Stationary Phase Specifications | Phases Compared According to Relative
Hydrophobicity | Phases Compared According to Relative Polarity | Categorization
of Phases According to Hydrophobicity and Polarity | Comparison of Column Efficiency for a Neutral Compound | Comparison of Column Efficiency for Basic Compounds | Phases Grouped According to Silanol Activity | Comparison of Phases According to Metal Activity Fourth Edition June 2008 Comparison Guide to C18 Reversed Phase HPLC Columns Comparison Data on Commonly Used C18 Phases a n a l y t i c a l
i n c . MAC-MOD Analytical 103 Commons Court P.O. Box 587 Chadds Ford, PA 19317 Phone: 1.800.441.7508 Fax: 1.610.358.5993 E-mail: info@mac-mod.com Web Site: www.mac-mod.com a n a l y t i c a l
i n c . Comparison Guide to C18 Reversed Phase HPLC Columns Introduction Tere are so many dierent C18 columns to choose from, that nding the right column for a particular separation can be very time consuming and expensive. Two apparently similar C18 phases can give very dierent results. For example, Figure 1 compares the separation of the same sample mixture on a Hypersil HyPurity C18 and a Symmetry C18 column under identical mobile phase conditions. Even though both columns are packed with base deactivated C18 stationary phases, the band spacing (selectivity) between peaks is very dierent on the two columns. Without more information, it is impossible to predict how the performance of dierent stationary phases will compare. Tis Comparison Guide to C18 Reversed Phase HPLC Columns provides basic comparison information on commonly used C18 columns to help you more easily identify similarities and dierences before investing time and money in chromatographic testing. Hopefully, this infor- mation will help you nd the right column for your application quicker. Only silica based C18 bonded phases are evaluated in this Guide. Other bonded phases, such as C8, CN, Phenyl and polar embedded phases, are excluded. Tis Guide does not identify an overall best column. Te column that works best for one application will not necessarily be the column that will work best for other applications. And, there certainly is not a single column that will work best for all applications. However, this Guide can help you identify columns that are likely to perform well so that at least you can narrow the number of columns for chromatographic testing. You may nd that this Guide helps you identify several columns that provide good separations and performance. It is always desirable to have more than one column identied for an application, especially if you are running routine assays. Increasingly, chromatographers are seeking to identify alternate brands of HPLC columns suitable for their assays. Having an alternate column choice for a method reduces the risk of down time due to column problems such as a change in selectivity from one manufactured lot to another or slow supplier delivery. Finding an alternate or back-up column that will provide acceptable selectivity and performance when substituted into a method can be as expensive and time consuming as nding the right column for developing an initial separation. It is our hope that this Guide will make that job easier by identifying columns with similar chromatographic characteristics.
