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New HPLC Column; InertSustain AQ C18 technical info

The document discusses the physical properties and benefits of the inertsustain aq-c18 HPLC and LC/MS columns, highlighting their exceptional retention of polar compounds, even under 100% water mobile phases. It contrasts these columns with conventional ODS columns, detailing the advantages in preserving peak shapes and reproducibility without the need for ion-pairing reagents. The document also presents various method development strategies for optimizing the analysis of polar compounds using these columns.

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InertSustain AQ-C18 1 HPLC, LC/MS Columns
Physical Properties Silica : ES Silica Gel Particle Size : 1,9 μm, 3 μm, 5 μm Surface Area : 350 m2/g Pore Size : 100 Å (10 nm) Pore Volume : 0.85 mL/g Bonded Phase : Octadecyl Groups Bonded-Phase Structure : * Monomeric InertSustain AQ-C18® Carbon Load : 13.0 % End-capping : Complete pH Range : 1 – 10 USP Code : L1 * Although it is classified as a monomeric type ODS, the bonding of ODS groups are optimized to retain highly polar compounds.
・Exceptional polar compound retention in reversed phase mode ・The usage of highly inert packing with superior column lifetime  True polar compound retention without using any secondary interaction  Improve separation between polar analytes and solvent peaks or sample matrices  Enhanced retention can also prevent ion suppression in LC/MS methods 3 Benefits of InertSustain AQ-C18
First Choice Column Conventional ODS Column 1. Use of ion-pairing reagents 2. Use of 100% water mobile phase Often experience shorter column lifetime, poor method reproducibility, increased instrument maintenance… What other options are left? In case there were no retention of polar compounds… 4 Method Development for Polar Compounds
・Selecting different ODS columns having different carbon loading % ・Selecting polar embedded ODS columns ・Selecting different stationary phase columns such as Phenyl or PFP columns 3. Use of different reversed phase columns 5 Method Development for Polar Compounds Second Choice Columns • Really hard to tell if the polar analytes will be retained or not. • Adsorbed peaks of target analytes occur due to secondary interaction.
6 Method Development for Polar Compounds 4. Use of different interactions Second Choice Columns • Use of high concentration buffer (harsh mobile phase). • Reproducibility issues. • Takes time to equilibrate columns prior to the analysis. ・Selecting mix mode columns (ion-exchange + ODS) ・Selecting HILIC columns (hydrophilic interaction)
7 Method Development for Polar Compounds How will InertSustain AQ-C18 columns help with your polar samples? ・Easier to develop and optimize analytical conditions ・Offer strong polar compound retention even under 100% water or water rich mobile phases
8 Method Development for Polar Compounds ・The usage of highly inert packing results in delivering symmetric peaks for virtually any type of compounds ・Eliminating the use of ion-pairing reagents improves method reproducibility, extends column lifetime and reduces instrument maintenance How will InertSustain AQ-C18 columns help with your polar samples?
Synthesis of ES silica gel Bonding of ODS groups End-capping InertSustain C18 9 GL Sciences’ Accumulated Bonding Technology GL Sciences’ accumulated bonding technology and know-how allows InertSustain AQ-C18 to provide exceptional polar compound retention. Specifically, optimization of bonding of the C18 groups at equal distance to the silica gel enable InertSustain AQ-C18 to offer significant retention for highly polar compounds even under water rich mobile phases. Bonding of ODS groups GL Sciences’ Accumulated Bonding Technology End-capping InertSustain AQ-C18
StrongerRetentionof Hydrophiliccompounds Stronger Retention of Hydrophobic compound 0,5 0,7 0,9 1,1 1,3 0,4 0,5 0,6 0,7 0,8 0,9 1 1,1 10 Retention Properties of InertSustain AQ-C18 InertSustain AQ-C18 Inertsil ODS-3 InertSustain C18 Inertsil ODS-4 Inertsil ODS-EP XSelect CSH C18 Synergi Fusion RP Altantis T3 CAPCELL PAK C18-AQ Inertsil ODS-SP Aquasil C18 RetentionFactorofacidic,basicandneutral compoundsusing100%watermobilephase Retention Factor of amylbenzene using methanol/water=80/20 Comparison of Retentivity for Hydrophilic compounds between various ODS columns Remarks: 1. The horizontal and vertical retention factor values were calculated with reference to the retention factor values obtained from InertSustain C18. 2. The horizontal values represents the retention factor of amylbenzene using methanol/water=80/20. 3. The vertical values represents the retention factor of acidic, basic and neutral compounds using 100% water mobile phase. (For further details, please see the next 2 slides)
Two different mobile phases were used in this testing, 0.1 % formic acid in H2O and 0.1 % phosphoric acid in H2O. And total of 6 compounds were used to determine the retention factor of these 6 compounds to confirm the retentivity of each column. Determination of Retentivity for Hydrophilic Compounds Testing Conditions 1. Testing Conditions 2. Column ： 5 μm, 150 × 4.6 mm I.D. Column ： 5 μm, 150 × 4.6 mm I.D. Eluent ： 0.1 % formic acid in H2O Eluent ： 0.1 % phosphoric acid in H2O Sample: Sample: 1. Pyridoxamine (Basic) 1. Pyridoxamine (Basic) 2. Dopamine (Basic) 2. Dopamine (Basic) 3. Acetic Acid (Acidic) 3. Acetic Acid (Acidic) 4. Succinic Acid (Acidic) 4. Succinic Acid (Acidic) 5. Uracil (Neutral) 5. Uracil (Neutral) 6. 5-methyluridine (Neutral) 6. 5-methyluridine (Neutral) 11
12 N NH O O N NH O O O OHOH N OH HO H2N HO HO NH2 O OH HO OH O O Succinic Acid (Acidic) Acetic Acid (Acidic) Uracil (Neutral) 5-Methyluridine (Neutral) Dopamine (Basic) Pyridoxamine (Basic) Determination of Retentivity for Hydrophilic Compounds
The following radar chart was created with reference to the retention factor values obtained from InertSustain C18 (conventional ODS). When the surface of silica is neutral and interactions between hydrophobic molecules are present under water rich mobile phases, the blue highlighted area will show larger area. Determination of Retentivity for Hydrophilic Compounds 13 0 0,5 1 1,5 0 0,5 1 1,5 0 0,5 1 1,5 0 0,5 1 1,5 Basic Acidic Neutral InertSustain C18 Pyridoxamine Uracil Succinic acid 5-Methyl uridine Dopamine Acetic Acid Pyridoxamine Uracil Succinic acid 5-Methyl uridine Dopamine Acetic Acid Pyridoxamine Uracil Succinic acid 5-Methyl uridine Dopamine Acetic Acid Pyridoxamine Uracil Succinic acid 5-Methyl uridine Dopamine Acetic Acid Atlantis T3 XSelect CSH C18 When the surface of the silica gel is basic: Retentivity = Acidic >> Basic (Neutral compounds are not influenced) When the surface of the silica gel is acidic: Retentivity = Basic >> Acidic (Neutral compounds are not influenced) InertSustain AQ-C18
0 2 4 6 8 Time (min) 0 2 4 6 8 Time (min) 1 2 1 2 3 Atlantis T3 3+4 65 4 5 6 Conditions Column ： 5 μm, 150 × 4.6 mm I.D. Eluent ：0.1% HCOOH in H2O Flow Rate ：1.0 mL/min Col. Temp. ：40 ℃ Detection ：UV 210 nm Sample 1. Pyridoxamine （B6) 2. Thiamin （B1） 3. Nicotinic Acid （B3） 4. Pyridoxal （B6） 5. Nicotinamide （B3） 6. Pyridoxine （B6） t0 t0 14 Comparison of Retention for Water-Soluble Vitamin B The use of ion-pairing reagents is a popular approach to retain water-soluble vitamins. Instead, 0.1% HCOOH in H2O was used to demonstrate and compare the retention for water-soluble vitamins. As proven below, InertSustain AQ-C18 delivered significant retention without the need for such reagents. InertSustain AQ-C18
0 10 20 Time (min) 0 10 20 Time (min) 0 10 20 Time (min) Atlantis T3 InertSustain C18 (Conventional ODS) Conditions Column ： 5 μm, 150 × 4.6 mm I.D. Eluent ： 0.1% HCOOH in H2O Flow Rate ：1.0 mL / min Col. Temp.：40 ℃ Detection ：UV 254 nm Sample 1. Cytidine 2. Uridine 3. Adenosine 4. Guanosine 5. 5-Methyl uridine t0 t0 t0 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 15 Comparison of Retention for Nucleosides Nucleosides are often analyzed in HILIC mode as they are highly hydrophilic . However, ODS columns also show some retention under 100% water mobile phase. In the following test, InertSustain AQ-C18 demonstrated to show simply stronger retention without the change of selectivity and analytical condition. InertSustain AQ-C18
0 2 4 6 8 10 Time (min) 0 2 4 6 8 10 Time (min) 0 2 4 6 8 10 Time (min) 1 3 2 4 56 1 32 4 5 6 1 2 4 5 6 3 Conditions Column : 5 μm, 150 × 4.6 mm I.D. Eluent ： A) ACN B) 0.1% HCOOH in H2O A/B = 20 /80 Flow Rate ：1.0 mL / min Col. Temp. ：40℃ Detection ：UV 280 nm Injection Vol. ：1 μL Sample： 1. Gallocatechin(-) (GC(-)) 2. Catechin(+) (C) 3. Epicatechin (-) (EC) 4. Epigallocatechin gallate(-) (EGCg) 5. Epicatechin gallate(-) (ECg) 6. Catechin gallate(-) (Cg) 100 μg/mL each 16 Comparison of Retention for Catechins (Neutral) As shown below, InertSustain AQ-C18 delivered stronger retention of catechins even under 20% organic solvent mobile phase with exceptional peak shapes, while competitive column brand failed. Furthermore, InertSustain AQ-C18 can prevent the co-elution between the targeted polar analytes and solvent peaks or sample matrices due to its enhanced retentivity. 2.0 2.2 2.4 2.6 Time(min) Elution with solvent peak Unresolved peaks 2.0 2.2 2.4 2.6 Time(min) Complete separation between solvent peak Baseline separation Atlantis T3 InertSustain C18 (Convetnional ODS) InertSustain AQ-C18
0 2 4 6 Time (min) Conditions Column : 5 μm, 150 × 4.6 mm I.D. Eluent ： A) ACN B) 0.1% HCOOH in H2O A/B = 20 /80 Flow Rate ：1.0 mL / min Col. Temp. ：40 ℃ Detection ：UV 270 nm Injection Vol. : 1 μL Sample 1. Chlorogenic acid 100 μg/mL (Acidic) 2. Caffeine 100 μg/mL (Basic) 3. Caffeic acid 100 μg/mL (Acidic) 0 2 4 6 Time (min) 0 2 4 6 Time (min) 1 2 3 1 2 3 1 2 3 t0 t0 t0 17 Comparison of Retention for Coffee Samples (Acidic, Basic) InertSustain AQ-C18 demonstrated stronger retention for hydrophilic coffee samples (acidic, basic) under 20% organic solvent mobile phase. Atlantis T3 InertSustain C18 (Convetnional ODS) InertSustain AQ-C18
Retention for Polar Compounds Highly Inert Packing Material InertSustain AQ-C18 Offer Both Superior Benefits 18 Exceptional Peak Shapes + Retention for Polar Compounds The structure of highly polar compounds contains ionic groups or metal complexes and they tend to be adsorbed to the packing material which results in tailing of peaks. In addition, polar embedded C18 columns or C18 columns with hydrophilic end-capping are available in the market for retaining highly polar compounds. However, they often show tailing or poor peak shapes due to the secondary interaction from their embedded functional groups. InertSustain AQ-C18 not only provide strong retention for highly polar compounds, but also delivery symmetric peak shapes for virtually any type of analytes. +
0 10 20 Time (min) Atlantis T3 1 2 3 0 10 20 Time (min) 0 10 20 Time (min) 0 10 20 Time (min) 1 2 3 1 2 3 Not eluted 1 0 10 20 Time (min) 1 CAPCELL PAK C18-AQ Synergi Fusion RP Aquasil C18 Conditions Eluent : A) CH3CN B) 25 mM K2HPO4 (pH 7.0) A/B = 30/70,v/v Flow Rate : 1 mL/min Col. Temp. : 40 ℃ Detection : UV 230 nm Sample : 1. Uracil 2. Berberine chloride 3. Dextromethorphan H3CO N CH3H N OCH3 OCH3 O O 2. Berberine chloride 3. Dextromethorphan 19 Dextromethorphan is a strong basic compound. Severe tailing can be confirmed when the packing material contains residual silanol groups. Comparison of Peak Shapes for Basic Compounds InertSustain AQ-C18 2, 3 Not eluted 2, 3 Not eluted
0 2 4 6 8 10 12 14 Time (min) 0 2 4 6 8 10 12 14 Time (min) 0 2 4 6 8 10 12 14 Time (min) 1 2 3 1 2 3 2 3 2 3 0 2 4 6 8 10 12 14 Time (min) 0 2 4 6 8 10 12 14 Time (min) 2 3 Conditions Eluent : A) CH3CN B) 0.1% H3PO4 in H2O A/B = 25/75,v/v Flow Rate : 1 mL/min Col. Temp. : 40 ℃ Detection : UV 254 nm Sample : 1. Brilliant Blue FCF 2. Phenol 3. Salicylic acid SO3 - N + N SO3 - - O3S 1. Brilliant Blue FCF 20 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. Comparison of Peak Shapes for Acidic Compounds Atlantis T3 CAPCELL PAK C18-AQ Synergi Fusion RP Aquasil C18InertSustain AQ-C18 1 Not eluted 1 Not eluted 1 Not eluted
1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 1: Split peak 1 2 3 1 2 1 2 3 2 1 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 1 Conditions Eluent : A) CH3CN B) 0.1%HCOOH in H2O A/B = 40/60 Flow Rate : 1mL/min Col. Temp. : 40 ℃ Detection : UV 310 nm Sample : 1. 8-Quinolinol 2. Hinokitiol 3. Piroctone Olamine O OH 2. Hinokitiol1. 8-Quinolino 3. Piroctone Olamine N O OH NH2 OH+ N OH 21 Comparison of Peak Shapes for Chelating Compounds Atlantis T3 CAPCELL PAK C18-AQ Synergi Fusion RP Aquasil C18InertSustain AQ-C18 1: Split peak 3 Not eluted 2 Not eluted 2, 3 Not eluted Hinokitiol and Piroctone Olamine are strong chelating compounds, 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.
