Agilent Poroshell 120 Columns for HPLC and UHPLC PERFORM RUGGED, FAST LC WITH CONFIDENCE

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1 Agilent Poroshell Columns for HPLC and UHPLC PERFRM RUGGED, FAST LC WITH CNFIDENCE

2 AGILENT PRSHELL CLUMNS CAN MAKE EVERY LC AND LC/MS IN YUR LAB WRK EVEN HARDER We choose Poroshell because of its rugged performance Poroshell provides reliably excellent performance it s the new standard in our lab For complicated samples, which I face most, Poroshell columns save me a lot of time Poroshell is my go-to column QUTES FRM PRSHELL USERS Poroshell columns provide exceptional efficiency for standard HPLC, and significantly boost performance from all instruments, whether you have older 4 bar or newer bar UHPLC systems. These columns take the technology introduced with our Poroshell columns to the next level giving you higher throughput and resolution for a wider range of small molecules and peptides than ever. Their advanced features include: Excellent lot-to-lot reproducibility Poroshell columns are manufactured using a proprietary, single-step porous shell process that dramatically reduces tiny differences between columns and lots Comparable speed and resolution to sub- µm columns and significant improvements over μm columns with much lower backpressure, taking HPLC and UHPLC performance to a new level of flexibility and efficiency Superior peak shape especially at ph 6-7, for faster, more accurate results Long column life Poroshell columns use a standard μm frit, and resist plugging with dirty samples Up to TWELVE chemistries, depending on the particle size, including SB-C8 and SB-C8 for low ph applications and Poroshell HPH-C8 and HPH-C8 for high ph applications Easy method transfer to ZRBAX bonded phases and within the Poroshell family, for highest productivity from lab to lab, around the world UHPLC guard column options that reduce your operating costs by extending the life of Poroshell columns Scalability within the Poroshell Family with column options in 4 μm and.7 μm configurations for optimal performance for your method

3 Contents What makes Poroshell columns different? Unique processes that make our superficially porous particles and bonded phases contribute to the excellent results you can achieve Page 4 A family of particle sizes and bonded phases for flexible selectivity and scalability Poroshell 4 μm and.7 μm columns are scalable to and from ZRBAX phases Page 6 Making your HPLC work harder Yes, you can achieve Fast LC performance at HPLC pressures Page 6 Making every LC and LC/MS work harder Perform high-speed, high-resolution separations on your instruments Page 9 Increasing the flexibility of your UHPLC methods Now you can perform very fast, high-efficiency separations under the widest range of separation conditions Page Easy method transfer Save time and money by moving methods from μm or. μm columns to Poroshell Page 4 Solutions to frustrating throughput and resolution problems Poroshell columns help you meet the challenges you face every day Page 8 New options for proteins and peptides Achieve faster peptide mapping and protein separations with Poroshell technology Page Infinitely better liquid chromatography Introducing the Agilent 9 Infinity II LC Page Specifications and ordering information Page 6 To learn more about Agilent Poroshell columns, visit agilent.com/chem/poroshell

4 HIGH EFFICIENCY AT LWER PRESSURES PRVEN CLUMN-T-CLUMN CNSISTENCY THAT S THE PRSHELL DIFFERENCE A key feature of Agilent Poroshell columns is their superficially porous microparticulate column packing. Poroshell columns are available with 4 μm and.7 μm particles to provide scalable performance. The particles have a solid silica core with a porous shell. This unique configuration gives you all the performance advantages of smaller totally porous particles, but with lower backpressures..8 μm totally porous Agilent Poroshell.7 μm Agilent Poroshell 4 μm. μm.7 μm.7 μm. μm Pore size Å, ideal for small molecules.8 μm.7 μm 4 μm How a Poroshell particle is made To create the best column for small molecule separations, we completely reinvented our superficially porous particle technology. Specifically, we imized the number of manufacturing steps to ensure maximum particle and chromatographic reproducibility. STEP Make the solid core Poroshell cores have a very smooth surface and a uniform particle size which contribute to a tight overall particle size distribution. As a result, you get a more tightly packed column bed and higher efficiency than with totally porous particles. STEP Apply the porous shell At Agilent, we apply the porous shell in one single step similar to the coacervation technique used to make traditional ZRBAX columns. This unique single-step process delivers higher yields and more column-tocolumn reproducibility than other vendors can. STEP Apply the bonded phase The family of Agilent Poroshell phases is expanding to align with the ZRBAX family for method development flexibility and assured scalability. 4

5 A comparison of particle size distributions between totally porous and Poroshell particles This graph demonstrates that Poroshell columns have the tightest final particle size distribution a direct result of starting with a tight core size distribution. Number 4 Poroshell.7 µm ZRBAX RX SIL.8 µm ZRBAX RX SIL. µm ZRBAX RX SIL. µm The tight final particle size distribution is a result of starting with a tight core size distribution. This helps imize diffusion and results in the high efficiency of superficially porous columns (µm) The standard measure of particle size distribution is the 9/ ratio, which should be below. The ZRBAX totally porous particles (.8 μm,. μm, and. μm) all have an acceptable particle size distribution. However, the Poroshell particle has a % tighter particle size distribution, which substantially improves column efficiency. Poroshell (.7 µm) LN B6 ZRBAX.8 µm ZRBAX. µm ZRBAX. µm %.4 µm.67 µm.7 µm 4.9 µm 9%.8 µm.4 µm 4.44 µm 6. µm 9%/% ratio Reproducible performance from lot to lot, year after year Poroshell particles are made with a proprietary porous particle manufacturing process, invented by Agilent. Instead of traditional multilayering, Poroshell columns are manufactured using a single-step coacervation process that produces a more consistent final particle and more reliable chromatographic results. The simpler the manufacturing process, the more consistent the column A single-step shell process creates a highly reproducible column, as you can see in this lot-to-lot comparison of five lots. Poroshell EC-C8,. x mm,.7 µm (p/n ) from five different lots 4 4 B B8 4 B4 4 B6 4 B4 To learn more about Agilent Poroshell columns, visit agilent.com/chem/poroshell

6 A VARIETY F BNDED PHASES MEANS YU NEVER HAVE T CMPRMISE N SELECTIVITY Poroshell columns are made at the same facility as the Agilent industry-leading ZRBAX column family. The bonding chemistries used with Poroshell columns mirror those of all ZRBAX columns, giving you the advantages of easier method transfer and assured scalability from lab to lab, around the world. All the selectivities you need to perfect your separation AN EXCELLENT FIRST CHICE H H N N N N N N N N Poroshell EC-C8 (USP L) and EC-C8 (USP L7)* You can count on this high-performance phase to deliver excellent peak shape and resolution for acids, bases, and neutrals. The chemistry is very similar to the ZRBAX Eclipse Plus phase, for easy method transferability. Exceptional peak shape, efficiency, resolution, and lifetime. Tip: Select the C8 phase first, and use the C8 phase for less retention with a variety of samples. H N N N N N N A variety of published methods from Agilent use Poroshell, allowing for easy method development. H H H H H H H C N N N C N Methods easily convert from [ZRBAX] Eclipse Plus columns to Poroshell we use them for all methods. QUTES FRM TXICLGY LAB USERS 6 6

