<html><head><meta http-equiv="content-type" content="text/html; charset=utf-8"></head><body dir="auto"><div>I think this was already answered. I think your sample is heterogeneous. Small variations on composition (I.e molar mass heterogeneity) will cause peak broadening that will appear as diffusion. If you include enough data in the fit. It should be apparent in the residuals. C(s) will add enough components to account for the heterogeneity (albeit with the same f/fo) and should give better residuals. Sedanal will not because fitting to a single species (I. E, the wrong model) should not give as good of a fit. The heterogeneity will cause Sedanal to return a value that is too large and therefore, a molar mass that is too small. <br><br>Walter Stafford<div><a href="mailto:wstafford3@walterstafford.com">wstafford3@walterstafford.com</a></div><div><br></div></div><div><br>On Aug 12, 2013, at 20:57, "John Sumida" <<a href="mailto:jpsumida@u.washington.edu">jpsumida@u.washington.edu</a>> wrote:<br><br></div><blockquote type="cite"><div><meta http-equiv="Content-Type" content="text/html; charset=us-ascii"><meta name="Generator" content="Microsoft Word 14 (filtered medium)"><style><!--
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</o:shapelayout></xml><![endif]--><div class="WordSection1"><p class="MsoPlainText">Dear RASMB,<o:p></o:p></p><p class="MsoPlainText"><o:p> </o:p></p><p class="MsoPlainText">The following was posted on the SEDFIT user list and it has been suggested that I also post this message to the RASMB list. Thus I am reposting my question to RASMB and am including some edits and additional information for clarity and correctness.<o:p></o:p></p><p class="MsoPlainText"><o:p> </o:p></p><p class="MsoPlainText">Objective<o:p></o:p></p><p class="MsoPlainText">I am trying to reconcile results obtained using two different approaches, SEDANAL and SEDFIT.<o:p></o:p></p><p class="MsoPlainText"><o:p> </o:p></p><p class="MsoPlainText">Observations<o:p></o:p></p><p class="MsoPlainText">In SEDFIT, analysis of sed velocity FDS data returns an s20w of 8.5s and MW=1.3 MDa for the major peak, comprising >80% of the total loading concentration, determined in a c(s) fit. Please note after checking with our collaborators this value is consistent with the MW returned from DLS and static light scattering experiments. Thus the SEDFIT result is consistent with the DLS and static light scattering data which estimate a MW of 1.1 MDa. <o:p></o:p></p><p class="MsoPlainText"><o:p> </o:p></p><p class="MsoPlainText">In SEDANAL, the simplest model necessary to fit the data well was a single species model. Using this model, analysis of the same data-set returns an s20w of 8.8s, similar to the value calculated in SEDFIT, but a MW of 655 kDa is calculated.<o:p></o:p></p><p class="MsoPlainText"><o:p> </o:p></p><p class="MsoPlainText">My question:<o:p></o:p></p><p class="MsoPlainText">Why does the value of the MW returned for SEDANAL and SEDFIT differ by a factor of ~2 when the s20w in each case are similar (8.5s versus 8.8s). My purpose here is that I believe the apparent difference being returned from these parallel analyses is saying something fundamentally important about the behavior of these materials in buffer and our assumptions thereof.<o:p></o:p></p><p class="MsoPlainText"><o:p> </o:p></p><p class="MsoPlainText">Background.