Mr. Fletcher vs. Mr. Daubert: A Jour­ney into the Surreal

By:  Fred­eric White­hurst, J.D., Ph.D.

Many a defense attor­ney has looked in amaze­ment at the court­room “iden­ti­fi­ca­tion” of Cannabis Sativa L. by a law enforce­ment offi­cer sport­ing a gilded pot metal badge and a high school diploma who has writ­ten a report that would turn a ninth grade Eng­lish teacher’s hair gray and deny that same offi­cer a high school diploma. Obvi­ously the award­ing of a Basic Law Enforce­ment Train­ing (BLET) diploma ele­vates such high school grad­u­ates to the doc­toral level in botany, enabling them to con­duct a visual leaf archi­tec­tural analy­sis and the court accepted alchemy of the Duquenois Levine test to reach a con­clu­sion that the green veg­etable mate­r­ial in that lit­tle baggy can be iden­ti­fied as mar­i­juana to the exclu­sion of all other plant mate­r­ial. On the face of it, this iden­ti­fi­ca­tion screams to our com­mon senses as not being pos­si­ble. How­ever, we find that in State v. Fletcher, 92 N.C. App. 50, 373 S.E. 2d 681 (N.C. App. 1988), our courts in North Car­olina are will­ing to accept this tes­ti­mony as evi­dence. Though State v. Ward, 364 N.C. 133 (2010) began a foray into rec­og­niz­ing the ridicu­lous­ness of the legit­i­macy of visual iden­ti­fi­ca­tion of con­trolled sub­stances in a world full of coun­ter­feit mate­ri­als, State v. Gar­nett, ___ N.C. App. ___, 706 S.E.2d 280 (2011) backed away from Ward, thus allow­ing an in-court iden­ti­fi­ca­tion of cannabis sativa to be admis­si­ble. This all flies in the face of a long list of sci­en­tific lit­er­a­ture and even State v. Tate, 300 N.C. 180 (1980), which found that the then known list of mate­ri­als caus­ing false pos­i­tives with this Duquenois chem­i­cal spot test ren­dered the test not sci­en­tif­i­cally reli­able or admis­si­ble as an means of iden­ti­fy­ing mar­i­juana. In North Car­olina we are now given a new stan­dard for admis­si­bil­ity of scientific/expert tes­ti­mony in courts of law. Rule 702 has entered the mod­ern era. As of Octo­ber 1, 2011, the amend­ments to Chap­ter 8C, Rule 702(a) read:

If sci­en­tific, tech­ni­cal or other spe­cial­ized knowl­edge will assist the trier-of-fact to under­stand the evi­dence or to deter­mine a fact in issue, a wit­ness qual­i­fied as an expert by knowl­edge, skill, expe­ri­ence, train­ing, or edu­ca­tion, may tes­tify thereto in the form of an opin­ion, or oth­er­wise, if all of the fol­low­ing apply:
1. The tes­ti­mony is based upon suf­fi­cient facts or data.
2. The tes­ti­mony is the prod­uct of reli­able prin­ci­ples and meth­ods.
3. The wit­ness has applied the prin­ci­ples and meth­ods reli­ably to the facts of the case.

So let us now con­sid­ered this “expert iden­ti­fi­ca­tion” of Cannabis Sativa L. by a BLET “Ph.D. botanist”. The expert must be qual­i­fied by knowl­edge, skill, expe­ri­ence, train­ing or edu­ca­tion. The law enforce­ment offi­cer who has seized green veg­etable mate­r­ial on ten or ten thou­sand occa­sions is not test­ing his qual­i­fi­ca­tions or his “iden­ti­fi­ca­tion pro­to­col”. He is sim­ply seiz­ing green veg­etable mate­r­ial. Let’s con­sider the data that he has at the point of seizure.
The most preva­lent form of seized and “iden­ti­fied as mar­i­juana” mate­r­ial that we see is the mate­r­ial that has been finely crushed. What this means is that the law enforce­ment offi­cer has seized a mate­r­ial which no longer has the macro­scopic char­ac­ter­is­tics of the orig­i­nal plant (if indeed the seized mate­r­ial is plant mate­r­ial). But is it plant mate­r­ial? What is a plant and there­fore veg­etable mate­r­ial? Ask any dic­tio­nary. The Amer­i­can Her­itage Dic­tio­nary defines a plant as “an organ­ism of the veg­etable king­dom, char­ac­ter­is­ti­cally hav­ing cel­lu­lose cell walls, grow­ing by syn­the­sis of inor­ganic sub­stances and lack­ing loco­mo­tion.” Good­ness gra­cious, look it up. We all have dic­tio­nar­ies. And at that point we are see­ing the flaw in the law enforce­ment officer’s qual­i­fi­ca­tions. We ask, “Do you carry a micro­scope?” I’m not being ridicu­lous. A plant mate­r­ial (veg­etable mate­r­ial) con­tains cells. With­out a micro­scope one can not see those cells. So what expe­ri­ence does the offi­cer have in con­duct­ing micro­scopic analy­sis? If none, then we must con­clude that he does not have the req­ui­site expe­ri­ence, train­ing or skill needed to qual­ify as an expert in iden­ti­fy­ing plant mate­r­ial. Of course he can opine that the mate­r­ial he has seized lacks loco­mo­tion but so does road kill, so do rocks, so does a fake plas­tic plant. At a recent CLE sem­i­nar at which I taught, before my lec­ture I went out into the hotel where the CLE was held and broke off a small part of a dec­o­ra­tive plas­tic pot­ted plant. Dur­ing the lec­ture I pulled this pur­loined “veg­etable” mat­ter from my coat pocket and handed it to the man in front of me and asked him what kind of plant it was. He advised that he knew it was a plant but did not know what kind of plant.

Let us get beyond the issue of whether the law enforce­ment offi­cer car­ries a micro­scope as well as or instead of a gun. Let’s sup­pose that the offi­cer in Gar­nett actu­ally had a micro­scope on the wit­ness stand and was able to see cell struc­ture. Let’s give him the ben­e­fit of the doubt that he was not will­ing to give to our client. What would be the next step in this “iden­ti­fi­ca­tion” that is being con­ducted by the offi­cer who is qual­i­fied by train­ing, skill, edu­ca­tion, etc.. We know that mar­i­juana is a seed bear­ing plant. What did the offi­cer see that indi­cated to him that this seized mate­r­ial was a seed bear­ing plant? Did he see any seeds at all? And if he did see objects that he thought were seed what qual­i­fi­ca­tions does he have to rec­og­nize these objects as seeds. Has he con­ducted any research what-so-ever in the visual iden­ti­fi­ca­tion of mar­i­juana seeds? The answer to that ques­tion is absolutely not. In fact there was no research in this area at all any­where until about 2009 when Jenna Fussell, John Thorn­ton and Fred­eric White­hurst authored “The Visual Char­ac­ter­i­za­tion and Iden­ti­fi­ca­tion of Cannabis sativa (Mar­i­juana) Seeds pub­lished in the Jour­nal of Foren­sic Iden­ti­fi­ca­tion 59 (5) 2009. So that leaves out the seeds issue and the offi­cer can not tes­tify that he has skill in iden­ti­fy­ing mar­i­juana seeds. Very likely at this point we have an offi­cer on the stand or at the point of seizure who has no expe­ri­ence or train­ing or skill at “iden­ti­fy­ing” mar­i­juana based upon the char­ac­ter­is­tics we shall call “plant” and “seeds.”

