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: a new column type (3) Characteristics of Polymer HILIC columns 2
Use of HPLC in general HPLC Reversed Phase (RP) (mainly ODS) 60-70% of samples can be applied Size Exclusion Ion Exchange (Ion Chromatography) Normal Phase Ligand Exchange etc. 3
(1) Comparison between Silica and Polymer-based RP Silica-based (ex:ods) Polymer-based (ex:odp) Higher resolution Reasonable price Relatively lower resolution Relatively higher price Narrow ph range (ph2-7) Wide ph range (ph2-12) Impossible to use under the low salt concentration Possible to use under the low salt concentration Short life time Long life time Impossible to inject large concentration sample Not enough separation of hydrophilic substances Need ion-pair reagent to separate basic substances Possible to inject large concentration sample Good separation of hydrophilic substances Possible to separate basic substances without any reagent 4
Problems of Silica-based RP columns Si O Si O Si OH O Si O Si O Residual silanol capping difficult to remove ionic adsorption Lot-to-lot differences effect of metal coodination, difficult to control residual silanols Si OH O Low durability against solvents Si OH Si O Si C18 Si OH O O OH Si O OH OH Si OH HO Si Si O Si O Si OH 5
Sample suitable for ODS analysis Molecular size Suitable for ODS Low Unsuitable for ODS High also sample including high MW impurities Polarity Compounds with different polarity Low to Moderate Compounds with similar polarity High or Ionic Solution Acidic or Neutral Basic other choice Polymer 6
(2) Characteristics of polymer-based RP columns C X CH X=OR, COOR, etc Si O Si O O O H Si a) Separation Almost the same b) Analysis of basic sample Good c) ph range in eluent Wide d) Effect of salt concentration in eluent Low e) Reproducibility Good 7
a) Separation performance (1) Polymer-based Silica-based Sample : 5µL 1. 0.2% Methylethyl ketone 2. 0.2% Diethyl ketone 3. 0.4% Di-n-propyl ketone 4. 0.6% Di-n-butyl ketone Eluent : CH 3 CN / H 2 O = 50 / 50 Flow rate : 1.0mL/min Detector : UV (262nm) Column temp. : 40 o C Almost the same separation 8
a) Separation performance (2) Sample : 0.2% each, 5µL 1. Lactitol 2. Erythritol Column : Shodex RSpak DE-413 ODS column from other manufacturer Eluent : 10mM H 3 PO 4 aq. Flow rate : 1.0mL/min Detector : RI Column temp. : 50 o C Better separation of hydrophilic samples 9
b) Analysis of basic compounds (1) Amine compounds R- N + - Residual O-Si Silanol adsorption Polymer-based column Silica-based column Less Tailing Tends to cause tailing ( ion-pair reagent needed) 10
b) Analysis of basic compounds (2) ODP-50 no need ion-pair reagent ODS with ion-pair reagent no need of ion-pair reagent (ODP-50 4D) Eluent : 50mM Phosphate buffer (ph10) /CH 3 CN=80/20 Flow rate : 0.6mL/min Column temp. : 30 (ODS) Eluent : 0.1% 1-Pentanesulfonic acid sodium salt aq./ch 3 CN=80/20 Flow rate : 1.4mL/min Column temp. : 60 o C 11
c) Better resolution in high ph (1) Durability in Alkaline condition for example... better selectivity Available ph 1 2 7 13 14 ODS ODP higher TPN Good for the separation of basic sample under alkaline conditions 12