Figure 1 Apparently Similar C18 Phases Can Give Very Different Chromatographic Results Mobile Phase: 60% CH 3 CN 40% 50mM KH 2 PO 4 , pH 3.2 Sample: 1. Uracil 5. N,N-Dimethylaniline 2. Pyridine 6. 4-Butylbenzoic acid 3. Phenol 7. Toluene 4. Dimethyl phthalate Both Hypersil HyPURITY C18 and Symmetry C18 are base deactivated phases. You would expect them to provide similar performance, and in some cases they do. However, in the example given here you can see signifcant differences in peak retention times, selectivity and even peak shape. Tis Guide provides the following comparison data on commonly used C18 phases: Stationary Phase Specifcations Specications provided by column manufacturers Phases Compared According to Relative Hydrophobicity Retention data for hydrophobic and neutral compounds Phases Compared According to Relative Polarity Categorization of Phases According to Hydrophobicity and Polarity Comparison of Column Efciency for a Neutral Compound Comparison of Column Efciency for Basic Compounds Also measures peak tailing Phases Grouped According to Silanol Activity Phases Compared According to Metal Activity
0 1 2 3 4 5 6 7 8 9 10 11 Time (min) 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 8 9 10 11 Tine (nin) 1 2 3 4 5 6 7 Comparison Guide to C18 Reversed Phase HPLC Columns | 1 Hypersil HyPURITY C18 Symmetry C18 0 1 2 3 4 5 6 7 Tine (nin) 1 2 3 Figure 3 Specifcations of C18 Stationary Phases Particle Pore Surface Carbon High Purity Stationary Phase Size (m) Size () Area (m2/g) Load (%) Endcapped Silica ACE C18 5 100 300 15.5 yes yes ACE C18-300 5 300 100 9.0 yes yes ACE C18-HL 5 90 400 20.0 yes yes Bondapak C18 10 125 330 10 yes no Capcell Pak AG C18 5 120 300 15 yes no Capcell Pak UG C18 5 120 300 15 yes yes Develosil ODS-HG 5 140 300 18 yes yes Develosil ODS-MG 5 100 450 15 yes yes Develosil ODS-UG 5 140 300 18 yes yes Exsil ODS 5 100 200 11 yes no Exsil ODS1 5 100 200 11 yes no Exsil ODSB 5 100 200 12 yes no Gemini C18 5 110 375 14 yes yes Hichrom RPB 5 110 340 14 yes yes Hypersil BDS C18 5 130 170 11 yes no Hypersil GOLD 5 180 200 10 yes yes Hypersil HyPurity C18 5 180 200 13 yes yes Hypersil ODS 5 120 170 10 yes no Inertsil ODS 5 100 350 14 yes no Inertsil ODS3 5 100 450 15 yes yes Inertsil ODS2 5 150 320 18.5 yes yes Kromasil C18 5 100 340 19 yes yes LiChrosorb RP-18 10 100 300 17 no no LiChrospher RP-18 5 100 350 21.6 no no Luna 5 C18(2) 5 100 400 17.5 yes yes Novapak C18 4 60 120 7.3 yes no Nucleosil C18 5 100 350 15 yes no Nucleosil C18 HD 5 100 - 20 yes yes Nucleosil C18AB 5 100 350 24 yes no Partisil ODS 10 85 350 5 no no Partisil ODS2 10 85 350 15 yes no Partisil ODS3 10 85 350 10.5 yes no Prodigy ODS2 5 150 310 18.4 yes yes Prodigy ODS3 5 100 450 15.5 yes yes Purospher RP18-e 5 80 500 - yes yes Resolve C18 5 90 200 10 no no SunFire C18 5 100 340 16 yes yes Symmetry C18 5 100 335 19 yes yes TSK ODS-120T 5 120 - 22 yes no TSK ODS-80TM 5 80 - 15 yes no Ultrasphere ODS 5 80 - 12 yes no Vydac 218MS 5 300 70 - yes no Vydac 218TP 5 300 70 8 yes no Vydac Selectapore 300M 5 300 70 - yes yes Vydac Selectapore 300P 5 300 70 - yes yes Vydac Selectapore 90M 5 90 250 - yes yes Waters Spherisorb ODS1 5 80 220 6.2 no no Waters Spherisorb ODS2 5 80 220 11.5 yes no Waters Spherisorb ODSB 5 80 220 11.5 yes no XTerra MS C18 5 125 - 15.5 yes YMC JSphere ODS H80 4 80 510 22 yes no YMC JSphere ODS M80 4 80 510 14 yes no YMC ODS A 5 120 300 17 yes no YMC ODS AM 5 120 300 17 yes no YMC Pro C18 5 120 335 16 yes yes Zorbax Extend C18 5 80 180 12.5 yes yes Zorbax ODS 5 70 330 20 yes no Zorbax Rx-C18 5 80 180 12 no yes Zorbax SB-C18 5 80 180 10 no yes Zorbax XDB-C18 5 80 180 10 yes yes Figure 2 High Purity Silicas Provide Better Peak Shape for Basic Compounds High Purity Silica (ACE C18) Acidic Silica (Waters Spherisorb ODS2) Mobile Phase: 60% CH 3 OH, 40% H 2 O Sample: 1. Uracil 2. Pyridine 3. Phenol Interaction between cationic compounds and acidic silanol sites on the surface of silica stationary phase supports can contribute to retention and peak tailing. Phases made with high purity silica (less acidic silica) generally can be expected to provide better peak shape for basic compounds. 