Let’s give the offi­cer the ben­e­fit of the doubt that he did not give our clients. Let’s assume that he has con­ducted research and has the skills needed to iden­tify a mate­r­ial as a plant based upon an accepted def­i­n­i­tion of the word “plant” and he has con­ducted the research and has the skills needed to iden­tify the “seeds” that he sees as seeds from a mar­i­juana plant. So next we reach the issue of flow­ers. Mar­i­juana has flow­ers. Those of us who have been involved in legal cases where “mar­i­juana” was seized have a com­mon sense that mar­i­juana has flow­ers. So we must ask the offi­cer what expe­ri­ence and skill he has in “iden­ti­fy­ing” mar­i­juana flow­ers. And not nec­es­sar­ily even the flow­ers on the grow­ing plant but the flow­ers in the end prod­uct, that which on the street is rec­og­nized as the qual­ity prod­uct because of its high tetrahy­dro­cannabi­nol (THC) con­tent. Who wants leaves and stems and stalks when one can pur­chase the bud/flower? But what is a flower? Can the offi­cer iden­tify the parts of a flower? What skill and train­ing has he got to make him qual­i­fied to ren­der an opin­ion that the mate­r­ial he is observ­ing is even flower mate­r­ial much less mar­i­juana flower mate­r­ial? “I just know it when I see it” is a great response but the obvi­ous query is “Just what is it that you are see­ing and how does that com­pare with the struc­ture of the mar­i­juana flower?” In “The Struc­ture of Eco­nomic Plants”, Pro­fes­sor Her­man Hay­ward tells us that “Hemp is dioe­cious and the num­ber of sta­mi­nate and pis­til­late plants is rel­a­tively con­stant under nor­mal con­di­tions.” And describ­ing the sta­mi­nate inflo­res­cence and flower, Hay­ward tells us that ”the flow­ers develop in small, droop­ing, branched pan­i­cles, which arise in the axils of foliage leaves. The flow­ers of the pan­i­cle may occur singly on slen­der pedicels or in groups, and usu­ally the ter­mi­nal branches bear three flow­ers, a median one and two lat­er­als which are sub­tended by bracts or stip­ules. The indi­vid­ual flow­ers are petalous with a deeply parted calyx hav­ing five greenish-yellow or red lobes that are wide­spread at matu­rity. There are five sta­mens, and the anthers are sus­pended from long thread-like fil­a­ments.” This, of course describes the flow­ers in their grow­ing state, not in the state that one finds them on the street. The expe­ri­enced law enforce­ment offi­cer who has the skill, train­ing, knowl­edge, etc. to qual­ify him as an expert who can offer an opin­ion that what he is look­ing at is mar­i­juana flower, of course knows all of this and more and will be able to describe all of those parts that he is see­ing that fit Hayward’s very clear descrip­tion of grow­ing mar­i­juana plant flow­ers. If not then he does not have the req­ui­site expe­ri­ence, train­ing or skill. And if the offi­cer can rec­og­nize all the parts of mar­i­juana flower are present within the mate­r­ial he has seized and is call­ing mar­i­juana, we must still ask if he has the req­ui­site expe­ri­ence and train­ing to not only rec­og­nize the parts but tell us that no other plants has such parts, not no other plant he has seen, but no other plant. He has been asked to iden­tify the plant as mar­i­juana, mean­ing that he is say­ing that the plant is mar­i­juana to the exclu­sion of all other plants or at least a rea­son­able por­tion of the other plants on the planet.

This is not cannabis. It is Kenaf of Kanap that contaons no THC

This is not cannabis. It is Kenaf of Kanap that con­taons no THC

But again, let’s give this offi­cer the ben­e­fit of the doubt that he was not will­ing to extend to our clients and progress to the next level of inquiry about his skill and expe­ri­ence and train­ing. Mar­i­juana is a dicotyle­don as opposed to being a mono­cotyle­don. Does the offi­cer know what this means? Surely with the proper train­ing, skill and expe­ri­ence he does. In fact, if he remem­bers his ninth grade biol­ogy class he will real­ize what these terms mean. A cotyle­don is the part of the plant that nour­ishes the embry­onic plant until it can suc­ceed on its own. In North Car­olina, an agri­cul­tural state, all of us should know about open­ing up peanuts or lima beans. Two halves, fat leaves, inside of which exists a tiny lit­tle plant. (If you haven’t opened up a lima bean you haven’t lived. Go do it now.) Those two halves are cotyle­dons and because there are two of them we call plants with two of them, dicotyle­dons. Plants such as corn have only one lit­tle fat leaf, one cotyle­don, the ker­nel of corn, that feed the lit­tle corn plant until it grows into the stalk on which the corn is pro­duced. So, assum­ing the offi­cer remem­bers his ninth grade biol­ogy class, he will have the req­ui­site knowl­edge and skill needed to rec­og­nize if this plant is a dicotyle­don, which mar­i­juana is. But how many other plants are there that are dicotyle­dons? Does he know this? Can he prop­erly edu­cate a trier-of-fact to the poten­tial num­ber of plants that might be mis­taken for mar­i­juana based on the char­ac­ter­is­tics we have con­sid­ered thus far?

