Rapid Resolution High Definition LC Columns

Transcription

1 Rapid Resolution High Definition LC Columns New Columns for Use with the 1290 Infinity LC Edward Kim Application Engineer August 11, 2010 Page 1

2 What are Rapid Resolution High Definition (RRHD) Columns? RRHD columns are the columns designed to be compatible with the 1290 Infinity LC and be backwards compatible to other Agilent instruments with lower pressure limits (though not the first choice) and fully UHPLC compatible RRHD columns can be used up to bar and represent a complete Agilent solution with the 1290 LC. They are 1.8um ZORBAX columns packed to withstand the higher pressures of the 1290 LC. Like other ZORBAX columns we will maximize compatibility across instruments and labs by maintaining scalability across particle sizes. Page 2

3 What is Different Between RRHT 1.8um Columns and RRHD 1.8um Columns The pressure limit of the column RRHD columns are designed for bar use, RRHT columns are designed for 600 bar use on the Rapid Resolution LC (1200 SL) RRHD columns use improved hardware and packing processes the packing pressure and process are proprietary improved hardware is being transferred to RRHT columns (for consistency) This impacts the backpressure the column 1. These columns operate at a higher backpressure than a 600 bar column higher pressure packing = higher packing density, more backpressure 2. A 50mm length RRHD can not be used on a 400 bar instrument but can be used on a 1200 RRLC (600 bar instrument) 3. The 100 and 150mm length columns are only recommended for use on the 1290 Infinity LC (or UHPLC instruments) Page 3

4 Pressure (bar) LC Beyond 600 bar Requires New RRHD Columns 700 Agilent ZORBAX RRHT Eclipse Plus C18, 2.1 x 100 mm, 1.8 um; PN , SN USUXU01664, LN B y = x R 2 = y = x R 2 = y = x R 2 = Acetonitrile/Water (60:40) Acetonitrile/Water (50:50) Methanol/Water (60:40) Flow Rate (ml/) Page 4

5 Comparison of Pressure Generated on RRHT and RRHD 1.8um Columns 40:60 water:acetonitrile, Flow Rate: 0.5 ml/, ambient temperature RRHT 1.8um 600 bar RRHD 1.8um 1200 bar 2.1 x 50mm 292 bar 350 bar 2.1 x 100mm 452 bar 566 bar 2.1 x 150mm 660 bar 750 bar The RRHD columns are ideal for use on the 1290 Infinity LC or on other UHPLC instruments. To withstand maximum pressure requires a denser packed bed that generates a little more backpressure. This is necessary for optimum lifetime and use up to 1200 bar. Change in pressure may vary a little until optimized columns ship! Page 5

6 RRHT, 600 bar and RRHD Column Comparison Blue = RRHT, Red=RRHD Columns: 2.1 x 50mm, 1.8um Flow: 0.6 ml/ Sample: Alkylphenones Expected Differences due to higher packing density: RRHD longer retention, same selectivity Higher Back Pressure of RRHD = 434 bar vs. Back Pressure of RRHT = 323 bar N=11,168 N = 11,041 Page 6

7 Gain N and Rs w/ Densely Packed RRHD columns Mobile Phase: 40:60 water:acetonitrile, Flow Rate: 0.5 ml/, Temp: ambient, Columns: RRHD/RRHT SB-C18, 2.1 mm ID, Lengths as noted Peaks: 1: uracil, 2acetophenone, 3 propiophenone, 4 butyrophenone, 5 valerophenone, 6 hexanophenone 50 mm RRHT 1.8um 600 bar Rs 3,2 : 4.16 N 6 : 9200 RRHD 1.8um 1200 bar Rs 3,2 : 5.64 N 6 : mm Rs 3,2 : 8.38 N 6 : Rs 3,2 : 9.02 N 6 : mm Rs 3,2 : N 6 : Rs 3,2 : N 6 : The additional packing results in more retention, then Rs and efficiency. Page 7

8 h Optimum Flow Rates and Efficiencies-Van Deemters RRHT Eclipse Plus C18, 2.1 x 100 mm, 1.8 um; PN , SN USUXU01664, LN B08101 Heptanophenone k=10.8 RRHD Eclipse Plus C18, 2.1 x 100 mm, 1.8 um; PN , SN DAZ01002, LN B Naphthalene k=4.0 Note pressure of more than 500 bar on 100mm length! With just acetonitrile and water new 1290 Infinity LC ideal DAZ01002 LFS Column performance report results N=20600 TF 1.02 P=324 F=0.3 N=24900 TF 1.08 P=520 F=0.489 N=24900 TF 1.08 P=550 F=0.520 N=22300 TF 1.01 P=535 F= Van Deemters are very similar, this is expected u (mm/s) 2.1 x 100 RRHD Optimum flow rate is about 0.5 ml/. (60% MeCN, Themostatted 26 C, ~LC Performance Report Conditions) Page 8 John W. Henderson Jr Title Agilent Month Restricted ##, 200X

