Agilent Technologies Use of Temperature to Increase Resolution in the Ultrafast HPLC Separation of Proteins with ZORBAX Poroshell 300SB-C8 HPLC Columns. Optimization of the Agilent 1100 HPLC System f
Contents
1. performance or use of this material Information descriptions and specifications in this publication are subject to change without notice Agilent Technologies Inc 2004 Printed in the USA February 5 2004 5988 9998EN ra a Agilent Technologies2. 0 20 min the tubing diameter connecting the column to the instrument was 0 17 mm id and a 13 uL flow cell was used Summary ZORBAX Poroshell technology provides exceptionally rapid HPLC separation of peptides and proteins The resulting sample peaks are both narrow peak width and small in volume To ensure that this separation tech nology is fully used one needs to consider instrument configura tion in the experimental setup The configurations shown to make a critical difference to chromatographic performance in these experiments are readily available for most modern HPLC systems Configuration items to evaluate when beginning a sepa ration include e The peak width setting of the detector The detector must sample fast enough to ade quately capture sample peaks present in the flow cell for very short times obtain gt 8 points per peak e Connecting tubing should be small enough in i d so as not to contribute to peak broad ening Tubing of 0 12 mm i d or less is required e Detector flow cells should be chosen with volumes small enough that they do not contribute significantly to www agilent com chem peak broadening For the Agilent 1100 DAD detector and 2 1 mm or 1 0 mm i d ZORBAX Poroshell columns the 1 7 uL flow cell was shown to provide optimal peak widths For a 0 5 mm i d ZORBAX Poroshell column the 500 nL flow cell is recom mended Note that a flow cell with too small of an i d can cause
3. Figure 2B the column contains superficially porous particles ZORBAX Poroshell times as well as reduced peak width of large molecules such as proteins Finally operating at elevated temperature dramatically reduces back pressure facilitat ing use of higher flow rates The sterically protected C18 groups of Poroshell 300SB C18 make it extremely stable at low pH and temperatures up to 80 C Flow Rate If one considers the optimal flow rate for obtaining the best combination of throughput and resolution with Poroshell the data suggest a flow rate of 1 or 2 mL min for the 2 1 x 75 mm column When desiring the same separation on a column of a dif ferent diameter flow rate should be adjusted relative to the cross sectional area of the column This maintains the same relative flow rate linear velocity For instance to keep the same sepa ration when switching from a 2 1 to a 1 0 mm i d column the flow rate should be reduced 5 fold The equation below is helpful in these conversions Diameter Flow Flow x Diameter Gradient Adjustment Flow rate and gradient time are linked by the following gradient relationship Gradient retention k Cx Vm Where k is the relative retention within a gradient tG is the gra dient time in minutes F is flow rate in mL min Vm is the volume in mL of mobile phase in the packed column and C isa constant provided that the sample and the change in org
4. Figure 4 1 7 pL and 13 uL flow cells respectively Combined Configuration Effect In an attempt to summarize the findings discussed in this work the peptide protein separation on ZORBAX Poroshell was used to demonstrate the combined effect of using a non optimal system configuration slow detector peak width setting larger tubing and a larger flow cell In Figure 6 the lower chro matogram shows the peptide protein separation obtained using non optimized settings a ZORBAX Poroshell 300SB C18 400 part number 661750 902 detector peak width of 0 20 min a 0 17 mm i d tubing and a 13 uL flow cell The upper chro matogram in Figure 6 shows the separation achieved when using the optimized system configura tion of a lt 0 01 min detector peak width setting a 0 12 mm i d tubing and a 1 7 uL flow cell The improvement in chromato graphic performance is striking In addition to much narrower peaks a variety of smaller peaks are unmasked Notice the shoul ders to the right of peak 1 and to the left of peak 2 The new peak appearing to the left of peak 3 is almost baseline resolved One 3005 2005 1004 Peak width 0 094 0 069 0 061 N 0 4 2005 ral Peak width a de JT 0 171 0 1 2 3 Figure 6 a min more peak appears between peaks 3 and 4 this may or may not have been unmasked The percent improvements in peak width for peaks observed in the optimized chromatogram com p
