Inertsil Technical Library

Transcription

1 HPLC COLUMN INERTSIL TECHNICAL LIBRARY H P L C C o l u m n Inertsil Technical Library Inertsil ODS-4, C8-4 Comparison of Performance List of Compared Columns Experimental Explanation, Analytical Conditions Comparison between commercially available ODS Columns Comparison between commercially available C8 Columns Comparison of Separation Pattern between ODS and C8 Columns Comparison of Retentivity Comprehensive Evaluation

2 Introduction of Inertsil 4 Series About Inertsil Inertsil, the name of our brand comes from the key word INERT, which is the most important factor in the separation analysis. GL Sciences Inertsil columns delivers the maximum performance of an HPLC column and we would like to support you and your critical analysis. Inertsil 4 series is the most latest generation columns and it provides the highest inertness in the world. A Must-Have column in your laboratory, Inertsil ODS-4 Reversed phase ODS and C8 columns play an important role in an HPLC separation industry. Out of those thousand kinds of ODS and C8 columns that exists in the market, we selected those typical brands that are often seen in the customers laboratory. Please go through the chromatograms and see the results for yourself. We trust Inertsil ODS-4 will be a Must-Have column in your laboratory. Comparison Table ODS(C8) Columns Column Serial No. Specific Surface Area (m /g) Pore Diameter (A ) Pore Volume (ml/g) Carbon Content (%) Inertsil ODS-4 8JF Atlantis T SunFire C XBridge CAPCELLPAK MGⅡ A4AD CAPCELLPAK MGⅢ A5AD Zorbax Eclipse Plus C8 USUXA YMC-Pack Pro C8 0450(W) Luna 5u C8() Cadenza 5CD-C8 HJMI TSKgel ODS-00V N Mightysil RP-8GP 808 5~80 0~40.0~. 9.5~.5 Hypersil GOLD 0845E Shim-pack VP-ODS Column Size:5um,50 4. mm I.D. C8 Columns Column Serial No. Specific Surface Area (m /g) Pore Diameter (A ) Pore Volume (ml/g) Carbon Content (%) Inertsil C8-4 9JF YMC-Pack Pro C Zorbax Eclipse Plus C Luna C8() USUTA SunFire C (W) XBridge C Column Size: 5um, mm I.D.

3 The effectiveness of several basic compounds for testing silica-based stationary phases was reviewed by applying them to recent columns for reversed-phase HPLC. Most octadecylsilylated (C8) stationary phases, prepared as a base-deactivated material from high-purity silica gel with endcapping, provided excellent peak shape and column efficiency for the bases including benzylamine and amitriptyline that once caused problems and were subsequently employed for testing silanol activities. However, a cyclic tertiary amine, dextrometorphan, was eluted as an acceptable peak from only a few columns at neutral ph. Such a more sensitive probe is expected to contribute to further improvement of the stationary phase for reversed-phase HPLC. Experimental Explanation, Analytical Conditions By conducting the 4 tests below, we can evaluate how high the inertness of the column and whether it can be used in a 00% aqueous mobile phase condition. To strictly evaluate all columns, the below mentioned samples were used, which are all known as strong adsorptive compounds. Basic Compound Test Dextromethorphan is a strong basic compound. Severe tailing can be confirmed when the packing material contains residual silanol groups. H CO H N CH Mobile Phase: A) CH CN B) 5 mm Phosphate buffer (ph.0) A/B = 40/0 Flow Rate:.0 ml/min Col. Temp.: 40 Detection: Injection Vol.: UV 0 nm ul. Phenol (0.05 mg/ml). Dextromethorphan HBr (0. mg/ml) Acidic Compound Test Sharp peaks can be obtained when analyzing Phenol or Salicylic Acid. However, as Brilliant Blue FCF has three sulfonic groups in its chemical structure, tailing will occur when the surface of the packing material is slightly basic. - O S N SO - N + SO - Mobile Phase: A) CH CN B) 0. % H PO 4 A/B = 5/5 Flow Rate:.0 ml/min Col. Temp.: 40 Detection: Injection Vol.: UV 54 nm ul. Brilliant Blue FCF (0.05 mg/ml). Phenol (0. mg/ml). Salicylic acid (0. mg/ml) Chelating Compound Test Hinokitiol is a strong chelating compound, which coordinately binds with the surface of the residual trace metal impurities, resulting in causing a severe tailing. However, the peak shape improves as the injection increases since the surface of the packing material of the adsorption active sites will be masked. O OH Mobile Phase: A) CH CN B) 0. % H PO 4 A/B = 40/0 Flow Rate:.0 ml/min Col. Temp.: 40 Detection: Injection Vol.: UV 54 nm ul. Hinokitiol (0. mg/ml) When analyzing hydrophilic compounds in a water rich mobile phase condition, once the pump is stopped, the hydrophobic group pushes out the mobile phase out from the pore, resulting in causing a dewetting phenomenon. In such condition, even after introducing the mobile phase again, sudden loss of retention will be experienced as the mobile phase cannot be introduced to the pore thoroughly. Testing Procedure: ) Introducing 00% water for 0 minutes. ) Conduct Analysis (Upper chromatogram in the following pages) ) Stop flow for 5 minutes. 4) Introducing 00% water again for 0 minutes. 5) Stop flow for 5 minutes again. ) Conduct Analysis (Lower chromatogram in the following pages) Mobile Phase: 00 % H O Flow Rate:.0 ml/min Col. Temp. : 40 Detection: UV 54 nm Injection Vol.: ul. Cytosine (0.0 mg/ml). Uracil (0.0 mg/ml). Guanine (0.0 mg/ml) 4. Thymine (0.0 mg/ml) 5. Adenine (0.00 mg/ml) References Test compounds for detecting the silanol effect on the elution of ionized amines in reversed-phase LC Nobuo Tanaka, Kensuke Okusa, Yuki Suita, Yukio Otsuka, Mineo Tahara, Tohru Ikegami, Masayoshi Ohira, Masakazu Takahashi Journal of Separation Science, Volume Issue, Pages 48-58

