HPLC Column Troubleshooting Gulf Coast Conference 04 John Palmer Page
HPLC System Components Pump Injector/Autosampler Column Detector Data System/Integrator Problems Can Be Related to All Components in the System Page
There Can Be Many Causes Start Asking Questions That Lead to The Cause st Did System Suitability or the Sample Fail? nd Review Method for Compliance - Is The Procedure Being Followed Properly? - Are Instrument Settings Correct? rd Ask More Questions! - When Did the System Last Function Properly? - Has Anything Been Changed? 4 th Review ALL parameters! - The Obvious Is Not Always the Cause - Was There More Than One Change? Page
Categories of Column and System Problems A. Pressure B. Peak shape C. Retention Page 4
Pressure Issues Column Observations High pressure Low Pressure Potential Problems - Plugged frit - Column contamination - Plugged packing - Leak - Flow Incorrect Page 5
Determining the Cause and Correcting High Back Pressure Check pressure with/without column Remove Guard Remove Column If Column pressure is high: Back flush column Clear dirty frit surface Wash column high molecular weight/adsorbed compounds precipitate from sample or buffer Replace Column Change frit Clear plugged frit PREVENT THIS! Page 6
Peak Splitting Caused By Disrupted Sample Path Flow Path Disrupted by Void Sample Allowed to Follow Different Paths Through Column Poorly Packed Bed Settles in Use High ph Dissolves Silica Normal Split or Double Peaks Double Peaks Tip: Similar Effect Can be Caused by Partially Plugged Frit Page 7
Split Peaks from Injection Solvent Effects Column: StableBond SB-C8, 4.6 x 50 mm, 5 mm Mobile Phase: 8% H O : 8% ACN Injection Volume: 0 ml Sample:. Caffeine. Salicylamide A. Injection Solvent 00% Acetonitrile B. Injection Solvent Mobile Phase 0 0 0 0 Tip: Injecting in a solvent stronger than the mobile phase can cause peak shape problems such as peak splitting or broadening Trick: Keep Organic Concentration in Sample Solvent < Mobile Phase Group/Presentation Title September Agilent 8, 04Month Restricted ##, 00X Page 8
Split Peaks from Column Contamination Column: StableBond SB-C8, 4.6 x 50 mm, 5 mm Mobile Phase: 60% 5 mm Na HPO 4, ph.0 : 40% MeOH Flow Rate:.0 ml/min Temperature: 5 C Detection: UV 54 nm Sample: Filtered OTC Cold Medication:. Pseudoephedrine. APAP. Unknown 4. Chlorpheniramine Injection Injection 0 Injection After Column Wash with 00% ACN 4 4 4 0 5 0 5 0 5 0 5 0 5 0 5 Tip: Column washing eliminates the peak splitting, which resulted from a contaminant on the column How could this be prevented? (Guard Column, SPE clean up of samples, Periodic column wash) Page 9
Peak Tailing - Column Contamination Tip: Quick Test to Determine if Column is Dirty or Damaged Trick: Reverse Column and Run Sample If Improved, Possible Cleaning Will Help - No improvement - Column Damaged and Needs to be Replaced QC test forward direction Plates TF. 769.08. 04.64. 77.69 4 55. QC test reverse direction Plates TF. 7906.4. 44.. 7999.9 4 7098.5 QC test after cleaning 00% IPA, 5 C Plates TF. 7448.06. 7.. 566. 4 9067.7 4 4 4 0.0.5 5.0 0.0.5 5.0 0.0.5 5.0 Column: StableBond SB-C8, 4.6 x 50 mm, 5mm Mobile Phase: 0% H O : 80% MeOH Flow Rate:.0 ml/min Temperature: R.T. Detection: UV 54 nm Sample:. Uracil. Phenol. 4-Chloronitrobenzene 4. Toluene Page 0
