Evaluation of Acclaim HPLC Columns using the National Institute of Standards and Technology Standard Reference Material 870

Transcription

1 pplication Update 6 Evaluation of cclaim HPL olumns using the National Institute of Standards and Technology Standard Reference Material 870 INTRODUTION The National Institute of Standards and Technology (NIST) Standard Reference Material 870 (SRM 870) is a mixture of five organic compounds dissolved in methanol that was formulated for characterizing general aspects of HPL column performance, including efficiency, void volume, methylene selectivity, retentiveness, and activity toward chelators and organic bases. This standard was designed primarily for testing 8 columns, but can be used to characterize 8 and some polar-embedded phases. Other possible uses include column classification to aid column selection during method development, as a control material for monitoring L column performance over time, and as a standard for lot-to-lot and column-to-column comparisons. The five organic compounds in SRM 870 are uracil, toluene, ethylbenzene, quinizarin, and amitriptyline. These compounds are used to evaluate the void volume, hydrophobic retention, methylene selectivity, activity toward chelating reagents, and activity toward bases, respectively. In this application update, SRM 870 was used to evaluate the cclaim 0 8, 0 8, P, P II, and 00 8 columns. These columns are prepared from high purity type silica and use advanced, and in some cases patented, bonding chemistries. The NIST formulation of SRM 870 was also reformulated so that the five compounds had similar peak heights at nm. Standards with one half and one fifth concentration of each compound in SRM 870 were prepared. Different separation conditions are also shown (i.e., a different mobile phase concentration for the cclaim 00 8 column and a different column temperature for the cclaim 0 8 column). These are believed to be more valuable for making lot-to-lot and column-to-column comparisons. EQUIPMENT Dionex Summit HPL system consisting of: LPG P680 pump or HPG P680 pump SOR-00 solvent rack SI-00 uto Sampler T-00 Thermostatted olumn ompartment UVD 70 UV-Vis Detector and UVD 0 Photodiode rray Detector hromeleon hromatography Management Software, Version 6.70 pplication Update 6

2 ONDITIONS olumns and Samples olumns cclaim 0 8 (P/N 099), 0 8 (P/N 09), P (P/N 06), and P II (P/N 0699), all.6 0 mm packed with -μm particles having a pore size of 0 Å; and.6 0 mm cclaim 00 8 column (P/N 06066) packed with -µm particles having a pore size of 00 Å. Samples The five-component standard was prepared based on the SRM 870 document with the concentration of each compound the same as SRM 870 (uracil 8 µg/g, toluene 00 µg/g, ethylbenzene 700 µg/g, quinizarin 9 µg/g and amitriptyline 800 µg/g, five compounds all dissolved in methanol). lternatively, this standard can be purchased from the NIST (*). nother standard with an amitriptyline concentration that was tenfold less than that in SRM 870 with the other concentrations unchanged was also prepared. These concentrations yield similar peak heights for all five compounds on the cclaim 0 8 column with detection at nm. * Order number: 870, L performance, set (), ( Source of hemicals HPL-grade toluene, ethylbenzene, and quinizarin were purchased from Fluka, HPL-grade uracil was purchased from ldrich, and HPL-grade amitriptyline was purchased from Sigma. hromatographic onditions Eluent: 80% methanol, 0% buffer [0% 0 mm KH PO and 0% 0 mm K HPO (v/v)], adjust ph to 7.00 accurately, as described in the SRM 870 documentation. Flow Rate:.0 ml/min Temperature: º and 0 º Inj. Volume: µl Detection: 0 nm, nm, and 80 nm RESULTS ND DISUSSION The cclaim 0 8, 0 8, P, P II, and 00 8 columns were evaluated with SRM 870 at º. These evaluations used both a standard with the specified concentrations of each of the five compounds in SRM 870 and a standard prepared with concentrations that yielded a similar peak height for all five compounds at nm when analyzed with an cclaim 0 8 column. Four injections were performed for each sample using the separation conditions specified on the certificate of analysis for SRM 870, and were averaged for all calculations. ll five peaks were separated on cclaim 8, P, and P II columns; however, quinizarin and amitriptyline were not baseline resolved on the cclaim 0 8 and 00 8 columns. Therefore, single component standards of amitriptyline and quinizarin were injected to calculate their associated column parameters. Figure shows typical chromatograms for the evaluation of the cclaim 0 8 with SRM 870. Using these chromatograms and the equations below, the column parameters that SRM 870 was designed to produce were calculated. The data for all four injections are shown in Table. Table shows the average values for all five columns. The tailing factor for amitriptyline is higher than we normally observe for cclaim columns. This is due to the column temperature of º recommended by NIST. cclaim columns are tested and exhibit their best peak shapes at 0 º as shown in Table by the tailing factor for amitriptyline at 0 º. Therefore for optimum chromatography, a 0 º operating temperature is recommended. Figures through 6 show the five columns evaluated with the standard that yields similar peak heights for the five test compounds at 0,, and 80 nm. Dionex recommends this concentration for column-to-column and lot-to-lot comparisons. To baseline resolve quinizarin and amitriptyline, the column temperature was changed to 0 º for the cclaim 0 8 column and the proportion of methanol and buffer was changed for the cclaim 00 8 column. Figures 7 and 8 show the chromatography with the altered conditions on the 0 8 and 00 8 columns, respectively. Evaluation of cclaim HPL olumns using the National Institute of Standards and Technology (NIST) Standard Reference Material 870 (SRM 870)