5. Poroshell particles are extremely hard 5 um silica spheres each consisting of a 0 25 um porous crust formed around a nonporous 4 5 um core See Figure 1 With ZORBAX Poroshell packed columns protein and peptide separations can be optimized simpli fied and enhanced by choosing appropriate detector settings tubing diameter and flow cell volume Porous shell Figure 1 HPLC particle Introduction It has long been known that high linear velocity high relative flow rate is effective in achieving ultrafast separation of small molecules 1 2 This use of high relative flow rates to achieve an extremely fast separation high velocity chromatography 3 is limited when using large mole cules for example proteins because of their smaller diffu sion constant As flow rate is increased a point is reached where mobile phase moves past a column particle faster than a large molecule can get into it and back out peak broadening is the result ZORBAX Poroshell tech nology was designed to circum vent this limitation on flow rate The reduced diffusion distance required for molecules separated on ZORBAX Poroshell means that a large molecule moves a much shorter distance into and out of the particle and can do this before the mobile phase has moved a significant distance down the column 4 5 This allows higher flow rates to be achieved before peak broadening occurs Figure 2 allows a visual compari son of separa
6. absorbance being averaged in with the current peak This gives diminished apparent resolution and peak heights that are easily restored by use of a narrower peak width setting The peak widths of the four sample com ponents shown in Figure 3 become smaller as the peak width setting of the detector is reduced from 0 2 min to lt 0 01 min Note the improvement in resolution between peak 3 and the shoulder to its left as the detector peak width setting is reduced For the experiment shown in Figure 3 a ZORBAX Poroshell 300 SB C18 column 1 0 mm x 75 mm containing 5 um parti cles was used The Agilent 1100 HPLC system is configured essentially as it was used for Figure 2 with the exception that a 500 nL DAD flow cell was used and detection was at 212 4 nm with the reference at 320 20 nm Mobile phases consisted of A H 0 with 0 1 TFA B ACN with 0 07 TFA Gradients were 5 to 60 B in 8 8 min at a flow rate of 113 uL min All tubing was of 0 12 mm i d Table 2 shows percent increases in peak width determined at half height of the peak observed as the peak width setting for detection is increased from lt 0 01 to 0 2 min comparing top and bottom chromatograms The observed peak width increased by about 35 It is clear that a proper peak width setting is 800 DAD Peak width setting Chromatographic 2 essential for achieving optimal iin peak width 3 ZORBAX Poroshell
7. diameter column a peak ana lyzed on a 2 1 mm i d column has one fifth the volume of the same peak eluting from a 4 6 mm i d column Peaks eluting from a 1 mm i d column used in many of these ZORBAX Poroshell mAU ZORBAX Poroshell 300SB C18 part number 661750 902 Tubing 0 12 mm id Peak width Symmetry 0 54 experiments elute in a volume that is one twentieth that of peaks eluting from a 4 6 mm i d column As peak volume decreases peak width becomes more and more sensitive to ECV effects Therefore when using a 1 mm i d ZORBAX Poroshell column additional attention should be paid to the ECV Effect of Tubing Diameter Figure 4 illustrates peak broad ening that results from use of column connecting tubing having too large an i d The upper chro matogram represents a separa tion of a peptide protein mixture ona 1 x 75 mm ZORBAX Poroshell 300SB C18 column with a connecting tube of 0 12 mm 0 005 in i d The lower chromatogram was obtained under identical conditions but the connecting tube was replaced with a 0 17 mm i d 0 007 inch tube of the same length Peak broadening and asymmetry effects from use of the nonoptimized tubing are clearly observed 2 1 0 069 0 061 0 EN 0 612 0 545 0 439 Tubing 0 17 mm id 0 086 0 388 Peak width 0 124 0 081 0 088 100 Symmetry 0 61 0 392 0 294 04 T T mm 2 3 7 8 min Figure 4 Effect of extra column tubing volume on peak widths of a
