More Speed and Resolution for Current Methods

Transcription

1 More Speed and Resolution for Current Methods A Simple How to Guide for Easier Method Transfer in the Current Lab Environment Edward Kim Application Engineer June 15, 2010 Page 1

2 Key Points for Successful Transfer 1. Set Your Goals--Speed, Resolution, Both? 2. Choosing The Column 3. Reducing The Column Length 4. Changing The Column i.d. 5. Adjusting The Injection Volume 6. Transferring to 1.8um Columns - Is and Grad 7. Increase Resolution 8. Speeding It Up Page 2

3 Rule of Thumb Maybe not always 100% Technically Correct but a very good easy alternative Page 3

4 Column Choice is Key to Overall Success Choose Bonded Phase as Close to Original Method as Possible Try Alternate Bonded Phases to Solve Peak Placement Problems Choose Column Chemistry for Optimum ph Stability and Column Life Page 4

5 Bonded Phase Structures Affect Selectivity and ph Ruggedness StableBond C18 Extend C18 C18 Si O C18 Si O Eclipse C18 StableBond CN Page 5

6 Start Method Development with RRHT Columns: Different ZORBAX RRHT C18 Bonded Phases for Max Selectivity 1 st choice Best Resolution Eclipse Plus C18 & Peak Shape 2 nd choice Good alternate selectivity due to non-endcapped StableBond SB-C18 Mobile phase: (69:31) ACN: water Flow 1.5 ml/min. Temp: 30 C Detector: Single Quad ESI positive mode scan Columns: RRHT 4.6 x 50 mm 1.8 um 3 rd choice Good efficiency & peak shape Resolution could be achieved 4 th choice Resolution not likely, Other choices better, for this separation ,3 4 4 Eclipse XDB-C min Extend-C18 Sample: 1. anandamide (AEA) 2. Palmitoylethanolamide (PEA) 3. 2-arachinoylglycerol (2-AG) 4. Oleoylethanolamide (OEA) Multiple bonded phases for most effective method development. Match to one you are currently using. Page 6

7 How To Match a Column to a ZORBAX RRHT Column General Phase Type Typical endcapped C18 or C8 bonded phases, newer columns Endcapped C18 or C8 columns, older generation Non-endcapped columns Older types of columns Aqueous type columns CN or Phenyl Starting ZORBAX Choice Eclipse Plus C18 or C8 Eclipse XDB-C18 or C8 StableBond C18 StableBond C18, C8 etc. SB-AQ SB-CN, SB-Phenyl Page 7

8 Choose Suitable Column Phase Acidic Stable Bond ph 1 6 SB can use increased temperature up to 100C Mobile Phase ph Intermediate Eclipse Plus /Eclipse XDB + ph 2 9 Eclipse can use increased temperature up to 60C Basic Extend ph Extend can use increased temperature up to 45C Page 8

9 The Complete List of RRHT 600 bar Columns Jan 1 Dimensions Eclipse Plus C18 Eclipse Plus C8 Eclipse XDB-C18 Eclipse XDB-C8 Extend-C x x x x x x x x x x x x x x x Dimensions SB-C18 SB-C8 SB-Phenyl SB-CN SB-AQ Rx-Sil 4.6 x x x x x x x x x x x x x x x (Main product page) (P/N List) 2 Recommended Starting Choices: They are Eclipse Plus C18 Columns. Page 9

10 First, Let s Work on Easy Speed Gains! Page 10

11 Page 11 IS O C R A T IC E L U T IO N

12 Simplified Method Transfer for Increased Isocratic Speed 1. Shorten the Column Length 2. Reduce Particle Size 3. Maintain Flow Rate 4. For More Speed. Wait, Hold the Line - Easy Gains First! Very Fast is Just a Few Slides Away! Page 12

13 Reducing Column Length and Particle Size Maintains Resolution While Reducing Assay Time Column Length (mm) Column Efficiency N(5 µm) Column Efficiency N(3.5 µm) Column Efficiency N(1.8 µm) ,500 21,000 35, ,500 14,000 23, ,500 17, ,200 7,000 12, N.A. 4,200 6, N.A. 2,100 2,500 Efficiency Analysis (N) Time Pressure Peak Volume Solvent Usage Analysis Time* - -33% -50% -67% -80% -90% Reduction in analysis time compared to 150 mm column; all columns 4.6-mm i.d. Shorter columns with small particles provide the efficiency of longer columns with larger particles Page 13

14 First, Reduce Column Length 5um 1.8um Reduce column length by factor of 3 Quite often original method will have more resolution than is actually needed and a reduction by 5 may be possible 3.5 um 1.8um Reduce column length by factor of 2 Quite often original method will have more resolution than is actually needed and a reduction by 3 is possible Page 14

15 Reduced Column Length and Particle Size Reduce Analysis Time Both Separations Performed on Same Agilent 1200 SL HPLC mau Original method ZORBAX LC column Extend-C x 150 mm, 5 μm min mau x faster Mobile phase: (70:30) MeOH: 50 mm pyrrolidine buffer Flow = 1.0 ml/min, Temp. : ambient ZORBAX RRHT column Extend C x 50 mm, 1.8 μm min Page 15

16 Reached Flow Rate Limit? Change Column I.D. Changing Column I.D. Will Allow Lower Flow Rate to Match Linear velocity Can improve speed potential lower initial flow rate used allows higher overheads within the flow rate range of the instrument Reduce Mobile Phase Use Can improve sensitivity for same mass loading, but must optimize the flow path volume to minimize dispersion. Page 16

17 How conversion works for flow Flow modification, for columns of different diameters Flow col. 1 Diam. Diam. column2 column1 2 Flow col. 2 i.e mm 1.0ml/min 0. 21ml/min 4.6mm Page 17

18 Reduce Column Diameter and Flow Rate by Same Factor 4.6 mm 3 mm Reduce flow rate by factor of 2.4 Reducing to 3 mm i.d. column allows use of higher linear flow rates as the 1200 SL pump will pump up to 5ml/min. Compared to 4.6 mm id column at 1 ml/min, you can operate a 3 mm id column at 0.42 ml/min and save solvent or reduce flow rate to MS detector, or you can keep the flow rate at 1 ml/min, and have 2.4-fold increase in linear velocity 4.6 mm 2.1 mm Reduce flow rate by factor of 4.8 Page 18

19 Quick Reference for Changing to Common Column Diameters Maintains Equivalent Linear Velocity for Different Column IDs: Column Type Column ID Flow Rate Analytical 4.6 mm 1.0 ml/min Solvent Saver 3.0 mm 0.42 ml/min Narrow Bore 2.1 mm 0.21 ml/min MicroBore 1.0 mm 47 ml/min Capillary 0.5 mm 12 ml/min Capillary 0.3 mm 4.2 ml/min Nano 0.1 mm 472 nl/min Nano mm 266 nl/min Flow rate column 2 = (diameter column 2) 2 /(diameter column 1) 2 x Flow rate column 1 Maintain equivalent mobile phase linear velocity when scaling down in column diameter. Page 19

20 Changing Both Column Length and Diameter Change from a 4.6 x 250 mm (5 um) to a 3.0 x 100 mm (3.5 um) Column. mau Mobile Phase: 25% methanol in 0.4% Formic Acid ZORBAX SB-C18, 4.6 x 250 mm, 5 mm, 1 ml/min Solvent Used: 34 ml mau min ZORBAX SB-C18, 3.0 x 100 mm, 3.5 mm, ml/min Solvent Used: 5.7 ml, decrease of 83% (decrease in analysis time of 57%) min Page 20

21 OK, Let s Run More Isocratic Speed Increase flow rate If flow rate limit of instrument* is reached, reduce column diameter and reduce flow rate to maintain linear velocity If pressure becomes problematic, increase mobile phase/column temperature Increase flow rate as necessary or desired to within 80-90% of flow or pressure limit * Flow Rate Limit is 5mL/min for Agilent Binary Pump, 10mL/min Agilent Quaternary Pump (P < 200 bar) Page 21

22 HETP (cm/plate) Speeding It Up-How Fast can We Go? Increase Linear Velocity (Flow Rate) mL/min 5.0 mm 3.5 mm 1.8 mm ml/min Interstitial linear velocity (u e - cm/sec) Step 1 (2x Original Flow) Increase the flow rate by 100% Reduce run time by 50% Reduce gradient time segments by 50% Example 1ml/min > 2ml/min 30 minutes > 15 minutes 35-65% over 30 mins > over 15 mins Page 22

23 HETP (cm/plate) Speeding It Up Faster! Increase Linear Velocity (Flow Rate) mL/min 5.0 mm 3.5 mm 1.8 mm ml/min Interstitial linear velocity (u e - cm/sec) Step 2 (3 x original flow) Increase the flow rate by another 100% Reduce original run time to 1/3 Reduce original Gradient time to 1/3 Example 1ml/min > 3ml/min 30 minutes > 10 minutes 35-65% over 30 mins > over 10 mins Is resolution OK? Pressure OK? Keep Going! Page 23

24 HETP (cm/plate) Speeding It Up-FASTER!! Increase Linear Velocity (Flow Rate) mm 3.5 mm 1.8 mm Step 3 (4 x original flow) Increase the flow rate by another 100% Reduce original run time to 1/4. Reduce original Gradient time to ¼ mL/min Example ml/min > 4ml/min 4.0 ml/min 30 minutes > 7.5 minutes Interstitial linear velocity (u e - cm/sec) 35-65% over 30 mins > over 7.5 mins Is resolution OK? Pressure OK? Keep Going! Page 24

25 Agilent 1200 Rapid Resolution System Speed Conventional LC UFLC RRLC F= 1.20ml/min T = 40 C Analysis Time = 11min Solvent Cons. = 13.2ml F = 4.80ml/min T = 40 C Analysis Time = 1.05min Solvent Cons. = 5.1ml R s = R s = 2.3 High Resolution: 4.6mm x 150mm 5.0µm min High Speed: 4.6mm x 50mm 5.0µm R s will be ~60% lower though min F= 1.00ml/min T = 40 C Analysis Time = 1.1min Solvent Cons. = 1.1ml High Speed & Resolution: 2.1mm x 50mm 1.8µm min F= 2.40ml/min T = 95 C Analysis Time: 0.4min Solvent Cons. = 1.0ml PW HH = 197msec > 20x faster! Max Speed at T = 95 o C 2.1mm x 50mm 1.8um min Page 25

26 8X Faster Analysis on RRHT Columns with Easy Method Transfer High Pressure not Required mau RRHT 4.6 x 50 mm 1.8 μm Flow Rate: 3 ml/min Pressure = 229 bar Column: ZORBAX Eclipse XDB-C18 Mobile Phase: 95% ACN: 5% Water Temp: Ambient Injection volume: 1 ul Conventional 4.6 x 150 mm 5 μm Flow Rate: 1 ml/min P = 37 bar min 13.5 min Sample: Vitamin E a, b, g-tocopherols in gel cap Eclipse XDB-C18 is a good first choice for many methods. min Page 26

27 Reduce Analysis Time by up to 95% using Rapid Resolution HT Columns R s (1,2) = N= x 250 mm, 5 mm N= R s (1,2) = x 100 mm, 3.5 mm N=11691 min N= R s (1,2) = 3.3 N= x 30 mm, 1.8 mm 1 ml/min min min N= N=6460 R s (1,2) = x 30 mm, 1.8 mm 2 ml/min min olumns: ZORBAX SB-C18 Mobile Phase: 50% 20 mm NaH 2 PO 4, ph 2.8: 50% ACN Flow Rate: 1 ml/min Temperature: RT Detection: UV 230 nm Sample: 1. Estradiol 2. Ethinyl estradiol 3. Dienestrol 4. Norethindrone Page 27

28 Viscosity Viscosity When to Stop When flow limit of pump is reached Consider using a smaller i.d. column When pressure is approaching maximum (e.g. 550 bar) Remember to allow for increase in pressure at viscosity maximum for mobile phase mixture When resolution is no longer satisfactory ACN/water 40C Methanol/water 40C %ACN %Methanol Page 28

29 Decreased Column Volume May Require Conversion for Injection Volume Keep Injection volume proportional to column volume Inj.Vol. col. 1 Volume Volume column2 column1 Inj.Vol. col. 2 Zorbax column volume = 3.14 x r 2 x L x 0.6 (r and L in cm) i.e. 20μl 0.4ml column2 col ml column1 μl col. 2 Page 29

30 Reduce injection volume Reduction to allow for diameter change 4.6 mm 3 mm = 0.4 x Original 4.6 mm 2.1 mm = 0.2 x Original x Reduction to allow for length change 150 mm 50 mm = 0.33 x Original 150 mm 100 mm = 0.67 x Original 100 mm 50 mm = 0.5 x Original e.g. Original 4.6mm x 150mm transferred to 2.1mm x 100mm = 0.2 x 0.67 = 0.13 x original injection volume Page 30

31 mau mau mau R = 16.5 N/m= Reduce Injection Volume as Column Volume is Reduced 4.6 x 150 mm, 5 um 5 ul inj. USP Requirements: L7 column, R s > 8 T f (5%) < 2.0 for each N=3000 or 20000/m R = 17.3 N/m=117,000 Rapid Resolution 4.6 x 100 mm, 3.5 umtailing factor for each of these six peaks is < ul inj R = 15.8 N/m= Rapid Resolution HT 4.6 x 50 mm, 1.8 um 1.7 ul inj. Mobile phase: ( 500:496:4) acetonitrile: water: H 3 PO 4 Flow = 2.0ml/min. isocratic Temp: ambient Detection: UV 272nm LC: Agilent 1100 Sample: resolution solution, fenoprofen (peak #1) with gemfibrozil prepared as described in USP High resolution and exceptional efficiency maintained for low cost updating to fast LC methods min min min Page 31

32 Page 32 GRADIENT ELUTION

33 Simplified Method Transfer for Increased Gradient Speed 1. Shorten the Column Length 2. Adjust Gradient Time by same Factor 3. Maintain Flow Rate 4. For More Speed Just Wait a Little Longer. Remember, Easy Gains First! Faster is Just a Few Slides Away! Page 33

34 What is Gradient Elution? Is It More Difficult to Increase Gradient Speed? Increasing the solvent strength = Increasing the % organic in the mobile phase Linear solvent strength gradient = % B per min is a constant %ACN 90 90% 70% 50% 30% min. } }} } t 1 = t 2 = t 3 = t 4 = 80% gradient change t G = 40 min. gradient time t G = 2%/min. gradient slope For every 20% change in ACN, t is 10 min. Page 34

35 0% B 0% B 100% B t g = 5 100% B t g = 10 Changing Gradient Time to Affect Retention (k*) and Resolution 100% B k* t g F S %B V m 1/k* = gradient steepness = b 0% B t g = % B F = change in volume fraction of B solvent S = constant F = flow rate (ml/min.) t g = gradient time (min.) V m = column void volume (ml) 0% B t g = Time (min) P1.PPT Group/Presentation Title Agilent Restricted Month ##, 200X

36 How conversion works for time Run Time or Gradient segment Time Adjustment* Time col. 1 Length Length column2 column1 Time col. 2 i.e. 150mm 25min. 250mm 15min. *assumes diameter is equal for columns 1 and 2 Page 36

37 Convert run time (and gradient) to shorter column with no flow increase Flow Rate Injection Volume Run time Gradient Times = As converted = As converted = Reduced by length change reduction factor = Reduced by length change reduction factor Reduction to allow for length change 150 mm 75 mm = 1/2 x Reduction e.g. 18mins 9mins 45-90% over 18mins 45-90% over 9mins Page 37

38 Short Columns Reduce Total Gradient Analysis Time A. 4.6 x 150 mm, 5 mm Eclipse XDB-C8 t G = 18 min 1 Gradient Separation of Cardiac Drugs B. 4.6 x 75 mm, 3.5 mm Eclipse XDB-C8 5 t G = 9 min 4.6 x 150 mm 4.6 x 75 mm Run Time 13 min 8 min Equilibration 15 min 7 min Time Total Analysis 28 min 15 min Time 3 48 samples/day 96 samples/day Time (min) Time (min) Page 38

39 Time to Have Fun More Gradient Speed Reduce Particle Size to Increase Efficiency and Peak Capacity Decrease Gradient Time and Increase Flow Rate by Same Factor If Flow Rate limit of instrument* is reached, reduce diameter of column and reduce flow rate to maintain linear velocity Repeat Flow Rate Increase to Flow or Pressure Limit If pressure becomes problematic, increase mobile phase/column temperature * Flow Rate Limit 5mL/min for Agilent Binary Pump, 10mL/min Agilent Quaternary Pump (P < 200 bar) Page 39

40 Faster Gradient Analysis of Cardiac Drugs What Changes Did We Make to Accomplish This? Optimized Column and Gradient 5 Run Time Equilibration 1 min Time Total Analysis 2.8 min Time 480 Samples/day min Column: Rapid Resolution Eclipse XDB- C8, 4.6 x 50 mm, 3.5 mm Mobile Phase: A: 25 mm Na 2 HPO 4, ph 3 B: MeOH Gradient: 42 90% B in 3 min Flow Rate: 3 ml/min Temperature:35 C Sample: Cardiac Drugs 1. Diltiazem 2. Dipyridamole 3. Nifedipine 4. Lidoflazine 5. Flunarizine Time (min) Page 40

41 Column Efficiency under Gradient Conditions Concept of Peak Capacity. Standard concept of column efficiency, N (plates), is only appropriate for isocratic conditions. A more useful concept for the case of gradient conditions is Peak Capacity - the number of peaks that can be separated (at a specified resolution) in a given amount of time. It is another measure of column efficiency. 4 P c = (1+ t G /w) for gradient conditions 5 mm 1.8 mm 5 2 peaks fit R s = +50% 3 peaks fit 50% more!! Page 41

42 Gradient Resolution and Shorter Columns - Faster Analyses for Complex Samples Higher Peak Capacity, P c = (1+ t G /w): 2.1x150mm, 5mm P/N min gradient 0.2 ml/min k* = 12.0 Detection: UV 214 nm Sample: HSA Tryptic Digest 120 peaks 60 min x50mm, 1.8mm P/N min gradient 0.5mL/min k* = 12.9 min 125 peaks 10 min.! x50mm, 1.8mm P/N min gradient 0.5mL/min k* = 38.6 min 156 peaks! 25 min Conditions: Mobile Phase A: Water w/ 0.1% TFA, B: ACN w/0.1% TFA, Gradient 2%B to 50%B, Temperature: 50C min Page 42

43 Peptide Map of Tryptic Digest of BSA run on Agilent RRHT Zorbax SB-C18, 2.1x50mm, 1.8µ Gradient time 10min Peak Capacity min 398 Longer Gradient Times Increase Peak Capacity 22.5 min 441 Page 43

44 Peak Capacity as a function of Gradient Time (t g ) and Column Length on 1.8 µm RRHT Columns Gradient Time 50 mm 100 mm 150 mm Tripling column length increases peak capacity by 55%. Increasing gradient time by 225% increase peak capacity by 25% Page 44

45 Flow Rate Limitations Due to Pressure? Higher Temperature Can Help! Page 45

46 Higher Temperature as an Aid to Faster Operation Higher Temperature: Temperature should always be considered as a parameter during speed optimization Provides more rapid mass transfer: Improves efficiency enhances resolution Decreases analysis time faster separations with no loss in resolution Decreases Mobile Phase Viscosity Lowers backpressure allows for higher flow rates, faster separations, greater efficiency and use of sub 2-micron columns Can change selectivity optimize resolution Be wary of on-column decomposition Faster flow rates shorten analyte residence time at elevated temperature and lead to less decomposition for labile compounds Page 46

47 Elevated Temperature Reduces Pressure Expands Column Choices Cardiac drugs Column: SB-C18, As described below Mobile Phase: A: 0.1% TFA, 5% MeCN, (v/v) B: 0.08% TFA, 95% MeCN (v/v) Sample: 0.1 mg/ml of cardiac drugs Temperature: 70 C Flow: 2 ml/min. gradient Detection: 230,16 nm R s = 4.3 Very high N column 4.6 x 250 mm, 3.5 µm P=221 bar R s = min R s = 3.6 R s = x 150 mm, 1.8 µm P=418 bar R s = R s = 3.4 % B mm mm mm x 50 mm, 1.8 µm P=164 bar min min Page 47

48 INCREASED RESOLUTION! When Do You Need It? How Do You Get It? Complex Samples Large No. of Peaks in Short Time Frame Closely Related Compounds Changes in Bonded Phase Have Not improved Resolution Changes in Mobile Phase Have Not Improved Resolution Temperature Has Not Helped Change Selectivity What Is Left That Will Improve Resolution? Page 48

49 mau Norm Reduce Particle Size and Maintain Column Length Increased N in Isocratic Separation DAD1 A, Sig=254,4 Ref =of f (051119A\SIG10003.D) DAD1 A, Sig=254,4 Ref=off (051004D\LC_J0002.D) 4.6 x 150, 5um 93 bar N = 7259 R s = 1.15 S/N = min min Column:150x4.6 mm 5µm Pressure: 93 bar N: 8213 Height: 1.25 mau S/N: 42.3 R s = 1.15 Height = 1.25 Noise = 24uAU t r = 14.9 min (1 st epimer) N ptp : mau 4.6 x 150, 3.5um 165 bar Column:150x4.6 mm 3.5µm N = Pressure: 165 bar N: R s = 1.37 S/N = 50 Height:1,34 mau S/N: 50.7 R s = 1.37 Height = 1.34 t r = 15.3 min (1 st epimer) Noise = 20uAU N ptp : mau N Norm N 1 d p DAD1 A, Sig=254,4 Ref=off (051007D\LC_X D) 10 1/dp 4.6 x 150, 1.8um 490 bar min Column:150 x 4.6 mm 1.8µm Pressure: 490 bar N: N = Height:1.78 R s = 1.80 (+57%) S/N: 43.6 S/N = 44 R s = 1.80 t r = 17.2 (1 st epimer) Height = 1.80 N ptp : Noise = 30uAU Page 49

50 1.8u Particles Reveal More Information and Improve Detection and Integration Customer Sample, Translation of Isocratic Impurity Methods, Zoom Critical Time Range (t = 7min) 4 Impurities 2 Not Baseline Separated! 7 Impurities 6 Not Baseline Separated! 7 Impurities All 7 Baseline Separated! 4.6 x 150, 5μm 93 bar N = 7259 R_S = 1.15 S/N = x 150, 3.5μm 165 bar N = R_S = 1.37 S/N = x 150, 1.8μm 490 bar N = R_S = 1.80 (+57%) S/N = 44 Up to 60% higher resolution without loss in sensitivity Page 50

51 u Particle Size Increases Gradient Resolution mau mau Agilent 1100 Agilent 1200 RRLC Increased Peak Capacity Zorbax 2.1x150mm SB C-18, 5µm min Zorbax 2.1x150mm SB C-18, 1.8µm Conditions for both experiments Pumps Solvent A: H 2 O + 0.1% TFA Solvent B: ACN + 0.1% TFA Gradient: 10% to 95% ACN in 40min, hold for 1min Flow Rate: 0.4ml/min Autosamplers Injection volume: 3µl Thermostatted Column Comp. Temperature: 50 C Detectors DAD 2µl cell and 20Hz, 220nm, Ref: Appl. Note by Edgar Naegele min Page 51

52 Particle Size - More Peak Capacity with 1.8 um RRHT Columns - Peptide Map of BSA mau Conditions: Columns: as listed, Mobile Phase: A:0.1% TFA in Water B:0.08% TFA in ACN Gradient: 5% B to 60%B in 25 min. Temperature: 80 C Sample: BSA tryptic digest Column used SB-C18, 2.1x150mm, 1.8µ Peak Capacity 673 Starting Pressure 380 bar 35% More peak capacity, more resolution mau SB-C18, 2.1x150mm, 3.5µ Peak Capacity 502 Starting Pressure 105 bar min Less peak capacity, less resolution Smaller particle size = sharper peaks = greater peak capacity min Page 52

53 Tools Rapid Resolution Compendium CD-ROM (Please Ask For A Copy Pub No EN) Application Examples Technology Overview Rapid Resolution in The Scientific and Trade Press and Conference Posters System Configurator (guide to reduce dead volume and extra column effects) Method Translator Program Page 53

54 Agilent HPLC Method Translator A Simple to Use Tool to Move Methods to RRHT Mini-Demo Method Translator Page 54

55 Summary Shorter 1.8um columns allow faster analysis with same resolving power as longer 5um and 3.5 um columns Change column diameter if necessary to allow for lower solvent consumption and higher accessible linear velocity Adjust the injection volume to maintain the same mass loading on the smaller column Adjust run time and gradient to allow for the shorter column to match k* for longer column or to increase k* vs. longer column. Speed up the flow rate until max flow or max pressure or loss of resolution Page 55

56 Summary Shorter 1.8um columns allow faster analysis with same resolving power as longer 5um and 3.5 um columns Change column diameter if necessary to allow for lower solvent consumption and higher accessible linear velocity Adjust the injection volume to maintain the same mass loading on the smaller column Adjust run time and gradient to allow for the shorter column to match k* for longer column or to increase k* vs. longer column. Speed up the flow rate until max flow or max pressure or loss of resolution Page 56

57 Page 57 Questions?