Testing Procedure: 1) 100 % water is introduced into column over 60 minutes. 2) Conduct Analysis (1st injection). 3) Stop flow for 15 minutes. 4) 100 % water is introduced again into column over 30 minutes. 5) Conduct Analysis (2nd injection). 6) Stop flow for 15 minutes again. 7) 100 % water is introduced again into column over 30 minutes. 8) Conduct Analysis (3rd injection). 9) Disconnect the column from the system and stored for 24 hrs. 10) 100 % water is introduced into column over 30 minutes. 11) Conduct Analysis (4th injection). Conditions Eluent : H2O (100%) Flow Rate : 1 mL/min Col. Temp. : 40 ℃ Detection : UV 254 nm Sample : 1. Cytosine 2. Uracil 3. Guanine 4. Thymine 5. Adenine 22 Comparison of Compatibility with 100% Water Mobile Phase 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. InertSustain AQ-C18 guarantees compatibility with 100% aqueous mobile phases without the risk of dewetting.
0 2 4 6 8 10 12 14 16 18 Time (min) 0 2 4 6 8 10 12 14 16 18 Time (min) 1.3% Retention Lost 0 2 4 6 8 10 12 14 16 18 Time (min) 4th Injection 3rd Injection 1st Injection 0 10 20 Time (min) 0 10 20 Time (min) 0 10 20 Time (min) Atlantis T3 23 Comparison of Compatibility with 100% Water Mobile Phase InertSustain AQ-C18 Testing Procedure: 1) 100 % water is introduced into column over 60 minutes. 2) Conduct Analysis (1st injection). 3) Stop flow for 15 minutes. 4) 100 % water is introduced again into column over 30 minutes. 5) Conduct Analysis (2nd injection). 6) Stop flow for 15 minutes again. 7) 100 % water is introduced again into column over 30 minutes. 8) Conduct Analysis (3rd injection). 9) Disconnect the column from the system and stored for 24 hrs. 10) 100 % water is introduced into column over 30 minutes. 11) Conduct Analysis (4th injection). Conditions Eluent : H2O (100%) Flow Rate : 1 mL/min Col. Temp. : 40 ℃ Detection : UV 254 nm Sample : 1. Cytosine 2. Uracil 3. Guanine 4. Thymine 5. Adenine 2.3% Retention Lost 19.8% Retention Lost 26.2% Retention Lost
InertSustain C18 InertSustainSwift C18 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 min 0.00 0.25 0.50 0.75 1.00(x100,000,000) 1:TIC(+) 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 min 0.00 0.25 0.50 0.75 1.00(x100,000,000) 1:TIC(+) 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 min 0.00 0.25 0.50 0.75 1.00(x100,000,000) 1:TIC(+) 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 min 0.00 0.25 0.50 0.75 1.00(x100,000,000) 1:TIC(+) Conditions System : Nexera LCMS-8030 plus system Column : 3 μm , 50 x 2.1 mm I.D Eluent :A) 0.1% HCOOH in H2O B) 0.1% HCOOH in CH3CN Flow Rate : 0.4 mL/min Col. Temp. : 40 ℃ Detection : LC/MS/MS (ESI) , Positive m/z 50 – 1,000 Time (min) B% 0 5 2 100 5 100 5.1 5 8 5 Atlantis T3 24 Comparison of Column Bleed for LC/MS Methods InertSustain AQ-C18
InertSustian AQ-C18 InertSustainC18 Inertsil ODS-3 Inertsil ODS-4 Inertsil ODS-SP Inertsil ODS-EP AtlantisT3CAPCELL PAK C18-AQ Synagi FusionRP AQUASIL C18 XSELECT CSH C18 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 2 3 4 5 6 7 8 9 10 立体選択性α(Tri/o‐Ter) 疎水性 k (Amylbenzene) Inertsustain C18-AQ Inertsustain C18 Inertsil ODS-3 Inertsil ODS-4 Inertsil ODS-SP Inertsil ODS-EP Atlantis T3 CAPCELL PAK C18 AQ Synergi Fusion RP Aquqsil C18 X SELECT CSH C18 The stereo-selectivity between InertSustain AQ-C18 and Inertsil ODS-3 are about the same. 25 Comparison of Selectivity Hydrophobicity Stereo-selectivity CAPCELL PAK C18-AQ :Unknown, but probably isn’t a polar embedded type Synergi Fusion RP :Polar embedded type AQUASIL C18 :Hydrophilic end-capping type XSELECT CSH C18 :Polar embedded (Surface of silica is positive charge)
0 2 4 6 8 10 12 14 Time(min) Conditions Column :3 μm, 150 × 2.1 mm I.D. Eluent ： A) CH3OH B) H2O A/B = 80/20, v/v Flow Rate ：0.3 mL / min Col. Temp. ：40 ℃ Detection ：UV 254 nm Injection Vol. ：1 μL Sample： 1. Uracil 2. Caffeine 3. Phenol 4. Butylbenzene 5. o-Terphenyl 6. Amylbenzene 7. Triphenylene 26 Comparison of Selectivity InertSustain AQ-C18 Inertsil ODS-3 InertSustain C18 Inertsil ODS-4InertSustainSwift C18 0 2 4 6 8 10 12 14 Time(min) 1 2 3 4 6 5 7 0 2 4 6 8 10 12 14 Time(min) 1 2 3 4 56 7 0 2 4 6 8 10 12 14 Time(min) 1 2 3 4 5,6 7 0 2 4 6 8 10 12 14 Time(min) 1 2 3 4 5 6 7 1 2 3 4 5,6 7
0 20 40 60 80 100 120 0 50 100 150 200 250 300 350 理論段数 保持時間 Results: Retention Time :Only 10.7% Retention Lost Efficiency (N) :Only 0.2% Efficiency Lost Durability Test at Low pH (pH 1.0) Analytical Conditions： Eluent :CH3CN/H2O = (65/35), v/v Flow Rate :1.0 mL / min Col. Temp. :40 ℃ Detection :UV 254 nm Sample ：Naphthalene 27 Extended Column Lifetime at Low pH Purging Conditions： Column :5 μm, 150 × 4.6 mm I.D. Eluent ： CH3CN/ H2O/ TFA (10/90/1), v/v/v (pH 1) Column Temp. : 60 ℃ Efficiency (N) Retention Time
0 20 40 60 80 100 120 0 50 100 150 200 250 300 350 理論段数維持率 保持時間 28 Extended Column Lifetime at High pH Durability Test at High pH (pH 9.5) Analytical Conditions： Eluent :CH3CN/H2O = (65/35), v/v Flow Rate :1.0 mL / min Col. Temp. :40 ℃ Detection :UV 254 nm Sample ：Naphthalene Purging Conditions： Column :5 μm, 150 × 4.6 mm I.D. Eluent ：50 mM TEA (pH 9.5)/CH3OH = 70/30, v/v Flow Rate :1.0 mL/min Column Temp. : 50 ℃ Efficiency (N) Retention Time Results: Retention Time :Only 5.8% Retention Lost Efficiency (N) :Only 0.6% Efficiency Lost
29 Summary First Choice Column Conventional ODS Column In case there were no retention of polar compounds… Second Choice Column InertSustain AQ-C18 *Easier to develop and optimize analytical conditions *Offer strong polar compound retention even under 100% water or water rich mobile phases *The usage of highly inert packing results in delivering symmetric peaks for virtually any type of compounds *Eliminating the use of ion-pairing reagents improves method reproducibility, extends column lifetime and reduces instrument maintenance
• InertSustain C18 First Choice Conventional ODS Column • InertSustain AQ-C18 For Retaining Highly Polar Compounds • InertSustainSwift C18 For Rapid Elution of samples • InertSustain Phenyl • InertSustain Phenylhexyl For Changing Elution Pattern and to Improve Separation via pi-pi- Interaction 30 Column Selection Guide on InertSustain Series
0 10 20 30 40 50 60 70 Time (min) 4 5 6 7 3 2 1 0 10 20 Time (min) 4 5 3 2 1 Conditions Column : 5 μm, 150 × 4.6 mm I.D. Eluent ： 20 mM NaH2PO4 Flow Rate ：1.0 mL / min Col. Temp. ：40℃ Detection ：UV 254 nm Inj. Vol. ：2μL Sample ： 100 ppm 1.IMP 2.Uric Acid 3. Hypoxanthine 4. Xanthine 5. AMP 6. Inosine 7. Adenosine Conditions Column : 5 μm, 150 × 4.6 mm I.D. Eluent ： 20 mM NaH2PO4 Flow Rate ：1.0 mL / min Col. Temp. ：40℃ Detection ：UV 254 nm Inj. Vol. ：2μL Sample ： 100 ppm 1. GMP 2. Uric Acid 3. Guanine 4. Xanthine 5. Guanosine GMP Channel AMP Channel Analysis of Metabolites of Purine Nucleosides 31 Applications
1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) Conditions Column : 5 μm, 150 × 4.6 mm I.D. Eluent ： 25mM NaH2PO4 in H2O Flow Rate ：1.0 mL / min Col. Temp. ：40℃ Detection ：UV 254 nm Inj. Vol. ：1μL Sample ： 500μg/mL 1. CMP 2. UMP 3. GMP 4. AMP 1 2 3 4 32 Applications Analysis of Highly Hydrophilic Nucleotide Monophosphate
0 10 20 Time (min) 4 5 6 3 2 1 Conditions Column : 5 μm, 150 × 4.6 mm I.D. Eluent ：20 mM NaH2PO4 Flow Rate ：1.0 mL / min Col. Temp. ：40℃ Detection ：UV 254 nm Inj. Vol. ：1 μL Sample ： 100 ppm 1. ATP 2. ADP 3. IMP 4. Hypoxanthine 5. AMP 6. Inosine 33 Applications Analysis of Fish Freshness, K-Value
min 0 5 10 15 20 mAU 0 100 200 300 mAU 0 100 200 300 PDA-210 nm 026 Conditions Column : 3 μm, 150 × 4.6 mm I.D. Eluent ： A: 0.1% H3PO4 in H2O B: ACN A/B = 99/1 (5 min)- 99/1 – (20min) – 80/20 (5min), 99/1 Flow Rate ：1.0 mL / min Col. Temp. ：40℃ Detection ：UV 220 nm PDA Inj. Vol. ：5μL Sample： 1. Pyridoxamine 2. Thiamin 3. Nicotinic Acid 4. Ascorbic acid 5. Nicotinamide 6. Pyridoxal 7. Pyridoxine 8. Pantonenic Acid 9. Folic Acid 10. Cyanocobalamin 11. Riboflavin 12. Biotin 1 2 3 4 5 6 7 8 9 10 11 t0 34 Applications Analysis of Water-Soluble Vitamins Without the use of ion-pairing reagents 12
0 2 4 6 8 10 12 14 Time (min) 1 3 4 5 2 Conditions Column : 5 μm, 150 × 4.6 mm I.D. Eluent ： 0.1% H3PO4 in H2O Flow Rate ：1.0 mL / min Col. Temp. ：40℃ Detection ：UV 210 nm Inj. Vol. ：1 μL Sample： 1. Norepinephrine 2. L-Adrenaline 3. Dopamine 4. L-DOPA 5. L-(-)-Tyrosine OH OH NH2 HO OH OH N H HO HO HO OH O NH2 OH OH NH2 HO OH O NH2 35 Analysis of Catecolamines Applications 1 3 4 5 2 5
36 Analysis of Organic Acids Applications 0 2 4 6 8 Time (min) 1 2 3 4 5 6 7 8 9 10 11 Conditions Column ： 5 μm, 150 × 4.6 mm I.D. Eluent ： 50 mM NaH2PO4 in H2O (pH 2.1, H3PO4) Flow Rate ：1.0 mL / min Col. Temp. ：40℃ Detection ：UV 210 nm Inj. Vol. ：10 μL Sample： 1. Oxalic acid 50 mg/L 2. Tartaric acid 500 mg/L 3. Formic acid 1000 mg/L 4. Malic acid 1000 mg/L 5. Lactic acid 1000 mg/L 6. Acetic acid 1000 mg/L 7. Maleic acid 10 mg/L 8. Citric acid 1000 mg/L 9. Pyroglutamic acid 100 mg/L 10. Succinic acid 1000 mg/L 11. Fumaric acid 10 mg/L