Please remem­ber that what the offi­cer seizes most often is crushed mate­r­ial, no longer hav­ing all the char­ac­ter­is­tics of the grow­ing plant. Dur­ing the prepa­ra­tion for dis­tri­b­u­tion, the flow­ers are left whole but the leaves are crushed into very small pieces. A “leaf archi­tec­tural analy­sis” is no longer pos­si­ble. Go out into the woods right now or out into your yard. Pick leaves off a num­ber of plants. Let them dry a bit and then crush them up. Ask your­self if you can then tell from which plant those crushed up leaves orig­i­nated. You might be able to dif­fer­en­ti­ate crush yard grass or corn leaves from oak leaves but can you dif­fer­en­ti­ate live oak leaves from red oak leaves from maple? Can you dif­fer­en­ti­ate oregano leaves from tea leaves just by look­ing at them? Does the offi­cer have the skill, train­ing and edu­ca­tion to do this? Hickey in the Jour­nal of Botany, 60(1): 17–33 (1973) describes for us this leaf archi­tec­tural analy­sis. Has the offi­cer the skill, knowl­edge or train­ing in this area? Leaves are clas­si­fied accord­ing to leaf ori­en­ta­tion, orga­ni­za­tion, shape, mar­gin, tex­ture, gland posi­tion, peti­ole, types of vena­tion, and ele­ments of tooth archi­tec­ture. When we fol­low Hicky we being to look at the form of leaf mar­gin; we see clas­si­fi­ca­tion con­cepts such as entire, lobed, toothed, cre­nate, erose, rev­o­lute or enrolled, sinuses, spac­ing and series. Does the offi­cer know what these con­cepts even mean? But wait! These are gen­er­ally not seized plants that we have in evi­dence but crushed up mate­r­ial. So how do we begin to con­duct Hickey’s sug­gested leaf archi­tec­tural analy­sis? What train­ing, expe­ri­ence or skill can the offi­cer tes­tify to to be qual­i­fied in the “iden­ti­fi­ca­tion” of mar­i­juana from a leaf archi­tec­tural analy­sis when the leaves are no longer in their nat­ural state? In his paper “Foren­sic Aspects of Cys­tolith Hairs of Cannabis and Other Plants”, Jour­nal of AOAC (Vol. 52, No.1, 1969), George Naka­mura noted that even with a micro­scopic analy­sis he could not dif­fer­en­ti­ate 82 dif­fer­ent seed bear­ing plants out of 31874 he con­sid­ered. He never con­sid­ered a leaf archi­tec­tural analy­sis because, obvi­ously once the leaves are crushed up into prod­uct for street sales, such an analy­sis would not be pos­si­ble. So how can a law enforce­ment offi­cer visu­ally iden­tify green veg­etable mate­r­ial as mar­i­juana even with a microscope?

The response this author has heard in the past has been that one need not be a botanist in order to under­stand botan­i­cal con­cepts. OK, but Rule of Evi­dence 702 now requires that

If sci­en­tific, tech­ni­cal or other spe­cial­ized knowl­edge will assist the trier-of-fact to under­stand the evi­dence or to deter­mine a fact in issue, a wit­ness qual­i­fied as an expert by knowl­edge, skill, expe­ri­ence, train­ing, or edu­ca­tion, may tes­tify thereto in the form of an opin­ion, or oth­er­wise, if all of the fol­low­ing apply:
1. The tes­ti­mony is based upon suf­fi­cient facts or data.
2. The tes­ti­mony is the prod­uct of reli­able prin­ci­ples and meth­ods.
3. The wit­ness has applied the prin­ci­ples and meth­ods reli­ably to the facts of the case.

Cannabis Sativa L. is an extremely com­plex organ­ism which shares many fea­tures in com­mon with many other plants. We are informed by Ivan Ross in “Med­i­c­i­nal Plants of the World, Chem­i­cal Con­stituents, Tra­di­tional and Mod­ern Med­i­c­i­nal Uses” Vol­ume 3 that over 500 known chem­i­cal con­stituents have been iso­lated and iden­ti­fied in mar­i­juana. We are informed by Robert Thorne in “How Many Speci­cies of Seed Plants Are There”, Taxon 51, August 2002, that there are con­ser­v­a­tively 258,650 known seed bear­ing plants. If we refer to the Ency­clo­pe­dia Bri­tan­nica we see esti­mates of 200,000 to 500,000 known seed bear­ing plants. When we iden­tify mar­i­juana we are say­ing that the data we have col­lected is suf­fi­cient to iden­tify the mate­r­ial to the exclu­sion of any other plant or at least to the exclu­sion of a rea­son­able num­ber of other plants. We are say­ing that the com­bi­na­tion of fea­tures we have observed are only seen in mar­i­juana. Does the iden­ti­fy­ing offi­cer have the skill, edu­ca­tion, knowl­edge or train­ing to be able to artic­u­late why the fea­tures he is observ­ing would not be observed in that com­bi­na­tion in any other plant mate­r­ial? And has he col­lected suf­fi­cient data to deter­mine the mate­r­ial he has iden­ti­fied as mar­i­juana is in fact mar­i­juana. In Gar­nett the offi­cer sim­ply looked at the green veg­etable mate­r­ial and did not deter­mine if it was a plant mate­r­ial in a reli­able man­ner. The opin­ion does not address the pro­to­col the offi­cer fol­lowed in deter­min­ing if the mate­r­ial was a seed bear­ing mate­r­ial, if the mate­r­ial was a flow­er­ing plant or a dicotyle­don, if the offi­cer con­ducted a leaf archi­tec­tural analy­sis or an analy­sis of any poten­tial flower mate­r­ial in the evi­dence. In the words of Rule 702, is it true that “The tes­ti­mony is based upon suf­fi­cient facts or data?”, to say that this com­bi­na­tion of fea­tures would be observed in no other plant. And what fea­tures are we talk­ing about. Green veg­etable mate­r­ial? Unique odor?

How does one deter­mine if the odor the offi­cer is detect­ing is unique to mar­i­juana? Does the offi­cer have the expe­ri­ence, train­ing, skills or edu­ca­tion to say that the odor that he is detect­ing (what­ever that is) is unique to mar­i­juana. The offi­cer may very well tes­tify that he has never smelled any­thing else that smells like mar­i­juana. How does one test that state­ment? The officer’s olfac­tory senses can be tested but have they been tested? Is there suf­fi­cient data from the olfac­tory analy­sis from the offi­cer that the court can deter­mine the admis­si­bil­ity of the evi­dence or even the weight of the evi­dence? In other words, is the tes­ti­mony the prod­uct of reli­able prin­ci­ples and meth­ods and did the offi­cer prop­erly apply those prin­ci­ples and meth­ods to the facts of the case. It would be vir­tu­ally impos­si­ble for the trial court to test the officer’s olfac­tory senses and I am not aware of any police agency that tests for olfac­tory senses in its offi­cers. Flem­ming et. al. in “Top­ics in Het­e­ro­cyclic Chem­istry”, Vol­ume 10, 1–42 (2007) inform us that there are 120 susquiter­penoid and ter­penoid com­pounds which com­pose the mate­ri­als which are detected as the “odor” of mar­i­juana. Any Google search of sites that pur­port to sell mar­i­juana or its seeds for pro­duc­tion adver­tise their prod­ucts accord­ing to effect on the body, appear­ance and odor. It is rea­son­able to believe that envi­ron­men­tal impact will alter the qual­i­ta­tive and quan­ti­ta­tive enve­lope of the chem­i­cal com­po­si­tion of Cannabis Sativa L.. One need only look at the results of the use of such prod­ucts as Mir­a­cle Grow on a home gar­den to real­ize that envi­ron­men­tal fac­tors play a very big role in plant com­po­si­tion. The Court may very well be left with no way of deter­min­ing if the odor detec­tion part of the mar­i­juana analy­sis pro­to­col is reli­able? And the Court would do well to ask this, “Why does or did the North Car­olina Crime Lab ever con­duct a mar­i­juana analy­sis if an offi­cer need only smell the green veg­etable mate­r­ial to prove it is mar­i­juana”? It defies logic.

