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</o:shapelayout></xml><![endif]--></head><body lang=EN-US link=blue vlink=purple><div class=WordSection1><p class=MsoNormal><span style='font-size:10.0pt;font-family:"Arial","sans-serif";color:blue'>John, to back up a bit, do you actually know what fraction of this glycoprotein is carbohydrate? If so, why not simply calculate the vbar, using the values discussed in Lewis, M. S. and Junghans, R. P. (2000). Ultracentrifugal analysis of molecular mass of glycoproteins of unknown or ill-defined carbohydrate composition. <i>Methods Enzymol</i>. 321, 136-149? This has always worked pretty well for me.<o:p></o:p></span></p><p class=MsoNormal><span style='font-size:10.0pt;font-family:"Arial","sans-serif";color:blue'><o:p> </o:p></span></p><p class=MsoNormal><span style='font-size:10.0pt;font-family:"Arial","sans-serif";color:blue'>Yes if you assume values for the <i>dn/dc</i> of the carbohydrate you could in principle use the synthetic boundary experiment to determine the <i>dn/dc</i> and the fraction carbohydrate by getting the protein concentration from the OD. But I would use the OD as measured by a bench-top spectrophotometer rather than from the XL-I. For the carbohydrate <i>dn/dc</i> in 2001 we suggested using the value for beta-cyclodextrin (0.145 as measured by Brent Kendrick at Amgen), but frankly whether that is really a relevant or accurate estimate is hard to know. <o:p></o:p></span></p><p class=MsoNormal><span style='font-size:10.0pt;font-family:"Arial","sans-serif";color:blue'><o:p> </o:p></span></p><p class=MsoNormal><span style='font-size:10.0pt;font-family:"Arial","sans-serif";color:blue'>John<o:p></o:p></span></p><p class=MsoNormal><span style='font-size:10.0pt;font-family:"Arial","sans-serif";color:blue'><o:p> </o:p></span></p><div><div style='border:none;border-top:solid #E1E1E1 1.0pt;padding:3.0pt 0in 0in 0in'><p class=MsoNormal><b>From:</b> John Sumida [mailto:jpsumida@u.washington.edu] <br><b>Sent:</b> Tuesday, September 30, 2014 11:41 AM<br><b>To:</b> jphilo@mailway.com; rasmb@list.rasmb.org<br><b>Subject:</b> RE: [RASMB] Reproducibility in densitometry measurement<o:p></o:p></p></div></div><p class=MsoNormal><o:p> </o:p></p><p class=MsoNormal><span style='font-size:12.0pt;font-family:"Courier New";color:blue'>Dear John,<o:p></o:p></span></p><p class=MsoNormal><span style='font-size:12.0pt;font-family:"Courier New";color:blue'><o:p> </o:p></span></p><p class=MsoNormal><span style='font-size:12.0pt;font-family:"Courier New";color:blue'>Thank you for your response and suggestions. We will try to work at higher protein concentration, though we are somewhat sample limited.<o:p></o:p></span></p><p class=MsoNormal><span style='font-size:12.0pt;font-family:"Courier New";color:blue'><o:p> </o:p></span></p><p class=MsoNormal><span style='font-size:12.0pt;font-family:"Courier New";color:blue'>Since you raise the question of knowing dn/dc and since the protein is glycosylated, for us knowing the correct value for dn/dc is a problem. A colleague of mine and I thought about comparing the interference determination of the synthetic boundary with the absorbance signal obtained in the same experiment. I have an extinction coefficient determined for the sequence composition (as per Gill et al. Analytical Biochem 1989) so we could determine the protein concentration (O.D. < 1.00). I could also assume that the protein portion of dn/dc = 0.187, and that portion corresponding to sugar was 0.146, (which I understand to be the value for dextran). <o:p></o:p></span></p><p class=MsoNormal><span style='font-size:12.0pt;font-family:"Courier New";color:blue'><o:p> </o:p></span></p><p class=MsoNormal><span style='font-size:12.0pt;font-family:"Courier New";color:blue'>Since the interference measurement will be sensitive to glycosylation whereas the absorption is not, I should be able to estimate a value for dn/dc that better reflects the degree of glycosylation by determining the relative contribution of the sugar component to dn/dc?