9 Why Is It Important to Know the Optimal Flow Rate? Efficiency is Highest at Optimal Flow Rate DAZ01002 LFS Column performance report results N=20600 TF 1.02 P=324 F= DAZ01002 N=25100 TF 1.08 P=550 F= Sub-Optimal Optimal 0.3 ml/ 0.5 ml/ N= 20,600 N= 25,100 P= 324 bar P = 550 bar At sub optimal flow rates the efficiency is lower and customers may not see the benefits of a 1.8um column or UHPLC technology. 2 Page 9 John W. Henderson Jr Title Agilent Month Restricted ##, 200X

10 What is Different Between RRHT 1.8um Columns and RRHD 1.8um Columns The column hardware on the inside We redesigned the hardware to improve available frit surface area to reduce the likelihood 2.1 mm ID columns will plug We increased the frit pore size to 0.5um to also reduce the likelihood a column will plug The column label on the outside We now use a colored label so customers can visually tell the difference between a 600 bar RRHT column and new RRHD column Available column dimensions 1. Initially the columns will only be available in 2.1 mm ID 2. We will test 3.0 mm ID mm ID will not be available due to technical limitations in this configuration, other 4.6 mm ID with lower pressure limits can be used on the 1290 Infinity LC with appropriate pressure limits entered Page 10

11 Examples of the RRHT and RRHD Columns RRHD columns have a light blue label RRHD columns have no visible groove on the column between end of the fitting and the column body. RRHT columns show this groove in the column between the end of the fitting and the column body. They also have a white label. Page 11

12 What is Different Between RRHT 1.8um Columns and RRHD 1.8um Columns The part numbers! These new columns will have new part numbers (table below) The column shipping with the 1290 Infinity LC will be the ZORBAX Rapid Resolution High Definition (RRHD) Eclipse Plus C18, 2.1 x 50mm, 1.8um PN Description RRHD Eclipse Plus C18, 2.1 x 150mm, 1.8um RRHD Eclipse Plus C18, 2.1 x 100mm, 1.8um RRHD Eclipse Plus C18, 2.1 x 50mm, 1.8um RRHD SB-C18, 2.1 x 150mm, 1.8um RRHD SB-C18, 2.1 x 100mm, 1.8um RRHD SB-C18, 2.1 x 50mm, 1.8um RRHD SB-C8, 2.1 x 150mm, 1.8um RRHD SB-C8, 2.1 x 100mm, 1.8um RRHD SB-C8, 2.1 x 50mm, 1.8um Page 12

13 What s Not Different Between the RRHT 1.8um and RRHD 1.8um Columns The ZORBAX packing materials These are still the same ZORBAX 1.8um packing materials with the same bonded phases - Eclipse Plus, StableBond etc. The packing material still has the same particle size distribution the change in pressure is only related to change in packing density, not particle size distribution We could have changed it, but there was no performance advantage We will start with our most popular C18 phases Eclipse Plus C18 and StableBond C18 Eclipse Plus delivers better peak shape than the BEH Hybrid material StableBond C18 is still the best column for low ph lifetime ZORBAX columns can deliver excellent performance Page 13

14 Updated Peak Shape Comparisons Why Compare Chromatograms with Dextromethorphan? Dextromethorphan is a strongly basic compound that generates the best results in LC at low ph. Eclipse Plus claims superior peak shape so comparisons for peak shape are most relevant Peak shape is a critical issue and poor peak shape is common with this compound. Peak shape continues to be a major driver in column choice for customers Because of this, efficiency can also be low on columns so it is a critical parameter to monitor. High efficiency and good peak shape provide the most quantitative accuracy Page 14

15 Peak Shape of Comparison of sub 2 um columns Eclipse Plus is Superior to Acquity BEH C18 Mobile Phase = 80% 25 mm phosphate (ph 3): 20% ACN, Flow Rate = 0.3 ml/ Detection: 205nm Analyte: Dextromethorphan ACQUITY BEH C x 50 mm, 1.7 um P = 228 Bar N = 10107, Peak Width = 0.137, Tf= 1.39 Tf = RRHT Eclipse Plus C x 50 mm, 1.8 um P = 202 Bar N = 10881, Peak Width = 0.124, Tf= 1.23 Tf = Page 15

