HPLC Column Troubleshooting
|
|
- Allen Stewart
- 5 years ago
- Views:
Transcription
1 HPLC Column Troubleshooting Gulf Coast Conference 04 John Palmer Page
2 HPLC System Components Pump Injector/Autosampler Column Detector Data System/Integrator Problems Can Be Related to All Components in the System Page
3 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
4 Categories of Column and System Problems A. Pressure B. Peak shape C. Retention Page 4
5 Pressure Issues Column Observations High pressure Low Pressure Potential Problems - Plugged frit - Column contamination - Plugged packing - Leak - Flow Incorrect Page 5
6 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
7 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
8 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 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
9 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 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
10 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 QC test reverse direction Plates TF QC test after cleaning 00% IPA, 5 C Plates TF 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
11 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
12 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
13 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
14 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
15 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
16 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
17 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
18 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
19 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
20 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
21 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 UDC 5 SOC 4 C -OC + + 7, - OC J.C. (975) J.C. 68(98) ph A B C ELUENT ph Retention and selectivity can change dramatically when ph is changed. Page
22 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 Buffered mobile phases enhance retention, resolution, and peak shape. September 8, 04 CI06C
23 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 ± 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
24 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%) USP Tf (5%) September 8, 04 CI06C
25 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 = Inconsistent and tailing peaks may occur when operating close to an analyte pka and should be avoided. Page 5
26 Lot-to-Lot Selectivity Change Related to ph Choice ph Lot ph.0 - Lot -Base 4-Base ph Lot ph.0 - Lot -Base 4-Base 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
27 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 ph The resolution of ionizable compounds can change markedly with ph changes even as small as ph units. September 8, 04 CI06C
28 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) P.PPT Page 8
29 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
30 Mobile Phase: Organic Component Column Mobile Phase Aqueous Component Organic Component Sample Instrument - % Organic Modifier Gradient Separations September 8, 04 CI06C
31 Even a Small Change in % Organic Modifier Can Change Resolution 59% MeOH % MeOH % MeOH 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
32 The Sample Experimental Variables That Impact Resolution Column Mobile Phase Sample Injection volume Sample solvent strength Instrument Gradient Separations September 8, 04 CI06C
33 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
34 Test For Injection Volume Robustness µl µl µl 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
35 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
36 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
37 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%) mm TEA USP TF (5%) TIme (min) Tip: Mobile phase modifier (TEA) competes with Sample for surface ion exchange sites at mid-range ph values Page 7
38 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%) ph 7.0 USP TF (5%) Tip: Reducing mobile phase ph reduces interactions with silanols and peak tailing. Page 8
39 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, t 5 R = 8.5 t R = Peak Shape and Retention of this sample of basic compounds improves at high ph where column has high IEX activity. Why? Page 9
40 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
41 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 Phantom peak from first injection Tip: The extremely low plates for moderately retained peaks are an indication of a very late eluting peak from a preceding run. Page 4
42 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
43 Column - Initial Column Aging/Equilibration Causes Retention/Selectivity Changes Column - Next Day Column - After Cleaning with % H PO 4 /Equilibration 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
44 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
45 Small Temperature Changes Can Cause Dramatic Changes in Resolution 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
46 CASE STUDIES Page 46
47 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 Time(min) 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
48 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
