Inertsil 3 Series The Inertsil 3 series from GL Sciencesʼ represents a major advance in performance over the original Inertsil 2 Series. Inertsil 3 Series phases are based on a purer, higher surface area silica which is specially manufactured to provide maximum bonded phase coverage. The result is a series of columns which provide excellent peak shapes using simple eluents while operating at low pressure. Inertsil Series 3 phases Phase Endcapped Particle Size (µm) Pore Size (Å) Surface Area (m 2 /g) Carbon Load (%) SIL-100Å - 3, 5 100 450 - ODS-3 Yes 2, 3, 4, 5 100 450 15 ODS-3V Yes 5 100 450 15 ODS-P No 5 100 450 29 ODS-EP No 5 100 450 9 Peptide C18 Yes 5 100 450 Proprietary ODS-Sprint Yes 3, 5 100 450 8.5 Sulfa C18 Yes 3, 5 100 450 15 C8-3 Yes 2, 3, 5 100 450 9 Ph-3 No 2, 3, 5 100 450 9.5 CN-3 No 5 100 450 14 NH2 No 5 100 450 8 Diol No 5 100 450 22 HILIC No 5 100 450 20 AX No 5 100 450 17 CX No 5 100 450 14 Inertsil ODS-3 Excellent reproducibility Exceptional stability and column lifetime Low column back pressure Ultra pure and rigid silica Rigorous physical property tests High inertness High reproducibility with longer column life Rigorous quality control of the physical properties of the base silica, proprietary bonding and endcapping chemistry, as well as excellent chromatographic tests for inertness and selectivity, contribute to the production of columns with good reproducibility and long column lifetimes. The inherent lower column back pressure of Inertsil ODS-3 enables a 5µm, 250mm x 4.6mm column to be operated below 10Mpa even when using water/methanol as the mobile phase. Now available in 2µm for UHPLC, see page 139 for ordering information Rigorous tests for base silica Inertsil base silica is manufactured using the purest reagents available, in a process that excludes contaminants during the formation of the silica bead structure. The resulting base silica is ultra pure and shows improved efficiency and sample recovery for samples that interact with metals. The silica particles are rigid and have a smooth surface, preventing structural fragmentation when operated at pressures in excess of 30Mpa. To ensure extremely low metal levels of the silica, analysis by Atomic Emission and ICP is applied to every batch. Scanning Electron Microscopy is also used for the assurance of the surface smoothness. Particle sizes are measured by Nitrogen Adsorption and Laser Ray Particle Analyzer. Every batch of synthesized silica gel is subject to rigorous inspection, and only those batches meeting the strict specifications are used for the subsequent bonding procedure. 142
Inertsil ODS-3 (continued) High inertness It is well known that residual silanol groups on the surface of the silica gel will cause severe peak tailing and adsorption, especially with nitrogen-containing compounds. In the production of Inertsil ODS-3, the silanols are eliminated through a proprietary bonding-endcapping technology developed at GL Sciences. To ensure maximum inertness, each batch of Inertsil ODS-3 must pass a new quality control program using 29 Si NMR. The 29 Si NMR spectrum (Fig. 1 next page) shows that isolated silanols on Inertsil ODS-3 packings have been completely eliminated, thereby ensuring excellent inertness and optimum performance of the phase. There are many new generation ODS phases on the market which still show residual silanols using this test. Inertsil ODS-3 shows excellent inertness for both basic and acidic compounds. In addition, chelating compounds also elute with good peak shape owing to the high purity of the base silica. As typical examples of these compound types, pyridine, ethylaniline, benzylamine and aminopyridine (basic