D.W. John­son and J.W. Gunn in Dan­ger­ous Drugs, Adul­ter­ants, Dilu­ents and Decep­tion in Street Sam­ples” , Jour­nal of Foren­sic Sci­ence, 1972 found that up to 20% of the alleged mar­i­juana sam­ples sub­mit­ted to the US Depart­ment of Justice’s Bureau of Nar­cotics and Dan­ger­ous Drugs lab were not in fact mar­i­juana. Though I have not found any more recent data describ­ing the occur­rence of coun­ter­feit mar­i­juana being sub­mit­ted by law enforce­ment offi­cers as mar­i­juana to crime labs, North Car­olina Gen­eral Statutes accept that coun­ter­feit con­trolled sub­stances are in fact a very real prob­lem and are deemed to be ille­gal. We must ask how many times law enforce­ment offi­cers sub­mit mate­r­ial they sus­pect of being mar­i­juana to a crime lab and the mate­r­ial is found to not be mar­i­juana. In other words, has any­one deter­mined the error rate for an analy­sis of sus­pected mar­i­juana con­ducted by a law enforce­ment offi­cer visu­ally and chem­i­cally ana­lyzed mar­i­juana or worse, sim­ply visu­ally deter­min­ing the green veg­etable mate­r­ial is marijuana.

In sum­mary, Fletcher meets Daubert (or in North Car­o­line Rule of Evi­dence 702) and leaves us very only with ques­tions, ques­tions that should be answered before a North Car­olina Court can accept an officer’s “iden­ti­fi­ca­tion” of mar­i­juana sim­ply by a visual or olfac­tory test. If a defen­dant wishes to stip­u­late that the mate­r­ial that was in his pos­ses­sion was mar­i­juana then the ques­tion is closed. How­ever, do we know even if defen­dants are capa­ble of deter­min­ing that they have not been sold coun­ter­feit mate­r­ial? Remem­ber that there is a new test of reli­a­bil­ity now in North Car­olina which must be sat­is­fied before sci­en­tific, tech­ni­cal or other spe­cial­ized knowl­edge will be admit­ted as evidence.

If sci­en­tific, tech­ni­cal or other spe­cial­ized knowl­edge will assist the trier-of-fact to under­stand the evi­dence or to deter­mine a fact in issue, a wit­ness qual­i­fied as an expert by knowl­edge, skill, expe­ri­ence, train­ing, or edu­ca­tion, may tes­tify thereto in the form of an opin­ion, or oth­er­wise, if all of the fol­low­ing apply:
1. The tes­ti­mony is based upon suf­fi­cient facts or data.
2. The tes­ti­mony is the prod­uct of reli­able prin­ci­ples and meth­ods.
3. The wit­ness has applied the prin­ci­ples and meth­ods reli­ably to the facts of the case.”
Street and/or court­room “iden­ti­fi­ca­tion” of green veg­etable mate­r­ial being mar­i­juana in no way meets that test.

Through­out this series of posts we have exam­ined the Thorton-Nakumura pro­to­col that is used through­out the United States for the pros­e­cu­tion of ille­gal pos­ses­sion mar­i­juana. A fair exam­i­na­tion of the ques­tion reveals that there is no valid­ity to the notion that the 3 test reg­i­men pro­duces a valid con­clu­sion that the unknown exam­ined in fact con­tains THC.

Here are those series of posts:

  1. What is the goal and the pur­pose of test­ing of unknowns gen­er­ally? How do we best design a test for marijuana?
  2. How is most mar­i­juana test­ing con­ducted in the United States?
  3. What is micro­scopic mor­pho­log­i­cal exam­i­na­tion? Is it a “good” test?
  4. What is the mod­i­fied Duquenois-Levine test? Is it a “good” test?
  5. What is Thin Layer Chro­matog­ra­phy? Is it a “good” test?
  6. Is the com­bi­na­tion of all three tests cre­ate a “good” test­ing scheme?
  7. Is there a bet­ter way to test for marijuana?

There is a bet­ter way: Mod­ern Instru­men­ta­tion

There is an instru­ment dri­ven way that is very spe­cific and pro­duces ver­i­fi­able data. This instru­ment dri­ven tech­nique has been val­i­dated and if use in the val­i­dated man­ner with a prop­erly trained oper­a­tor uti­liz­ing proper sam­ple col­lec­tion, proper sam­ple selec­tion, proper sam­ple prepa­ra­tion, per­fect instru­men­ta­tion, and legit­i­mate inter­pre­ta­tion of the data will arrive at a valid result. This mod­ern day instru­men­ta­tion is called the Gas Chro­matog­ra­phy with Mass Spec­trom­e­ter (GC-MS).

Gas Chromatography with Mass Spectrometer

Gas Chro­matog­ra­phy with Mass Spectrometer

When the lab­o­ra­tory ana­lyst is pro­fi­ciency tested on unknowns and then graded to see whether or not they can con­duct a proper analy­sis of an unknown, the ana­lyst uses GC-MS. When the lab­o­ra­tory wants to check that the known that they have pur­chased from a third party ven­dor that is used and needed in Thin Layer Chro­matog­ra­phy (TLC) to com­pare against the unknown, the lab­o­ra­tory requires that it be ver­i­fied by GC-MS.

So when the lab­o­ra­tory really wants to know or really needs to know whether or not some­thing con­tains delta 9 THC, it uses the most spe­cific device avail­able that pro­duces ver­i­fi­able data. The ver­i­fi­able data are the print­outs that result from the analy­sis. This is called a Total Ion Cur­rent (TIC) chro­matogram and the result­ing spec­trum that is com­pared against an adju­di­cated known that is pro­duced by the National Insti­tutes of Stan­dards and Tech­nol­ogy (NIST).