49 Selectivity between particle sizes of Eclipse Plus C8, 4.6 x 50 mm, using same test solutes mau mau mau DAD B, Sig=80,6 Ref=60,0 (F:\EPC8SELECTIVITY\ANALINE00005.D) DAD B, Sig=80,6 Ref=60,0 (F:\EPC8SELECTIVITY\P5MICRON0000.D) DAD B, Sig=80,6 Ref=60,0 (F:\EPC8SELECTIVITY\ANALINE00009.D) 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 aniline #DIV/0! toluidine chloroaniline naphthylamine dichlorobenzidine um, UXF009, B08 tr k alpha uracil aniline #DIV/0! toluidine..6 chloroaniline naphthylamine dichlorobenzidine um, UXG088, B080 tr k alpha uracil aniline #DIV/0! o-toluidine chloroaniline naphthylamine ,'-dichlorobenzidine John W. Henderson JrGroup/Presentation 49 Title Agilent Month Restricted ##, 00X
50 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
51 One channel premixed mobile phase shows similar α whereas previous two channels doesn t show similar α! mau 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, NEP bar one channel tr k alpha uracil aniline #DIV/0! o-toluidine chloroaniline naphthylamine ,'-dichlorobenzidine mau 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, B bar one channel tr k alpha uracil aniline #DIV/0! o-toluidine chloroaniline naphthylamine ,'-dichlorobenzidine 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 min John W. Henderson JrGroup/Presentation 5 Title Agilent Month Restricted ##, 00X
52 Repeat experiment of two channel isocratic mobile phase confirms different selectivity probably due to wrong composition of mobile phase being delivered mau 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, NEP bar two channel (nd exp) tr k alpha uracil aniline #DIV/0! o-toluidine chloroaniline naphthylamine ,'-dichlorobenzidine mau 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 min two channel (nd exp) tr k alpha uracil aniline #DIV/0! o-toluidine chloroaniline naphthylamine ,'-dichlorobenzidine min John W. Henderson JrGroup/Presentation 5 Title Agilent Month Restricted ##, 00X
53 Typical Case History: Lack of Efficiency on Poroshell 00 Agilent Publication Customer Results 5
54 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
55 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
56 APPENDIX Page 56
57 Important Buffer Systems Buffer Selection Buffer pk a ph Range UV Cutoff (A > 0.5) Trifluoroacetic acid << (0.5) nm (0.%) KH PO 4 /phosphoric acid..-. <00 nm (0.%) tri-k-citrate/hydrochloric acid nm (0 mm) Potassium formate/formic acid nm (0 mm) tri-k-citrate /hydrochloric acid nm (0 mm) Potassium acetate/acetic acid nm (0 mm) tri-k-citrate /hydrochloric acid nm (0 mm) Ammonium formate (50 mm) Bis-tris propane HCl/Bis-tris propane nm (0 mm) Ammonium acetate (50 mm) KH PO 4 /K HPO <00 nm (0.%) Tris HCl/Tris nm (0 mm) Bis-tris propane HCl/Bis-tris propane nm (0 mm) Ammonium hydroxide/ammonia nm (0 mm) Borate (H BO /Na B 4 O 7 0 H O) Glycine HCl/glycine methylpiperidine HCl/-methylpiperidine nm (0 mm) Diethylamine HCl/diethylamine Triethylamine HCl/triethylamine <00 nm (0 mm) Pyrollidine HCl/pyrollidine Adapted from Practical HPLC Method Development, nd Edition, Snyder, L.R., Kirkland, J.J. and Glajch, J.L., page 99. September 8, 04 CI06C
58 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 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 Nylon filter membranes, 47 mm, 0.45 µm pore size, p/n (not for proteins!) September 8, 04 CI06C
59 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
60 Using Buffers Successfully Shutdown State and Instrument Flushing Next day use using same buffers Pump mobile phase very slowly (for example, 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
61 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
62 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 Page 6
63 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 Buffers are critical to good retention and peak shape in many separations. Page 6
64 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 Page 64
Barry E. Boyes, Ph.D. Consumables and Accessories Business Unit May 10, 2000
Barry E. Boyes, Ph.D. Consumables and Accessories Business Unit May 0, 000 HPLC Column Troubleshooting What Every HPLC User Should Know :00 a.m. EST Telephone Number: 86-650-06 Chair Person: Tim Spaeder
More informationTips and Tricks of HPLC System Troubleshooting
Tips and Tricks of HPLC System Troubleshooting Agilent Technologies, Inc. LC Tips And Tricks Seminar Series Page 1 Trouble Shooting Steps You Have Recognized There is a Problem! How Do You Fix It? 1 st
More informationAdvanced Topic: Tips and Tricks of HPLC Separations and Troubleshooting. Rita Steed LC Advanced Topics Seminar Series August 25, 2010.
Advanced Topic: Tips and Tricks of HPLC Separations and Troubleshooting Rita Steed LC Advanced Topics Seminar Series August 25, 200 Page Troubleshooting Steps You Have Recognized There is a Problem! How
More informationEliminating Unwanted Variability in LC Methods: More Uptime, More Sample Throughput
Eliminating Unwanted Variability in LC Methods: More Uptime, More Sample Throughput HPLC Separation Robustness and Ruggedness Group/Presentation Title June 20, Agilent 2017Month Restricted ##, 200X Page
More informationTips and Tricks of Faster LC Analysis Without Capital Investment It may be easier than you think!
Tips and Tricks of Faster LC Analysis Without Capital Investment It may be easier than you think! Agilent Technologies Page Tips and Tricks of HPLC System Troubleshooting Agilent Technologies Page Trouble
More informationHPLC Column Troubleshooting: Is It Really The Column? LC Columns and Consumables. Robyn C. Woolley Online Application Engineer July 24, 2007.
