InertSustainSwift C8

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2 Physical Properties Silica Particle Size Surface Area Pore Size Pore Volume Bonded Phase End-capping Carbon Loading ph Range USP Code :ES (Evolved Surface) Silica Gel :.9 μm, μm, μm :00 m /g :00 Å (0 nm) :.00 ml/g :Octyl Groups :Complete :6 % :~0 :L

3 Benefits of Highly inert packing material results in less tailing of peaks for virtually any type of analytes Extreme resistance to low and high ph mobile phases Rapid elution of samples in isocratic methods Endlessly reproducible from column-to-column and batch-to-batch

4 Comparison of Retentivity Between & InertSustain C8 The retentivity of is weaker than InertSustain C8 for neutral and especially for hydrophobic compounds. The weak retentivity is generated due to the difference in physical properties between (surface area: 00 m/g, carbon load: 6 %) and InertSustain C8 (surface area: 0 m/g, carbon load: 8 %). Highly Hydrophilic eutral Compounds Hydrophobic Compounds InertSustain C8 6 InertSustain C Column size : μm, 0 x.6 mmi.d. Eluent : A) CH OH B) H O A / B = 80/0, v / v Flow Rate :.0 ml/min Col. Temp. : 0 Detection : UV nm.uracil.caffeine.phenol.n-butylbenzene. n-amylbenze 6. o-terphenyl. Triphenylene Column size : μm, 0 x.6 mmi.d. Eluent : A) CH C B) H O A / B = 90/0, v / v Flow Rate :.0 ml/min Col. Temp. : 0 Detection : UV 00 nm. Vitamin A. Vitamin D. Vitamin E(α). Vitamin K

5 Comparison of Retentivity Between & Other C8 columns offer moderate retentivity while comparing with GL Sciences C8 columns. InertSustain C Column size : μm, 0 x.6 mmi.d. Eluent : A) CH OH B) H O A / B = 80/0, v / v Flow Rate :.0 ml/min Col. Temp. : 0 Detection : UV nm.uracil.caffeine.phenol.n-butylbenzene. n-amylbenze 6. o-terphenyl. Triphenylene Inertsil C8-, n-butylbenzene. n-amylbenze Inertsil C8-, Inertsil WP00 C8,6 6. o-terphenyl. Triphenylene 0 0 0

6 Comparison of Retentivity Between & Other C8 columns This example shows a difference in selectivity and retentivity between C8 and C8 columns which were tested under the same analytical conditions. C8 Columns C8 Columns InertSustainSwift C8 6 6, InertSustain C InertSustain C Inertsil C8-,6 Inertsil ODS-, Inertsil C8-,6 Inertsil ODS- 6 Inertsil WP00 C , Inertsil WP00 C

7 Comparison Between InertSustainSwift C8 & C8 Under this analytical conditions, the retentivity is similar between both columns while the InerSustainSwift C8 provided better separation, which can be used to optimize selectivity or analysis time. Carbon Load: 6.0 % InertSustainSwift C8 Carbon Load: 9.0 % 6, Time(min) Time(min) Column size : μm, 0 x.6 mm I.D. Eluent : A) CH OH B) H O A/B = 80/0, v/v Flow Rate :.0 ml/min Col. Temp. : 0 Detection : UV nm Sample:.Uracil.Caffeine.Phenol.n-Butylbenzene.n-Amylbenze 6.o-Terphenyl.Triphenylene Sample o., n-butylbenzene and Sample o., 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 o.6, o-terphenyl and Sample o., 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.