0 1 2 3 4 5 Tine (nin) 1 2 3 2 | Comparison Guide to C18 Reversed Phase HPLC Columns Figure 4 C18 Phases Compared According to Relative Hydrophobicity Stationary Phase Specifcations Stationary phase specications provide basic information that can be helpful in deciding which phases to select for evaluation. For example, phases with high surface area and high carbon load will generally retain hydrophobic compounds longer than phases with low surface area and low carbon load. If you are analyzing macromolecules, such as peptides and proteins, a wider pore (200 300 ) phase usually provides better performance than a phase with small pores. New high purity silicas usually provide better peak shape for basic compounds than older, more acidic silicas (see Figure 2). Stationary phase specications, however, will not give you enough information to accurately predict retention or band spacing (selectivity). Tis is especially true when separating polar compounds. Inertsil ODS3 LiChrospher RP-18 YMC JSphere ODS H80 Nucleosil C18 Kromasil C18 Develosil ODS-MG Partisil ODS2 LiChrosorb RP-18 Symmetry C18 Inertsil ODS Develosil ODS-UG Purospher RP18-e YMC Pro C18 Prodigy ODS3 Zorbax ODS Nucleosil C18HD SunFire C18 YMC ODS AM YMC ODS A Inertsil ODS2 Cosmosil C18-AR Prodigy ODS2 TSK ODS-80TM ACE C18 Luna C18(2) Genesis C18 Nucleosil C18AB Waters Spherisorb ODS2 Zorbax Extend C18 Develosil ODS-HG Resolve C18 Zorbax RX C18 Zorbax XDB C18 Waters Spherisorb ODSB Hypersil BDS C18 Hypersil HyPurity C18 Ultrasphere ODS TSK ODS-120T Partisil ODS3 Hichrom RPB Waters Spherisorb ODS1 Bondapak C18 Exsil ODS Capcell Pak AG C18 YMC JSphere ODS M80 Capcell Pak UG C18 Zorbax SB C18 Exsil ODSB XTerra MS C18 ACE C18-300 Hypersil ODS Hypersil GOLD Novapak C18 Exsil ODS1 Vydac SelectaPore 90M Vydac SelectaPore 300P Vydac 218TP Partisil ODS Vydac 218MS Vydac SelectaPore 300M
Mobile Phase: 90% CH 3 OH, 10% H 2 O Elution Order: 1. Dimethyl phthalate 2. Toluene 3. Biphenyl 4. Phenanthrene Column Temperature: 24C Comparison Guide to C18 Reversed Phase HPLC Columns | 3 lner:s|| ODS3 Furospner RF-!8e Kronas|| C!8 YMC 'Spnere ODS H80 ACE C!8-HL Deve|os|| ODS-MC Synne:ry C!8 Frod|gy ODS3 SunF|re C!8 Zorbax ODS L|Cnrospner RF-!8 Luna C!8(2) Zorbax Ex:end C!8 lner:s|| ODS2 Nuc|eos|| C!8 Cen|n| C!8 Nuc|eos|| C!8 HD Zorbax XDE-C!8 Frod|gy ODS2 Deve|os|| ODS-UC Zorbax Rx C!8 lner:s|| ODS \a:ers Spner|sorb ODS2 Far:|s|| ODS2 ACE C!8 YMC ODS A YMC Fro C!8 YMC ODS AM XTerra MS C!8 Capce|| Fa| AC C!8 U|:raspnere ODS Reso|ve C!8 Zorbax SE-C!8 TSK ODS-80TM \a:ers Spner|sorb ODSE H|cnron RFE L|Cnrosorb RF-!8 Deve|os|| ODS-HC Exs|| ODS Nuc|eos|| C!8AE Hypers|| ODS Hypers|| EDS YMC 'Spnere ODS M80 TSK ODS-!20T Far:|s|| ODS3 Hypers|| HyFURlTY C!8 \a:ers Spner|sorb ODS! Vydac Se|ec:apore 90M Novapa| C!8 Hypers|| COLD Exs|| ODS! ACE C!8-300 Eondapa| C!8 Exs|| ODSE Vydac 2!8MS Vydac 2!8TF Vydac Se|ec:apore 300F Vydac Se|ec:apore 300M Far:|s|| ODS 0.0 0.5 1.0 k (ToIuene) 1.5 2.0 2.5 Figure 5 C18 Phases Ranked According to Retention for Toluene Mobile Phase: 80% CH 3 OH 20% 25mM KH 2 PO 4 , pH 6.0 Sample: Toluene Temperature: 24C Phases Compared According to Relative Hydrophobicity Hydrophobicity is measured as the retention of a hydrophobic solute, phenanthrene. Figure 4 gives a comparison of hydrophobicity with the C18 phases listed according to hydrophobicity. Notice, however, that the retention for dimethyl phthalate, the least hydrophobic solute in the mixture, cannot always be predicted from the hydrophobicity ranking. Some low hydrophobicity phases actually have greater retention for dimethyl phthalate than some high hydrophobicity phases. We nd that this is not unusual when separating polar compounds. Phases that are signicantly more retentive for hydrophobic analytes may show only slightly more retention for polar compounds than low hydrophobicity phases, and sometimes they show less.