Column Cleaning Flush with stronger solvents than your mobile phase. Reversed-Phase Solvent Choices in Order of Increasing Strength Use at least 5 ml of each solvent for long analytical columns This Is Time Consuming Often Performed Offline Mobile phase without buffer salts 00% Methanol 00% Acetonitrile 75% Acetonitrile:5% Isopropanol 00% Isopropanol 00% Methylene Chloride* 00% Hexane* Must Reverse to Re-Equilibrate Page
Changing a Frit May Not Be a Good Idea May not be possible with new generation columns May damage high performance columns Column Inlet Frit Column Body Compression Ferrule Wear gloves Do not allow bed to dry Do not touch the column - body heat will extrude packing Do not overtighten Female End Fitting Male End Fitting Tip: Prevention is a Much Better Idea! Page
The Trick: Prevention Techniques - A Better Choice! Filter samples Filter buffered mobile phases Use column protection - In-line filters - Guard columns Easy Sample clean-up (i.e. SPE) Appropriate column flushing Not As Easy Page
Inexpensive Filters Prevent Column Frit Plugging Regenerated Cellulose (RC) Recommended Universal hydrophilic membrane, compatible with most solvents - aqueous and organic High purity, extremely low extractables and binding More Uniform Surface Different than Other Cellulose Filters!! In-line Filters Easy to Use and replace Frits Available in 0.,0.5 and.0µ Porosity Much Less expensive than a Column Easier and Faster to Replace than a Column Frit Page 4
Mini-UniPrep Filtering Vials Pre-assembled filtration devices for removing particulate matter from samples. Single disposable unit can replace the combination of syringe filters, syringes, auto-sampler vials, transfer containers, septa and caps. Quick, environmentally conservative way to filter samples prior to HPLC analysis. Manufactured by Whatman, a division of GE Healthcare Page 5
Separation Conditions and the Effect on Retention* Flow Rate +/- % +/- % Tr Temp +/- deg C +/- to % Tr %Organic +/- % +/- 5 to 0% Tr ph +/- 0.0% +/- 0 to % Tr *excerpted from Troubleshooting HPLC Systems, J. W. Dolan and L. R. Snyder, p 44. 6
Mobile Phase: Aqueous Component Experimental Variables That Impact Resolution Column Mobile Phase Aqueous Component - Importance of Buffers - Considerations for Buffer Selection - Buffer ph - Buffer Concentration Organic Component Sample Instrument Gradient Separations Your opportunity to improve robustness and ruggedness September 8, 04 CI06C
Mobile Phase ph and ph Buffers Why Are These So Important in HPLC? ph Effects Ionization - Silica Surface of Column - Sample Components of Interest Buffers - Resist Changes in ph and Maintain Retention - Improve Peak Shape for Ionizable Compounds Effects Column Life - Low ph strips Bonded Phase - High ph Dissolves Silica Page 8
Why Worry About ph? ph, pka and Weak Bases R NH + R N + H + K a = [R N][H + ] [R NH + ] CHOCH CH N + H CH CH CHOCH CH N + CH CH H + K a = x 0-9 pk a = 9 At ph 9 the sample exists as protonated and unprotonated diphenhydramine in a ratio of :. Peak shape can be poor. At ph 0 9% of the sample exists as unprotonated diphenhydramine. At ph 8 9% of the sample exists as protonated diphenhydramine. Page 9
Why Worry About ph? ph, pka and Weak Acids RCOOH RCOO - + H + COOH COO _ K a = [RCOO - ][H + ] [RCOOH] + H + K a = 6.4 x 0-5 pk a = 4. At ph 4. the sample exists as benzoic acid and the benzoate ion in a ratio of :. Peak shape can be poor At ph 5. 9% of the sample exists as the benzoate ion. RP retention decreases. At ph. 9% of the sample exists as benzoic acid. RP retention increases. Page 0
