loaded design constantly pushes the tubing against the receiving port, delivering a reproducible connection with no dead volume for consistent chromatographic performance Stem length is adjustable through the spring, which makes the fitting compatible with all types of LC columns. 6

h High efficiency separations All 3 van Deemter terms are reduced with SPP SPP = superficially porous particle van Deemter equation: h A B/ C B A A Separation Speed (v) Lower h = higher efficiency! C C A term eddy diffusion Particle size & packing quality Narrow particle size distribution B term longitudinal diffusion Less mobile phase in the column Reduced diffusion C term mass transfer Shorter diffusion paths More effect on larger molecules 7

Higher efficiencies using SPP Additional efficiency can be generated through the use of superficially porous particles (SPP) rather than a totally porous particle (TPP) SPP particle For Maximum pressure Typical pressure Efficiency 1.9 µm Highest UHPLC performance 1300 bar 2.7 µm UHPLC performance at lower pressures 600 bar Similar to sub-2 µm totally porous 50% of sub-2 µm totally porous 4 µm Improved HPLC performance 600 bar Typically < 200 bar ~120% of sub-2 µm totally porous ~90% of sub-2 µm totally porous ~200% of 5 µm totally porous 8

Higher efficiencies using SPP Seed column feedback ZORBAX RRHD Eclipse XDB C18 3.0 x 50 mm, 1.8 µm InfinityLab Poroshell 120 EC-C18 3.0 x 50 mm, 1.9 µm the efficiency of the new Poroshell column was superior to Zorbax once achieved a good resolution in the separation of isomers of estradiol, essential for the validation of a method for monitoring such analytes. Thus, we will start to use the new Poroshell column in the ongoing validations in anabolic - Residue Laboratory Veterinary Medication LANAGRO / MG 9

Fast LC Aromatic acids 5991-7518EN 10

Advantages of fast LC Save time and reduce solvent use 5991-7518EN 11

Ultra-fast LC Aromatic acids 5991-7518EN 12

High resolution LC Tanshinones in Danshen (Salvia miltiorrhiza) High efficiency column increases the number of peaks that can be resolved (n c ) and Improves the accuracy of fingerprinting 5991-7559EN 13

The advantage of longer columns Total phenolic acids in Danshen (Salvia miltiorrhiza) 5991-7559EN 14

It s not only about efficiency R s = N 4 1 k k + 1 To increase resolution: Increase retention (k) Change selectivity (α) Increase efficiency (N) Change particle to impact N, smaller diameter and SPP for higher N Change mobile phase or column chemistry to impact k and α 15

Selectivity is also important Best all around InfinityLab Poroshell 120 EC-C18 1.9 µm, 2.7 µm, 4 µm InfinityLab Poroshell 120 EC-C8 1.9 µm, 2.7 µm, 4 µm InfinityLab Poroshell 120 Phenyl-Hexyl 1.9 µm, 2.7 µm, 4 µm Best for high ph mobile phases InfinityLab Poroshell HPH-C18 1.9 µm, 2.7 µm, 4 µm Best for alternative selectivity InfinityLab Poroshell 120 PFP 1.9 µm, 2.7 µm, 4 µm Best for more polar compounds InfinityLab Poroshell 120 HILIC 1.9 µm, 2.7 µm, 4 µm A range of different chemistries provides the selectivity options to develop methods quickly 16

Characterizing selectivity Tanaka and Hydrophobic subtraction model (HSM) Tanaka HSM Parameter Details CH2 H Hydrophobicity Separation based on differences in analyte hydrophobicity T/O S* Steric interaction Separation based on differences in analyte shape C/P A Hydrogen-bond acidity Separation based on hydrogen bonding by basic analytes B Hydrogen-bond basicity Separation based on hydrogen bonding by acidic analytes B/P (ph 2.7) C (ph 2.8) Ion-exchange capacity Separation based on ion exchange by analyte at ph <3 B/P (ph 7.6) C (ph 7.0) Ion-exchange capacity Separation based on ion exchange by analyte at ph 7 k PB EB Hydrophobic retention Retention of a neutral analyte HSM also features the F s factor to describe the similarity of two column selectivities. A small F s indicates that two columns are very similar, while a large factor indicates that two columns are very different. Further details at: http://www.hplccolumns.org/ 17