Alternative Test for Hydrophobicity Toluene can also be used as a probe to measure hydrophobicity. Notice that the ranking of C18 phases according to retention for toluene (Figure 5) is slightly dierent from the ranking according to retention for phenanthrene (Figure 4). 4 | Comparison Guide to C18 Reversed Phase HPLC Columns YMC Fro C!8 Deve|os|| ODS-UC Luna C!8(2) Cen|n| C!8 Exs|| ODSE Zorbax XDE-C!8 Deve|os|| ODS-MC ACE C!8 Capce|| Fa| UC C!8 Deve|os|| ODS-HC H|cnron RFE ACE C!8-300 SunF|re C!8 Frod|gy ODS2 Hypers|| HyFur|:y C!8 lner:s|| ODS3 Kronas|| C!8 Nuc|eos|| C!8 HD ACE C!8-HL Hypers|| COLD Frod|gy ODS3 Zorbax Ex:end C!8 lner:s|| ODS XTerra MS C!8 lner:s|| ODS2 Far:|s|| ODS2 Synne:ry C!8 YMC ODS AM YMC ODS A Nuc|eos|| C!8AE Capce|| Fa| AC C!8 Far:|s|| ODS3 Vydac Se|ec:apore 90M Nuc|eos|| C!8 U|:raspnere ODS Vydac Se|ec:apore 300M \a:ers Spner|sorb ODSE Furospner RF!8-e TSK ODS-!20T YMC 'Spnere ODS M80 YMC 'Spnere ODS H80 Zorbax Rx-C!8 Zorbax SE-C!8 L|Cnrosorb RF-!8 Novapa| C!8 TSK ODS-80TM Eondapa| C!8 Vydac 2!8MS \a:ers Spner|sorb ODS2 Exs|| ODS L|Cnrospner RF-!8 Vydac Se|ec:apore 300F Vydac 2!8TF Hypers|| EDS C!8 Far:|s|| ODS Zorbax ODS Exs|| ODS! \a:ers Spner|sorb ODS! Reso|ve C!8 Hypers|| ODS 0 1 2 k (Fyridine) / k (FhenoI) 3 4 5 High FoIarity Low FoIarity Figure 6 C18 Phases Ranked According to Polarity Phases Ranked According to Relative Polarity Tere have been several chromatographic tests suggested for measuring polarity of stationary phases. Although there is no one test that we think provides a denitive measurement, we have chosen to use the ratio of k values for pyridine and phenol as our measure of relative polarity for these C18 phases. Figure 6 ranks stationary phases according to relative polarity using the test conditions given. In this ranking, there is not necessarily a signicant dierence between consecutive listings. If a dierent mobile phase condition was used for the test, e g., a lower mobile phase pH, or if dierent probes were used, the ranking may be somewhat dierent. However, phases at the high polarity end of the ranking and phases at the low polarity end of the ranking are likely to test that way under most polarity tests conditions. Terefore, this ranking can be used to identify relative dierences and similarities in polarity that can aect selectivity for polar compounds Since silanol activity is a major contributor to phase polarity, the test conditions used here to measure polarity have also been used by some chromatographers as an indication of silanol activity. Tis seems consistent with the fact that most phases at the high polarity end of the ranking use more acidic silicas as stationary phase supports where phases at the low polarity end of the ranking use less acidic (high purity) silicas. However, there are other factors that contribute to the retention of pyridine and phenol that prevent us from using their relative retention as a reliable measure of silanol activity. For example, Inertsil ODS has moderate polarity but shows signicant silanol activity in other tests. Also, we see that Prodigy ODS2 tests with similar polarity as the ACE C18, but in tests for silanol activity, the ACE C18 shows signicantly less silanol activity (see Figures 10 and 13). Mobile Phase: 60% CH 3 OH 40% H 2 O Polarity = k Pyridine / k Phenol Temperature: 24C Comparison Guide to C18 Reversed Phase HPLC Columns | 5 High Polarity/ High Polarity/ High Polarity/ Low Hydrophobicity Moderate Hydrophobicity High Hydrophobicity Exsil ODS1 Exsil ODS LiChrosorb RP-18 Hypersil ODS Hypersil BDS C18 LiChrospher RP-18 Novapak C18 Resolve C18 Partisil ODS TSK ODS-80TM Vydac 218MS Bondapak C18 Vydac 218TP Waters Spherisorb ODS1 Vydac Selectapore 300P Waters Spherisorb ODS2 Zorbax ODS Moderate Polarity/ Moderate