HPLC Column Troubleshooting: Is It Really The Column? LC Columns and Consumables Robyn C. Woolley Online Application Engineer July 24, 2007 Page 1 Troubleshooting in HPLC ma U 20 00 15 00 10 005 0 0 0
More informationChoosing Columns and Conditions for the Best Peak Shape
Choosing Columns and Conditions for the Best Peak Shape What is Good Peak Shape and Why is it Important? Good peak shape can be defined as a symmetrical or gaussian peak and poor peak shape can include
More informationHPLC Separation Robustness and Ruggedness Assessing the Effects of Experimental Variables During Method Development
HPLC Separation Robustness and Ruggedness Assessing the Effects of Experimental Variables During Method Development Rita Steed Application Engineer Agilent Technologies, Inc. November 19, 2015 Method Development
More informationWilliam E. Barber, Ph.D. Applications Chemist October
William E. Barber, Ph.D. Applications Chemist ctober 0 00 Secrets of Good Peak Shape in HPLC Time: :00 p.m. CET Telephone Number: + 44 0 76 088 Chairperson: John Vis The Secrets of Good Peak Shape in HPLC
More informationWelcome to our E-Seminar: Choosing HPLC Columns for Faster Analysis Smaller and Faster
Welcome to our E-Sear: Choosing HPLC Columns for Faster Analysis Smaller and Faster High Throughput/Fast LC Requires. Short columns 0 mm or shorter Small particle sizes. µm Rapid Resolution or new.8 µm
More informationThe Secrets of Rapid HPLC Method Development. Choosing Columns for Rapid Method Development and Short Analysis Times
The Secrets of Rapid HPLC Method Development Choosing Columns for Rapid Method Development and Short Analysis Times Rapid Analysis Is More Than Run Time It is developing a method to meet a goal and developing
More informationLC and LC/MS Column Selection Flow Chart
LC and LC/MS Column Selection Flow Chart To use the column selection diagram below, simply follow the path for your analyte and mobile phase. At the far right, follow your final column selection to the
More informationHPLC Method Development with Eclipse Plus: Standard Practices and New Columns. Agilent Technologies
HPLC Method Development with Eclipse Plus: Standard Practices and New Columns Agilent Technologies What are Some Standard Method Development Practices?. Follow preferred method development scheme and do
More informationLC Column Troubleshooting. Do you have an equivalent column for my LC column?
LC Column Troubleshooting Do you have an equivalent column for my LC column? LC columns from different suppliers may have very different retention properties even if the bonding is the same. Although there
More informationToo Polar for Reversed Phase What Do You Do?
Too Polar for Reversed Phase What Do You Do? June 20, 2013 Mark Powell Columns and Consumables Technical Support Page 1 C8 or C18 Doesn t Always Do the Job Typical reversed phase conditions involve water/buffer
More informationHigh Resolution Fast LC
High Resolution Fast LC Easier Than You Think Rita Steed LC Columns Application Engineer May 9, 2013 What is High Resolution Fast LC? Maintain Resolution with Faster Run Time Increased Resolution with
More informationNew To HPLC Avoiding Beginner Pitfalls. Rita Steed LC Columns Application Engineer April 24, 2013
New To HPLC Rita Steed LC Columns Application Engineer April 24, 2013 Outline Instrument Connections Performance Detector Column Characteristics Lifetime Method Conditions Mobile phase ph Temperature Sample
More informationpenta-hilic UHPLC COLUMNS
penta-hilic UHPLC COLUMNS penta-hilic Highly retentive, proprietary penta-hydroxy-ligand Excellent peak shape for polar compounds with a variety of functional groups: acids, bases, zwitterions strong and
More informationHigh Performance Liquid Chromatography
Updated: 3 November 2014 Print version High Performance Liquid Chromatography David Reckhow CEE 772 #18 1 HPLC System David Reckhow CEE 772 #18 2 Instrument Basics PUMP INJECTION POINT DETECTOR COLUMN
More informationHigh Performance Liquid Chromatography
Updated: 3 November 2014 Print version High Performance Liquid Chromatography David Reckhow CEE 772 #18 1 HPLC System David Reckhow CEE 772 #18 2 1 Instrument Basics PUMP INJECTION POINT DETECTOR COLUMN
