Dear all, thanks for the wealth of replies. I'll try to summarize and give some answers and clarification. Here the original post:
Dear all, I was wondering if somebody could help me out by suggesting the "best" column to be used in a Static Light Scattering (I guess it would be the same for a Multi Angle Light Scattering) instrument. We were suggested using a silica-based column, with very high separation properties, but it seems that these columns are highly sensitive to (even slightly) basic pH's. Even running the column in PBS, it looks like injecting samples at pH 8.0 ruins the column resin, making it unusable. On the other hand, GE Healthcare columns would require a huge amount of material to be loaded. What are you guys using in your instruments? Thanks a lot in advance for the feedback, best, ciao Sebastiano Here's replies and comments: Laurent Terradot wrote: > Dea Sebastiano, > I encountered a similar problem and lost one of these columns. (We use > Dawn-Treos/OptiReX in my lab with an akata purifier) > I am now using GE superdex columns (S200 and S75) and load around 100ul of > protein at 10mg/ml. That' s a lot but I fractionate and recover it. The > results are good. > > If money is not a problem, Wyatt make their own columns, which might be > better for you. I have heard good things about it. > I 'll be happy to have info if you have some feed back later. > Best regards, > > Laurent Well that's one of our problems. We are using a Viscotek SLS instrument on their own HPLC machine, and don't have a fractionation unit in line, since we thought we could be using analytical amounts, without recovering. In this respect, 100 ul at 10 mg/ml are way to much. However, protein amount can be much lower, it seems: Nikos Pinotsis wrote: > Hi Sebastiano, > > we have used both silica based and superdex columns and we found the second > as a better option for general usage. > Most of our proteins are on basic buffer and about 50% of them stick on the > silica based column. On the other side a superdex 200 10/30 from GE performed > quite well, we got quite accurate results for both a ~250kDa hexameric enzyme > as also for a 0.3 mg/ml injection of an insulin sample. > Eventually I think depends on the applications you prefer to have. > > cheers > Nikos Engin Özkan wrote: > On 3/8/11 5:03 AM, Sebastiano Pasqualato wrote: >> On the other hand, GE Healthcare columns would require a huge amount of >> material to be loaded. >> > What do you mean by a huge amount of material? You would not be using a 16/60 > column (125 ml column volume) for an analytical experiment. How about a > Superdex 10/300 (used to be called 10/30) or a 5/150 column. These have > column volumes at 25 ml and 3 ml, respectively, have great resolution, and > probably already compatible with your proteins and buffers. > > We used to use HPLC columns, but some proteins would never elute from these > columns. Then we switched to good old Superdex 200 10/300, and it works like > a charm every time. We inject <100 ul material at concentrations around 1 > mg/ml (depending on the molecular weight of the protein in question). The > only issue is we have to run these columns at 0.35 ml/min flow rates (instead > of the default 0.5 ml/min), since our HPLC has a lot of back pressure for > FPLC columns. > > Best, > Engin Engin Özkan wrote: > And as others have said shedding of dextran is a problem with GE columns > (this was confirmed to me by GE people), but after extensive system > equilibration, we do not see a problem significant enough to ever hurt our > light scattering measurements. With respect to the choice of which GE Column to use, it looks there's a consensus for Superdex/Superose 10/300 columns. Some have suggested Superdex 5/150 columns, which would allow much less material to be loaded (they have a diameter of ~5 mm, as the suggested silica column), but others have pointed out that the resolution power is way less than those of the 30 cm columns, and this is indeed our experience, too, and the reason we dropped those columns. Sally Pham Thanh Van wrote: > But I doubt if GE 5/150 column has the same resolution with > the 10/30 one. When I had mixture of several species, the 10/30 always > gave better resolution for me, and this is crucial in obtaining > accurate information about oligomerization state and/or absolute > molecular weight of the protein. So I think if choosing