log k«retention ph vs. Selectivity for Acids and Bases.5 6 Column: Nucleosil-C8 Mobile Phase: 45% ACN/55% phosphate buffer Sample: Bile Acids SCD 40 Column: mbondapak-c8 Mobile Phase: 60% 5 mm phosphate buffer 40% Methanol. Salicylic acid 5. Phenobarbital. Phenacetin 4. Nicotine 5. Methamphetamine.0 0.5 UDC 5 SOC 4 C -OC + + 7, - OC + + 0 0 J.C. (975) 49 4 0.0-0.5 J.C. 68(98) + + 4 5 6 7 8 ph A B C 0 0 5 7 9 ELUENT ph Retention and selectivity can change dramatically when ph is changed. Page
Why Use Buffered Mobile Phases? Column: ZORBAX Rapid Resolution Eclipse XDB-C8, 4.6 x 75 mm,.5 µm Mobile Phase: 44% A : 56% methanol Flow Rate:.0 ml/min Temperature: 5 C Detection: UV 50 nm Sample:. ketoprofen. ethyl paraben. hydrocortisone 4. fenoprofen 5. propyl paraben 6. propranolol 7. ibuprofen, 4, 6, 7 A = ph 7.0 water A = ph 7.0, 5 mm phosphate buffer 4 5 5 6 7 0 4 0 4 5 Buffered mobile phases enhance retention, resolution, and peak shape. September 8, 04 CI06C
Considerations For Buffer Selection Buffer Type Inorganic vs. organic buffers choice can affect resolution, column lifetime and MS compatibility Buffer ph Select buffer based on desired ph and optimum buffer ph range. Measure ph of buffer solution before mixing with organic modifier. Compare resolution at desired ph ± 0. 0. ph units. Buffer Concentration and Ionic Strength Start at 0 5 mm. Prepare buffer according to accepted procedures. Avoid overshoot and readjustment when setting ph. Compare resolution at desired buffer concentration ± 5 0 mm. September 8, 04 CI06C
Changes in Buffer Concentration Can Affect Retention, Peak Width and Peak Shape Column: ZORBAX Eclipse XDB-C8, 4.6 x 50 mm, 5 µm Mobile Phase: 40% phosphate buffer (ph 7.0) : 60% ACN Flow Rate:.5 ml/min Temperature: 40 C Sample: Tricyclic Antidepressants,. Desipramine. Nortriptyline. Doxepin 4. Imipramine 5. Amitriptyline 6. Trimipramine 0 mm Phosphate 5 mm Phosphate USP Tf (5%)..6..65..6 4..77 5..8 6.. 4 5 6 4 5 6 USP Tf (5%)..4..50.. 4..9 5..6 6..00 0.0.5 5.0 7.5 0.0.5 5.0 September 8, 04 CI06C
Effect of ph on Peak Shape at or Near the Sample pka Column: ZORBAX SB-C8 4.6 x 50 mm, 5 mm Flow Rate:.0 ml/min. Mobile Phase: 40% 5 mm KH PO 4 : 60% ACN Temperature: RT CH CHCOOH ph 4.4 ph.0 CH CH(CH ) Ibuprofen pk a = 4.4 0 4 5 6 7 8 9 0 0 4 5 6 7 8 9 0 Inconsistent and tailing peaks may occur when operating close to an analyte pka and should be avoided. Page 5
Lot-to-Lot Selectivity Change Related to ph Choice ph 4.5 - Lot ph.0 - Lot -Base 4-Base 4 0 4 6 8 0 4 6 8 0 4 6 8 0 4 6 8 ph 4.5 - Lot ph.0 - Lot -Base 4-Base 4 0 4 6 8 0 4 6 8 0 4 6 8 0 4 6 8 ph 4.5 shows selectivity change from lot-to-lot for basic compounds ph.0 shows no selectivity change from lot-to-lot Indication of poorly controlled ionization Group/Presentation Title September Agilent 8, 04Month Restricted ##, 00X Page 6
Test for ph Robustness Column: ZORBAX Rapid Resolution Eclipse XDB-C8, 4.6 x 75 mm,.5 µm Mobile Phase: 44% 5 mm phosphate, ph 7.00 : 56% methanol Flow Rate:.0 ml/min Temperature: 5 C Detection: UV 50 nm Sample:. ketoprofen. ethyl paraben. Hydrocortisone 4. fenoprofen 5. propyl paraben 6. Propranolol 7. ibuprofen ph 7.00 4 5 7 6 0 4 5 6 ph 7.5 4 5 6 7 0 4 5 6 The resolution of ionizable compounds can change markedly with ph changes even as small as 0.05 0.5 ph units. September 8, 04 CI06C
Don t Forget - Match Column to ph of Mobile Phase for Maximum Column Lifetime low ph and high temperature (ph 0.8, 90 C) Purge Solvent: 50% methanol/water with.0% TFA Solute: Toluene Kirkland, J.J. and J.W. Henderson, Journal of Chromatographic Science, (994) 47-480. 0000P.PPT Page 8
