HPLC & UHPLC Workshop PerkinElmer Korea 009 9 18 009 Perkin Elmer HPLC Basic Principle PerkinElmer Korea Kim, Wang-Yu PhD 009 Perkin Elmer
Chromatography Chromatography h Chromos(color) + graphein(write) = 1906 Michael Tswett. (Liquid-Solid). Molecular components in a mixture are separated because of their affinities for two substances called phases. ( ) 3 HPLC 4
HPLC Chromatogram Injection Port computer high pressure Pump Colum mn Detector 5 Chromatography chromatography Gas chromatography Liquid Chromatography Gas-Solid Gas-Liquid Adsorption Partition Ion exchange Size Exclusion Affinity Gel Filtration Gel Permeation 6
High Performance Liquid Chromatography 7 Partition chromatography 8
(reverse phase) O O Si O O H + Cl CH 3 Si (CH ) 17 CH 3 CH 3 O CH 3 O Si O Si (CH ) 17 CH 3 O CH 3 Pore Si Si CH 3 Si -O-Si-(CH ) - CH 17 3 Si CH 3 Si Si 9 10
Adsorbent( Solvent Pentane Hexane Iso-octa ne Cyclohex xane Carbon tetrachlo oride l-chlorob butane Xylene Toluene Chlorobe enzene Benzene e Ethyl eth her Dichloro methane Chlorofo orm 1,-Dich hloroethane Methyl ethyl ketone Acetone Dioxane 1-Pentan nol Tetrahyd drofuran Methyl t -butyl ether Ethyl ace etate Dimethy yl sulfoxide Diethyla mine Acetonit rile 1-Butano ol Pyridine -Metho oxyethanol n-propyl l alcohol Isopropy yl alcohol Ethanol Methano ol Ethylene e glycol Dimethy yl formami ide Water (Al O 3 ) 0.00 0.00-0.01 0.01 0.04 0.17-0.18 0.6-0.30 0.6 0.0-0.30 0.30-0.31 0.3 0.38 0.36-0.4 0.36-0.40 0.44-0.49 0.51 0.56-0.58 0.56-0.61 0.61 0.45-0.6 0.3-0.6 0.58-0.6 0.6-0.75 0.63 0.5-0.65 0.70 0.71 0.74 0.78-0.8 0.78-0.8 0.88 0.95 1.11 - - (SiOH) 0.00 0.00-0.01 0.01 0.03 0.11 0.0-0. 0.3 0.5 0.38-0.43 0.3-0.3 0.6 - - 0.47-0.53 0.49-0.51-0.53 0.48 0.38-0.48 - - 0.50-0.5 - - - - 0.60-0.70-0.73 - - - (C 18 ) P _ 0.0 _ 0.1 _ 0.1 _ 0. _ 1.6 _ 1.0 _.5 _.4 _.7 _.8 _ 3.1 _ 4.1 _ 3.5 _ 5.7 8.8 5.1 11.7 4.8 - - 3.7 4.0 -.5-4.4-7. - - 3.1 5.8-3.9-5.3-5.5 10.1 4.0 8.3 3.9 3.1-1.0 5.1 - - 7.6 6.4-10. 11 Chromatography ( ) HPLC HPLC 1 HPLC column
Isocratic vs Gradient 13 Polarity Functional Group Polarity Comparisons Polarity Functional Group Structure Bonding Types Intermolecular Forces Displayed Low Methylene σ London R (CH ) Phenyl σ, π London R Halide R F, Cl, Br, I σ London, Dipole-Dipole Ether R O σ London, Dipole-Dipole, H-bonding R O - Nitro R N + O σ, π London, Dipole-Dipole, H-bonding Ester R O σ, π London, Dipole-Dipole, H-bonding O R O Aldehyde σ, π London, Dipole-Dipole, H-bonding R H Ketone R O σ, π London, Dipole-Dipole, H-bonding R Amino R NH σ, π London, Dipole-Dipole, H-bonding, Acid-base chemistry Hydroxyl R OH σ London, Dipole-Dipole, H-bonding O High Carboxylic Acid R σ, π London, Dipole-Dipole H-bonding Acid-base OH 14
Absorption Fluorescence Absorption Fluorescence Luminescence 15 Ion exchange( ) chromatography eletrostatic force polystyrene or silica gel 16
Ion exchage column Strong Cation Exchanger Polystyrene or Silica R1 (CH CH ) SO 3 - R4 N + R R3 Strong Anion Exchanger Polystyrene or Silica CH CH CH NR 3 + - OOC R Application : 17 Size exclusion( ) chromatography 18
Size exclusion 19 High Performance Liquid Chromatography 0
Mass transfer( ) 1 Longitudinal diffusion( )
B u Eddy-diffusion( ) Initial Band Width Final Band Width 3 Van Deemter Equation B A 4
Column: C18, 4.6 x 150 mm (5 µm) Mobile Phase: 8% H O : 18% ACN Injection Volume: 0 µl Sample: 1. Caffeine. Salicylamide RT(retention time) Column temperature : 30 degree Detection : 54 nm INJECT calibration : Integration : 5 Chromatogram RT(retention time) R R1 R1 0 0 R1 R α R1 R INJECT 6
