The LC Column Playbook

Transcription

1 The LC Column Playbook Mark Powell Applications Engineer Technical Support 1

2 Overview How do we achieve our goal: Resolution? Efficiency Particle size Column length Selectivity Bonded phase Mobile phase Retention Polar compounds Polar bonded phase HILIC 2

3 Resolution R s tr2 tr1 1/ 2 ( Wb 2 Wb 1) Separation t r2 -t r1 Peak width W b1,2 Resolution describes the ability of a column to separate the peaks of interest. Resolution describes whether you have achieved base line separation or not. 3

4 h Resolution Baseline Separations R s = 1.5 t If we consider peaks of equal height: 1 - minimum for a measureable separation.6 - required to discern a valley between two equal-height peaks considered to be a baseline separation 1.7 or greater - desirable for rugged methods 4

5 Resolution Equation R s 1 4 N 1 k 1 k Efficiency Selectivity Retention Improve resolution by improving any of these parameters: Efficiency describes the separation power of the column Selectivity has the highest influence on the resolution. Small changes in selectivity can lead to big changes in resolution Retention has only a significant influence at small k values 5

6 Efficiency N L d p Parameters influencing column efficiency: Column length (increasing column length increases efficiency) Particle size (decreasing particle size increases efficiency) 6

7 Retention Factor k = t R t t t R = retention time for sample peak t = retention time for unretained peak The retention factor measures the period of time that the sample component resides in the stationary phase relative to the time it resides in the mobile phase. It is calculated from the retention time divided by the time for an unretained peak. 7 Confidentiality Label

8 Selectivity (Separation Factor) k k 2 1 k 1 k 2 Selectivity Retention factor of 1 st peak Retention factor of 2 nd peak Selectivity is a measure of the time or distance between the maxima of two peaks. If α = 1, the two peaks have the same retention time and co-elute. Parameters influencing selectivity: Stationary phase Mobile phase Temperature 8

9 Resolution Selectivity impacts resolution most Change stationary phase Change mobile phase Plates are easiest to increase Resolution as a function of selectivity, column efficiency or retention If you double the column length, you will obtain more theoretical plates But you only get a square root of 2, or 1.4x improvement in the resolution 9

10 How to Improve Resolution? High plate number (N) provides: Sharp and narrow peaks Better detection Ability to resolve complex samples But resolution increases only with the square root of the plate number R S ~ N Plate number increase is limited by experimental conditions Time Pressure (instrument) 1

11 How to Improve Resolution? Increase N with longer column N L d p 11

12 Poroshell 12 Particle Sizes.75um.5um.35um 1.2um 1.7um 2.5um.35um.5um.75um InfinityLab Poroshell µm Highest UHPLC performance InfinityLab Poroshell µm UHPLC performance at lower pressure InfinityLab Poroshell 12 4 µm Improved HPLC performance 12

13 Resolution Decreasing Particle Size Increases Efficiency mau mau DAD1 A, Sig=24,8 Ref=off (AEM_PLUTO_TRANSFER\CATECHINS_19UM-P12-ECC18_ _ D) DAD1 A, Sig=24,8 Ref=off (AEM_PLUTO_TRANSFER\CATECHINS_27UM-P12-ECC18_ _ D) 1.9 µm 2.7 µm min Columns: InfinityLab Poroshell 12 EC-C18, 2.1 x 5 mm, 1.9 um InfinityLab Poroshell 12 EC-C18, 2.1 x 5 mm, 2.7 um InfinityLab Poroshell 12 EC-C18, 2.1 x 5 mm, 4 um Mobile phase A:.2% formic acid in water Mobile phase B: Acetonitrile Gradient: 5-16% B in 7 min Flow rate:.5 ml/min Detection: 24 8 Hz Sample:1 μl of.6 mg/ml each of gallic acid, gallocatechin, epigallocatechin, catechin, caffeine, epicatechin, epigallocatechin gallate, gallocatechin gallate, epicatechin gallate, catechin gallate -1 mau DAD1 A, Sig=24,8 Ref=off (AEM_PLUTO_TRANSFER\CATECHINS_4UM-P12-ECC18_ _ D) min Performance vs pressure µm Particle Pressure R smin 1.9 µm 226 bar µm 131 bar µm min µm 53 bar µm Higher N improves resolution as particle size is decreased µm Pressure N L d p 13