<o:p></o:p></span></p><p class=MsoNormal><span style='font-size:12.0pt;font-family:"Courier New";color:blue'><o:p> </o:p></span></p><p class=MsoNormal><span style='font-size:12.0pt;font-family:"Courier New";color:blue'>Thanks again for your time in considering my question and your willingness to share your knowledge on RASMB.<o:p></o:p></span></p><p class=MsoNormal><span style='font-size:12.0pt;font-family:"Courier New";color:blue'><o:p> </o:p></span></p><p class=MsoNormal><span style='font-size:12.0pt;font-family:"Courier New";color:blue'>Best regards,<o:p></o:p></span></p><p class=MsoNormal><span style='font-size:12.0pt;font-family:"Courier New";color:blue'>John Sumida<o:p></o:p></span></p><p class=MsoNormal><span style='font-size:12.0pt;font-family:"Courier New";color:blue'>University of Washington<o:p></o:p></span></p><p class=MsoNormal><span style='font-size:12.0pt;font-family:"Courier New";color:blue'><o:p> </o:p></span></p><div><div style='border:none;border-top:solid #B5C4DF 1.0pt;padding:3.0pt 0in 0in 0in'><p class=MsoNormal><b><span style='font-size:10.0pt;font-family:"Tahoma","sans-serif"'>From:</span></b><span style='font-size:10.0pt;font-family:"Tahoma","sans-serif"'> RASMB [mailto:rasmb-bounces@list.rasmb.org] <b>On Behalf Of </b>John Philo<br><b>Sent:</b> Tuesday, September 30, 2014 10:12 AM<br><b>To:</b> 'John Sumida'; rasmb@list.rasmb.org<br><b>Subject:</b> Re: [RASMB] Reproducibility in densitometry measurement<o:p></o:p></span></p></div></div><p class=MsoNormal><o:p> </o:p></p><p class=MsoNormal><span style='font-size:10.0pt;font-family:"Arial","sans-serif";color:blue'>John, it has been a long, long time since I last did any of these measurements, so regrettably I don’t remember any details, but I do remember all too well that it takes quite a while to figure out how to load the cell reproducibly without any bubbles, and that to get reproducible results you really have to obsessive about doing everything exactly the same way. <o:p></o:p></span></p><p class=MsoNormal><span style='font-size:10.0pt;font-family:"Arial","sans-serif";color:blue'><o:p> </o:p></span></p><p class=MsoNormal><span style='font-size:10.0pt;font-family:"Arial","sans-serif";color:blue'>If someone failed to clean your cell well and you’ve got some junk stuck on the surfaces that might explain your problems, so perhaps you should try a more aggressive cleaning.<o:p></o:p></span></p><p class=MsoNormal><span style='font-size:10.0pt;font-family:"Arial","sans-serif";color:blue'><o:p> </o:p></span></p><p class=MsoNormal><span style='font-size:10.0pt;font-family:"Arial","sans-serif";color:blue'>Remember that the protein samples will always behave differently with regard to bubbles because the protein itself lowers the surface tension.<o:p></o:p></span></p><p class=MsoNormal><span style='font-size:10.0pt;font-family:"Arial","sans-serif";color:blue'><o:p> </o:p></span></p><p class=MsoNormal><span style='font-size:10.0pt;font-family:"Arial","sans-serif";color:blue'>But I definitely agree with Holger that you are using quite low protein concentrations. Back in my Amgen days we would typically use concentrations of at least several mg/mL, i.e. an order of magnitude higher than you are attempting. <o:p></o:p></span></p><p class=MsoNormal><span style='font-size:10.0pt;font-family:"Arial","sans-serif";color:blue'><o:p> </o:p></span></p><p class=MsoNormal><span style='font-size:10.0pt;font-family:"Arial","sans-serif";color:blue'>Holger is also quite right that accuracy for the protein concentrations is often a limiting factor. Determining the extinction coefficient via the synthetic boundary approach assumes you know the correct refractive increment, and that will vary significantly depending on the buffer composition.