Dr. Tina Settineri: So, today we're going to talk about some new hybrid electrodes and column mounting hardware for our micro LC/MS applications on the ExpressHT Ultra system from Eksigent.

To talk about, first, micro LC and some of the advantages of particularly micro UHPLC with the ExpressHT, this product provides micro LC that is reliable and reproducible, chromatography at high pressure with our patented Microfluidic Flow Control. This provides for the highest levels of accuracy and reproducibility in your chromatography.

It enables fast gradient separations due to our very small mixing volume relative to the size of the columns that we use on the system. It provides lower total cost of ownership versus a traditional higher flow UHPLC due to our simplified pump design with less parts to exchange or repair, less wear and tear, fewer things to replace over time.

And because we're flowing in the microflow rate regime, there's a significantly reduced solvent consumption and solvent waste; so, something like 95 percent less slower mobile phase is used, and so you don't have the cost for both purchasing, as well as storage and disposal of that mobile phase solvent. And of course, we're flowing at lower flow rates, and going hand in hand with that is small volume injections. And those low flow rates provide reduced solvent load on your mass spectrometer, so it doesn't get dirty as fast, so there's less cleaning or less common--what you need to be cleaning. And for specific applications where you are really limited in your sample volumes, this is a very good choice.

So, here's the ExpressHT Ultra system and what it looks like sitting on top of our Triple Quad 5500--or this could be a QTRAP 5500. In this case it's a Triple Quad.

Some of the specs are listed here. It's 10,000 psi max for the UHPLC. It supports columns in the range of one millimeter, 0.5 and 0.3 millimeter ID columns. And for small molecule analysis, the cycle time is typically less--can be less than one minute. It has a very compact footprint, as you can see, but yet has a high-throughput autosampler for large sample capacity.

And the way we've designed it with some of the stuff especially that we're going to talk about today, it allows very close coupling with the mass spectrometer into the Turbo V source or DuoSpray source on a 5600, for the S-quality [sp] chromatography with minimum dispersion of your peaks. And of course, we have integrated software for running the LC/MS systems together.

Now, why are we providing some new hardware for this? Well, micro LC can definitely be a challenge for bioanalytical LC/MS/MS due to the fact that we're flowing at very low flow rates, and the slower you flow the more chance you have for peak dispersion and less you can use really narrow tubing and minimize all your connection--or the lengths between your connections. And particularly after the column, we want to minimize the size and the length of that tubing to minimize this post-column dispersion or broadening of your peaks.

This isn't as much of a problem with higher flow LC/MS--or LC because you're flowing so much faster and it's easier to keep your peak widths small at these faster flow rates. But, as long as you do this controlling of the post-column dispersion, you can get extremely high quality chromatography equal, or even better in some cases, to high flow UHPLC.

And so, one component of that is the electrodes that are in the ion-spray source, in this case the Turbo V source or the DuoSpray source. What we've done is create some smaller ID electrodes to minimize the peak dispersion and improve your chromatography as we go to lower flows. So, what we've had in the past is the 65 micron ID small bore electrode for use in flow rates of around 60 to 250 microliters a minute, so typically used with half and one millimeter columns--or we found this--with half-millimeter columns, having something a little bit narrower would provide better quality chromatography.

So, this is what is involved with our new smaller ID electrodes. So, we've made these electrodes for flow rates, you know, below about 60 microliters a minute to improve your chromatography for 0.3 and 0.5 millimeter ID columns.

In addition, as I mentioned, trying to mount the column coming out of the LC as close as possible to the source, we've added some new hardware to enable this in a simpler and easier way. So, the column sits in a column heater or a column oven, and we've created some basic hardware to right and mount the column as close as possible when it's in that heater to the Turbo V or DuoSpray sources; again, so that your distance going from the LC to the column and the column, then, into the source of the mass spectrometer is as small as possible to minimize any dispersion that can occur.

And additionally, the previous electrode was made of stainless steel. And sort of long residence time of molecules in stainless steel can sometimes have surface interactions with that stainless steel, so we will start making these new electrodes out of fused silica, or in this case PEEKsil, rather than the stainless steel to minimize any possibility of that from happening.

So, the new solution that we have with these two smaller ID electrodes are what we call hybrid electrodes, so it's a hybrid of this PEEKsil combined with a stainless steel tip.