Retention and selectivity 18

HSM Fs values for InfinityLab Poroshell 120 F s = 12. 5 H 2 H 1 2 + 100 S 2 S 1 2 + 30 A 2 A 1 2 + 143 B 2 B 1 2 + 83 C 2 C 1 2 1 2 Chemistry H S* A B C (ph 2.8) C (ph 7.0) EB K F s (ph 7.0) F s (ph 2.8) EC-C18 1.02 0.008-0.13-0 0.161 0.123 6.9 0.00 0.00 EC-C8 0.88 0.011-0.23 0.023 0.127 0.09 4.8 5.92 5.96 HPH C18 1.03 0.005-0.14-0.01 0.073-0.004 6.1 10.65 7.46 Phenyl-Hexyl 0.75-0.08-0.39 0.018 0.136 0.14 3.6 12.99 13.08 PFP 0.63-0.06-0.46 0.015-0.038 0.741 2.9 52.96 21.14 Reversedphase only so HILIC not included Hydrophobic Subtraction Model (HSM) Data provided by Dwight Stoll EC-C18 HSM F s Values EC-C8 Phenyl-Hexyl HPH C18 PFP 0.00 10.00 20.00 30.00 40.00 50.00 60.00 Fs (ph 2.8) Fs (ph 7.0) 19

Benzodiazepines 5991-7539EN 20

HILIC Hydrophilic interaction liquid chromatography ACN ACN ACN ACN ACN ACN 1. ACN ACN CH 2 CHCH 3 ACN NH 3 H 2 O CH H 2 O 2 CHCH 3 H 2 O H 2 O H 2 O H 2 O - H 2 O + H O 2 O - NH - H O 3 O 2 O 2. O - + ACN 1. Partitioning in and out of adsorbed water layer 2. Ion exchange with silanols 21

HILIC separation Free amino acids 5991-7541EN 22

Free amino acids Separation of isobaric compounds 5991-7541EN 23

Retention Time Impact of mobile phase ph on selectivity Change in retention with mobile phase ph 12 10 8 6 More retention for non-charged analytes (i.e. acids at low ph and bases at high ph) Acetylsalicylic acid (pka 3.5) Pyridine (pka 5.2) Codeine (pka 8) Procainamide (pka 9.2) Amphetamine (pka 9.9) Caffeine (pka 14) Column: InfinityLab Poroshell HPH-C18 4 2 0 ph 2.5 ph 6.5 ph 8 ph 11.5 Mobile Phase: 45% Methanol, 55% 20 mm Phosphate Buffer 24

Approaches for longer lifetime at high ph Approach Totally porous silica-hybrid particles Bonding chemistry on superficially porous silica particles Integration of organic compound into the porous layer of SPP (prior to bonding) Comments Do not have the efficiency of superficially porous particles Do not have the lifetime of silica-hybrid particles Combine the advantages of silica-hybrid and superficially porous particles Utilizes a proprietary technology for particle synthesis Core P120 Particle Treated P120 25

Value of UHPLC Improve throughput and decrease cost-per-sample Increase the accuracy and precision of analysis results and reduce re-work 26

Issues getting the best value from UHPLC Issue Consequence Impact on value Columns give poor peak shape (tailing) Columns generate very high pressure Columns cannot maintain high efficiency under UHPLC conditions Decreased resolution leading to lower accuracy and precision of analysis results Cannot be run at optimum flow rate leading to slower runs or lower resolution Need to be replaced often Increased costs due to rework Increased costs due to lower throughput or increase in re-work Increased costs due to column purchase, disruption and re-work 27