<o:p></o:p></span></p><p class=MsoNormal><span style='font-size:10.0pt;font-family:"Arial","sans-serif";color:blue'><o:p> </o:p></span></p><p class=MsoNormal><span style='font-size:10.0pt;font-family:"Arial","sans-serif";color:blue'>John<o:p></o:p></span></p><p class=MsoNormal><span style='font-size:10.0pt;font-family:"Arial","sans-serif";color:blue'><o:p> </o:p></span></p><div><div style='border:none;border-top:solid #E1E1E1 1.0pt;padding:3.0pt 0in 0in 0in'><p class=MsoNormal><b>From:</b> RASMB [<a href="mailto:rasmb-bounces@list.rasmb.org">mailto:rasmb-bounces@list.rasmb.org</a>] <b>On Behalf Of </b>John Sumida<br><b>Sent:</b> Tuesday, September 30, 2014 9:06 AM<br><b>To:</b> 'HGSR (Holger Martin Strauss)'; <a href="mailto:rasmb@list.rasmb.org">rasmb@list.rasmb.org</a><br><b>Subject:</b> Re: [RASMB] Reproducibility in densitometry measurement<o:p></o:p></p></div></div><p class=MsoNormal><o:p> </o:p></p><p class=MsoNormal><span style='font-family:"Courier New";color:blue'>Dear Holger,<o:p></o:p></span></p><p class=MsoNormal><span style='font-family:"Courier New";color:blue'><o:p> </o:p></span></p><p class=MsoNormal><span style='font-family:"Courier New";color:blue'>Thank you for your detailed comments. Our experience with degassing the sample is consistent with yours: does not appear to make much of a difference. We have not tried removing the syringe during measurement – Anton Paar rep recommended that we leave it in the plug during the sample measurement. We will try removing the syringe and see if that helps.<o:p></o:p></span></p><p class=MsoNormal><span style='font-family:"Courier New";color:blue'><o:p> </o:p></span></p><p class=MsoNormal><span style='font-family:"Courier New";color:blue'>What concentration range to you normally use for your measurments?<o:p></o:p></span></p><p class=MsoNormal><span style='font-family:"Courier New";color:blue'><o:p> </o:p></span></p><p class=MsoNormal><span style='font-family:"Courier New";color:blue'>Thank you for taking the time to consider my question.<o:p></o:p></span></p><p class=MsoNormal><span style='font-family:"Courier New";color:blue'><o:p> </o:p></span></p><p class=MsoNormal><span style='font-family:"Courier New";color:blue'>Best regards,<o:p></o:p></span></p><p class=MsoNormal><span style='font-family:"Courier New";color:blue'>John Sumida.<o:p></o:p></span></p><p class=MsoNormal><span style='font-family:"Courier New";color:blue'><o:p> </o:p></span></p><div><div style='border:none;border-top:solid #B5C4DF 1.0pt;padding:3.0pt 0in 0in 0in'><p class=MsoNormal><b><span style='font-size:10.0pt;font-family:"Tahoma","sans-serif"'>From:</span></b><span style='font-size:10.0pt;font-family:"Tahoma","sans-serif"'> RASMB [<a href="mailto:rasmb-bounces@list.rasmb.org">mailto:rasmb-bounces@list.rasmb.org</a>] <b>On Behalf Of </b>HGSR (Holger Martin Strauss)<br><b>Sent:</b> Tuesday, September 30, 2014 1:29 AM<br><b>To:</b> John Sumida; <a href="mailto:rasmb@list.rasmb.org">rasmb@list.rasmb.org</a><br><b>Subject:</b> Re: [RASMB] Reproducibility in densitometry measurement<o:p></o:p></span></p></div></div><p class=MsoNormal><o:p> </o:p></p><p class=MsoNormal><span lang=EN-GB style='font-size:10.0pt;font-family:"Verdana","sans-serif";color:#3F31F7'>Dear