GE columns for > light scattering experiment, I would go for 10/30. > > Best wishes, > Sally. M T wrote: > Dear Sebastiano, > > In our case we don't have magical column... We are using small silica columns > from shodex or wyatt and for proteins that cannot be happy in pH<7.5 we have > a 25 mL superdex 200 from GE. Maybe you can try to use a very small superdex > 200 from GE (ref : 17-1089-01) but I think that the maximum flow rate of > these columns is too low to be suitable with your system. The peoples of > Wyatt told us that if you are at a too low flow rate (under 0.5 or 0.2 I > don't remember exactly) the dilution in the flow cell of your analyzing > system will be more important than the flow rate effect and then the peaks > will be very broad... So I have no miracle column to suggest. An other > possibility could be to find a compagny able to pack custom columns, if it > exist, and to ask them to use your matrix (and to give them some superdex 200 > gel), or to find a way to pack some superdex 200 gel in a jacket of an old > silica column... > > Michel. Some of you are happy with silica columns purchased by Wyatt (and maybe produced by Agilent?): Federico Forneris wrote: > Hi Sebastiano, > > we are very happy with the SEC columns from Wyatt coupled to our FPLC/MALLS > machine. > http://www.wyatt.com/solutions/hardware/sec-columns-for-multi-angle-light-sc > attering.html > > The separation range is (more or less) comparable to GE standard spdx > 200/75/superose 6, and the columns are rock solid. The quality of separation > is excellent in analytical runs, we routinely use them loading sample > volumes of 10-25 microliters at concentrations around 1mg/ml. We did not > encounter reproducibility problems/column damage so far with buffers at pH > in the 6-8.5 range (8.5 is actually the high pH limit recommended by Wyatt). Sally Pham Thanh Van wrote: > I should say clearer that I did a comparison between GE (Superdex 200 > 10/30) and the Wyatt column of the same separation range on AKTA > connected with MALS. The Wyatt column gave better resolution for size > exclusion chromatography as well as signal-noise ratio for light > scattering. So I think it is worth for you to try it. They offer > money-back guarantee. Dima Klenchin wrote: >> they buy it from some small company, idea being the applicability to >> SLS, optimized for minimized bleeding/sheding or material >> from the column, which will show up only in the light scattering >> detector. > > I don't know. Every single spec of Wyatts columns looks exactly the same as > "Bio SEC-5" series of columns available from Agilent. tommi kajander wrote: > Hi, > Silica is ok 6.5-7-7.2, we use that range even pH 7.4 but i wouldn't really > go there. depends on the manucfacturer data sheet of course. > 10/300 GE S200 is quite ok with few micrograms, it depends more on your > instrument sensitivity and dead vol. What system > are you using??? we have analytical scale HPLC and Wyatt. some columns shead > more particles than others thats the biggest limitations > in sensitivity for light scattering, but for us signals are okish even for > old S200 column. > > best, > Tommi > i have to say some of our proteins stick to their columns (happened during > the demo), whereas they dontt to TOSOH, > on the other hand some proteins, as mentioned dont like silica at all --- so > you have to try, or if you know GE columns work for you > (i have some proteins that like to stick to sacharide based things...) can go > with them quite ok. Few tens of micrograms is > certainly enough on a 10/300 S200 for instance... narrow bore silica columns > (eg 4.8 mm TOSOH) appear to be bit limiting since > the flow rate would be better to be bit faster than what you can get there > (<0.4 ml/min vs 0.5-1ml/min on S200 superdex) > > Tommi We have been trying with a Tosoh bioscience silica column, and that is exactly the column that gave us a problem in the phase being ruined by the injections of samples at pH 8.0. So in the end, it looks like both silica Wyatt column and GE Healthcare columns can be a good choice, depending on the instrument setup and applications. Thanks a lot for the very interesting feedback, ciao Sebastiano -- Sebastiano Pasqualato, PhD Crystallography Unit IFOM-IEO Campus Cogentech - Consortium for Genomic Technologies via Adamello, 16 20139 - Milano Italy tel +39 02 9437 5172 fax +39 02 9437 5990