More informationHPLC Method Development: Standard Practices and New Columns
HPLC Method Development: Standard Practices and New Columns Edward Kim Application Engineer Agilent Technologies, Inc. March 8, 29 What are Some Standard Method Development Practices?. Follow preferred
More informationImprove Peak Shape and Productivity in HPLC Analysis of Pharmaceutical Compounds with Eclipse Plus C8 Columns Application
Improve Peak Shape and Productivity in HPLC Analysis of Pharmaceutical Compounds with Eclipse Plus C8 Columns Application Pharmaceuticals Authors John W. Henderson Jr., Nona Martone, and Cliff Woodward
More informationAcclaim Mixed-Mode WCX-1
Acclaim Mixed-Mode WCX-1 Product Manual for the Acclaim Mixed-Mode WCX-1 Column Page 1 of 28 PRODUCT MANUAL for the Acclaim Mixed-Mode WCX-1 Columns 4.6 x 150 mm, P/N (068353) 4.6 x 250 mm, P/N (068352)
More informationC18 Column. Care & Use Sheet
C18 Column Care & Use Sheet HALO Description HALO C18 is a high-speed, high-performance liquid chromatography column based on a new Fused-CoreTM particle design. The Fused-Core particle provides a thin
More informationpenta-hilic UHPLC COLUMNS
penta-hilic UHPLC COLUMNS Highly retentive, proprietary penta-hydroxy-ligand penta-hilic Excellent peak shape for polar compounds with a variety of functional groups: acids, bases, zwitterions strong and
More informationInertSustainSwift C8
Physical Properties Silica Particle Size Surface Area Pore Size Pore Volume Bonded Phase End-capping Carbon Loading ph Range USP Code :ES (Evolved Surface) Silica Gel :.9 μm, μm, μm :00 m /g :00 Å (0 nm)
More informationfor Acclaim Mixed-Mode HILIC-1 Column
for Acclaim Mixed-Mode HILIC-1 Column Product Manual for ACCLAIM Mixed-Mode HILIC-1 Page 1 of 17 Product Manual for ACCLAIM Mixed-Mode HILIC-1 Column 5µm, 4.6 x 250mm, P/N 066844 5µm, 4.6 x 150mm, P/N
More informationValidated HPLC Methods
Validated HPLC Methods To Tweak or Not to Tweak Slide 4 Adjustments of operating conditions to meet system suitability requirements may be necessary. USP 23 p1776 Slide 5 001816S1.PPT What is the line
More informationPerformance characteristics of the Agilent 1290 Infinity Quaternary Pump
Performance characteristics of the Agilent 129 Infinity Quaternary Pump Technical Overview Author A.G.Huesgen Agilent Technologies, Inc. Waldbronn, Germany Abstract This Technical Overview presents Proof
More informationfor Acclaim Mixed-Mode WCX-1
for Acclaim Mixed-Mode WCX- Product Manual for the Acclaim Mixed-Mode WCX- Column Page of 8 Product Manual for Acclaim Mixed-Mode WCX- Columns Acclaim Mixed-Mode WCX-, 3µm, Analytical column, 3.0 x 50mm
More informationHPLC Method Development: Standard Practices and New Columns. Agilent Technologies February 2007
HPLC Method Development: Standard Practices and New Columns Agilent Technologies February 7 What are Some Standard Method Development Practices?. Follow preferred method development scheme and do hands-on
More informationTechnical Procedure for Concentration Determination of Methamphetamine in Liquids via HPLC
Technical Procedure for Concentration Determination of 1.0 Purpose This procedure specifies the required elements for the preparation and use of the Agilent 1100/1200 series High Performance Liquid Chromatograph
More informationLC Method Development and Transfer
LC Method Development and Transfer I would like to reduce solvent usage and waste generation for our lab s analyses and methods. Which type of column should I choose? The 3.0 mm ZORBAX Solvent Saver (
More informationAgilent s New Weak Anion Exchange (WAX) Solid Phase Extraction Cartridges: SampliQ WAX
Agilent s New Weak Anion Exchange (WAX) Solid Phase Extraction Cartridges: SampliQ WAX Technical Note Agilent s SampliQ WAX provides Applications for strongly acidic, acidic and neutral compounds Excellent
More informationFast Separation of Vastly Different Compounds by Isocratic HPLC
Fast Separation of Vastly Different Compounds by Isocratic HPLC Aimee N. Heyrman and Yury Zelechonok Resolution Systems, Inc. 590 E. 32nd St. Holland. MI, 49423 SIELC Technologies, 15 E. Palatine Rd, Suite
More informationTips and tricks to increase HPLC column lifetime. Dr. Frank Michel