Selectivity α R 0 α = = R 1 0 R R 1 α 7 Separation of water soluble vitamins Mobile Phase: 15% MeOH 85% (10 mm hexanesulfonate, Separations of Water-Soluble Vitamins 1% HOAc, 0.13% TEA in H 0) using different tc8 and C18 columns Flow Rate: 1.5 ml/min at 35 C 1. Vitamin C. Niacin 3. Niacinamide 4. Pyridoxine 5. Thiamine 6. Folic Acid 7. Riboflavin 8 Ref.: M.W. Dong and J. L. Pace, LC.GC, 14(9), 794-803, 1996.
Efficiency Inject t R t N = 16 ( R t ) = 5.54 ( R W ) b W 1/ W 1/ HETP = L/N W b 9 N = 5000 N = 10,000 N = 0,000 1 3 5 7 9 11 13 15 Time (min) 30
Resolution( R s = 1 N 4 α -1 α k 1 + k R = 0.4 R = 0.5 R = 0.6 R = 0.7 R = 08 0.8 R=10 1.0 R=15 1.5 31 3
HPLC column & separation PerkinElmer Korea Kim, Wang-Yu PhD 009 Perkin Elmer HPLC Chromatogram Injection Port computer high pressure Pump Colum mn Detector 34
35 Short (30-50mm) - short run times, low backpressure Long (50-300mm) - higher resolution, long run times Narrow (.1mm) - higher detector sensitivity Wide (10-mm) - high sample loading Narrow columns enable to save solvent waste. Short columns enable to reduce the analyssis time. 36
4.6 mm.1 mm L = 50 mm L = 150 mm 37 Spherical particles offer reduced back pressures and longer column life when using viscous mobile phases like 50:50 MeOH:HO. 38
Smaller particles offer higher efficiency, but also cause higher backpressure. Choose 3µm particles for resolving complex, multi-component samples. Otherwise, choose 5 or 10µm packings. P (1/d p ) P = Pressure d p = Particle Size 39 High surface area generally provides greater retention, capacity and resolution for separating complex, multi-component samples. Low surface area packings generally equilibrate quickly, especially important in gradient analyses. 40
Larger pores allow larger solute molecules to be retained longer through h maximum exposure to the surface area of the particles. Choose a pore size of 60~150Å or less for sample MW 000. Choose a pore size of 300Å or greater for sample MW > 000. 41 Higher carbon loads generally offer greater resolution and longer run times. Low carbon loads shorten run times and many show a different selectivity. 4
Endcapping reduces peak-tailing of polar solutes that interact excessively with the otherwise exposed, mostly acidic silanols. Non-endcapped packings provide a different selectivity than do endcapped packings, especially for such polar samples. 43 Brownlee(from PerkinElmer) Validated 44
Question : What column? 45 Answer : C18 Phenyl 46
Question : What column? Which two sample components have the most similar structures? Draw them, then circle the structural t differences between them. Anthracene 3-Hexylanthracene (CH) 5 CH 3 Note: The structural difference between these two compounds is the hydrophobic hexyl side chain. This suggests a non-polar C18 or C8 column would interact with this area of difference to help provide separation of these two compounds. Recommended bonded phase (silica based materials only) mark one Normal phase silica NH CN Reversed phase C18 C8 Ph CN 47 : Column problem A. : plugged frit, column contamination B. : split peak, peak tailing, broad peak C. : Equilibration, flow change, selectivity change 48
49 Column: C18, 4.6 x 150 mm, 5 µm Mobile Phase: 8% H O : 18% ACN Injection Volume: 30 µl Sample: 1. Caffeine. Salicylamide A. Injection Solvent B. Injection Solvent 100% Acetonitrile il Mobile Phase 1 0 10 Time (min) 0 10 Time (min) 50