compounds), formic acid and acetic acid (acids) and oxine copper (chelating compound) are generally employed for QC tests. Good peak shapes are obtained on Inertsil ODS-3 columns. Conversely, there are many columns on the market which are referred to as new generation columns but show significant tailing for these compounds (see Fig. 2). High durability A long column lifetime is one of the most important factors contributing to cost efficiency of an analysis. In general, routine use in the ph range of 2 to 7.5 is recommended for silica-based packings. The durability of Inertsil ODS-3 at ph 2 and ph9 has been tested. The graph below shows the variations in retentivity of naphthalene on Inertsil ODS-3 compared with other ODS columns. Inertsil ODS-3 was stable at ph 2 (0.1% TFA) over 500 hours. Figure 1. Comparison of commercially available ODS packings, using 29 Si NMR spectra (referenced from previous page) Figure 2. Comparison between ODS-3 and commercially available ODS columns Comparison between ODS-3 and commercially available ODS columns Formatic Acid and Acetic Acid TEST Column Size: 250mm x 4.6mm Eluent: 0.1% H 3 PO 4 (v/v) Flow: 1.0mL/min Temp: 40 C Detection: UV210nM Inj. Vol: 4µL Sample: 1. Formic Acid 0.1% (v/v) 2. Acetic Acid 0.1% (v/v) Inertsil ODS-3 ODS-A ODS-B ODS-C ODS-D 143
Inertsil ODS-3V A validated Inertsil column Validated LC analysis requires validated instruments and LC columns. Unless your column is manufactured in a validated process, especially when changing from old to new columns, your analytical results may vary significantly and a time-consuming preparation of validation documents has to be completed on all such occasions. Inertsil ODS-3V columns are already validated and include a Manufacturerʼs Validation Certificate so that you can eliminate unnecessary work. GL Sciences has developed a program to manufacture, test and guarantee validated high performance LC columns that offer complete reliability from column to column, batch to batch, time after time. 1) the absence of surface silanols 2) minimal metallic residues 3) inertness to basic compounds 4) reproducible separations In Figure 1 the separation of a series of antidepressant drugs demonstrates the inert nature of Inertsil ODS-3. Figure 1. Separation of antidepressant drugs on Inertsil ODS-3V 1 2 1. Norephedrine 2. Nortriptyline 3. Toluene 4 4. Imipramine 5. Amitriptyline 3 5 Each lot of packing undergoes a series of critical analytical tests to confirm: Inertsil ODS-P Polymerically bonded C18 Improved separation of planar compounds Useful for carotenoid anaylsis Separation of the 16 EPA PAHs Column dimensions: 250mm x 4.6 (mm) Flow rate : 1ml/min. Eluent : CH 3 OH - 25mM KH 2 PO 4, ph 6 (60:40) Temperature : 25 C Figure 2. Separation of toad venom 3 1. Bufalin 2 Inertsil ODS-P is a polymerically bonded phase which is densely bonded with octadecylsilane groups. It shows unique separation capabilities and offers an alternative selectivity to Inertsil ODS-3. Inertsil ODS-P is particularly suited to the separation of polycyclic aromatic hydrocarbons, and shows additional retentivity for planar compounds. 1 4 2. Cinobufagin Figure 2 shows the separation of components from toad venom. Figure 3 shows the separation of terphenyl isomers on the three types of ODS phases. On the polymeric Inertsil ODS-P phase, the planar p-terphenyl is retained longer than the non-planar o- and m- isomers. Figure 3. Separation of terphenyl isomers minutes Column: Inertsil ODS-P (250mm x 4.6mm) Eluent: CH 3 CN - 0.1% H 3 PO 4 (45:55) Flow rate: 1ml/min Temperature: 40 C o-terphenyl (non-planar) m-terphenyl (non-planar) p-terphenyl (planar) Column: Inertsil ODS-P (150mm x 4.6mm) Eluent: CH 3 CN - H 2 O (85:15) Flow rate: 1ml/min Temperature: 40 C p- o- m- m- p- o- m- o- p- minutes minutes minutes Monomeric (Intersil ODS-3) Intermediate (Intersil ODS-2) Polymeric (Intersil ODS-P) 144