7 Poroshell Phenyl-Hexyl (USP L)* This phase provides alternative selectivity for phenyl groups, and is very similar to ZRBAX Eclipse Plus Phenyl-Hexyl for easy method transfer. H C H H N CH CH Poroshell PFP (USP L4)* An alternative selectivity for halogenated compounds and polar analytes. F H CH H HIGH ph APPLICATINS Poroshell HPH-C8 (USP L) and HPH-C8 (USP L7) The silica in this special chemistry has been modified with a proprietary process to increase stability at high ph. H H H H Poroshell Bonus-RP (USP L6) Bonus-RP is polar-embedded to improve peak shape for basic compounds at low- and mid-ph. This phase is the same as ZRBAX Bonus-RP. S H H H H F F H H N CH CH H C N CH H H H N CH H LW ph APPLICATINS StableBond SB-C8 (USP L) and SB-C8 (USP L7) StableBond performs well with acids, bases, and neutrals with superior lifetime at low ph. What s more, these phases transfer readily from ZRBAX SB-C8 and ZRBAX SB-C8 phase chemistries. H Poroshell HILIC* With its unbonded silica, Poroshell HILIC retains and separates small polar analytes. Poroshell EC-CN (USP L) Similar to ZRBAX Eclipse XDB-CN, this cyano phase simplifies method transfer. H N H N H H H H N H C H CH HCl Poroshell SB-Aq This proprietary phase provides an alternate selectivity option, and is ideal for polar compounds and high aqueous conditions. Its chemistry is the same as ZRBAX SB-Aq. H H H H H H H H H H H H H * Now available in 4 μm and.7 μm particle sizes. Visit agilent.com/chem/discoverporoshell for videos, Application Notes, and more or to order now 7 7

8 Agilent Poroshell EC-C8 and Poroshell SB-C8 provide different selectivity for optimizing separations Poroshell SB-C8,. x mm,.7 µm (p/n 6897-) Mobile phase: % H, 6% CH CN Flow rate: ml/ Temperature: ºC 4 MS acquisition: Dynamic MRM Compound Precursor ion Fragmentor voltage Anandamide (AEA) 48 Palmitoylethanolamide (PEA) -Arachidonoylglycerol (-AG) 79 leoylethanolamide (EA) 6 MS Source: Gas temp: ºC Gas flow: L/ Nebulizer: 4 psi Capillary: 4, V Poroshell EC-C8,. x mm,.7 µm (p/n 6997-) Analytes:. Anandamide (AEA). -Arachidonoylglycerol. Impurity 4. Palmitoylethanolamide (PEA). leoylethanolamide (EA) 4 Agilent Poroshell EC-C8 is less retentive for faster analysis of nonpolar compounds Mobile phase: Flow rate: Temperature: 6 ºC Detection: Sample: 6% CH CN, 4% H.8 ml/ 4 nm µl of RRLC checkout sample (p/n 88-69), alkylphenones Poroshell EC-C8,. x mm,.7 µm (p/n )..... Poroshell EC-C8,. x mm,.7 µm (p/n ).... 8

9 A choice of particle sizes help you make the best choice for your method development Easy drop-in replacement for existing μm traditional LC methods This separation of a phenol mix shows the scalability of selectivity going from μm Eclipse Plus C8 to Poroshell 4 μm and.7 μm columns. It also demonstrates a significant performance improvement in peak capacity. Additionally, the 4 μm column offers imal backpressure increases over existing μm columns, and the.7 μm offers significantly lower backpressure than sub- μm columns. Phenol mix gradient at. ml/ ZRBAX Eclipse Plus C8 µm Poroshell EC-C8 4 µm Poroshell EC-C8.7 µm Columns: Instrument: Mobile phase: Flow rate: Temperature: C Detection: Sample: All columns 4.6 x mm Agilent 6 Infinity LC, pulse damper and mixing column bypassed A:.% Formic acid B: MeH +.% formic acid.4 ml/ 6 nm Phenol mix ZRBAX Eclipse Plus C8.8 µm Phenol mix gradient scaled at flows between. and. ml/ Gradient: 4-8% MeH/4 Peak Capacity. 4.. Pressure (bar) Flow Rate (ml/) Flow Rate (ml/) Poroshell EC-C8 4 µm Poroshell EC-C8.7 µm ZRBAX Eclipse Plus C8.8 µm ZRBAX Eclipse Plus C8 µm To learn more about Agilent Poroshell columns, visit agilent.com/chem/poroshell 9

10 ENABLE HIGH-PERFRMANCE SCREENING METHDS WITH PRSHELL HPH-C8 AND HPH-C8 A robust method development process is critical to ensuring that your method is long lasting, stable, and reliable. Because the retention and selectivity of ionizable compounds (such as acids and bases) can change significantly with varying ph, it is becog standard practice to use low, medium, and high ph analyses during method development. Poroshell HPH-C8 and HPH-C8 are made by chemically modifying Poroshell particles using proprietary technology to give high ph stability. That means you can use the Poroshell family for all your Fast LC method development needs, regardless of mobile phase ph. Reliable separations for varying ph levels Here, a method using low, mid, and high ph separates the same mixture of acids, bases, and neutrals. The highest resolution for all compounds was obtained under higher-ph conditions, and so, high ph would be the best choice going forward ph, mm NH 4 HC ph 4.8, mm NH 4 CH C ph, mm NH 4 HC Sample:. Procainamide. Caffeine. Acetyl salicylic acid 4. Hexanophenone deg.. Dipyrimadole 6. Diltiazem 7. Diflunisal 8. Hexanophenon

11 Excellent stability at High ph Count on Poroshell HPH chemistries for consistent performance and longevity, even when using high-ph mobile phases. Here,, injections of a separation mixture containing acidic, basic, and neutral compounds were performed under extreme ph conditions on an Agilent Poroshell HPH-C8 and a non-agilent high ph column. Notice the non-agilent column s loss of resolution between the Nortryptyline and Heptanophenone while the Poroshell HPH-C8 maintains resolution. Column: Agilent Poroshell HPH-C8,. x mm,.7 µm (p/n ) Injection Injection Injection, Injection, Instrument: Mobile phase: 6 Infinity Binary LC A: mm Ammonium bicarbonate adjusted to ph. in water B: Acetonitrile Column: other vendor High ph. x mm, µm Flow rate: Gradient:.4 ml/ Time % B 9. Sample:. Methyl salicylate. 4 Chlorocinnamic acid. Acetophenone 4. Quinine. Nortryptyline 6. Heptanophenone 7. Amitriptyline 4 6 Injection Injection Injection, Injection,