John,<o:p></o:p></span></p><p class=MsoNormal><span lang=EN-GB style='font-size:10.0pt;font-family:"Verdana","sans-serif";color:#3F31F7'><o:p> </o:p></span></p><p class=MsoNormal><span lang=EN-GB style='font-size:10.0pt;font-family:"Verdana","sans-serif";color:#3F31F7'>Your observations sound very familiar to our experiences with the DMA5000 and I would love to also hear from other users. Some comments from our experiences:<o:p></o:p></span></p><p class=MsoNormal><span lang=EN-GB style='font-size:10.0pt;font-family:"Verdana","sans-serif";color:#3F31F7'><o:p> </o:p></span></p><p class=MsoListParagraph style='text-indent:-.25in'><span lang=EN-GB style='font-size:10.0pt;font-family:"Verdana","sans-serif";color:#3F31F7'>-</span><span lang=EN-GB style='font-size:7.0pt;font-family:"Times New Roman","serif";color:#3F31F7'> </span><span lang=EN-GB style='font-size:10.0pt;font-family:"Verdana","sans-serif";color:#3F31F7'>It is an extremely sensitive instrument requiring a corresponding experimental standard (which is higher than with a 4000 or 4500)<o:p></o:p></span></p><p class=MsoListParagraph style='text-indent:-.25in'><span lang=EN-GB style='font-size:10.0pt;font-family:"Verdana","sans-serif";color:#3F31F7'>-</span><span lang=EN-GB style='font-size:7.0pt;font-family:"Times New Roman","serif";color:#3F31F7'> </span><span lang=EN-GB style='font-size:10.0pt;font-family:"Verdana","sans-serif";color:#3F31F7'>Make sure that the calibration is <i>exactly</i> as the measurement. For example, don’t leave the syringe in the plug under the measurement/calibration. It will affect the resonator, at the 5<sup>th</sup> and 6<sup>th</sup> decimal place. Likewise with the plugs or tubing at the other end. We fill our samples, and then remove every tubing, plug, etc. That’s also how we calibrate, but others might have found a better way.<o:p></o:p></span></p><p class=MsoListParagraph style='text-indent:-.25in'><span lang=EN-GB style='font-size:10.0pt;font-family:"Verdana","sans-serif";color:#3F31F7'>-</span><span lang=EN-GB style='font-size:7.0pt;font-family:"Times New Roman","serif";color:#3F31F7'> </span><span lang=EN-GB style='font-size:10.0pt;font-family:"Verdana","sans-serif";color:#3F31F7'>Gas bubbles can be a problem. Make sure that the temperature of the samples and solvent is similar to your measurement temperature before you dilute and fill them. Likewise, degass preferably at the measurement temperature. We use the small vacuum pump from our ITC-instrument, but in our hands, degassing did not make a huge difference. You can also degass the solvent before you dilute the protein, which can be done more thoroughly than with a protein in it. Any other experiences out there?<o:p></o:p></span></p><p class=MsoListParagraph style='text-indent:-.25in'><span lang=EN-GB style='font-size:10.0pt;font-family:"Verdana","sans-serif";color:#3F31F7'>-</span><span lang=EN-GB style='font-size:7.0pt;font-family:"Times New Roman","serif";color:#3F31F7'> </span><span lang=EN-GB style='font-size:10.0pt;font-family:"Verdana","sans-serif";color:#3F31F7'>0.1-0.3 mg/mL is a rather low concentration and small changes in the concentration due to adsorption of your sample to surfaces (from syringes, etc) will have a relatively large effect. But there might be a reason for the rather low concentration.<o:p></o:p></span></p><p class=MsoListParagraph style='text-indent:-.25in'><span lang=EN-GB style='font-size:10.0pt;font-family:"Verdana","sans-serif";color:#3F31F7'>-</span><span lang=EN-GB style='font-size:7.0pt;font-family:"Times New Roman","serif";color:#3F31F7'> </span><span lang=EN-GB style='font-size:10.0pt;font-family:"Verdana","sans-serif";color:#3F31F7'>Do you determine the density increment or the vbar? A single solution/solvent pair is sufficient for vbar and the highest concentration usually is the most precise measurement. You can of course increase the precision of your parameter by repeated measurements.