3 x 100 mm Poroshell 120 Columns
Contents
1. 3 0 pti m ize th e Wh 0 e Ex p e ri m e nt s00 If Dimension X is too long leaks will occur Mixing Chamber fa E Ain SU all 3 X EcipsePls35um an T a T 7 7 P Instrument Contributes to Column Performance m m eee aT Mirianin o Using Aglent 1200SL w80 Hz Detection n m 0 10 20 30 40 50 60 70 80 90 100 i 56bar 05 mimin i F Data Collection Rate Hz Column part number 695775 902 Sample i E I i hoeh NA Wt AM A F System Optimization 1 ul QC Mix Uracil Phenol k 0 5 4 Chloronitrobenzene k 2 Napthalene k 3 8 55 MeCN 45 Water 0 55 ml min micro flow cell a If Dimension X is too Pen adead Doe J F E C 4 Sesh ata 5min volume or mixing chamber will t aa occur 1 Optimize the data collection rate of the detector Set the data For best results when using small columns collect data at 40 He A llection r he f in h that signal to noi N ioni i i collection rate to the fastest setting such that signal to noise S N Slower data collection is practical when analytes retain longer K gt 4 LC troubleshooting poor peak shape improperly is not adversely affected Mobile Data Eea eee in Phase Device swaged tubing Before troubleshooting 2 Choose the flow cell carefully The standard flow cell on the C h 0 ce of F ow Ce Problem 1 Poroshell 120 columns can achieve similar efficiencies as sub 2 Agilent 1200 has a volume of 13 pl This may diminish the Extra Column Volume sample volume connecting Sip AR j micron columns2. Optimization of HPLC Instrumentation for Use with Poroshell 120 Columns N rs se a a a BEE aoe aa 3 Se Agilent Technologies Willen L Cong Anne Wee Jonn W rlancdarson Je Agilan feennolocdias 2390 Canraryille rel Wilmineron Dal USATI Pittcon 2010 4 a Orlando FL USA By d i d z Ontini z fD Coll z Data Collection Rate Combined with P Col C a Ontimi FI Rat p Flow Cell Choice p Superficially porous HPLC columns such as Poroshell 120 are Optimize Detector Speed Larger Flow Cells used at Fast Data Collection speeds can e a em Optimal Flow Rate for Poroshell Faster than for 5 able to deliver similar performance to sub two micron materials be used with Larger Columns as shown with this g niii or 3 5 micron Columns it Replaces Sa na 3 x100 mm Poroshell 120 EC C18 Column This reduces extra column volume by half at substantially lower pressures While modern HPLC Optimize detector settings by adjusting the scan rate and or the time constant to the fastest possible s
3. 3 i the AYS end ferrule was flush with end of tubing causing a void biggest contributors but needle seats should be replaced with smaller p p dispersion an appropriate flow cell should be used z soo x r i Soliton ed i volume seats Red tubing has half the volume of green tubing Chromatography Optimization The volume of a Standard flow cells for an Agilent 1100 or 1200 nt it i i cia gigas 4 Take care to make proper connections w 1 r o Uu J j system is 10 UL i i i z E 5 Optimize flow rates for best performance Start with flow rate 2 ml min on a For best results replace standard flow cells with 5 uL flow cells s000 a 4 6 mm column 0 85 ml min on a 3 0 or 0 42 ml min on 2 1 mm column 1 Optimize the flow rate to achieve the desired efficiency N 2 uL when using 2 1 mm ID columns Stainl Steel and Polymer Fittin or peak capacity Pc ae 7 R s aa OTAI FINS Tubing Volume ul 2 If adapting an older method to a new Poroshell 120 column Columnparnumber693 79 902 Which type is used and when properly scale the gradient and injection voume tothe naw Flow Cell Choices with a 2 1 x100 mm Poroshell a eS ga 45 Water 0 55 ml min micro flow cell Sianissa Siso a5 fittings are the best choice for reliable high Bi H e pful 4 efe re n ces smaller column to quickly transfer the method and avoid 120 EC C18 early eluting compounds are less effected by extra column volume than with Agilent uses Swagelok type fittings w
4. ettings such that signal to 3mm flow cell 6 mm flow cell 10 mm flow cell sig eb DERRAT TTMS f f p g g l l 2 instrumentation and data systems are able to capture the noise S N is not adversely affected a al a o eT a 0 12 280 SS Red 1endpre swaged 01090 87610 3 2 io benefits of these particles attention to the instrumentation The Peakwidth control in ChemStation enables you to select the a A ee a ot C ik Se a EaR Proveepianosherore configuration is important if optimal results are desired peak width response time for your analysis The peak width as E nao taa i e n POSSIBLE 8 Temperature 25 C i o defined in the ChemStation software is the width of a peak at oak smoli a ie ae Der anae eaa i _ ene Configuration should be optimized with regard to the speed half height Set the peak width to the narrowest expected peak gt ri S 3 i tenons cp msc msm A S oK05 8 sic 8 bien 0 17 180 SS Green 1endpre swaged 1313 87305 4 1 7 ea ee with which peaks pass through the detector and the small peak in your sample With Poroshell 120 column expect narrow coo fe S E oop fT 5 ono T S at a een GREEN TUBING HAS hs ee eee peaks similar to those generated with sub 2 micron columns h h pe i ame mR eee 2x VOLUME OF RED a volumes produced in the system Peak volumes of 50 to 75 Set the detector to the fastest setting then to the second fastest m ox 15 S Green a tis a TUBING FOR EQUAL eaters Suggested z z 500 m 500 6 hour f