Inertsil ODS-EP C18 with polar embedded group Compatible with 100% aqueous eluents Alternative selectivity to C18 Ideal for LC-MS Inertsil ODS-EP contains a polar functional group embedded between the silica surface and the C18 group. The embedded polar group makes the C18 phase stable in 100% aqueous eluents without phase collapse. This phase is also extremely base deactivated and provides superior peak shape for acids and bases in organic eluents as well as acidified eluents typically used in LC-MS. Figure 1. Naphthalene sample for retention and peak shape comparison Column : Inertsil ODS-EP, 5µm, 150mm 4.6mm Mobile Phase: Acetonitrile:Water = 64:35 Detection: UV254 nm Peak identification 1. Acetophenone 2. Benzene 3. Toluene 4. Naphthalene Figure 2. Selectivity test sample comparison Column : Inertsil ODS-EP, 5µm, 150mm 4.6mm Mobile Phase: Acetonitrile:Water = 70:30 Detection: UV265 nm Peak identification 1. Ethylbenzene 2. Naphthalene 3. n-propylbenzene 4. n-butylbenzene 5. Anthracene Figure 3. Tanaka test sample (Comparison of hydrogen bonding capacity, hydrophobicity and selectivity) Column : Inertsil ODS-EP, 5µm, 150mm 4.6mm Mobile Phase: Methanol:Water = 80:20 Detection: UV254 nm Peak identification 1. Uracil 2. Caffeine 3. Phenol 4. n-butylbenzene 5. n-amylbenzene 6. o-terphenyl 7. Triphenylene 145
Inertsil HILIC Hydrophilic Interaction Chromatography is a variation of normalphase mode where a high concentration of non-polar solvent is used in a mobile phase. It is an ideal method for separating and retaining highly polar analytes unretained by traditional reversedphase mode. HILIC columns become widely used for critical separations in demanding drug metabolism, drug discovery and combinatorial chemistry. In addition, volatile mobile phases are used for HILIC mode and afford enhanced ESI-MS sensitivity resulting in lower limits of detection. Application for HILIC mode - Amino acids - Water-soluble vitamins - Drug metabolites and drug discovery - Combinatorial chemistry - Promoted retention of compounds unretained by reversedphase mode Advantages of HILIC mode - Low column back pressure analyses can be performed due to high organic solvent composition - A high organic solvent concentration of the mobile phase will lead to a high sensitivity LC-MS analysis - The benefit of less ion suppression effect in LC-MS analysis - Precision determination for polar compounds due to the fast elution of hydrophobic contents Figure 2. Water soluble vitamins Column : Inertsil HILIC 5µm, 250mm 3.0mm Eluent : Acetonitrile (0.1% TFA)/H 2 O (0.1% TFA) = (90/10), (80/20), (60/40), 20/80) Flow Rate : 0.4 ml/min Detection: UV 254 nm Sample Size: 20µL Sample: 1. Thiamine (8ppm) 2. Nicotinamide (8ppm) 3. L-Ascorbic acid (80ppm) 4. Riboflavin (8ppm) Figure 1. Separation of oligosaccharide Column : Inertsil HILIC, 5µm, 150mm 4.6mm Mobile Phase : Acetonitrile/H 2 O (85:15, w/w) Detection: RI Peaks: 1. Maltose 2. Maltotriose 3. Maltotetraose 4. Maltopentaose 5. Maltohexaose 6. Maltoheptaose Figure 3. Amino acids Column : Inertsil HILIC 5µm, 250mm 3.0mm Eluent : Acetonitrile 10mM KH 2 PO4 (10%ACN) = (85/15),(80/20),(70/30),(50/50),(30/70) Flow Rate : 0.4 ml/min Detection: UV 210 nm Sample Size: 1mL Sample: 1. Phenylalanine (100ug/mL) 2. Methionine (500ug/mL) 3. Valine (4mg/mL) 4. Alanine (4mg/mL) 5. Serine (8mg/mL) 146