<o:p></o:p></p><p class="MsoPlainText" style="margin-left:.5in;text-indent:-.25in;mso-list:l1 level1 lfo3"><!--[if !supportLists]--><span style="mso-list:Ignore">1.<span style="font:7.0pt "Times New Roman""> </span></span><!--[endif]-->Rotor speed was 30 krpm, vbar=0.917 (measured), solution density = 1.00506 g/cm3 (measured), and viscosity =0.0100281 Poise (measured). Temperature = 20oC.<o:p></o:p></p><p class="MsoPlainText" style="margin-left:.5in;text-indent:-.25in;mso-list:l1 level1 lfo3"><!--[if !supportLists]--><span style="mso-list:Ignore">2.<span style="font:7.0pt "Times New Roman""> </span></span><!--[endif]-->The material being studies is a polymer micelle with a CMC of 14 micrograms/ml. <o:p></o:p></p><p class="MsoPlainText" style="margin-left:.5in;text-indent:-.25in;mso-list:l1 level1 lfo3"><!--[if !supportLists]--><span style="mso-list:Ignore">3.<span style="font:7.0pt "Times New Roman""> </span></span><!--[endif]-->The polymer was run over a series of concentrations ranging from 0.2 mgs/ml to 1.00 mgs/ml. <o:p></o:p></p><p class="MsoPlainText" style="margin-left:1.0in;text-indent:-.25in;mso-list:l1 level2 lfo3"><!--[if !supportLists]--><span style="mso-list:Ignore">a.<span style="font:7.0pt "Times New Roman""> </span></span><!--[endif]-->Thus under the conditions of the experiment I am at least 14 times the CMC at the lowest concentration.<o:p></o:p></p><p class="MsoPlainText" style="margin-left:.5in;text-indent:-.25in;mso-list:l1 level1 lfo3"><!--[if !supportLists]--><span style="mso-list:Ignore">4.<span style="font:7.0pt "Times New Roman""> </span></span><!--[endif]-->A major peak is observed in the initial c(s) distribution comprising >80% of the total loading and the position of this peak (7.7 s-exp; s20w=8.5) does not shift with concentration. <o:p></o:p></p><p class="MsoPlainText" style="margin-left:.5in;text-indent:-.25in;mso-list:l1 level1 lfo3"><!--[if !supportLists]--><span style="mso-list:Ignore">5.<span style="font:7.0pt "Times New Roman""> </span></span><!--[endif]-->From the analysis of raw SV data, SEDANAL returns an experimental sedimentation coefficient of 7.9s and a calculated s20w of 8.8s. <o:p></o:p></p><p class="MsoPlainText" style="margin-left:.5in;text-indent:-.25in;mso-list:l1 level1 lfo3"><!--[if !supportLists]--><span style="mso-list:Ignore">6.<span style="font:7.0pt "Times New Roman""> </span></span><!--[endif]-->A global analysis in SEDPHAT was performed over the entire concentration range, transforming the initial c(s) distribution into a set of 8 discrete species. <o:p></o:p></p><p class="MsoPlainText" style="margin-left:1.0in;text-indent:-.25in;mso-list:l1 level2 lfo3"><!--[if !supportLists]--><span style="mso-list:Ignore">a.<span style="font:7.0pt "Times New Roman""> </span></span><!--[endif]-->Four of these 8 species survived a critical chi square analysis suggesting that these four species were important in retaining the quality of the fit. <o:p></o:p></p><p class="MsoPlainText" style="margin-left:1.0in;text-indent:-.25in;mso-list:l1 level2 lfo3"><!--[if !supportLists]--><span style="mso-list:Ignore">b.<span style="font:7.0pt "Times New Roman""> </span></span><!--[endif]-->Of these four discrete species, three were grouped beneath the major peak observed in the initial c(s) analysis.<o:p></o:p></p><p class="MsoPlainText" style="margin-left:1.0in;text-indent:-.25in;mso-list:l1 level2 lfo3"><!