16 Peak Shape Comparison of 2.5 & 1.8 um Columns Eclipse Plus Delivers Peak Shape and Efficiency Mobile Phase = 80% 25 mm phosphate (ph 3): 20% ACN, Flow = 0.3 ml/ (2.1mm), 0.27 ml/ (2.0mm ID) Detection: 205nm Sample: Dextromethorphan XBRIDGE C x 50 mm, 2.5 um P = 134 Bar N = 6094, PW = 0.153, TF= Luna HST C x 50 mm, 2.5 um P = 128 Bar N = 4953, PW = 0.225, TF= Shim Pak XR C x 50 mm, 2.5 um P = 159 Bar N = 6940, PW = 0.195, TF= Eclipse Plus C x 50 mm, 1.8 um P = 202 Bar N = 10881, PW = 0.124, Tf = Acquity and XBridge are the same packing material, yet we see worse performance on the XBridge maybe not as optimized packing process as for Acquity? Page 16

17 Comparing RRHD to Competition on 1290 Infinity Mobile Phase: 40:60 water:acetonitrile, 0.1% Formic Acid Flow Rate: 0.5 ml/, Temp: ambient, Det: UV 254 nm Columns: RRHD SB-C18, 2.1 x 50mm, 1.8um and competitive Peaks: 1: uracil, 2acetophenone, 3 propiophenone, 4 butyrophenone, 5 valerophenone, 6 hexanophenone mau RRHD SB-C x 50mm, 1.8um USCES α 3,2 : 1.36 Rs 3, N 6 : TF 6 : 1.04 mau mau Acquity HSS C18 SB 2.1 x 50 mm 1.8um 5 Acquity BEH C x 50 mm, 1.7um α 3,2 : 1.22 Rs 3, N 6 : 9400 TF 6 : 1.17 α 3,2 : 1.29 Rs 3, N 6 : TF 6 : StableBond is still the best column for low ph lifetime on the market! 6 Page 17

18 These Columns Are Now Available! Customers receiving beta test or pre-release LC s can receive beta test or pre-release columns These customers are a critical part of our beta testing program and we want feedback from all of them Feedback to date has been excellent though mostly verbal A RRHD column will ship with every 1290 Infinity LC Part number RRHD Eclipse Plus C18, 2.1 x 50mm, 1.8um This allows quick evaluation of performance and uses our best perforg bonded phase for peak shape Eclipse Plus Page 18

19 The Technical Details. Status Report Currently we have made more than 100 columns available in a working hardware design We are finalizing hardware design these are small details on the internal part of the hardware and impact nothing visible to the customer Every column configuration is tested multiple times to a imum of 5000 injections to be considered optimized We have good packing processes for some of the phases that meet these requirements (one shown earlier) We will continue to optimize the packing process for the multiple bonded phase and configurations so that they are ready for introduction We will continue to add part numbers and phases each month after initial intro but this will be driven by requests/customer needs and sales Page 19

20 RRHD Columns Tested to Minimum Lifetime of 5000 Injections at 1000 Bar RRHD 2.1 x 100mm, 1.8um Bar 1000 N Pressure QC Plates Injections Page 20

21 Overlay of First and Last Injections of QC Test for Lifetime of RRHD 2.1 x 100mm, 1.8um Column Column QC Test: Mobile Phase: ACN:water Flow Rate: 0.3 ml/ Sample: Uracil, Phenol, 4- Chloronitrobenzene, Naphthalene mau Inj #1 = N 4 = 21,480 Inj #5000 N 4 = 21, The QC chromatogram shows consistent performance over the 5000 injections. Page 21

22 Column Test of RRHT Column with new RRHD Type Hardware Pressure of RRHT Column with New Hardware Consistent pressure for over 5500 injections (test ended). Trend line is straight! Conditions: Mobile Phase: 70% Methanol/30% water Flow Rate: 2mL/ flow Temp: RT Detection: 254nm, VWD Injection volume: 2ul Sample: QC sample 1B - Uracil, phenol, 4-Cl nitrobenzene, toluene Run time 1.1 utes Column: RRHT Eclipse Plus C18, 1.8um Pressu re Linear (Press ure) Page 22

23 How Do They Compare On Performance. Use and Lifetime at 1000 bar or more Current feedback from beta testers is excellent Roche feedback indicated use at over 1000 bar with continued good performance Column was taken above 1000 bar though focus is on 1000 bar for routine use and higher pressure as the room left for pressure changes, shifts due to gradients etc. We are expecting more data to be available very soon in this area and we will update as it is available Page 23

24 Gain Efficiency and Resolution with Long Length mm = 250 mm 1000 bar Mobile Phase 20:80 water:acetonitrile, F=0.5 ml/, ambient temperature, ZORBAX RRHD SB-C18, 1.8um columns mau mm (P:560bar) α 3,2 : 1.15 Rs 3,2 : 4.92 N 6 : Pw 2 : Pw 6 : mau Uracil 2. Acetophenone 3. Propiophenone 4. Butyrophenone 5. Valerophenone 6. Hexanophenone mm (P: 960 bar) α 3,2 : 1.15 Rs 3,2 : 6.75 N 6 : Pw 2 : Pw 6 : Highest Rs in less than 3 utes, 250mm, 1.8um at 1000 bar Page 24

