When optimization is discussed throughout this talk, it s in terms of performing more samples, in less time, while minimizing investment in capital

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3 When optimization is discussed throughout this talk, it s in terms of performing more samples, in less time, while minimizing investment in capital instruments. 3

4 Efficiency is a measure of peak width. The narrower the peak, the more efficient it is, ostensibly meaning your entire separation is more efficient. Peak capacity is directly proportional to efficiency because peak capacity is a measurement of how many fullyresolved peaks you can fit in a fixed period of time. When you have narrow (efficient) peaks, you can fit more peaks in a given period of time. Additionally, since more efficient peaks are narrower, they re taller, which improves sensitivity. We re looking for Princess Leia, not Jabba. 4

5 So what does efficiency have to do with optimization? If you don t have an efficient separation, you ll ultimately end up with higher instrument costs due to lower instrument capacity and higher consumables cost due to low sensitivity. 5

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7 HPLC columns consist of a metal tube filled with a tightly packed bed of silica particles. These silica particles are porous, so as the mobile phase moves through the column, it moves both around and through the silica particles. The illustration here is of a fully porous silica particle. Due to the pore structure, the path of mobile phase through the particle is kind of long and meandering. Additionally, these pores are coated in stationary phase (e.g. C18, biphenyl, etc.). 7

8 Knowing that solutes diffuse in solvent over time (more time, more diffusion), if we think about that long, meandering path of mobile phase and analytes through our fullyporous silica particles, we can see that the longer the path through the silica particle, the wider our analyte band will be when it elutes due to diffusion and prolonged interactions with the stationary phase. A wider analyte band means a broader, less efficient, peak. One way to increase efficiency in HPLC is to reduce the path length through the fully porous silica particle simply by making the particle smaller. Columns with smaller particle size (usually < 2µm) are called UHPLC (Ultra high performance liquid chromatography) columns. 8

9 If we go back and take a close look at the packed bed inside an HPLC column, we can see that although the particles are packed very tightly together, they re spherical, which means that there will still be space between the particles. Larger particles mean larger spaces (called interstitial spaces), which translates to easier movement of the mobile phase through the HPLC column and lower backpressures. HPLC columns can be used on instruments with a maximum pressure of ~4,500 psi. 9

10 When we look at UHPLC columns, we can see that due to the smaller particle size, the interstitial spaces in the packed bed are much smaller. This increases resistance of mobile phase movement through the column, resulting in much higher backpressures. UHPLC columns require special UHPLC instruments capable of pressures above 10,000 psi. 10

11 So if we decide to increase our efficiency (optimize) through instrumentation, that means we can go with UHPLC instrumentation and columns. UHPLC definitely results in much more efficient (and therefore faster) analyses, but the drawbacks to UHPLC instrumentation include high instrument cost, and increased risk of column clogging due to the smaller interstitial spaces in UHPLC columns. 11

12 Aside from the UHPLC approach, there is another way we can increase the efficiency of our HPLC analyses. We know that we can decrease the path length of our mobile phase and analytes through our silica particles simply by making them smaller. But we can also decrease that path length by manufacturing silica particles that aren t entirely porous. These superficially porous particles consist of a solid core with a porous outer shell. Since the center of the particle is solid, the path length through the particle is greatly decreased without the need for a significantly smaller particle than those used in HPLC columns. Remember, larger particles mean lower backpressure, so we get the efficiency of UHPLC without the backpressure or the need to invest in expensive UHPLC instruments. 12

13 So let s take a look at a conventional HPLC analysis for cannabis potency. We get good resolution between all of our analytes on affordable, conventional instrumentation, but our analysis time (including re equilibration) is about 30 minutes. This means that the maximum number of injections we can perform in a 24 hour period with this method is 48. If we include 6 injections of calibrators and quality control samples for each analysis day, this brings our maximum number of samples to be analyzed in 24 hours down to 42. If we need to analyze more than 42 samples per day, we re going to have to buy a second instrument to increase capacity. While HPLC instruments are much less costly to purchase, operate, and maintain than UHPLC instruments, they re still not cheap. 13

