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A Chromatographic Comparison of Silica-C18 HPLC Columns

A Chromatographic Comparison of Silica-C18 HPLC Columns

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A Chromatographic Comparison of Silica-C18 HPLC Columns

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  1. A Chromatographic Comparison of Silica-C18 HPLC Columns Charles H. Jersild Alltech Associates, Inc. 2051 Waukegan Road • Deerfield, IL 60015 Phone: 1-800-ALLTECH • Web Site: PP044

  2. Introduction • Silica-based C18 columns are the most commonly used columns for HPLC. The number of available silica-based C18 columns has risen greatly in the past few years and now stands at more than 150. The proliferation of C18 columns has made it difficult to choose the right column for an application or as an appropriate backup column. • Typically chromatographers choose HPLC columns by comparing the packing media specifications supplied by the manufacturer (i.e. surface area, particle size and shape, carbon load, endcapping, pore diameter, pore volume, bonding density, bonding type, etc.). This type of comparison, although sometimes useful, can not be used to accurately predict a column’s performance. Column selectivity and peak shape are largely influenced by the underlying silica, and to a lesser extent, the bonding procedure used rather than the packing material’s physical characteristics. Therefore, the best way to compare columns is by their chromatographic performance.

  3. In 1995, Steffeck and co-workers1 developed a chromatographic test and graphed the results in order to simplify comparisons of silica-based C18 and C8 HPLC columns based on their retention, symmetry, and selectivity for polar and non-polar compounds. The test mix used in that work contained acidic, basic, and neutral probes. In this study, we used a similar approach using a different test mix, and we ran the tests at two different pH values (pH 2.5 and pH 7). Additionally, we used a test mixture containing polyaromatic hydrocarbons to assess the columns’ shape selectivity as described by Sander and Wise2. • To compare column performance for a wide variety of compounds under both acidic (pH 2.5) and neutral (pH 7.0) conditions we used the test mixture shown in Figure 1. It includes polar and non-polar probes that are acidic, basic, or neutral. Each probe has a specific purpose in the test mix. Uracil is used as a void-volume marker. 3-Butylpyridine is a basic amine that tests the silanol activity toward bases. Tailing of this peak is an indicator of interaction with acidic silanols.

  4. Phenol is a weak acid used in conjunction with 3-butylpyridine to test the activity of the underlying silica. Their elution order is an indicator of the degree of silanol exposure. Columns with little silanol exposure retain phenol longer than 3-butylpyridine. A reversal in elution order indicates that there is a significant population of active silanols. 4-Phenylbutyric acid is a carboxylic acid that tests silanol activity towards acids. This peak tails when silanol activity towards acids is significant. 4-Phenylbutylamine is a strong base that tests silanol activity towards amines. N,N-diethyl-m-toluamide (DEET) is another polar probe. The selectivity of this weakly basic molecule with a neutral probe such as propylbenzene is good indicator for the degree of endcapping on a bonded phase. Non-endcapped phases give lower alpha values for this pair than do endcapped phases. Quinizarin is a chelator. It is used to check for residual metals in the packing. When residual metals are present, quinizarin will either tail or irreversibly adsorb onto the column packing. Propylbenzene and butylbenzene are neutral probes that test the capacity and hydrophobic selectivity of the bonded phase.

  5. To assess column shape selectivity we used an empirical test described by Sander and Wise2. The test uses Standard Reference Material® 869a (SRM 869a) shown in Figure 2, which contains three polyaromatic hydrocarbons (PAH). We added acetone as a void volume marker. The relative retention of Benzo[a]pyrene (BaP) and 1,2:3,4:5,6:7, 8-tetrabenzonaphthalene (TBN) is a measure of columns’ shape selectivity. The shape selectivity values obtained using this test have been shown to be an indicator of columns’ bonding type (monomeric or polymeric)2. • We tested 116 silica-based C18 (or similar) HPLC columns using the test mixes previously described. Sample retention, selectivity and peak shape for these probes reflect the hydrophobicity of the bonded phase, the activity of the underlying silica, and the bonding type. Retention and peak shape were compiled into charts, and shape selectivity data was tabulated to simplify column comparisons.