3 hromatography and alculations 00-0,00 olumn: cclaim 0 8 Temperature: Peaks:. Uracil 8 µg/g. Toluene 00. Ethylbenzene 700. Quinizarin 9. mitriptylene 80 Parameters for an cclaim 0 8 using SRM 870 alculations. k' (ethylbenzene) = (t R t 0 ) / t 0 = ( )/.077 =. k' = apacity factor t R = 6.7 (Retention time of ethylbenzene) t 0 =.077 (Dead time: retention time of uracil). symmetry (quinizarin) = (LW % +RW % ) / ( LW %) = ( ) / ( 0.6) =. symmetry: Peak tailing factor LW % : 0.6 (left peak width in % of the peak height) RW % : 0. (right peak width in % of the peak height) k' (amitriptyline) = (t R t 0 ) / t 0 = ( ) /.077 =. k' = apacity factor t R = 0.8 (Retention time of amitriptyline) t 0 =.077 (Dead time: retention time of uracil) symmetry (amitriptyline) = (LW % + RW % ) / ( LW % ) = ( ) / ( 0.7) =. symmetry: Peak tailing factor LW % : 0.7 (left peak width in % of the peak height) RW % : 0. (right peak width in % of the peak height) Figure. hromatography of SRM 870 with an cclaim 0 8. ) nm, ) 0 nm, ) 80 nm. Table. Test Results for an cclaim 0 8 Evaluated With SRM 870 Silanol ctivity Samples Hydrophobicity apacity Factor (k') for Ethylbenzene helating Tailing Factor ( s ) for Quinizarin apacity Factor (k') for mitriptyline Tailing Factor ( s ) for mitriptyline verage.... pplication Update 6

4 Evaluation of Five cclaim olumns with SRM 870 olumn Table. Test Results for cclaim olumns Evaluated With SRM 870 Hydrophobicity apacity Factor (k ' ) for Ethylbenzene helating Tailing Factor ( s ) for Quinizarin apacity Factor (k ' ) for mitriptyline Silanol ctivity Tailing Factor ( s ) for mitriptyline 0 Shape Selectivity onding Density (µmols/m ) cclaim cclaim cclaim P cclaim P II N/ cclaim hromatography of the Modified Standard on the Five cclaim olumns The concentration of the amitriptyline in NIST SRM 870 does not yield ideal chromatography for column-to-column and lot-to-lot comparisons. This is due to column overloading by amitriptyline, as seen with the cclaim P and P II phases, which have lower capacities than the other phases tested. To remedy this situation, a standard was prepared with the amitriptyline concentration reduced 0-fold while the concentrations of the other four compounds were kept the same. This reformulated standard produced chromatograms with comparable peak heights for all five components at nm. Figures thorough 6 show chromatography of this modified standard on each of the five cclaim columns listed in Table. hromatography of the Optimized onditions on cclaim 0 8 and 00 8 olumns s shown in Figures and 6, quinizarin and amitriptyline were not baseline separated on cclaim 0 8 and 00 8 columns both at the recommended temperature of º and at an 80:0 ratio of methnol/ buffer. s mentioned above, cclaim columns are tested at 0 º and, for optimum chromatography, a 0 º operating temperature is recommended. Therefore the standard was also analyzed at 0 º. Experiments demonstrated that SRM 870 could be separated on all of the five evaluated cclaim columns at 0 º. Figure 7 shows the chromatography of SRM 870 with cclaim 0 8 column, from which we can see that baseline separation 70 0, olumn: cclaim 0 8 Temperature: Peaks:. Uracil 8 µg/g. Toluene 00. Ethylbenzene 700. Quinizarin 9. mitriptyline Figure. hromatography of the modified standard with the cclaim 0 8 column. ) nm, ) 0 nm, ) 80 nm. Evaluation of cclaim HPL olumns using the National Institute of Standards and Technology (NIST) Standard Reference Material 870 (SRM 870)