<o:p></o:p></span></p><p class=MsoListParagraph style='text-indent:-.25in'><span lang=EN-GB style='font-size:10.0pt;font-family:"Verdana","sans-serif";color:#3F31F7'>-</span><span lang=EN-GB style='font-size:7.0pt;font-family:"Times New Roman","serif";color:#3F31F7'> </span><span lang=EN-GB style='font-size:10.0pt;font-family:"Verdana","sans-serif";color:#3F31F7'>I’m not aware that a particular salt or concentration can have an effect – but this is more testimony to my ignorance (and bliss, for that matter).<o:p></o:p></span></p><p class=MsoListParagraph style='text-indent:-.25in'><span lang=EN-GB style='font-size:10.0pt;font-family:"Verdana","sans-serif";color:#3F31F7'>-</span><span lang=EN-GB style='font-size:7.0pt;font-family:"Times New Roman","serif";color:#3F31F7'> </span><span lang=EN-GB style='font-size:10.0pt;font-family:"Verdana","sans-serif";color:#3F31F7'>Accuracy of the concentration measurements is a huge issue and my impression is that the DMA5000’s precision is better than the accuracy of most concentration measurements. Are you using interference optics for the concentration determination? Are you sure that the additional sample handling steps do not change your concentration? Can you run N-determination or AA-analysis in parallel (though they also have their issues)?<o:p></o:p></span></p><p class=MsoListParagraph style='text-indent:-.25in'><span lang=EN-GB style='font-size:10.0pt;font-family:"Verdana","sans-serif";color:#3F31F7'>-</span><span lang=EN-GB style='font-size:7.0pt;font-family:"Times New Roman","serif";color:#3F31F7'> </span><span lang=EN-GB style='font-size:10.0pt;font-family:"Verdana","sans-serif";color:#3F31F7'>Finally, instruments from a certain period have a CPU-board that will give out after a certain time (in our case, after the warranty expired). I seem to be remember that our measurements were less stable in the weeks before the instrument became unusuable, but I have no documentation to this end. Check with your local AntonPaar subsidiary.<o:p></o:p></span></p><p class=MsoListParagraph style='text-indent:-.25in'><span lang=EN-GB style='font-size:10.0pt;font-family:"Verdana","sans-serif";color:#3F31F7'>-</span><span lang=EN-GB style='font-size:7.0pt;font-family:"Times New Roman","serif";color:#3F31F7'> </span><span lang=EN-GB style='font-size:10.0pt;font-family:"Verdana","sans-serif";color:#3F31F7'><o:p></o:p></span></p><p class=MsoNormal><span lang=EN-GB style='font-size:10.0pt;font-family:"Verdana","sans-serif";color:#3F31F7'>There is of course the experimental alternative of using 18OH2, which avoids the issue of concentration accuracy. But you will still need accuracy in your density and s-values. Moreover, defining the solution conditions wrt species concentrations becomes more of an issue.<o:p></o:p></span></p><p class=MsoNormal><span lang=EN-GB style='font-size:10.0pt;font-family:"Verdana","sans-serif";color:#3F31F7'><o:p> </o:p></span></p><p class=MsoNormal><span lang=EN-GB style='font-size:10.0pt;font-family:"Verdana","sans-serif";color:#3F31F7'>Cheers, Holger<o:p></o:p></span></p><p class=MsoNormal><span lang=EN-GB style='font-size:10.0pt;font-family:"Verdana","sans-serif";color:#3F31F7'><o:p> </o:p></span></p><div><div style='border:none;border-top:solid #B5C4DF 1.0pt;padding:3.0pt 0in 0in 0in'><p class=MsoNormal><b><span style='font-size:10.0pt;font-family:"Tahoma","sans-serif"'>From:</span></b><span