14 Particle Size: When to Use What Size Technique / product Performance 4 µm HPLC 2.7 µm UHPLC 1.9 µm Improved HPLC performance Pressure rating: 6 bar Typical pressure: Often < 2 bar Efficiency: ~2% of 5 μm totally porous UHPLC performance at lower pressure Pressure rating: 6 bar Typical pressure: 5% of sub-2 μm totally porous Efficiency: ~9% of sub-2 μm totally porous Highest UHPLC performance Pressure rating: 13 bar Typical pressure: Similar to sub-2 μm totally porous Efficiency: ~12% of sub-2 μm totally porous 14

15 High Resolution Separations Smaller Particle Size with Longer Columns 15

16 How to Improve Resolution? Change Selectivity: Bonded phase Phases other than C18/C8 Phenyl-Hexyl, Polar-embedded, CN, PFP, HILIC Different interactions with polar or non-polar compounds Other types of interactions with a bonded phase (π-interactions, etc.) Mobile phase Mobile phase organic modifier (ACN, MeOH etc.) Mobile phase ph over a wide ph range ph 1-12 if needed Temperature 16

17 Agilent InfinityLab Poroshell phases Best all around Best for low ph mobile phases Best for high and mid ph mobile phases Best for alternative selectivity Best for more polar compounds HILIC for polar compounds Poroshell 12 Poroshell 12 Poroshell Poroshell 12 Poroshell 12 Poroshell 12 EC-C18 SB-C18 HPH-C18 Phenyl-Hexyl SB-Aq HILIC 1.9 µm, 2.7 µm, 4 µm 2.7 µm 1.9 µm, 2.7 µm, 4 µm 1.9 µm, 2.7 µm, 4 µm 2.7 µm 1.9 µm, 2.7 µm, 4 µm Poroshell 12 Poroshell 12 Poroshell Poroshell 12 Poroshell 12 Poroshell 12 EC-C8 SB-C8 HPH-C8 Bonus-RP EC-CN HILIC-Z 1.9 µm, 2.7 µm, 4 µm 2.7 µm 2.7 µm, 4 µm 1.9 µm, 2.7 µm, 4 µm 2.7 µm 2.7 µm Poroshell 12 Poroshell 12 PFP HILIC-OH5 2.7 µm 2.7 µm 17

18 Why is Changing the Bonded Phase Effective? Different interactions for polar and non-polar compounds. Exploit other interactions with bonded phase (e.g., pi-pi) Changing the bonded phase can improve selectivity/resolution, reduce analysis time Having numerous different bonded phases available on the same particle makes development easier Fast Poroshell methods make development faster 18

19 Poroshell Selectivity Antioxidants (ACN) EN 19 Title Confidentiality label Regulatory statement (if applicable)

20 Poroshell Selectivity Antioxidants (MeOH) EN 2 Title Confidentiality label Regulatory statement (if applicable)

21 Poroshell Selectivity Beta Blockers mau Poroshell 12 EC-C min mau Poroshell 12 SB-C min mau Poroshell 12 Phenyl Hexyl min mau Poroshell 12 Bonus RP Atenolol, 2. Pindolol, 3. Naldolol, 4. Metoprolol, 5. Acebutolol, 6. Propranolol, 7. Alprenolol 1 mm ph 3.8 NH 4 HCO 2, Methanol; 9 % B to 3 % B over 12 min,.35 ml/min, 2.1 x 1 mm min 21

22 NSAID Separation with a Methanol Gradient mau PFP mau EC-C , min mau Bonus RP min mau Phenyl Hexyl 2 3 5, min APAP, 2. Phenacetin, 3. Piroxicam, 4. Tolmetin, 5. Ketoprofen, 6. Naproxen, 7. Sulindac, 8. Diclofenac, 9. Diflunisal Time % Organic mL/min 254 nm min 22

23 Agilent Poroshell phases Best all around Best for low ph mobile phases Best for high and mid ph mobile phases Best for alternative selectivity Best for more polar compounds HILIC for polar compounds Poroshell 12 Poroshell 12 Poroshell Poroshell 12 Poroshell 12 Poroshell 12 EC-C18 SB-C18 HPH-C18 Phenyl-Hexyl SB-Aq HILIC 1.9 µm, 2.7 µm, 4 µm 2.7 µm 1.9 µm, 2.7 µm, 4 µm 1.9 µm, 2.7 µm, 4 µm 2.7 µm 1.9 µm, 2.7 µm, 4 µm Poroshell 12 Poroshell 12 Poroshell Poroshell 12 Poroshell 12 Poroshell 12 EC-C8 SB-C8 HPH-C8 Bonus-RP EC-CN HILIC-Z 1.9 µm, 2.7 µm, 4 µm 2.7 µm 2.7 µm, 4 µm 1.9 µm, 2.7 µm, 4 µm 2.7 µm 2.7 µm Poroshell 12 Poroshell 12 PFP HILIC-OH5 2.7 µm 2.7 µm 23

24 Comparing Low, Mid, & High ph 24

25 Agilent Poroshell phases Best all around Best for low ph mobile phases Best for high and mid ph mobile phases Best for alternative selectivity Best for more polar compounds HILIC for polar compounds Poroshell 12 Poroshell 12 Poroshell Poroshell 12 Poroshell 12 Poroshell 12 EC-C18 SB-C18 HPH-C18 Phenyl-Hexyl SB-Aq HILIC 1.9 µm, 2.7 µm, 4 µm 2.7 µm 1.9 µm, 2.7 µm, 4 µm 1.9 µm, 2.7 µm, 4 µm 2.7 µm 1.9 µm, 2.7 µm, 4 µm Poroshell 12 Poroshell 12 Poroshell Poroshell 12 Poroshell 12 Poroshell 12 EC-C8 SB-C8 HPH-C8 Bonus-RP EC-CN HILIC-Z 1.9 µm, 2.7 µm, 4 µm 2.7 µm 2.7 µm, 4 µm 1.9 µm, 2.7 µm, 4 µm 2.7 µm 2.7 µm Poroshell 12 Poroshell 12 PFP HILIC-OH5 2.7 µm 2.7 µm 25

26 Aliphatic Acids on Poroshell SB-Aq EN Page 26

27 HILIC Hydrophilic Interaction Chromatography HILIC offers more retention than reversed-phase for very polar bases Polar stationary phase: Silica Amine Amide Zwitterionic Diol Others Polar mobile phase: Water is the strong solvent Typically ACN/water Buffer controls ionization of analyte and stationary phase Typically ammonium acetate or ammonium formate Retention/elution is from least to most polar Page 27

28 Advantages to using HILIC Uses a standard LC system and common reversed-phase solvents Separates cations, anions and polar neutrals in a single run Retains polar analytes where reversed-phase methods may not MS compatible eluents and easy setup Improved ionization and sensitivity in MS mode Compatible with MS in both positive and negative analysis modes

29 HILIC vs C18 Page 29

30 NEW INFINITYLAB POROSHELL 12 HILIC COLUMNS HILIC-Z: Zwitterionic stationary phase 2.7 µm Poroshell particles 2.1mm, 3.mm and 4.6mm ID 5mm, 1mm and 15mm lengths HILIC-OH5: poly-hydroxy fructan stationary phase 2.7 µm Poroshell particles 2.1mm and 4.6mm ID 5mm, 1mm and 15mm lengths 3 Confidentiality Label

31 Fast re-equilibration and excellent peak shape Polar pesticides mau 14 DAD1 B, Sig=38,8 Ref=off (AEM_ICECAP\PARAQUAT_DIQUAT_GLYPHOSATE_P12-ZIC-PEEK_ _ D) DAD1 B, Sig=38,8 Ref=off (AEM_ICECAP\PARAQUAT_DIQUAT_GLYPHOSATE_P12-ZIC-PEEK_ _ D) 12 1 InfinityLab Poroshell HILIC-Z 2.1 x 1mm, 2.7µm min Repeat Injections (back-to-back) Sample Diquat and Paraquat Gradient 6 min Equilibration 4 min Total Runtime 1 min Excellent baseline reproducibility with only 1.5 min re-equilibration between injections!