Mobile Phase: 40% 5 mm KH PO 4 : 60% ACN Flow Rate: 1.0 ml/min. Temperature: RT. ph 4.4 ph 3.0 CH 3 CHCOOH CH CH(CH 3 ) Ibuprofen pk a = 4.4 0 1 3 4 5 6 7 8 9 10 0 1 3 4 5 6 7 8 9 10 Time (min) Time (min) [RCOO - ][H + ] RCOOH RCOO - + H + K a = [RCOOH] 51 May be caused by: Column aging Column contamination Insufficient equilibration Poor column/mobile phase combination Change in mobile phase Change in flow rate Other instrument issues 5
UHPLC Basic principle PerkinElmer Korea Kim, Wang-Yu PhD 009 Perkin Elmer HPLC Chromatogram Injection Port computer high pressure Pump Colum mn Detector 54
Chromatogram( ) R RT(retention time) R1 0 R1 INJECT R 55 Benefits of UHPLC UHPLC Green Productivity Higher throughput Much Less Solvent Better detection Isoflavones in nutriceutical products Can be analyzed with UHPLC over six times faster, with higher sensitivity, using over 90% less mobile phase solvent compared with conventional HPLC methods. Conventional HPLC 56 0 5 10 15 0 min. save time and money
Common Fears of Migrating to UHPLC New Technology Cost of new hardware Cost of ownership and operation Ruggedness of sub-µm columns High pressure Training i Software Method conversion Migration to UHPLC is easier than most people p think. New hardware will pay for itself by increasing productivity and reducing operating costs. 57 Don t Be Afraid of UHPLC!!! Principles and Theory of HPLC Resolution Equation 1 k R s = N 1 4 α 1 + k ( ) Efficiency particle size column length solvent velocity Retention solvent strength th stationary phase composition Selectivity type of stationary phase mobile phase composition additives 58 baseline separation is the goal for identification and quantification
UHPLC High Resolution and Speed R s 1 k = N 4 1 + α k ( 1 ) Efficiency Retention capacity Selectivity 1 Efficiency is inversely N α proportional to particle dp size (d p ) L Efficiency is directly N α proportional to column dp length (L) 59 the strength of UHPLC is increased efficiency by reducing particle size Van Deemter Equation Smaller particles size means Faster chromatography Increased Efficiency Enhanced Resolution/Selectivity Wider range of flow velocities Less solvent consumption Smaller particles size also means Higher operating pressure Filtration of MP and samples (0.µm) Low system volume required Lower sample loading Higher skill level of operator 60 the benefits justify the challenge
Van Deemter Equation - Contributions to Band Broadening H H = A + B u + Cu Van Deemter equation describes column efficiency as a function of mobile phase Actual Plot velocity in terms of solute: eddy diffussion i (A) molecular diffusion (B) mass transfer (C) C Term Optimum efficiency A Term B Term Linear Velocity (u) 61 Van Deemter Equation - Contributions to Band Broadening H = A + B u + Cu Eddy Diffusion H 3 Column Faster 1 Time Slower A Term Independent d of mobile phase velocity (u) A decreases as particle size (d p ) decreses Linear Velocity (u) 6
Van Deemter Equation - Contributions to Band Broadening H = A + B u + Cu Molecular Diffusion H Describes diffusion of solute in the mobile phase Effect of B becomes negligible at sufficient flow rate A Term B Term Linear Velocity (u) 63 Van Deemter Equation - Contributions to Band Broadening H = A + B u + Cu H Describes the transfer of solute into and out of the stationary phase (sorption and desorption). C decreases with particle size (d p ) so slope of C*u also decreases with particle size. Mass Transfer stationary silica mobile phase phase support A Term B Term Linear Velocity (u) 64