So, as you can see in the picture in the middle, these are about 200 millimeters long. And the tip on the end, just about one centimeter of that, is the stainless steel, and all the rest is PEEKsil, so there's very little time that any of your sample is going to be exposed to stainless steel. And that's where you spray out of right into the--you know, so that whole piece fits into the Turbo V source, so that's spraying right into the mass spectrometer. It also has an integrated grounding union to allow grounding, of course, to the source in an easy and robust way. And as you can see in this bottom graphic, these electrodes fit easily into the Turbo V probe housing.

Also, you'll note that there's two different sizes that we've made specific for certain flow rate ranges, which work with the different typical micro LC columns. So, a 25 micron ID electrode is ideal for the 0.3 millimeter columns, and the flow rate's listed there, and the 50 micron ID electrode is ideal for the 0.5 millimeter ID columns, for the flow rate range of 20 to 100 microliters a minute.

So, the next slide shows a detailed connection setup with part numbers listed here. It's probably more detail than you need, but it shows a nice simple graphic of the tubing coming from the LC at the top, going into the column that's in that little box, which is the column heater, and then there's tubing that comes out of that. That's your post-column region, so the very small piece of PEEKsil tubing that we recommend that then goes into the electrode that's shown on the bottom for--you know, on both sides here, either the orange for the 25 micron ID or the grey/black for the 50 micron ID. And we've got typical diameters and fittings, etc., listed here that go all together for use with the systems.

So, this is a little bit more detail of what it looks like now once the electrode's in the source, and we've got the grounding listed there. So, these arrows is pointing to--there's grounding clamp that goes on the neck of the Turbo V source, and if you look to the--that picture on the far right, that, then, connects to some hardware that is used, then, to hold that column heater box.

And then, the grounding starts at that integrated grounding union on the top of the electrode, and then has a pin into the clamp. And then, if you're not using the clamp for whatever reason because you don't need to use the column heater or you want a different setup, you can also ground just directly to the source housing, which is shown on the bottom.

And again, as you can see in the picture on the far right, the column, then, is mounted very, very close to the top of the electrode there on the source, so you've got the smallest possible place where dispersion can occur.

And just one more last slide of pictures. On the next slide is the components of the column of a mounting clamp and rod kit for the either the DuoSpray or Turbo V source of where the grounding occurs. And an example on the bottom for our older style triple quads and QTRAPS, you would do a--sort of a sideways mount to connect to the source on the mass spec from where your LC would sit, and that's what that picture shows.

So, now some data examples to show you what these new electrodes and hardware bring you. So, first is a simple example with a flow injection experiment at 10 microliters a minute just injecting a sample and seeing what sort of peak you get, flowing, again, at this 10 microliters a minute into the Turbo V source and the counts that you get, and what that peak shape looks like depending on the size electrode that you're using.

So, the standard Turbo V electrode is in black, and that's just about 100 micron ID electrodes that comes with the system that's great for flow rates of, you know, 100 microliters a minute on up or 200 microliters a minute to two mils a minute. But, you can see for 10 microliters a minute it gives us a very, very broad peak that kind of scans over several seconds, shown here on the graphic on the bottom.

So, the next slide, which I introduced at the beginning, was this 65 micron ID electrode, which we've had for a while, but well suited for flow rates, say, of 60 to 250 microliters a minute. And again, flowing at 10, you see you're sacrificing peak shape and pretty broad peak widths.

A 50 micron ID electrode improves that some. So, now you're about at 0.3 seconds full width at half mass as we go to the 25 micron ID electrode, which is the smallest and clearly gives the narrowest peaks and shows that it's best suited for 10 microliters a minute flow rate.

We have just a simple table to clarify--to have all in one place, you know, what typical flow rates I use with what column IDs and what electrodes I recommended to be used with those. And we've sort of spoken about this throughout the presentation, but here it is all in one place to make it simple and clear.

So, you could use the--either the 25 or the 50 micron ID electrode for use with a half-millimeter column. It sort of depends on the exact flow rate you pick and the samples you're running and the particular column you have--the column length. If you can get it in the right pressure for your LC, then you can--that sometimes affects which size you might use. And then, for the one millimeter ID column, again, you can use the 50 micron or the 65 micron ID electrode. So, here's just a simple example showing a comparison of the same 11 compound drug mix, either with the original 65 micron stainless steel electrode on the top, and then on the bottom the same sample--everything else run exactly the same, but using the 50 micron ID electrode.

So, now your peak widths basically go from three seconds at base to two seconds at base. So, without doing anything else, you've narrowed your peaks and improved your chromatography. So, if you really did need to have improved separation, for example if the mass spec could not distinguish all the compounds or wasn't fast enough to do that, this would enable you to get that without changing anything else.