Here are other blog posts on the GC-MS process:

  1. Introduction-The dif­fer­ent con­fig­u­ra­tions and the Elec­tron Impact process
  2. What types of mass ana­lyz­ers are there?
  3. What type of detec­tors are there?
  4. What types of analy­sis can be done?
  5. How do you read the output?
  6. How do they come to a qual­i­ta­tive mea­sure using software?
  7. How do they quan­ti­tate the results?
  8. Do you need chro­matog­ra­phy if you are using Mass Spectrometry?
  9. Other top­ics of inter­est about GC-MS

In the analy­sis of  unknowns that are seized, the process of deriva­ti­za­tion can be used to volatilize the sam­ple for intro­duc­tion the the GC-MS. For exam­ple, the ana­lyst can use MTBSTFA (N-methyl-N-(tert-butyldimethylsilyl)-trifluoroacetamide) or BSTFA/TMCS N,O-bis (trimethylsi­lyl) trifluoroacetamide/ Trimethylchlorosi­lane or MSTFA: N-methyl-N-trimethylsilyltrifluoroacetamide to deriv­a­tive the unknown.

A pop­u­lar tech­nique includes:

A sam­ple prepa­ra­tion that includes 500 mg of dry and homog­e­nized herbal cannabis are extracted with 5 ml methanol : chlo­ro­form (9:1 v/v) by the fol­low­ing pro­ce­dure: 10 sec­onds on a vor­tex, 15 min. ultra­sonic bath includ­ing again vor­tex­ing after 5, 10 and 15 min­utes, then cen­trifu­ga­tion. The sam­ple then needs to go through decar­boxy­la­tion. A 200 μl of the above extract are trans­ferred into a deriva­ti­za­tion ves­sel. The sol­vent is evap­o­rated under nitro­gen gas to dry­ness. The sam­ple is decar­boxy­lated for 15 min­utes at 210°C. The residue is dis­solved in 200 μl methanol : chlo­ro­form (9:1 v/v). The prepa­ra­tion of the final solu­tion next involves tak­ing the above decar­boxy­la­tion solu­tion and dilut­ing it with methanol by a fac­tor of 100 (in two steps, each 100 μl + 900 μl) and is then used for the analysis.

Also, THC is very much amenable to Head­space Solid Phase Microex­trac­tion (HS-SPME). It is a non-derivatization-based tech­nique where there is a extrac­tion from the solid dose itself. It is a more direct mea­sure as one is not chem­i­cally chang­ing the sam­ple. Dif­fer­ent types of fil­ters and fibers can be used such as poly­di­methyl­silox­ane 100 μm.

GC based test­ing for THC has been dis­cussed in  the sci­en­tific lit­er­a­ture since 1971. In 1991 the UNDCP study dis­cussed both GC-based meth­ods and HPLC meth­ods to iden­tify THC.

Other for­eign gov­ern­ments require much more test­ing than we do here in the United States. For exam­ple, Canada requires four tests includ­ing two instrument-based analy­sis that pro­duce ver­i­fi­able infor­ma­tion such as a spectrum.

There is a bet­ter way, but it is not used. This cer­tainly can­not be fair and just. It is time that we as cit­i­zens demand proof in the court­room and end the myth of spe­cific iden­ti­fi­ca­tion of mar­i­juana in crim­i­nal courtrooms.

The way to close this series is by an apt obser­va­tion made by Dr. Fred­eric White­hurst, PhD JD who wrote:

Many a defense attor­ney has looked in amaze­ment at the court­room “iden­ti­fi­ca­tion” of Cannabis Sativa L. by a law enforce­ment offi­cer sport­ing a gilded pot metal badge and a high school diploma who has writ­ten a report that would turn a ninth grade Eng­lish teacher’s hair gray and deny that same offi­cer a high school diploma. Obvi­ously the award­ing of a Basic Law Enforce­ment Train­ing (BLET) diploma ele­vates such high school grad­u­ates to the doc­toral level in botany, enabling them to con­duct a visual leaf archi­tec­tural analy­sis and the court accepted alchemy of the Duquenois Levine test to reach a con­clu­sion that the green veg­etable mate­r­ial in that lit­tle baggy can be iden­ti­fied as mar­i­juana to the exclu­sion of all other plant material.

The mod­ern day pros­e­cu­tion for the unlaw­ful pos­ses­sion of mar­i­juana is based upon a three test régime involv­ing micro­scopic mor­pho­log­i­cal exam­i­na­tion, mod­i­fied Duquenois-Levine col­ori­met­ric test­ing, and Thin Layer Chro­matog­ra­phy. Each of these three tests are non-specific for THC which is the phar­ma­co­dy­nam­i­cally active ingre­di­ent which makes mar­i­juana ille­gal. The ques­tion becomes is this three test bat­tery col­lec­tively con­clu­sively spe­cific to arrive at a valid con­clu­sion that the unknown seized and tested is in fact mar­i­juana (con­tains THC) and there is no pos­si­bil­ity of a false positive?

In this series of posts we are going to exam­ine this seemly sim­ple question:

  1. What is the goal and the pur­pose of test­ing of unknowns gen­er­ally? How do we best design a test for marijuana?
  2. How is most mar­i­juana test­ing con­ducted in the United States?
  3. What is micro­scopic mor­pho­log­i­cal exam­i­na­tion? Is it a “good” test?
  4. What is the mod­i­fied Duquenois-Levine test? Is it a “good” test?
  5. What is Thin Layer Chro­matog­ra­phy? Is it a “good” test?
  6. Is the com­bi­na­tion of all three tests cre­ate a “good” test­ing scheme?
  7. Is there a bet­ter way to test for marijuana?

Part 6: Is the com­bi­na­tion of all three tests cre­ate a “good” test­ing scheme?

The gov­ern­ment would choose to argue that the com­bi­na­tion of these three tests results in a valid con­clu­sion that this tested unknown is mar­i­juana (con­tains THC). All  three roads leads, so they say, lead to THC. The more sophis­ti­cated ver­sion of this argu­ment is the Venn dia­gram. Per­haps some­thing like the below:

Venn diagram that the prosecution argues
Venn dia­gram that the pros­e­cu­tion argues

This is sim­ply not proven or sup­ported by the pub­lished empir­i­cal research. Even if it were true there is no evi­dence how wide or big that “D” area is. It could be very wide and large con­tain­ing many com­pounds such as this below exam­ple below where the result of this type of test­ing would be a pos­i­tive for O, T, H, P, M, A, B, X, K, Y and not for B alone:

What could be in there? We don't know.
What could it be? We don’t know.

There is no evi­dence that these dis­tinct tests ever intercept.