14 If we move our analysis over to a superficially porous column on the same conventional instrumentation as the previous slide we can see that our total cycle time (including reequilibration) drops to 7 minutes. This brings our total 24 hour sample load for the same instrument up to 205 injections. In order to analyze that many samples in one day using the conventional column and method shown in the slide above, you would have to purchase, operate, and maintain 5 instruments. This is the advantage of optimization. 14

15 We can see the same benefits for pesticide analyses as well. 15

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17 So how do we gain efficiency for our GC analyses? And why do we even need GC when we have our fancy HPLC with its superficially porous particle columns and hyper expensive MS/MS? The short answer to this is that there are some things that the mighty HPLC just can t do. Small solvent molecules do not retain on HPLC columns and these small molecules are not readily detectable by UV detectors. This means that residual solvent analysis must be performed by GC. Really, analysis of small, volatile, compounds is what GC was designed for. The only way to reliably analyze very volatile compounds like propane, butane, and methanol is to use headspace GC (HS GC). This technique involves placing a sample (solid or liquid) inside a 10 20mL vial, then capping and heating the vial until the volatile molecules in the sample partition out of the sample to a point where they reach equilibrium between the liquid/gas phase. After equilibrium is established, some of the gas phase in the vial (the headspace) is injected into the GC. The beauty of headspace analysis is that you get absolutely no interference from heavier matrix interferences because they never partition into the headspace. Additionally, no matrix crud is injected into the GC, resulting in very little maintenance of the column or inlet in headspace instruments. 17

18 The title of the previous slide was Increasing GC Efficiency Through Technique. The reason for this is that in contrast to HPLC columns, GC columns are all extremely efficient. Just looking at the columns, we can see that we have 15 30m of GC column to work with, whereas we only have m of HPLC column from which to extract the best separation possible. 18

19 Yes. For headspace analyses, the place where you can get the most increase in efficiency is in your inlet. The reason for this is that for the most part, all of our volatile analytes for residual solvent analysis are in their tightest bands as soon as they leave the headspace vial. In traditional GC analyses involving liquid injections, in general, analytes are introduced onto the GC column below their boiling point, so as soon as they re injected as a gas, they recondense on the head of the GC column. This is called focusing, and it allows tight analyte bands to re form at the head of the column before they re eluted. In the case of both propane and butane, the head of the GC column is already significantly higher than their boiling point, so they have no opportunity to focus as discrete analyte bands. So if we re going to analyze propane and butane (and to a lesser extent methanol, pentane, and isopropanol), we need to make sure that our analyte bands are as tight as we can get them before they are introduced to the column, because they ll only get wider after that. And remember, efficiency is all about peak (a.k.a. analyte band) width. The wider our peaks, the lower our efficiency. 19

20 The best way to increase efficiency for headspace analyses is to minimize the time analytes spend getting from our headspace vial, through the inlet, and onto the column (this is called sample transfer). This is especially true for headspace instruments that employ a transfer line rather than a syringe. While transfer line headspace autosamplers work very well, analytes have a lot of opportunity to expand in the gas phase inside that long transfer line. If you have a syringe headspace autosampler, the following slides will help with your analyses too, so don t stop reading. One way to increase speed of sample transfer is to reduce the volume of our GC inlet. Most GC inlets use liners with a 4mm internal diameter. This is fine for liquid injections, but for our analyte molecules in the gas phase exiting a narrow (<0.53mm) transfer line or syringe needle, 4mm is cavernous. We all know that gases expand quickly to fill available space, so performing a headspace injection into a 4mm liner automatically broadens our analyte bands. If we switch to a 1mm (or 2mm for some syringe samplers) liner, we ve reduced the volume in our inlet by a factor of 16 for a 1mm liner and by 4 for a 2mm liner. Aside from the advantage of reducing the overall volume available for expansion in the inlet, remember that no matter which liner we choose, we re running a set flow of carrier gas through that liner. This means that for liners with smaller IDs, the velocity of the gas through the liner increases, which further reduces the time it takes for sample transfer. 20