Van Deemter Equation - Contributions to Band Broadening H = A + B u + Cu H Van Deemter equation terms A and C decrease as particle size decreases. Sub-µm columns are more efficient over a broader range of flow rates. Optimum efficiency ATerm Linear Velocity (u) B Term CTerm A Term B Term 65 UHPLC Basics Sub-µm Particle Columns Increase Column Efficiency ( N 1 d p ) Improve Resolution ( R 1 ) s d p Increase Column Backpressure ( P d ) 1 p 150mmx46mmx50µm 4.6 x 5.0 50mmx1mmx19µm.1 x 1.9 N Constant (~ 10,000 plates) R s Constant t P (~ 6X) t R (~ 8X) 66 faster analysis at higher pressure
Requirements for a UHPLC System System must handle pressures up to 15,000 psi to in order to utilize sub -µm particle size columns. Ultra high pressure pumps such as the Flexar FX-10 and FX-15. Fast autosamplers with injection valves that can handle pressures >15,000 psi. Column oven elevated temperature reduces mobile phase viscosity and system backpressure. Fast detectors such as the Flexar FX UV/Vis with a sampling rate of 100 Hz. Reduced extra-column volume is critical to minimize band broadening in UHPLC. Low ID Tubing Short tubing length Low volume detector flow cell Contributions of Band Broadening col 67 Flexar FX UHPLC systems are up to the task Extra column volume Extra-Column-Volume = sample volume + connecting tubing volume + fitting volume + detector cell volume Tubing volume 68
Band Broadening Comparison 85.00 80.00 75.00 1400 3360 Conventional system IBW = µl 70.00 65.00 60.00 100.00 0.0 0.1 0. 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1. 1.3 1.4 1.5 1.6 1.7 1.8 1.9 90.00 5700 80.00 3800 Optimized UHPLC system IBW = 1 µl 70.0000 60.00 0.0 0.1 0. 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1. 1.3 1.4 1.5 1.6 1.7 1.8 1.9 69 minimizing extra-column volume significantly reduces band broadening General Steps of Method Migration 1. Choose a HPLC analysis that is appropriate for migration to UHPLC.. Select an UHPLC column with the best potential for successful migration. 3. Calculate UHPLC method parameters (Flow Rate, Injection Volume, etc.). 4. Run transferred method. 5. Optimize transferred method. Other Considerations: Use only HPLC grade or UHPLC grade mobile phases. Filter all mobile phases, standards and samples with 0.µm membranes. Utilize elevated temperature to reduce MP viscosity and column backpressure. Prepare samples and standards in initial MP conditions. 70 careful planning maximizes potential for success
Choosing the Right Column Choose a column with the same type of stationary phase to maintain retention order. The optimal column ID for UHPLC is.1mm because mobile phase consumption and frictional heating are significantly reduced. Choose column dimensions that will have a similar efficiency as the HPLC column currently used. A good rule of thumb for choosing a UHPLC column length when currently using a 4.6mm ID, 5.0µm particle HPLC column is: HPLC Column Dimensions 50mmx46mm 4.6 UHPLC Column Dimensions 30 mm x.1 mm 150 mm x 4.6 mm 50 mm x.1 mm 50 mm x 4.6 mm 100 mm x.1 mm 71 the right column choice will simplify method migration Tools Available to Simplify Method Migration Many good references are available with detailed descriptions of method transfer including a PerkinElmer white paper entitled Guidelines for the Use of UHPLC Instruments by D. Guillarme & J. Veuthey. White paper is available at: http://las.perkinelmer.com/content/applicationnotes/wtp_guidelinesforuhplcinstruments.pdf There are five major method parameters to convert when transferring a method from HPLC to UHPLC. Injection Volume (V inj ) Flow Rate (F) Isocratic Step Time (t iso ) Gradient Step Time (t grad ) Gradient Slope 7 method transfer calculator is available from PerkinElmer