Don t Forget - Match Column to ph of Mobile Phase for Maximum Column Lifetime High ph and Room Temperature (ph RT) Mobile Phase: 50%ACN: 50% Water : 0.% TEA (~ ph ) Initial After 0 injections Tip: Use Columns Designed for chosen ph Page 9
Mobile Phase: Organic Component Column Mobile Phase Aqueous Component Organic Component Sample Instrument - % Organic Modifier Gradient Separations September 8, 04 CI06C
Even a Small Change in % Organic Modifier Can Change Resolution 59% MeOH 0 5 57.5% MeOH 0 4 56% MeOH 0 4 4 4 5 4 6 6 5 R s (5,6) =.5 R s (5,6) =.7 R s (5,6) =. 6 4 Column: XDB-C8 ZORBAX Rapid Resolution Eclipse 4.6 x 75 mm,.5 µm Mobile Phase: A: 5 mm phosphate, ph 7.00 (0 mm TEA) B: methanol (0 mm TEA) Flow Rate:.0 ml/min Temperature: 5 C controlled Injection: Detection: 5 ml 75 nm Sample:. ketoprofen. ethyl paraben. hydrocortisone 4. fenoprofen 5. propyl paraben 6. propranolol Verify that resolution doesn t change significantly around desired conditions (for example, %B ± %). September 8, 04 CI06C
The Sample Experimental Variables That Impact Resolution Column Mobile Phase Sample Injection volume Sample solvent strength Instrument Gradient Separations September 8, 04 CI06C
Injection Volume and Sample Solvent Strength Injection Volume Lack of ruggedness typically seen when V inj is increased to improve signal-to-noise (S/N) ratio, or, when column size is decreased. Use minimum V inj for required repeatability and limit of detection. Compare resolution, peak shape and repeatability at 0.X, X and 5X V inj. Sample Solvent Strength Match % organic modifier in mobile phase (or weaker). If stronger sample solvent needed (solubility, stability), keep V inj to minimum. Compare resolution, peak shape and width at desired solvent strength ±50% relative. September 8, 04 CI06C
Test For Injection Volume Robustness 4 5 0 µl 6 0 4 5 5 µl 5 4 0 4 5 µl 4 5 6 7 0 4 5 6 R s (6,7) =.6 7 R s (6,7) =. 7 R s (6,7) =.5 Column: ZORBAX Rapid Resolution Eclipse XDB-C8 4.6 x 75 mm,.5 µm Mobile Phase: 44% 5 mm phosphate, ph 7.00 Flow Rate: 56% methanol.0 ml/min Temperature: 5 C Detection: Sample: UV 50 nm. ketoprofen. ethyl paraben. hydrocortisone 4. fenoprofen 5. propyl paraben 6. propranolol 7. ibuprofen Varying injection volume can sometimes reveal lack of robustness for resolution and peak shape. September 8, 04 CI06C
Peak Tailing, Broadening and Loss of Efficiency May be caused by: Column secondary interactions Column contamination Column aging Column loading Extra-column effects Page 5
Peak Shape: Tailing Peaks Symmetry >. Causes Some Peaks Tail: Secondary - Retention Effects. Residual Silanol Interactions. Small Peak Eluting on Tail of Larger Peak. Normal Tailing All Peaks Tail: Extra-Column Effects. Build up of Contamination on Column Inlet. Bad Column. Normal Tailing Page 6
Peak Tailing Identifying Column Secondary Interactions Column: Alkyl-C8, 4.6 x 50 mm, 5mm Mobile Phase: 85% 5 mm Na HPO 4 ph 7.0 : 5% ACN Flow Rate:.0 ml/min Temperature: 5 C Sample:. Phenylpropanolamine. Ephedrine. Amphetamine 4. Methamphetamine 5. Phenteramine No TEA USP TF (5%)..9..9..6 4..5 5..57 4 5 4 5 0 mm TEA USP TF (5%)..9..8..0 4..6 5..4 0.0.5 5.0 TIme (min) 0.0.5 5.0 Tip: Mobile phase modifier (TEA) competes with Sample for surface ion exchange sites at mid-range ph values Page 7
