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Agilent ZORBAX Extend-C18 Datasheet

1. not harmful to the column reverse flow should be avoided except to attempt removal of inlet pluggage see Column Care section e Anew column contains a mixture of methanol and wa ter Initially care should be taken not to pass any mo bile phase through the column that might cause a pre cipitate Extend C18 is compatible with water and all common organic solvents e The use of an Extend C18 guard column is recom mended to protect the Extend C18 column and extend its useful lifetime e Avoid use of this column below pH 2 or above pH 11 5 optimum operating range is pH 2 11 5 see pH Guide lines section e Maximum operating pressure for columns up to 9 4 mm ID is 400 bar 6000 psi e Maximum operating temperature is 60 C NOTE Extend C18 columns are designed for stability over a wide pH range When using these silica based columns under conditions of pH gt 6 maximum column lifetime is obtained by operation at temperatures not exceeding 40 C Mobile Phase Selection The bonded stationary phase is nonpolar in nature and is best used with mobile phases such as metha nol water or acetonitrile water mixtures Increasing the amount of organic component usually reduces the retention time of the sample Due to the relatively high viscosity of recommended mobile phases increased efficiency can be achieved with the use of column temperatures in the range of 40 60 C however best column lifetime is achieved w
2. separations organic buffers should be used and the operating temperature should not exceed 40 C Highest column lifetime is obtained at lower temperatures such as 25 C Useful organic buffers and their properties are summarized below Organic base pK Effective pH use range pyrrolidine 11 3 10 3 12 3 triethylamine 10 7 9 7 11 7 1 methyl piperidine 10 3 9 3 11 3 glycine 9 8 8 8 10 8 TRIS 8 1 7Z1 9 1 ammonia 9 2 8 2 10 2 Other organic buffers such as bis tris propane also ap pear to be acceptable alternatives All of these buffers can be made by titrating with HCl to the desired pH Borate buffers apparently also can be used but less experience is available with these materials When ever possible carbonate and phosphate buffer should be avoided at both intermediate and high pH since these materials enhance the solubility of the silica support to cause column failure compared to the or ganic buffers listed above Most experience has been obtained with 1 methyl piperidine buffers which is useful at pH 11 where most basic compounds are es sentially transformed into free bases Studies indicate that methanol is a preferred organic modifier for high pH applications since the rate of silica support dissolution and subsequent column deg radation is slower with this solvent than with acetoni trile Methanol also can produce better peak shapes when the column is used at intermediate pH No preference for organic modif
3. vents Solvents such as methanol acetonitrile or a 95 5 mixture of dichloromethane and methanol should remove most highly retained compounds In extreme cases dimethyl sulfoxide or dimethylfor mamide at low flow rates may also be used for this purpose When switching between solvents with vastly different polarities it may be necessary to first purge the column with a mutually miscible solvent such as isopropanol Since columns have 1 16 terminations a short 1 4 wrench should be used in assembling fittings to pre vent overtightening the ferrules Overtightening the fittings can damage the fitting and necessitate re placement Storage Recommendations Long term storage of silica based bonded phase col umns should be in a pure organic solvent preferably an aprotic liquid such as 100 acetonitrile If the col umn has been previously used with a buffered mobile phase the buffer should first be removed by purging the column with 20 30 column volumes of a 50 50 mixture of methanol or acetonitrile and water fol lowed by 20 30 column volumes of the pure solvent Before storing the column the end fittings should be tightly capped with end plugs to prevent the packing from drying out Columns may be safely stored for short periods in most mobile phases However to protect equipment it is desirable to remove salts from the instrument and column by purging the column with the same mobile phase without the buffer e g using 60 40 A
4. 4 nm Sample Volume 5 pL Peak Identity 1 Uracil 2 Phenol 3 4 Chloro nitrobenzene 4 Toluene 5 uglmL 200 g mL 25 uglmL 850 ygimL 2 Retention Time min pH Guidelines The Extend C18 column consists of porous Zorbax Rx SIL silica with a double endcapped stationary phase containing about 12 carbon The bidentate feature results from the covalent bonding of the silane to the silica support with two siloxane groups see above schematic The monofunctionality of the bonded ligand one silanol reacted with one equivalent of C18 ensures that the stationary phase can be repro ducibly synthesized within narrow limits This bidentate bonding to the silica support also strongly contributes to the stability of the stationary phase at all pH values hydrolysis at low pH is inhibited and degradation at intermediate and high pH by silica dis solution is moderated When using organic buffers it has been documented that the longevity of such a col umn at pH 11 0 can exceed 30 000 column volumes without significant separation degradation This sta bility is similar to traditional silica based columns but only when they are operated at low or intermediate pH Although this column is primarily proposed for high pH separations it is highly competitive for low and intermediate pH applications The Extend C18 column has been used up to pH 11 5 with good results providing certain operating param eters are followed For high pH