Injection Volume The injection volume (V inj ) must be adjusted to avoid column overload as well as maintain sensitivity and reduce extra-column band broadening. As a rule, injected volumes should not exceed 1-5% of the column volume. The column volume is a function of the column length (L) and internal diameter (d c ) but is independent of stationary phase particle size. V dc ij inj = Vij inj 1 dc L L 1 1 V inj = Injection Volume d c = Cl Column Diameter L = Column Length 150 mm x 4.6 mm x 5.0 µm 50 mm x.1 mm x 1.9 µm V inj1 = 15.0 µl V inj = 1.0 µl 15X Decrease in Injection Volume 73 conserve precious sample and expensive standards Flow Rate The flow rate (F) for UHPLC must be adjusted to maintain a similar mobile phase linear velocity (u) used in the HPLC column. The linear velocity within a column is directly proportional to the column diameter (d c ) but also depends on the particle size (d p ) of the stationary phase. Therefore u*d p must be maintained at a constant value to account for changes in column diameter and particle size. F = F 1 d c d p 1 d c d p 1 F = Flow Rate d c = Column Diameter d p = Particle Diameter 150mmx46mmx50µm 4.6 x 5.0 50mmx1mmx19µm.1 x 1.9 F 1 = 10mL/min 1.0 F = 054mL/min 0.54 ~X Decrease in Flow Rate 74 save on mobile phase consumption
Isocratic Step Times % B The ratio between the isocratic step time (t iso ) and the column dead time must be maintained between HPLC and UHPLC conditions. Time The column dead time depends on the flow rate, column diameter and length. t F d 1 c L iso = t iso 1 F d L c 1 1 F = Flow Rate d c = Column Diameter L = Column Length 150 mm x 4.6 mm x 5.0 µm 50 mm x.1 mm x 1.9 µm t iso1 = 5.0 min t iso = 0.6 min 75 5 min 0.6 min Isocratic Step Gradient Slope The initial and final MP compositions in % B any HPLC gradient step should be maintained in the UHPLC method. Time d c 1 slope = slope 1 d c The slope and time of the gradient step in the UHPLC method must be adjusted so the product of the gradient slope and dead time remain constant between the traditional HPLC method and the UHPLC method. t grad L L 1 F F 1 ( % B ) final % B 1 initial 1 = slope 76 Example: slope t grad 4. 6 mm 150 mm 0. 54 ml/min = 0. 75% / min 5. 83% / min.1mm 50mm 1. 0mL/min = ( 85% % 70% % ) = = 5. 83% / min. 6min 0 min.6 min Gradient Step
Transferred Gradient Program 90 HPLC 85 90 UHPLC 85 %ACN 80 75 %ACN 80 75 70 70 65 0 5 10 15 0 5 30 35 40 45 Time(min) 65 0 1 3 4 5 6 7 8 9 Time(min) Comparison of HPLC and the transferred (calculated) UHPLC gradient programs Step HPLC Time UHPLC Time %ACN 1 0 0 70 5 0.6 70 3 5 3. 85 4 9 3.7 85 5 30 3.8 70 6 45 5.7 70 8X Decrease in Predicted Runtime 77 45 min Run 5.7 min Run UHPLC Applications 34.00 3.00 Nutraceutical ti Application: Ginsenosides id from Ginseng Peak List 1. Rg1 1. Re 3 3. Rf 30.0000 4. Rb1 4 6 5. Rc 8.00 5 7 1 6. Rb 6.00 4.00.00 7. Rd 3 6 4 0.00 18.00 5 PE Brownlee C 18 7 50 5.0 55 5.5 60 6.0 65 6.5 70 7.0 75 7.5 80 8.0 85 8.5 90 9.0 50 mm x.1 mm, 1.9 µm 0.5 1.0 1.5.0.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 16.00 14.00 1.00 10.00 PE Brownlee C 18 150 mm x 4.6 mm, 5 µm 0 4 6 8 10 1 14 16 18 0 4 6 8 30 3 34 36 38 40 4 44 46 48 50 5 54 56 58 60 78 5X Throughput Improvement