8. peak broadening at high relative flow rates A few minutes spent checking the instrument setup will ensure outstanding ZORBAX Poroshell analyses References 1 K M Kirkland D A McCombs and J J Kirkland Rapid high resolution high performance liquid chromatographic analysis of antibiotics 1994 J Chromatogr A 660 327 337 2 R D Ricker J W Henderson and B A Bidlingmeyer Prac tical Aspects of achieving ultra fast HPLC Poster 436 Presented at the 37th Eastern Analytical Symposium 1998 Nov 15 20 Somerset N J Agilent Technologies Publication 5989 0095EN www agilent com chem 3 B A Bidlingmeyer and R D Ricker High Velocity Chromatography of Bio macro molecules Agilent Technologies Publication 5988 2400EN www agilent com chem 4 J J Kirkland F A Truszkowski C H Dilks Jr and G S Engel Superficially porus silica microspheres for fast high performance separa tion of macromolecules 2000 J Chromatogr A 890 3 5 J J Kirkland F A Truszkowski and R D Ricker Atypical silica based column packings for high performance liquid chromatography 2002 J Chromatogr A 965 25 34 For More Information For more information on our products and services visit our Website at www agilent com chem Search Poroshell Agilent shall not be liable for errors contained herein or for incidental or consequential damages in connection with the furnishing
9. peptide protein mixture separated on ZORBAX Poroshell Peptide protein mixture separated using a ZORBAX Poroshell 300 SB C18 column 1 0 mm x 75 mm containing 5 um particles Chromatograms were aligned in the data system by peak elution time for comparison The Agilent 1100 HPLC system configured as in Figure 3 with the exception that the tubing diameter connecting the column to the instrument was variable 0 12 or 0 17 mm i d and a 1 7 L flow cell was used in the DAD The DAD peak width setting was lt 0 01 min Table 3 shows the percent change in both peak width and tailing Symmetry for peaks in the peptide protein sample ana lyzed under the two conditions in Figure 4 The increase in peak width when using the larger diameter tubing is consistent for all peaks and averages around 29 indicating a significant effect of the larger tubing i d Peak symmetry also changes sig nificantly in the direction of more peak tailing The first peak appears to have improved in symmetry when the wider tubing was used but this may be a result of the narrower peak in the upper chromatogram unmasking a shoulder peak at its trailing base This would give the peak observed with smaller i d tubing an unusually large tail and correspondingly small sym metry value For the remaining three peaks 2 through 4 increase in symmetry averages almost 47 The beneficial effects of using a fast detector peak width setting and small volume co
10. performance 4 2004 0 20 min 0 118 z 0 109 0 116 Jow 0 JU EN on Table 2 Effect of Detector Response 4004 Time Setting on the Peak Widths of a 200 0 10 mi 0 107 0 086 0 083 Aow ide i i i min L JA J Peptide Protein Sample Mixture aan TO Analyzed on ZORBAX Poroshell 600 4004 Increase 2004 0 03 min 0 101 _0 082 0 078 I O Peak in peak width 0 1 18 aan me eee 600 4 2 35 4004 a 3 51 200 4 lt 0 01 min 0 100 0 081 0 077 0 079 0 ie 4 35 0 1 2 3 4 5 6 7 8 9 min Values for percent increase in peak width are calcu lated using peak width at half height for peaks in the Figure 3 Effect of increasing detector response time settings on peak widths of test top 0 20 min and bottom lt 0 01 min chromatograms sample peaks 4 of Figure 3 The next two figures will focus on the effects of extra column volume ECV relative to peak volume and how this may broaden peaks Extra Column Volume ECV the volume of all HPLC system components between the injector and the detector excluding the column This typ ically includes the sample loop connecting tubing fittings and flow cell Tubing diameter and flow cell volume will be the focus of the next two experiments Sample peaks in HPLC elute in a system dependent volume Peak volume is reduced when peaks elute from a higher efficiency column narrow bands defined by a large theoretical plate number meter Peak volume is also reduced when using a smaller