21 Aside from reducing the volume of our inlet, we can also speed sample transfer by employing a split injection. Split injections involve introducing extra flow through the inlet, a portion of which flows out the split vent of your GC. This means that you ll also be splitting off a portion of your analytes, reducing the amount of analyte you re introducing to the column. For trace analyses, a lot of folks think that splitless injection (not splitting any analyte off) is the best approach because of the small amount of analyte present in the sample in the first place. However, when it comes to HS GC, splitless is not always better especially for very volatile analytes like butane. During split injection, you re dumping more flow through your inlet than you would be during a splitless injection. More flow means more velocity through the inlet, which translates to faster sample transfer. We can see in the chromatograms above that by simply increasing our split from 5:1 to 10:1, we ve vastly improved our chromatography. And since we now have narrower peaks, those peaks are taller, so even though we re introducing roughly half the sample during a 10:1 split injection as we are during a 5:1 split injection, we haven t lost much in the way of peak height/sensitivity. Another way to increase inlet flow that s not covered in this talk is to run your analysis at a fast linear velocity. In developing our cannabis residual solvents application, we took an approach similar to labs performing blood alcohol analyses. We used an 80cm/sec linear velocity for our carrier gas, which is pretty far outside the optimal linear velocity range for helium. However, the amount of efficiency we gain through faster sample transfer outweighs our efficiency loss due to the fast carrier gas velocity. 21

22 So here we are. Here s the beautiful chromatogram. This was generated by ProVerde labs using the conditions listed above. For full details of this analysis, type FFAN2009 into the search box at then click resources. The interesting thing to note here is that ProVerde was using nitrogen carrier gas, which has a very slow optimal linear velocity (8 16cm/sec). We ran this application at 80cm/sec nitrogen and still got great chromatography because when it comes to this kind of headspace analysis, we have efficiency to pretty much throw away in terms of the column, but we need to scrape out all we can in the injection. 22

23 Here are the beautiful calibration curves for the residual solvents. 23

24 So. We ve purchased an instrument because we have to do residual solvent analyses. Let s see what else we can do on this instrument. Terpenes are perfect to run by GC FID because of the following reasons: There are tons of terpenes potentially present in cannabis. Since they re all in the same compound class, separating all of them is extremely challenging. This means you need a really efficient analysis. While HPLC is pretty efficient, GC can t be beat with its really long columns. There s no reason to try terpenes with MS. Many of them share the same molecular weights and they all pretty much share the same spectral fragments. Terpenes don t respond very well to UV detectors Terpenes are volatile (you can smell them, right?) Since they re composed primarily of carbon and hydrogen, they respond very well on FID detectors almost as well as they do on MS 24

25 Instrument consolidation is another way we can optimize. If we consolidate two lessfrequently used methods onto one instrument, not only do we free up our workhorse instruments for the high volume testing, but we minimize our overall instrument investment. True consolidation means that you need to use the same GC, sampler, column, and liner. There should be no downtime for hardware changeover between methods. 25

26 So that s what we did with terpenes. We used headspace sampling to gain a semiquantitative profile of a comprehensive set of cannabis terpenes, the same column, and same liner. In this case, since terpenes are less volatile and will focus on the head of our column and we need all the efficiency we can get for these difficult separations, we reduced our carrier velocity to something closer to nitrogen s optimal linear velocity. For full details of this analysis, type FFAN2045 into the search box on then click resources. 26

27 Tl;dr: Use superficially porous HPLC columns to drastically reduce your analysis time for cannabis potency while eliminating the need for costly UHPLC instrumentation. Use headspace GC FID for residual solvent and terpene analyses. You can set up both of these analyses on the same instrument with the same consumables. Gain efficiency by using a 1mm liner, 10:1 (minimum) split ratio, and fast (80cm/sec) linear velocity for the residual solvents. 27

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