12 PTIMIZE EVERY SEPARATIN WITH A CHICE F RTHGNAL PHASES Selectivity is the most powerful tool for optimizing HPLC separations. Poroshell EC-C8 is the best place to start your method development, because of its exceptional flexibility. However, if you are working with challenging analytes, the Poroshell family has many additional chemistries to choose from. For example, our Poroshell PFP columns are engineered with a pentafluorophenyl ligand, which provides an orthogonal separation mechanism with traditional reversed-phase columns. By specifically targeting polar retention mechanisms, PFP phases can separate analytes based on small differences in structure, substitution, and steric access to polar moieties. The resulting selectivity for positional isomers, halogenated compounds, and polar analytes is particularly useful when analyzing complex mixtures and small-molecule pharmaceuticals. Comparative analysis of NSAIDs This separation was completed with four Poroshell chemistries using acetonitrile. Each run was only five utes long. nly Poroshell PFP resolved all compounds, although both Poroshell EC-C8 and Poroshell Phenyl-Hexyl columns eluted the compounds in the same order. The PFP and Bonus-RP columns had very similar elution orders, with the exception of the last two peaks , ,6,9 4,7,6 9 8 Poroshell PFP Poroshell EC-C8 Poroshell Bonus-RP Poroshell Phenyl-Hexyl Columns: Poroshell PFP, 4.6 x mm,.7 µm (p/n ) Poroshell EC-C8, 4.6 x mm,.7 µm (p/n ) Poroshell Bonus-RP, 4.6 x mm,.7 µm (p/n ) Poroshell Phenyl-Hexyl, 4.6 x mm,.7 µm (p/n ) Instrument: 6 Infinity Binary LC Mobile phase: A: mm NH 4 HC, ph. B: Acetonitrile Flow rate: Detection: Gradient: ml/ UV, 4 nm Time % rganic Fluorinated HPLC Phases: Looking Beyond C8 for Reverse-Phase HPLC M. Przybyciel, LCGC Europe 9() pp 9-8, 6. Sample:. APAP. Phenacetin. Piroxicam 4. Tolmetin. Ketoprofen 6. Naproxen 7. Sulindac 8. Diclofenac 9. Difunisal

13 Selectivity the most powerful tool for optimizing HPLC separations Positional isomers ( compounds) Mobile phase A, Water (.% acetic acid), B, Acetonitrile Sample:.,4 Dimethoxyphenol.,6 Dimethoxyphenol., Dimethoxyphenol 4.,6 Difluorophenol.,4 Difluorophenol 6., Difluorophenol 7.,4 Difluorophenol 8. Degradation product,6 dimethoxyphenol 9., Dimethylphenol.,6 Dimethylphenol.,6 Dichlorophenol. 4 Chloro methyl phenol. 4 Chloro methyl phenol 4.,4 Dichlorophenol., Dichlorophenol Time %B 6 Flow rate: ml/ Detection: 7 nm Column dimensions: 4.6 x mm Poroshell PFP 4 7,8, , Poroshell EC-C8 Poroshell Phenyl-Hexyl Poroshell EC-C This separation illustrates the benefits of the PFP phase chemistry. Here, positional isomers are analyzed using four different chemistries, with the PFP offering the best resolution. To learn more about Agilent Poroshell columns, visit agilent.com/chem/poroshell

14 Analysis of beta blockers: a comparison of Poroshell phases This challenging separation demonstrates how different selectivities produce different results. verall, the Bonus-RP phase delivered the best peak shape and resolution. This was especially true for nadolol, which appeared as a split peak with the C8 and Phenyl-Hexyl phases Poroshell Bonus-RP Poroshell Phenyl-Hexyl Poroshell EC-C Poroshell SB-C8 - Columns: Poroshell Bonus-RP,. x mm,.7 µm (p/n ) Poroshell Phenyl-Hexyl,. x mm,.7 µm (p/n ) Poroshell EC-C8,. x mm,.7 µm (p/n ) Poroshell SB-C8,. x mm,.7 µm (p/n ) Instrument: 6 Infinity Binary LC Mobile phase: A: mm NH 4 HC, ph.8 B: MeH Flow rate:.4 ml/ Temperature: 4 C Detection: 6 nm Gradient: % B to % B/ Sample:. Atenolol. Pindolol. Nadolol 4. Metoprolol. Acebutolol 6. Propranolol 7. Alprenolol 4

15 With the Poroshell 4 µm columns, you can still take advantage of the flexibility of additional phase chemistries. With five chemistries available, choose the phase that takes advantage of key analyte interactions, such as the pi-pi interactions shown here with a steroid separation. Isocratic Test Column: Poroshell 8 or PH, 4.6 x mm, 4 µm Mobile Phase: 64% MeCN or MeH 6% Water w/.% acetic acid Sample:. Triamcinolone. Prednisolone. Corticosterone 4. Estradiol. DES 6. Dienestrol 7. Deoxycorticosterone Flow rate:. ml/ Temperature: ºC Detection:, 4 nm 4,, Poroshell EC-C8 4 µm Poroshell Phenyl-Hexyl 4 µm Steroids separation on both the Poroshell EC-C8 4 µm and Poroshell Phenyl-Hexyl 4 µm chemistries. You can see that better resolution was achieved on the Poroshell Phenyl-Hexyl due to the pi-pi interactions with the analytes and the stationary phase. To learn more about Agilent Poroshell columns, visit agilent.com/chem/poroshell

16 FAST LC/UHPLC PERFRMANCE FRM A STANDARD HPLC? AGILENT PRSHELL MAKES IT PSSIBLE With Poroshell columns, you can achieve up to 9% or more of the efficiency you would expect from a sub- μm Fast LC/UHPLC column but at HPLC pressures (below 4 bar). This ability to perform fast separations at low pressures with both the 4 μm and.7 μm column options can dramatically enhance your productivity. Now, you can run more samples in less time using your lab s existing HPLC systems as the following examples illustrate. Plus, you ll be ready to transfer your method seamlessly to an Agilent Infinity Series of your choice when you re ready, for even more productivity. UHPLC efficiency with less pressure For this sample of neutral alkylphenones, the Poroshell column delivered >9% of the efficiency of the.8 μm column. Note, too, that the pressure on the Poroshell column is about % of the pressure on the.8 μm column. Mobile phase: 6% Acetonitrile, 4% water Flow rate:.8 ml/ Injection volume: 4 µl Temperature: 6 ºC Detection: DAD Sig = 4,4 nm Ref = 6, nm Sample preparation: RRLC checkout sample (p/n 88-69) spiked with µl mg/ml thiourea in water: acetonitrile (6:) Poroshell EC-C8,. x mm,.7 µm (p/n 6997-) N =,, Press = 8 bar >9% of the efficiency of.8 µm Eclipse Plus C8,. x mm,.8 µm (p/n ).48.7 N = 7,9, Press = 86 bar

