Agilent Technologies Effect of Flow Rate on Ultra-fast Separations of Proteins on Porous Superficially Porous Particle Columns
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1. The authors Cliff Woodward and Robert Ricker are application bio chemists based at Agilent Technologies Wilmington Delaware Agilent shall not be liable for errors contained herein or for incidental 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 2003 Printed in the USA October 13 2003 5988 9943EN l a Agilent Technologies2. 5 um particle size of ZORBAX Poroshell columns helps keep back pressures within desirable limits Proteins analyzed by HPLC are almost always separated using a mobile phase gradient To produce the top and bottom chromatograms shown here gradient time was adjusted to correspond with flow rate In general keeping gradient volume flow rate multiplied by the gradient time constant results in chro matograms with the same relative elution pattern The separation looks the same but occurs within shorter run times as flow rate is increased Protein sep arations on ZORBAX Poroshell often benefit from increasing flow rate alone with no adjustment to gradient time This is because increasing gradient volume flow rate multiplied by gradient time decreases the gradient slope thereby increasing relative retention k and resolution Rs The resolution of a 5 min run is achieved in 0 5 min The additional benefit of using a rapid high resolution separation in initial method development should not be overlooked A series of scouting runs may be made in one fifth to one tenth the usual time This allows the analyst to get through the method development and into the actual analyses much more quickly This speed in method development is especially useful when using mass spectrometry for detection For More Information For more information on our products and services visit our Web site at www agilent com chem Search Poroshell
3. Instrument Agilent 1100 WPS with AutoBypass Column ZORBAX Poroshell 300SB C18 2 1 x 75 mm 5 pm p n 660750 902 Temperature 70 C Flow rate As indicated Detection UV 215 nm Highlights ZORBAX Poroshell 300SB C18 allows the use of higher mobile phase linear velocities with no loss in resolution resulting in shorter run times Because of the thin superficially porous layer ZORBAX Poroshell 300SB C18 allows rapid equilibra tion of slowly diffusing large molecules ZORBAX Poroshell 300SB C18 uses proven StableBond tech nology to maintain stability at low pH and elevated temperatures ZORBAX Poroshell 300SB C18 columns are ideally suited to the rapid LC MS analysis of intact proteins LC MS is now a standard technique used in protein chemistry laboratories Sample peak Protein name 1 Neurotensin 2 RNAse A 3 Lysozyme 4 Myoglobin Mobile phase A B Il 95 H20 5 ACN with 0 1 TFA 5 H20 95 ACN with 0 07 TFA Il Agilent Technologies www agilent com chem When analyzing small molecules by HPLC increase of flow rate can be of great advantage in achieving faster separations If the absolute flow rate becomes too great the column diameter and flow can be reduced while the same relative flow rate linear velocity is maintained Small molecules have high diffusion con stants and can move into and out of the pores of HPLC particles very quickly These molecules tend to elute in with the same pea
4. Effect of Flow Rate on Ultrafast Separation of Proteins Comparison of e e e e ZORBAX Poroshell Technology to o e Totally Porous Columns TTY Application a e pd e Biochemical e e e J r Cliff Woodward and Robert D Ricker In the modern protein laboratory speed resolution and sensitivity are some times competing goals Frequently the analyst is working under conditions of limited sample size many samples and a need for resolution of contaminants in the shortest possible time ZORBAX Poroshell column technology addreses these goals in a unique way providing the analyst with a powerful tool for the rapid separation and analysis of proteins J Pi loss e porous 800 4 3 mL min 2 1x75 mm 5 um 400 4 0 100 B in 0 67 min K 0 joe 2 mL min x 7 0 100 B in 1 min a a F 0 75 800 1 mL min 4 400 0 100 B in 2 min M N K 0 r 7 J 4 1 5 800 0 5 mL min 1 42 3 400 4 0 100 B in 4 min M A 0 i i 3 0 min Aligned mAU gt 3 mL min Sustained efficiency and resolution ZORBAX Poroshell 800 0 100 B in 0 67 min a 300SB C18 400 5 X i N J p n 660750 902 4 2 mL min 0 5 800 0 100 B in 1 min f 400 5 of A r L 4 L J 1 mL min 0 75 800 0 100 B in 2 min k 400 5 o A oe 0 5 mL min 1 5 300 0 100 B in 4 min 2 3 j 1 a a ee 0 r r a e 3 0 min Aligned Conditions
5. k width even as flow is increased The result is extremely fast high resolution separations with typical flow and solvent consumption For bio macromolecules such as proteins attempts to shorten analysis time by increasing the flow rate can lead to band broadening This occurs as a result of smaller diffusion constants for large molecules As mobile phase moves faster and faster past the HPLC particles these large molecules cannot move into and out of porous particles before significant mobile phase moves past top chro matogram The result is increasingly broad peaks as flow rate linear velocity is increased ZORBAX Poroshell technology facilitates rapid analysis and method develop ment of large molecules by use of its shortened diffusion path and its tolerance of high linear velocity high relative flow rate ZORBAX Poroshell particles con sist of a solid silica core covered by a thin totally porous crust Large molecules which diffuse very slowly compared to small molecules can move into and out of the thin crust in a very short time Flow rate can now be increased to decrease run time without significant peak broadening bottom chromatogram ZORBAX Poroshell columns are typically used at flow rates five to ten times those used with a column of the same dimensions but containing totally porous particles The result is run times that are five to ten times shorter than that typically possi ble Finally the short length and
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Agilent Technologies Effect of Flow Rate on Ultra fast Separations of Proteins on Porous Superficial