Inertsil ODS-Sprint Well balanced retention of both polar and non polar compounds Superior inertness and base deactivation Compatible with completely aqueous or neat organic eluents Low operating back pressure 20mm x 2.1mm column with new Sprint HG hardware is ideal for rapid LC-MS separations Moderate retentivity GL Sciencesʼ newly developed chemical bonding technology allows Inertsil ODS-Sprint to retain hydrophilic compounds, but elutes hydrophobic compounds faster. It offers 1/2 of Inerstil ODS-3 retention for hydrophobic compounds (see Figure 1). Figure 1. Elution of hydrophobic compounds Column : Inertsil ODS-SP, 5µm, 250mm 4.6mm Mobile Phase: CH 3 OH/H 2 O = 80/20, v/v Detection: UV254 nm) Inertsil ODS-Sprint is an important new addition to the Inertsil 3 column series. Inertsil ODS-Sprint is extremely base deactivated and optimally bonded to retain polar compounds without excessive retention of non-polar compounds. This allows you to achieve improved separations faster. ODS-Sprint operates at low back pressure even at high flow rates, shows outstanding longevity and is reproducible from column to column and batch to batch. To complement this new phase, GL Sciencesʼ has created a zero-dead-volume column hardware format, known as Sprint-HG hardware, further enhancing peak symmetry. For additional information, please contact Canadian Life Science. Short equilibration time A highly uniformed low carbon loading ODS enables shorter equilibration time for gradient analysis and can be used for highthroughputs. The time for purging and cleaning the column can be reduced in isocratic analysis. In Figure 2, Inertsil ODS-SP shows a complete equilabration after 10 mins with stable retentivity. Figure 2. Equilibration time comparison Column : Inertsil ODS-SP, 5µm, 250mm 4.6mm Mobile Phase: CH 3 CN/H 2 O = 5/95 (5min hold) - (7min) - 100/0 (5min hold),v/v Detection: UV215 nm Sample: 1. Methamidophos 2. Acephate 147
Inertsil ODS-Sprint Sharp peak shape Sharp peak shapes can be observed due to GL Sciencesʼ newlydeveloped bonding technology and column hardware. 3µm packed columns are also available for further sharp peak shapes. Inertsil ODS-sprint does not show any tailing with complete base line separation, resulting in excellent quantitative determination. Peak tailing of large volume compounds (unreacted derivatizing reagent) disturbed the detection of the target compound on Competitor E, F and G columns (see Figure 3). Figure 3. Analysis of derivatized products Column : Inertsil ODS-SP, 5µm, 150mm 4.6mm Mobile Phase: CH 3 CN/H 2 O = 55/45, v/v Sample 1. DNPH-Formadehyde Detection: UV360 nm) 2. DNPH-Acetoaldehyde No adsorption of peaks GL Sciencesʼ intensive endcapping is performed to eliminate residual silanols, creating an exceptionally inert column for reverse-phase chromatography. Excellent peak shapes for a wide range of polar analytes, including both basic and acidic compounds, can be obtained. Inertsil ODS-Sprint shows excellent peak shapes of basic and acidic compounds. Other competitorʼs columns are showing tailing peaks of both compounds. Some columns may be able to analyze basic compounds, but show peak tailing for acidic compounds (see Figure 4). Figure 4. Analysis of basic compounds Column : Inertsil ODS-SP, 5µm, 250mm 4.6mm Mobile Phase: CH 3 OH/20mM Phosphate buffer (ph 7.6) = 10/90, v/v Sample Detection: UV254 nm 1. 4-aminopyridine 2. 3-aminopyridene 3. 2-aminopyridine Stable performance To retain water-soluble organic compounds, the use of mobile phases that contain little or no organic modifier are sometimes needed. Under these highly aqueous conditions, Inertsil ODS-Sprint shows excellent reproducible retention behaviour and high resistance to buffer or acid additives from lot-to-lot (see Figure 5). Figure 5. Stop-flow test using 100% water eluent Testing procedure 1) 1 st analysis conducted after introducing eluent for 20 minutes 2) Stop flow for 15 minutes 3) 2 nd analysis conducted after introducing eluent for 10 minutes 4) Stop flow for 15 minutes Column : Inertsil ODS-SP, 5µm, 250mm 4.6mm Sample Mobile Phase: H 3 O 1005 1.Cytosine 2. Uracil 3. Guanine Detection: UV254 nm) 4. Thymine 5. Adenine 148