Tips and tricks to increase HPLC column lifetime Dr. Frank Michel Overview How to influence HPLC column life time? HPLC method Helpful accessories Best practice 2 HPLC Method Mobile Phase Mobile phase
More informationReversed Phase Chromatography: Mobile phase Considerations
Reversed Phase: Mobile Phase Considerations Dr. www.forumsci.co.il/hplc Example of a Reversed Phase Method with UV Detection 1 Chromatographic Process B+A Mobile phase Elution through the Column-movie
More informationProduct Bulletin. ACE LC/MS and Rapid Analysis HPLC Columns. HPLC Columns
Product Bulletin HPLC Columns ACE LC/MS and Rapid Analysis HPLC Columns 0 mm, 30 mm, 35 mm and 50 mm column lengths.0,., 3.0, 4.0 and 4.6 mm column diameters Configured for High Sample Throughput Specially
More informationSolid Phase Extraction Method Development Tips and Tricks
Solid Phase Extraction Method Development Tips and Tricks Carol Haney Ball, Ph.D. Application Scientist Agilent Technologies, Inc. February 3, 2009 Sources of Error Generated and Time Spent During a Typical
More informationProPac WCX-10 Columns
ProPac WCX-10 Columns Guidance for column use Tips to maximize column lifetime ProPac WCX-10 Column Tips and Tricks This guide provides essential information and invaluable guidelines for mobile phases,
More informationState-of-the-art C18 HPLC Columns
An HPLC GL Sciences Newest and Most Advanced ODS Phase-New For 00 State-of-the-art C HPLC s Improved Peak Shapes and Heights Enhancing Sensitivity High Resolution Fast Equilibration Compatible with 00%
More informationReversed Phase Solvents
Part 1. General Chromatographic Theory Part 2. verview of HPLC Media Part 3. The Role of the Mobile Phase in Selectivity Part 4. Column Care and Use Reversed Phase Solvents 2 Solvents for RP Chromatography
More informationMethod Development Kits
Method Development Kits sales representative for ordering information or contact...77 Method Development Kits First column choice for Pharma Development USP L C Method Development Kits Maximize retention
More informationDo you need trouble-free speed? The NEW Chromolith HR
Do you need trouble-free speed? The NEW Chromolith HR EMD Millipore is a division of Merck KGaA, Darmstadt, Germany ... then speed-up to Chromolith HighResolution... Revolutionary monolithic silica columns
More informationProntoSIL C18-EPS Reversed-Phase HPLC Columns
ProntoSIL C8-EPS Reversed-Phase HPLC Columns Provides excellent separation of polar compounds Better peak shape for acids and bases Stabilized bonded phase for rugged, robust HPLC methods More retentive
More informationDo you need trouble-free speed? The NEW Chromolith HR
Do you need trouble-free speed? The NEW Chromolith HR EMD Millipore is a division of Merck KGaA, Darmstadt, Germany ... then speed-up to Chromolith HighResolution... Revolutionary monolithic silica columns
More information[ CARE AND USE MANUAL ]
CONTENTS I. INTRODUCTION II. III. IV. GETTING STARTED a. Column Installation b. Column Equilibration c. Initial Column Efficiency Determination COLUMN USE a. Guard Columns b. Sample Preparation c. ph Range
More informationThe new Agilent 1290 Infinity Quaternary LC
The new Agilent 1290 Infinity Quaternary LC How binary pump-like performance can enhance your lab efficiency Sebastian Krahe Produktspezialist HPLC Agilent Technologies Page 1 The 1290 Infinity Quaternary
More informationAgilent 1290 Infinity Quaternary LC Stepwise Transfer to Methods with MS-Compatible Mobile Phases
Agilent 129 Infinity Quaternary LC Stepwise Transfer to Methods with MS-Compatible Mobile Phases Technical Overview Author A.G.Huesgen Agilent Technologies, Inc. Waldbronn, Germany Abstract The Agilent
More informationP R O D U C T B U L L E T I N. Fused-Core particle technology for hyper-fast and super-rugged HPLC columns
P R O D U C T B U L L E T I N Fused-Core particle technology for hyper-fast and super-rugged HPLC columns HALO column packings are not made the typical way. Instead, the particles packed into HALO columns
More informationBasic principles of HPLC
Introduction to the theory of HPLC HPLC (High Performance Liquid Chromatography) depends on interaction of sample analytes with the stationary phase (packing) and the mobile phase to effect a separation.