Polar compounds coordinates to trace metal impurities in the packing resulting in providing poor peak shapes and low sensitivity. The same phenomenon is observed on water-soluble pesticides, however, InertSustain AQ-C18 delivers high sensitivity with exceptional peak shape, while other brand show adsorption of peak and lower sensitivity. Conditions System : Nexera LCMS-8030 plus system Column : InertSustain AQ-C18 (3 μm, 50 × 2.1 mm I.D) Eluent : A) 0.1% HCOOH in H2O B) 0.1% HCOOH in CH3CN Flow Rate : 0.4 mL/min Col. Temp. : 40 ℃ Detection : LC/MS/MS (ESI) , Positive Injection Vol.: 2 μL Sample : 1.Oxine Cu 200 μg/L Time (min) B% 0 5 1.50 30 1.51 5 3.00 5 0.0 0.5 1.0 min 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0(x10,000) 0.0 0.5 1.0 min 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0(x10,000) RT= 0.85minRT= 1.18min t0 t0 1 1 37 Analysis of Water-Soluble Pesticides Applications Atlantis T3InertSustain AQ-C18 Difference in Sensitivity
Conditions System : Nexera LCMS-8030 plus system Column size : 3 μm HP, 150 x 2.1 mm I.D Eluent : A) 5 mM Ammonium formate in H2O B) CH3CN Flow Rate : 0.3 mL/min Col. Temp. : 40 ℃ Detection : LC/MS/MS (ESI) , Positive Injection Vol.: 2 μL Sample : 1.ATP 500 μg/L 2.ADP 500 μg/L 3.AMP 500 μg/L Time (min) B% 0 2 0.5 2 3.0 25 3.01 2 7.00 2 38 Applications Analysis of Nucleotides via LC/MS/MS 0.0 2.5 min 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0(x10,000) 0.0 2.5 min 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0(x10,000) RT=1.54 minRT= 1.41 min 1 2 t0 Adsorption to the stainless steel hardware t0 3 Higher Sensitivity without adsorption Samples such as nucleotides have several phosphate groups which is sensitive to stainless steel hardware. As shown below, the combination of highly inert packing material of InertSustain AQ-C18 and usage of a new Steel-Coated-PEEK hardware (metal-free) deliver excellent peak shapes with higher sensitivity for phosphate compounds WITHOUT the formation of phosphate-iron complexes found with stainless steel column hardwares. Stainless Steel PEEK Packing Material Stainless Steel Hardware Packed with InertSustain AQ-C18 Steel-Coated-PEEK hardware Packed with InertSustain AQ-C18 Stainless Steel Packing Material
39 Inertsil ODS-SP Inertsil ODS-4 InertSustain AQ-C18 InertSustain C18 Carbon Loading 8.5 % 11 % 13 % 14 % Silica Gel 3 Series Silica Gel 3 Series Silica Gel ES Silica Gel ES Silica Gel Surface Area (m2/g） 450 450 350 350 Bonding Distance of C18 Groups Unadjusted Unadjusted Optimized Unadjusted Retention for Polar Analytes under Water Rich Mobile Phases ★★★★ ★★★★ ★★★★★ ★★★ Silica Gel C18 groups 39 Retention Mechanism of InertSustain AQ-C18 InertSustain AQ-C18 columns are designed to achieve strong retention for highly polar compounds, which is the most challenging goals in developing reversed phase methods. The optimization of bonding of the C18 groups at equal distance to the silica gel enable InertSustain AQ-C18 to offer significant retention for highly polar compounds even under water rich mobile phases.
Methanl/Water Acetonitrile/Water 0 Water 100% Organic 33 % Silica surface Gray zone: The border between mobile and stationary phases Distance from the silica surface Silica surface Abundance Schure, Mark R., Jake L. Rafferty, Ling Zhang, and J. Ilja Siepmann. 2013. How reversed-phase liquid chromatography works. LCGC North America 31 (8) (JAN 01): 630-7. Density profiles of C18 chains, methanol, water, and acetonitrile for pure water (W), pure methanol (M), pure acetonitrile (A), and solvent mixtures given as mole percent. It appears that very little solvent is present in the interior of the chain system for highly aqueous mobile phases and there are simply no solvent molecules to displace the solute. The solute, therefore, has little to no competition with the solvent for interaction sites within the chain interior region, resulting in less retention of analytes. http://www.chromatographyonline.com/how-reversed-phase-liquid- chromatography-works-0 Cited Literature Organic 67 % Organic 100 % Distance from the silica surface 40
Estimated Model of InertSustain AQ-C18 created by GL Sciences The black C18 line is flattened, which indicates the C18 groups are straightened out instead of being tangled up at the surface of silica. 41 Estimated Model of InertSustain AQ-C18 created by GL Sciences W (Water Rich) Gray zone, the border between mobile phase and stationary phase are broader/wider, which results in having more interaction between the C18 groups and analytes under water rich mobile phases.
42 Comparison of Retention Properties for Polar Compounds N NH O O N NH O O O OHOH N OH HO H2N O OH HO OH O O Succinic Acid (Acidic) Acetic Acid (Acidic) Uracil (Neutral) 5-Methyluridine (Neutral) Pyridoxamine (Basic) HO HO NH2 Dopamine (Basic)
0 0,5 1 1,5 Pyridoxamine Dopamine Acetic Acid Succinic acid Uracil 5-Methyluridine Atlantis T3 0 0,5 1 1,5 Pyridoxamine Dopamine Acetic Acid Succinic acid Uracil 5-Methyluridine Synergi Fusion RP 0 0,5 1 1,5 Pyridoxamine Dopamine Acetic Acid Succinic acid Uracil 5-Methyluridine InertSustain AQ-C18 Basic Acidic Neutral Pyridoxamine Urasil Succinic acid 5-Methyl uridine Dopamine Acetic Acid 43 0 0,5 1 1,5 Pyridoxamine Dopamine Acetic Acid Succinic acid Uracil 5-Methyluridine CAPCELL PAK C18-AQ 0 0,5 1 1,5 Pyridoxamine Dopamine Acetic Acid Succinic acid Uracil 5-Methyluridine XSELECT CSH C18 0 0,5 1 1,5 Pyridoxamine Dopamine Acetic Acid Succinic acid Uracil 5-Methyluridine AQUASIL C18 Comparison of Retention Properties for Polar Compounds
0 0,5 1 1,5 Pyridoxamine Dopamine Acetic Acid Succinic acid Uracil 5-Methyluridine InertSustain AQ-C18 0 0,5 1 1,5 Pyridoxamine Dopamine Acetic Acid Succinic acid Uracil 5-Methyluridine InertSustain C18 0 0,5 1 1,5 Pyridoxamine Dopamine Acetic Acid Succinic acid Uracil 5-Methyluridine Inertsil ODS-3 0 0,5 1 1,5 Pyridoxamine Dopamine Acetic Acid Succinic acid Uracil 5-Methyluridine Inertsil ODS-4 0 0,5 1 1,5 Pyridoxamine Dopamine Acetic Acid Succinic acid Uracil 5-Methyluridine Inertsil ODS-SP 0 0,5 1 1,5 Pyridoxamine Dopamine Acetic Acid Succinic acid Uracil 5-Methyluridine Inertsil ODS-EP Basic Acidic Neutral Pyridoxamine Urasil Succinic acid 5-Methyl uridine Dopamine Acetic Acid 44 Comparison of Retention Properties for Polar Compounds
Conditions Column : 5 μm, 150 × 4.6 mm I.D. Eluent : CH3OH/H2O = 80/20, v/v Flow Rate : 1.0 mL/min Col. Temp. : 40 ℃ Detection : UV 254 nm Injection Vol : 2.5 μL Sample ： 1. Uracil 2. Toluene 3. Ethylbenzene 4. Propylbenzene 5. n-Butylbenzene 6. n-Amylbenzene Sample No.6, n-Amylbenzene was used to determine the hydrophobic property of columns. R CH3 CH2CH3 CH2CH2CH3 CH2CH2CH2CH3 CH2CH2CH2CH2CH3 Hydrophobic Property of Column Conditions Eluent : A) CH3CN B) 25 mM K2HPO4 (pH 7.0, KH2PO4) A/B = 30/70,v/v Flow Rate : 1mL/min Col. Temp. : 40 ℃ Detection : UV 230 nm Injection Vol : 2.5 μL Sample ： 1:Uracil 2:Berberine chloride 3:Dextromethorphan H3CO N CH3H N OCH3 OCH3 O O Dextromethorphan is a strong basic compound. Severe tailing can be confirmed when the packing material contains residual silanol groups. 4. Berberine chloride 5. Dextromethorphan Basic Compound Test (2) Conditions Column : 5 μm, 150 × 4.6 mm I.D. Eluent : : A) CH3CN B) 25mM K2HPO4 (pH 7.0, KH2PO4) A/B = 60/40,v/v Flow Rate : 1.0 mL/min Col. Temp. : 40 ℃ Detection : UV 220 nm Injection Vol : 1μL Sample ： 1. Chlorpheniramine 2. Triprolidine 3. Homochlorcyclizine 4. Hydroxyzine 5. Clemastine 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 1, 2, 3 and 5. N Cl N H3C N N Cl N H N CH3 Cl N H N O OH CH3 Cl O N H CH3 1. Chlorpheniramine 2. Triprolidine 3. Homochlorcyclizine 4. Hydroxyzine 5. Clemastine Basic Compound Test (1) 45 Comparison of Performance
Conditions Column : 5 μm, 150 × 4.6 mm I.D. Eluent : A) CH3CN B) 0.1 %HCOOH A/B = 40/60,v/v Flow Rate : 1 mL/min Col. Temp. : 40 ℃ Detection : UV 310 nm Injection Vol : 2 μL Sample ： 1. 8-Quinolinol 2. Hinokitiol 3. Piroctone Olamine Traditional chelating compound test used phosphate buffer, which made it easy to elute these type of chelating compounds. In this test, formic acid mobile phase is used as it is regularly used in LC/MS methods and hard to detect in such condition. Piroctone Olamine is used in this test, which is now widely used as antimicrobial agents. O OH Chelating Compound Test 2. Hinokitiol 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. SO3 - N + N SO3 - - O3S Conditions Column : 5 μm, 150 × 4.6 mm I.D. Eluent : A) CH3CN B) 0.1% H3PO4 A/B = 25/75,v/v Flow Rate : 1 mL/min Col. Temp. : 40 ℃ Detection : UV 254 nm Injection Vol : 2 μL Sample ： 1.Brilliant Blue FCF 2. Phenol 3.Salicylic acid Acidic Compound Test 1. Brilliant Blue FCF Conditions Column : 5 μm, 150 × 4.6 mm I.D. Eluent : 0.1 %HCOOH Flow Rate : 1 mL/min Col. Temp. : 40 ℃ Detection : UV 210 nm Injection Vol : 2 μL Sample ： 1. Pyridoxamine (VB6) 2. Thiamin (VB1) 3. Nicotinic Acid (VB3) 4. Pyridoxal (VB6) 5. Nicotinamide (VB3) 6. Pyridoxine (VB6) Polar Compound Test (Water-Soluble Vitamins) 3. Piroctone Olamine N OH 1. 8-Quinolinol The use of ion-pairing reagents is a popular approach to retain water-soluble vitamins. Instead, 0.1% HCOOH in H2O was used to demonstrate and compare the retention for water-soluble vitamins. Pyridoxamine Thiamin Nicotinic Acid Pyridoxal Nicotinamide Pyridoxine 46 Comparison of Performance