  6. Figure 1 - Test Probes used at pH 2.5 and pH 7 Uracil 3-Butylpyridine 4-Phenylbutylamine* 4-Phenylbutyric acid** Phenol Propylbenzene N,N-Diethyl- meta-toluamide Quinizarin Butylbenzene * 4-Phenylbutylamine was not used for testing at pH 2.5 ** 4-Phenylbutyric acid was not used for testing at pH 7

  7. Figure 2 - Shape Selectivity Test Probes Benzo[a]pyrene, BaP 1,2:3,4:5,6:7,8- Tetrabenzonaphthalene, TBN Phenanthro[3,4-c]phenanthrene, PhPh

  8. Materials and Equipment • Instrumentation • – On-Line Degassing System, Alltech Associates, Inc. (Deerfield, IL) • – Alltech Model 525 HPLC Pump, Alltech Associates, Inc. • – Alltech Model 580 Autosampler, Alltech Associates, Inc. • – 12-Position Electrically Actuated Valve, Valco Instruments, Co., Inc. (Houston, TX) • – JB-1000 Column Oven, Jordi Associates, Inc. (Bellingham, MA) • – Linear Model UVis-205 Absorbance Detector, ThermoQuest Corp. (San Jose, CA) • Data Collection • – Chrom Perfect™ Magellan software version 3.54, Justice Innovations (Mountain View, CA)

  9. Reagents and Samples – HPLC Grade water was produced in-house using a Millipore (Bedford, MA) Elix® purification system. – HPLC Grade Acetonitrile was purchased from VWR Scientific Products (S.Plainfield, NJ). – Uracil, phosphoric acid, potassium phosphate, monobasic anhydrous, and potassium phosphate, dibasic trihydrate were purchased from Sigma (St. Louis, MO). – N,N-diethyl-m-toluamide, quinizarin, propylbenzene, butylbenzene, 4-phenylbutyric acid, 4-phenylbutylamine, 3-butylpyridine, and phenol were purchased from Aldrich Chemical Co. (Milwaukee, WI). – Standard Reference Material® 869a (SRM 869a) was purchased from National Institute of Standards & Technology (Gaithersburg, MD). HPLC Columns – Table 1 lists the columns used and their suppliers. All of the columns had dimensions of 150mm (length) x 4.6mm (ID) except the Resolve™ C18 (3.9mm ID). The particle size of all the packings was 5µm with the exception of Genesis® C18 (4µm) and µBondapak® C18 (10µm).

  10. Table 1 - Listing of Columns Tested • The columns tested in this study and where they were obtained are listed below: • Alltech Associates, Inc. (Deerfield, IL) • Adsorbosil® C18, Adsorbosphere® C18, Adsorbosphere® HS C18, Adsorbosphere® UHS C18, Adsorbosphere® XL C18, Adsorbosphere® XL C18-B, Allsphere™ ODS-1, Allsphere™ ODS-2, Alltima™ C18, Alltima™ C18-LL, alphaBond™ C18, Spheri-5® ODS, Spheri-5® RP-18, Brownlee™ Validated™ C18, Econosil™ C18, Econosphere™ C18, Exsil™ ODS, Exsil™ ODS-B, Hypersil® 100 C18, Hypersil® BDS C18, Hypersil® ODS, Inertsil® ODS-2, Inertsil® ODS-3, Kromasil™ C18, LiChrosorb® C18, LiChrosorb® RP • Select B, LiChrospher® 100 RP-18 (endcapped), LiChrospher® 100 RP-18, LiChrospher® RP Select B, Nucleosil® C18, Nucleosil® C18AB, Nucleosil® C18HD, Nucleosil® PROTECT, Partsil® ODS-2, Perkin Elmer HS C18, Perkin Elmer Reduced Activity C18, Partisil™ ODS-3, Platinum™ EPS C18, Platinum™ C18, Ultrasphere® C18, Waters Spherisorb® ODS-1, Waters Spherisorb® ODS-2, Partisphere™ RTF • Bio-Rad® Laboratories (Hercules, CA) • Bio-Sil® HL 90-5 • Eichrom Technologies, Inc. (Darien, IL) • SynChropak® RPP-100, SynChropak® SCD-100 • ES Industries (West Berlin, NJ) • AquaSep™ C8 • Higgins Analytical, Inc. (Mountain View, CA) • CLIPEUS C18, HAISIL 100 C18, TARGA C18 • Jones Chromatography (Lakewood, CO) • APEX® Basic, APEX® II ODS, APEX® ODS, Genesis® C18 • Keystone Scientific, Inc. (Bellefonte, PA) • AQUASIL C18, BetaBasic® C18, BETASIL® C18, PRISM® RPN, PRISM® RP • MAC-MOD Analytical, Inc. (Chadds Ford, PA) • ACE® 5 C18, Eclipse® XDB-C18, HydroBond™ PS C18, ProntoSIL C18-H, ProntoSIL ODS-AQ, Zorbax® Bonus RP, Zorbax® Extend C18, Zorbax® ODS, Zorbax® Rx C18, Zorbax® SB-C18