5 olumn: cclaim 0 8 Temperature: Peaks:. Uracil 8 µg/g. Toluene 00. Ethylbenzene 700. mitriptyline 80. Quinizarin 9 olumn: cclaim P II Temperature: Peaks:. Uracil 8 µg/g. Toluene 00. Ethylbenzene 700. Quinizarin 9. mitriptyline 80,000 00, Figure. hromatography of the modified standard with the cclaim 0 8 column. ) nm, ) 0 nm, ) 80 nm. Figure. hromatography of the modified standard with the cclaim P II column. ) nm, ) 0 nm, ) 80 nm. olumn: cclaim P Temperature: Peaks:. Uracil 8 µg/g. Toluene 00. Ethylbenzene 700. mitriptyline 80. Quinizarin 9 olumn: cclaim 00 8 Temperature: Peaks:. Uracil 8 µg/g. Toluene 00. Ethylbenzene Quinizarin 9. mitriptyline 80,00-0, Figure. hromatography of the modified standard with the cclaim P column. ) nm, ) 0 nm, ) 80 nm. Figure 6. hromatography of the modified standard with the cclaim 00 8 olumn. ) nm, ) 0 nm, ) 80 nm. pplication Update 6

6 0 0, olumn: cclaim 0 8 Temperature: Peaks:. Uracil 8 µg/g. Toluene 00. Ethylbenzene 700. Quinizarin 9. mitriptyline Figure 7. hromatography of SRM 870 with the cclaim 0 8 at º. ) nm, ) 0 nm, ) 80 nm. 00 0, olumn: cclaim 0 8 Temperature: 0 Peaks:. Uracil 8 µg/g. Toluene 00. Ethylbenzene 700. Quinizarin 9. mitriptyline 80 Figure 7. hromatography of SRM 870 with the cclaim 0 8 at 0. ) nm, ) 0 nm, ) 80 nm Table. Test Results for an cclaim 0 8 with Different oncentrations of SRM 870 at and 0 cclaim 0 8 olumn apacity Factor (k ) symmetry (EP) Plates (EP) Ethylbenzene SRM 870 Quinizarin * mitriptyline * Ethylbenzene / SRM 870 Quinizarin ** mitriptyline ** / SRM 870 Ethylbenzene Quinizarin ** mitriptyline ** djusted SRM 870 with omparable Peak Height Ethylbenzene Quinizarin.9.7.0** mitriptyline ** * Obtained by injecting single component standard ** Obtained by manual integration 6 Evaluation of cclaim HPL olumns using the National Institute of Standards and Technology (NIST) Standard Reference Material 870 (SRM 870)

7 00 0, olumn: cclaim 00 8 Temperature: Peaks:. Uracil 8 µg/g. Toluene 00. Ethylbenzene 700. mitriptyline 80. Quinizarin Figure 8. hromatography of a modified standard with the cclaim 00 8 column at a methanol/buffer ratio of 7:. ) nm, ) 0 nm, ) 80 nm. 9 of quinizarin and amitriptyline was achieved at 0 º. nalysis results at nm on cclaim 0 8 column are summarized in Table. It was found that on an cclaim 00 8 column, quinizarin and amitriptyline could be separated on baseline at the methanol/buffer ratio of 7: at º. typical chromatogram is shown in Figure 8. lthough SRM 870 was developed to assist in characterizing, comparing, and monitoring 8 columns, it can also be used to evaluate a number of other phases. This pplication Update describes modifications to SRM 870 component concentrations and separation conditions to best test Dionex s cclaim 0 8, 0 8, P, P II, and 00 8 columns. When conditions were adapted, the cclaim columns demonstrated excellent separation, capacity, and efficiency. The reformulated SRM 870, when used with the conditions described in this pplication Update, is a powerful tool for monitoring the performance of these columns and comparing their performance to other columns. PREUTIONS This test mixture contains small amounts of organic compounds known to be toxic. are should be exercised during handling and use. Use proper methods for disposal of waste. The cclaim 00 8 column is packed with -µm particles, so it has higher backpressure than the others evaluated in this document. Typically, MeN is the organic solvent in the mobile phase for separations using the 00 8 columns. If methanol is used in the mobile phase, be careful of higher backpressures, compared to MeN, when increasing the flow rate. REFERENES. ertificate of nalysis, Standard Reference Material (SRM) 870, National Institute of Standards and Technology. pplication Update 6 7

8 SI-00 is a trademark and cclaim, hromeleon, and Summit are registered trademarks of Dionex orporation. Standard Reference Material is a registered trademark of the National Institute of Standards and Technology. Passion. Power. Productivity. Dionex orporation 8 Titan Way North merica U.S. (87) Europe ustria () 66 elgium () 9 Denmark () sia Pacific P.O. 8 ox 60 Evaluation anada (90) of cclaim HPL France () olumns Germany using (9) 66 the 99 National 0 Italy (9) 0 6 Institute 67 of Standards The Netherlands () Switzerland () and Technology South merica (NIST) Standard Reference Material 870 (SRM 870) Sunnyvale, U.K. and Ireland () razil () 7 0 (08) ustralia (6) 90 hina (8) 8 8 India (9) 87 Japan (8) Korea (8) 6 80 Singapore (6) LPN 8 / Dionex orporation