17 Choose Agilent Poroshell for high efficiency HPLC In this analysis of soft drink components, the Poroshell column achieved: Poroshell EC-C8,. x mm,.7 µm (p/n 6997-) N =,8 P = 4 bar >9% of the efficiency of a sub- μm column x the efficiency of the. μm column Pressure below 4 bar, while the pressure on sub- μm columns is above 4 bar The low backpressure achieved with the methanol mobile phase is especially significant, because methanol generates more pressure than acetonitrile. 4 6 ZRBAX RRHT Eclipse Plus C8,. x mm,.8 µm (p/n ) N =,64 P = 46 bar Column:. x mm,.7 µm Mobile phase: A: 6%,.% Formic acid B: % Methanol isocratic Flow rate:. ml/ Injection volume: µl Temperature: 6 C Detection: UV, nm ZRBAX Rapid Resolution Eclipse Plus C8,. x mm,. µm (p/n 9996-) N =,896 P = bar Page 7 Sample:. Saccharin. Caffeine. p-hydroxybenzoic acid Aspartame. Dehydroacetic acid 6. Benzoic acid To learn more about Agilent Poroshell columns, visit agilent.com/chem/poroshell 7

18 Why use a guard column? Simply put, guard columns can save your lab money by extending the life of your analytical column. Installing a less expensive guard column, especially when analyzing dirty samples, prevents damage caused by particulate matter and strongly adsorbed material. As a guide, you should replace your guard column when the plate number, pressure, or resolution changes by more than %. However, you will need to make an exact deteration based on your application. The example here is an accelerated lifetime test with : water:similac with. mg sulfachloropyridazine and sulfamethoxazole. Mobile phase: A:.% Formic acid in water B: Acetonitrile Flow rate:.6 ml/ Gradient: Hold % B for, ramp to 4% B in Injection volume: µl Temperature: C Detection: Sig = 4, 4 nm; Ref = ff Instrument: Infinity Series Sample preparation: ml Water +. ml similac + ml. mg/ml sulfachloropyridazine and sulfamethoxazole Peak Width () Peak Width () Without a guard column: Analytical column failure after just 8 injections. With a guard column: The guard column fails after 8 injections preserving the analytical column. Injection no Sulfachloropyridazine Injection no. Replace the analytical column Sulfamethoxazole Replace only the less-expensive guard column Fast LC applications stay fast with Agilent Fast Guard columns Agilent Fast Guard columns for UHPLC are rugged and reliable at high pressures, and are fully compatible with Agilent Fast LC and UHPLC columns. They can also be installed without any special tools. Watch the video to learn how easy it is to install Agilent Fast Guard columns: agilent.com/chem/poroshell 8

19 A RUGGED CHICE FR HIGH RESLUTIN LC/MS AND LC/MS/MS Agilent Poroshell columns can make your LC/MS and LC/MS/MS systems work even harder. Their porous outer layer and solid core limit diffusion distance and improve separation speed, while their narrow particle size distribution improves efficiency and resolution. ther advantages include: Quick and efficient resolution of critical isobaric compounds Better resolution of closely eluting peaks More compounds resolved in a single analysis Improved LC/MS accuracy and identification A standard µm frit that resists plugging with dirty samples Separation of cholesterol and other sterols using Poroshell EC-C8 columns with LC/MS/MS Note that adequate resolution was obtained, even at the : cholesterol:lathosterol. This is critical for effective quantitation, because the two compounds have the same molecular weight. Sample:. Calcifediol. Desmosterol. -Cholesten--one 4. Lathosterol. Cholesterol 6. Campesterol 7. Stigmasterol 8. Cholestanol 9. Sitosterol Column: Poroshell EC-C8,. x mm,.7 µm (p/n 6997-) Mobile phase: 8% Acetonitrile, % methanol Flow rate:.6 ml/ Injection volume: µl Temperature: C Detection: APCI, positive ion To learn more about Agilent Poroshell columns, visit agilent.com/chem/poroshell 9

20 D-Separation of Vita D /D on Agilent Poroshell EC-C8 Poroshell provides a very fast LC/MS/MS analysis of vita D /D in plasma. Isocratic conditions were varied to compare speed of separation with chromatographic resolution. Column: Poroshell EC-C8,. x mm,.7 µm (p/n ) Mobile phase: A: H +.% formic acid B: MeH +.% formic acid Flow rate:. ml/ Injection volume: μl Temperature: C Autosampler temp: C Needle wash: Flush port (::, IPA: MeH:H ) Isocratic analysis: A: %, B: 8% Analysis time: x x Counts vs. Acquisition Time () 8% MeH 8% MeH *.7 -H Vita D -H Vita D Counts vs. Acquisition Time () Rugged performance even after, injections This test confirms the outstanding longevity of Poroshell columns, with little performance degradation after, injections. Stability is expressed in the consistency of the retention times (%RSD). Analyte %RSD (RT) Analyte %RSD (RT) Analyte %RSD (RT) Morphine.7 Meperidine.4 Triazolam Codeine.4 Zolpidem. Naltrexone. Hydrocodone.4 Fentanyl. Chlordiazepoxide. MDMA. EDDP. Desmethyl diazepam. Norfentanyl. Nitrazepam. Cocaethylene. Heroin. Propoxyphine. -nor-9-carboxy-delta-9-thc Methylphenidate. Buprenorphine.

21 AGILENT PRSHELL CLUMNS HELP YU INCREASE THE FLEXIBILITY F YUR UHPLC METHDS Because Poroshell columns have a pressure limit of 6 bar, you can successfully apply them to your UHPLC methods including those that use very long columns, higher flow rates, and viscous solvents. Agilent Poroshell EC-C8 for fast UHPLC separations This example shows a fast separation using a mobile phase that generates higher pressures. In the top chromatogram, a. mm id column was used, with a flow rate of. ml/ and a pressure below 4 bar making this a typical LC separation. Although the top separation was fast (just under 6 utes), the middle and bottom chromatograms show that you can reduce run times to under utes by increasing the flow rate. These faster analyses will take your pressure to 4 to 6 bar. Explore the Agilent Infinity Series flexible upgrade options to help you take advantage of UHPLC capabilities More viscous solvents like methanol can be used at HPLC or UHPLC pressures. Flow rate =. ml/, P = bar, N BA = 4,97 Flow rate =.7 ml/, P = 4 bar Flow rate =. ml/, P = 9 bar More viscous solvents like methanol can be used Column: Poroshell EC-C8. x mm,.7 µm (p/n 6997-) Mobile phase: A: 6%,.% Formic acid B: % Methanol isocratic Flow rate: see chromatograms Injection volume: µl Temperature: 6 ºC Detection: Sig =, 4 nm, Ref = ff Sample:. Saccharin. Caffeine. p-hydroxybenzoic acid 4. Aspartame. Dehydroacetic acid 6. Benzoic acid P P P To learn more about Agilent Poroshell columns, visit agilent.com/chem/poroshell