25 High Resolution Separation with Long 1.8um Columns Mobile Phase A: water B:acetonitrile, F=0.25 ml/, ambient temperature, Columns: ZORBAX RRHD, SB-C18, 2.1 x mm, 1.8um mau 200 DAD1 A, Sig=254,4 Ref=off (E:\090422JWH\PRESSURE D) PMP1, PMP1B, Pressure Gradient % B/15. Pressure bar Low point of gradient about 480 bar Long column or columns in series provide max Rs Page 25

26 Fast Analysis on ZORBAX RRHD Eclipse Plus C18 mau 600 DAD1 D, DAD1C, DAD: Signal C, 280 nm/bw:10 nm Ref 400 nm/bw:50 nm (G:\1290\AOX JASMIN\AOX-AM2-45C-101.D) DAD1 E, DAD1D, DAD: Signal D, 255 nm/bw:10 nm Ref 400 nm/bw:50 nm (G:\1290\AOX JASMIN\AOX-AM2-45C-101.D) THBP Eclipse Plus C18, 1.8um, Pmax 1070 bar Data Courtesy of: Gerd Vanhoenacker Research Institute for Chromatography Kennedypark Kortrijk Belgium 300 PG TBHQ NDGA BHA OG Ionox-100 DG BHT AP Page 26

27 Fast Analysis on Agilent Zorbax Rapid Resolution High Definition Eclipse Plus C18 Method Column ZORBAX RRHD Eclipse Plus C x 50 mm, 1.8 µm (SN USDAY01001) Mobile phase A=0.02% H 3 PO 4, B=acetonitrile/methanol 3/1 Flow rate 1.6 ml/ Gradient % B Injection volume 1 µl Detector Sig=280/10 nm, Ref=400/50, 80 Hz Sig=255/10 nm, Ref=400/50, 80 Hz Temperature 45 C Sample Antioxidant mixture, ca. 100 ppm Data Courtesy of: Gerd Vanhoenacker Research Institute for Chromatography Kennedypark Kortrijk Belgium Page 27

28 Customer Example Generated on 1290 with RRHD Eclipse Plus C18 3 ute analysis, Zorbax Eclipse + C18 2.1x100mm, 1.8um Seiko TOF, 100pg on-column, 10 Hz Acquisition. 162/220 Pesticides Identified using TOF, Page 28

29 What Applications Are Planned on the RRHD Columns? CSD Led Information Note: A consolidated applications plan is available and covered in another presentation Applications Categories for CSD 1. Proof of Performance 1. Lifetime, Efficiency 2. Resolution/Peak Capacity Message 1. Resolution per time based on improving resolution with different gradient times and column lengths 3. Comparisons to Acquity Page 29

30 CSD Initial Applications Plan for RRHD Columns General App Samples Columns Lifetime at 1000 and 1100 bar Formic acid mp, dirtier sample All Efficiency -simple tests of each dimension Van Deemters, consideration of frictional heating, limited to 2.1 mm and 3.0 mm ID? QC and Waters acid base neutral mix Phenones - RRLC checkout sample 2.1 x 50mm, 2.1 x 100mm 2.1 x 50mm, 2.1 x 100mm Efficiency - 2 columns in series Neutrals - and complex pharma mix 2.1 x 100mm Comparisons to Acquity Demonstrate use on an Acquity system bar means any 1000 bar LC (Prove fittings work appropriately) Efficiency x 150mm in 10/20/30 ute increments - demonstrate a short and long run with efficiency/rs per time Widest range of sample types, show off selectivity Duplicate Acquity app - target big pharma examples so that we can sell columns where they already have experience Several samples of different complexity - peptide map (most complex), pharma sample with impurities, sample with at least 5 components with at least one closely eluting 2.1 x 50mm, 2.1 x 100mm 2.1 x 50mm, 2.1 x 100mm 2.1 x 150mm Page 30

31 Summary New ZORBAX RRHD Rapid Resolution High Definition LC columns for the 1290 Infinity LC are designed for the demands of this UHPLC and any other UHPLC. RRHD columns are made from the same ZORBAX 1.8um particles and bonded phases in the ZORBAX RRHT columns for the RRLC. RRHD columns deliver long lifetime and high resolution over an expanded performance range compared to the RRHT columns Practically speaking they can go to the high pressure limits with new hardware design and packing processes. Page 31