5. CN H O to remove a 60 40 ACN 0 02 M phosphate buffered mo bile phase Re equilibration is rapid with the original mobile phase when using this approach and any dan ger of corrosion from the salts is eliminated Ordering Information Agilent Part No Extend C18 Columns 3 5 pm 1 0mmIDx 30mm 761600 902 1 0mmIDx 50mm 765600 902 1 0mmIDx 150mm 763600 902 2 1mmIDx 50mm 735700 902 2 1mmIDx 100mm 761753 902 3 0mmIDx 50mm 735954 302 3 0mmIDx 150mm 763954 302 46mmIDx 50mm 735953 902 46mmIDx 75mm 766953 902 46mmIDx 100mm 764953 902 46mmIDx 150mm 763953 902 Extend C18 Columns 5 pm 2 14mmIDx 50mm 760450 902 2 1mmIDx 150mm 773700 902 46mmIDx 50mm 746450 902 46mmIDx 150mm 773450 902 46mmIDx 250mm 770450 902 Guard Columns 2 1mmIDx 12 5 mm 4 pack 821125 930 46mmIDx 12 5 mm 4 pack 820950 930 Guard Column Hardware Kit 820888 901 Agg Agilent Technologies For more information on our products visit our Agilent Technologies home page on the World Wide Web at www agilent com chem supplies Printed in USA 07 30 03 Part No 820641 002 D For Technical Support in the US and Canada call 1 800 227 9770 or call your local Agilent sales office
6. General Description Zorbax Extend C18 incorporates a patented biden tate organosilane combined with double endcapping to protect its ultra pure Type B silica support from dissolution at high pH Extend C18 is specially de signed for stable use with high pH mobile phases and is particularly well suited for separating highly basic compounds as free bases In addition the structure of the unique bidentate C18 bonded stationary phase also is useful for separations at low and intermediate pH with excellent stability and chromatographic separation properties Extend C18 packing is made by first chemically bonding a dense monolayer of propylene bridged bidentate C18 silane stationary phase to a specially prepared ultra high purity 299 995 SiO Zorbax Rx SIL porous silica micro sphere support A schematic of the structure of this attached bidentate silane to the silica support is shown below O O The bidentate C18 bonded phase is double endcapped using proprietary reagents and procedures to obtain maximum deactivation of the silica support surface The combination of the bidentate C18 and the exhaus tive endcapping produces a unique highly hydropho bic stationary phase that greatly reduces the rate of silica dissolution that normally occurs with silica based column packings at intermediate and high pH This feature then allows the Extend C18 packing to be used routinely with certain high pH mobile phases for high efficiency separat
7. iate and high pH Successful operation and good column lifetime at pH 11 5 and 40 C with a buffer made from Aldrich pyrrolidine also has been demon strated Good column stability also has been found with a pH 10 5 mobile phase of ammonia methanol which is a useful medium for high pH separations when using a mass spectrometer as a detector An overview of bidentate chemistry and chromatography was recently published in Anal Chem 70 1998 4344 4352 Instrument Guidelines Many HPLC instruments including Agilent instru ments use Vespel rotor seals in their injection valves both manual and autosampler injection valves Vespel is recommended for use up to pH 9 5 At pH s above 9 5 Vespel rotor seals will start to degrade pos sibly causing plugging of downstream components in the flow path including the HPLC column Therefore Vespel rotor seals should be replaced with Tefzel rotor seals which are stable up to pH 12 5 for applications using pH s above 9 5 The appropriate part numbers are listed below for Agilent instruments Agilent Autosampler Tefzel Rotor Seal P N Model 1090 1535 4900 Model 1050 0101 1849 Model 1100 0100 1849 Agilent Manual Injector Tefzel Rotor Seal P N Model 1090 Rheodyne 7010 1535 4900 Model 1090 Rheodyne 7125 0101 0620 Model 1050 Rheodyne 7125 0101 0620 Model 1100 Rheodyne 7125 0101 0620 Operational Guidelines e The direction of flow is marked on the column e While it is