22 . Agilent Poroshell for HPLC and UHPLC comparison of EPA 8 separation on short and long columns Poroshell columns give you the flexibility to choose longer columns for higher resolution. Here, you can see that as the column gets longer, resolution improves and pressure increases (up to UHPLC pressures for the longest column). Note that column length affects resolution not by the batch of material used in the column proving that Poroshell columns deliver reproducible performance mm length P = bar SN USCFZ7, batch B mm length P = 8 bar SN USCFX69, batch B4 mm length P = 68 bar SN USCFW49, batch B Balancing column length, resolution, and analysis time are important for any separation. Column: Poroshell EC-C8,.7 µm Mobile phase: % Methanol, 7% water Flow rate: ml/ Temperature: 44 ºC Agilent Poroshell columns in series deliver the highest efficiency at HPLC and UHPLC pressures Because low backpressure is one of the advantages of Poroshell columns, you can couple several columns in series to achieve the highest separation power per unit time. This enables better separation of complex samples. Peak no. Compound Plates k Acetophenone 4,.9 Benzene 9, Toluene 4,8.6 Three Poroshell EC-C8, 4.6 x mm,.7 µm (p/n ) columns in series for very high efficiency. Poroshell Columns in Series 4.6 x mm,.7µm Flow rate: ml/ 7. N ~ 8, (peak 4). Max pressure: 6 bar Flow rate:. ml/ 7. N ~, (peak 4). Max pressure: 478 bar Flow rate:.8 ml/ N ~, (peak 4). Max pressure: 7 bar Maximum efficiency at.8 ml/

23 Fast analysis on an Agilent Poroshell EC-C8 of compounds found in analgesics Here, we used a high flow rate to speed up the separation of common analgesic compounds using a Poroshell column Maximum pressure 4 bar, Column: Poroshell EC-C8, 4.6 x mm,.7 µm (p/n ) Mobile phase: A: Water +.% formic acid B: Acetonitrile Flow rate:. ml/ Injection volume: µl Temperature: 4 C Detection: DAD, 4 nm Sample:. Acetaophen. Caffeine. -Acetamidophenol 4. Acetamide. Phenacetin 6. Sulindac 7. Piroxicam 8. Tolmetin 9. Ketoprofen. Diflusinal. Diflunisal Keep your LC system running at its best with Agilent A-Line supplies More ways to get more done through: Convenience Simplicity Increased efficiency Learn more: agilent.com/chem/a-line To learn more about Agilent Poroshell columns, visit agilent.com/chem/poroshell

24 CMPLEX METHD TRANSFERS MADE SIMPLE Many methods developed on longer µm C8 columns can be moved to Poroshell columns quickly and easily, especially with the newly available Poroshell 4 µm columns. New changes to the USP regulations are making it easier to transfer conventional methods to newer technologies like Aglient Poroshell. This enables chromatographers to significantly increase throughput and reduce costs. n the following pages, we will show you how five separations, including USP methods, can be repeated on Poroshell columns and can be completed - times faster than the same separations on μm columns. Transfer methods between Agilent Poroshell and ZRBAX for time savings or scalability In this example, a complex method was transferred from a ZRBAX Eclipse Plus C8 mm, μm column to a mm Poroshell EC-C8 column. All conditions were kept the same, except for the gradient time, which was adjusted for the shorter column. As you can see, both separations are the same. However, the bottom chromatogram was generated in just over 7 utes instead of utes for the top chromatogram an excellent productivity improvement. Keep in d that both separations were run on an older Agilent Series instrument proving that even gradient methods can be transferred while keeping the pressure below 4 bar bar bar Column: ZRBAX Eclipse Plus C8, 4.6 x mm, µm (p/n ) Flow rate: ml/ Sample:. Sulfadiazine. Sulfathiazole. Sulfapyridine 4. Sulfamerazine. Sulfamethazine Column: Flow rate: Sulfamethazole 7. Sulfamethoxypyridazine 8. Sulfachloropyridazine 9. Sulfamethoxazole. Sulfadimethoxine Poroshell EC-C8, 4.6 x mm,.7 µm (p/n ) ml/ Time %B 8 Time %B 8. Mobile phase: A:.% Formic acid in water B:.% Formic acid in acetonitrile 4

25 The Agilent Poroshell, 4 μm column expansion This addition provides the Poroshell platform with a scalable solution for chromatographers and method developers. This robust platform extension, initially consisting of the EC-C8, EC-C8, Phenyl-Hexyl, PFP, and HILIC chemistries, helps you enter the Poroshell family in a very simple manner with the ease of drop-in method replacements. Column pressures are % less than the.7 μm Poroshell, and efficiencies are nearly double those of traditionally totally porous μm columns. Chromatographers looking for moderate performance increases can adopt the 4 μm Poroshell columns into their method with simplicity. USP method for naproxen tablets 4.x faster analysis on Agilent Poroshell at HPLC pressures This naproxen separation demonstrates how easy it can be to convert a method to Poroshell columns without changing the flow rate or mobile phase. The st chromatogram shows a USP analysis on an Agilent ZRBAX Eclipse Plus C8 column, which delivers sharp peaks, three times the needed efficiency, and a resolution of ~4. In the nd and rd chromatograms, the Poroshell EC-C8 4 μm columns ( mm and mm) provide greater efficiency and speed of the original method as easy, drop-in replacements. And because the pressure is only 6 bar for the mm column and 98 bar for the mm column, this isocratic method is an excellent HPLC option. In the 4th chromatogram, the Poroshell EC-C8.7 μm column ( mm) provides greater efficiency and resolution at nearly x the speed of the original method. The Poroshell EC-C8 column ( mm), in the th chromatogram still meets the requirements for efficiency and resolution, but is 4. times faster than the μm column. mau 6 4 mau 6 4 System suitability method requirement: N > 4,, Rs >. mau 6 4 mau mau N =,69 x Efficiency 9 bar N = 9,4 6 bar x Faster, same backpressure N =,86 x Faster 98 bar N =,46 N =, Poroshell EC-C8, 4.6 x mm (L),.7 µm N =,8 (p/n ) N =, 6.7 µl injection Rs =.6 P= bar 4.x Faster Mobile phase: Flow rate: :49: MeCN:H :acetic acid. ml/ Poroshell EC-C8, 4.6 x mm (L),.7 µm (p/n ).67 µl injection Rs = 7. P = 8 bar Poroshell is an excellent choice for faster methods at HPLC pressures. Sample:. Naproxen. Butyrophenone ZRBAX Eclipse Plus C8, 4.6 x mm, µm USP Prescribed Column (p/n ) L/dp =, 8 9 Poroshell, 4.6 x mm [L], 4 µm (p/n ) L/dp = 7, 8 9 Poroshell, 4.6 x mm [L], 4 µm (p/n ) L/dp =, 8 9 Watch the video to learn how to transfer a naproxen method to Poroshell columns, and optimize your LC system for the best results. Go to agilent.com/chem/poroshellvideo