More informationHypersil BDS Columns TG 01-05
TG 0-0 Hypersil BDS Columns Introduction Hypersil BDS columns have gained a reputation over the years as one of the most robust, reproducible and reliable HPLC column brands available. This Technical Guide
More informationA C18 Silica Column With Exceptional Temperature and ph Stability
A C18 lica Column With Exceptional Temperature and ph Stability Brian A. Jones 1, Stephanie J. Marin 1, Jody Clark 1, Nathan L. Porter 1, J. Andreas Lippert 2, and Todd M. Johnson 2 1. Selerity Technologies,
More informationShodex TM ODP2 HP series columns
HPLC Columns Shodex TM ODP2 HP series columns Better retention of highly polar substances Technical notebook No. 6 Contents 1. Introduction 1-1. Specifications 1-2. Eluent Compatibility of ODP2 HP Series
More informationfor Acclaim Trinity TM P1
for Acclaim Trinity TM P Product Manual for Acclaim TRINITY P Columns Page of 4 Product Manual for Acclaim Trinity TM P Columns 07556 Acclaim Trinity P, 3µm, 4.6 x 00mm 07556 Acclaim Trinity P, 3µm, 4.6
More informationMethod Development in Solid Phase Extraction using Non-Polar ISOLUTE SPE Columns for the Extraction of Aqueous Samples
Technical Note 101 Method Development in Solid Phase Extraction using Non-Polar ISOLUTE SPE Columns for the Extraction of Aqueous Samples This technical note includes by specific information on the extraction
More informationCAPCELL PAK C18 IF2 Type http://hplc.shiseido.co.jp/e/column/html/if2_index.htm Page 1 of 2 List of Sales Representatives Contact Shiseido Technical Materials Catalog List HPLC Columns HPLC Instruments
More informationPackings for HPLC. Packings for HPLC
Summary of packings for HPLC In analytical HPLC, packings with particle sizes of 3 to 10 µm are preferred. For preparative separation tasks, also particles with diameters larger than 10 µm are applied.
More informationComparison of different aqueous mobile phase HPLC techniques
June 009 ewsletter Pharmaceutical Analysis: What is Your Problem? SIELC Technologies, Inc., Prospect Heights, IL 0070 Pharmaceutical analysis involves liquid chromatography of various compounds, from active
More informationcolumns Acclaim Mixed-Mode WCX-1 for Separating Basic Molecules
columns Acclaim Mixed-Mode WCX- for Separating Basic Molecules The Acclaim Mixed-Mode WCX- is a novel, high-efficiency, silica-based column specially designed for separating various basic analytes. This
More information(HILIC) Bill Champion Agilent Technologies, Inc opt 3/opt3/opt 2 HILIC - Agilent Restricted
Hydrophilic Interaction Chromatography (HILIC) Bill Champion Agilent Technologies, Inc. 800-227-9770 opt 3/opt3/opt 2 william_champion@agilent.com Page 1 HILIC A method of recent attention Bill Champion
More informationPacked Column for Ultra-Fast Reversed-Phase Liquid Chromatography, TSKgel Super-ODS. Table of Contents
No. 089 SEPARATION REPORT Packed Column for Ultra-Fast Reversed-Phase Liquid Chromatography, TSKgel Super-ODS Table of Contents 1. Introduction 1 2. Column Specification 1 3. Features of Packing Materials
More informationThermo Scientific Accucore XL HPLC Columns. Technical Manual
Thermo Scientific Accucore XL HPLC Columns Technical Manual Thermo Scientific Accucore XL HPLC Columns Based on Core Enhanced Technology using µm solid core particles, Accucore XL HPLC columns allow users
More informationLC Technical Information
LC Technical Information Method Transfer to Accucore.6 μm Columns Containing solid core particles, which are engineered to a diameter of.6μm and a very narrow particle size distribution; Accucore HPLC
More informationFAQ's. 1 - Which column would be the most appropriate for my application?
1 - Which column would be the most appropriate for my application? Due to the complexity of the chiral recognition mechanism, it is not possible yet to establish rules for the selection of the best chiral
More informationAlltech Alltima HP Introduction
Alltech Introduction High-Stability, High-Purity, High-Performance, Low-Bleed Columns for Demanding Applications Better Peak Symmetry high-purity silica eliminates peak tailing problems Long Column Life
More informationAnalysis of Metals, Halides, and Inorganic Ions Using Hydrophilic Interaction Chromatography
Application Note Inorganic Ions, Water Testing, Minerals, Metals, Basic Chemicals Analysis of Metals, Halides, and Inorganic Ions Using Hydrophilic Interaction Chromatography Authors Anne Mack, Adam Bivens
More informationHPLC Separation Fundamentals. Ed Kim Application Engineer Agilent Technologies, Inc January 14, 2009
HPLC Separation Fundamentals Ed Kim Application Engineer Agilent Technologies, Inc January 14, 2009 Separation fundamentals Presentation Outline Major HPLC modes Key Equations Resolution van Deemter Common
