The future of liquid chromatography

[AA]

To Yury,

Your simulation shows the benifit quite clearly, the separation is faster and the peak width is narrower (ie an increase in resolution). All this from a column that is 1/3 the length at half the flow rate.

[AA]

Make that 1/3 shorter....

[tom jupille]

First off, I should never, ever post on the forum late at night (that's when I get angstroms and nanometers confused ).

Now, back to the fray!

Yury, you're right about selectivity, of course, but it's something of a red herring, because the uplc discussion about efficiency applies to any selectivity. You're also right about the current waters system falling short of what we'd really like to have, but it is a step in that direction. As it happens, I I do drive a "sports car" (Toyota MR-2), and while it won't do 200 mph, it can make over 150 mph (240 kph for all of you in parts of the world that don't use archaic measuring systems). The extra power and handling have saved my butt on at least a couple of occasions by allowing me to maneuver out of harm's way. The same argument could be made about pressure and efficiency in an LC system; you may not use them at the limit most of the time, but they can be very handy.

Now, I'll do a flip-flop: at the cutting-edge, high-performance aircraft (e.g., the SR-71 spy plane) can fly at Mach 3 and altitudes in excess of 25,000 m, but the vast majority of air travel is done at about Mach 0.9 and 10,000 m -- limits which have remained unchanged since the early 1970s. Is there a parallel with HPLC here?

[Ron]

I have noticed in posts by Tom and Uwe among others references to van Dempter minima and plate heights and theoretical calculations based on the classical equations. I have noticed in the GC world when going to microbore columns, 100 microns or less in interior diameter, the pressure drops across the column are much higher than when using standard capillaries and separation efficiency does not seem to follow the predictions made using classical van Dempter plots. I have also seen significant shifts in retention index using the same test probes on the same column phase when going to higher pressure drops across the column. Others I have talked to have seen the same type of effect. Is it reasonable to apply the equations that have worked well for the current generation of HPLC systems and columns and assume that they will have the same predictive accuracy when dealing with much smaller particle sizes and much larger pressure drops?

I may be mistaken about this as I have no direct experience with the UPLC, but couldn't there be difficulty in translating methods from standard particle sizes and pressure drops and getting equivalent performance? I realize that this will not be an issue for many people on this forum, but what about when it is time to translate and validate QA/QC methods?

[tom jupille]

Ron, LC is more benign than GC in that regard, because the compressibility is much less, so you get essentially the same linear velocity across the entire column length. Arguably as you go to ultra-high pressures you may start to change some equilibria, but off the top of my head, I don't know if that would be significant at the pressures we're talking about here.

[Uwe Neue]

Yuri, I am also quite capable to show silly simulations where the performance of a 5 micron and a 1.7 micron column are the same. Let me go back to what I said before: unless you are making columns that are held together by ferrules, you can feel free to make small particles and participate in this new market of very small particles. Nobody is hindering you or any other column manufacturer to enter, and some are moving in this direction already.
Also, Yuri, another poster has already pointed out that your claim that UPLC is very expensive is not correct.
Finally, the optimum operating conditions for any column are close to the minimum of the van-Deemter curve. One does not need to depart by a factor of 5 from the minimum of the curve. I invite you again to read the relevant papers, as I did in 1976…

[Uwe Neue]

Kostas:

I don’t know about the Agilent claim of increased efficiency with lower backpressures with a broader particle size distribution. I studied this in the 90’s, and the real story is exactly the other way round. Look in my book on page 84 together with the associated text. If you don’t buy this, you can look at publications from Istvan Halasz from the early 70’s.

At this moment, I think it is way to early to start to dream about 0.1 micron particles. It took 30 years to get from 10 micron particles to 1.5 micron particles. At this rate, we will need to wait until the second half of this century for 0.1 micron particle to become useful for HPLC.

Tom:

Yes, the PDA is the standard detector, and the standard UPLC instrument comes with a fast PDA with a very small flow cell.

