Viscous Heat Generation in UPLC

[Victor]

I agree with Kostas. Unmgvar- I think you are confused about capillary HPLC. People are using typically 15cm columns packed with 3um particles for capillary HPLC-with column diameters down to 75um or thereabouts. These to an approximation give just about the same performance (column efficiency) as a 15cm routine diameter HPLC column (15cm x 0.46cm diameter) packed with 3um particles. The situation is not analogous to capillary GC where stationary phases are coated on the inner walls of the column (i.e. no packing) and much higher efficiencies can result, principally because very long columns can be used.

One of the main driving forces for capillary HPLC is to use low flow rates which give high sensitivity in some detectors-principally mass spectrometry.

Alternatively UPLC uses smaller particles in columns which are of diameters within the normal range, principally to achieve the same separations as in conventional hplc but in a shorter analysis time. Perhaps Waters will introduce a capillary HPLC which works with UPLC particles (1.7um). Maybe they already have such an instrument; but maybe there are some practical considerations which need to be solved.

[Kostas Petritis]

Victor,

They already have one and it's called nano-aquity. They introduced it at the beginning of 2005....

[unmgvar]

Victor,
Maybe the angle from which i look at the matter confuses things.

i might call it a "customer approach". I am looking for solutions to a given set of problems. I look at the different aproaches not necessarely by how they work but by what they solve. so in priciple it does not matter to me for example if the "high flow" (UPLC) or "low flow" (nano) are cotracdictory somewhat. what matters is which one give a broader solution, present and future.

if we look at it "anthropologically", UPLC is a logical next step. for the given stationnary phase, the increase of surface area by decreasing particle size while being able to keep or even increase flow rates is an next evolutionnary step, of doing better.
just for argument sake thou, in the mid 19th century Jules vernes wrote the book: "a trip to the moon". for him it was obvious with the knowledge that he had that the only logical way to go to moon was with a big enough cannon. UPLC is a "bigger" canon basically. what i am interested to know is:
is it the too big of a cannon, the last step before or do we have enough steps before that, so as that what is gained compare to what is invested is really worth it (too high maintenace cost, consumable costs and life time and so on...).

currently according to one of Waters own pubished articles on the matter there are limitations, and it does look to me has if for future column development of every particle size, there will be a need for many hardware upgrade/changes.

it is true that theoretically their is no connection between column i.d. and particle size, but as we saw it on the praticle side their is, especially regarding temp. effects. this causes it to very much take inot account the internal volume of the columns
those volume channges also affect the dwell volume of the entire instrument from pump to detector. gradient formation, sample loading, sample purification steps, detection capacity especially.
for exemple the ultimate 3000 of dionex for nano and cap work, can be retrofittedd in order to accomodate working in the different ranges.

also the nano acquity system is for low flow. See Uwe response in the smallest particle stationary phase for LC topic.


my two scent worth wild guess is that the vendor that will be capable of producing more reliable monolith column that can be put on a UPLC system will have the better solution.
the polymerisation process of monolith column seems to permit an better ratio for surface area of stationnary phase to internal volume of mobile phase. but reliable production and a better control of the polymerisation process is not quite there yet.

[Kostas Petritis]

Unmgvar,

I do not agree with everything you say but I think that the home message you make is that people has to evaluated what is gained compare to what is invested that will vary depending on what everyone wants to achieve (i.e. for some people high-througput might be a priority for someother not etc...). In your case Uwe offered to demonstrate to you what UPLC can do for your method(s)...

I guess that this evaluation is made not only from the customers but also from other HPLC manufacturers which will have to make a decision if they need to invest or not to it. In general people starts thinking more of investing in a technology when they see an industry adoption/shift towards the same technology...

Remember that there is always a premium that you have to pay if you want to be an early adopter of a new technology (which companies justifies if they are unique in the market in order to pay back some of the R&D money they spent etc). Prices will go down with time and if there are any technical challenges will be resolved.

[Uwe Neue]

unmgvar:
It is nice of you to recognize that UPLC is the next step. Of course, further increases in speed or pressure will require further hardware improvement, but how about we first use the improvements that are available with UPLC today. Remember, I offered to you to calculate how UPLC will improve either your separation power or your analysis time, if you give me a complete description of your classical HPLC conditions.
Monoliths are far too limited to compete with UPLC. They have severe pressure limitations due to the polymeric wall, and they have too large an i.d. They are no better than what one can do with 3.5 micron particles. While this is not bad, it is not UPLC - nowhere close. UPLC uses 1.7 micron particles, and takes full advantage of this.

[unmgvar]

Kostas,

i'll even add to what you say with that remark (which i personally hate):

"i can't use it or try it, it is not in USP or EP"

it is incredible how most of my collegues from the generic pharma industry in my country fear novelties and innovation. can blame them to much, after all they are part of a huge "me too" concept. Most of them are totally afraid to try and validate something that has not been reviewed by USP and EP.

Still the question remaining is: will UPLC give a long term solution or is it simply the easiest next step because everybody is using HPLC? i personnaly will not take a certain solution for solving today's problem only. i will invest on something that will answer my needs for several years to come.