110.00 100.00 90.00 80.00 70.00 UHPLC Applications Nutraceutical ti Application: Isoflavones in Soy 3 1 4 3 5 4 6 Peak List 1. Daidzin. Glycitin 3. Genistin 4. Daidzein 5. Gylcitein 6. Genistein 60.0000 50.00 0 0.5 1.0 1.5.0.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 PE Brownlee C 18 50 mm x.1 mm, 1.9 µm 40.0000 1.5.0.5 3.0 5 6 30.00 0.0000 10.00 PE Brownlee C 18 150 mm x 4.6 mm, 5 µm 0 4 6 8 10 1 14 16 18 0 4 6 8 30 3 34 36 38 40 4 79 6X Throughput Improvement` Benefits from Migration to UHPLC Improve Productivity Expected analysis time (t ana) is directly proportional to change in column dead volume. 1 = HPLC Method So: d p L = UHPLC Method t ana = t ana 1 d p L 1 1 d p = Particle Diameter L = Column Length 150 mm x 4.6 mm x 5.0 µm 50 mm x.1 mm x 1.9 µm Example: t ana 1. 9um 50mm = 45min 5. 7min 5. 0um 150mm = ~ 8X Throughput Improvement 80 10 Runs/Shift 84 Runs/Shift
Benefits from Migration to UHPLC Decrease Mobile Phase Consumption For successful method migration the product of mobile phase velocity (u) and particle size (d p ) should be maintained at constant value. [ (u d p ) 1 =(u d p ) ] The migration of flow rate is dependent on particle size and internal column diameter. Example: F dc d F = F1 d d c 1 p p 1 d c = Column Diameter F = Flow Rate. 1mm 5. 0um ml/min 0. 54 ml/min 4. 6mm 1. 9um = 1 = MP Consumption HPLC = 1.0 ml/min x 45 min = 45 ml MP Consumption UHPLC = 0.54 ml/min x 5.7 min = 3.1 ml ~93% Decrease in MP Consumption Flow Rate ~ 50% 81 Use Less MP and Reduce Waste Disposal Summary Migration to UHPLC requires an LC system optimized for UHPLC, not just a new column Conversion of HPLC method conditions is simple with the available UHPLC conversion calculators Increase productivity up to 9-fold by decreasing runtimes Decrease consumption of mobile phase by up to 93% and lower waste disposal cost Green Technology Method development/optimization is much faster due to short UHPLC runtimes 8 Instruments will pay for themselves by increasing productivity and reducing operating costs. Don t Be Afraid of UHPLC
Flexar TM : New HPLC PerkinElmer Korea Kim, Wang-Yu PhD from PerkinElmer 83 009 Perkin Elmer Flexar. More choices in LC analysis. 18,000 PSI 84 the most choices in pump pressures
Flexar Features Bottle tray with integrated degassing and SW comm link Inter-component drain management Built on rugged PerkinElmer LC technology, recognized for reliability Tubing management for streamlined chromatography Elegant, ergonomic user interface with streamlined, consistent look & footprint Controlled by both Chromera and TotalChrom 85 designed with form and function in mind Flexar Solvent Manager Three versions available Without degassing 3-channel degassing 5-channel degassing Can be combined with any Flexar pumps Solvent delivery tubes conveniently managed through rear manifold. Can hold up to five 1 liter solvent bottles Built-in communications link and vacuum degasser Stackable design, with Flexar tube management and builtin inter-component drain system Removable tray to contain breach of complete bottle 86 more than just a bottle tray
Flexar Autosampler Easy Open Access 87 absolutely accessible for easy loading and service Flexar Autosamplers: World-class Performance Excellent Injection Repeatability Above 5µL injections, %RSDs of 0.5% For 5µL Linjections, in µl-pickup mode, %RSDs are also typically 0.5% Area %RSDs fixed loop mode of 0.3% For µl injections, in µl-pickup mode, (10µL loop) area %RSDs are typically 0.5% Anthracene area precision: 0.4% RSD 5µL injection Very low carryover: Pressure assist air purge after each run (1) 300 ppm caffeine n=30 (3) 30µg/L caffeine () Blank injection 88 what carry over?