26 Fast low pressure analysis Here, a method for analyzing non-nutritive food and beverage additives was transferred from a μm ZRBAX Eclipse Plus C8 column to a Poroshell EC-C8 column, reducing the analysis time from over utes to less than utes. Solvent consumption was reduced by more than 8% and resolution of the critical pair improved from.8 to.. ZRBAX Eclipse Plus, µm, P max = bar Mobile phase: A: mm Ammonium acetate, ph 4.8 B: Acetonitrile Flow rate: ml/ Temperature: C Gradient: 4% B at t o, ramp to % B in. Critical pair Rs Poroshell EC-C8,.7 µm 4 Mobile phase: A: mm ammonium acetate, ph 4.8 B: Acetonitrile Flow rate:.8 ml/ Temperature: C Gradient: 4% B at t o, ramp to % B in. Critical pair Rs. Faster analysis of simvastatin on Poroshell Here, a -ute USP method for simvastatin tablets was easily transferred to a Poroshell column, with x faster results. Note that we reduced the column length by 7%, allowing a Poroshell EC-C8, 7 mm column to be substituted for a mm column while still being considered a method adjustment. The Poroshell EC-C8 phase is similar to other USP L phases, and so the results are similar, but faster. ZRBAX Eclipse Plus C8, 4.6 x mm, µm (p/n ) Mobile phase: 6% CH CN, %.9 g/l Na H 4 P 4, ph Flow rate:. ml/ for µm column.8 ml/ for.7 µm Poroshell column 4.47 Temperature: 4 ºC Detection: DAD Sig = 8, 8 Ref = 6, nm Poroshell EC-C8, 4.6 x 7 mm,.7 µm (p/n ) USP Requirement µm (. ml/).7 µm (.8 ml/) T R n/a k >..96. N > 4 6,99 4,49 T f <..9. 6

27 Separation of morphine and metabolites using a Poroshell HILIC column An increasing number of labs are using HILIC early in drug discovery and development for several reasons: To achieve MS compatibility To improve retention of polar compounds and their more polar degradation products To increase LC/MS sensitivity The separation of morphine and metabolites is one example of a fast, efficient HILIC LC/MS method. Here, you can see that these polar compounds are completely resolved in under utes with excellent peak shape and efficiency on the Poroshell HILIC column. A reversed-phase method with high aqueous would have limited retention. Poroshell HILIC,. x mm,.7 µm (p/n ) x Sample. Normorphine. Morphine. M6G 4. MG Counts (%) vs. Acquisition Time () Mobile phase: A: mm NH 4 HC, ph. B: Acetonitrile: mm NH 4 HC, ph. (9:) Flow rate:.8 ml/ Temperature: C Pressure: 7 to bar System: 9 Infinity LC and 64 Triple Quadrupole LC/MS 4 Time %B.44.9 Analysis of vita B and related compounds using a Poroshell HILIC,. x mm,.7 µm column HILIC eliates the need for ion-pair reagents, such as the hexane sulfonic acid that is typically used in mobile phases for separating B vitas. It also increases LC/MS compatibility and retention. Poroshell HILIC,. x mm,.7 µm (p/n ) x Counts (%) vs. Acquisition Time () 4 Sample. 4 Aobenzoic acid. Nicotinamide. Riboflavin 4. Nicotinic acid Mobile phase: Acetonitrile: mm NH 4 HC, ph. (9:) Flow rate:.7 ml/ Temperature: C Pressure: 4 bar System: 9 Infinity LC and 64 Triple Quadrupole LC/MS To learn more about Agilent Poroshell columns, visit agilent.com/chem/poroshell 7

28 AGILENT PRSHELL CLUMNS: PWERFUL EVERYDAY PRBLEM SLVERS A μm column inlet frit stands up to your dirtiest samples Sub- μm particles offer significant speed and resolution advantages, but are susceptible to clogging with dirty samples because a. µm frit must be used at the column inlet. Poroshell columns solve this problem with a standard μm frit that resists plugging with dirty samples including unfiltered plasma. Sample loading of basic compounds on Poroshell columns is comparable to sub- µm columns Small, non-porous particles have low surface area available for sample interaction, and are limited in their sample loading capability. However, Poroshell columns are designed with a larger surface area for greater sample loading. In fact, the loading capacity of Poroshell columns is comparable to.8 μm columns even for the most difficult basic compounds. The peak shape you need for your most accurate results Poroshell columns provide exceptional peak shape especially at ph 6 to 7 when compared to other superficially porous columns. Agilent and Infinity Series are easily optimized for Poroshell columns The inherent properties of Poroshell columns make them ideal for most HPLC and UHPLC instruments, including the new 9 Infinity II. For and Infinity Series LC systems, all you need are or configuration changes (such as flow rate, connector tubing length and id, flow-cell volume, and detector peak-width setting) to achieve superior results with lower pressures and higher efficiencies. 8

29 Agilent Poroshell resists plugging with µm frit Even with dirty samples, such as unfiltered plasma, Poroshell columns show great resistance to plugging. Here, we precipitated the proteins, but did not centrifuge or filter the sample. Even under these conditions, there was no pressure increase, even after, injections. Column: Injection volume: Sample: Poroshell EC-C8,. x mm,.7 µm (p/n ) µl injections Precipitated plasma: parts plasma, 7 parts :8 water:mecn with.% formic acid with part diflunisal in : water:mecn µg/ml (final concentration diflunisal µg/ml) shaken and allowed to settle utes Not centrifuged and not filtered Diflunisal in plasma Pressure Solvent A: Water with. % TFA Solvent B: MeCN with.8 % TFA Flow Rate: ml/ µl injection Diflusinal in Plasma Injections Time %B Efficiency (N) End press Plates Instrument: Infinity RRLC (SL) Achieve comparable sample loading to totally porous particles In this example, we loaded nortriptyline (a basic compound) onto several Agilent columns. Note that the Poroshell.7 μm column has the same loading capacity as the.8 μm column, and that the. μm column has a broader starting peak width that can compromise resolution. The loads on these columns are typical, proving that Poroshell columns can be used with confidence in basic separations. Peak Width Base loading with nortriptyline Concentration of Nortriptyline (mg/ml) Poroshell EC-C8,. x mm,.7 µm (USCFX84) (p/n 6997-) ZRBAX Eclipse Plus C8,. x mm,.8 µm (USUYB44) (p/n ) ZRBAX Eclipse Plus C8,. x mm,. µm (USUXV48) (p/n ) 8% mm Na HP 4 buffer, ph., % acetonitrile Temperature: ºC Detection: nm To learn more about Agilent Poroshell columns, visit agilent.com/chem/poroshell 9

30 Agilent Poroshell columns deliver superior peak shape for better results with basic compounds Here is another basic compound separation, proving how Poroshell columns outperform the other vendors for challenging analytes Poroshell Tf =.48 ther vendor S Tf =.68 ther vendor P Tf = Column: Poroshell EC-C8, 4.6 x mm,.7µm (p/n ) Mobile phase: mm 4% Na HP 4, ph 7, 6% acetonitrile Flow rate:. ml/ Temperature: 4 C Detector: DAD, 4 nm, µl flow cell Sample: µl injection of µg/ml amitriptyline, µg/ml uracil in H :CH CN (9:) k =.9, N=7,6 ther vendor T 4.6 x mm N=6,9, Tf= k =6., N=6, ther vendor S 4.6 x mm N=,, Tf= k =8.6, N=6, Poroshell EC-C8, 4 µm 4.6 x mm (p/n ) N=,4, Tf=. Column: Poroshell EC-C8, 4.6 x mm, 4 µm Mobile phase: mm 4% K HP 4 / KH P 4 ph 7, 6% methanol Flow rate:. ml/ Sample:. UraciI. Propranolol. Butyl Paraben 4. Dipropylpthalate. Amitriptyline