Monolith technology has been promising for quite a while. However, the manufacturer has only been able to cook up a column dimension that is not suitable, because it requires high flow rates to take full advantage of the column design. The best flow rates for fast gradients are outside the range of what one can do with a standard HPLC instrument, and they have not yet managed to come up with a smaller i.d. column. Until this is changing, I do not see the monoliths to be a threat for UPLC.

There are effects in UPLC that are outside what one has encountered in HPLC. This includes compressibility effects and temperature effects. Some can be circumvented with the proper instrument design, others have a rather minor effect.

The key effects for the user is the performance advantage of UPLC, which can be demonstrated in theory and has been demonstrated in practice. It can either be performance, or it can be speed. I personally lean towards looking at the speed component, because I am an impatient person.

[HW Mueller]

It appears plausible what many have said that ~20 peaks is the maximum that one can separate with a normal HPLC column. What are the expectations for lets say a column requiring 40Kbar? A factor of three more compounds separated, maybe a factor of 10?? If that is correct than what good does UPLC do, other than being an intersting specialty, for proteomics or lots of other biological samples (for instance, almost everything in a clinical chemistry lab)?

[Kostas Petritis]

Uwe,

I know (most) of the theory. About the Agilent particles, I just said what is claimed by them and nothing else... I am not aware if they have a scientific explanation or if it is just an emperical observation. About your book it has been a while now that I am thinking to buy it but haven't do it yet. Maybe I should wait until the next HPLC conference so I could have a free copy of your book, with dedication: "To my chromatography forum friend, Kostas."... just kidding
About the 0.1 um particle, I was refering to non-porous particles. There already 1 micron non-porous C18 particles...

HWM,

Resolution gain is one advantage, separation speed (high-throughput) is another.

[Uwe Neue]

Ron:

The translation of UPLC methods to HPLC methods is an interesting issue. If your UPLC separation is such that you can drive a truck through the spaces between the peaks, then a translation to HPLC will not be a problem, or only be a small problem. However, if the methods developed require the higher resolution at the short analysis time of UPLC, there is only one way to do this: to purchase a UPLC instrument for the QC department.

Another solution could very well be the development of a fast UPLC method, which can then be translated to a slower HPLC method with larger particles and shorter columns of the same type as the UPLC columns used for the fast method. This is of course a compromise, but the impediments to this approach do not appear to be large. The user can then decide, if it is cheaper to purchase a UPLC instrument for the QC lab or to spend the labor on the longer method.

[HW Mueller]

But in more than 5 years combinatorial chemistry and "high throughput" has almost exclusively been high money wastage. Useful medication has practically not emerged. We need intelligent chemistry again, including, of course, more chemistry in analytics (it seems to me that biology has peaked already).
I have nothing against speed, but it should most likely not be the mainstream.

[bert]

But in more than 5 years combinatorial chemistry and "high throughput" has almost exclusively been high money wastage. Useful medication has practically not emerged.

Maybe not because of "high throughput" but despite.

regards Bert

[Yury Zelechonok]

To Uwe or/and AA
Can you give us specific numbers for instrumentation price lets say a basic UPLC unit with gradient pump, degasser, autosampler, column heater, and a UV detector (DAD if there is no variable wavelength). I also would need probably a device or program for PC to make it alive. How much for that? I was told by Waters representative that this is $70K+ price range. They did not tell me how big the +. But it is definitely not minus. And last but not least what is a price tag for a full service contract including parts let say for a year within continental US?

[HW Mueller]

bert, watch what is being done with oligonucleotide aptamer, maybe even with RNAi or "Antisense", or even with antibodies, diabodies, etc. More intelligent approaches seem to yield more results. Now if there was less bio, more chemistry in these endeavors ...... who knows.
(It is my belief that these things are also quite slow, though more promising than combinatorial.., because chemical knowledge is not up to the problems involved, yet). (On combinatorial methods: At least one prominent pharmacist has expressed the thought that small molecule medication has passed the zenith).

[Uwe Neue]

Above, I have pointed out that you can do things faster with UPLC, and that this is where my preference and my head is. On the other hand, we have applications in metabonomics, where you don't see anything (or very little barely) with a standard analysis, but with the 3-fold higher resolvinf power of a UPLC gradient you find the needles in the haystack.