c) Better resolution in high ph (2) Basic sample (local anesthetic drug) Working condition of Polymer column! ph 3 ph 7 ph 11 Column: Shodex Asahipak ODP-50 4D Eluent: 25mM Phosphate buffer/ch 3 CN (60/40) Flow Rate: 0.6mL/min Detector: UV(254nm) Column temp.: 30 o C 13
d) Effect of salt concentration in analysis of ionic drugs ODS-A ODS-C ODP-50 Sample : 1mL/mL Scopolamine,50µL 10mM 25mM Low concentration for ionic drugs excellent for LC/MS 1mM Column : Shodex Asahipak ODP-50 4D ODS Columns from other manufacturer Eluent : Phosphate buffer(ph7.0)/ch 3 CN=60/40 Flow rate : 0.6mL/min Detector : UV (254nm) Column temp. : 30 o C 14
e) Reproducibility Lot A Lot B Lot C CV (%) Rt (min) 1 2.504 2.505 2.482 0.425 2 3.148 3.152 3.123 0.409 3 4.878 4.894 4.847 0.400 4 7.961 8.001 7.920 0.415 Lot A Lot B Lot C CV (%) Control of batch differences Peak area (UV x sec) Peak area (%) 1 36616 37614 36414 1.422 2 32168 33166 31934 1.648 3 48560 50090 48075 1.756 4 64814 66686 64276 1.583 Lot A Lot B Lot C CV (%) 1 20.102 20.055 20.151 0.195 2 17.659 17.683 17.673 0.056 3 26.658 26.706 26.605 0.155 4 35.581 35.555 35.571 0.030 15
- an example: a new column type When ODS is not suitable Problem High MW impurity High polarity (ionic) Solution ODP2 HP ex) direct analysis of drugs in biological fluids 16
Characteristics of polymer-based RP ODP2 column ODP2 HP Polyhydroxymethacrylate gel ca. 40A ODS Silica gel ca. 100-120A drug with low Mw protein Less adsorption of proteins More adsorption of proteins Advantages: Hydrophilic surface of packing material Small pore size Proteins will be removed without sample pre-treatment less time-consuming 17
a) Analysis of high polarity substances Strong retention of high polarity substances Low Retention High log k' 1.00 0.50 0.00-0.50-1.00 ODS(A) ODS(B) ODP2 HP ODP-50-1 0 1 2 log P High Polarity Low Column : Shodex ODP2 HP-4D (4.6mmID x 150mm) Eluent : H 2 O/CH 3 CN=75/25 Flow rate : 1.0mL/min Column temp. : 40 o C 18
b) Analysis of hydrophilic compound ODP2 HP-4D 1 2 Better separation for hydrophilic materials 3 hydrophilic 123 Polymer-based RP column (Shodex Existing column) Sample : 5µL 1. Uracil 30mg/L 2. Theobromine 75mg/L 3. Caffeine 130mg/L 4. Phenol 300mg/L 5. Methyl benzoate 350mg/L 6. Toluene 1000mg/L 7. Naphthalene 150mg/L 4 5 6 N: 13,200 7 4 5 6 N: 5,700 7 0 5 10 15 min 0 5 10 15 min Column : Shodex ODP2 HP-4D (4.6mmID x 150mm) Eluent : H 2 O/CH 3 CN=55/45 Flow rate : 0.6mL/min Detector : UV(254nm) Column temp. : 40 o C Column : Shodex Asahipak ODP-50 4D (4.6mmID x 150mm) Eluent : H 2 O/CH 3 CN=35/65 Flow rate : 0.6mL/min Detector : UV(254nm) Column temp. : 40 o C 19
c) Removing proteins Pressure change ratio [%] 160 150 140 130 120 110 100 ODS(A) ODP2 HP-2B ODP2 HP-2B No adsorption no C18 end-capping Hydrophilic surface 90 0 20 40 60 80 100 120 140 Injection number 0 0.5 1.0 1.5 2.0 min Proteins are not adsorbed in ODP2 HP and the pressure did not increase. Column : ODP2 HP-2B (2.0mmID x 50mm), ODS(A) (2.0mmID x 50mm) Eluent : 1mM CH 3 COONH 4 aq./ch 3 CN=90/10 Flow rate : 0.2mL/min Detector : UV (220nm) Column temp. : 30 o C Longer column life Sample : 5µL BSA 7.0mg/mL 20