Maybe these three tests have nothing in common
Maybe these three tests have noth­ing in common

While in ear­lier posts, we have exam­ined the pro­pri­ety and suit­abil­ity of these three sep­a­rate and dis­tinct tests in the iden­ti­fi­ca­tion of THC, we can­not prop­erly judge them in iso­la­tion. We must remem­ber and in fair­ness this is a 3 test process and technique.

There are no mean­ing­ful or robust stud­ies pub­lished that truly val­i­dates this three test pro­ce­dure as result­ing in a spe­cific qual­i­ta­tive mea­sure for THC.

What com­pounds the issue of the pos­si­bil­ity of error is that as the ana­lyst is not using the same sam­ple through­out each of these 3 tests. It is in fact, three dif­fer­ent sam­ples of the orig­i­nal unknown. The ana­lyst pre­sumes that the unknown is homoge­nous. Fur­ther assumed is that the sam­pling and the sam­ple selec­tion of the unknown con­ducted by the ana­lyst result in iden­ti­cal homo­ge­neous sam­ples. These are not jus­ti­fied sci­en­tific assump­tions (An assump­tion is not drawn from evi­dence; it is a hypoth­e­sis {my assump­tion can be tested by look­ing at the dic­tio­nary}. A pre­sump­tion implies a basis in evi­dence {the legal pre­sump­tion of innocence})

Each of these tests are wholly destruc­tive in nature by their very process.

  • Just because the sought after fea­tures of the micro­scopic mor­pho­log­i­cal exam­i­na­tion were present in the first sam­ple doesn’t nec­es­sar­ily mean that that this sam­ple will con­tain the sought after fea­tures in the mod­i­fied Duquenois-Levine and the TLC exam­i­na­tion because they are not exam­ined for in this sample.
  • Just because the sought after fea­tures of the mod­i­fied Duquenois-Levine exam­i­na­tion were present in the sec­ond sam­ple doesn’t nec­es­sar­ily mean that the fea­tures sought after in the micro­scopic mor­pho­log­i­cal exam­i­na­tion are there (because it was not exam­ined) and the sought after fea­tured of the TLC exam­i­na­tion are there (because it was not exam­ined) in this sec­ond sample.
  • Just because the sought after fea­tures of the TLC exam­i­na­tion were present in the third sam­ple doesn’t nec­es­sar­ily mean that the fea­tures sought after in the micro­scopic mor­pho­log­i­cal exam­i­na­tion are there (because it was not exam­ined) and the sought after fea­tured of the mod­i­fied Duquenois-Levine exam­i­na­tion are there (because it was not exam­ined) in this third sample.

All pros­e­cu­tions for the unlaw­ful pos­ses­sion of mar­i­juana requires as an essen­tial ele­ment of the crime for the gov­ern­ment to advance proof that the unknown sub­mit­ted for test­ing is in fact mar­i­juana (con­tains THC). Can the gov­ern­ment actu­ally do that based upon its typ­i­cal test­ing method?

In this series of posts we are going to exam­ine this seemly sim­ple question:

  1. What is the goal and the pur­pose of test­ing of unknowns gen­er­ally? How do we best design a test for marijuana?
  2. How is most mar­i­juana test­ing con­ducted in the United States?
  3. What is micro­scopic mor­pho­log­i­cal exam­i­na­tion? Is it a “good” test?
  4. What is the mod­i­fied Duquenois-Levine test? Is it a “good” test?
  5. What is Thin Layer Chro­matog­ra­phy? Is it a “good” test?
  6. Is the com­bi­na­tion of all three tests cre­ate a “good” test­ing scheme?
  7. Is there a bet­ter way to test for marijuana?

Part 5: What is Thin Layer Chro­matog­ra­phy? Is it a “good” test?

Thin Layer Chro­matog­ra­phy test­ing

What is it?

Thin Layer Chro­matog­ra­phy (TLC) is a chro­mato­graphic tech­nique. It is a com­bi­na­tion of a col­ori­met­ric test and is mea­sured in terms of separation.

Mechan­i­cally how is it preformed?

TLC depends on the sta­tion­ary phase, often a glass plate coated with sil­ica (it must be prop­erly  des­ic­cated or dried) and the mobile phase which is com­prised of a sol­vent mix­ture made of toluene and diethy­lamine typ­i­cally. When placed on a prop­erly dried plate and using a prop­erly mixed and pre­pared sol­vent, the sol­vent (the mobile phase) will be wicked up by the sil­ica with cap­il­lary action and travel up the plate. A sam­ple from the unknown is selected. It is mashed up in some mechan­i­cal process and in some processes call for it to be dis­solved. It is placed in the des­ig­nated spot. A test is com­pared by per­form­ing the reac­tion of the plate of a known sam­ple from an adju­di­cated source. This com­par­i­son is made with the analyst’s eyes. There will be dif­fer­ent spots on the plate at dif­fer­ent posi­tions from the ori­gin with var­i­ous inten­si­ties of color. The height and the color change are visu­al­ized. The spots typ­i­cally need to be visu­al­ized with a chem­i­cal spray such as Fast Blue B Salt (50 mg in 20 ml of NaOH (0.1 N)) or par­tic­u­lar lighting.

How is the typ­i­cal crime lab­o­ra­tory ana­lyst trained to con­duct this?

Once again, the ana­lyst is not trained in the fun­da­men­tals of how or why this process works. If you handed them a pen and paper and ask them to dia­gram and explain with specifics the chro­mato­graphic process, they would likely be baf­fled. Also for­eign to them would be the specifics as to why the sol­vent used to elute up the plate has to be in a spe­cific ratio and not another. In essence, it is another sub­jec­tive test as it is based upon per­cep­tion of color by the ana­lyst and the per­cep­tion of this height devel­op­ing on the plate ver­sus the adju­di­cated known.

The ana­lysts are not aca­d­e­m­i­cally trained in the the­ory of this tech­nique of chro­matog­ra­phy, and are not taught about cross-reactivity and false pos­i­tives or other sources of errors.

Is this a ver­i­fi­able test?

Much like the mod­i­fied Duquenois-Levine test, TLC is poten­tially ver­i­fi­able. Dig­i­tal cam­eras exist. Pho­tographs can be taken. Heck, even video can be taken to show how it is con­ducted on a par­tic­u­lar sam­ple from the unknown and this com­par­i­son to the adju­di­cated known. How­ever, the mod­ern prac­tice is to not take a sin­gle pho­to­graph, to not take advan­tage of video tech­nol­ogy and pro­duce noth­ing ver­i­fi­able in court that the test was even con­ducted or that the analyst’s per­cep­tion of the change did hap­pen and was cor­rectly interpreted.