Flexar LC Column Oven Three versions Heat only Peltier (heat and cool) Peltier with column selection/switching (TotalChrom only Chromera coming) Built-in i leak alarm Integrated solvent pre-heater/chiller minimizes temperature gradients Better column performance More repeatable retention times Temperature range of 30ºC to90ºc (5ºC to 90ºC for Peltier), controlled to within 0.ºC throughout entire temperature range Stackable design, with Flexar tube management and builtin inter-component drain system Large, easily accessible column compartment holds even 30-cm column format 89 Precise temperature control for improved retention time stability. Flexar PDA Detectors FX PDA UHPLC Detector Excellent performance, HPLC or UHPLC Improved noise specifications (<0.9 x 10 5 ), while maintaining excellent signal-to-noise, noise wide linear range and low drift Best-in-class spectral integrity delivers improved peak identification and purity FX version: High efficiency.4ul flow cell with detector electronics optimized for flow cell IRIS Spectral Processing Software PDA LC Detector 90...Deeper insights with more powerful methods development.
Flexar UV/VIS Detectors FX UV/VIS UHPLC Detector World-class UV/VIS detection Dual beam optical design with choice of tungsten or deuterium light sources with wavelength range of 190-700 nm Up to 50 pts/sec 10 µl flow cell standard d compatible with a wide range of optional flow cells Fast, sharp UHPLC performance High efficiency.4 µl flow cell for optimum UHPLC peak resolution 100 pts/sec detection to capture even the fastest t UHPLC peaks UV/VIS LC Detector 91 sensitive, selective, speed, dynamic range Flexar Refractive Index and Fluorescence Detectors Refractive Index Detector Combine sensitivity and specificity Best sensitivity in the market Now under Chromera control and can conveniently be combined with UV/VIS detection (PAH) 9 Rugged general-purpose detection Highly stable and sensitive LC and GPC detector for compounds that do not have high UV absorbance such as polymers, sugars, organic acids and triglycerides Internal temperature of flow cell for baseline stability Autozero and autopurge of reference cell make it easy to use Fluorescence Detector now fully controlled under Chromera!
Flexar UHPLC Systems FX-10 UHPLC System FX-15 UHPLC System FX-10: High Value UHPLC Micro Binary 10,000 psi pump package FX-15: Ultimate in UHPLC Dual reciprocating i 18,000 psi pump High efficiency FX UHPLC autosampler Column oven for retention time reproducibility and viscosity reduction High speed FX UV/VIS detector (100 pt/sec) with high efficiency.4µl flow cell 93 You don t have to pay more to achieve so much. Flexar FX-15 UHPLC Pump 15,0000 psi operation for the most demanding UHPLC applications with up to 10x productivity improvement up to 5mL/min at 18,000 psi! Green productivity mobile phase solvent consumption reduced by 10-15x Optical Sensor synchronizes injection with piston position Maximum retention time repeatability for UHPLC Dual reciprocating 15,000 psi pump Smoother more precise flow for retention time repeatability High pressure Ti-tip check valves and pulse dampeners Rated to >15,000 psi operation for highest throughput UHPLC Integrated t piston wash function Keeps precision pumps clean even with buffers 94 the ultimate in UHPLC
Flexar FX-15 Pump for Ultimate UHPLC Exclusive Ultra High Pressure Pulse Dampeners Rated to >18,000 p.s.i. Significantly reduces pulsation. Innovative Check T Prevents channel-to-channel cross talk. No back flow to affect composition. Improved retention time repeatability. 95 FX-15 UHPLC Pump Piston Drive Automatic Piston Wash SAPPHIRE PISTON Greatly Increases Seal Life (particularly with buffers) MOBILE PHASE FLOW OF FLUSH SOLUTION PISTON MOVEMENT No Auxiliary Pump Required (self flush) PRIMARY HIGH-PRESSURE SEAL SECONDARY SELF-FLUSH SEAL Dedicated recirculation flush solvent 15 µl displacement Self-centering piston saddle Captive spring for fast head removal 96
Flexar. Reach for more choices in LC analysis. FX-15 15,000 psi Autosamplers FX-10 pump package FX-15 Pumps FX-10 10,000 psi PDA Flexar 6,000 psi UV/VIS RI Manual injection systems FL Ovens Chromera software Direct access to all system status parameters User configurable Direct access status panel for to all critical parameters instrument controls and settings Direct access to any area in the software 98
Chromera Report Refine the report style based upon the needs of the data Choose from a gallery of report styles Create and save the specific layouts needed for automated reporting 99