  11. Table 1 - Listing of Columns Tested (Cont’d) • MetaChem Technologies (Torrance, CA) • Abzelute™ ODS DB, Carbosorb™ C18, MetaSil™ AQ C18, MetaSil™ Basic, MetaSil™ ODS, MetaSil™ MonoChrom™ C18, Polaris™ C18A • Phenomenex (Torrance, CA) • Columbus™ C18, Cosmosil™ C18AR, Cosmosil™ C18MS, Kingsorb™ C18, Luna® C18, Luna® C18(2), PrimeSphere™ C18, Prodigy™ ODS-2, Prodigy™ ODS-3 • Restek Corp. (Bellefonte, PA) • Allure™ C18, Ultra C18, Ultra IBD • The Separations Group, Inc. (Hesperia, CA) • Vydac® 201SP C18 • SGE Incorporated (Austin, TX) • Wakosil™ C18RS • Supelco (Bellefonte, PA) • Discovery® Amide C16, Discovery® C18, Supelcosil™ ABZ+, Supelcosil™ LC18 DB, Supelcosil™ LC18, Suplex pKb-100 • Thermo Hypersil, Inc. (Runcorn, U.K.) • HyPURITY® Advance, HyPURITY® Elite • TosoHaas (Montgomeryville, PA) • TSK™- GEL ODS80TS • Varian, Inc. (Walnut Creek, CA) • ChromSpher 5 C18, Microsorb 100, OmniSpher C18 • Waters Corp. (Milford, CT) • J’Sphere™ ODS M80, Novapak® C18, Resolve™ C18, Symmetry® C18, SymmetryShield™ C18, • µBondapak® C18, XTerra™ RP18, XTerra™ MS18, YMCbasic™, YMCPack ODS-A™, YMCPack ODS-AL™, • YMC Pack ODS-AM™, YMCPack ODS-AQ™, YMCPack PRO C18™

  12. Experimental • Each column was subjected to three different chromatographic tests. Test 1 used the mixture of acidic, basic, and neutral probes listed in Figure 1 and a pH 2.5 potassium phosphate buffer/acetonitrile mobile phase. Test 2 used a similar test mix and a pH 7.0 potassium phosphate buffer/acetonitrile mobile phase. Test 3 used the SRM® 869 test mix for shape selectivity determination. Test conditions are described in more detail in the HPLC Conditions section. • Mobile phase was prepared in one large batch for each test. Concentrated stock solutions of the individual sample probes used in tests 1 and 2 were prepared by dissolving reagents in mobile phase. Known amounts of these stock solutions were mixed and diluted in mobile phase to make-up the test mixes. The shape selectivity test mix (SRM® 869a) was obtained pre-mixed and we added acetone as a void marker. The concentrations of individual probes in all of the test mixes were varied such that we could use peak areas to help in identifying peaks. Individual samples of the test probes having the same concentrations as in the test mixes were also prepared. We injected these individual test probes when necessary for peak identification and to isolate peaks for tailing factor calculations.

  13. All of the columns used in this study were previously unused and were either freshly packed by Alltech Associates, Inc. or recently purchased. Prior to each experiment, the columns were flushed with at least 15 column volumes of mobile phase. Each test mix was injected twice on each column. Reproducibility of the injections was checked to confirm that columns were fully equilibrated. The testing was done using an automated system capable of unattended testing of 12 columns in succession. • We calculated capacity factors using k´= (tr – t0)/t0, where tr is the retention time of the peakof interest and t0 is experimentally determined void time. We calculated tailing factors (T) using the United States Pharmacopoeia formula, T = W0.05/2f, where W0.05 is the peak width at 5% peak height and f is the distance at 5% height from the leading edge of the peak to a perpendicular drawn from the peak maximum to the baseline. Shape selectivity or TBN/BaP was calculated as the ratio of capacity factors k´TBN/k´BaP.

  14. Test 1 • HPLC Conditions • Mobile Phase: Acetonitrile:20mM Potassium Phosphate, pH 2.5 (65:35) • Flowrate: 1.0mL/min • Column Temperature: 30°C • Detection: UV at 254nm • Injection Volume: 10µL • Sample (mg/mL): Uracil (0.025), 3-butylpyridine (0.067), phenol (0.90), 4-phenylbutyric acid (2.1), N,N-diethyl-m-toluamide (0.66), quinizarin (0.20), propylbenzene (5.0), and butylbenzene (6.7) dissolved in mobile phase

  15. Test 2 • HPLC Conditions • Mobile Phase: Acetonitrile:20mM Potassium Phosphate, pH 7.0 (65:35) • Flowrate: 1.0mL/min • Column Temperature: 30°C • Detection: UV at 254nm • Injection Volume: 10µL • Sample (mg/mL): Uracil (0.05), 3-butylpyridine (0.02), phenol (1.0), 4-phenylbutylamine (4.0), N,N-diethyl-m-toluamide (1.0), quinizarin (0.20), propylbenzene (4.0), and butylbenzene (4.0) dissolved in mobile phase

  16. Test 3 • HPLC Conditions • Mobile Phase: Acetonitrile:Water (85:15) • Flowrate: 2.0mL/min • Column Temperature: 25°C • Detection: UV at 254nm • Injection Volume: 10µL • Sample: SRM® 869a (contains: Benzo[a]pyrene, 1,2:3,4:5,6:7,8-tetrabenzonaphthalene, and phenanthro[3,4-c]phenanthrene), and Acetone (added as void marker)

  17. Results and Discussion • Test 1 • Figure 3a shows a chromatogram that is typical of those obtained with base-deactivated columns. Figure 3b shows a chromatogram that is typical of those obtained using columns developed before base-deactivation techniques were available. These two chromatograms differ in 3 aspects: peak order, peak shape, and retention. In Figure 3a, the basic probe, 3-butylpyridine, elutes before phenol. In Figure 3b, 3-butylpyridine elutes after phenol. Figure 3a shows sharp, symmetrical peaks for all of the test probes. In Figure 3b, 3-butylpyridine and the metal chelator, quinizarin, tail significantly. In Figure 3a, the overall retention times are higher than the retention times in Figure 3b.

  18. Peak order and peak shape of the polar compounds is related to the activity of the underlying silica. In Figure 3a, 3-butylpyridine elutes prior to phenol with good peak shape on the base-deactivated column. In Figure 3b, the same peak elutes later with poor peak symmetry. From these results we can conclude that the base-deactivated column (3a) is less active towards polar amines than the non base-deactivated column (3b). Neither of the columns showed activity towards acids as they both give good peak shape for the acidic probe (4-phenylbutyric acid). • The retention and peak shape of the chelator, quinizarin, is related to the amount of metal impurity in the underlying silica. Most base-deactivated phases have low levels of metal impurities, so the quinizarin peak shape is symmetrical as in Figure 3a. Some of the older type silicas have higher levels of metal impurities. These columns tend to give poor peak shapes for quinizarin (see Figure 3b), or in some cases irreversibly adsorb the compound.

  19. Test 2 • Figures4a and 4b show chromatograms obtained at pH 7 with the same base-deactivated and non-base-deactivated columns shown in Figures 3a and 3b. • The chromatogram in Figure 4a in typical of what was observed for base-deactivated type columns. Peak shapes were all symmetrical except for 4-phenylbutylamine, which has an odd peak shape that we’re unable to explain. The elution order is slightly different at pH 7.0 than it was at pH 3.0. 3-Butylpyridine elutes later in the chromatogram, indicating the increased silanol activity that is expected at higher pH. • The chromatogram in Figure 4b is typical of what was observed for many of the non-base-deactivated column types. Quinizarin, the metal chelator, did not elute from this column at pH 7.0, an indication that the packing material contains a significant amount of metal impurities. Also typical is the poor peak symmetry and longer retention of the basic probes (3-butylpyridine, and 4-phenylbutylamine).

  20. Column Comparisons Charts* based on Tests 1 and 2 • Figure 5 is a graphical presentation of data from 25 of the columns we tested at pH 2.5, while Figure 6 presents data from the same 25 columns at pH 7.0.On the x-axes are test probe capacity factors (k’), while y-axes show the columns in descending order of butylbenzene capacity factors (k’). The right-hand side of each chart lists test probes that that did not elute and test probes that tailed (T  2.0) under our test conditions. • These charts are helpful for choosing back-up or alternative columns. We can choose a good backup column by finding columns with similar retention and selectivity. Alternatively, if we are looking for a replacement for a column that can not separate a sample adequately, we can choose one with different retention and selectivity characteristics. • *Note: Due space limitations, charts containing data for all 116 columns tested were not included here. Charts containing data for all of the columns tested will be included in reprints of this poster.

  21. When choosing a column to separate a specific sample, it is important to compare based on only those test probes that are similar to the sample components. If the sample contains only non-polar components, then compare columns based on the retention and selectivity of the hydrophobic probes (propyl- and butylbenzene). If the sample is a polar amine or acid then compare columns based on the 3-butylpyridine and DEET, or 4-phenylbutyric acid probes. Additionally, it is best to compare based on the appropriate pH. If you intend to work at acidic pH, then use the pH 2.5 comparison chart (Figure 5). If you plan to work at neutral pH then use the pH 7 comparison chart (Figure 6).

  22. Test 3 • We determined column shape selectivity from the results of test 3. A shape selectivity value was calculated for each column using the formula TBN/BaP= k´TBN/k´BaP, where TBN/BaP is shape selectivity, k´TBN is the capacity factor of 1,2:3,4:5,6:7,8-tetrabenzonaphthalene, and k´BaP is the capacity factor for Benzo[a]pyrene. Shape selectivity values for all columns are listed in Table 2. Sander and Wise2 have shown that these shape selectivity values are good indicators of C18 bonding phase type. Phases with shape selectivity values  1.7 reflect monomeric-like C18 bonding. An example is the Platinum™ C18 column with a shape selectivity value of 1.84 (the chromatogram is shown in Figure 7a). Phases with shape selectivity values  1 reflect polymeric-like C18 bonding. An example is the Nucleosil® C18AB column with a shape selectivity value of 0.69 (the chromatogram is shown in Figure 7b). Shape selectivity values between 1 and 1.7 reflect C18 phases of intermediate type bonding. These may be densely loaded monomeric phases or lightly loaded polymeric phases. An example is the Inertsil® ODS-2 column with a shape selectivity value of 1.36 (the chromatogram is shown in Figure 7c).

  23. Figures 3 - Chromatograms at pH 2.5 3a Base-deactivated 3b Non base-deactivated 9238 9239 1. Uracil 2. 3-butylpyridine 3. Phenol 4. 4-phenylbutyric acid 5. N,N-diethyl-m-toluamide (DEET) 6. Quinizarin 7. Propylbenzene 8. Butylbenzene Column: Alltima™ C18, 5m, 150 x 4.6mm Column: Adsorbosphere™ C18, 5m, 150 x 4.6mm

  24. Figures 4 - Chromatograms at pH 7 4a Base-deactivated 4b Non base-deactivated 1. Uracil 2. Phenol 3. 4-phenylbutylamine 4. N,N-diethyl-m-toluamide (DEET) 5. 3-butylpyridine 6. Quinizarin 7. Propylbenzene 8. Butylbenzene 9324 9325 Column: Alltima™ C18, 5m, 150 x 4.6mm Column: Adsorbosphere® C18, 5m, 150 x 4.6mm

  25. Figure 5 - Column Comparison at pH 2.5

  26. Figure 6 - Column Comparison at pH 7

  27. Table 2 - Shape Selectivity Shape Shape Shape Packing Selectivity Packing Selectivity Packing Selectivity Abzelute™ ODS DB 1.98 HAISIL 100 C18 1.92 PRISM® RP 1.58 ACE® 5 C18 1.95 HydroBond™ PS C18 2.08 PRISM® RPN 1.28 Adsorbosil® C18 1.60 Hypersil® 100 C18 1.99 Prodigy™ ODS-2 1.93 Adsorbosphere® C18 1.78 Hypersil® BDS C18 1.63 Prodigy™ ODS-3 2.14 Adsorbosphere® HS C18 1.99 Hypersil® ODS 1.90 ProntoSIL C18-H 2.03 Adsorbosphere® UHS C18 1.11 HyPURITY® Advance 1.30 ProntoSIL ODS-AQ 1.62 Adsorbosphere® XL C18 1.57 HyPURITY® Elite 1.68 Resolve™ C18 1.86 Adsorbosphere® XL ODS-B 1.76 Inertsil® ODS-2 1.36 Supelcosil™ ABZ+ 1.10 Allsphere™ ODS-1 1.54 Inertsil® ODS-3 2.24 Supelcosil™ LC18 1.91 Allsphere™ ODS-2 1.76 J'Sphere™ ODS M80 2.06 Supelcosil™ LC18 DB 1.94 Alltima™ C18 1.83 Kingsorb™ C18 2.13 Suplex pKb 100 1.17 Alltima™ C18-LL 1.82 Kromasil™ C18 1.73 Symmetry® C18 1.60 Allure™ C18 1.74 LiChrosorb® C18 1.67 SymmetryShield™ C18 1.49 alphaBond™ C18 1.94 LiChrosorb® RP Select B 1.71 SynChropak® RPP-100 1.99 APEX® Basic 0.70 LiChrospher® RP Select B 1.66 SynChropak® SCD-100 1.57 APEX® II ODS 1.90 Lichrospher® RP18 1.60 TARGA C18 2.07 APEX® ODS 1.78 Lichrospher® RP18 (endcap) 1.76 TSK™-GEL ODS80TS 2.12 AquaSep™ C8 1.73 Luna® C18 2.14 Ultra C18 1.76 AQUASIL C18 1.66 Luna® C18 (2) 2.13 Ultra IBD 1.23 BetaBasic® C18 1.69 MetaSil™ AQ C18 1.44 Ultrasphere® C18 1.90 BETASIL® C18 1.72 MetaSil™ Basic 1.61 Vydac® 201SP C18 1.98 Bio-Sil® HL 90-5 1.82 MetaSil™ ODS 1.46 Wakosil™ C18 RS 2.09 Brownlee™ Spheri-5® ODS 1.55 Microsorb 100 C18 1.93 Waters Spherisorb® ODS-1 1.39 Brownlee™ Spheri-5® RP-18 1.94 MonoChrom™ C18 1.60 Waters Spherisorb® ODS-2 1.80 Brownlee™ Validated™ C18 1.85 Novapak® C18 1.90 XTerra™ MS18 2.03 Carbosorb™ C18 1.99 Nucleosil® C18 1.78 XTerra™ RP18 1.74 ChromSpher 5 C18 1.39 Nucleosil® C18 AB 0.69 YMCbasic™ 1.82 CLIPEUS C18 2.00 Nucleosil® C18 HD 1.79 YMCPack ODS-AL™ 1.82 Columbus™ C18 2.14 Nucleosil® PROTECT 1.52 YMCPack ODS-AM™ 1.94 Cosmosil™ C18AR 1.47 OmniSpher C18 1.38 YMCPack ODS-AQ™ 2.08 Cosmosil™ C18MS 1.93 Partisil® ODS-2 1.32 YMCPack ODS-A™ 1.94 Discovery® Amide C16 1.62 Partisil® ODS-3 1.94 YMCPack PRO C18™ 2.13 Discovery® C18 1.75 Partisphere™ RTF C18 1.10 Zorbax® Bonus RP 1.84 Eclipse® XDB-C18 2.11 PerkinElmer HS C18 2.04 Zorbax® Extend C18 1.93 Econosil™ C18 1.57 PerkinElmer Red. Activity C18 1.52 Zorbax® ODS 1.77 Econosphere™ C18 1.94 Platinum™ C18 1.84 Zorbax® Rx C18 1.54 Exsil™ ODS 1.76 Platinum™ EPS C18 1.21 Zorbax® SB C18 2.16 Exsil™ ODS-B 0.76 Polaris™ C18A 1.72 µBondapak® C18 1.88 Genesis® C18 1.96 PrimeSphere™ C18MC 1.83