4 ODS Columns Comparison () Basic Compound Test Acidic Compound Test Chelating Compound Test Inertsil ODS-4 Basic Compound : Acidic Compound: Chelating Compound: Dewetting: : Phenol : Dextromethorphan : Brilliant Blue FCF : Phenol : Salicylic acid. Hinokitiol : 9.8 % 0 Atlantis T Basic compound: Acidic compound: Chelating compound: Dewetting: No peak 5.9 % 0 SunFire C8 Basic compound: Acidic compound: Chelating compound: Dewetting: :No peak 9.4 % 0 Inertsil ODS-4 Atlantis T SunFire C8 Proved superior peak shapes for all various compounds along with extreme stability under 00% aqueous mobile phases. Inertsil ODS-4 is just compatible with any type of compounds in any mobile phase conditions delivering highly stable chromatograms for qualitative and quantitative analysis. Compatible for the analysis of hydrophilic compounds, but not for the analysis of chelating compounds. Overall, it is not an easy-to-use column as adsorption of peaks can be concerned especially when conducting a trace analysis.

5 Basic Compound Test Acidic Compound Test Chelating Compound Test XBridge Basic compound: Acidic compound: Chelating compound: Dewetting: :No peak No peak 4.0 % 0 CAPCELLPAK MG II Basic compound: Acidic compound: Chelating compound: Dewetting: :No peak 4.% 0 CAPCELLPAK MG III Basic compound: Acidic compound: Chelating compound: Dewetting: :No peak.% 0 XBridge CAPCELLPAK MG II CAPCELLPAK MG III Adsorption of acidic and chelating compounds were confirmed. In addition, it is not compatible with 00% aqueous mobile phase as it showed a sudden loss of retention due to a dewetting phenomenon. Compatible for analysis of hydrophilic compounds, but caution needs to be taken when analyzing acidic and chelating compounds. Can obtain high reproducibility and strong retentivity under 00% aqueous mobile phases. Precision of analysis can be concerned as adsorbed peaks were confirmed in the analysis of basic and acidic compounds.

6 ODS Columns Comparison () Basic Compound Test Acidic Compound Test Chelating Compound Test Inertsil ODS-4 Basic compound: Acidic compound: Chelating compound: Dewetting: : Phenol : Dextromethorphan : Brilliant Blue FCF : Phenol : Salicylic acid : Hinokitiol 9.8% 0 Zorbax Eclipse Plus C8 Basic compound: Acidic compounds: Chelating compound: Dewetting: :No peak No peak.% 0 YMC-Pack Pro C8 Basic compound: Acidic compound: Chelating compound: Dewetting: :No peak No peak 4.% 0 Inertsil ODS-4 Zorbax Eclipse Plus C8 YMC-Pack Pro C8 Proved superior peak shapes for all various compounds along with extreme stability under 00% aqueous mobile phases. Inertsil ODS-4 is just compatible with any type of compounds in any mobile phase conditions delivering highly stable chromatograms for qualitative and quantitative analysis. Overall, it is not an easy-to-use column. Retentivity is relatively high and excellent peak shapes were obtained for hydrophilic compounds. However, quantitative analysis can be concerned since the adsorption of acidic and chelating compounds were confirmed.

7 Basic Compound Test Acidic Compound Test Chelating Compound Test Luna 5u C8 () Basic compound: Acidic compound: Chelating compound: Dewetting: No peak 8.0% 0 Cadenza 5CD-C8 Basic compound: Acidic compound: Chelating compound: Dewetting: 85.9% TSKgel ODS-00V 0 Basic compound: Acidic compound: Chelating compound: Dewetting:.5% 0 Luna 5u C8 () Cadenza 5CD-C8 TSKgel ODS-00V Compatible for the analysis of basic compounds. On the other hand, adsorption of chelating compounds and unstable results were confirmed for the analysis of hydrophilic compounds under 00% aqueous mobile phases. Compatible for the analysis of acidic compounds, but not for basic, acidic and hydrophilic compounds under 00% aqueous mobile phases. Compatible for the analysis of acidic and hydrophilic compounds under 00% aqueous mobile phases. Caution needs to be taken when analyzing chelating compounds.

8 ODS Columns Comparison () Basic Compound Test Acidic Compound Test Chelating Compound Test Mightysil RP-8GP Basic compound: Acidic compound: Chelating compound: Dewetting: No Peak : 8.4% 0 Hypersil GOLD Basic compound: Acidic compound: Chelating compound: Dewetting: : 4.8% 0 Shim-pack VP-ODS Basic compound: Acidic compound: Chelating compound: Dewetting: :No Peak : 5.% 0 Mightysil RP-8GP Hypersil GOLD Compatible for the analysis of acidic compounds, but not for basic, chelating and hydrophilic compounds under 00% aqueous mobile phases. Compatible for the analysis of chelating compounds. As the retentivity of hydrophilic compounds were weak, separation of multi-compounds may be difficult to achieve. Shim-pack VP-ODS Overall, it is not an easy-to-use column.

9 Comparison of C8 Columns Basic Compound Test Acidic Compound Test Chelating Compound Test Inertsil C8-4 : Phenol : Dextromethorphan Basic compound: : Brilliant Blue FCF : Phenol : Salicylic acid Acidic compound: Chelating compound: Dewetting: : Phenol : Hinokitiol 4.4% SunFire C8 Basic compound: Acidic compound: Chelating compound: Dewetting: :No peak 8.8% Xbridge C8 Basic compound: Acidic compound: Chelating compound: Dewetting: :No Peak :No Peak.% Inertsil C8-4 SunFire C8 XBridge C8 Delivers the same separation pattern and extreme inertness to any type of compounds just like Inertsil ODS-4 along with extreme stability under 00% aqueous mobile phases for qualitative and quantitative analysis. Compatible for the analysis of basic compounds, but adsorption of acidic compounds and unstable results were obtained under 00% aqueous mobile phases. Compatible for the analysis of basic compounds. As the retentivity is relatively weak when comparing to other commercially available C8 columns, separation of multi-compounds may be difficult to achieve.

10 Basic Compound Test Acidic Compound Test Chelating Compound Test Zorbax Eclipse Plus C8 Basic compound: Acidic compound: Chelating compound Dewetting: :No Peak :No Peak 5.% YMC-Pack Pro C8 Basic compound: Acidic compound: Chelating compound: Dewetting: :No Peak :No Peak 8.5% Luna C8 () Basic compound: Acidic compound: Chelating compound: Dewetting: :No Peak 80.9% Zorbax Eclipse Plus C8 YMC-Pack Pro C8 Adsorption of peaks were confirmed and was not compatible under 00% aqueous mobile phases as the retention was lost. Overall, it is not an easy-to-use column. Luna C8() Only compatible for the analysis of basic compounds.

11 Comparison of Separation Pattern between ODS and C8 columns Shortening the analysis time without changing the separation pattern It is well known to use a C8 column when observing the retentivity of an ODS column to be too strong. It would be ideal to obtain the same separation pattern without changing the mobile phase conditions. Inertsil C8-4 provides the same separation pattern and extreme inertness to any type of compounds just like Inertsil ODS-4, which enables easy method transfer from ODS-4 to C8-4. Commercially available ODS and C8 columns show dramatically different selectivity even though it is from the same brand/series. For example, the elution position of Sample No., Triphenylene were different between commercially available ODS and C8 columns from the same brand/series. This indicates that the stereoselectivty between those ODS and C8 columns are different as the bonding density of the functional group is high on their C8 column when comparing from their ODS column. Therefore, caution needs to be taken when changing the column from their ODS to C8 column. Experimental Explanation of Selectivity Test Sample No., Uracil cannot be retained in a reversed phase mode when using the mobile phase condition described on the right. Therefore, Uracil was used to determine the t0 position. t0: Void Volume Sample No.,, Caffeine and Phenol are used to confirm the amount of residual silanol on the surface of the silica gel. Caffeine elutes later against Phenol when there is a lot of residual silanol on the surface of the silica gel. Sample No.4, n-butylbenzene and Sample No., n-amylbenzene were used to determine the hydrophobic property of the column. n-amylbenzene elutes later against n-butylbenzene when the hydrophobicity of the column is high. Stereoselectivity is indicated by Sample No.5, o-terphenyl and Sample No., Triphenylene. O-Terphenyl has a twisted tertiary structure and Triphenylene has a planar structure. Triphenylene elutes later against o-terphenyl when the stereoselectivity of the column is high. Mobile Phase: A) CH OH B) H O A/B = 80/0 Flow Rate:.0 ml/min Col. Temp.: 40 Detection: UV 54 nm Injection Vol.: 5 ul ) Uracil (0.005 mg/ml) ) Caffeine (0.04 mg/ml) ) Phenol (0.08 mg/ml) 4) n-butylbenzene (. mg/ml) 5) o-terphenyl (0.04 mg/ml) ) n-amylbenzene (. mg/ml) ) Triphenylene (0.04 mg/ml) O HN O NH. Uracil O N N OH O N N. Caffeine. Phenol 4. n-butylbenzene 5. o-terphenyl. n-amylbenzene. Triphenylene It is necessary to set the bonding density of the function group of an ODS and C8 column to obtain the same separation pattern. Theoretically, the carbon loading ratio shall be 0.44 =8/8. The carbon loading ratio of Inertsil 4 series is 0.45 and we have successfully obtained the same separation pattern between ODS-4 and C8-4. Now, not only the analysis time can be shortened, but the same separation pattern can be obtained using Inertsil 4 series.

12 Inertsil 4 Series SunFire XBridge ODS-4 ODS ODS Carbon Loading Ratio 5/=0.45 Carbon Loading Ratio.5/=0.,, Carbon Loading Ratio.5/=0.,, 4 5+,, C8-4 0 C8 0 C8,, 4 5+,, 4 5 4,5,,, Zorbax Eclipse Plus ODS,, + Carbon Loading Ratio 8/8=.0 ODS,, YMC-Pack Pro Carbon Loading Ratio 0/=0. ODS,, Luna () Carbon Loading Ratio.5/.5= C C8 0 C8,,,, 5 4 5,,

13 Comparison of Retentivity List of ODS(C8)Columns Cadenza 5CD-C8 Shim-pack VP-ODS YMC-Pack Pro C8 SunFire C8 CAPCELLPAK MGⅡ Inertsil ODS-4 Mightysil RP-8GP CAPCELLPAK MGⅢ TSKgel ODS-00V Luna 5u C8() Atlantis T Zorbax Eclipse Plus C8 XBridge Hypersil GOLD Retention Time of n-amylbenzeneの保持時間 (min) (min) List of C8 Columns YMC-Pack Pro C8 Luna C8() SunFire C8 Zorbax Eclipse Plus C8 Inertsil C8-4 Xbridge C n-amylbenzene の保持時間 (min) Retention Time of n-amylbenzene (min)

14 Comprehensive Evaluation of ODS(C8) Columns Column Basic compound Acidic compound Chelating compound Dewetting Inertsil ODS-4 Atlantis T SunFire C8 Xbridge CAPCELLPAK MGⅡ CAPCELLPAK MGⅢ Zorbax Eclipse Plus C8 YMC-Pack Pro C8 Luna 5u C8() Cadenza 5CD-C8 TSKgel ODS-00V Mightysil RP-8GP Hypersil GOLD Shim-pack VP-ODS Comprehensive Evaluation of C8 Columns Column Basic compound Acidic compound Chelating compound Dewetting Inertsil C8-4 YMC-Pack Pro C8 Zorbax Eclipse Plus C8 Luna C8() SunFire C8 Xbridge C8

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