11. Optimization of the Agilent 1100 HPLC System for Superior Results J s e e e o e oe 2 e ee5ue rcemica a 9 e e e J e e Abstract ZORBAX Poroshell superficially porous particle technol ogy allows the fastest most rugged HPLC separation of proteins to date A 0 25 um porous silica crust formed around a nonporous 4 5 pm diameter silica core results in extremely hard spherical 5 um ZORBAX Poroshell parti cles Molecules diffusing the short distance into and out of the thin porous crust are rapidly separated While the inherent properties of ZORBAX Poroshell columns make them usable with most modern HPLC instruments the use of superficially porous particle columns of small diameter 2 1 mm 1 0 mm and 0 5 mm i d for the separation of proteins and peptides places certain demands on the instrument configuration This technical overview briefly addresses temperature flow rate and gradient adjustment and then details sev eral of the more important parameters that can influence separation performance These include detector peak width setting extra column volume and flow cell volume j Agilent Technologies with ZORBAX Poroshell Columns Technical Overview Cliff Woodward and Robert D Ricker ZORBAX Poroshell Column Features ZORBAX Poroshell 300SB columns come in a vari ety of internal diameters and phases all being extremely useful for the fast separation of proteins and peptides
12. anic concentration during the gradient are unchanged from run to run Combining this for mula with the understanding that superficially porous Poroshell particles may be used at high linear velocities one can choose gradient and flow rate conditions that optimize use of Poroshell technology The formula was used in Figure 2 to keep the relative elution pat terns constant while changing flow rate For instance k is kept constant by using a 2 min tG x F gradient at 1 mL min and a 1 min gradient 2 mL min The equation also allows one to increase resolution through increased k and to adjust for different column lengths Peak Width Setting Because ZORBAX Poroshell sep arations occur very quickly peaks are not present in the detector for long periods of time In the examples shown in Figure 2 measured peak widths vary from 0 01 min 0 6 s to 0 06 min 3 6 s The rate of detector response makes a very serious contribution to observed peak width under these condi tions but it also contributes to band broadening under less demanding conditions A more systematic demonstration of this is shown in Figure 3 Peak widths in the 9 min separa tion shown are approximately 0 1 min 6 s Use of a large diode array detector DAD peak width setting causes data to be averaged for too long a time before it is reported to the data system for calculations and storage This can result in another peak or some baseline
13. ared to those in the deoptimized chromatogram are shown in Table 5 By optimizing system configura tion peak widths improved by 80 to 180 up to a 3 fold improvement The result is a greatly improved resolving power that can be used to pro vide enhanced qualitative and quantitative data Table 5 Combined Effect of Several Deoptimized System Parameters on the Peak Shape of a Peptide Protein Sample Mixture Analyzed on ZORBAX Poroshell Change in Peak peak width 1 79 2 135 3 182 4 148 Values for percent change in peak width are calcu lated using peak width at half height for peaks in the top and bottom chromatograms of Figure 6 Effect of deoptimized instrument configuration on chromatographic results Peptide protein mixture separated using a ZORBAX Poroshell 300 SB C18 column 1 0 mm x 75 mm containing 5 um particles Chromatograms were aligned in the data system by peak elution time for comparison The Agilent 1100 HPLC system was used in the fully optimized configuration upper chromatogram and with all three non optimized parameters lower chromatogram The system configuration was essentially the same as that for Figure 3 with the following exceptions For the upper chromatogram the detector peak width setting was lt 0 01 min the tubing diameter connecting the column to the instrument was 0 12 mm id and the 1 7 uL flow cell was used For the lower chromatogram the detector peak width setting was
14. e also very impor tant in achieving optimal perfor mance for Poroshell A very brief discussion of these parameters follows Temperature Temperature has a number of benefits in rapid separations using Poroshell First change of temperature can be used to achieve more desirable selectivity Operation at elevated tempera ture results in reduced retention 300SB C18 mAU 1200 Totally porous particle A 800 3 mL min 400 0 100 B 0 4 0 67 min N 02 Of 035 o4 OMS 05min min 1200 5 800 2 mL min v4 X 4004 0 100 B M NK ak q 1min 12007 0 75 min 800 5 1 mL min K X 400 0 100 B M AR J 2min SS oo i 1200 7 4 1 5 min 4 2 3 800 0 5 mL min 4005 0 100 B I pi Ami 0 T mn T T T T T T T T T T T 3 min ZORBAX Poroshell 300SB C18 mAU Superficially porous particle 1200 5 part number 660750 902 B 800 4 3mL min X 4o04 9 100 B 0 67 min a C 0 Tore T T T T T T T T T T T T T T 1 ran 0 35 0 4 0 45 0 5 min min 8004 2mL min aoo 0 100 B m A AA 0 T T T Z T T T T T T 12004 0 75 min 800 5 1mL min x 400 0 100 B SET S cel So a 7 1200 1 5 min 2 3 4 800 0 5mL min j x 4004 0 100 B opin J e eee Looe 3 min Figure 2 Effect of linear velocity relative flow rate on peak widths of a peptide protein mixture on wide pore SB C18 columns In Figure 2A the column contains totally porous particles while in