  28. Figures 7 - Shape Selectivity Chromatograms 7b Nucleosil® C18 AB, 5µm, 150 x 4.6mm 7a Platinum™ C18, 5µm, 150 x 4.6mm 7c Inertsil® ODS-2, 5µm, 150 x 4.5mm 9321 9320 9326 1. Acetone 2. Benzo[a]pyrene, (BaP) 3. Phenanthro[3,4-c]phenanthrene, (PhPh) 4. 1,2:3,4:5,6:7,8-Tetrabenzonaphthalene, (TBN)

  29. Conclusion • One-hundred and sixteen silica-based C18 (or similar) columns were compared using 3 different chromatographic tests. The chromatographic data was compiled into charts and tables for easy comparison of columns. These charts and tables simplify selection of backup or alternative columns based on chromatographic performance rather than on the physical characteristics of the packing material.

  30. Acknowledgements • Alltech Associates, Inc. acknowledges that this technical communication represents a considerable investment in time and effort on the part of many dedicated professionals. • Author(s): Charles H. Jersild • Laboratory Contribution(s): Charles H. Jersild, Michelle Cornelius • Editorial Contribution(s): Raymond J. Weigand, Robert J. Ziegler • Graphics Preparation: Kimberly Volk, Elizabeth Fisher, and • Julia Poncher • The trademarks referred to herein are the property of their respective owners.

  31. References • 1. R.J. Steffeck, “A Comparison of Silica-Based C18 and C8 HPLC Columns to Aid Column Selection”, LC•GC 13 (9), 720-726 (1995). • 2. Sander and Wise, “Evaluation of Shape Selectivity in Liquid Chromatography”, LC•GC, 8 (5), 378-390 (1990).

  32. Figure 5 - Column Comparison at pH 2.5

  33. Figure 5 - Column Comparison at pH 2.5 (cont’d)

  34. Figure 5 - Column Comparison at pH 2.5 (cont’d)

  35. Figure 5 - Column Comparison at pH 2.5 (cont’d)

  36. Figure 6 - Column Comparison at pH 7

  37. Figure 6 - Column Comparison at pH 7 (cont’d)

  38. Figure 6 - Column Comparison at pH 7 (cont’d)

  39. Figure 6 - Column Comparison at pH 7 (cont’d)