Agilent Technologies Separation of Explosives in EPA 8330: Column Choices Optimize Speed Resolution Solvent Use
Contents
1. between 5 3 5 and 1 8 um particles is uniform EPA 8330 explosives residues are typically ana lyzed by a 4 6 mm x 250 mm 5 um C18 column 1 but can be improved by newer technology smaller 1 8 um or 3 5 um ZORBAX particles and Extend C18 bonded phase Many different Extend C18 columns can be chosen the combination of column length diameter and particle size to pro vide a satisfactory separation and each separation exemplifies a newer column technology s benefit and supports the end user s choice of speed resolution and solvent usage High efficiency 1 8 um particles in 100 mm length columns reduce analysis time and have about the same efficiency compared to 5 um particles in 250 mm columns Therefore they are helpful by saving time in method development or generating more data in a limited amount of time But these columns will generate a higher back pressure that some people may not desire It is still possible to obtain the same resolution but using a longer 3 5 um column The end result is an analysis time still shorter than that achieved with a 250 mm 5 um column a Agilent Technologies Experimental The Agilent 1200 Rapid Resolution LC RRLC system e G1312B binary pump SL with mobile phase A 5 mM ammonium formate in water B methanol e G1376C automatic liquid sampler ALS SL e G1316B Thermally Controlled Column TCC Compartment SL using the low volume heat exchanger kit PN G1316 800032. 5 um columns used in the Kinghorn and EPA methods The methanol composition of the mobile phase was lowered incrementally from 50 to 25 until all 14 were reasonably resolved A critical pair peaks 6 and 7 persisted as partially resolved Further decreasing organic strength would result in exces sive retention of peaks 12 13 and 14 Temperature was then optimized A one degree temperature increase 41 C provided enough selectivity to resolve the critical pair Figure 1 demonstrates temperature s selectivity effect on these compounds Rs7 0 77 Rs7 1 50 Figure 1 Temperature optimizes critical pair resolution Extend C18 provides ample selectivity for the 14 nitroaromatics and nitramines identified in the EPA 8330 method excellent resolution is obtained in a reasonable time Figure 2 shows the separa tion using a RRHT 4 6 mm x 100 mm 1 8 um Extend C18 Resolution of all peaks is baseline or better Rs gt 1 5 High resolution makes it easier to quantify the analytes For example the EPA 8330 method warns 2 4 DNT and 2 6 DNT elute at similar retention times Rs lt 1 5 and a large concentration of one isomer may mask the other therefore if it is not apparent that both isomers are present an isomeric mixture should be reported 1 When baseline resolution is obtained retention times differ significantly avoiding peak masking If higher resolut
3. e G1365C multiwavelength detector MWD at 254 nm with a G1315 60024 micro flow cell 3 mm path 2 uL volume response time set ting of 0 5 s ZORBAX columns e Rapid Resolution High Throughput RRHT Extend C18 4 6 mm x 100 mm 1 8 um PN 728975 902 e Rapid Resolution RR Extend C18 4 6 mm x 100 mm 3 5 um PN 764953 902 e Solvent Saver Plus Extend C18 3 0 mm x 100 mm 3 5 um PN 764953 302 The sample is a 1 1 mix of EPA 8330 Mix A cat no 47283 and EPA 8830 Mix B cat no 47284 from Sigma Aldrich Bellefonte PA diluted in methanol water Results and Discussion Selectivity or the relative band spacing between two peaks is different among C18 columns In many cases the difference is small so adjusting mobile phase organic strength can fine tune the retention to achieve comparable resolution between one C18 column and an alternative C18 column Temperature may also influence selectiv ity and small adjustments in temperature can fine tune the resolution For complex mixtures fine tuning organic strength and temperature could be used to improve resolu tion and ultimately make a method more robust Determining the combination of temperature organic and what column stationary phase is best is frequently discovered by experimentation This is time consuming at the very least and often daunting Fortunately research narrows the test ing Consider an explosive residue standard of 14 nitroaromatic a