32 Phenylalanine Leucine Isoleucine Methionine Tyrosine Valine Proline Alanine Cysteine Glycine Serine Glutamic Acid Aspartic Acid Histidine Arginine Lysine Cystine Threonine Excellent retention, peak shape and sensitivity Underivatized Amino Acids by LC/MS x1 2 +ESI MRM Frag=25.V CF=. DF=. CID@5. (76. -> 3.1) Amino_2.1x1 (P12-ZIC_#4-PEEK)_#3.d Counts (%) vs. Acquisition Time (min) InfinityLab Poroshell HILIC-Z 2.1 x 1mm, 2.7µm A: ph 3. 2mM ammonium formate B: ACN Gradient method on LC/QQQ 32 1/1/217

33 Consistent analytical reproducibility and long column lifetimes High ph Sugar Analysis ELS1 A, Voltage (TCW29B_2.1X1_23\ _ \6-12.D) mv 1 16 column volumes min ELS1 A, Voltage (TCW29B_2.1X1_23\ _ \6-182.D) mv column volumes min InfinityLab Poroshell HILIC-Z 2.1 x 1mm, 2.7µm A: 1.% Ammonium hydroxide ph 12. B: CH3CN 9%B for 4 min,.4 ml/min, 35 C ELSD: 6C/3.5psi

34 Alternate Selectivity of HILIC Phases - Retention of Inorganic Ions mv Br - I - ClO 3 - Cl - BrO 3 - Li + Na + K + H 2 PO Mg 2 + Ca 2 + InfinityLab Poroshell 12 HILIC 2.1x1mm 2.7µm min Calcium chloride Lithium bromide Magnesium chloride Potassium bromate Potassium iodide Potassium phosphate Sodium bromide Sodium chlorate mv 25 2 I - ClO 3 - Mg 2 + InfinityLab Poroshell 12 HILIC- OH5 2.1x1mm 2.7µm 15 Br BrO 3 - Cl Li + Na + Ca 2 + H 2 PO 4 - K + min Method conditions for both applications: A: 1 mmph 3 Ammonium Formate, B: Acetonitrile, hold 91% B for 1 min, 91-6% B in 1 min, 3 min reequilibration,.4 ml/min, 3 C, 1 ul injection, ELSD 4C/3.5psi/3Hz

35 Selectivity Across HILIC phases Water soluble vitamins DAD1 A, Sig=26,8 Ref=off (AEM_HILIC_...INS\AEM_HILIC_VITAMINS-B-C \HILIC-OH5-4.D) InfinityLab Poroshell 12 column 2.1 x 1 mm 2.7 μm mau B6 B1 HILIC-OH5 B3 B2 B1 B9 C B12 mau DAD1 A, Sig=26,8 Ref=off (AEM_HILIC_...INS\AEM_HILIC_VITAMINS-B-C \HILIC-Z-4.D) B1 B1,B3 HILIC-Z min B6 B2 C B12 B9 mau DAD1 A, Sig=26,8 Ref=off (AEM_HILIC_...AMINS\AEM_HILIC_VITAMINS-B-C \HILIC-4.D) B1 B2 B HILIC B3 B9 B1 B12 C min min A: 1 mm Ammonium Acetate +.5% Acetic Acid (ph ~4.6) in H 2 O, B: CH 3 CN.5 ml/min, 87% B for 1 min, 87-5% B in 4 min, 3 min re-equilibration 1 μl injection, 4 o C, UV detection at 26 nm, 8 Hz 35 1/1/217

36 Water-Soluble Vitamins Thiamine, B1 Riboflavin, B2 Nicotinic Acid, B3 Pantothenic Acid, B5 Pyridoxine, B6 Biotin, B7 Folic Acid, B9 PABA, B1 Ascorbic Acid, C Cyanocobalamin, B12 36

37 HILIC and Choice of Sample Solvent

38 Resolution How to reach your goal? Choose a column with the right balance of particle size and length Take advantage of selectivity changes Mobile phase ph Bonded phase Take advantage of polar phases or HILIC for hard to retain analytes Thank you! LC-column-support@agilent.com 38