Fortis Method Development Options

Transcription

1 Method Dev Fortis Method Development Options Choice of Stationary phase functionality Based on Ultra pure silica Reversed Phase (RP) and Normal Phase (NP) options Fortis Ho - Polar endcapped C8 - Increased polar retention - Organic acids - Catecholamines Fortis Phenyl - Unique di-phenyl structure - Separate Positional Isomers - Metabolite profiling Fortis C8 - General HPLC use - Method dev. from ph - - Acids, Bases and Neutral Fortis Cyano - Cyano functionality - RP or NP use - Explosives - Pesticides Fortis HILIC - High Polar Retention - Highly Pure Silica - Carboxylic acids - Nucleotides Fortis C8 - Reduced Hydrophobicity - Lipids - Steroids 9 Tel:

2 mau Method Dev mau 5 mau Getting Started : Method development typically starts with a C8 or C8 column, both provide Hydrophobic retention with good peak shapes for neutral, acidic and basic analytes. Generally if retention of polar molecules is also needed then a polar endcapped stationary phase such as Fortis Ho is a good starting choice. If selectivity is insufficient then Phenyl or Cyano stationary phases are a good alternative, they will change selectivity and even elution order since they work on dipole characteristics as opposed to just hydrophobicity. Fortis Cyano is good in normal phase (NP) conditions for polar analytes with COOH, NH, NHR or NR groups. If small polar molecules still do not retain then HILIC chromatography is a suitable alternative. 5 5 Fortis Ho Extra Polar Retention 5 5 mau mau 5 5 Fortis Phenyl Change Selectivity Fortis C mau 5 5 Fortis Cyano Extra Selectivity 5 5 mau 5 5 Fortis C8 Lower Hydrophobicity Acidic, Neutral & basic analytes - Fortis C8 - Fortis C8 - Fortis Phenyl Polar acidic molecules - Fortis Ho - Fortis HILIC - Fortis Cyano in NP mode Polar basic molecules - Fortis C8 operated at high ph - Fortis Phenyl - Fortis Ho Alternate Selectivity - Fortis Phenyl - Fortis Cyano Tel:

3 Fortis Phenyl C8 Fortis C8 Superior Peak Shapes ph Range - Based on Ultra Pure Silica Fully Scalable Fortis C8 is a pure silica based stationary phase with unique high and low ph performance. Whether carrying out simple compound screens or complex metabolite identification Fortis C8 will provide the best in peak shape, resolution and extended ph range for method development flexibility..5μ 3μ 5μ μ L C8 38m/g 7% Optimised Peak Shape Whatever the compound functionality the optimised hydrophobic bonding of Fortis C8 leads to peak symmetries being near perfect whatever the analyte type. Basic, Acidic and Neutral analyte performance is first class across the ph spectrum. Superior Peak Shapes - Higher Efficiencies Greater Reproducibility p/n: F8-553 Fortis C8 x4.6mm 3μ Mobile Phase: A - H O +.% Formic acid B - ACN +.% Formic acid Gradient: 5-4% in min ml/min Temp: C. Protriptyline. Nortriptyline 3. Amitriptyline 4. Trimipramine 54nm Extreme ph range Fortis C8 has the ability to not only operate at low ph like other silica based phases, but also to operate at high ph to aid with basic analyte retention and performance. Fortis C8 Asy -.97 N -,5 Rs The ability to quickly equilibrate from formic acid or TFA into ammonia or bicarbonate aids in method development. Mass transfer, loadability and precision of a silica matrix are all maintained. Higher Efficiency than Hybrids - Greater Reproducibility Retain polar basic analytes Hybrid C8 Asy -. N- 7,865 Rs Columns: 5x4.6mm 3μ Mobile Phase: 5:5.% NH 3 : MeCN.ml/min Temp: 5 C 3nm Tel:

4 ph Fortis Phenyl C8 Extended ph Range p/n: F8-553 Fortis C8 x4.6mm 3μ Mobile Phase: A - H O +.% Formic acid B - ACN +.% Formic acid Inflammation and Infection Gradient: - 5% in min ml/min Temp: C 54nm. Dexamethasone. Neomycin Sulphate 3. Acetic acid Fortis C8 5x4.6mm 5μ p/n: F8-535 Antipsychotic Mobile Phase: A - 5mM NH 4 OAc B - ACN Gradient: - 4% in min ml/min Temp: C. Quetiapine 54nm ph Selective Estrogen Modulator Fortis C8 5x3.mm 3μ p/n: F8-333 Mobile Phase: 3:7 H O + mm ammonium bicarbonate : MeOH.4ml/min Temp: 5 C MS Detection Raloxifene Glucuronides ph Data Courtesy of : Pharmaceutical company, USA Tel:

5 Fortis C8 Fortis C8 High ph Stability The unique bonding of Fortis C8 enables stability at extremes of ph to be maintained. Run continuously in.% ammonia Fortis C8 shows no deterioration in efficiency over a 9 day period. Optimised Resolution Only by optimising all factors of stationary phase design can the analyst be assured of the best possible chromatography. Fortis C8 s unique bonded character ensures that not only is reproducibility and robustness assured, but also that resolution is of the highest level. Only by obtaining sharp peak shapes for many analyte types can this sort of resolution be achieved. Analysed here are 35 transitions of pesticide residues. Good LC resolution leads to excellent sensitivity in MS detection. 6.e5 5.5e5 5.e5 4.5e5 4.e5 3.5e5 3.e5.5e5.e5.5e5 35 Pesticide Transitions Fortis C8 5x.mm 3μ.e5 3.6 Data Courtesy of : Central Science Laboratories, UK 5.e Tel:

6 (.83) 5.5 (.) 8.5 (.5) 94.5 (.) 8.5 (.) MDA/. Fortis C8 Fortis C8 Drugs of Abuse p/n: F8-373 Fortis C8 5x3.mm 3μ Mobile Phase: A - H O +.% ammonia B - MeOH +.% ammonia Gradient: Temp: C.4ml/min MS Detection Amphetamine/.7 MDMA Ecstasy/.667 Methamphetamine/.97 MDEA/..5. MDA (3,4-Methylenedioxyamphetamine). Amphetamine 3. MDMA (Ecstasy) 4. Methaphetamine MDEA (3,4-Methylenedioxy-N-ethylamphetamine) Data Courtesy of : Forensic Laboratory, UK Hydrophobicity Fortis C8 high surface area combined with the optimised C8 ligand bonding provides high retention for compounds. This is advantageous in a number of ways: - Higher retention of analytes, more organic modifier can be used to elute, therefore greater MS sensitivity. - Higher retention of analytes, more organic leads to shorter dry-down in fraction collection. - Higher retention of analytes, more chance of resolution Fortis C8 Column Length F8-3xx F8-5xx F8-7xx - Column Diameter 3. F8-33xx F8-35xx F8-37xx F8-53xx F8-55xx F8-57xx F8-59xx Fortis C8 Guards Length Column Diameter. F8-xxG 4.6 F8-5xxG Replace xx - for.5µm - 3 for 3µm - 5 for 5µm - for µm Tel:

7 Fortis Phenyl Fortis Phenyl Unique Selectivity Separate Positional Isomers No MS bleed, stable hydrophobic ligand Enhanced Polar Retention Fortis Phenyl is designed to provide characteristics which will enhance selectivity. It provides the analyst with extra retention of compounds containing aromatic functionality. Extra selectivity and retention can be found for polar substrates, along with metabolite profiling..5μ 3μ 5μ μ L Diphenyl 3% 38m/g Unique Functionality Fortis Phenyl is based upon a unique di-phenyl functionality. Three controlled mechanisms of interaction can occur. π-π (phenyl rings) This allows for unique resolution of closely related species, and metabolites. No complex mobile phases are necessary simplifying method development. Steric Selectivity (diphenyl arrangement) Hydrophobicity (alkyl chain) π-π High Selectivity Resolution enhanced Sharp Peak Shapes - Highly Stable phenyl ligand Separate Positional Isomers Selectivity of compounds normally difficult to resolve on a hydrophobic alkyl chain stationary phase is simplified by the π- π interactions provided by the phenyl functionality. In this application two hydroxyestradiol steroids exhibit resolution from each other, which is not achievable on alkyl chain phases. No complex mobile phases are necessary. Fortis Phenyl 5x4.6mm 5μ p/n: FPH-575 Mobile Phase: 4:6 H O : MeOH ml/min Temp: C nm. 4-Hydroxyestradiol (mw=88.38) HO HO OH CH 3 H H H OH CH High Selectivity Resolution enhanced Sharp Peak Shapes. -Hydroxyestradiol (mw=88.38) 75 HO OH H H H Tel:

8 Fortis Phenyl Di-phenyl vs C8 Selectivity of the Fortis di-phenyl is radically different to that of a C8 stationary phase. Fortis Phenyl In this pharmaceutical mixture we can see an increase in retention of the parent drug, whilst the degradents are all eluted quickly, removing them from co-elution with the parent. 7 Selectivity such as this can be extremely useful, combined with the ability to separate closely related species such as metabolites and positional isomers. Fortis C8 5 8 Data Courtesy of : Major Pharmaceutical company, USA PET Tracer - PK95 Antiarrhythmic 353. (.) 39. (.). Dechlorinated PK95. PK95 8. Quinidine. Dihydroquinidine Fortis Phenyl 5x4.6mm 5μ p/n: FPH-575 Mobile Phase: 4 : 6 H O : ACN ml/min Temp: 5 C MS Detection Data Courtesy of : Wolfson Molecular Imaging Centre p/n: Mobile Phase: Fortis Phenyl 5x4.6mm 5μ FPH : 3 H O +.% formic acid MeOH ml/min Temp: 5 C 35nm Fortis Phenyl Column Length FPH-3xx FPH-5xx FPH-7xx - Column Diameter 3. FPH-33xx FPH-35xx FPH-37xx FPH-53xx FPH-55xx FPH-57xx FPH-59xx Fortis Phenyl Guards Length Column Diameter. FPH-xxG 4.6 FPH-5xxG Replace xx - for.5µm - 3 for 3µm - 5 for 5µm - for µm Tel: µm Fortis Phenyl page 5 6

9 Fortis Ho Fortis Ho Retention of Polars Enhanced Resolution % Aqueous Compatible Fully Scalable Fortis Ho is designed to aid in the separation and retention of polar analytes. Complex mobile phase systems can be bypassed if sufficient retention can be provided by the stationary phase chemistry. Fortis Ho is designed to supply additional interaction with polar molecules which allows their successful retention. 3μ 5μ μ Polar Endcapping L 8% 38m/g Retention of Polar analytes Fortis Ho is optimised not only to help retain and resolve polar analytes, but also to be complimentary in resolution to Fortis C8. Hydrophilic as well as Hydrophobic interaction s Alternative Selectivity No MS bleed Column : Fortis Ho 5x4.6mm 5μ p/n : FHO-575 Mobile Phase: H O +.% Formic acid Flow :.8ml/min Temp : C 7nm. Dopamine. Serotonin 3. DOPAC 4. 5-HIAA Neurotransmitters Alternative Selectivity - Organic acids (x,) Fortis Ho s unique bonded character ensures that not only is reproducibility and robustness assured, but also that resolution is of the highest level (.) 9. (.) 73. (.) Methylmalonic acid Fortis Ho 5x.mm 3μ p/n: FHO-73 Mobile Phase: 5mM Ammonium Formate +.5% Formic acid.4ml/min Temp: 6 C MS Detection (x,) 7. (.). 9. (.) 73. (.) Succinic acid Tel:

10 Fortis C8 Reduced Hydrophobicity over C8 Excellent Peak Shapes Fully Scalable Fortis C8 is designed to provide characteristics similar to Fortis C8 but specifically for situations where less hydrophobicity is required. The same gains in peak shape, efficiency, resolution and scalability are available providing increased productivity to the analyst..μ 3μ 5μ μ L7 C8 38m/g 3% Optimised Peak Shape Fortis C8 is optimised to provide the best possible peak shapes and efficiency. Fortis C8 5x4.6mm 5μ p/n: F8-575 Basic, Acidic and Neutral analyte performance is first class. Mobile Phase: A - H O +.% Formic acid B - MeOH +.% Formic acid Gradient: - 9% in min 8 Higher Efficiencies Greater Reproducibility Symmetrical peak shapes - Lower Hydrophobicity ml/min Temp: 5 C. Lamotrigine 54nm Anticonvulsant Antiplatelet Fortis C8 5x4.6mm 5μ p/n: F8-575 Mobile Phase: ml/min Temp: 5 C 4 : 6 H O +.% formic acid ACN nm. Valproate Semisodium Fortis C8 Bipolar disorders Fortis C8 5x4.6mm 5μ p/n: F8-575 Mobile Phase: 5 : 75 H O : ACN ml/min Temp: 5 C 54nm. Clopidogrel Tel:

11 Fortis Cyano Fortis Cyano Retention of Polars Alternative Selectivity Normal Phase or Reverse Phase system Rapid Equilibration.μ 3μ 5μ Cyano 7% Fortis Cyano allows the use of aqueous reversed phase conditions to provide less retention for compounds too heavily retained on C8 functionality. However, it can also be used in normal phase solvent systems to retain and separate polar analyte species. Cyano columns are particularly useful for polar species, Fortis Cyano is now also available in.µm particle size for UHPLC work. L 38m/g Herbicides Fortis Cyano is optimised not only to help retain and resolve polar analytes, but also to be complimentary in resolution to other Fortis phases. Column : Fortis Cyano 5x.mm 3μ p/n : FCN-33 Mobile Phase: 8: H O : ACN +.% Acetic acid Flow :.ml/min Herbicides Temp : C Normal phase as well as Reversed phase use Alternative Selectivity Rapid Equilibration. Banvel. Int Std 3.,4-D 8nm 4. MCPA 5. PCOC 6.,4-DCP 7.,4-DP 8. CMPP 9.,4-DB. MCPB To see more applications on Fortis Cyano turn to page 6. To learn more about Fortis Cyano.µm see page 7. Fortis Cyano Column Length FCN-3xx FCN-5xx FCN-7xx - Column Diameter 3. FCN-33xx FCN-35xx FCN-37xx FCN-53xx FCN-55xx FCN-57xx F8-59xx Fortis Cyano Guards Length Column Diameter. FCN-xxG 4.6 FCN-5xxG Replace xx - for.µm - 3 for 3µm - 5 for 5µm 9 Tel:

12 Fortis HILIC Fortis HILIC Retention of Polars Increased MS sensitivity Alternate selectivity Reduced extraction (SPE) and dry down times. 3μ, 5μ HILIC na Fortis HILIC (Hydrophilic Interaction Chromatography) is designed to aid in the separation and retention of very polar analytes. Extended retention is afforded by the partitioning, ion-exchange and hydrogen bonding that can occur on a HILIC stationary phase. Fortis HILIC can increase sensitivity in MS analysis and provide alternate selectivity to that achieved with reversed phase C8. L3 38m/g Nucleosides Fortis HILIC is optimised to help retain and resolve polar analytes. By use of high concentrations of organic solvent polar analytes partition with the stationary phase. Column : Fortis HILIC 5x4.6mm 5μ p/n : FHI-535 Mobile Phase: 95:5 ACN : mm NH 3 OAc Flow : ml/min Nucleoside s Temp : C Polar Retention Alternative Selectivity Rapid Equilibration. Uracil 54nm. Uridine 3. Cytosine 4. Guanosine Hydrophilic Interaction Chromatography (HILIC) works in a similar way to normal phase chromatography. A polar surface combined with a non-polar mobile phase, typically ACN, allows for partition of the polar analytes and hence retention and separation. Water is used in low concentration as the strong solvent in order to elute the compounds. 8 k 6 4 Adenine Theobromine Uracil Toluene % organic Fortis HILIC Column Length FHI-3xx FHI-5xx FHI-7xx - Column Diameter 3. FHI-33xx FHI-35xx FHI-37xx FHI-53xx FHI-55xx FHI-57xx FHI-59xx Fortis HILIC Guards Length Column Diameter. FHI-xxG 4.6 FHI-5xxG Replace xx - 3 for 3µm - 5 for 5µm - for µm Tel: