New HPLC Column Options for UHPLC
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1 New HPLC Column Options for UHPLC Rapid Resolution High Definition Columns and More Chor Koon Tan, PhD Business Development Manager Columns & Supplies Division Asia-Pacific Agilent Technologies Page 1
2 UHPLC Why Use Different Columns? UHPLC instruments include a variety of high pressure LC choices including those designed for high throughput/fast LC. These instruments operate at pressures >4 bar/6 psi. The desire is to have small particle size columns <2-3um for improved efficiency, resolution and productivity on these instruments. These provide the flexibility to operate above 4 bar for many applications for speed or for enhanced resolution with long 1.8um columns Agilent s new 129 Infinity LC can operate up to 12 bar. This requires columns designed for up to 12 bar operating conditions the new RRHD Columns! Page 2
3 The 129 Infinity Has a Wide Useable UHPLC Range Page 3 Month June ##, 6, 2X 25
4 Productivity that Can Be Achieved Using RRHD Columns with the 129 Infinity LC 1. Fast analysis especially with somewhat complex samples 1. Gradient analysis with 4-1 components 2. More resolution in a limited amount of time 1. Choosing different column lengths for the resolution desired 2. Maximizing peak capacity in gradient separations of complex samples RRHD Rapid Resolution High Definition Page 4
5 Fast Analysis on ZORBAX Rapid Resolution High Definition Eclipse Plus C DAD1 D, DAD1C, DAD: Signal C, 28 nm/bw:1 nm Ref 4 nm/bw:5 nm (G:\129\AOX JASMIN\AOX-AM2-45C-11.D) DAD1 E, DAD1D, DAD: Signal D, 255 nm/bw:1 nm Ref 4 nm/bw:5 nm (G:\129\AOX JASMIN\AOX-AM2-45C-11.D).415 THBP RRHD Eclipse Plus C18, 2.1 x 5mm, 1.8um, Pmax 17 bar This 1 component sample requires a gradient that goes over 1 bar. This is a sub 2ute separation! PG.272 TBHQ.48 NDGA BHA OG 1.55 Ionox-1 Data Courtesy of: Gerd Vanhoenacker Research Institute for Chromatography Kennedypark Kortrijk Belgium DG BHT AP Page 5
6 Fast Analysis on Agilent Zorbax Rapid Resolution High Definition Eclipse Plus C18 Method Column ZORBAX RRHD Eclipse Plus C x 5 mm, 1.8 µm (SN USDAY11) Mobile phase A=.2% H 3 PO 4, B=acetonitrile/methanol 3/1 Flow rate 1.6 ml/ Gradient % B Injection volume 1 µl Detector Sig=28/1 nm, Ref=4/5, 8 Hz Sig=255/1 nm, Ref=4/5, 8 Hz Temperature 45 C Sample Antioxidant mixture, ca. 1 ppm A 1.6 ml/ flow rate is very fast, and allows a fast separation. Data Courtesy of: Gerd Vanhoenacker Research Institute for Chromatography Kennedypark Kortrijk Belgium Page 6
7 Where Does this Example Fall in the 129 Infinity Range This app falls in the Power Range Page 7 Month June ##, 6, 2X 25
8 Choose Column Length for Analysis Time and Rs Mobile Phase: 4:6 water:acetonitrile, Flow Rate:.5 ml/, Temp: ambient, Columns: RRHD SB-C18, 2.1 mm ID, Lengths as noted Peaks: 1: uracil, 2 acetophenone, 3 propiophenone, 4 butyrophenone, 5 valerophenone, 6 hexanophenone 5 mm RRHD 1.8um 12 bar Rs 3,2 : 5.64 N 6 : 1,7 Pressure: 35 bar mm Rs 3,2 : 9.2 N 6 : 21,1 Pressure: 566 bar 15 mm Rs 3,2 : N 6 : 34,1 Pressure:75 bar The 15mm length RRHD column becomes a good option for high resolution with the 129 Infinity LC. Page 8
9 Gain Efficiency and Resolution with Long Length mm = 25 mm 11 bar Mobile Phase 25:75 water:acetonitrile, Flow Rate:.5 ml/, ambient temperature, ZORBAX RRHD SB-C18, 1.8um columns mm (P:582 bar) α 3,2 : 1.19 Rs 3,2 : 6.42 N 6 : 31,4 Pw 2 :.16 Pw 6 : Uracil 2. Acetophenone 3. Propiophenone 4. Butyrophenone 5. Valerophenone 6. Hexanophenone mm (P:195 bar) α 3,2 : 1.2 Rs 3,2 : 8.76 N 6 : 5,2 Pw 2 :.18 Pw 6 : High Rs in 3.5 utes, 25mm, 1.8um at 11 bar Resolution is fine on 15mm column, but for even more Rs longer column is used. Page 9
10 Where Does this Example Fall in the 129 Infinity Range This app falls in the Power Range Page 1 Month June ##, 6, 2X 25