15. low cell sees the total absorbance in the flow cell so that peaks may appear overlapped broadened and reduced in height In Figure 5 these effects are evi dent for peak 3 and the small shoulder to its left With a smaller volume flow cell 1 7 uL second chromatogram the peak and shoulder are quite well sepa rated In contrast the peak and shoulder merge together when the 13 uL flow cell bottom chro matogram is used Relatively high flow rates may create abnormal turbulence in flow cells of certain dimensions This may explain why for 1 0 mm i d columns the 1 7 uL flow cell not the 500 nL flow cell provides optimal peak widths The 1 7 uL flow cell is also the best choice for 2 1 mm i d columns How ever when using 0 5 mm i d columns the 500 nL flow cell provides the best performance data not shown Peak width 0 099 N 0 al 0 or 0 0 1 7 uL Peak width 0 096 0 ool 0 061 0 N m me 4 5 uL min Peak width 0 099 AL nosol 0 067 j 0 066 A 13 uL min Peak width TNE uzh 0 120 0 123 04 N N 1 2 3 4 Figure 5 5 T 6 7 8 min Effect of flow cell volume on peak widths of a peptide protein mixture separated on ZORBAX Poroshell Peptide protein mixture separated using a ZORBAX Poroshell 300 SBC18 column 1 0 mm x 75 mm containing 5 um particles Chro matograms were aligned in the data system by peak elution time for comparison The Agilen
16. nnecting tubing were discussed In the Table 3 Effect of Column Connector Tubing on the Peak Shape of a Peptide Protein Sample Mixture Analyzed on ZORBAX Poroshell Changein Change in Peak peak width symmetry value 1 29 12 2 25 58 3 33 33 4 28 49 Values for percent change in peak width are calculated using peak width at half height for peaks in the top 0 12 mm i d tubing and bottom 0 17 mm id tubing chromatograms of Figure 4 Percent change in sym metry values was derived in a similar way using pseudomoment analysis as reported by the Agilent ChemStation next section the effect of differ ent flow cells on peak shape will be shown for a separation of peptide and protein standards run on a ZORBAX Poroshell 300SB C18 column As was described in the previous sec tion sample peaks in HPLC elute in a system dependent volume For identical injection volume peaks eluting from 1 mm i d ZORBAX Poroshell columns are one twentieth the volume of those eluting from a 4 6 mm i d column These small volume peaks are very sensitive to ECV In the following experiments ECV was changed by use of different sized flow cells Effect of Flow Cell Volume When the flow cell is much larger than the peak volumes it is more likely that subsequent peaks or baseline absorbance will enter the cell before the cur rent peak has left The detector ZORBAX Poroshell 300SB C18 part number 661750 902 500 nanoliter f
17. se of the gradient adjustments Resolution remains high especially at flow rates of 2 mL min or less A flow rate of 2 mL min on these 2 1 mm i d columns corresponds to a flow of 10 mL min ona 4 6 mm i d column Table 1 allows a quantitative comparison of the results from Figure 2 Using the Agilent ChemStation peak width at half height was determined for peaks 3 and 4 separated at all flow rates Peaks 1 and 2 were excluded for simplicity and because they were insufficiently resolved at higher flow rates on the column having totally porous particles Values in the table represent absolute peak width rather than the relative peak width depicted in the figure Therefore as flow is increased one expects peak width to drop proportionately for example increasing flow from 1 to 2 mL min should cut the peak width 50 Any band broaden ing is apparent as peak widths that decrease at less than the expected rate The value for Mean Increase in pw1 2 is meant to show this more clearly First peak widths were normal ized by multiplying their value times the flow rate Then the percent increase in normalized peak widths relative to their value at a flow rate of 0 5 mL min was reported Mean relative peak width increases quickly with flow rate when using superficially porous parti cles reaching 74 at 2 mL min and 106 at 3 mL min In con trast when using the Poroshell column mean relative peak width increases b