So, this slide shows an example from a customer that was a tester of our electrodes. And he runs derivatized amino acids in a clinical research lab. And two of the amino acids, leucine and isoleucine, are isobaric and cannot be distinguished in the mass spectrometer, so you do need to separate them very cleanly because he's doing quantitative work. They need to be completely separated in the LC before they go into the mass spec. And so, that is what limits the speed with which he can run the analysis for this set of, say, 20 derivatized amino acids.

So, on the top it shows completely optimized gradient for separation of all of these compounds with the ability, of course, of separating this leucine and isoleucine pair in the red box. And you can see the gradient was about seven minutes long--or the separation was seven minutes long using the 65 micron ID electrode. And this was as fast as you could get it using the ExpressHT system we had and the 65 micron ID electrode.

But, by going to this smaller electrode on the bottom, he was able to actually get good enough separation of all the compound and complete separation of the leucine/isoleucine pair, but cut the gradient time down to 4.3 minutes, which was a significant improvement that he was very happy about because this improves his throughputs because he has lots and lots of samples to run per day. You can see this was using a half-millimeter column in the flow rate range of 28 to 35 microliters a minute during the run.

And again, there's no compromise in sensitivity. This was run everything else being equal. If anything, the intensity went up a little bit because his peaks got a little bit sharper. But, overall, you could say that, you know, the sensitivity was about the same. So, you're not giving anything up by doing this, and if anything you're going to get better in addition to the improved resolution.

So, another example showing a mixture of pesticides. Again, just a very simple experiment comparing the analysis of the peak separation at 40 microliters a minute using a 65 micron ID electrode on a 3200 QTRAP system with the ExpressHT on the top, and then on the bottom comparing directly, running everything else the same, but using the 25 micron ID hybrid electrode.

You can see, again, the peaks get narrower and a little bit taller, and so your overall MS intensity is a little bit higher, maybe a factor of two at the most there, so about a 2x increase in your signal response. So, if you've got peaks that you're trying to--that don't separate well or need improved peak shape, this can often really help that.

Another example here with peptide quantitation, also an application that people like to do at microflow rates on the low end of around, say, typically 10 microliters a minute because you get improved throughput versus running in the nanoflow regime for a peptide quant. And this example just shows a standard of Beta Galactosidase peptides, and then trying to do the quantitation curve looking at LLQ for a select number of peptides in this mixture. And again, this was done using the 25 micron ID electrode. And the LLQ was around 200 atom moles of the peptide on column, so it was 100 atom mole per microliter injection injecting two microliters of the sample with the system onto the column with pretty good accuracies all the way down to the bottom. So, a couple of examples more. This one just comparing to trying to use the electrode on a mass spec using a traditional Shimadzu LC with a CTC autosampler and using the same column and flow rate, and the 50 micron ID electrode in this case, at 40 microliters a minute. So, you can see on the top, or the ExpressHT Ultra, this pesticide mixture of about eight--seven or eight compounds could be separated for quantitative work in just over one minute. We didn't need have to have baseline separation of all the peaks because the mass spec can, of course, handle that.

But, to get an equivalent type separation on the Shimadzu system, you have about one-minute delay per run simply because of the additional delay volume and perhaps the mixer, etc., from the components of the Shimadzu LC that are different from the ExpressHT Ultra. So, this just demonstrates how being designed specifically as a micro LC system enables you to really minimize any delay volume that can occur and delay time, and so you can get--do the fastest possible run-to-run time.

So, this just demonstrates that even at these very low flow rates at 10 microliters a minute with the 50 micron ID electrode how you can get good quality robustness. This is a five-day analysis of quantitative work with an API 5000, and we're separating compounds for quantitation. It shows good quality robustness with consistent peak areas for the four different compounds listed here across over 800 injections.

And you can see on the right hand side of the box listed here with the different compounds and the CVs obtained for the raw peak areas were all less than 10 percent and, heck, most of them were 6 percent or less for this set. So, pretty good robustness at this low flow rate over a long period of time with these electrodes.

So, in summary, what we've talked about in this presentation is two new hybrid electrodes that we've launched to help minimized post-column dispersion for microflow LC on the ExpressHT system. These electrodes are made up of PEEKsil tubing with a short--very short stainless steel tip to minimize any possible surface interaction of analytes going through the electrode.

And when combined with the new column heater mounting hardware that allows the placement of the columns as close as possible to the source, this enables improved chromatographic resolution that results in narrower peaks, increased peak heights and general improvements in your chromatographic resolution for 0.5 and 0.3 millimeter ID columns.

And of course, all this is optimized specifically for the ExpressHT Ultra system.