Atlantis T3 47 Comparison of Performance (1/4) InertSustain AQ-C18 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 0 10 20 Time (min) 0 10 20 Time (min) 0 2 4 6 8 10 12 14 Time (min) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 0 10 20 Time (min) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 0 10 20 Time (min) 0 10 20 Time (min) 0 2 4 6 8 10 12 14 Time (min) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 0 10 20 Time (min) Polar Compound Test Basic Compound Test (1) Basic Compound Test (2) Acidic Compound Test Chelating Compound Test Hydrophobic Property 1 2 3 4 5 6 1 2 34 5 1 2 3 1 2 3 1 2 3 1 2 3 4 5 1 2 5 3,4 6 12 3 4 5 3, 5: Severe tailing of peaks 1 2 3 3:Adsorption of peak 1 2 3 1 2 3 1:Split peak 1 2 3 4 5
0 10 20 Time (min) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 0 2 4 6 8 10 12 14 Time (min) 0 10 20 Time (min) 0 10 20 30 Time (min) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) CAPCELL PAK C18-AQ 48 Comparison of Performance (2/4) InertSustain AQ-C18 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 0 10 20 Time (min) 0 10 20 Time (min) 0 2 4 6 8 10 12 14 Time (min) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 0 10 20 Time (min) Polar Compound Test Basic Compound Test (1) Basic Compound Test (2) Acidic Compound Test Chelating Compound Test Hydrophobic Property 1 2 3 4 5 6 1 2 34 5 1 2 3 1 2 3 1 2 3 1 2 3 4 5 1,2 3 5 4 6 12 3 4 5 1 2 3: Not eluted 2 3 1 2 3 1:Split peak 3:Adsorption of peak 1 2 3 4 5 1,2 were unretained 1: Not eluted3, 5: Severe tailing of peaks
1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 0 10 20 30 40 Time (min) 0 10 20 Time (min) 0 2 4 6 8 10 12 14 Time (min) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 0 10 20 Time (min) Synergi Fusion RP 49 Comparison of Performance (3/4) InertSustain AQ-C18 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 0 10 20 Time (min) 0 10 20 Time (min) 0 2 4 6 8 10 12 14 Time (min) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 0 10 20 Time (min) Polar Compound Test Basic Compound Test (1) Basic Compound Test (2) Acidic Compound Test Chelating Compound Test Hydrophobic Property 1 2 3 4 5 6 1 2 34 5 1 2 3 1 2 3 1 2 3 1 2 3 4 5 3 5 4 6 1 2 3 4 5 1 2 2: Adsorption of peak 3: Not eluted 2 3 1 2 2:Adsorption of peak 3:Not eluted 1 2 3 4 5 2: Not eluted 5: Tailing of peak 1: Not eluted1, 2, 3, 5: Severe tailing of peaks 1
1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 0 10 20 30 40 Time (min) 0 10 20 Time (min) 0 2 4 6 8 10 12 14 Time (min) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 0 10 20 Time (min) Aquasil C18 50 Comparison of Performance (4/4) InertSustain AQ-C18 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 0 10 20 Time (min) 0 10 20 Time (min) 0 2 4 6 8 10 12 14 Time (min) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 0 10 20 Time (min) Polar Compound Test Basic Compound Test (1) Basic Compound Test (2) Acidic Compound Test Chelating Compound Test Hydrophobic Property 1 2 3 4 5 6 1 2 34 5 1 2 3 1 2 3 1 2 3 1 2 3 4 5 3,4 5 2 6 4 1 2, 3: Not eluted 2 3 1 1:Tailing of peak 2, 3:Not eluted 1 2 3 4 5 2,6 are retained, but show slight adsorption 1: Not eluted1, 2, 3, 5: Not eluted 1
1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 0 10 20 Time (min) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 0 10 20 Time (min) 0 10 20 Time (min) 0 2 4 6 8 10 12 14 Time (min) InerSustain C18 51 Comparison of Performance between GL Sciences’ Columns (1/4) InertSustain AQ-C18 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 0 10 20 Time (min) 0 10 20 Time (min) 0 2 4 6 8 10 12 14 Time (min) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 0 10 20 Time (min) Polar Compound Test Basic Compound Test (1) Basic Compound Test (2) Acidic Compound Test Chelating Compound Test Hydrophobic Property 1 2 3 4 5 6 1 2 34 5 1 2 3 1 2 3 1 2 3 1 2 3 4 5 5 1 3,4 5 4 1 2 3 1 1 2 3 4 5 1,2 6 2 3 2 3 1 2 3
0 10 20 Time (min) 0 2 4 6 8 10 12 14 Time (min) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 0 10 20 Time (min) 0 10 20 Time (min) Inertsil ODS-4 52 Comparison of Performance between GL Sciences’ Columns (2/4) InertSustain AQ-C18 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 0 10 20 Time (min) 0 10 20 Time (min) 0 2 4 6 8 10 12 14 Time (min) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 0 10 20 Time (min) Polar Compound Test Basic Compound Test (1) Basic Compound Test (2) Acidic Compound Test Chelating Compound Test Hydrophobic Property 1 2 3 4 5 6 1 2 34 5 1 2 3 1 2 3 1 2 3 1 2 3 4 5 5 1 3,4 5 4 1 2 3 1 1 2 3 4 5 1 6 2 3 2 3 1 2 3 2
0 2 4 6 8 10 12 14 Time (min) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 0 10 20 Time (min) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 0 10 20 Time (min) 0 10 20 Time (min) Inertsil ODS-3 53 Comparison of Performance between GL Sciences’ Columns (3/4) InertSustain AQ-C18 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 0 10 20 Time (min) 0 10 20 Time (min) 0 2 4 6 8 10 12 14 Time (min) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 0 10 20 Time (min) Polar Compound Test Basic Compound Test (1) Basic Compound Test (2) Acidic Compound Test Chelating Compound Test Hydrophobic Property 1 2 3 4 5 6 1 2 34 5 1 2 3 1 2 3 1 2 3 1 2 3 4 5 3 1 5 5 4 1 2 3 1 1 2 3 4 5 1,2 6 2 3 2 3 1: Not eluted 2 4 1,2 were unretained 1:Split peak 3:Not eluted
1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 0 10 20 Time (min) 0 10 20 Time (min) 0 2 4 6 8 10 12 14 Time (min) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 0 10 20 Time (min) Inertsil ODS-SP 54 Comparison of Performance between GL Sciences’ Columns (4/4) InertSustain AQ-C18 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 0 10 20 Time (min) 0 10 20 Time (min) 0 2 4 6 8 10 12 14 Time (min) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 0 10 20 Time (min) Polar Compound Test Basic Compound Test (1) Basic Compound Test (2) Acidic Compound Test Chelating Compound Test Hydrophobic Property 1 2 3 4 5 6 1 2 34 5 1 2 3 1 2 3 1 2 3 1 2 3 4 5 5 1 5 4 1 2 3 1 1 2 3 4 5 1 6 2 3 2 3 2 3,4 2 3:Not eluted 1
55 Ordering Information Analytical Columns UP Type End-fittings End-fitting Format 3.3 mm 1/16” Waters End-fittings HP Series Particle Size：3 μm Max. Operating Pressure： 50 MPa (500 Bar) Length/ I.D. (mm) 2.1 3.0 4.6 30 5020-89920 5020-89926 5020-89932 50 5020-89921 5020-89927 5020-89933 75 5020-89922 5020-89928 5020-89934 100 5020-89923 5020-89929 5020-89935 150 5020-89924 5020-89930 5020-89936 250 5020-89925 5020-89931 5020-89937 2.4 mm※ End-fittings are 1/16” Waters-compatible. ※ UHPLC compatible end-fittings are also available upon request for UHPLC systems (Ex: UPLC®) to avoid dead volume. ※ Indicate “UP Type end-fittings” when ordering. (Please note that UP type is not available for a 4.6 mm I.D. column) UPLC® is a registered trademark of Waters Corporation. UP Type End-fittings 2.4 mm Particle Size：1.9 μm Max. Operating Pressure： 80 MPa (800 Bar) Length/ I.D. (mm) 2.1 3.0 50 5020-89938 5020-89941 100 5020-89939 5020-89942 150 5020-89940 5020-89943
56 Ordering Information Analytical Columns ※ End-fittings are 1/16” Waters-compatible. ※ Other column sizes available upon request.
57 Ordering Information Analytical Columns ※ End-fittings are 1/16” Waters-compatible. ※ Other column sizes available upon request.
58 Ordering Information Guard Column for UHPLC (Max. Operating Pressure 80 MPa, 800 Bar) ※ For connection details, please refer to the diagram below.
59 Ordering Information Cartridge Guard Columns ※ End-fittings are 1/16” Waters-compatible. ※ Maximum operating pressure is 20 MPa, 200 Bar.
60 Ordering Information Preparative Columns Preparative Columns
61 Ordering Information Capillary Columns ※ End-fittings are 1/16” Valco (10-32 UNF). ※ 1/32” Valco (6-40 UNF) end-fittings can also be arranged upon request. ※ Indicate “1/32” end-fittings” when ordering.
62 Ordering Information Connection Kits for Capillary EX-Nano and EX Columns
63 Ordering Information Metal-Free PEEK Columns ※ End-fittings are UP type end-fittings. ※ For further product details, please contact your local GL Sciences’ representative.
64 InertSearch Application Notes Access to the latest pharmaceutical, life science, environmental and food applications at www.glsciences.com/tech/inertsearch

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New HPLC Column; InertSustain AQ C18 technical info

  • 1.
  • 2.