11 High Resolution Separation with Long 1.8um Columns Mobile Phase A: water B:acetonitrile, F=.25 ml/, ambient temperature, Columns: ZORBAX RRHD, SB-C18, 2.1 x 15+1 mm, 1.8um 2 DAD1 A, Sig=254,4 Ref=off (E:\9422JWH\PRESSURE27.D) PMP1, PMP1B, Pressure Gradient 1-1% B/15. Pressure 8-9 bar Low point of gradient about 48 bar Long column or columns in series provide max Rs with this complex sample smoked food flavoring. Page 11
12 Achieving Optimal Resolution for a Gradient with RRHD Column Choices Columns range in length from 5 15mm. Column length and gradient time can be changed to optimize the resolution and retention of the analytes. k* = t G F S Φ V m These 3 parameters can be easily changed. t G = gradient time F = flow rate V m = column volume (change by changing column length) Page 12
13 Separation of Licorice Root on RRHD Columns 3 ute gradient on each length Rs: Mobile Phase: 1-1% B /3 A:.1% formic acid (fa) B: acetonitrile with.1% fa F=.4 ml/ Ambient temperature 28nm UV 129 Infinity RRHD SB-C x 5 mm, 1.8um Pmax=366 bar n c = Rs: 1.37 RRHD SB-C x 1 mm, 1.8um Pmax=595 bar n c = Rs: 2.4 RRHD SB-C x 15 mm, 1.8um Pmax=768 bar n c = Page 13
14 Separation of Licorice Root on RRHD Columns 3 ute gradient on each length Mobile Phase: 1-1% B /3 A:.1% formic acid (fa) B: acetonitrile with.1% fa F=.4 ml/ Ambient temperature 28nm UV 129 Infinity 5 mm 1 mm 15 mm 7 peaks 8 peaks 9 peaks Rs: Rs: 1.37 Rs: Increasing peak capacity led to increased Rs for this sample. Page 14
15 Peak Capacity Comparisons Length and Gradient Time nc= tg/wbaseline of last eluting peak 1 Licorice Root sample Gradient Time Column Length 1 utes 2 utes 3 utes Shaded boxes indicate comparable peak capacities 1. Neue, U.D., et. al, Advances in Chromatography, 41, 93 (21) Page 15
16 RRHD 1.8um Columns The part numbers! These new columns will have new part numbers (table below) The column shipping with the 129 Infinity LC will be the ZORBAX Rapid Resolution High Definition (RRHD) Eclipse Plus C18, 2.1 x 5mm, 1.8um PN Description RRHD Eclipse Plus C18, 2.1 x 15mm, 1.8um RRHD Eclipse Plus C18, 2.1 x 1mm, 1.8um RRHD Eclipse Plus C18, 2.1 x 5mm, 1.8um RRHD SB-C18, 2.1 x 15mm, 1.8um RRHD SB-C18, 2.1 x 1mm, 1.8um RRHD SB-C18, 2.1 x 5mm, 1.8um RRHD SB-C8, 2.1 x 15mm, 1.8um RRHD SB-C8, 2.1 x 1mm, 1.8um RRHD SB-C8, 2.1 x 5mm, 1.8um Page 16
17 Why Two Different C18 Columns? Eclipse Plus C18 and StableBond C18 Different selectivity Eclipse Plus C18 vs. StableBond C18 Optimum peak shape Achieved with endcapped C18 Eclipse Plus C18 Optimum stability at low ph Achieved with non-endcapped C18 StableBond C18 Additional bonded phases will be converted to the RRHD format Page 17
18 Different ZORBAX RRHT/RRHD C18 Bonded Phases for Maximum Selectivity Mobile phase: (69:31) ACN: water Flow 1.5 ml/. Temp: 3 C Detector: Single Quad ESI positive mode scan Columns: RRHT 4.6 x 5 mm 1.8 um Sample: 1. anandamide (AEA) 2. Palmitoylethanolamide (PEA) 3. 2-arachinoylglycerol (2-AG) 4. Oleoylethanolamide (OEA) 1 st choice Best Resolution & Peak Shape Eclipse Plus C nd choice Good alternate selectivity due to non-endcapped StableBond SB-C Multiple bonded phases for most effective method development. Page 18
19 Peak Shape of Comparison of sub 2 um columns Eclipse Plus is Superior to Acquity BEH C18 Mobile Phase = 8% 25 mm phosphate (ph 3): 2% ACN, Flow Rate =.3 ml/ Detection: 25nm Analyte: Dextromethorphan ACQUITY BEH C x 5 mm, 1.7 um P = 228 Bar N = 117, Peak Width =.137, Tf= 1.39 Tf = RRHT Eclipse Plus C x 5 mm, 1.8 um P = 22 Bar N = 1881, Peak Width =.124, Tf= 1.23 Tf = Many columns are competitive and slight improvements in peak shape can be important to maximum sensitivity. Page 19