Peak Tailing Low ph Minimizes Secondary Interactions for Amines Column: Alkyl-C8, 4.6 x 50 mm, 5mm Mobile Phase: 85% 5 mm Na HPO 4 : 5% ACN Flow Rate:.0 ml/min Temperature: 5 C Sample:. Phenylpropanolamine. Ephedrine. Amphetamine 4. Methamphetamine 5. Phenteramine ph.0 USP TF (5%) 4.. 4 5 4 5 ph 7.0 USP TF (5%) 4..5 0.0.5 5.0 0.0.5 5.0 Tip: Reducing mobile phase ph reduces interactions with silanols and peak tailing. Page 8
Peak Tailing High ph Eliminates Secondary Interactions for Amines Column: ZORBAX Extend-C8, 4.6 x 50 mm, 5 m m Mobile Phase: See Below Flow Rate:.0 ml/min Temperature: RT Detection: UV 54 nm Sample:. Maleate. Scopolamine. Pseudoephedrine 4. Doxylamine 5. Chlorpheniramine 6. Triprolidine 7. Diphenhydramine ph 7 0% 0 mm Na HPO 4 70% MeOH ph 0% 0 mm TEA 70% MeOH, 4 7 4 7 t 5 R = 8.5 t R =.4 5 6 6 0 5 0 5 0 Peak Shape and Retention of this sample of basic compounds improves at high ph where column has high IEX activity. Why? Page 9
Peak Shape: Broad Peaks All Peaks Broadened: Loss of Column Efficiency. Column Void. Large Injection Volume. Some Peaks Broadened: Late Elution from Previous Sample (Ghost Peak). - High Molecular Weight. - Sample - Protein or Polymer. 40 Page 40
Unknown Phantom Peaks Column: Extend-C8, 4.6 x 50 mm, 5 mm Mobile Phase: 40% 0 mm TEA, ph : 60% MeOH Flow Rate:.0 ml/min Temperature: R.T. Detection: UV 54 Sample:. Maleate. Pseudoephedrine. Chlorpheniramine Sample : Chlorpheniramine maleate Peak : maleate Sample : Chlorpheniramine maleate and Pseudoephedrine Peak : maleate Peak : pseudoephedrine Peak : chlorpheniramine (from st injection) Plates. 59. 9879. 779 Phantom peak from first injection 0 5 0 0 5 0 5 Tip: The extremely low plates for moderately retained peaks are an indication of a very late eluting peak from a preceding run. Page 4
Changes in Retention Can Be Chemical or Physical May be caused by: Column aging Column contamination Insufficient equilibration Poor column/mobile phase combination Change in mobile phase Change in flow rate Different Gradient Delay Volumes Page 4
Column - Initial Column Aging/Equilibration Causes Retention/Selectivity Changes Column - Next Day Column - After Cleaning with % H PO 4 /Equilibration 0 5 9 5 0 5 9 5 The primary analyte was sensitive to mobile phase aging/ conditioning of the column The peak shape was a secondary issue (metal chelating compound) resolved by de-activating the active metal contamination Group/Presentation Title September Agilent 8, 04Month Restricted ##, 00X Page 4
Column Temperature Adequate Temperature Control is Essential Laboratory temperatures can vary by ±5 C or more. Column temperature changes affect resolution and repeatability. Useful tool for changing selectivity, retention and efficiency when developing separations. Important parameter to control during method development and validation. Compare resolution, peak width and peak shape at desired temperature ±5 C. September 8, 04 CI06C
Small Temperature Changes Can Cause Dramatic Changes in Resolution 5 4 6 0 0.5.5.5.5 0 0.5.5.5.5 5 5 4 6 0 0.5.5.5.5 4 6 0 C 5 C 0 C Column: ZORBAX Rapid Resolution Eclipse XDB-C8 4.6 x 75 mm,.5 µm Mobile Phase: Isocratic, 8%B : 7%A Flow Rate:.0 ml/min. A: 5/95 methanol/ph 7.00 buffer 5 mm, 0 mm TEA B: 80/0 methanol/ph 7.00 buffer 5 mm, 0 mm TEA Temperature: See Figure Injection: 5 ml Detection: 75 nm Sample:. ketoprofen. ethyl paraben. hydrocortisone 4. fenoprofen 5. propyl paraben 6. propranolol Column temperature control will produce the most consistent results. September 8, 04 CI06C
CASE STUDIES Page 46