--[if !supportLists]--><span style="mso-list:Ignore">c.<span style="font:7.0pt "Times New Roman""> </span></span><!--[endif]-->The calculated weight averaged s20w for these three species was 8.1s. <o:p></o:p></p><p class="MsoPlainText" style="margin-left:1.0in;text-indent:-.25in;mso-list:l1 level2 lfo3"><!--[if !supportLists]--><span style="mso-list:Ignore">d.<span style="font:7.0pt "Times New Roman""> </span></span><!--[endif]-->The s20w values were checked and confirmed with a manual calculation, SEDNTERP verstion 1.09, as well as the calculate “s(20,w) from s(xp) in SEDFIT.<o:p></o:p></p><p class="MsoPlainText" style="margin-left:.5in;text-indent:-.25in;mso-list:l1 level1 lfo3"><!--[if !supportLists]--><span style="mso-list:Ignore">7.<span style="font:7.0pt "Times New Roman""> </span></span><!--[endif]-->Using the ratio of s/D in the Svedberg equation and values for diffusion estimated by assuming 655 kDA, (the MW returned in SEDANAL), I calculate a MW of 655 kDa. Thus I believe that the value for diffusion being estimated in SEDANAL is consistent with the 655 kDA molecular weight.<o:p></o:p></p><p class="MsoPlainText" style="margin-left:1.0in;text-indent:-.25in;mso-list:l1 level2 lfo3"><!--[if !supportLists]--><span style="mso-list:Ignore">a.<span style="font:7.0pt "Times New Roman""> </span></span><!--[endif]-->Estimates of the diffusion coefficient in SEDFIT and SEDNTERP (and thus presumable also from SEDANAL) are not very different. <o:p></o:p></p><p class="MsoPlainText" style="margin-left:1.0in;text-indent:-.25in;mso-list:l1 level2 lfo3"><!--[if !supportLists]--><span style="mso-list:Ignore">b.<span style="font:7.0pt "Times New Roman""> </span></span><!--[endif]-->The diffusion coefficient estimate in SEDFIT is 3.19E-7 cm2/sec and the value calculated in SEDNTERP 3.67E-7 cm2/sec (Teller approximation).<o:p></o:p></p><p class="MsoPlainText" style="margin-left:.5in;text-indent:-.25in;mso-list:l1 level1 lfo3"><!--[if !supportLists]--><span style="mso-list:Ignore">8.<span style="font:7.0pt "Times New Roman""> </span></span><!--[endif]-->Using the calculate M(s) function in SEDFIT and providing the experimental sedimentation coefficient noted above I need to input an ff0<1 (0.8967) in order to arrive at the 655kDA MW returned by SEDANAL. <o:p></o:p></p><p class="MsoPlainText" style="margin-left:1.0in;text-indent:-.25in;mso-list:l1 level2 lfo3"><!--[if !supportLists]--><span style="mso-list:Ignore">a.<span style="font:7.0pt "Times New Roman""> </span></span><!--[endif]-->I understand that this is nonsensical as ff0 cannot be less than 1. <o:p></o:p></p><p class="MsoPlainText" style="margin-left:1.0in;text-indent:-.25in;mso-list:l1 level2 lfo3"><!--[if !supportLists]--><span style="mso-list:Ignore">b.<span style="font:7.0pt "Times New Roman""> </span></span><!--[endif]-->Notably SEDNTERP version 1.09 also calculates a frictional ratio<1; namely SEDNTERP calculate ffp=0.9449. <o:p></o:p></p><p class="MsoPlainText" style="margin-left:1.0in;text-indent:-.25in;mso-list:l1 level2 lfo3"><!--[if !supportLists]--><span style="mso-list:Ignore">c.<span style="font:7.0pt "Times New Roman""> </span></span><!--[endif]-->The c(s) analysis in SEDFIT returns an ff0=1.47.<o:p></o:p></p><p class="MsoPlainText"><o:p> </o:p></p><p class="MsoPlainText">Thus to summarize:<o:p></o:p></p><p class="MsoPlainText" style="margin-left:.5in;text-indent:-.25in;mso-list:l0 level1 lfo1"><!