18. t 1100 HPLC system configured as in Figure 3 with the exception that the peak width setting was lt 0 01 min The flow cell volume was variable as indicated and included appropriate Agilent flow cells The DAD peak width setting was lt 0 01 min Tubing was 0 12 mm i d Some confusion may be avoided by pointing out the effect that the flow cell s path length has on peak area in Figure 5 The flow cells used in the upper and lower chromatograms 500 nL and 13 uL respectively have path lengths of 1 cm The other two flow cells 1 7 uL and 4 5 uL have path lengths of only 0 6 mm The shorter path length propor tionately reduces observed absorbance hence the smaller peaks for the two middle chromatograms Table 4 lists the percent increase in peak width as a function of flow cell volume for a 1 0 mm i d ZORBAX Poroshell column Cal culations were made using the peak widths at half height of sample peaks detected using the 1 7 uL and 13 uL flow cells With peak widths up to twice as large it is clear that flow cell configu ration can make a significant dif ference in chromatographic performance Table 4 Effect of Detector Cell Volume on the Peak Shape of a Peptide Protein Sample Mixture Analyzed on ZORBAX Poroshell Change in Peak peak width 1 36 2 74 3 96 4 75 Values for percent change in peak width are calcu lated using peak width at half height for peaks in the second and fourth chromatograms of
19. tions resulting from reverse phase HPLC RP HPLC on totally porous and superficially porous Poroshell 5 um Schematic of a 5 um superficially porous ZORBAX Poroshell particles at various flow rates The chromatograms have been aligned along the time axis for example the chromatogram obtained at a flow rate of 3 mL min has been stretched 6 fold relative to the chro matogram obtained at 0 5 mL min so that relative peak widths and band spacing are more apparent The sample mixture used throughout this technical over view consisted of neurotensin peak 1 RNase A peak 2 lysozyme peak 3 and myoglobin peak 4 each at approximately 0 25 mg mL in mobile phase A The upper set of chromatograms Figure 2A were obtained using a 300SB C18 totally porous pack ing in a 2 1 x 75 mm column As flow rate is increased from 0 5 to 3 mL min the relative width of peaks 1 through 4 increases sig nificantly The first two sample components broaden to the extent that they merge into a single peak The relative elution times stay constant because of the gradient adjustments but resolution is lost because of peak broadening The lower set of chromatograms Figure 2B were obtained using ZORBAX Poroshell 300SB C18 packing in a 2 1 x 75 mm column format The relative peak width remains almost constant as flow rate is increased from 0 5 to 3 mL min The relative elution times stay constant as in Figure 2A becau
20. y only 14 at 2 mL min and by 39 at 3 mL min These results confirm observa tions made for the aligned chromatograms of Figure 2 Table 1 Effect of Flow Rate on Peak Width and Resolution when Using Totally Porous and Superficially Porous Particles Totally Porous Particle Mean Flow rate Peak width increase mL min 1 2 height in pw1 2 Resolution Peak3 Peak4 Peak 3 and Peak 4 0 5 0 0158 0 0188 0 9 9 1 0 0100 0 0121 28 75 2 0 0070 0 0080 74 5 7 3 0 0057 0 0061 106 41 Superficially Porous Particle Poroshell Mean Flow rate Peak width increase mL min 1 2 height in pw1 2 Resolution Peak3 Peak4 Peak 3 and Peak 4 0 5 0 0129 0 0171 0 10 0 1 0 0075 0 0083 7 10 1 2 0 0037 0 0048 14 8 2 3 0 0031 0 0038 39 7 0 Virtually all modern HPLC instruments can take advantage of Poroshell separation technol ogy however it is important to check the different aspects of instrument configuration dis cussed in this document before starting a separation Under standing the mechanism of extremely fast separations using Poroshell Figures 1 2 helps one to see the need for instrument settings that yield optimal per formance As previously men tioned three main issues will be addressed here e The response time peak width setting of the detector e The effect of tubing diameter volume The effect of flow cell volume The choice of temperature flow rate and adaptation of gradient parameters ar
Download Pdf Manuals
Related Search