4. Mobile phase A 5 mM NH COOH pH 6 120 4 J B MeOH 75A 25B 1004 Flow rate 1 1 mL min 804 Temperature 41 C 604 Solvent used 17 6 mL Pressure 220 bar Figure 3 Rapid resolution options for EPA 8330 explosive standard on Extend C18 Table 2 Column Dimensions Highlight Resolution Speed and Solvent Savings RRHT 4 6 mm id 1 8 pm Resolution Rs 7 6 2 3 Resolution Rs 8 7 1 6 Resolution Rs 9 8 2 3 Analysis time 26 min Solvent consumption 44 2 mL analysis Table 2 suggests that another column configura tion could be valuable for this analysis Solvent Saver HT Extend C18 3 0 mm x 100 mm 1 8 um column PN 728975 302 This would produce high resolution like the RRHT column and produce time and solvent savings from the smaller column diam eter The Solvent Saver HT Extend C18 column was not evaluated in this work Conclusions Highly efficient 1 8 um short columns 100 mm are ideal for method development compared to 5 um 150 mm or 250 mm columns because shorter analysis time increases productivity and allows more analyses to be performed in a fixed time frame Selectivity is manipulated by changing stationary phase mobile phase and temperature An isocratic HPLC method for complex mixtures of explosive materials was quickly created from highly efficient 100 mm columns Extend C18 s unique selectivity and temperature optimization The selectivity and column configurations make Extend C18 a com
5. Separation of Explosives in EPA 8330 Column Choices Optimize Speed Resolu p tion and Solvent Use a Pi o e Resist ee pplication a s s a 7 Environmental Authors John W Henderson Jr and William J Long Agilent Technologies Inc 2850 Centerville Road Wilmington DE 19808 1610 USA Abstract ZORBAX Extend C18 columns separate the explosive compounds in EPA method 8330 and the variety of column configurations available allows customized HPLC meth ods based on resolution speed and even solvent usage For example a fast method for the explosive materials standard EPA 8330 uses 1 8 um short length columns The method was then customized using two other Extend C18 column configurations Each column highlights a combination of resolution speed and or solvent savings The advantage is being able to choose which combination of resolution speed and solvent usage is needed by simple column substitution Introduction The ZORBAX Rapid Resolution High Throughput RRHT 1 8 um LC column line has over 120 column choices including 11 bonded phases and silica three column diameters and six lengths In addition there are another 150 Rapid Resolu tion 3 5 um column choices allowing customiza tion of HPLC methods to meet the analyst s tai lored objectives Many ZORBAX column choices are available because the stationary phase chem istry both silica support and bonded phase
6. ion is the most important objective then the Extend C18 4 6 mm x 100 mm 1 8 um column using the condi tions in Figure 2 is an excellent choice mAU 4 a TE PPs Rs 2 27 60 4 Rs 1 58 50 4 Rs 2 31 40 305 205 26D 10 3 2NT 4NT 3NT E 1 T T T T T j 0 5 10 15 20 25 min Column Rapid Resolution HT Extend C18 4 6 mm x 100 mm 1 8 pm Mobile phase A 5 mM NH COOH ph 6 B MeOH 75A 25B Flow rate 1 7 mL min Temperature 41 C Injection 2 uL x 0 5 pg ea mL Pressure 520 bar Figure 2 EPA 8330 explosive standard high resolution separation on Extend C18 Table 1 names the 14 explosives and their abbrevi ations used in the figures Table 1 EPA 8330 Explosives and Their Abbreviations Name Abbreviation Cyclotetramethylene tetranitramine HMX Cyclotrimethylene trinitramine RDX 1 3 5 trinitrobenzene 135TNB 1 3 dinitrobenzene 13DNB Nitrobenzene NB 2 4 6 trinitrophenyl N methylnitramine tetryl 2 4 6 trinitrotoluene TNT 2 amino 4 6 dinitrotoluene 2A DNT 4 amino 2 6 dinitrotoluene 4A DNT 2 4 dinitrotoluene 24 DNT 2 6 dinitrotoluene 26 DNT 2 nitrotoluene 2NT 4 nitrotoluene 4NT 3 nitrotoluene 3NT If higher throughput is important isocratic meth ods can be sped up by increasing flow rate The 25 methanol mobile phase flowing 1 7 mL min through the 4 6 mm x 100 mm 1 8 um column gen erates a system pressure of about 500 bar leaving a small range to increase flow rate An alternative is to substitute the 1 8 um c