Peak shape Issue Consequence Impact on value Columns give poor peak shape (tailing) Decreased resolution leading to lower accuracy and precision of analysis results Increased costs due to rework Superior peak shapes improve the accuracy and precision of your analytical results 1290 LC with ULD kit, 2.1 x 50 mm columns, 40% 20 mm Sodium Phosphate ph 7, 55 or 60% Acetonitrile, 0.5 ml/min, 0.5 ul, 25C, 254 nm, 80 Hz 28

0.192 1.528 1.096 0.825 1.534 0.930 1.035 0.503 0.191 0.915 0.191 0.800 1.107 0.413 0.481 0.686 0.715 What superior peak shape looks like Poroshell 120 EC-C18 1.9 µm SPP 1.7 µm DAD1 A, Sig=254,8 Ref=off (AEM_WATERS_QA\AEM_WATERS_QA2015-11-12 09-52-46\PLUTO-0000003.D) DAD1 A, Sig=254,8 Ref=off (AEM_WATERS_QA\AEM_WATERS_QA 2015-11-12 08-04-28\17KINETEX-00003.D) mau mau 400 350 300 60% ACN 250 250 200 150 200 100 50 0 150 0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.25 DAD1 A, Sig=254,8 Ref=off (AEM_WATERS_QA\AEM_WATERS_QA 2015-11-12 08-04-28\17KINETEX-00006.D) min mau 100 200 55% ACN 150 50 100 50 0 0 0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.25 min 0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.25 min Low peak tailing, increased resolution 1290 LC with ULD kit, 2.1 x 50 mm columns, 40% 20 mm Sodium Phosphate ph 7, 55 or 60% Acetonitrile, 0.5 ml/min, 0.5 ul, 25C, 254 nm, 80 Hz 29

Manageable pressure Issue Consequence Impact on value Columns generate very high pressure Cannot be run at optimum flow rate leading to slower runs or lower resolution Increased costs due to lower throughput or increase in re-work Manageable pressure allows you to use your columns at optimum flow rate and/or use longer columns without exceeding the pressure rating of your UHPLC instruments 1290 LC with ULD kit, 2.1 x 50 mm columns, 40% 20 mm Sodium Phosphate ph 7, 55 or 60% Acetonitrile, 0.5 ml/min, 0.5 ul, 25C, 254 nm, 80 Hz 30

Longest column lifetime Issue Consequence Impact on value Columns cannot maintain high efficiency under UHPLC conditions Need to be replaced often Increased costs due to column purchase, disruption and re-work Longer column lifetime reduces your costs for column replacement and the associated disruption and re-work 31

Column lifetime at high mobile phase ph Working at high mobile phase ph provides additional selectivity options but the column must be stable under these conditions Compatibility with high ph mobile phases means that additional selectivity options are available - allowing you to improve the accuracy and precision of your analytical results Columns: 2.1 x 50 mm, Isocratic ph 10, 50 C 10 mm Ammonium Bicarbonate, 0.4 ml/min, Sample: Butyl benzene 32

Economic value of robust UHPLC columns Long lifetime not only reduces column spend fewer columns are needed for the same amount of work but also reduces costs due to the disruption caused and re-work required when columns fail This represents a significant economic value to your laboratory 33

Getting the best value from UHPLC Feature Advantage Economic value High purity silica, quality novel chemistries Robust superficially porous 1.9 um particles, optimized column loading Robust superficially porous 1.9 um particles, optimized column loading, novel chemistries Superior peak shape Manageable pressure Long column lifetime Decreased costs due to re-work Decreased costs due to lower throughput or increase in re-work Decreased costs due to column purchase, disruption and re-work 34

Scalable family of particles 0.75um 0.5um 0.35um 1.2um 1.7um 2.5um 0.35um 0.5um 0.75um InfinityLab Poroshell 120 1.9 µm Highest UHPLC performance InfinityLab Poroshell 120 2.7 µm UHPLC performance at lower pressure InfinityLab Poroshell 120 4 µm Improved HPLC performance www.agilent.com/chem/discoverporoshell 35