--[if !supportLists]--><span style="mso-list:Ignore">1.<span style="font:7.0pt "Times New Roman""> </span></span><!--[endif]-->Relatively similar s20w values are determined in both SEDFIT and SEDANAL for the same data-set.<o:p></o:p></p><p class="MsoPlainText" style="margin-left:1.0in;text-indent:-.25in;mso-list:l0 level2 lfo1"><!--[if !supportLists]--><span style="mso-list:Ignore">a.<span style="font:7.0pt "Times New Roman""> </span></span><!--[endif]-->8.5s from SEDFIT<o:p></o:p></p><p class="MsoPlainText" style="margin-left:1.0in;text-indent:-.25in;mso-list:l0 level2 lfo1"><!--[if !supportLists]--><span style="mso-list:Ignore">b.<span style="font:7.0pt "Times New Roman""> </span></span><!--[endif]-->8.8s from SEDANAL <o:p></o:p></p><p class="MsoPlainText" style="margin-left:.5in;text-indent:-.25in;mso-list:l0 level1 lfo1"><!--[if !supportLists]--><span style="mso-list:Ignore">2.<span style="font:7.0pt "Times New Roman""> </span></span><!--[endif]-->SEDFIT calculates a MW almost exactly 2 times the value of the MW returned by SEDANAL for the same s20w.<o:p></o:p></p><p class="MsoPlainText" style="margin-left:.5in;text-indent:-.25in;mso-list:l0 level1 lfo1"><!--[if !supportLists]--><span style="mso-list:Ignore">3.<span style="font:7.0pt "Times New Roman""> </span></span><!--[endif]-->The estimates of diffusion in both programs are quite similar<o:p></o:p></p><p class="MsoPlainText" style="margin-left:.5in;text-indent:-.25in;mso-list:l0 level1 lfo1"><!--[if !supportLists]--><span style="mso-list:Ignore">4.<span style="font:7.0pt "Times New Roman""> </span></span><!--[endif]-->The frictional ratio ff0 in the SEDFIT analysis is 1.47 whereas the calculated ffp in SEDNTERP based on the observe MW in SEDANAL is <1. <o:p></o:p></p><p class="MsoPlainText" style="margin-left:.5in;text-indent:-.25in;mso-list:l0 level1 lfo1"><!--[if !supportLists]--><span style="mso-list:Ignore">5.<span style="font:7.0pt "Times New Roman""> </span></span><!--[endif]-->The frictional ratio calculated in SEDNTERP, assuming the MW=1.3 MDa, is 1.54<o:p></o:p></p><p class="MsoPlainText" style="margin-left:.5in;text-indent:-.25in;mso-list:l0 level1 lfo1"><!--[if !supportLists]--><span style="mso-list:Ignore">6.<span style="font:7.0pt "Times New Roman""> </span></span><!--[endif]-->There does appear to be heterogeneity in the major peak observed in the c(s) analysis.<o:p></o:p></p><p class="MsoPlainText"><o:p> </o:p></p><p class="MsoPlainText">Thank you in advance for your comments and suggestions. I apologize for the length of this post, but in fairness, I am attempting to provide information that would enable an informed response.<o:p></o:p></p><p class="MsoPlainText"><o:p> </o:p></p><p class="MsoPlainText">Best regards<o:p></o:p></p><p class="MsoPlainText">John Sumida<o:p></o:p></p><p class="MsoPlainText">University of Washington<o:p></o:p></p><p class="MsoPlainText">Analytical Biopharmacy Core<o:p></o:p></p><p class="MsoNormal"><span style="font-size:14.0pt"><o:p> </o:p></span></p></div></div></blockquote><blockquote type="cite"><div><span>_______________________________________________</span><br><span>RASMB mailing list</span><br><span><a href="mailto:RASMB@list.rasmb.org">RASMB@list.rasmb.org</a></span><br><span><a href="http://list.rasmb.org/listinfo.cgi/rasmb-rasmb.org">http://list.rasmb.org/listinfo.cgi/rasmb-rasmb.org</a></span><br></div></blockquote></body></html>