Uwe,

we should be more precise, i said "anthropologically", UPLC is a logical next step

also i will agree with you and Kostas that technologies should be given time to solve the problems they face.
Still i wil be interested in knowing how column manufacturer will succeed in packing ever small particle size in ever small column i.d. with ever increasing pressure. will the column casing hold it unless made of titanium? will the Silica change it's structural geometry?
because of ever smaller i.d. for the column we need to improve the detection. How will manufacturers resolve the detection issues? let's not forget that the higher the Hz at wich the detector work the noiser they get and so we actually get a worst S/N ratio.

true, monolith column are not as good yet. but like you say, you need to give time to solve some of the challenges.
but if you can increase the degree of control over the polymerisation process, you will increase the amount of material in the column, increasing surface area, and still keeping pressure lower then in current UPLC. mobile phase consuption will drop as well.
monolith column add another thing as well. they permit to separate by combining 3 types of ways:
1. absorption-the silica provides that.
2. partition- gradient applications
3. Size exclusion- the macro structure of channels created by the polymerisation process.

and 3 is way better then 2. monolith columns are way better at separating peptides then the particle size columns. and let's not forget that today everybody is including peptides or peptide based molecules in their research.

[Uwe Neue]

unmgvar:

Jorgenson has worked with silica at pressures of 60 000 psi at least. Of course, one needs to think about how to construct such particles and columns, but I have not seen any fundamental difficulties.
Smaller detection volumns are used already today. It is called capillary chromatography, and instruments are available for this (e.g. Cap-LC). Other manufacturers have equipment as well. One can use smaller particles for two things: one is extreme speed, the other is improved performance at standard speeds. Only for extreme speeds does one need significant improvements in data rates, but for the current state of the technology, the data rate is not the limiting factor.

Also, I am surprised that you exclude SEC from UPLC. There are some issues with shear of very high molecular weight samples, but for normal proteins, I do not see an issue.

[adam]

I'm not sure you guys are being fair to monolithic columns. I think they offer quite a bit. The best way to look at this is in terms of the equivalent particle diameter concept. And it turns out that different diameters need to be used for performance vs backpressure (I can give a few references if you want).

The current state of the art (I am working off the top of my head - but I am pretty sure this is in the ballpark) is that monoliths give you the performance of a packed column with 1 um particles, with the backpressure of a packed column with a 10 um particle. That's pretty impressive. When you consider that backpressure is inversely proportional to particle diameter it mean that, as a rough rule, monoliths can give the same performance with 100 times lower pressure.

I haven't seen that many disadvantages. They may be more pressure limited but given the above conclusion, it doesn't really matter much. And this will probably improve over time anyway.

[Newman768]

When I think of UPLC and (future) monolithic columns I always ask myself, which seperation enhancements should be possible, if the uplc column heater is able to cool the column down to -20°C or so...
I have seen a seperation with utlc at - 20°C and since that I always hope that this might get possible with hplc.
A lot of people speak about increasing the temperature to enhance seperation - my experience is that with increasing temp the seperation more often decreases. So it is a logical way to think about low temp "low" pressure seperations - and that seems to be able only with monolithic columns.

[MG]

One gripe I had with monoliths, the last time I looked, was that it was hard to find smaller i.d. columns. As if I want to flow 5 mL/min into my mass spec just to gain speed. Seems as though 1 mm i.d. monoliths would be ideal for conventional LC-MS (run at flow rates similar to 2 mm packed columns), but last time I looked, maybe 6 months ago, I did not see this size for sale.

[adam]

MG

Dionex sells micro-scale monoliths.

Adam

[Kostas Petritis]

Polymeric monolithic columns...

[Uwe Neue]

Adam, your description of a monolith is off by a few orders of magnitude...

Based on the data published by Guiochon and Kele, the performance of a Chromolith is that of about a 5 micorn particle (only for an unretained peak it looked cloaser to a 3 micron particle) while the backpressure was that of a 9 micron particle.

That ain't bad, but it does not get you anywhere, if you can't get to the advantage that the lower pressure offers, because a normal HPLC does not give you enough flow to get there.

There are some other disadvantages as well, but this is really a major issue. You can get with small particles packed into short columns of the correct dimensions better results than from the monolith.

[Uwe Neue]

Newman: Columns can be cooled to wherever you want. Just stick them into a bath at the temperature that you want. The reason that commercial column coolers do not go to -20 degrees is that most separations are done with reversed-phase HPLC, and you do not want to get into a spot where the water freezes inside your column.

However, due to slower diffusion, low temperatures are only useful for special occasions, and they are definitely not the tool to use if you want speed.

[Alex Buske]

I have watched at the silica monoliths since 1999. They have certainly lower backpressures. Since the introduction the column formats havent changed. From my opinion a 2x200mm column would be sensible with regards to conventional LC equipment.
The particulate columns show astonishing differences in their plates/backpressure. I think there is some room for improvment.
As long as "ultra pressure" equipment has limited availibility it will be restricted to special applications.
For us the "separation power" is important and I actually dont see better resolutions at sub-2µm particles in 10cm columns to good 3µm Particles in 15 cm or even 25 cm columns. Speed is also important once separation has been achieved.
Alex