    Physical Properties Silica :ES Silica Gel Particle Size : 1,9 μm, 3 μm, 5 μm Surface Area : 350 m2/g Pore Size : 100 Å (10 nm) Pore Volume : 0.85 mL/g Bonded Phase : Octadecyl Groups Bonded-Phase Structure : * Monomeric InertSustain AQ-C18® Carbon Load : 13.0 % End-capping : Complete pH Range : 1 – 10 USP Code : L1 * Although it is classified as a monomeric type ODS, the bonding of ODS groups are optimized to retain highly polar compounds.
  • 3.
    ・Exceptional polar compoundretention in reversed phase mode ・The usage of highly inert packing with superior column lifetime  True polar compound retention without using any secondary interaction  Improve separation between polar analytes and solvent peaks or sample matrices  Enhanced retention can also prevent ion suppression in LC/MS methods 3 Benefits of InertSustain AQ-C18
  • 4.
    First Choice Column ConventionalODS Column 1. Use of ion-pairing reagents 2. Use of 100% water mobile phase Often experience shorter column lifetime, poor method reproducibility, increased instrument maintenance… What other options are left? In case there were no retention of polar compounds… 4 Method Development for Polar Compounds
  • 5.
    ・Selecting different ODScolumns having different carbon loading % ・Selecting polar embedded ODS columns ・Selecting different stationary phase columns such as Phenyl or PFP columns 3. Use of different reversed phase columns 5 Method Development for Polar Compounds Second Choice Columns • Really hard to tell if the polar analytes will be retained or not. • Adsorbed peaks of target analytes occur due to secondary interaction.
  • 6.
    6 Method Development forPolar Compounds 4. Use of different interactions Second Choice Columns • Use of high concentration buffer (harsh mobile phase). • Reproducibility issues. • Takes time to equilibrate columns prior to the analysis. ・Selecting mix mode columns (ion-exchange + ODS) ・Selecting HILIC columns (hydrophilic interaction)
  • 7.
    7 Method Development forPolar Compounds How will InertSustain AQ-C18 columns help with your polar samples? ・Easier to develop and optimize analytical conditions ・Offer strong polar compound retention even under 100% water or water rich mobile phases
  • 8.
    8 Method Development forPolar Compounds ・The usage of highly inert packing results in delivering symmetric peaks for virtually any type of compounds ・Eliminating the use of ion-pairing reagents improves method reproducibility, extends column lifetime and reduces instrument maintenance How will InertSustain AQ-C18 columns help with your polar samples?
  • 9.
    Synthesis of ESsilica gel Bonding of ODS groups End-capping InertSustain C18 9 GL Sciences’ Accumulated Bonding Technology GL Sciences’ accumulated bonding technology and know-how allows InertSustain AQ-C18 to provide exceptional polar compound retention. Specifically, optimization of bonding of the C18 groups at equal distance to the silica gel enable InertSustain AQ-C18 to offer significant retention for highly polar compounds even under water rich mobile phases. Bonding of ODS groups GL Sciences’ Accumulated Bonding Technology End-capping InertSustain AQ-C18
  • 10.
    StrongerRetentionof Hydrophiliccompounds Stronger Retention ofHydrophobic compound 0,5 0,7 0,9 1,1 1,3 0,4 0,5 0,6 0,7 0,8 0,9 1 1,1 10 Retention Properties of InertSustain AQ-C18 InertSustain AQ-C18 Inertsil ODS-3 InertSustain C18 Inertsil ODS-4 Inertsil ODS-EP XSelect CSH C18 Synergi Fusion RP Altantis T3 CAPCELL PAK C18-AQ Inertsil ODS-SP Aquasil C18 RetentionFactorofacidic,basicandneutral compoundsusing100%watermobilephase Retention Factor of amylbenzene using methanol/water=80/20 Comparison of Retentivity for Hydrophilic compounds between various ODS columns Remarks: 1. The horizontal and vertical retention factor values were calculated with reference to the retention factor values obtained from InertSustain C18. 2. The horizontal values represents the retention factor of amylbenzene using methanol/water=80/20. 3. The vertical values represents the retention factor of acidic, basic and neutral compounds using 100% water mobile phase. (For further details, please see the next 2 slides)
  • 11.
    Two different mobilephases were used in this testing, 0.1 % formic acid in H2O and 0.1 % phosphoric acid in H2O. And total of 6 compounds were used to determine the retention factor of these 6 compounds to confirm the retentivity of each column. Determination of Retentivity for Hydrophilic Compounds Testing Conditions 1. Testing Conditions 2. Column ： 5 μm, 150 × 4.6 mm I.D. Column ： 5 μm, 150 × 4.6 mm I.D. Eluent ： 0.1 % formic acid in H2O Eluent ： 0.1 % phosphoric acid in H2O Sample: Sample: 1. Pyridoxamine (Basic) 1. Pyridoxamine (Basic) 2. Dopamine (Basic) 2. Dopamine (Basic) 3. Acetic Acid (Acidic) 3. Acetic Acid (Acidic) 4. Succinic Acid (Acidic) 4. Succinic Acid (Acidic) 5. Uracil (Neutral) 5. Uracil (Neutral) 6. 5-methyluridine (Neutral) 6. 5-methyluridine (Neutral) 11
  • 12.
  • 13.
    The following radarchart was created with reference to the retention factor values obtained from InertSustain C18 (conventional ODS). When the surface of silica is neutral and interactions between hydrophobic molecules are present under water rich mobile phases, the blue highlighted area will show larger area. Determination of Retentivity for Hydrophilic Compounds 13 0 0,5 1 1,5 0 0,5 1 1,5 0 0,5 1 1,5 0 0,5 1 1,5 Basic Acidic Neutral InertSustain C18 Pyridoxamine Uracil Succinic acid 5-Methyl uridine Dopamine Acetic Acid Pyridoxamine Uracil Succinic acid 5-Methyl uridine Dopamine Acetic Acid Pyridoxamine Uracil Succinic acid 5-Methyl uridine Dopamine Acetic Acid Pyridoxamine Uracil Succinic acid 5-Methyl uridine Dopamine Acetic Acid Atlantis T3 XSelect CSH C18 When the surface of the silica gel is basic: Retentivity = Acidic >> Basic (Neutral compounds are not influenced) When the surface of the silica gel is acidic: Retentivity = Basic >> Acidic (Neutral compounds are not influenced) InertSustain AQ-C18
  • 14.
    0 2 46 8 Time (min) 0 2 4 6 8 Time (min) 1 2 1 2 3 Atlantis T3 3+4 65 4 5 6 Conditions Column ： 5 μm, 150 × 4.6 mm I.D. Eluent ：0.1% HCOOH in H2O Flow Rate ：1.0 mL/min Col. Temp. ：40 ℃ Detection ：UV 210 nm Sample 1. Pyridoxamine （B6) 2. Thiamin （B1） 3. Nicotinic Acid （B3） 4. Pyridoxal （B6） 5. Nicotinamide （B3） 6. Pyridoxine （B6） t0 t0 14 Comparison of Retention for Water-Soluble Vitamin B The use of ion-pairing reagents is a popular approach to retain water-soluble vitamins. Instead, 0.1% HCOOH in H2O was used to demonstrate and compare the retention for water-soluble vitamins. As proven below, InertSustain AQ-C18 delivered significant retention without the need for such reagents. InertSustain AQ-C18
  • 15.
    0 10 20 Time(min) 0 10 20 Time (min) 0 10 20 Time (min) Atlantis T3 InertSustain C18 (Conventional ODS) Conditions Column ： 5 μm, 150 × 4.6 mm I.D. Eluent ： 0.1% HCOOH in H2O Flow Rate ：1.0 mL / min Col. Temp.：40 ℃ Detection ：UV 254 nm Sample 1. Cytidine 2. Uridine 3. Adenosine 4. Guanosine 5. 5-Methyl uridine t0 t0 t0 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 15 Comparison of Retention for Nucleosides Nucleosides are often analyzed in HILIC mode as they are highly hydrophilic . However, ODS columns also show some retention under 100% water mobile phase. In the following test, InertSustain AQ-C18 demonstrated to show simply stronger retention without the change of selectivity and analytical condition. InertSustain AQ-C18
  • 16.
    0 2 46 8 10 Time (min) 0 2 4 6 8 10 Time (min) 0 2 4 6 8 10 Time (min) 1 3 2 4 56 1 32 4 5 6 1 2 4 5 6 3 Conditions Column : 5 μm, 150 × 4.6 mm I.D. Eluent ： A) ACN B) 0.1% HCOOH in H2O A/B = 20 /80 Flow Rate ：1.0 mL / min Col. Temp. ：40℃ Detection ：UV 280 nm Injection Vol. ：1 μL Sample： 1. Gallocatechin(-) (GC(-)) 2. Catechin(+) (C) 3. Epicatechin (-) (EC) 4. Epigallocatechin gallate(-) (EGCg) 5. Epicatechin gallate(-) (ECg) 6. Catechin gallate(-) (Cg) 100 μg/mL each 16 Comparison of Retention for Catechins (Neutral) As shown below, InertSustain AQ-C18 delivered stronger retention of catechins even under 20% organic solvent mobile phase with exceptional peak shapes, while competitive column brand failed. Furthermore, InertSustain AQ-C18 can prevent the co-elution between the targeted polar analytes and solvent peaks or sample matrices due to its enhanced retentivity. 2.0 2.2 2.4 2.6 Time(min) Elution with solvent peak Unresolved peaks 2.0 2.2 2.4 2.6 Time(min) Complete separation between solvent peak Baseline separation Atlantis T3 InertSustain C18 (Convetnional ODS) InertSustain AQ-C18
  • 17.
    0 2 46 Time (min) Conditions Column : 5 μm, 150 × 4.6 mm I.D. Eluent ： A) ACN B) 0.1% HCOOH in H2O A/B = 20 /80 Flow Rate ：1.0 mL / min Col. Temp. ：40 ℃ Detection ：UV 270 nm Injection Vol. : 1 μL Sample 1. Chlorogenic acid 100 μg/mL (Acidic) 2. Caffeine 100 μg/mL (Basic) 3. Caffeic acid 100 μg/mL (Acidic) 0 2 4 6 Time (min) 0 2 4 6 Time (min) 1 2 3 1 2 3 1 2 3 t0 t0 t0 17 Comparison of Retention for Coffee Samples (Acidic, Basic) InertSustain AQ-C18 demonstrated stronger retention for hydrophilic coffee samples (acidic, basic) under 20% organic solvent mobile phase. Atlantis T3 InertSustain C18 (Convetnional ODS) InertSustain AQ-C18
  • 18.
    Retention for Polar Compounds HighlyInert Packing Material InertSustain AQ-C18 Offer Both Superior Benefits 18 Exceptional Peak Shapes + Retention for Polar Compounds The structure of highly polar compounds contains ionic groups or metal complexes and they tend to be adsorbed to the packing material which results in tailing of peaks. In addition, polar embedded C18 columns or C18 columns with hydrophilic end-capping are available in the market for retaining highly polar compounds. However, they often show tailing or poor peak shapes due to the secondary interaction from their embedded functional groups. InertSustain AQ-C18 not only provide strong retention for highly polar compounds, but also delivery symmetric peak shapes for virtually any type of analytes. +
  • 19.
    0 10 20 Time(min) Atlantis T3 1 2 3 0 10 20 Time (min) 0 10 20 Time (min) 0 10 20 Time (min) 1 2 3 1 2 3 Not eluted 1 0 10 20 Time (min) 1 CAPCELL PAK C18-AQ Synergi Fusion RP Aquasil C18 Conditions Eluent : A) CH3CN B) 25 mM K2HPO4 (pH 7.0) A/B = 30/70,v/v Flow Rate : 1 mL/min Col. Temp. : 40 ℃ Detection : UV 230 nm Sample : 1. Uracil 2. Berberine chloride 3. Dextromethorphan H3CO N CH3H N OCH3 OCH3 O O 2. Berberine chloride 3. Dextromethorphan 19 Dextromethorphan is a strong basic compound. Severe tailing can be confirmed when the packing material contains residual silanol groups. Comparison of Peak Shapes for Basic Compounds InertSustain AQ-C18 2, 3 Not eluted 2, 3 Not eluted
  • 20.