style='font-size:10.0pt;font-family:"Tahoma","sans-serif"'> RASMB [<a href="mailto:rasmb-bounces@list.rasmb.org">mailto:rasmb-bounces@list.rasmb.org</a>] <b>On Behalf Of </b>John Sumida<br><b>Sent:</b> 30 September 2014 00:50<br><b>To:</b> <a href="mailto:rasmb@rasmb.org">rasmb@rasmb.org</a><br><b>Subject:</b> [RASMB] Reproducibility in densitometry measurement<o:p></o:p></span></p></div></div><p class=MsoNormal><span lang=EN-GB><o:p> </o:p></span></p><p class=MsoNormal><span style='color:black'>Dear RASMB,<o:p></o:p></span></p><p class=MsoNormal><span style='color:black'><o:p> </o:p></span></p><p class=MsoNormal><span style='color:black'>We are trying to perform a measurement of v-bar using an Anton Paar DMA5000. The measurement is of a glycosylated protein. The densitometer was calibrated against a pure dI water standard and we’ve resorted to air checks between measurements to control for our cleaning protocol: 3 x detergent wash, 3 x dI water rinse, 3 x ethanol rinse, and air drying for 4 minutes or until the reading on the densitometer is 0.0012 g/cm3.<o:p></o:p></span></p><p class=MsoNormal><span style='color:black'><o:p> </o:p></span></p><p class=MsoNormal><span style='color:black'>We determined the extinction coefficient by synthetic boundary and believe we have accurately measured the concentration of our samples. Stock sample was dialyzed for a little over 48 hours against PBS and dilutions of the stock into dialysate were made in order to generate four solutions ranging from 0.1 to 0.3 mgs/mL.<o:p></o:p></span></p><p class=MsoNormal><span style='color:black'><o:p> </o:p></span></p><p class=MsoNormal><span style='color:black'>We are getting a values ranging between ~0.60 – 0.70, and are observing that the density measurements are not reproducible beyond the fourth decimal place. <o:p></o:p></span></p><p class=MsoNormal><span style='color:black'><o:p> </o:p></span></p><p class=MsoNormal><span style='color:black'>The reason for the lack of reproducibility appears to be that over time, while the sample is in the U-tube, small gas bubbles are observed inside the tube, either due to auto-hydrolysis of the solution or due to the small vibration of the tube or something else I am not aware of. We do not observe this for water alone. Samples were degassed, each for 10 minutes.<o:p></o:p></span></p><p class=MsoNormal><span style='color:black'><o:p> </o:p></span></p><p class=MsoNormal><span style='color:black'>My questions: <o:p></o:p></span></p><p class=MsoNormal><span style='color:black'>Has anyone observed similar issues when performing this measurement?<o:p></o:p></span></p><p class=MsoNormal><span style='color:black'>Is there an optimal buffer system for this measurement – perhaps at a salt concentration less than 100mM?<o:p></o:p></span></p><p class=MsoNormal><span style='color:black'>Is there, perhaps, a subtlety in this measurement that I need to be aware of?<o:p></o:p></span></p><p class=MsoNormal><span style='color:black'><o:p> </o:p></span></p><p class=MsoNormal><span style='color:black'>Thank you for any and all comments returned. They are greatly appreciated.<o:p></o:p></span></p><p class=MsoNormal><span style='color:black'><o:p> </o:p></span></p><p class=MsoNormal><span style='color:black'>Best regards<o:p></o:p></span></p><p class=MsoNormal><span style='color:black'>John Sumida<o:p></o:p></span></p><p class=MsoNormal><span style='color:black'>University of Washington</span><span style='font-size:12.0pt;font-family:"Courier New"'><o:p></o:p></span></p><p class=MsoNormal style='text-autospace:none'><span style='color:black'><o:p> </o:p></span></p></div></body></html>