5. ith front and back overloading Flow Call Cholcas 2 1 x 100 mm Poroshell 120 columns ferrules which give best sealing performance au 1 Agilent 1200 Series Diode Array and Multiple Wavelength Detector throughout our LC system use this on the instrument __ SP User Manual G1315 90011 February 2006 3 Minimize sample dispersion in the column 25000 Extra Column Effects on 3 x 100 mm Poroshell 120 Columns connections i e valves heaters etc ry p p Den 25000 fid 2 Maintaining Your Agilent LC and LC MS Systems 5990 4957EN 1 Usean injection solvent that is weaker than the mobile phase 20000 t 30 loss of efficiency with d PEEK lt 400 b iti ideaiwhere P especially when using an isocratic method a a 10 mm standard flow cell om a tr z lt 400 bar system pressure fittings are ideal where au 3 The Influence of Sub Two Micron Particles on HPLC Performance ue l Z 15000 2 With 2 1 mm columns itis eonnectiens are changed frequently i e connecting 5988 9251EN gt a _ k 0 5 2 Gradients can minimize dispersion but be aware of possible effects i dicts best to use a 3 mm flow ian s Bio compatibility is needed 4 Step by step upgrade of Agilent 1100 Series LC systems to Agilent 0000 I Bk 2 5 Bk 1 6 b Be 1200 Series Gooey heen LC systems for higher performance j asas cell 10000 Pressure is less critical Part 1 5989 6336EN Ak 3 This work was performed on an Agilent 1200 RALC P rs Polyketo
6. itti lower volume as compared to conventional 5 and 3 5 micron setting and evaluate if the signal to noise levels are different r K P Column part number i w e deen tine ea ees i Diameter Starting Flow Rate i R 695975 302 i paa j 5 for Optimization particle filled columns of similar size are found The most ae oa e 0 e oe a ae 1 gt k gt 10 relevant system parameters are detector response time Data Collection Rate Importance TEE renol fe 01 4 TE OEE E A E ete NGGNAD SE E EE E howl 4 6mm 2 ml min i eak Capacity Ofr orosnell 120 IS SII SAUN TE of chromatographic performance will be shown in which each smn _Tinvt ot Data Collection Rate usinga2 1 x 100mm Poroshell 120 EC C18 Soman Guu Rae Te 13 pl G1315 60022 What Happens If the Connections Poorly Made fea RRHT but saai i imi optimi h RFID I 1315 6002 of these parameters is optimized and de optimized The R A Seu ule wit tag 6mm 5u G1315 60025 Wanao BT ym l i Micro with RFID tag 3mm 2 ul G1315 60024 ge Peak Capacity wl iin sta nn combined effect of using all three in an optimized and a de z a n Ferrule cannotseat properly j Pressure gt 600 Bar Aa ee he ee o a x HU M i optimized mode will emphasize the results Guidelines will be Z 15000 ME l _ lt ETEN O o F os m a i PANNEN iven to help the chromatographer achieve best results E n S SEA NES A a g P grap e i Minimize Extra Column Volume E A pepe T awa d i i i EConpetrti825wm T i
7. ne fittings can be used up to 600 bar p 5 Ste oy stepi pgrade of Agilent 1100 Series LC systems to Agilent It indudeda G1312 BP np a G1367A Autosampler anda G1315C DAD SL S000 Use this fitting on column connections with Poroshell 120 a peor e ruan LC systems for higher performance Detect Dat collected and l q Stati 7 B PN 5042 8957 Some typical column connectors shown here ar a awas pr using a eo oo 2 4 6 8 0 n o 6 Optimize Data SEN ng Rate to Take Advantage of RRHT 3 01 Flow Cell Path Length mm Column part number 695775 902 0 1 2 3 4 5 6 7 8 Columns 5989 5810E Tubing Volume ul 1 ul QC Mix Uracil Phenol k 0 5 4 Chloronitrobenzene k 2 Napthalene k 3 8 55 MeCN 45 Water 0 55 ml min micro flow cell g
8. with substantially less Pressure i i j URI JL Le performance achievable using Poroshell 120 columns Smaller tube volume fitting volume detector cell volume i o 2 Excellent results can be achieved achieved on Agilent 1200 RRLC volume flow cells such as the semi micro 6 mm 5 ul or micro 3 2 1 x 100 mm Poroshell 120 Columns early eluters are Altoriroublesheoung or 1290 Infinity mm 2 ul are recommended for best performance more severely affected by extra column broadenin i ae ng tadenoy to uce age fw clin otro asl onan i il 3 Inordrtoachievebest performance 3 A w eat anit ae of ie a detect the compounds of interest is common However as Pa aa a i iE R eat een a oes 1 1 Optimize data collection rate 40 Hz detector with fast response time possible for all connections Use Red 0 12 mm id tubing instea shown in this work they can also be a source of extra column ee J 2 Use the smallest flow cell that you have 3mm micro flow cell works best of Green 0 17 mm as it has only half of the volume of the larger volume Choose the flow cell carefully soon z i when using small columns green tubing Use proper fittings for all connections Make zero Whi 4 a a tubing connecting ALS and column was swaged improperly on 3 Minimize extra column volume flow cell and connecting tubing are the ile detector speed can compensate for excessive flow cell a i dead volume connections These will ensure best peak shape
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