Example: Change in Retention/Selectivity Unintended Mobile Phase Variation Tip: The Source of the Problem is Often Not the Obvious Change Column Column Column - Fresh mobile phase 0 4 6 Time(min) 0 4 5 6 7 0 4 6 Time(min) I have experimented with our mobile phase, opening new bottles of all mobile phase components. When I use all fresh ingredients, the problem ceases to exist, and I have narrowed the problem to either a bad bottle of TEA or phosphoric acid. Our problem has been solved. Group/Presentation Title September Agilent 8, 04Month Restricted ##, 00X Page 47
Case History: Problem Selectivity Does Not Appear the Same from Column-to-Column Details: Columns with the Same Bonded Phase were used They were the same dimensions, just different particle sizes (and therefore different lots of material) They were tested on the same day on the same instrument and with the same mobile phase Isocratic elution; binary (two channel) pump generated mobile phase Problem: The selectivity was different on each of the columns 48
0.99.89 0.784 4.897.07.900 0.07 0.69.90 0.00 0.75 0.996.7 4.98 0.9 0.60.9.907 0.7 0.96.65 Selectivity between particle sizes of Eclipse Plus C8, 4.6 x 50 mm, using same test solutes mau 5 0 5 0 5 0-5 mau 5 0 5 0 5 0-5 mau 5 0 5 0 5 0-5 DAD B, Sig=80,6 Ref=60,0 (F:\EPC8SELECTIVITY\ANALINE00005.D) 4 5 0 4 5 DAD B, Sig=80,6 Ref=60,0 (F:\EPC8SELECTIVITY\P5MICRON0000.D) 0 4 5 DAD B, Sig=80,6 Ref=60,0 (F:\EPC8SELECTIVITY\ANALINE00009.D) 0 4 5 6 N=800 N=700 m.p.: A: water, B: acetonitrile (60:40 A:B) Flow :.5 ml/min Temp: 5 C Detection: UV 80nm,6, ref=60,0 Flow cell: 6mm, 5uL Data rate: 0.s Inj. Vol ul N=9900 min min min 5 um, UXE00, NEP06500 tr k alpha uracil 0.9 0.00 aniline 0.7.45 #DIV/0! toluidine 0.96..60 chloroaniline..4.40 naphthylamine.6 4.6.4 dichlorobenzidine.9.48.70.5 um, UXF009, B08 tr k alpha uracil 0. 0.00 aniline 0.7.4 #DIV/0! toluidine..6 chloroaniline.9.0.4 naphthylamine.7 4.7.4 dichlorobenzidine 4..00.75.8 um, UXG088, B080 tr k alpha uracil 0. 0.00 aniline 0.78.60 #DIV/0! o-toluidine.07.57.60 4-chloroaniline.9.6.4 -naphthylamine.9 5..47,'-dichlorobenzidine 4.9 5..88 John W. Henderson JrGroup/Presentation 49 Title Agilent Month Restricted ##, 00X
What Questions Should We Ask and What Should We Try to diagnose problem? Compare more columns see if they are different Try another LC Premix the mobile phase 50
0.848 0.47 0.48 0.5 0.87.4 0.90 4.07 4.07 0.7 0.74 0.97 0.970.668.666 0.49 0.5 0.5.4. 0.88 0.88 0.696 0.695.87.879 0.946 0.944.65.6 One channel premixed mobile phase shows similar α whereas previous two channels doesn t show similar α! mau 0 5 0 5 DAD B, Sig=80,6 Ref=60,0 (E:\ECLIPSE SELECTIVIY INVESTIGATION\ONECHANNEL0000.D) DAD B, Sig=80,6 Ref=60,0 (E:\ECLIPSE SELECTIVIY INVESTIGATION\ONECHANNEL0000.D) 5 um, UXE00, NEP06500 46 bar one channel tr k alpha uracil 0.9 0.00 aniline 0.69.8 #DIV/0! o-toluidine 0.94.4.6 4-chloroaniline..7.4 -naphthylamine.6 4.55.4,'-dichlorobenzidine.87.4.7 0-5 mau 0 5 0 5 0 5 0 4 DAD B, Sig=80,6 Ref=60,0 (E:\ECLIPSE SELECTIVIY INVESTIGATION\ONECHANNEL00005.D) DAD B, Sig=80,6 Ref=60,0 (E:\ECLIPSE SELECTIVIY INVESTIGATION\ONECHANNEL00006.D).8 um, UXG088, B080 46 bar one channel tr k alpha uracil 0.9 0.00 aniline 0.7.45 #DIV/0! o-toluidine 0.97.4.6 4-chloroaniline.4.8.40 -naphthylamine.67 4.76.45,'-dichlorobenzidine 4.07.0.74 0-5 m.p.: A: water, acetonitrile (60:40 v/v) Flow :.5 ml/min Temp: 5 C Detection: UV 80nm,6, ref=60,0 Flow cell: 6mm, 5uL Data rate: 0.s Inj. Vol ul 0 4 5 6 min John W. Henderson JrGroup/Presentation 5 Title Agilent Month Restricted ##, 00X