Sulfa C18 Analysis of antibiotic drugs and drug residues in products of animal origin has become an important element of ensuring food safely. Inertsil Sulfa C18 is suitable not only for the analysis of sulfa drugs but also for the screening of synthetic antibacterial agents which are directed in the food safety investigative guidelines. Figure 1. Analysis of 15 sulfa reagents Figure 2. Inertness comparison Column : Inertsil Sulfa, 5µm, 250mm 4.6mm Mobile Phase: 0.1% H 3 PO 4 v/v Detection: UV210 nm Sample Size: 4 µl Sample 1) Formic Acid 0.1% (v/v) 2) Acetic Acid 0.1% (v/v) Antibacterial agents, including sulfa drugs, consist of polar functional groups which can cause adsorption on the packing material. High inertness to basic and acidic compounds enables Inertsil Sufa C18 to effectively analyze antibacterial agents. Each batch of Inertsil Sulfa C18 is tested for the effective separation of sulfa drugs, as shown in Figure 1 below, and is provided with a copy of the chromatogram. System: GL-7400 LC SYSTEM Column : Inertsil SULFA C18, 3µm, 150mm 4.6mm Eluent : A) CH 3 CN/ CH 3 COOH = 777.66/3.15, w/w B) CH 3 COOH/H 2 O = 3.15/997, w/w A/B - 0/100-33min- 33/67-3min- 90/10-4min- 90/10 Flow Rate: 1.0mL/min Detection: UV 270 nm Injection Vol: 10µL Sample: 1. Sulfanilamide 8. Sulfamethoxypyridazine 2. Sulfacetamide 9. Sulfamonomethoxine 3. Sulfadiazine 10. Sulfachlorpyridazine 4. Sulfathiazole 11. Sulfamethoxazole 5. Sulfapyridine 12. Sulfabenzamide 6. Sulfameradine 13. Sulfadimethoxine 7. Sulfacymidine 14. Sulfaquinoxalin 15. Sulfanitran Column : Inertsil Sulfa, 5µm, 250mm 4.6mm Mobile Phase: 0.1% CH 3 OH/ H 2 O 30/70 Detection: UV254 nm Sample Size: 4 µl Sample 1) Pyridine 0.09 mg/ml 2) Phenol 0.41 mg/ml Figure 3. Analysis of antibacteria agents directed in the food sanitation investigation guideline 149
Inertsil Diol Unique selectivity in both reversed-phase and normal-phase Applications for amino acids, water-soluble vitamins and sugars The Inertsil Diol phase is a dihydroxypropyl bonded silica which can be used in both reversed-phase and normal-phase modes. The retentivity is higher than that of silica in normal-phase mode. Figure 1 shows the analysis of free amino acids on an Inertsil Diol column. Inertsil Peptide C18 The new Inertsil Peptide C18 column was developed for the analysis of Peptide mapping. 100Å pore size high purity silica gels are bonded with octadecyl groups and the residual silanols are drastically reduced. Metabolic analysis to Peptide analysis are performed with great reproducibility on Inertsil Peptide C18. Figure 2 shows the application of this column for the analysis of cytochrome C tryptic digest. Figure 2. Analysis of cytochrome C on Inertsil Peptide C18 Figure 1. Analysis of free amino acids Column: Inertsil Diol (250mm x 4.6mm) Eluent: CH 3 CN - H 2 O (80:20) Flow rate: 1ml/min Temperature: 40 C 1. Phenylalanine 2. Methionine 3. Valine 4. Dopa 5. Alanine 6. Serine 7. Citrulline Inertsil AX (Anion Exchange), CX (Cation Exchange) High theoretical plate number Silica base anion exchange columns High reproduciblity Exceptional stability and long column lifetime AX bonded phase structure Improved stability versus conventional SAX and SCX columns such as Partisil SAX, SCX CX bonded phase structure Figure 3. Adenosine and adenosine mono phosphate 150