The advantage of a scalable family of particles Aromatic acids 5991-7518EN 36

An SPP column for everyone Technique / product Performance 1.9 µm UHPLC 2.7 µm HPLC 4 µm Highest UHPLC performance Pressure rating: 1300 bar Typical pressure: Similar to sub-2 μm totally porous Efficiency: ~120% of sub-2 μm totally porous UHPLC performance at lower pressure Pressure rating: 600 bar Typical pressure: 50% of sub-2 μm totally porous Efficiency: ~90% of sub-2 μm totally porous Improved HPLC performance Pressure rating: 600 bar Typical pressure: Often < 200 bar Efficiency: ~200% of 5 μm totally porous 37

Use all the instruments in your laboratory Instruments UHPLC only Maximum pressure: High (> 600 to 1000+ bar) Dispersion volume: Very low We recommend InfinityLab Poroshell 120 1.9 μm InfinityLab Poroshell 120 2.7 μm HPLC and UHPLC Maximum pressure: Low to high (400 to 1000+ bar) Dispersion volume: Medium to very low InfinityLab Poroshell 120 2.7 μm InfinityLab Poroshell 120 4 μm HPLC only Maximum pressure: Low to mid (400 to 600 bar) Dispersion volume: High to low InfinityLab Poroshell 120 4 μm InfinityLab Poroshell 120 2.7 μm 38

Summary UHPLC is used either for very fast or very high resolution separations High efficiency is a key requirement for UHPLC and high pressure a key consequence Minimized instrument dispersion volume is key for successful UHPLC < 2 um SPP provide very high efficiency separations Methods can be developed quickly and easily with a range of chemistries, including phases for high ph work UHPLC columns providing a unique combination of superior peak shape manageable pressure and long lifetime represents significant economic value to your lab You can select the best column for your needs/instruments from a scalable family of particles 39

Find out more Learn more www.agilent.com/chem/discoverporoshell Get support www.agilent.com/chem/cstechsupport Contact a local Agilent customer center in your country: www.agilent.com/chem/contactus USA and Canada: 1-800-227-9770, agilent_inquiries@agilent.com Europe: info_agilent@agilent.com Asia Pacific: inquiry_lsca@agilent.com 40

Ordering details Size (mm) EC-C18 EC-C8 Phenyl-Hexyl HPH-C18 PFP HILIC 3.0 x 150 693675-302 693675-306 693675-312 693675-502 693675-308 693675-301 3.0 x 100 695675-302 695675-306 695675-312 695675-502 695675-308 695675-301 3.0 x 50 699675-302 699675-306 699675-312 699675-502 699675-308 699675-301 2.1 x 150 693675-902 693675-906 693675-912 693675-702 693675-408 693675-901 2.1 x 100 695675-902 695675-906 695675-912 695675-702 695675-408 695675-901 2.1 x 50 699675-902 699675-906 699675-912 699675-702 699675-408 699675-901 Size (mm) EC-C18 EC-C8 Phenyl-Hexyl HPH-C18 PFP HILIC 3.0 x 5 823750-940 823750-941 823750-943 823750-945 823750-942 823750-944 2.1 x 5 821725-940 821725-941 821725-943 821725-945 821725-942 821725-944 All InfinityLab Poroshell 120 1.9 μm columns are supplied with a pre-programmed Column ID 41

Column ID Usability, traceability and security All InfinityLab Poroshell 120 1.9 µm columns are shipped with preinstalled and pre-programmed Column ID Column ID Clip that holds column ID to column 42

Understand key details and use of your column Field Example Description Poroshell EC-C18 Length [mm] 100 Diameter [mm] 4.6 Particle size [µm] 2.7 Maximum pressure [bar] 600 Number of injections [counter] Product number 695975-902T Serial number USABC12345 Batch number B12345 Maximum temperature [ C] 60 Maximum measured temperature [ C] [updated from instrument] Minimum ph 2.0 Maximum ph 8.0 Void volume [ml] 1.00 First injection date [updated from instrument] Recent injection date [updated from instrument] Usability Easily find column details Traceability Always know exactly which column is/was installed Security Protect against the use of methods incompatible with the column 43

Agilent InfinityLab www.agilent.com/chem/infinitylab 44