Polarity/ Moderate Polarity/ Low Hydrophobicity Moderate Hydrophobicity High Hydrophobicity Vydac Selectapore 300M Capcell Pak AG C18 ACE C18-HL Vydac Selectapore 90M Inertsil ODS Nucleosil C18 Xterra MS C18 Inertsil ODS2 Partisil ODS2 Nucleosil C18 AB Symmetry C18 Partisil ODS3 YMC JSphere ODS H80 Prodigy ODS3 Purospher RP18-e TSK ODS-120T Ultrasphere ODS Waters Spherisorb ODSB YMC JSphere ODS M80 YMC ODS A YMC ODS AM Zorbax Extend C18 Zorbax Rx-C18 Zorbax SB-C18 Low Polarity/ Low Polarity/ Low Polarity/ Low Hydrophobicity Moderate Hydrophobicity High Hydrophobicity ACE C18-300 ACE C18 Develosil ODS-MG Exsil ODSB Capcell Pak UG C18 Inertsil ODS3 Hypersil GOLD Develosil ODS-HG Kromasil C18 Develosil ODS-UG Gemini C18 Hichrom RPB Hypersil HyPurity C18 Luna C18(2) Nucleosil C18 HD Prodigy ODS2 SunFire C18 YMC Pro C18 Zorbax XDB-C18 Figure 7 C18 Phases Grouped According to Hydrophobicity and Polarity Phases are listed in alphabetical order by category. Figure 8 Chromatographic Comparison of Stationary Phases from Different Categories ACE C18 Low Polarity/Moderate Hydrophobicity Hypersil BDS C18 High Polarity/Moderate Hydrophobicity Symmetry C18 Moderate Polarity/High Hydrophobicity Mobile Phase: 80% CH 3 OH, 20% 25mM KH 2 PO 4 , pH 6.0 Sample: 1. Norephedrine 4. Imipramine 2. Nortriptyline 5. Amitriptyline 3. Toluene Categorization of phases according to hydrophobicity and polarity. Te hydrophobicity and polarity data can be used to group phases with similar characteristics into categories. Te following criteria was used for the categories: Hydrophobicity High > 2.0 Moderate 1.30 to 1.99 Low < 1.30 High > 1.00 Moderate 0.50 to 0.99 Low < 0.50 k pyridine k phenol Polarity k for phenanthrene Figure 8 provides an example of how columns from dierent polarity/hydrophobicity categories will compare. In this separation of antidepressants, Symmetry C18 (high hydrophobicity) is slightly more retentive than Hypersil BDS-C18 (moderate hydrophobicity), and the band spacing of ACE C18 (low polarity) is more similar to Symmetry C18 (moderate polarity) than it is to Hypersil BDS C18 (high polarity). 6 | Comparison Guide to C18 Reversed Phase HPLC Columns N/Meter (ToIuene) Exs|| ODS! ACE C!8 YMC Fro C!8 ACE C!8-300 Zorbax SE-C!8 ACE C!8-HL Nuc|eos|| C!8 \a:ers Spner|sorb ODS! YMC ODS A Kronas|| C!8 H|cnron RFE Zorbax XDE-C!8 Hypers|| ODS Exs|| ODS Synne:ry C!8 Deve|os|| ODS-UC \a:ers Spner|sorb ODSE SunF|re C!8 \a:ers Spner|sorb ODS2 Hypers|| COLD C!8 Zorbax Rx-C!8 Luna C!8(2) Nuc|eos|| C!8AE Zorbax ODS Deve|os|| ODS-HC Nuc|eos|| C!8 HD YMC ODS AM Exs|| ODSE Vydac Se|ec:apore 300M Zorbax Ex:end C!8 L|Cnrospner RF-!8 Vydac Se|ec:apore 90M U|:raspnere ODS Vydac 2!8MS Hypers|| EDS C!8 Cen|n| C!8 L|Cnrosorb RF-!8 lner:s|| ODS Hypers|| HyFURlTY C!8 Deve|os|| ODS-MC YMC 'Spnere ODS H80 Vydac 2!8TF Frod|gy ODS3 TSK ODS-80TM lner:s|| ODS3 Novapa| C!8 YMC 'Spnere ODS M80 Capce|| Fa| UC C!8 TSK ODS-!20T Vydac Se|ec:apore 300F Far:|s|| ODS3 Capce|| Fa| AC C!8 Frod|gy ODS2 Far:|s|| ODS XTerra MS C!8 Reso|ve C!8 Furospner RF!8-e Far:|s|| ODS2 Eondapa| C!8 lner:s|| ODS2 0.0 20,000 40,000 60,000 80,000 100,000 120,000 Figure 9 Comparison of Column Effciency for a Neutral Compound Column effciency reported as Plates per meter (N/Meter) Comparison of Column Effciency for a Neutral Compound Column eciency is reported as plates per meter (N/Meter). Using a neutral compound (toluene) for the measurement greatly reduces the eects of secondary retention on the measurement of N and allows us to obtain data that is a better indication of the following factors: Particle size Smaller average packing particle size = Larger N Particle size distribution Broader particle size distribution = Smaller N Packing efciency Better packing procedures = Larger N Mobile Phase: 