    0 2 46 8 10 12 14 Time (min) 0 2 4 6 8 10 12 14 Time (min) 0 2 4 6 8 10 12 14 Time (min) 1 2 3 1 2 3 2 3 2 3 0 2 4 6 8 10 12 14 Time (min) 0 2 4 6 8 10 12 14 Time (min) 2 3 Conditions Eluent : A) CH3CN B) 0.1% H3PO4 in H2O A/B = 25/75,v/v Flow Rate : 1 mL/min Col. Temp. : 40 ℃ Detection : UV 254 nm Sample : 1. Brilliant Blue FCF 2. Phenol 3. Salicylic acid SO3 - N + N SO3 - - O3S 1. Brilliant Blue FCF 20 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. Comparison of Peak Shapes for Acidic Compounds Atlantis T3 CAPCELL PAK C18-AQ Synergi Fusion RP Aquasil C18InertSustain AQ-C18 1 Not eluted 1 Not eluted 1 Not eluted
  • 21.
    1.0 2.0 3.04.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 1: Split peak 1 2 3 1 2 1 2 3 2 1 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 1 Conditions Eluent : A) CH3CN B) 0.1%HCOOH in H2O A/B = 40/60 Flow Rate : 1mL/min Col. Temp. : 40 ℃ Detection : UV 310 nm Sample : 1. 8-Quinolinol 2. Hinokitiol 3. Piroctone Olamine O OH 2. Hinokitiol1. 8-Quinolino 3. Piroctone Olamine N O OH NH2 OH+ N OH 21 Comparison of Peak Shapes for Chelating Compounds Atlantis T3 CAPCELL PAK C18-AQ Synergi Fusion RP Aquasil C18InertSustain AQ-C18 1: Split peak 3 Not eluted 2 Not eluted 2, 3 Not eluted Hinokitiol and Piroctone Olamine are strong chelating compounds, 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.
  • 22.
    Testing Procedure: 1) 100% water is introduced into column over 60 minutes. 2) Conduct Analysis (1st injection). 3) Stop flow for 15 minutes. 4) 100 % water is introduced again into column over 30 minutes. 5) Conduct Analysis (2nd injection). 6) Stop flow for 15 minutes again. 7) 100 % water is introduced again into column over 30 minutes. 8) Conduct Analysis (3rd injection). 9) Disconnect the column from the system and stored for 24 hrs. 10) 100 % water is introduced into column over 30 minutes. 11) Conduct Analysis (4th injection). Conditions Eluent : H2O (100%) Flow Rate : 1 mL/min Col. Temp. : 40 ℃ Detection : UV 254 nm Sample : 1. Cytosine 2. Uracil 3. Guanine 4. Thymine 5. Adenine 22 Comparison of Compatibility with 100% Water Mobile Phase 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. InertSustain AQ-C18 guarantees compatibility with 100% aqueous mobile phases without the risk of dewetting.
  • 23.
    0 2 46 8 10 12 14 16 18 Time (min) 0 2 4 6 8 10 12 14 16 18 Time (min) 1.3% Retention Lost 0 2 4 6 8 10 12 14 16 18 Time (min) 4th Injection 3rd Injection 1st Injection 0 10 20 Time (min) 0 10 20 Time (min) 0 10 20 Time (min) Atlantis T3 23 Comparison of Compatibility with 100% Water Mobile Phase InertSustain AQ-C18 Testing Procedure: 1) 100 % water is introduced into column over 60 minutes. 2) Conduct Analysis (1st injection). 3) Stop flow for 15 minutes. 4) 100 % water is introduced again into column over 30 minutes. 5) Conduct Analysis (2nd injection). 6) Stop flow for 15 minutes again. 7) 100 % water is introduced again into column over 30 minutes. 8) Conduct Analysis (3rd injection). 9) Disconnect the column from the system and stored for 24 hrs. 10) 100 % water is introduced into column over 30 minutes. 11) Conduct Analysis (4th injection). Conditions Eluent : H2O (100%) Flow Rate : 1 mL/min Col. Temp. : 40 ℃ Detection : UV 254 nm Sample : 1. Cytosine 2. Uracil 3. Guanine 4. Thymine 5. Adenine 2.3% Retention Lost 19.8% Retention Lost 26.2% Retention Lost
  • 24.
    InertSustain C18 InertSustainSwift C18 0.01.0 2.0 3.0 4.0 5.0 6.0 7.0 min 0.00 0.25 0.50 0.75 1.00(x100,000,000) 1:TIC(+) 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 min 0.00 0.25 0.50 0.75 1.00(x100,000,000) 1:TIC(+) 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 min 0.00 0.25 0.50 0.75 1.00(x100,000,000) 1:TIC(+) 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 min 0.00 0.25 0.50 0.75 1.00(x100,000,000) 1:TIC(+) Conditions System : Nexera LCMS-8030 plus system Column : 3 μm , 50 x 2.1 mm I.D Eluent :A) 0.1% HCOOH in H2O B) 0.1% HCOOH in CH3CN Flow Rate : 0.4 mL/min Col. Temp. : 40 ℃ Detection : LC/MS/MS (ESI) , Positive m/z 50 – 1,000 Time (min) B% 0 5 2 100 5 100 5.1 5 8 5 Atlantis T3 24 Comparison of Column Bleed for LC/MS Methods InertSustain AQ-C18
  • 25.
    InertSustian AQ-C18 InertSustainC18 Inertsil ODS-3 InertsilODS-4 Inertsil ODS-SP Inertsil ODS-EP AtlantisT3CAPCELL PAK C18-AQ Synagi FusionRP AQUASIL C18 XSELECT CSH C18 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 2 3 4 5 6 7 8 9 10 立体選択性α(Tri/o‐Ter) 疎水性 k (Amylbenzene) Inertsustain C18-AQ Inertsustain C18 Inertsil ODS-3 Inertsil ODS-4 Inertsil ODS-SP Inertsil ODS-EP Atlantis T3 CAPCELL PAK C18 AQ Synergi Fusion RP Aquqsil C18 X SELECT CSH C18 The stereo-selectivity between InertSustain AQ-C18 and Inertsil ODS-3 are about the same. 25 Comparison of Selectivity Hydrophobicity Stereo-selectivity CAPCELL PAK C18-AQ :Unknown, but probably isn’t a polar embedded type Synergi Fusion RP :Polar embedded type AQUASIL C18 :Hydrophilic end-capping type XSELECT CSH C18 :Polar embedded (Surface of silica is positive charge)
  • 26.
    0 2 46 8 10 12 14 Time(min) Conditions Column :3 μm, 150 × 2.1 mm I.D. Eluent ： A) CH3OH B) H2O A/B = 80/20, v/v Flow Rate ：0.3 mL / min Col. Temp. ：40 ℃ Detection ：UV 254 nm Injection Vol. ：1 μL Sample： 1. Uracil 2. Caffeine 3. Phenol 4. Butylbenzene 5. o-Terphenyl 6. Amylbenzene 7. Triphenylene 26 Comparison of Selectivity InertSustain AQ-C18 Inertsil ODS-3 InertSustain C18 Inertsil ODS-4InertSustainSwift C18 0 2 4 6 8 10 12 14 Time(min) 1 2 3 4 6 5 7 0 2 4 6 8 10 12 14 Time(min) 1 2 3 4 56 7 0 2 4 6 8 10 12 14 Time(min) 1 2 3 4 5,6 7 0 2 4 6 8 10 12 14 Time(min) 1 2 3 4 5 6 7 1 2 3 4 5,6 7
  • 27.
    0 20 40 60 80 100 120 0 50 100150 200 250 300 350 理論段数 保持時間 Results: Retention Time :Only 10.7% Retention Lost Efficiency (N) :Only 0.2% Efficiency Lost Durability Test at Low pH (pH 1.0) Analytical Conditions： Eluent :CH3CN/H2O = (65/35), v/v Flow Rate :1.0 mL / min Col. Temp. :40 ℃ Detection :UV 254 nm Sample ：Naphthalene 27 Extended Column Lifetime at Low pH Purging Conditions： Column :5 μm, 150 × 4.6 mm I.D. Eluent ： CH3CN/ H2O/ TFA (10/90/1), v/v/v (pH 1) Column Temp. : 60 ℃ Efficiency (N) Retention Time
  • 28.
    0 20 40 60 80 100 120 0 50 100150 200 250 300 350 理論段数維持率 保持時間 28 Extended Column Lifetime at High pH Durability Test at High pH (pH 9.5) Analytical Conditions： Eluent :CH3CN/H2O = (65/35), v/v Flow Rate :1.0 mL / min Col. Temp. :40 ℃ Detection :UV 254 nm Sample ：Naphthalene Purging Conditions： Column :5 μm, 150 × 4.6 mm I.D. Eluent ：50 mM TEA (pH 9.5)/CH3OH = 70/30, v/v Flow Rate :1.0 mL/min Column Temp. : 50 ℃ Efficiency (N) Retention Time Results: Retention Time :Only 5.8% Retention Lost Efficiency (N) :Only 0.6% Efficiency Lost
  • 29.
    29 Summary First Choice Column ConventionalODS Column In case there were no retention of polar compounds… Second Choice Column InertSustain AQ-C18 *Easier to develop and optimize analytical conditions *Offer strong polar compound retention even under 100% water or water rich mobile phases *The usage of highly inert packing results in delivering symmetric peaks for virtually any type of compounds *Eliminating the use of ion-pairing reagents improves method reproducibility, extends column lifetime and reduces instrument maintenance
  • 30.
    • InertSustain C18First Choice Conventional ODS Column • InertSustain AQ-C18 For Retaining Highly Polar Compounds • InertSustainSwift C18 For Rapid Elution of samples • InertSustain Phenyl • InertSustain Phenylhexyl For Changing Elution Pattern and to Improve Separation via pi-pi- Interaction 30 Column Selection Guide on InertSustain Series
  • 31.
    0 10 2030 40 50 60 70 Time (min) 4 5 6 7 3 2 1 0 10 20 Time (min) 4 5 3 2 1 Conditions Column : 5 μm, 150 × 4.6 mm I.D. Eluent ： 20 mM NaH2PO4 Flow Rate ：1.0 mL / min Col. Temp. ：40℃ Detection ：UV 254 nm Inj. Vol. ：2μL Sample ： 100 ppm 1.IMP 2.Uric Acid 3. Hypoxanthine 4. Xanthine 5. AMP 6. Inosine 7. Adenosine Conditions Column : 5 μm, 150 × 4.6 mm I.D. Eluent ： 20 mM NaH2PO4 Flow Rate ：1.0 mL / min Col. Temp. ：40℃ Detection ：UV 254 nm Inj. Vol. ：2μL Sample ： 100 ppm 1. GMP 2. Uric Acid 3. Guanine 4. Xanthine 5. Guanosine GMP Channel AMP Channel Analysis of Metabolites of Purine Nucleosides 31 Applications
  • 32.
    1.0 2.0 3.04.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) Conditions Column : 5 μm, 150 × 4.6 mm I.D. Eluent ： 25mM NaH2PO4 in H2O Flow Rate ：1.0 mL / min Col. Temp. ：40℃ Detection ：UV 254 nm Inj. Vol. ：1μL Sample ： 500μg/mL 1. CMP 2. UMP 3. GMP 4. AMP 1 2 3 4 32 Applications Analysis of Highly Hydrophilic Nucleotide Monophosphate
  • 33.