Again, there are no crime lab­o­ra­to­ries that I am aware of that use the ACE-V (Analy­sis, Com­par­i­son, Eval­u­a­tion, and Ver­i­fi­ca­tion) tech­nique that one would find in fin­ger­print iden­ti­fi­ca­tion with a dou­ble check in real time by a fel­low bench ana­lyst. In essence, it is checked one time, by one per­son with no dou­ble check by another, and noth­ing pro­duced that proves that the analy­sis was done or that the fea­tures that are reported as present were in fact objec­tively present.

Is there empir­i­cal valid­ity stud­ies that prove that this is a spe­cific and con­fir­ma­tory test?

Once again, this type of test­ing has not been proven to be a val­i­dated method to test specif­i­cally for THC. There are well-known and dis­cov­ered false pos­i­tives which includes cof­fee, basil and even tobacco products.

Once again, the same issues that are dis­cussed prove true with this test­ing. This TLC test is pre­formed on a totally dif­fer­ent sam­ple from the unknown that is not sub­jected to the micro­scopic mor­pho­log­i­cal exam­i­na­tion or the mod­i­fied Duquenois-Levine col­ori­met­ric test.

In this series of posts we are going to exam­ine this seemly sim­ple question:

  1. What is the goal and the pur­pose of test­ing of unknowns gen­er­ally? How do we best design a test for marijuana?
  2. How is most mar­i­juana test­ing con­ducted in the United States?
  3. What is micro­scopic mor­pho­log­i­cal exam­i­na­tion? Is it a “good” test?
  4. What is the mod­i­fied Duquenois-Levine test? Is it a “good” test?
  5. What is Thin Layer Chro­matog­ra­phy? Is it a “good” test?
  6. Is the com­bi­na­tion of all three tests cre­ate a “good” test­ing scheme?
  7. Is there a bet­ter way to test for marijuana?

Part 4: What is the mod­i­fied Duquenois-Levine test? Is it a “good” test?

Mod­i­fied Duquenois-Levine test­ing

What is it?

It is tech­ni­cally referred to as a col­ori­met­ric test. In short, it is a color test. A totally dif­fer­ent selec­tion of the unknown is sam­pled and sub­jected to this test. A reagent is added to the unknown. The reagent is made up of a com­bi­na­tion of vanillin, acetalde­hyde, and ethanol in a spe­cific ratio of these com­po­nent materials.

Mechan­i­cally how is it preformed?

This totally sep­a­rate sam­ple from the unknown is placed into typ­i­cally a test tube. A cer­tain amount of the Duquenois reagent is added (typ­i­cally about 10 drops). The tube is closed. The tube is agi­tated (shaken) for an unspec­i­fied period. The tube is reopened. Con­cen­trated hydrochlo­ric acid is then added (usu­ally about 20 drops). The tube is closed. It is agi­tated (shaken) again. Any color change is then noted. The tube is reopened. Chlo­ro­form is added. The tube is closed. It is again agi­tated (shaken) or vor­texed (mixed). The ana­lyst is look­ing for a color change (thought to be vio­let or pur­ple) and a sep­a­ra­tion into two layers.

This is the end result of the modified Duquenois-Levine test. This picture is after the HCl and the chloroform are added. There is a deep purple color at the top and then a pink color at the bottom.

This is the end result of the mod­i­fied Duquenois-Levine test. This pic­ture is after the HCl and the chlo­ro­form are added. There is a deep pur­ple color at the top and then a pink color at the bottom.

How is the typ­i­cal crime lab­o­ra­tory ana­lyst trained to con­duct this test?

They are trained in the process and the steps in the mechan­i­cal process. No crime lab­o­ra­tory ana­lyst is ever instructed by a doc­toral level ana­lyt­i­cal chemist as to why this process results in any sort of color change or the way the phys­i­cal sep­a­ra­tion occurs in the col­ors with the addi­tion of chlo­ro­form or why it these changes hap­pen at all.

In essence, it is a sub­jec­tive test as it is based upon per­cep­tion of color by the ana­lyst and the per­cep­tion of this devel­op­ing of a sep­a­rate layer once chlo­ro­form is added.

It is a sec­ond sam­ple that is tested. Prior to the test­ing by mod­i­fied Duquenois-Levine tech­nique, there is no micro­scopic mor­pho­log­i­cal exam­i­na­tion con­ducted on this sam­ple. There is no TLC analy­sis on this sample.

It is tested one time and then dis­carded. No other test­ing is per­formed on this sample.

The ana­lysts are not aca­d­e­m­i­cally trained in the the­ory of the reagent use, and are not taught about cross-reactivity and false pos­i­tives or other sources of errors.

Is this a ver­i­fi­able test?

Poten­tially, yes. Dig­i­tal cam­eras exist. Pho­tographs can be taken just like the one above. Heck, even video can be taken to show how it is con­ducted on a par­tic­u­lar sam­ple from the unknown. How­ever, the mod­ern prac­tice is to not take pho­tos. They video­tape noth­ing.  The lab­o­ra­tory pro­duces noth­ing ver­i­fi­able in court that the test was even con­ducted or that the analyst’s per­cep­tion of the change in color did hap­pen and was cor­rect or that there was this sep­a­ra­tion once the chlo­ro­form is added.

Again, there are no crime lab­o­ra­to­ries that I am aware of that use the ACE-V (Analy­sis, Com­par­i­son, Eval­u­a­tion, and Ver­i­fi­ca­tion) tech­nique that one would find in fin­ger­print iden­ti­fi­ca­tion with a dou­ble check in real time by a fel­low bench ana­lyst. In essence, it is checked one time, by one per­son with no dou­ble check by another, and noth­ing pro­duced that proves that the analy­sis was done or that the fea­tures that are reported as present were in fact objec­tively present.

Is there empir­i­cal valid­ity stud­ies that prove that this is a spe­cific and con­fir­ma­tory test?