90% CH 3 OH 10% H 2 O Sample: Toluene Temperature: 24C Comparison Guide to C18 Reversed Phase HPLC Columns | 7 N/Meter (Fyridine) ACE C!8 Deve|os|| ODS-UC YMC Fro C!8 ACE C!8-300 SunF|re C!8 Hypers|| COLD Deve|os|| ODS-MC H|cnron RFE Zorbax XDE-C!8 Deve|os|| ODS-HC Capce|| Fa| UC C!8 Exs|| ODSE Cen|n| C!8 lner:s|| ODS3 \a:ers Spner|sorb ODSE lner:s|| ODS ACE C!8-HL YMC ODS A Zorbax Ex:end C!8 Luna C!8(2) Nuc|eos|| C!8 HD YMC ODS AM Vydac Se|ec:apore 300M XTerra MS C!8 TSK ODS-!20T Kronas|| C!8 Zorbax Rx-C!8 Far:|s|| ODS3 Frod|gy ODS3 Eondapa| C!8 Vydac Se|ec:apore 90M Furospner RF!8-e lner:s|| ODS2 YMC 'Spnere ODS M80 Hypers|| HyFURlTY C!8 Nuc|eos|| C!8 Zorbax SE-C!8 Far:|s|| ODS Frod|gy ODS2 Novapa| C!8 TSK ODS-80TM Vydac Se|ec:apore 300F L|Cnrosorb RF-!8 YMC 'Spnere ODS H80 Vydac 2!8MS Vydac 2!8TF Far:|s|| ODS2 Nuc|eos|| C!8AE Capce|| Fa| AC C!8 Synne:ry C!8 U|:raspnere ODS \a:ers Spner|sorb ODS2 Hypers|| EDS C!8 Exs|| ODS! Exs|| ODS \a:ers Spner|sorb ODS! Zorbax ODS Hypers|| ODS L|Cnrospner RF-!8 Reso|ve C!8 0 10,000 20,000 30,000 40,000 50,000 60,000 70,000 80,000 ACE Hypersil HyFUPlTY SynneLry Figure 10 Comparison of Column Effciency for a Basic Compound: Pyridine Figure 11 Comparison of Peak Shape Mobile Phase: 60% CH 3 OH 40% H 2 O Sample: Pyridine Temperature: 24C ACE C18 gave the best peak shape and highest column effciency for pyridine. Comparison of Column Effciency for a Basic Compound Measuring column eciency using a neutral compound is not very useful in predicting column performance when separating ionic compounds. Interaction between polar solutes and silanol sites on the stationary phase can cause tailing peaks and poor column eciency. To gain a better understanding of column performance with basic compounds, columns were tested using pyridine and amitriptyline as probes. Although columns are ranked somewhat dierently on the two tests, phases at the higher end of the ranking scale can be expected to give better peak shape and higher resolution for basic compounds than phases at the lower end of the scale. Not surprisingly, stationary phases that use high purity silicas exhibit better peak shape and higher column eciency than stationary phases that use more acidic silicas as their stationary phase supports. Mobile Phase: 60% CH 3 OH 40% H 2 O Sample: Pyridine Temperature: 24C 8 | Comparison Guide to C18 Reversed Phase HPLC Columns Figure 12 Comparison of Column Effciency For a Basic Compound: Pyridine ACE C18 65,200pl/m ACE C18-300 52,100pl/m SunFire C18 46,500pl/m Zorbax XDB C18 39,400pl/m Gemini C18 36,100pl/m Inertsil ODS3V 35,900pl/m Luna C18(2) 21,900pl/m XTerra MS C18 18,600pl/m Zorbax SB-C18 10,000pl/m Symmetry C18 3,100pl/m Hypersil BDS C18 600pl/m Zorbax ODS 100pl/m Column effciency measured at 10% pyridine peak height to account for peak tailing effects Column Dimensions: 250 x 4.6mm, 5m Sample: 1) uracil 2) pyridine 3) phenol Mobile Phase: 60:40 MeOH/H 2 O Temperature: 24C Flow: 1.0ml/min Comparison Guide to C18 Reversed Phase HPLC Columns | 9 N/Meter (AmitriptyIine) ACE C!8-300 ACE C!8 Hypers|| COLD YMC 'Spnere ODS M80 Cen|n| C!8 ACE C!8-HL YMC Fro C!8 SunF|re C!8 Deve|os|| ODS-UC Luna C!8(2) Frod|gy ODS3 H|cnron RFE Vydac Se|ec:apore 300M Nuc|eos|| C!8 HD lner:s|| ODS3 Deve|os|| ODS-MC Frod|gy ODS2 Capce|| Fa| UC C!8 Hypers|| HyFURlTY C!8 XTerra MS C!8 Hypers|| EDS C!8 Synne:ry C!8 Deve|os|| ODS-HC Furospner RF!8-e Kronas|| C!8 Nuc|eos|| C!8AE YMC ODS A Vydac Se|ec:apore 90M Zorbax XDE-C!8 YMC ODS AM Zorbax Ex:end C!8 Vydac Se|ec:apore 300F Vydac 2!8MS YMC 'Spnere ODS H80 Exs|| ODS! Zorbax SE-C!8 lner:s|| ODS2 lner:s|| ODS Exs|| ODSE Novapa| C!8 TSK ODS-80TM \a:ers Spner|sorb ODSE TSK ODS-!20T Vydac 2!8TF L|Cnrospner RF-!8 \a:ers Spner|sorb ODS2 Far:|s|| ODS Far:|s|| ODS3 L|Cnrosorb RF-!8 