    0 10 20 Time(min) 4 5 6 3 2 1 Conditions Column : 5 μm, 150 × 4.6 mm I.D. Eluent ：20 mM NaH2PO4 Flow Rate ：1.0 mL / min Col. Temp. ：40℃ Detection ：UV 254 nm Inj. Vol. ：1 μL Sample ： 100 ppm 1. ATP 2. ADP 3. IMP 4. Hypoxanthine 5. AMP 6. Inosine 33 Applications Analysis of Fish Freshness, K-Value
  • 34.
    min 0 5 1015 20 mAU 0 100 200 300 mAU 0 100 200 300 PDA-210 nm 026 Conditions Column : 3 μm, 150 × 4.6 mm I.D. Eluent ： A: 0.1% H3PO4 in H2O B: ACN A/B = 99/1 (5 min)- 99/1 – (20min) – 80/20 (5min), 99/1 Flow Rate ：1.0 mL / min Col. Temp. ：40℃ Detection ：UV 220 nm PDA Inj. Vol. ：5μL Sample： 1. Pyridoxamine 2. Thiamin 3. Nicotinic Acid 4. Ascorbic acid 5. Nicotinamide 6. Pyridoxal 7. Pyridoxine 8. Pantonenic Acid 9. Folic Acid 10. Cyanocobalamin 11. Riboflavin 12. Biotin 1 2 3 4 5 6 7 8 9 10 11 t0 34 Applications Analysis of Water-Soluble Vitamins Without the use of ion-pairing reagents 12
  • 35.
    0 2 46 8 10 12 14 Time (min) 1 3 4 5 2 Conditions Column : 5 μm, 150 × 4.6 mm I.D. Eluent ： 0.1% H3PO4 in H2O Flow Rate ：1.0 mL / min Col. Temp. ：40℃ Detection ：UV 210 nm Inj. Vol. ：1 μL Sample： 1. Norepinephrine 2. L-Adrenaline 3. Dopamine 4. L-DOPA 5. L-(-)-Tyrosine OH OH NH2 HO OH OH N H HO HO HO OH O NH2 OH OH NH2 HO OH O NH2 35 Analysis of Catecolamines Applications 1 3 4 5 2 5
  • 36.
    36 Analysis of OrganicAcids Applications 0 2 4 6 8 Time (min) 1 2 3 4 5 6 7 8 9 10 11 Conditions Column ： 5 μm, 150 × 4.6 mm I.D. Eluent ： 50 mM NaH2PO4 in H2O (pH 2.1, H3PO4) Flow Rate ：1.0 mL / min Col. Temp. ：40℃ Detection ：UV 210 nm Inj. Vol. ：10 μL Sample： 1. Oxalic acid 50 mg/L 2. Tartaric acid 500 mg/L 3. Formic acid 1000 mg/L 4. Malic acid 1000 mg/L 5. Lactic acid 1000 mg/L 6. Acetic acid 1000 mg/L 7. Maleic acid 10 mg/L 8. Citric acid 1000 mg/L 9. Pyroglutamic acid 100 mg/L 10. Succinic acid 1000 mg/L 11. Fumaric acid 10 mg/L
  • 37.
    Polar compounds coordinatesto trace metal impurities in the packing resulting in providing poor peak shapes and low sensitivity. The same phenomenon is observed on water-soluble pesticides, however, InertSustain AQ-C18 delivers high sensitivity with exceptional peak shape, while other brand show adsorption of peak and lower sensitivity. Conditions System : Nexera LCMS-8030 plus system Column : InertSustain AQ-C18 (3 μm, 50 × 2.1 mm I.D) Eluent : A) 0.1% HCOOH in H2O B) 0.1% HCOOH in CH3CN Flow Rate : 0.4 mL/min Col. Temp. : 40 ℃ Detection : LC/MS/MS (ESI) , Positive Injection Vol.: 2 μL Sample : 1.Oxine Cu 200 μg/L Time (min) B% 0 5 1.50 30 1.51 5 3.00 5 0.0 0.5 1.0 min 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0(x10,000) 0.0 0.5 1.0 min 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0(x10,000) RT= 0.85minRT= 1.18min t0 t0 1 1 37 Analysis of Water-Soluble Pesticides Applications Atlantis T3InertSustain AQ-C18 Difference in Sensitivity
  • 38.
    Conditions System : NexeraLCMS-8030 plus system Column size : 3 μm HP, 150 x 2.1 mm I.D Eluent : A) 5 mM Ammonium formate in H2O B) CH3CN Flow Rate : 0.3 mL/min Col. Temp. : 40 ℃ Detection : LC/MS/MS (ESI) , Positive Injection Vol.: 2 μL Sample : 1.ATP 500 μg/L 2.ADP 500 μg/L 3.AMP 500 μg/L Time (min) B% 0 2 0.5 2 3.0 25 3.01 2 7.00 2 38 Applications Analysis of Nucleotides via LC/MS/MS 0.0 2.5 min 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0(x10,000) 0.0 2.5 min 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0(x10,000) RT=1.54 minRT= 1.41 min 1 2 t0 Adsorption to the stainless steel hardware t0 3 Higher Sensitivity without adsorption Samples such as nucleotides have several phosphate groups which is sensitive to stainless steel hardware. As shown below, the combination of highly inert packing material of InertSustain AQ-C18 and usage of a new Steel-Coated-PEEK hardware (metal-free) deliver excellent peak shapes with higher sensitivity for phosphate compounds WITHOUT the formation of phosphate-iron complexes found with stainless steel column hardwares. Stainless Steel PEEK Packing Material Stainless Steel Hardware Packed with InertSustain AQ-C18 Steel-Coated-PEEK hardware Packed with InertSustain AQ-C18 Stainless Steel Packing Material
  • 39.
    39 Inertsil ODS-SP InertsilODS-4 InertSustain AQ-C18 InertSustain C18 Carbon Loading 8.5 % 11 % 13 % 14 % Silica Gel 3 Series Silica Gel 3 Series Silica Gel ES Silica Gel ES Silica Gel Surface Area (m2/g） 450 450 350 350 Bonding Distance of C18 Groups Unadjusted Unadjusted Optimized Unadjusted Retention for Polar Analytes under Water Rich Mobile Phases ★★★★ ★★★★ ★★★★★ ★★★ Silica Gel C18 groups 39 Retention Mechanism of InertSustain AQ-C18 InertSustain AQ-C18 columns are designed to achieve strong retention for highly polar compounds, which is the most challenging goals in developing reversed phase methods. The optimization of bonding of the C18 groups at equal distance to the silica gel enable InertSustain AQ-C18 to offer significant retention for highly polar compounds even under water rich mobile phases.
  • 40.
    Methanl/Water Acetonitrile/Water 0 Water 100% Organic33 % Silica surface Gray zone: The border between mobile and stationary phases Distance from the silica surface Silica surface Abundance Schure, Mark R., Jake L. Rafferty, Ling Zhang, and J. Ilja Siepmann. 2013. How reversed-phase liquid chromatography works. LCGC North America 31 (8) (JAN 01): 630-7. Density profiles of C18 chains, methanol, water, and acetonitrile for pure water (W), pure methanol (M), pure acetonitrile (A), and solvent mixtures given as mole percent. It appears that very little solvent is present in the interior of the chain system for highly aqueous mobile phases and there are simply no solvent molecules to displace the solute. The solute, therefore, has little to no competition with the solvent for interaction sites within the chain interior region, resulting in less retention of analytes. http://www.chromatographyonline.com/how-reversed-phase-liquid- chromatography-works-0 Cited Literature Organic 67 % Organic 100 % Distance from the silica surface 40
  • 41.
    Estimated Model of InertSustainAQ-C18 created by GL Sciences The black C18 line is flattened, which indicates the C18 groups are straightened out instead of being tangled up at the surface of silica. 41 Estimated Model of InertSustain AQ-C18 created by GL Sciences W (Water Rich) Gray zone, the border between mobile phase and stationary phase are broader/wider, which results in having more interaction between the C18 groups and analytes under water rich mobile phases.
  • 42.
    42 Comparison of RetentionProperties for Polar Compounds N NH O O N NH O O O OHOH N OH HO H2N O OH HO OH O O Succinic Acid (Acidic) Acetic Acid (Acidic) Uracil (Neutral) 5-Methyluridine (Neutral) Pyridoxamine (Basic) HO HO NH2 Dopamine (Basic)
  • 43.
    0 0,5 1 1,5 Pyridoxamine Dopamine Acetic Acid Succinic acid Uracil 5-Methyluridine AtlantisT3 0 0,5 1 1,5 Pyridoxamine Dopamine Acetic Acid Succinic acid Uracil 5-Methyluridine Synergi Fusion RP 0 0,5 1 1,5 Pyridoxamine Dopamine Acetic Acid Succinic acid Uracil 5-Methyluridine InertSustain AQ-C18 Basic Acidic Neutral Pyridoxamine Urasil Succinic acid 5-Methyl uridine Dopamine Acetic Acid 43 0 0,5 1 1,5 Pyridoxamine Dopamine Acetic Acid Succinic acid Uracil 5-Methyluridine CAPCELL PAK C18-AQ 0 0,5 1 1,5 Pyridoxamine Dopamine Acetic Acid Succinic acid Uracil 5-Methyluridine XSELECT CSH C18 0 0,5 1 1,5 Pyridoxamine Dopamine Acetic Acid Succinic acid Uracil 5-Methyluridine AQUASIL C18 Comparison of Retention Properties for Polar Compounds
  • 44.
    0 0,5 1 1,5 Pyridoxamine Dopamine Acetic Acid Succinic acid Uracil 5-Methyluridine InertSustainAQ-C18 0 0,5 1 1,5 Pyridoxamine Dopamine Acetic Acid Succinic acid Uracil 5-Methyluridine InertSustain C18 0 0,5 1 1,5 Pyridoxamine Dopamine Acetic Acid Succinic acid Uracil 5-Methyluridine Inertsil ODS-3 0 0,5 1 1,5 Pyridoxamine Dopamine Acetic Acid Succinic acid Uracil 5-Methyluridine Inertsil ODS-4 0 0,5 1 1,5 Pyridoxamine Dopamine Acetic Acid Succinic acid Uracil 5-Methyluridine Inertsil ODS-SP 0 0,5 1 1,5 Pyridoxamine Dopamine Acetic Acid Succinic acid Uracil 5-Methyluridine Inertsil ODS-EP Basic Acidic Neutral Pyridoxamine Urasil Succinic acid 5-Methyl uridine Dopamine Acetic Acid 44 Comparison of Retention Properties for Polar Compounds
  • 45.
    Conditions Column : 5μm, 150 × 4.6 mm I.D. Eluent : CH3OH/H2O = 80/20, v/v Flow Rate : 1.0 mL/min Col. Temp. : 40 ℃ Detection : UV 254 nm Injection Vol : 2.5 μL Sample ： 1. Uracil 2. Toluene 3. Ethylbenzene 4. Propylbenzene 5. n-Butylbenzene 6. n-Amylbenzene Sample No.6, n-Amylbenzene was used to determine the hydrophobic property of columns. R CH3 CH2CH3 CH2CH2CH3 CH2CH2CH2CH3 CH2CH2CH2CH2CH3 Hydrophobic Property of Column Conditions Eluent : A) CH3CN B) 25 mM K2HPO4 (pH 7.0, KH2PO4) A/B = 30/70,v/v Flow Rate : 1mL/min Col. Temp. : 40 ℃ Detection : UV 230 nm Injection Vol : 2.5 μL Sample ： 1:Uracil 2:Berberine chloride 3:Dextromethorphan H3CO N CH3H N OCH3 OCH3 O O Dextromethorphan is a strong basic compound. Severe tailing can be confirmed when the packing material contains residual silanol groups. 4. Berberine chloride 5. Dextromethorphan Basic Compound Test (2) Conditions Column : 5 μm, 150 × 4.6 mm I.D. Eluent : : A) CH3CN B) 25mM K2HPO4 (pH 7.0, KH2PO4) A/B = 60/40,v/v Flow Rate : 1.0 mL/min Col. Temp. : 40 ℃ Detection : UV 220 nm Injection Vol : 1μL Sample ： 1. Chlorpheniramine 2. Triprolidine 3. Homochlorcyclizine 4. Hydroxyzine 5. Clemastine 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 1, 2, 3 and 5. N Cl N H3C N N Cl N H N CH3 Cl N H N O OH CH3 Cl O N H CH3 1. Chlorpheniramine 2. Triprolidine 3. Homochlorcyclizine 4. Hydroxyzine 5. Clemastine Basic Compound Test (1) 45 Comparison of Performance
  • 46.
    Conditions Column : 5μm, 150 × 4.6 mm I.D. Eluent : A) CH3CN B) 0.1 %HCOOH A/B = 40/60,v/v Flow Rate : 1 mL/min Col. Temp. : 40 ℃ Detection : UV 310 nm Injection Vol : 2 μL Sample ： 1. 8-Quinolinol 2. Hinokitiol 3. Piroctone Olamine Traditional chelating compound test used phosphate buffer, which made it easy to elute these type of chelating compounds. In this test, formic acid mobile phase is used as it is regularly used in LC/MS methods and hard to detect in such condition. Piroctone Olamine is used in this test, which is now widely used as antimicrobial agents. O OH Chelating Compound Test 2. Hinokitiol 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. SO3 - N + N SO3 - - O3S Conditions Column : 5 μm, 150 × 4.6 mm I.D. Eluent : A) CH3CN B) 0.1% H3PO4 A/B = 25/75,v/v Flow Rate : 1 mL/min Col. Temp. : 40 ℃ Detection : UV 254 nm Injection Vol : 2 μL Sample ： 1.Brilliant Blue FCF 2. Phenol 3.Salicylic acid Acidic Compound Test 1. Brilliant Blue FCF Conditions Column : 5 μm, 150 × 4.6 mm I.D. Eluent : 0.1 %HCOOH Flow Rate : 1 mL/min Col. Temp. : 40 ℃ Detection : UV 210 nm Injection Vol : 2 μL Sample ： 1. Pyridoxamine (VB6) 2. Thiamin (VB1) 3. Nicotinic Acid (VB3) 4. Pyridoxal (VB6) 5. Nicotinamide (VB3) 6. Pyridoxine (VB6) Polar Compound Test (Water-Soluble Vitamins) 3. Piroctone Olamine N OH 1. 8-Quinolinol The use of ion-pairing reagents is a popular approach to retain water-soluble vitamins. Instead, 0.1% HCOOH in H2O was used to demonstrate and compare the retention for water-soluble vitamins. Pyridoxamine Thiamin Nicotinic Acid Pyridoxal Nicotinamide Pyridoxine 46 Comparison of Performance
  • 47.
    Atlantis T3 47 Comparison ofPerformance (1/4) InertSustain AQ-C18 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 0 10 20 Time (min) 0 10 20 Time (min) 0 2 4 6 8 10 12 14 Time (min) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 0 10 20 Time (min) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 0 10 20 Time (min) 0 10 20 Time (min) 0 2 4 6 8 10 12 14 Time (min) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 0 10 20 Time (min) Polar Compound Test Basic Compound Test (1) Basic Compound Test (2) Acidic Compound Test Chelating Compound Test Hydrophobic Property 1 2 3 4 5 6 1 2 34 5 1 2 3 1 2 3 1 2 3 1 2 3 4 5 1 2 5 3,4 6 12 3 4 5 3, 5: Severe tailing of peaks 1 2 3 3:Adsorption of peak 1 2 3 1 2 3 1:Split peak 1 2 3 4 5
  • 48.
    0 10 20 Time(min) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 0 2 4 6 8 10 12 14 Time (min) 0 10 20 Time (min) 0 10 20 30 Time (min) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) CAPCELL PAK C18-AQ 48 Comparison of Performance (2/4) InertSustain AQ-C18 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 0 10 20 Time (min) 0 10 20 Time (min) 0 2 4 6 8 10 12 14 Time (min) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 0 10 20 Time (min) Polar Compound Test Basic Compound Test (1) Basic Compound Test (2) Acidic Compound Test Chelating Compound Test Hydrophobic Property 1 2 3 4 5 6 1 2 34 5 1 2 3 1 2 3 1 2 3 1 2 3 4 5 1,2 3 5 4 6 12 3 4 5 1 2 3: Not eluted 2 3 1 2 3 1:Split peak 3:Adsorption of peak 1 2 3 4 5 1,2 were unretained 1: Not eluted3, 5: Severe tailing of peaks
  • 49.
    1.0 2.0 3.04.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 0 10 20 30 40 Time (min) 0 10 20 Time (min) 0 2 4 6 8 10 12 14 Time (min) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 0 10 20 Time (min) Synergi Fusion RP 49 Comparison of Performance (3/4) InertSustain AQ-C18 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 0 10 20 Time (min) 0 10 20 Time (min) 0 2 4 6 8 10 12 14 Time (min) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 0 10 20 Time (min) Polar Compound Test Basic Compound Test (1) Basic Compound Test (2) Acidic Compound Test Chelating Compound Test Hydrophobic Property 1 2 3 4 5 6 1 2 34 5 1 2 3 1 2 3 1 2 3 1 2 3 4 5 3 5 4 6 1 2 3 4 5 1 2 2: Adsorption of peak 3: Not eluted 2 3 1 2 2:Adsorption of peak 3:Not eluted 1 2 3 4 5 2: Not eluted 5: Tailing of peak 1: Not eluted1, 2, 3, 5: Severe tailing of peaks 1
  • 50.
    1.0 2.0 3.04.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 0 10 20 30 40 Time (min) 0 10 20 Time (min) 0 2 4 6 8 10 12 14 Time (min) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 0 10 20 Time (min) Aquasil C18 50 Comparison of Performance (4/4) InertSustain AQ-C18 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 0 10 20 Time (min) 0 10 20 Time (min) 0 2 4 6 8 10 12 14 Time (min) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 0 10 20 Time (min) Polar Compound Test Basic Compound Test (1) Basic Compound Test (2) Acidic Compound Test Chelating Compound Test Hydrophobic Property 1 2 3 4 5 6 1 2 34 5 1 2 3 1 2 3 1 2 3 1 2 3 4 5 3,4 5 2 6 4 1 2, 3: Not eluted 2 3 1 1:Tailing of peak 2, 3:Not eluted 1 2 3 4 5 2,6 are retained, but show slight adsorption 1: Not eluted1, 2, 3, 5: Not eluted 1
  • 51.
    1.0 2.0 3.04.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 0 10 20 Time (min) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 0 10 20 Time (min) 0 10 20 Time (min) 0 2 4 6 8 10 12 14 Time (min) InerSustain C18 51 Comparison of Performance between GL Sciences’ Columns (1/4) InertSustain AQ-C18 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 0 10 20 Time (min) 0 10 20 Time (min) 0 2 4 6 8 10 12 14 Time (min) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 0 10 20 Time (min) Polar Compound Test Basic Compound Test (1) Basic Compound Test (2) Acidic Compound Test Chelating Compound Test Hydrophobic Property 1 2 3 4 5 6 1 2 34 5 1 2 3 1 2 3 1 2 3 1 2 3 4 5 5 1 3,4 5 4 1 2 3 1 1 2 3 4 5 1,2 6 2 3 2 3 1 2 3
  • 52.
    0 10 20 Time(min) 0 2 4 6 8 10 12 14 Time (min) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 0 10 20 Time (min) 0 10 20 Time (min) Inertsil ODS-4 52 Comparison of Performance between GL Sciences’ Columns (2/4) InertSustain AQ-C18 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 0 10 20 Time (min) 0 10 20 Time (min) 0 2 4 6 8 10 12 14 Time (min) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 0 10 20 Time (min) Polar Compound Test Basic Compound Test (1) Basic Compound Test (2) Acidic Compound Test Chelating Compound Test Hydrophobic Property 1 2 3 4 5 6 1 2 34 5 1 2 3 1 2 3 1 2 3 1 2 3 4 5 5 1 3,4 5 4 1 2 3 1 1 2 3 4 5 1 6 2 3 2 3 1 2 3 2
  • 53.
    0 2 46 8 10 12 14 Time (min) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 0 10 20 Time (min) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 0 10 20 Time (min) 0 10 20 Time (min) Inertsil ODS-3 53 Comparison of Performance between GL Sciences’ Columns (3/4) InertSustain AQ-C18 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 0 10 20 Time (min) 0 10 20 Time (min) 0 2 4 6 8 10 12 14 Time (min) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 0 10 20 Time (min) Polar Compound Test Basic Compound Test (1) Basic Compound Test (2) Acidic Compound Test Chelating Compound Test Hydrophobic Property 1 2 3 4 5 6 1 2 34 5 1 2 3 1 2 3 1 2 3 1 2 3 4 5 3 1 5 5 4 1 2 3 1 1 2 3 4 5 1,2 6 2 3 2 3 1: Not eluted 2 4 1,2 were unretained 1:Split peak 3:Not eluted
  • 54.
    1.0 2.0 3.04.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 0 10 20 Time (min) 0 10 20 Time (min) 0 2 4 6 8 10 12 14 Time (min) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 0 10 20 Time (min) Inertsil ODS-SP 54 Comparison of Performance between GL Sciences’ Columns (4/4) InertSustain AQ-C18 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 0 10 20 Time (min) 0 10 20 Time (min) 0 2 4 6 8 10 12 14 Time (min) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time (min) 0 10 20 Time (min) Polar Compound Test Basic Compound Test (1) Basic Compound Test (2) Acidic Compound Test Chelating Compound Test Hydrophobic Property 1 2 3 4 5 6 1 2 34 5 1 2 3 1 2 3 1 2 3 1 2 3 4 5 5 1 5 4 1 2 3 1 1 2 3 4 5 1 6 2 3 2 3 2 3,4 2 3:Not eluted 1
  • 55.
    55 Ordering Information Analytical Columns UPType End-fittings End-fitting Format 3.3 mm 1/16” Waters End-fittings HP Series Particle Size：3 μm Max. Operating Pressure： 50 MPa (500 Bar) Length/ I.D. (mm) 2.1 3.0 4.6 30 5020-89920 5020-89926 5020-89932 50 5020-89921 5020-89927 5020-89933 75 5020-89922 5020-89928 5020-89934 100 5020-89923 5020-89929 5020-89935 150 5020-89924 5020-89930 5020-89936 250 5020-89925 5020-89931 5020-89937 2.4 mm※ End-fittings are 1/16” Waters-compatible. ※ UHPLC compatible end-fittings are also available upon request for UHPLC systems (Ex: UPLC®) to avoid dead volume. ※ Indicate “UP Type end-fittings” when ordering. (Please note that UP type is not available for a 4.6 mm I.D. column) UPLC® is a registered trademark of Waters Corporation. UP Type End-fittings 2.4 mm Particle Size：1.9 μm Max. Operating Pressure： 80 MPa (800 Bar) Length/ I.D. (mm) 2.1 3.0 50 5020-89938 5020-89941 100 5020-89939 5020-89942 150 5020-89940 5020-89943
  • 56.
    56 Ordering Information Analytical Columns ※End-fittings are 1/16” Waters-compatible. ※ Other column sizes available upon request.
  • 57.
    57 Ordering Information Analytical Columns ※End-fittings are 1/16” Waters-compatible. ※ Other column sizes available upon request.
  • 58.
    58 Ordering Information Guard Columnfor UHPLC (Max. Operating Pressure 80 MPa, 800 Bar) ※ For connection details, please refer to the diagram below.
  • 59.
    59 Ordering Information Cartridge GuardColumns ※ End-fittings are 1/16” Waters-compatible. ※ Maximum operating pressure is 20 MPa, 200 Bar.
  • 60.
  • 61.
    61 Ordering Information Capillary Columns ※End-fittings are 1/16” Valco (10-32 UNF). ※ 1/32” Valco (6-40 UNF) end-fittings can also be arranged upon request. ※ Indicate “1/32” end-fittings” when ordering.
  • 62.
    62 Ordering Information Connection Kitsfor Capillary EX-Nano and EX Columns
  • 63.
    63 Ordering Information Metal-Free PEEKColumns ※ End-fittings are UP type end-fittings. ※ For further product details, please contact your local GL Sciences’ representative.
  • 64.
    64 InertSearch Application Notes Accessto the latest pharmaceutical, life science, environmental and food applications at www.glsciences.com/tech/inertsearch