8. ier has been noted for low pH applications 1 Methyl piperidine as supplied by Aldrich Chemical Co Inc M7 260 9 has been successfully used as a buffering material with the Extend C18 column e g see J Chromatogr A 797 1998 111 While this chemical is reputed to have 99 purity prolonged use can result in some impurities building up on the inlet changing column characteristics Should this occur a thorough purge of the column with 50 methanol 50 0 05 trifluoroacetic acid generally restores the col umn to original performance Extend C18 guard col umns are recommended to protect the analytical col umn from any contamination source Ultraviolet transmittance of the organic buffers in the above table is useful down to about 220 nm The exception is ammonia which can be used at lower wavelengths The operating pH of buffers used for separating basic compounds should be at least one pH unit preferably 1 5 pH units above the pK of the basic compound of interest See J J Kirkland Current Issues in HPLC Technology LC GC Supplement May 1997 S46 Figure 13 Best stability at pH gt 6 is obtained using organic buffers and temperatures not exceeding 40 C as sug gested in recent J Chromatogr A papers on higher pH separations with silica based columns 797 1998 111 762 1997 97 728 1996 259 691 1995 3 Phosphate and carbonate buffers should be avoided for best stability of all silica based columns at inter med
9. ions The Extend C18 is espe cially suited for separating highly basic compounds that produce poor peak shapes on most columns The uniform spherical Extend C18 particles are based on ultra high purity Zorbax Rx SIL that has a nominal surface area of 180 m g and a narrow controlled pore size of 80A This special Zorbax silica support Type Agilent Zorbax Extend C18 Datasheet B is designed to eliminate or reduce strong adsorp tion of basic and highly polar compounds Columns are loaded to a stable uniform bed density using a proprietary high pressure slurry loading technique to give maximum column efficiency Column Characteristics A typical Quality Control test chromatogram for a 4 6 mm ID x 150 mm column is shown in Figure 1 The actual QC test and performance of your column is de scribed on the Column Performance Report enclosed with your column Safety Considerations e All points of connection in liquid chromatographic sys tems are potential sources of leaks Users of liquid chromatographic equipment should be aware of the toxicity or flammability of their mobile phases e Because of the small particle size dry Zorbax packings are respirable Columns should only be opened in a well ventilated area FIGURE 1 Extend C18 QC Chromatogram OPERATING CONDITIONS Column Extend C18 4 6 mm ID x 150 mm 5 pm Mobile Phase 85 Methanol 15 Water Flow Rate 1 0 mL min Temperature Ambient 23 C Detector UV ABS 25
10. ith operation at lt 40 C Gradient elution techniques for this packing often use 5 methanol or acetonitrile as the initial solvent and 100 methanol or acetoni trile as the final solvent Additional information on solvent selection may be found in Chapters Six and Seven Introduction to Modern Liquid Chromatogra phy Second Edition L R Snyder and J J Kirkland John Wiley amp Sons 1979 and Chapters Six Seven and Eight Practical HPLC Method Development Sec ond Edition L R Snyder J L Glajch and J J Kirkland John Wiley amp Sons 1997 Applications Extend C18 can be used with basic neutral or acidic compounds Ionizable compounds basic acidic of ten can be satisfactorily separated at pH 3 with this column The Extend C18 column also can be used with intermediate pH 5 8 mobile phases for com pounds that are not stable at low pH or for separa tions that have band spacing problems However Ex tend C18 is especially suited for separating basic com pounds at high pH 9 11 5 to obtain stable separa tions with excellent peak shapes and column effi ciency For optimum results and long term stability and reproducibility the use of 10 50 mM buffers is always recommended when separating ionizable com pounds For separations at low or intermediate pH basic modifiers such as triethylamine or dimethyl octylamine usually are not required to achieve effi cient separations with symmetrical peaks In rare case
11. s 10 20 mM of triethylamine or 5 10 mM di methyloctylamine might be needed in the low or inter mediate pH separation of certain highly basic com pounds No basic modifiers are required when high pH separations are performed as suggested Column Care The inlet frit on these columns has a nominal porosity of 2 um Samples that contain particulate matter which is larger than 2 um will plug the column inlet frit and should be filtered before injection into the column Zorbax guard columns and a hardware kit are recommended for use with such samples see Part Numbers If solvent flow appears to be restricted high column back pressure check first to see that solvent flow is unobstructed up to the column inlet If the column has the restriction there may be particulate matter on the inlet frit An initial attempt should be made to remove any inlet debris by back flushing 25 30 mL of mobile phase through the column If this fails to re turn the column to near its original back pressure the inlet frit should be changed To remove the frit care fully loosen the nut at the inlet taking care not to turn the end fitting itself Then carefully remove the fitting taking care not to disturb the column bed The frit should drop out when the fitting is tapped sharply on a hard surface Install a new frit and carefully tighten the fitting To remove strongly retained materials from the col umn flush the column with stronger less polar sol

Agilent ZORBAX Extend-C18 Datasheet

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