Eondapa| C!8 Nuc|eos|| C!8 Zorbax Rx-C!8 U|:raspnere ODS Capce|| Fa| AC C!8 Exs|| ODS Hypers|| ODS Far:|s|| ODS2 Reso|ve C!8 \a:ers Spner|sorb ODS! Zorbax ODS 0 10,000 20,000 30,000 40,000 50,000 60,000 70,000 Figure 13 Comparison of Column Effciency for a Basic Compound: Amitriptyline Figure 14 Top 10 Columns Ranked According to Peak Shape and Effciency Sample: Amitriptyline Mobile Phase: 80% CH 3 OH 20% 0.025M Phosphate, pH 6.0 Sample: Pyridine Mobile Phase: 60% CH 3 OH 40% H 2 O Note: All columns are packed with 5 micron size particles. Column ranking does differ in the two tests of column eff- ciency for a basic compound (Figures 10 and 13). However, of the 14 columns that ranked in the top 10 on at least one of the tests, 6 ranked in the top 10 on both tests. Plate count is measured at 10% of peak height to include peak tailing in the calculation. Both tests use mobile phases at neutral pH to encour- age interaction between the basic probes and silanols on the stationary phase. Mobile Phase: 80% CH 3 OH 20% 0.025M KH 2 PO 4 , pH 6.0 Sample: Amitriptyline Temperature: 24C 0 10,000 20,000 30,000 40,000 50,000 60,000 70,000 ACE C!8-300 ACE C!8 Hypers|| COLD YMC 'Spnere ODS M80 Cen|n| C!8 ACE C!8-HL YMC Fro C!8 SunF|re C!8 Deve|os|| ODS-UC Luna C!8(2) ACE C!8 Deve|os|| ODS-UC YMC Fro C!8 ACE C!8-300 SunF|re C!8 Hypers|| COLD Deve|os|| ODS-MC H|cnron RFE Zorbax XDE-C!8 Deve|os|| ODS-HC N/Meter (AmitriptyIine) 0 10,000 20,000 30,000 40,000 50,000 60,000 70,000 N/Meter (Fyridine) 0 10,000 20,000 30,000 40,000 50,000 60,000 70,000 ACE C!8-300 ACE C!8 Hypers|| COLD YMC 'Spnere ODS M80 Cen|n| C!8 ACE C!8-HL YMC Fro C!8 SunF|re C!8 Deve|os|| ODS-UC Luna C!8(2) ACE C!8 Deve|os|| ODS-UC YMC Fro C!8 ACE C!8-300 SunF|re C!8 Hypers|| COLD Deve|os|| ODS-MC H|cnron RFE Zorbax XDE-C!8 Deve|os|| ODS-HC N/Meter (AmitriptyIine) 0 10,000 20,000 30,000 40,000 50,000 60,000 70,000 N/Meter (Fyridine) 10 | Comparison Guide to C18 Reversed Phase HPLC Columns Figure 15 Comparison of Column Effciency For a Basic Compound: Amitriptyline ACE C18-300 65,300pl/m ACE C18 63,300pl/m Gemini C18 48,500pl/m SunFire C18 45,000pl/m Luna C18(2) 43,200pl/m Inertsil ODS3V 30,500pl/m Hypersil HyPURITY C18 24,800pl/m XTerra MS C18 24,400pl/m Hypersil BDS C18 22,700pl/m Symmetry C18 21,800pl/m Zorbax SB-C18 4,900pl/m Waters Spherisorb ODS2 1,300pl/m Column effciency measured at 10% amitriptyline peak height to account for peak tailing effects Column Dimensions: 250 x 4.6mm, 5m Sample: 1) norephedrine 2) nortriptyline 3) toluene 4) imipramine 5) amitriptyline Mobile Phase: 80:20 MeOH/0.025M KH 2 PO 4 , pH 6.0 Temperature: 24C Flow: 1.0ml/min Comparison Guide to C18 Reversed Phase HPLC Columns | 11 Feak Asymmetry Higher 5iIanoI Activity Lower 5iIanoI Activity 1.03 8.99 8.3 6.50 3.19 3.07 3.05 3.05 2.74 2.21 2.07 1.93 1.88 1.78 1.61 1.52 1.26 1.12 ACE C!8 XTerra MS C!8 lner:s|| ODS3 Luna C!8(2) Hypers|| E||:e C!8 D|scovery C!8 \a:ers Spner|sorb ODS! lner:s|| ODS2 Synne:ry C!8 Hypers|| EDS-C!8 Far:|s|| ODS2 Zorbax XDE-C!8 Novapa| C!8 Zorbax SE-C!8 Nuc|eos|| C!8 Vydac Se|ec:apore 300M Cosnos|| C!8 AR-ll Eondapa| C!8 1 2 4 6 8 10 Feak Asymmetry Higher MetaI Activity Lower MetaI Activity 1.07 3.07 2.56 2.42 2.41 2.27 1.46 1.43 1.37 1.34 1.31 1.27 1.26 1.24 1.16 1.12 1.09 1.07 ACE C!8 Zorbax XDE-C!8 Hypers|| E||:e C!8 XTerra MS C!8 Cosnos|| C!8 AR-ll lner:s|| ODS3 Hypers|| EDS-C!8 lner:s|| ODS2 Synne:ry C!8 Vydac Se|ec:apore 300M D|scovery C!8 Zorbax SE-C!8 Luna C!8(2) \a:ers Spner|sorb ODS! Nuc|eos|| C!8 Novapa| C!8 Eondapa| C!8 Far:|s|| ODS2 1.0 1.5 2.0 2.5 3.0 3.5 Figure 16 Comparison of Metal Activity Using the NIST Test: Asymmetry for Quinizarin Mobile Phase: 80% CH 3 OH 20% 5mM Potassium Phosphate, pH 7.0 Sample: Quinizarin Flow Rate: 2 mL/min Temperature: 