More informationInertSustainSwift C8
HPLC, LC/MS Columns InertSustainSwift TM C8 New! InertSustainSwift C8 is an octyl group (C8) bonded column offering the same extreme inertness to any type of compounds just like InertSustainSwift C8, which
More informationDilution(*) Chromatography
WA20264 Poster # 184, HPLC 2002, Montreal, 4-5 June 2002 At-Column Column-Dilution for Preparative Dilution(*) Chromatography Cecilia Mazza, Jie Cavanaugh, Ziling Lu,Tom Sirard,Tom Wheat and Uwe Neue Waters
More informationACQUITY UPLC HSS Columns
CONTENTS I. INTRODUCTION II. II. GETTING STARTED a. Column Connectors b. Column Installation c. Column Equilibration d. ecord Installation e. Initial Column Efficiency Determination f. VanGuard Pre-columns
More informationHICHROM. Chromatography Columns and Supplies. LC COLUMNS SIELC Primesep. Catalogue 9. Hichrom Limited
HICHROM Chromatography Columns and Supplies LC COLUMNS SIELC Primesep Catalogue 9 The Markham Centre, Station Road Theale, Reading, Berks, RG7 4PE, UK Tel: +44 (0)8 90 660 Fax: +44 (0)8 9 484 Email: sales@hichrom.co.uk
More informationSunniest C18 Sunniest RP-AQUA Sunniest C8 Patent pending
Patent pending Website : www.chromtech.net.au E-mail : info@chromtech.net.au telno : 0 97 0... in AUSTRALIA A Novel Bonding Technique (patent pending) An unique trifunctional silyl-reagent was developed
More informationNew Product Update. HPLC Columns
New Product Update HPLC Columns ZORBAX MicroBore HPLC Columns ZORBAX Extend-C18 Columns ZORBAX Bonus-RP Columns Inertsil ODS-2 Cartridge Columns ZORBAX Carbohydrate Analysis Columns ZORBAX Eclipse dsdna
More informationPractical Applications of Method Translation Using the Agilent Method Translation Tool Rita Steed Inside Application Engineer Agilent Technologies
Practical Applications of Method Translation Using the Agilent Method Translation Tool Rita Steed Inside Application Engineer Agilent Technologies Title Objectives Demonstrate Agilent Method Translation
More informationHigh Pressure/Performance Liquid Chromatography (HPLC)
High Pressure/Performance Liquid Chromatography (HPLC) High Performance Liquid Chromatography (HPLC) is a form of column chromatography that pumps a sample mixture or analyte in a solvent (known as the
More informationRapid Screening and Analysis of Components in Nonalcoholic Drinks Application
Rapid Screening and Analysis of Components in Nonalcoholic Drinks Application Food Author Michael Woodman Agilent Technologies, Inc. 285 Centerville Road Wilgton, DE 1988-161 USA Abstract Soft drinks,
More informationA Look at Column Choices. Ed Kim Application Engineer Agilent Technologies, Inc February 12, 2009
A Look at Column Choices Does one-size-fit-all? Ed Kim Application Engineer Agilent Technologies, Inc February 12, 2009 Seminar utline lica Surface Chemistry & Physical Characteristics ti lica Bonding
More informationValue of Eclipse Plus C18 and ph Selectivity to Analyze Amine-Containing Drugs
Value of Eclipse Plus C18 and ph Selectivity to Analyze Ae-Containing Drugs Application Pharmaceuticals Authors John W. Henderson Jr., William J. Long, and Cliff Woodward Agilent Technologies 285 Centerville
More informationHPLC analysis of VFAs, Furfural and HMF
HPLC analysis of VFAs, Furfural and HMF Introduction The following chromatographic analysis is developed in order to analyse volatile fatty acids and carbohydrate degradation products; furfural, hydroxymethylfurfural
More informationAdvantages of polymerbased. an alternative for ODS
Advantages of polymerbased HPLC columns an alternative for ODS Yukiko Higai Shodex, 1 Content (1) Comparison between Silica and Polymer-based RP columns (2) Characteristics of Polymer RP columns - an example:
More informationKromasil. for your analytical HPLC. Kromasil. The way to peak performance in liquid chromatography
Kromasil for your analytical HPLC Kromasil The way to peak performance in liquid chromatography Kromasil is known, worldwide, for its high performance and excellent total economy in preparative and industrial
More information[ Care and Use Manual ]
XSelect High Strength Silica (HSS) hplc Columns Contents I. INTRODUCTION II. CONECTING THE COLUMN TO THE HPLC SYSTEM a. Column Connection b. Column Connectors and System Tubing Considerations c. Band Spreading
More informationACQUITY UPLC Peptide BEH C 18, 130Å and 300Å Columns
CONTENTS I. INTRODUCTION II. III. GETTING STARTED a. Column Connectors b. Column Installation c. Column Equilibration d. ecord Installation e. Initial Column Efficiency Determination COLUMN USE a. Sample