7. nd nitramine compounds Trace residues of these explosives were analyzed by time of flight LCMS by Kinghorn et al using an Extend C18 4 6 mm x 250 mm 5 um column and a methanol water gradient at a temperature of 40 C 2 Additionally EPA method 8330 describes an HPLC method for the 14 compounds using an isocratic methanol water mobile phase and a C18 column Temperature is not specified but the method states If column temperature control is not employed special care must be taken to ensure that temperature shifts do not cause peak misidentification 1 In both methods a lack of selectivity required a TOF detector or additional analysis by an orthogo nal stationary phase to confirm peak identity We separated the 14 compounds with enough reso lution to make the MS detector or secondary analy sis by a different stationary phase redundant The above methods narrowed our method develop ment starting conditions to e Extend C18 from successful Kinghorn method e Isocratic mobile phase A 5 mM ammonium for mate B Methanol so new method is similar to EPA 8330 The ammonium formate was selected based on recommendations from a pre existing method The difference between water and 5 mM ammonium formate was not investigated e 40 C controlled temperature to ensure constant selectivity RRHT column configuration 4 6 mm x 100 mm 1 8 um for rapid analyses with efficiency com parable to the 4 6 mm x 250 mm
8. ntal or consequential damages in connection with the furnishing performance or use of this material Information descriptions and specifications in this publication are subject to change without notice Agilent Technologies Inc 2007 Printed in the USA November 21 2007 5989 7632EN Agilent Technologies
9. olumn with a 3 5 um column Pressure decreases substantially allowing faster flow rates Figure 3 overlays two Extend C18 chromatograms The top chromatogram is a 4 6 mm x 100 mm column with 3 5 um particles at a 2 5 mL min flow rate Compared to Figure 2 the 32 increase in flow rate reduces analysis time by roughly 40 The price for the considerable time savings is less resolution of closely neighboring peaks Resolution is still sufficient as a resolution factor Rs of 1 25 for equally sized peaks means 99 4 of peak area is not overlapped If one peak is 1 32 as tall as the other an Rs of 1 0 still means 99 2 of the peak areas do not overlap 3 Figure 3 s bottom chromatogram is a different column substitution replacing the 4 6 mm id column with the Solvent Saver 3 0 mm id column Flow rate was reduced from 2 5 to 1 1 mL min for equivalent mobile phase linear velocity The out come is similar retention and resolution but only half of the solvent is consumed Table 2 summarizes the customization benefits Column Rapid Resolution HT Extend C18 4 6 mm x 100 mm 3 5 ym Mobile phase A 5 mM NH COOH pH 6 mAU HMX B MeOH 75A 25B 40 Flow rate 2 5 mL min J 135TNB Temperature 41 C 4 Tetryl Solvent used 44 2 mL 30 J 13DNB 2ADNT Pressure 280 bar 204 z ZSDNT 2NT 4NT 3NT 0 104 T T T T T T T T T T 0 2 4 6 10 12 14 min mAU 1604 140 Column Solvent Saver Plus Extend C18 3 0 mm x 100 mm 3 5 um J
10. pelling choice for the analysis of explosive substances named in EPA method 8330 Extend C18 s selectivity provides ample resolution with negligible peak coelution this may eliminate an additional analysis to confirm peak identity RR Solvent Saver Plus 4 6 mm id 3 5 pm 3 0 mm id 3 5 pm 1 3 1 3 1 6 1 4 1 5 1 6 16 min 16 min 40 mL analysis 17 6 mL analysis The ZORBAX column family including Extend C18 has consistent stationary phase chemistry between 3 5 and 1 8 um particles enabling simple column substitution for method customization The high resolution 4 6 x 100 1 8 um configuration however requires flexibility to work at operating pressures above 400 bar The chromatographer can choose benefits such as higher resolution faster analysis time or less solvent usage based on column dimensions References 1 EPA Method 8330 Nitroaromatics and Nitramines by High Performance Liquid Chromatography HPLC revision 0 Septem ber 1994 2 R Kinghorn C Milner J Zweigenbaum Analy sis of Trace Residues of Explosive Materials by Time of Flight LC MS Agilent publication 5989 2449EN 2005 3 L R Snyder J J Kirkland Introduction to Modern Liquid Chromatography 2nd ed pp 38 42 1979 For More Information For more information on our products and services visit our Web site at www agilent com chem www agilent com chem Agilent shall not be liable for errors contained herein or for incide
Download Pdf Manuals
Related Search
Agilent Technologies Separation of Explosives in EPA 8330: Column Choices Optimize Speed Resolution