0.87 0.87 0.68 0.67.40.400 0.00 4.99 4.985 0.78 0.78.074.075.98.96 Repeat experiment of two channel isocratic mobile phase confirms different selectivity probably due to wrong composition of mobile phase being delivered mau 0 5 0 5 DAD B, Sig=80,6 Ref=60,0 (E:\ECLIPSE SELECTIVIY INVESTIGATION\TWOCHANNEL00005.D) DAD B, Sig=80,6 Ref=60,0 (E:\ECLIPSE SELECTIVIY INVESTIGATION\TWOCHANNEL00006.D) 5 um, UXE00, NEP06500 45 bar two channel (nd exp) tr k alpha uracil 0.9 0.00 aniline 0.7.48 #DIV/0! o-toluidine 0.98.8.60 4-chloroaniline.5..9 -naphthylamine.67 4.76.44,'-dichlorobenzidine 4.0.90.7 0-5 mau 5 0 5 0 5 0-5 0 4 5 6 DAD B, Sig=80,6 Ref=60,0 (E:\ECLIPSE SELECTIVIY INVESTIGATION\TWOCHANNEL0000.D) DAD B, Sig=80,6 Ref=60,0 (E:\ECLIPSE SELECTIVIY INVESTIGATION\TWOCHANNEL0000.D).8 um, UXG088, B080 8bar m.p.: A: water, B: acetonitrile (60:40 A:B) Flow :.5 ml/min Temp: 5 C Detection: UV 80nm,6, ref=60,0 Flow cell: 6mm, 5uL Data rate: 0.s Inj. Vol ul 0 4 5 6 min two channel (nd exp) tr k alpha uracil 0. 0.00 aniline 0.78.60 #DIV/0! o-toluidine.07.57.60 4-chloroaniline.4.67.4 -naphthylamine.9 5.40.47,'-dichlorobenzidine 4.98 5.60.89 min John W. Henderson JrGroup/Presentation 5 Title Agilent Month Restricted ##, 00X
Typical Case History: Lack of Efficiency on Poroshell 00 Agilent Publication Customer Results 5
Looked Bad What Did We Do?. Customer needed to understand that the column used. x 75mm, Poroshell 00 column---is a low volume column, and therefore, low column volume guidelines should be followed. What about the instrument and sample?. Turns out that instrument was quaternary pump 00, not set up appropriately for low volume use (difficult to optimize completely) 4. Customer provided with details on how to achieve a better low volume set-up, based on 00-RRHT Kit documents 5. Customer now achieving acceptable results 54
Conclusions HPLC column problems are evident as High pressure (prevention better than the cure) Undesirable peak shape Changes in retention/selectivity Often these problems are not associated with the column and may be caused by instrument and chemistry issues. ph of mobile Phase Instrument Connections Detector Settings Metal Contamination Start With the Correct Questions Find the Answers The Answers will Lead to Solutions Page 55
APPENDIX Page 56
Important Buffer Systems Buffer Selection Buffer pk a ph Range UV Cutoff (A > 0.5) Trifluoroacetic acid << (0.5).5-.5 0 nm (0.%) KH PO 4 /phosphoric acid..-. <00 nm (0.%) tri-k-citrate/hydrochloric acid.06.-4. 0 nm (0 mm) Potassium formate/formic acid.8.8-4.8 0 nm (0 mm) tri-k-citrate /hydrochloric acid 4.7.7-5.7 0 nm (0 mm) Potassium acetate/acetic acid 4.8.8-5.8 0 nm (0 mm) tri-k-citrate /hydrochloric acid 5.4 4.4-6.4 0 nm (0 mm) Ammonium formate.8.8-4.8 9. 8.-0. (50 mm) Bis-tris propane HCl/Bis-tris propane 6.8 5.8-7.8 5 nm (0 mm) Ammonium acetate 4.8.8-5.8 9. 8.-0. (50 mm) KH PO 4 /K HPO 4 7. 6.-8. <00 nm (0.%) Tris HCl/Tris 8. 7.-9. 05 nm (0 mm) Bis-tris propane HCl/Bis-tris propane 9.0 8.0-0.0 5 nm (0 mm) Ammonium hydroxide/ammonia 9. 8.-0. 00 nm (0 mm) Borate (H BO /Na B 4 O 7 0 H O) 9.4 8.-0. Glycine HCl/glycine 9.8 8.8-0.8 -methylpiperidine HCl/-methylpiperidine 0. 9.-. 5 nm (0 mm) Diethylamine HCl/diethylamine 0.5 9.5-.5 Triethylamine HCl/triethylamine.0 0.0-.0 <00 nm (0 mm) Pyrollidine HCl/pyrollidine. 0.-. Adapted from Practical HPLC Method Development, nd Edition, Snyder, L.R., Kirkland, J.J. and Glajch, J.L., page 99. September 8, 04 CI06C
Separation Ruggedness Buffer Preparation. Dissolve salt in organic-free water in - or -L beaker. Use appropriate volume to leave room for ph adjustment solution. Equilibrate solution to room temperature for maximum accuracy.. Calibrate ph meter. Use -level calibration and bracket desired ph. Use appropriate audit solution to monitor statistical control (for example, potassium hydrogen tartrate, saturated solution, ph =.56).. Adjust salt solution to desired ph. Minimize amount of time electrode spends in buffer solution (contamination). Avoid overshoot and readjustment (ionic strength differences can arise). 4. Transfer ph-adjusted buffer solution quantitatively to volumetric flask, dilute to volume, and mix. 5. Filter through 0.45 µm filter. Discard first 50 00 ml filtrate. Rinse solvent reservoir with small volume of filtrate and discard. Fill reservoir with remaining filtrate or prepare premix with organic modifier. Agilent Solvent Filtration Kit, 50-mL reservoir, 000-mL flask, p/n 50-0577 Nylon filter membranes, 47 mm, 0.45 µm pore size, p/n 90-0895 (not for proteins!) September 8, 04 CI06C
Using Buffers Successfully Initial Column and System Equilibration In an appropriate vessel, test highest % organic/buffer ratio to verify that buffer will not precipitate. With stirring, add organic to buffer first, not vice versa. Equilibrate column with, in order: 00% organic modifier (if brand new) mobile phase minus buffer buffered mobile phase containing highest % organic modifier (gradient high end) buffered mobile phase containing lowest % organic modifier (gradient low end). Inject standard or sample several times until RTs stable, or for gradient methods, precede former with or blank gradients. September 8, 04 CI06C
Using Buffers Successfully Shutdown State and Instrument Flushing Next day use using same buffers Pump mobile phase very slowly (for example, 0.0 0.mL/min). When flushing column or for longer term column storage Flush with 0/80 organic/water, then 80/0 organic/water or 00% organic. Instrument flushing Replace column with capillary tubing. Leave disconnected from detector. Flush pumps with water, then connect capillary tubing to detector. Inject water - times at maximum injection volume setting. Flush all pumps with 00% organic for long term storage. September 8, 04 CI06C
Importance of ph and Buffers A Practical Example Why the Sample Dictates Use What Happens When Buffer Used Effectively What Happens When Buffer Ignored or Used Improperly Page 6
Importance of ph and Buffers - A Practical Example Optimized Isocratic Conditions for Cardiac Drugs 4 Column: StableBond SB-C8, 4.6 x 50 mm, 5 mm Mobile Phase: 45% 5 mm NaH PO 4, ph.0 55% MeOH Flow Rate:.0 ml/min. Temperature: 5 C Detection: UV 54 nm Sample: Cardiac Drugs. Diltiazem. Dipyridamole. Nifedipine 4. Lidoflazine 5 5. Flunarizine 4 5 0 4 5 6 7 8 9 0 4 0 4 5 6 Page 6
I Don t Have Time to Make Buffers or Adjust ph Column: StableBond SB-C8 4.6 x 50 mm, 5 mm Mobile Phase: A: 0% H O B: 80% MeOH Flow Rate:.0 ml/min. Temperature: 5 C UV Detection: 54 nm Sample: Cardiac Drugs Even at very high % MeOH Most Components Strongly Retained with Poor peak Shape Due to IEX at Surface 0 5 0 5 0 5 Buffers are critical to good retention and peak shape in many separations. Page 6
What If You Work Outside the Buffer Range? Unsuitable Peak Shape Columns: StableBond SB-C8 4.6 x 50 mm, 5 mm Mobile Phase:A: 0% 5 mm NaH PO 4, ph 4.8 unbuffered B: 70% MeOH Flow Rate:.0 ml/min. Temperature: 5 C UV Detection: 54 nm Sample: Cardiac Drugs. Diltiazem. Dipyridamole. Nifedipine 4. Lidoflazine 5. Flunarizine 5 4 0 5 0 5 0 5 Page 64