Intensity uau 1100000 1000000 900000 800000 Intensity Intensity 700000 600000 500000 400000 300000 200000 100000 300000 200000 100000 100000 80000 60000 40000 20000 0 0.12 100000 Blank ODP2 HP UV (220nm) Blank Barbital Inject Peak to Area MS:100% Barbital ODP2 HP with BSA 0 1.0 2.0 3.0 4.0 80000 Time (min) 60000 Barbital 40000 Peak Area :99% TIC SIM m/z 182.5-183.5 Barbital 20000 m/z =183.2 Peak Area : : 100% 0 0.0 1.0 2.0 3.0 4.0 Peak Hight : 100% Time (min) d) LC/MS Intensity Intensity uau Barbital 100000 80000 0 1.0 with 2.0 BSA 3.0 4.0 60000 Time (min) Barbital 40000 Peak Area :71% TIC m/z 182.5-183.5 SIM Barbital 20000 300000 0 200000 0.0 m/z =183.2 Peak Area : : 107% 1.0 2.0 3.0 4.0 Peak Hight : 102% 100000 Intensity 1100000 1000000 900000 100000 80000 800000 60000 700000 40000 600000 20000 500000 0 400000 300000 200000 BSA ODS(A) Blank Barbital Peak Inject Area :100% to MS ODS(A) Time (min) with BSA UV (220nm) Barbital recovery ratio [%] Sample : 1. Barbital 500µg/L 2. BSA 7.0mg/L Pore size: 40Å SEC effect to remove protein 0 1.0 2.0 3.0 4.0 Time (min) 0 1.0 2.0 3.0 4.0 Column : ODP2 HP-2B (2.0mmID x 50mm) Eluent : 10mM CH 3 COONH 4 aq./ch 3 CN=70/30 Flow rate : 0.2mL/min Detector : UV (220nm), ESI-MS Column temp. : 30 o C 0 Time (min) Sample pre-treatment There is no ion suppression by protein. 21
(3) Characteristics of Polymer-based amino column (HILIC) a) Enables sugar analysis at room temp. (alkali atmosphere) b) Long life time/stability. Regeneration possible with alkali solvents c) Reduced noise level because of less bleeding 22
a) Sugar analysis at room temp. Comparison with an amide column at low temp. Amino column Alkali atmosphere due to amino groups No anomers even at room temp. Amide column Not alkali atmosphere (acryl amide groups) Need high temp. to avoid anomers Column : Shodex Asahipak NH2P-50 4E (4.6mmID x 250mm), Amide column from another manufacturer (4.6mmID x 250mm) Eluent : CH 3 CN / H 2 O = 75 / 25 Flow rate : 1.0mL/min Detector : Shodex RI Column temp. :30deg-C 23
b) Long life time/stability Comparison with a silica-column in Life time Polymer -based Silica -based 24
c) Reduced noise level less bleeding Silica Amino column Source: LMS CO., LTD. Japan Noise level: 30pA Lower limit (S/N=3) Absolute amount 100 ng Absolute amount 500 ng Polymer Amino Column 100 times better Noise level: 0.8pA Lower limit (S/N=3) Absolute amount 1 ng Absolute amount 500 ng Reduced noise level because of less bleeding 25
Poly- and oligosaccharides Sample : 0.5% Hydrolyzed dextran, 20 µl Column : Shodex Asahipak NH2P-50 4E (250mmLx4.6mmID) Eluent : (A) CH3CN / H2O = 50 / 50 (B) 0 to 7min CH3CN=57/43 to 50/50 (Linear gradient) 7 to 16min CH3CN=50/50 Flow rate : 1.0mL/min Detector : ELSD Column temp. : 30oC Sample : 0.5%, 20 µl Gradient mobile phase enables a better separation of hydrolyzed dextran in Mw. ELSD detection is effective. 26
Summary: Polymer vs ODS 1. Separation performance similar Good separation for hydrophilic substances 2. Better separation of basic substances Possible to analyze trace level of basic substances No need of ion-pair reagent 3. Alkaline solvent can be used Wide selection of solvent for analyses 4. Low influence of salt Better peak shape under low concentration of salt lower ion suppression 5. Good Reproducibility 27
Thank you for your attention! for details >>> www.shodex.de www.bioanalytics.co.il 28