More informationThis method describes the identification of the following prohibited colorants in cosmetic products:
A. IDENTIFICATION BY TLC 1. SCOPE AND FIELD OF APPLICATION This method describes the identification of the following prohibited colorants in cosmetic products: Names C I number Pigment Orange 5 12075 Metanil
More informationAnalysis - HPLC A.136. Primesep 5 µm columns B.136
Primesep 5 µm columns Primesep columns feature double functionality of the bonding i.e : alkyl chain with anionic or cationic group, chelating group. This feature creates unique selectivities when using
More information[ CARE AND USE MANUAL ]
XBridge Peptide BEH C 18, 130Å and 300Å Columns CONTENTS VIII. ADDITIONAL INFORMATION I. GETTING STARTED II. III. IV. a. Column Installation b. Column Equilibration c. Initial Column Efficiency Determination
More informationCOMPARISON GUIDE. to C18 Reversed Phase HPLC Columns. Comparison Data on 60 Commonly Used C18 Phases. Stationary Phase Specifications
COMPARISON GUIDE to C18 Reversed Phase HPLC Columns Comparison Data on 60 Commonly Used C18 Phases Stationary Phase Specifications Phases Compared According to Relative Hydrophobicity Phases Compared According
More information[ Care and Use Manual ]
high Strength Silica hplc Columns Contents I. INTRODUCTION II. CONECTING THE COLUMN TO THE HPLC SYSTEM III. Waters Small Particle Size (3.5 m) Columns Fast Chromatography IV. Column Equilibration V. Column
More informationGetting More Resolution from Your LC Michael Woodman Columns and Consumables September 10, 2008
Getting More Resolution from Your LC Michael Woodman Columns and Consumables September 10, 2008 Page 1 What are the Parameters Affecting Resolution? Isocratic Separations: physical and chemical separation
More informationApplication Note. Author. Abstract. Pharmaceutical QA/QC. Siji Joseph Agilent Technologies, Inc. Bangalore, India
Effective use of pharmacopeia guidelines to reduce cost of chromatographic analysis Optimized, cost-effective HPLC analysis of atorvastatin by varying column dimensions within the USP allowed limts
More informationDoes one-size-fit-all?
A Look at Column Choices: Does one-size-fit-all? Bill Champion Agilent Technologies, Inc. 800-227-9770 opt 3/opt3/opt 2 william_champion@agilent.com Page 1 Separation Techniques Hydro - philic Volatile
More informationFigure 1. Structures for Vitamin B2 and Vitamin B1.
CH 461 & CH 461H F 18 Name Experiment 2C Integrated Laboratory Experiment DETERMINATION OF RIBOFLAVIN: A COMPARISON OF TECHNIQUES PART C. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY The purpose of this experiment
More informationHigh Performance Liquid Chromatography
STANDARDBASE techniques: High Performance Liquid Chromatography Drenthe College, The Netherlands 1. Introduction HPLC. High Performance Liquid Chromatography High Performance Liquid Chromatography (HPLC)
More informationXSEL EC T C HA RGED SU RFAC E HYBRID (C SH) COLUMNS
[ CARE AND USE MANUAL ] XSEL EC T C HA RGED SU RFAC E HYBRID (C SH) COLUMNS CONT ENT S I. G E T T ING STA RT E D a. Column Installation b. Column Equilibration c. Initial Column Efficiency Determination
More informationHICHROM. Chromatography Columns and Supplies. LC COLUMNS Zorbax. Catalogue 9. Hichrom Limited
HICHROM Chromatography Columns and Supplies LC COLUMNS Zorbax Catalogue 9 1 The Markham Centre, Station Road Theale, Reading, Berks, RG7 4PE, UK Tel: +44 (0)118 930 3660 Fax: +44 (0)118 932 3484 Email:
More informationSeparation of Explosives in EPA 8330: Column Choices Optimize Speed, Resolution, and Solvent Use. Application. Authors. Abstract.
Separation of Explosives in EPA 833: Column Choices Optimize Speed, Resolution, and Solvent Use Application Environmental Authors John W. Henderson Jr. and William J. Long Agilent Technologies, Inc. 28
More informationHigh Performance Liquid Chromatography
High Performance Liquid Chromatography What is HPLC? It is a separation technique that involves: Injection of small volume of liquid sample Into a tube packed with a tiny particles (stationary phase).
More informationFigure 1. Structures for Vitamin B2 and Vitamin B1.
CH 461 & CH 461H F 15 Name Experiment 2C Integrated Laboratory Experiment DETERMINATION OF RIBOFLAVIN: A COMPARISON OF TECHNIQUES PART C. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY The purpose of this experiment
More informationEfficiently analyze your most challenging compounds. Deliver high-quality results faster. Agilent ZORBAX Column Selection Guide for HPLC
Efficiently analyze your most challenging compounds. Deliver high-quality results faster. Agilent ZORBAX Column Selection Guide for HPLC Agilent ZORBAX Column Selection Guide for HPLC Here it is your single
More information