The interaction of Phenyl-Hexyl stationary phases with aromatic compo - PDF document

The interaction of Phenyl-Hexyl stationary phases with aromatic compounds has been proven by Kirkland and other authors1,2. This Application Note explores the in” uence of methanol, acetonitrile, and a mixture of both for the separation of nitro-aromatics on an Agilent Phenyl-Hexyl phase. In addition, the EPA method 8330A/B based on a C18 phase was used for further separation techniques. Solvent and gradient scouting for the two columns was done using the Agilent 1290 In“ nity Quaternary Method Development Solution. Applying the EPA method and the method developed using the Phenyl-Hexyl column, all nitro-aromatics could be separated. A UV spectral library was used to identify the compounds in the different chromatograms. The 1290 In“ nity Quaternary Method Development Phenyl-Hexylcoelution of peaks 8, 9, and 6, 7 min 15 20 25 mAU 40 60 80 100 120 13+206+7 8+9 Environmental in selectivity. This does not mean that using a Pheny-Hexyl column. To evaluate with the Method Scouting Wizardthrough a spectral library. The results were compared to results using the EPA nity LC Method Development Solution Quaternary PumpG4204AAuto samplerG4226AALS coolerG1330BDiode array detectorG4212A Compounds20 nitro-aromatics were purchased from Dr. Ehrenstorfer, Column 1Agilent Poroshell 120 Phenyl Hexyl, 4.6 × 100 mm, 2.7 m, p/n 695975-912 Column 2Agilent Poroshell 120 C-18, 4.6 × 100 mm, 2.7 m, p/n 685975-902Mobile phases(A) Water, (B) Acetonitrile, (C) MethanolGradientsee Figure 1Flow rate0.8 mL/min for C18 phase and 1 mL/min for Phenyl-Hexyl phaseInjection volume3 LColumn temperature45 °CDetection214,235,254/10 nm, Reference 400/80 nm, 20 Hz, 10-mm cell, every second Table 1. Instrumentation used. 3 MinutesA waterB ACN 08020107030301090311090 MinutesA waterB ACNC MeOH 07552010705253015580315590 MinutesA waterB ACN 08020107030301090311090 Gradient 3 MinutesA waterB ACNC MeOH 0755201070525305590315590 Gradient 5Gradient 6 MinutesA waterB ACNC MeOH 0802010703017.99505018.00455503020575315590 MinutesA waterB ACNC MeOH 0755151070525305590315590 MinutesA waterB ACNC MeOH 07552010855253015580315590 Peak ID numberName of compoundStructure Peak 1NitroguanidinPeak 2Octogen (HMX)Peak 3Hexogen (RDX)Peak 41,3,5 Trinitrobenzene NNH HOO N+N+N+O_H3C NO N+N+N+O_O_O_ O2N Table 2. Compounds analyzed, the peak ID number is used in the chromatograms. 4 Peak ID numberName of compoundStructure Peak 52-Amino-6-nitrotoluenePeak 61.2-DinitrobenzenePeak 71,3-DinitrobenzenePeak 82-Amino-4-NitrotoluenePeak 9NitrobenzenePeak 10TetrylPeak 112,4,6-Trinitrotoluene (TNT)Peak 124-Amino-2,6-dinitrotoluene H2NN+O_ OO N+N+_O_O OO N+N+O _ O_ H2NO N+O _ O N+O _ CH3 O2NO2N CH3O2N OCH3O H2NN+O _ N+_O 5 Peak ID numberName of compoundStructure Peak 132,6-DinitrotoluenePeak 142-NitrotoluenePeak 154-NitrotoluenePeak 163-NitrotoluenePeak 17NitropentaPeak 182,4,6-Trinitrophenol Peak 192-Amino-4,6-dinitrotoluenePeak 202,4-Dinitrotoluene NNO CH3 CH3 CH3 O N+O _ O_O_N+N+ON+O_O OH O2N OO N+O _ N+_OH3C CH3 6 software C.01.05 and Method Scouting Wizard € Analyzing the 20 nitro-aromatics € Analyzing the compounds using € Analyzing the compounds using the EPA method using a binary € Comparison of both separation uence of methanol or cant differences in selectivity. The peak numbers in Table 2. Peaks 5, 8, 9, acetonitrile and methanol as with the Method Scouting Wizard min 10 12 14 16 18 20 22 24 mAU 100 150 200 250 300 ACN 13, 208+91110 14;151614+15 MeOH + 5 %ACN 8+98+914+1511 106+714+151219 9 2 11 10 7 14+15 9 1 10 12 14 16 18 20 22 24 mAU 100 150 200 250 300 Figure 3. Analysis of nitro-aromatics using methanol and a mixture of 5 % acetonitrile in methanol as organic phase. scouting. A set of different binary and ushing, and column storage methods.A mixture of methanol and acetonitrile was tested for its in” uence on the separation. In Figure 3, the chromatogram cant differences in selectivity, cation using a UV library cation of compounds was done using a UV spectral library (Figures5 cation tool. Every second spectra was taken during the analysis. This also cation of coeluting peaks. 100 150 200 250 300 350 400 18 Peaks18 Peaks17 Peaks17 Peaks 8+96+7 Gradient 3Gradient 4Gradient 5Gradient 6Gradient 7 uence of different gradients on selectivity. cation of 2-Amino-4,6-dinitrotoluene, fully resolved peak.