31 FAST, CNFIDENT SEPARATINS F PRTEINS AND PEPTIDES Agilent Poroshell technology is an excellent choice for separating and characterizing complex biomolecules, including both intact and digested proteins. Agilent AdvanceBio RP-mAb columns focus on the unique challenges of monoclonal antibody characterization. For mapping peptides in protein digests, use AdvanceBio Peptide Mapping columns which are pretested with a challenging peptide mix to ensure optimal performance for your peptide mapping application. High speed, high-resolution separation of monoclonal antibodies Large biomolecules such as monoclonal antibodies are typically separated slowly to reduce potential peak broadening of these slow-diffusing analytes. However, the Poroshell technology used in AdvanceBio RP-mAb columns reduces the diffusion distance, thus allowing higher flow rates and steeper gradients even on 6 bar systems. The wide 4Å diameter of the pores in the thin layer provides full access to the bonded phase by large monoclonal antibody molecules, ensuring the best possible chromatography. The choice of robust bonded phases designed for monoclonal antibody separations, C4, SB-C8, and a unique diphenyl, provide a range of selectivities that allow resolution to be optimized. Column: AdvanceBio RP-mAb C4. x mm,. µm (p/n ) Mobile phase: A:.% TFA in water:ipa (98.) B: IPA:ACN:mobile phase A (7::) Flow rate:. ml/ Temperature: 8 C Characterization in less than utes Detector: Gradient: Sample: UV, 4 nm -8% B in 4, wash at 9% B, reequilibration at % B µl injection of humanized recombinant Herceptin variant IgG intact from Creative Biolabs ( mg/ml) Agilent has one of the broadest families of biocolumns available, including AdvanceBio columns to help you advance accuracy and productivity for bioseparations. Learn more at agilent.com/chem/advancebio

32 BioConfirm Molecular Feature Extractor of Stratagene mab trypsin peptide map Using the BioConfirm Molecular Feature Extractor, we can demonstrate % sequence coverage on both the light and heavy chains of the same monoclonal antibody. Q-TF Instrument Parameters Source ESI positive Gas temperature: ºC Drying gas: L/ Nebulizer: 4 psi Vcap: 4, V Fragmentor: V Skimmer: 6 V ctapole RF: 7 V MS: 4 Hz Mass range: - m/z Reference mass: Acq. mode: Extended dynamic range mode ( GHz) x Counts vs. Acquisition Time () Column: Poroshell EC-C8,. x mm,.7 µm (p/n 6897-) Mobile phase: A: Water,.% formic acid B: ACN,.% formic acid Flow rate:. ml/ Tempurature: 4 C Detection: Gradient: Time %B 4 6 Q-TF, ESI positive Shown in table Time %B Insulin analysis: transfer from a.8 μm ZRBAX StableBond column to a Poroshell column for increased efficiency The Poroshell SB-C8 column provided double the efficiency of the ZRBAX RRHD SB-C8 8Å due to the larger pore size and more rapid diffusion in the Å pores. Poroshell columns are ideal for small protein insulin or other peptides, providing higher efficiency at lower pressure. ZRBAX SB-C8, 4.6 x mm,.8 µm (p/n ). Porcine insulin. A- desamido insulin 4 N =, N = 6,69 Tf =.4 N =. bar Poroshell SB-C8, 4.6 x mm,.7 µm (p/n ) 6 4 N =,86 N =,7 Tf =. Tf =. 9 bar

33 Peptide map of a biosimilar EP The top chromatogram shows a peptide map of a highly glycosylated EP from a biosimilar. Note the excellent resolution achieved for small peptide fragments using UV detection. The bottom chromatogram shows the same separation using mass spectroscopy to detere the sequence coverage (%). UV detection is used for comparing peptide maps, while MS is ideal for identifying ao acid substitutions and modifications. So, you can easily confirm protein identity, and identify any posttranslational modifications, using the AdvanceBio Peptide Mapping column. Column: AdvanceBio Peptide Mapping,. x mm,.7 µm (p/n 67-9) Flow Rate:.4 ml/ Injection: µl (. mg/ml) Temp: ºC Detection: Gradient: nm A: water (.% FA); B: ACN (.% FA), -8, -4% B; 8-, 4-6% B; -4, 6-9% B x Cpd : Counts vs. Acquisition Time () EP digest, LC/MS TF % sequence coverage achieved using MassHunter Workstation software Agilent AdvanceBio Peptide Mapping columns offer the same Fast LC advantages of Poroshell columns, and are batch-tested with a rigorous peptide mix to ensure suitability and reproducibility. AdvanceBio Peptide Mapping column choices include the new mm length for maximum resolution of the most complex peptide maps. Learn more at agilent.com/chem/advancebio or request Publication No EN. To learn more about Agilent Poroshell columns, visit agilent.com/chem/poroshell

34 Which Fast LC column is best for you? Agilent offers the widest range of Fast LC columns, including Poroshell, ZRBAX Rapid Resolution High Definition (RRHD) columns,.8 µm (stable to bar) and ZRBAX Rapid Resolution High Throughout (RRHT),.8 µm (stable to 6 bar). We bond all of these columns with similar stationary phases for assured scalability. With all of these choices, you have flexibility in creating a method to optimize your situation. Your Lab Situation Agilent Recommends Rationale Both UHPLC (+ bar) and HPLC instruments (e.g. Agilent 9 Infinity LC and 6 Infinity LC 6 bar) nly 4-6 bar HPLCs Agilent Infinity Series, Agilent (4 bar) as well as the Infinity LC or 6 Infinity LC (6 bar) A mix of UHPLC instruments (Agilent 9 Infinity LC, other + bar instruments) and some HPLC instruments (e.g. Infinity Series). Poroshell, 4 μm and.7 μm. ZRBAX RRHD.8 µm. Poroshell, 4 μm and.7 μm. ZRBAX Eclipse Plus. µm and µm. ZRBAX RRHD.8 µm. Poroshell,.7 µm Poroshell is an easy column to use on both instrument types. ZRBAX RRHD will help you optimize the capabilities of the 9 Infinity LC for UHPLC. With Poroshell 4 μm and.7 μm columns, you can enhance the performance of older 4 bar instruments, and also get even better performance from newer 6 bar UHPLC instruments. For established methods that you cannot transfer, the ZRBAX Eclipse Plus column provides exceptional peak shape and performance. ZRBAX RRHD can deliver optimum performance on all of these instruments. Poroshell can be used on the 6 bar instruments to optimize their performance. Want the best results from your sample analysis? Start with the best Sample Prep Reduce the need for repeated runs, and imize interferences that can compromise separation, detection, and quantitation. The Agilent Sample Preparation Portfolio combines innovative products, stateof-the art manufacturing, and stringent quality-control monitoring to ensure reliable, consistent results. agilent.com/chem/samplepreparation 4

35 PUSH YUR UHPLC PERFRMANCE T INFINITE LIMITS AND RUN YUR CNVENTINAL METHDS WITH CNFIDENCE Whether you need a workhorse LC system for routine analysis or the most sophisticated, high-resolution LC/MS system, the Agilent Infinity Series has what you re looking for. Together with Poroshell columns, the Infinity Series delivers ultimate resolution and sensitivity, while helping you boost your separation power. They also ensure easy method transferability between systems without redevelopment or revalidation. Infinitely more AFFRDABLE Infinitely more CNFIDENT Infinitely more PWERFUL Learn why Agilent s Infinity Series is infinitely better at agilent.com/chem/infinity Infinity LC 6 Infinity LC 9 Infinity II LC To learn more about Agilent Poroshell columns, visit agilent.com/chem/poroshell

36 SPECIFICATINS AND RDERING INFRMATIN Agilent Poroshell 4 µm columns NEW Size (mm) EC-C8 EC-C8 PFP Phenyl-Hexyl HILIC 4.6 x x x x x x x x x x x x Guard columns for 4 µm columns Size (mm) EC-C8 4.6 x x x NEW Agilent Poroshell.7 µm columns HIGH ph Size (mm) EC-C8 EC-C8 SB-C8 SB-C8 NEW HPH-C8 NEW HPH-C8 4.6 x x x x x x x x x x x x x x x

37 Agilent Poroshell.7 µm columns (continued) Size (mm) Phenyl-Hexyl SB-Aq Bonus-RP HILIC EC-CN NEW PFP 4.6 x x x x x x x x x Note: Poroshell columns have a 6 bar/9, psi pressure limit. Agilent Poroshell Fast Guards for UHPLC Size (mm) EC-C8 EC-C8 SB-C8 Phenyl-Hexyl NEW PFP HPH-C8 4.6 x x x Size (mm) HPH-C8 SB-C8 SB-Aq Bonus-RP HILIC EC-CN 4.6 x x x Note: Guards supplied as /pk. Agilent Poroshell bonded phase specifications Bonded Phase Pore Size Temp. Limits ph Range Endcapped Carbon Load Surface Area EC-C8 Å 6 ºC.-8. Double % m /g EC-C8 Å 6 ºC.-8. Double % m /g SB-C8 Å 9 ºC.-8. No 9% m /g SB-C8 Å 8 ºC.-8. No.% m /g HPH-C8 Å 6 ºC.-. Double Proprietary 9 m /g HPH-C8 Å 6 ºC.-. Double Proprietary 9 m /g Phenyl-Hexyl Å 6 ºC.-8. Double 9% m /g SB-Aq Å 8 ºC.-8. No Proprietary m /g Bonus-RP Å 6 ºC.-9. Triple 9.% m /g HILIC Å 6 ºC.-8. No N/A m /g EC-CN Å 6 ºC.-8. Double.% m /g PFP Å 6 ºC.-8. Yes.% m /g Specifications represent typical values. Unique chemistries increase high-ph stability 7

38 Agilent Poroshell μm columns Description Size (mm) SB-C8 SB-C8 SB-C Extend-C8 Narrow Bore. x MicroBore. x Capillary. x Guard Cartridge, 4/pk. x Guard Hardware Kit MicroBore Guard, /pk. x Note: Poroshell columns have a 4 bar/6, psi operating pressure limit. Agilent Poroshell bonded phase specifications Bonded Phase Pore Size Temp. Limits ph Range Endcapped Poroshell SB-C8, C8, C Å 9 ºC.-8. No Poroshell Extend Å 4 C above ph 8 6 C below ph 8.-. Yes Specifications represent typical values. Agilent AdvanceBio RP-mAb columns Agilent AdvanceBio Peptide Mapping columns Description Part Number Description Part Number C4, 4.6 x mm,. μm C4, 4.6 x mm,. μm C4, 4.6 x mm,. μm C4,. x mm,. μm C4,. x mm,. μm C4,. x 7 mm,. μm C4,. x mm,. μm SB-C8, 4.6 x mm,. μm SB-C8, 4.6 x mm,. μm SB-C8, 4.6 x mm,. μm SB-C8,. x mm,. μm SB-C8,. x mm,. μm SB-C8,. x 7 mm,. μm SB-C8,. x mm,. μm Diphenyl, 4.6 x mm,. μm Diphenyl, 4.6 x mm,. μm Diphenyl, 4.6 x mm,. μm Diphenyl,. x mm,. μm Diphenyl,. x mm,. μm Diphenyl,. x 7 mm,. μm Diphenyl,. x mm,. μm x mm,.7 μm x mm,.7 μm 69-. x mm,.7 μm x mm,.7 μm x mm,.7 μm mm Fast Guard* mm Fast Guard* mm Fast Guard* 87-9 * Fast Guards extend column lifetime without slowing down the separation or affecting resolution. Agilent AdvanceBio Peptide Mapping specifications Bonded Phase Pore Size Temp. Limits ph Range* Endcapped C8 Å 6 ºC.-8. Double Specifications represent typical values only. Agilent AdvanceBio RP-mAb specifications Bonded Phase Pore Size Temp. Limits ph Range* Endcapped AdvanceBio RP-mAb C4 4Å 9 ºC.-8. Yes AdvanceBio RP-mAb SB-C8 4Å 9 ºC.-8. No AdvanceBio RP-mAb Diphenyl 4Å 9 ºC.-8. Yes Specifications represent typical values. * Columns are designed for optimal use at low ph. At ph 6-8, highest column stability for all silica-based columns is obtained by operating at temperatures <4 C and using low buffer concentrations in the range of.-. M. 8

39 Agilent AdvanceBio ligonucleotide columns Description Part Number. x mm,.7 µm x mm,.7 µm x mm,.7 µm mm Fast Guard x mm,.7 µm x mm,.7 µm x mm,.7 µm mm Fast Guard 87-9 ligonucleotide Resolution Standard 9-98 ligonucleotide Ladder Standard 9-99 Agilent AdvanceBio ligonucleotide specifications Bonded Phase Pore Size Temp. Limits ph Range* Endcapped C8 Å 6 ºC.-. Double Agilent innovation: The first high-ph stable superficially porous particle LC column for oligonucleotide analysis Agilent AdvanceBio Glycan Mapping columns Description Part Number 4.6 x mm,.7 μm x mm,.7 μm x mm,.7 μm x mm,.7 μm x mm,.7 μm x mm,.7 μm mm,.7 μm, Fast Guard Agilent AdvanceBio Glycan Mapping specifications Bonded Phase ID (mm) Particle Size (µm) Endcapped ph Stability perating Temperature Pressure limit Amide HILIC. and 4.6.8, fully porous No -7 6 C bar Amide HILIC. and 4.6.7, superficially porous No -7 6 C 6 bar To learn more about Agilent Poroshell columns, visit agilent.com/chem/poroshell 9