8 Comparison Between InertSustainSwift C8 & C8 Many chromatographers prefer a C8 column when an ODS phase shows excessive retention values. In this example, the provides shorter analysis time with better selectivity on the impurities eluted after sample no. due to the shorter alkyl chain length of the stationary phase. Carbon Load: 6.0 % InertSustainSwift C8 Carbon Load: 9.0 % Time(min) Time(min) Column size : μm, 0 x.6 mm I.D. Eluent : A) CH C B) H O A/B = 90/0, v/v Flow Rate :.0 ml/min Col. Temp. : 0 Detection : UV 00 nm. Vitamin A. Vitamin D. Vitamin E(α). Vitamin K 8

9 % Initial Retention Time Durability Testing at Low ph columns are stable over a wide ph range, just like InertSustain C8. Low ph Resistance Test Hours of Purging (h) InertSustain C8 Purging Solvent : 0. % TFA (ph.0)/chc = 90/0, v/v Column Temp. : 60 Sample : aphthalene Testing Procedure ) Purging solvent is introduced into column. ) The column is then flushed with 0 % CHC. ) aphthalene is used to verify the % initial retention time. 9

10 Efficiency () Durability Testing at High ph columns are stable over a wide ph range, just like InertSustainSwift C8. High ph Resistance Test Purging Solvent : 0 mm TEA (ph 9.0)/CHOH = 0/0, v/v Column Temp. : 0 Sample : aphthalene Testing Procedure ) Purging solvent is introduced into column. ) The column is then flushed with 0 % CHOH. ) aphthalene is used to verify the efficiency () Hours of Purging (h) InertSustain C8 0

11 % Initial Retention Time Comparison of Durability Between & Other C8 columns columns are stable over a wide ph range, just like InertSustain C8. Low ph Resistance Test Hours of Purging (h) InertSustain C8 Inertsil C8- Inertsil C8- Purging Solvent : 0. % TFA (ph.0)/chc = 90/0, v/v Column Temp. : 60 Sample : aphthalene Testing Procedure ) Purging solvent is introduced into column. ) The column is then flushed with 0 % CHC. ) aphthalene is used to verify the % initial retention time.

12 Efficiency () Comparison of Durability Between & Other C8 columns columns are stable over a wide ph range, just like InertSustain C8. High ph Resistance Test Purging Solvent : 0 mm TEA (ph 9.0)/CHOH = 0/0, v/v Column Temp. : 0 Sample : aphthalene Testing Procedure ) Purging solvent is introduced into column. ) The column is then flushed with 0 % CHOH. ) aphthalene is used to verify the efficiency () Hours of Purging (h) InertSustain C8 Inertsil C8- Inertsil C8- Triart C8 Luna C8

13 Comparison of Strong Basic Compound Test Dextromethorphan is a strong basic compound. Severe tailing can be confirmed when the packing material contains residual silanol groups. Hypersil BDS C Eluent : A) CH C B) mm K HPO (ph.0, KH PO ) A/B = 0/0, v/ v Flow Rate :.0 ml/min Col. Temp. : 0 Detection : UV 0 nm Sample :.Uracil.Pyridine.Phenol.Berberine chloride.dextromethorphan H CO O O OCH H CH OCH. Berberine chloride. Dextromethorphan

14 Comparison of Strong Basic Compound Test A basic compound test using antihistamine drugs are highly basic, which can show tailing of peaks and different elution pattern on columns with insufficient end-capping. Columns with insufficient end-capping will show later elution of sample,, and. Hypersil BDS C Eluent : A) CH C B) mm K HPO (ph.0, KH PO ) A/B = 60/0, v / v :.0 ml/min Flow Rate Col. Temp. : 0 Detection : UV 0 nm Sample :. Chlorpheniramine. Triprolidine. Homochlorcyclizine. Hydroxyzine. Clemastine Cl H C Cl H CH Cl. Chlorpheniramine. Triprolidine. Homochlorcyclizine. Hydroxyzine. Clemastine H O OH Cl O CH H CH

15 Comparison of Strong 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. Hypersil BDS C8 RT()=6.min RT()=.6min Eluent : A) CH C B) 0.% H PO A/B = /, v/v Flow Rate :.0 ml/min Col. Temp. : 0 Detection : UV nm : Brilliant Blue FCF : Phenol : Salicylic acid - O S SO - + SO -. Brilliant Blue FCF

16 Comparison of Selectivity Sample o., n-amylbenzene was used to determine the hydrophobic property of columns. The overall retentivity of offers similar retentivity to Hypersil BDS C8. Hypersil BDS C8,:Incomplete Separation Eluent : A) CH OH B) H O A/B = 80/0, v/v Flow Rate :.0 ml/min Col. Temp. : 0 Detection : UV nm Sample:.Uracil.Caffeine.Phenol.n-Butylbenzene. n-amylbenze 6. o-terphenyl. Triphenylene. n-butylbenzene. n-amylbenze 6. o-terphenyl. Triphenylene 6

17 Comparison of Compatibility with 00 % Water Mobile Phase Sample o., n-amylbenzene was used to determine the hydrophobic property of columns. The overall retentivity of offers similar retentivity to Hypersil BDS C8. Hypersil BDS C8 First Injection Third Injection Variation: 99. % Variation: Under 0 % with peaks adsorbed Eluent : 00 % H O Flow Rate :.0 ml/min Col. Temp. : 0 Detection : UV nm Sample :.Cytosine.Uracil.Guanine.Thymine.Adenine Testing Procedure ) 00 % water is introduced into column over 60 minutes. ) Conduct analysis, upper chromatogram. ) Stop flow for minutes. ) 00 % water is introduced again into column over 0 minutes. ) Stop flow for minutes again. 6) Conduct analysis, lower chromatogram.

18 Comparison of Performance 8

19 List of Columns Brand Dimension (mm) Particle Size (μm) Surface Area (m /g) Pore Size (A ) Pore Volume (ml/g) Carbon Loading (%) ph Range InertSustainSwfit C InertSustain C Thermo Fisher Scientific: Hypersil BDS C YMC;Triart C Phenomenex;Luna C8() YMC;YMC-Pack Pro C CERI; L-column C Waters;SunFire C Waters;XBridge C Agilent; Zorbax Eclipse plus C

20 Explanation of Analytical Tests and Selectivity Test Sample o., n-butylbenzene and Sample o., 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 o.6, o-terphenyl and Sample o., 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. Basic Compound Test () Dextromethorphan and Berberine chloride are strong basic compounds. Severe tailing can be confirmed when the packing material contains residual silanol groups. O O. n-butylbenzene. n-amylbenzene 6. o-terphenyl. Triphenylene OCH OCH H CO. Berberine chloride. Dextromethorphan H CH Eluent : A) CH OH B) H O A/B = 80/0, v / v Flow Rate :.0 ml/min Col. Temp. : 0 Detection : UV nm Sample :. Uracil. Caffeine. Phenol. n-butylbenzene. n-amylbenzene 6.o-Terphenyl. Triphenylene Eluent : A) CH C B) mm K HPO (ph.0, KH PO ) A/B = 0/0, v/ v Flow Rate :.0 ml/min Col. Temp. : 0 Detection : UV 0 nm Sample :.Uracil.Pyridine.Phenol.Berberine chloride.dextromethorphan Basic Compound Test () A basic compound test using antihistamine drugs and are highly basic, which can show tailing of peaks and different elution pattern on columns with insufficient end-capping. Column with insufficient end-capping will show later elution of sample,, and. Cl H C Cl H CH Cl. Chlorpheniramine. Triprolidine. Homochlorcyclizine. Hydroxyzine. Clemastine H O OH Cl O CH H CH Eluent : A) CH C B) mm K HPO (ph.0, KH PO ) A/B = 60/0, v / v :.0 ml/min Flow Rate Col. Temp. : 0 Detection : UV 0 nm Sample :. Chlorpheniramine. Triprolidine. Homochlorcyclizine. Hydroxyzine. Clemastine 0

21 Explanation of Analytical Tests and 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. - OS - SO +.Brilliant Blue FCF SO - Eluent : A) CH C B) 0.% H PO A/B = /, v/v :.0 ml/min Flow Rate Col. Temp. : 0 Detection : UV nm Sample :.Brilliant Blue FCF.Phenol.Salicylic acid Chelating Compound Test Hinokitiol is a strong chelating compound, which coordinately binds with the surface of residual trace metal impurities, resulting in severe tailing. However, the peak shape improves as the injection increases since the surface of the packing material of the adsorption active sites eventually become masked. O OH Eluent : A) CH C B) 0.%H PO A/B = 0/60, v/v Flow Rate :.0 ml/min Col. Temp. : 0 Detection : UV nm Inject Vol. : μl, 0ppm Sample :. Phenol. β-thujaplicin (Hinokitiol). β-thujaplicin (Hinokitiol) Dewetting Test When analyzing hydrophilic compounds under water rich mobile phase condition, once the pump is stopped, the hydrophobic bonded group pushes the aqueous mobile phase out off the pore in an irreversible fashion, in what has become known as the dewetting phenomenon. Testing Procedure: ) 00 % water is introduced into column over 60 minutes. ) Conduct Analysis (Upper chromatogram in the following pages) ) Stop flow for minutes. ) 00 % water is introduced again into column over 0 minutes. ) Stop flow for minutes again. 6) Conduct Analysis (Lower chromatogram in the following pages) Eluent : 00 % H O Flow Rate :.0 ml/min Col. Temp. : 0 Detection : UV nm Sample :.Cytosine.Uracil.Guanine.Thymine.Adenine

22 Comparison of Performance (/9) Selectivity Test Basic Compound Test () Basic Compound Test () Acidic Compound Test Chelating Compound Test Dewetting Test Variation: 99. % Hypersil BDS C8 6, :Adsorption observed Severe tailing of peaks Variation:. %

23 Comparison of Performance (/9) Selectivity Test Basic Compound Test () Basic Compound Test () Acidic Compound Test Chelating Compound Test Dewetting Test Variation: 99. % Luna C8, :Adsorption observed Severe tailing of peaks :Adsorption observed Variation: 9. %

24 Comparison of Performance (/9) Selectivity Test Basic Compound Test () Basic Compound Test () Acidic Compound Test Chelating Compound Test Dewetting Test Variation: 99. % XBridge C8 6 :ot eluted :Adsorption observed Variation: 66.8 %

25 Comparison of Performance (/9) Selectivity Test Basic Compound Test () Basic Compound Test () Acidic Compound Test Chelating Compound Test Dewetting Test Variation: 99. % SunFire C8 :Adsorption observed :ot eluted :Adsorption observed 6, Variation: 8. %

26 Comparison of Performance (/9) 6 Selectivity Test Basic Compound Test () Basic Compound Test () Acidic Compound Test Chelating Compound Test Dewetting Test Variation: 99. % ZORBAX Eclipse Plus C8 :Adsorption observed :ot eluted :Adsorption observed Variation: 8. %

27 Comparison of Performance (6/9) Selectivity Test Basic Compound Test () Basic Compound Test () Acidic Compound Test Chelating Compound Test Dewetting Test Variation: 99. % Triart C Variation: 89.9 %

28 Comparison of Performance (/9) 8 Selectivity Test Basic Compound Test () Basic Compound Test () Acidic Compound Test Chelating Compound Test Dewetting Test Variation: 99. % YMC-Pack Pro C8 :Adsorption observed Severe tailing of peaks :ot eluted :Severe tailing of peak Variation: 6. %

29 Comparison of Performance (8/9) 9 Selectivity Test Basic Compound Test () Basic Compound Test () Acidic Compound Test Chelating Compound Test Dewetting Test Variation: 99. % L-column C8, :Severe tailing of peaks :Severe fronting of peak Variation: 0. %

30 Comparison of Performance (9/9) 0 Selectivity Test Basic Compound Test () Basic Compound Test () Acidic Compound Test Chelating Compound Test Dewetting Test Variation: 99. % InertSustain C Variation: 98.9 %

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