20 Resolution on RRHD vs. Other Sub 2um on 129 Infinity Mobile Phase: 4:6 water:acetonitrile,.1% Formic Acid Flow Rate:.5 ml/, Temp: ambient, Det: UV 254 nm Columns: RRHD SB-C18, 2.1 x 5mm, 1.8um and competitive Peaks: 1: uracil, 2acetophenone, 3 propiophenone, 4 butyrophenone, 5 valerophenone, 6 hexanophenone RRHD SB-C x 5mm, 1.8um USCES157 4 Acquity HSS C18 SB 2.1 x 5 mm 1.8um 5 Acquity BEH C x 5 mm, 1.7um α 3,2 : 1.36 Rs 3, N 6 : 17 TF 6 : 1.4 α 3,2 : 1.22 Rs 3, N 6 : 94 TF 6 : 1.17 α 3,2 : 1.29 Rs 3, N 6 : 11 TF 6 : 1.14 StableBond offers greatest Rs and is still the best column for low ph lifetime on the market! 6 Page 2
21 Are There Other Column Types for UHPLC? Review of Van Deemter Curves for Efficiency Column types for UHPLC depending on speed, resolution, and throughput requirements 1. RRHD columns 1.8um columns for high productivity up to 12 bar 2. RRHT columns 1.8um columns for fast LC and with a 6 bar pressure limit Ideal for fast LC and for working at the 6 bar pressure limit Available in lengths from 2mm to 15 mm, with 2, 3, 5 and 75mm lengths ideal for fast high resolution separations 4.6 mm ID columns ideal for use at this lower pressure limit, with 3. and 2.1 mm ID columns also available 3. Upcog Poroshell 12 columns Page 21
22 Van Deemters for RRHT and RRHD Columns RRHT Eclipse Plus C18, 2.1 x 1 mm, 1.8 um; PN , SN USUXU1664, LN B811 Heptanophenone k=1.8 RRHD Eclipse Plus C18, 2.1 x 1 mm, 1.8 um; PN , SN DAZ12, LN B913 Naphthalene k=4. Note pressure of more than 5 bar on 1mm length! With just acetonitrile and water new 129 Infinity LC ideal h DAZ12 LFS Column performance report results N=26 TF 1.2 P=324 F=.3 N=249 TF 1.8 P=52 F=.489 N=249 TF 1.8 P=55 F=.52 N=223 TF 1.1 P=535 F= Van Deemters are basically the same for RRHT and RRHD, this is expected u (mm/s) 2.1 x 1 RRHD Optimum flow rate is about.5 ml/. (6% MeCN, Themostatted 26 C, ~LC Performance Report Conditions) Page 22 John W. Henderson Jr Title Agilent Month Restricted ##, 2X
23 Why Is It Important to Know the Optimal Flow Rate? Efficiency is Highest at Optimal Flow Rate DAZ12 LFS Column performance report results N=26 TF 1.2 P=324 F= DAZ12 N=251 TF 1.8 P=55 F= Sub-Optimal Optimal.3 ml/.5 ml/ N= 2,6 N= 25,1 P= 324 bar P = 55 bar At sub optimal flow rates the efficiency is lower and all of the benefits of a 1.8um columns or UHPLC technology may not be apparent. Page 23 John W. Henderson Jr Title Agilent Month Restricted ##, 2X
24 Van Deemter for Poroshell 12 and RRHT Plate Height H (cm) H (cm) vs Flow rate (ml/) (6:4 ACN:H 2 O, all columns 4.6mm x 5mm) Poroshell 12 SB-C18, 2.7µm RRHT Zorbax SB-C18, 1.8 µm 2.7 µm Poroshell µm totally porous particles Flow Rate (ml/) Superficially porous particles and 1.8um particles have similar efficiencies and are both choices for UHPLC. Page 24
25 Poroshell 12 Particle technology SOLID CORE < 3 µm < 2 µm Poroshell 12 Superficially porous Totally porous Fully porous sub-twomicron (STM) T. Maloney, J. Cunliffe, J. Sep. Sci. 27, 3, G. Guiochon et al, Journal of Chromatography A, 1157 (27) G. Guiochon et al, Journal of Chromatography A, 1166 (27) 3 46 Yu Zhang et al, Journal of Chromatography A, 1216 (29) Page 25
26 Fast Analysis of Analgesics on Poroshell 12 High Flow Rates, for Fast Analysis are Possible 1. Acetaophen 2. Caffeine 3. 2-acetamidophenol 4. Acetanilide 5. Phenacetin 6. Salicylic acid ml/ 142 bar ml/ 211 bar 2. ml/ 279 bar 2.5 ml/ 347 bar Conditions: Column: Poroshell 12 SB-C18, 4.6 x 5mm, 2.7um Mobile Phase:.2% Formic Acid 8% water:2% ACN Temperature: 25 C, Detection: 275 nm, Sample Injection: 2ul Page 26
27 Conclusions New Rapid Resolution High Definition (1.8um) columns are one solution for UHPLC systems and are unique in the marketplace because of their ability to be used over >1 bar. This makes them ideal for use with the 129 Infinity LC. Other UHPLC columns are also available including the RRHT, 6 bar columns with over 14 choices. The 4.6 mm ID columns represent a unique choice for easy to use UHPLC performance. The upcog release of the Poroshell 12 columns will provide a 3 rd option for UHPLC type columns because a Page 27
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