23C Figure 17 Comparison of Silanol Activity Using the NIST Test: Asymmetry for Amitriptyline Mobile Phase: 80% CH 3 OH 20% 5mM Potassium Phosphate, pH 7.0 Sample: Amitriptyline Flow Rate: 2 mL/min Temperature: 23C Additional Comparison Tests Tere are numerous suggestions from dierent scientic groups about how to best characterize stationary phases. Most of these tests have merit, but the fact that the ranking of columns will often dier among the dierent tests shows the diculty in devising a denitive test that will predict column behavior in all, or even most circumstances. Te National Institute of Standards & Technology (NIST) has developed test conditions (Standard Reference Material 870) that do a particularly good job of characterizing stationary phases according to metal activity and silanol activity. Te presence of metals on the surface of stationary phases can have a signicant eect on chromatographic performance. Even trace levels of metal impurities can contribute to peak tailing of some compounds. In addition, subtle lot-to-lot variations in the amount of trace metals are another cause of poor column reproducibility. Te NIST test uses peak asymmetry of quinizarin, a strong metal chelating agent, to measure metal activity. Figure 16 ranks stationary phases according to metal activity using the NIST test. To test silanol activity, the NIST test uses amitriptyline, as does the test used to generate the data in Figure 13. However, the NIST test species a mobile phase pH of 7.0 rather than 6.0, and measures peak asymmetry rather than plate count to determine silanol activity. Te lower the asymmetry value of the amitriptyline peak (less tailing) the less silanol activity. Figure 17 ranks stationary phases according to silanol activity using the NIST test. 12 | Comparison Guide to C18 Reversed Phase HPLC Columns Figure 18 Grouping of C18 Columns According to Silanol Activity Material ACE C18 ACE C18-300 Develosil ODS-MG Hypersil GOLD Hypersil HyPURITY C18 Inertsil ODS3 Luna C18(2) Nucleosil C18 HD SunFire C18 XTerra MS C18 YMC Pro C18 ACE C18-HL Capcell Pak UG C18 Develosil ODS-HG Develosil ODS-UG Gemini C18 Hichrom RPB Inertsil ODS2 Kromasil C18 Prodigy ODS2 Prodigy ODS3 Purospher RP18-e Symmetry C18 YMC ODS A YMC ODS AM Zorbax Extend C18 Zorbax XDB-C18 Capcell Pak C18 SG Exsil ODSB Hypersil BDS C18 Inertsil ODS Nova-Pak C18 Nucleosil C18AB Partisil ODS3 Synchropak CR101 TSK ODS-120T TSK ODS-80TM Bondapak C18 Vydac 218MS Vydac 218TP Vydac Selectapore 300M\ Vydac Selectapore 300P Vydac Selectapore 90M Waters Spherisorb ODSB YMC JSphere ODS H80 YMC JSphere ODS M80 Zorbax Rx-C18 Zorbax SB-C18 Capcell Pak C18 AG Exsil ODS Exsil ODS1 Hypersil ODS LiChrosorb RP-18 LiChrospher RP-18 Nucleosil C18 Partisil ODS Partisil ODS2 Resolve C18Ultrasphere ODS Waters Spherisorb ODS1 Waters Spherisorb ODS2 Zorbax ODS Phases Grouped According to Silanol Activity Amitriptyline and pyridine are both good test probes to use for measur- ing silanol activity of stationary phases. Even a small amount of silanol exposure by the stationary phase can cause measurable peak broadening and peak asymmetry on one or both of these compounds. Chromato- graphic tests using these two probes are the primary measurements used to group these C18 phases according to silanol activity. In general, phases identied as having very low silanol activity will give the highest column eciency in the pyridine and amitriptyline tests (Figures 10, 13 and 17). S I L A N O L
A C T I V I T Y V e r y
L o w L o w M o d e r a t e H i g h Comparison Guide to C18 Reversed Phase HPLC Columns | 13 MAC-MOD Analytical 103 Commons Court P.O. Box 587 Chadds Ford, PA 19317 Phone: 1.800.441.7508 Fax: 1.610.358.5993 E-mail: info@mac-mod.com Web Site: www.mac-mod.com a n a l y t i c a l