Nope. In fact the empir­i­cal stud­ies clearly show the oppo­site. It is not spe­cific for THC. The reac­tion is not unique to THC. In fact, the stud­ies clearly show that it is not even mean­ing­fully selec­tive for THC. Chi­nese moth­er­wort if tested, by this method will turn vio­let. But that is not all. There are a great many plants that yield sim­i­lar color results when Duquenois-Levine test­ing is applied. Yet ana­lysts are taught that is color change is diag­nos­tic of THC. Naka­mura him­self pub­lished and acknowl­edges that M. J. de Faubert Maun­der listed 25 species of plants which exhib­ited vio­let to pur­ple col­ors in the Duqenois test, and were extractable in chlo­ro­form, which is the dis­tin­guish­ing fea­tures of the Duquenois Levine test. (502) Naka­mura tested 23 of those species noted by M. J. de Faubert Maun­der. Using the Duquenois-Leine test, he found that the vio­let to pur­ple color when test­ing the leaves of the fol­low­ing (502):

  • cof­fee
  • a species of gum copal called Caplafer conjugata
  • gum Kawri
  • wood sage
  • Thuja occi­den­talis
  • San­darac

He pub­lished that he tested and found vio­let to pur­ple color reac­tions using the Duquenois-Levine test in other plants as well, and not just the leafy por­tions of these plants. (502). That list includes:

  • cala­mus
  • cul­ver root
  • gin­ger
  • gum animi
  • gum copal
  • gum myrrh
  • henna
  • let­tuce opium
  • san­dal wood
  • tolu
  • wood betony
  • liquorice
  • nut­meg
  • poi­son flag

The United Nations study on mar­i­juana con­cludes that Duquenois-Levine test on sub­stances com­pletely unre­lated to mar­i­juana will give false pos­i­tives too. This includes Arthemisia Gran­cunulus, Euca­lyp­tus Glab­u­lus Labill and sev­eral other herbs that are very com­mon such as rose­mary, thyme, and sage.

None of these items above con­tain THC. Yet, they fea­ture these color changes and some even have the sep­a­ra­tion when chlo­ro­form is added that the ana­lysts are trained to look for when apply­ing these reagents.

While we have exam­ined the pro­pri­ety and suit­abil­ity of this mod­i­fied Duquenois-Levine test­ing in the iden­ti­fi­ca­tion of THC, we can­not judge this Duquenois-Levine test in iso­la­tion. We must remem­ber and in fair­ness this is a 3 test process and tech­nique. What com­pounds the issue of the pos­si­bil­ity of error is that as the ana­lyst is not using the same sam­ple through­out each of these 3 tests. It is in fact, three dif­fer­ent sam­ples are taken of the unknown and each test gets its own sam­ple with no sam­ple receiv­ing the ben­e­fit of all three tests. The ana­lyst pre­sumes that the sam­pling and the sam­ple selec­tion of the unknown is homo­ge­neous mean­ing that each sam­ple will be exactly iden­ti­cal. This is not a jus­ti­fied sci­en­tific assump­tion (An assump­tion is not drawn from evi­dence; it is a hypoth­e­sis {my assump­tion can be tested by look­ing at the dic­tio­nary}. A pre­sump­tion implies a basis in evi­dence {the legal pre­sump­tion of inno­cence}) Just because the sought after fea­tures of the micro­scopic mor­pho­log­i­cal exam­i­na­tion were present in the first sam­ple, that doesn’t nec­es­sar­ily mean that it will be present in the next sam­ple that the ana­lyst now sub­jects to mod­i­fied Duquenois-Levine testing.

Naka­mura tested and found that mace and nut­meg, which yielded col­ors sim­i­lar to that obtained with mar­i­juana with the Duquenois-Levine test, could be “cred­i­bly con­fused” with mar­i­juana based on micro­scopic appear­ance. (502)

In 2006, more than 829,000 peo­ple were arrested in this coun­try for marijuana-related offenses alone. Since 1937 with the pas­sage and adop­tion of the Mar­i­huana Tax Act, mar­i­juana has been effec­tively pro­hib­ited in the United States.

Lit­er­ally mil­lions upon mil­lions of peo­ple have been accused of pos­sess­ing mar­i­juana. The ques­tion becomes are these con­vic­tions sci­en­tif­i­cally supported?

In this series of posts we are going to exam­ine this seemly sim­ple question:

  1. What is the goal and the pur­pose of test­ing of unknowns gen­er­ally? How do we best design a test for marijuana?
  2. How is most mar­i­juana test­ing con­ducted in the United States?
  3. What is micro­scopic mor­pho­log­i­cal exam­i­na­tion? Is it a “good” test?
  4. What is the mod­i­fied Duquenois-Levine test? Is it a “good” test?
  5. What is Thin Layer Chro­matog­ra­phy? Is it a “good” test?
  6. Is the com­bi­na­tion of all three tests cre­ate a “good” test­ing scheme?
  7. Is there a bet­ter way to test for marijuana?

Part 2: How is most mar­i­juana test­ing con­ducted in the United States?

So what hap­pens in Amer­ica, here, now and today in the iden­ti­fi­ca­tion of marijuana?

  • Are the tests pre­sump­tive or confirmatory?
  • Are the meth­ods used verifiable?
  • Does it result in a spe­cific or selec­tive identification?

Let’s take a look at how mar­i­juana is tested in the United States today in the super-majority of cases. It all begins with a police offi­cer or another per­son who seizes an unknown sub­stance. It’s green. It’s veg­etable like. It looks like mar­i­juana to them. Per­haps they do some sort of quick and dirty test on the side of the road using a fast col­ori­met­ric test and there is a change in color. This is a true unknown. We don’t know what it is. We can­not con­clude what it is based upon sim­ply look­ing at it with our own two eyes or even by our sense of smell. This is why we have foren­sic sci­en­tists and fur­ther test­ing because those types of obser­va­tions may lead to an improper or incor­rect result.

attempts at identifying marijuana at the gross or macro level can be misleading

Attempts at iden­ti­fy­ing mar­i­juana at the gross or macro level can be misleading

A basic fun­da­men­tal ques­tion becomes: What makes mar­i­juana ille­gal to pos­sess? What makes mar­i­juana ille­gal is that it con­tains the phar­ma­co­dy­nam­i­cally sub­stance known as Delta-9 Tetrahy­dro­cannabi­nol (THC).

Given that THC is what makes mar­i­juana ille­gal, then log­i­cally what would be best is if we were design tests that react exclu­sively to THC and noth­ing else and pro­duces ver­i­fi­able data that iden­ti­fies that the green veg­etable sub­stance in fact specif­i­cally con­tains THC.

Because we can’t tell what it is just by look­ing at it or just by smelling it, then we must sub­ject this unknown to test­ing. Largely in the United States there is a régime or process of how crime lab­o­ra­to­ries con­duct this test­ing. The triad of test­ing that is con­ducted are:

  1. Micro­scopic mor­pho­log­i­cal examination
  2. Mod­i­fied Duquenois-Levine testing
  3. Thin Layer Chromatography

This is gen­er­ally referred to as the Thorton-Nakumura protocol.

In order to answer our ques­tions of speci­ficity, ver­i­fi­ca­tion, and con­fir­ma­tory we need to look at each of these three test­ing tech­niques. In our next blog posts over the next sev­eral weeks each of these tests will be scrutinized.

The great Bard once penned:

All: God save your majesty!

Cade: I thank you, good people—there shall be no money; all shall eat and drink on my score, and I will apparel them all in one liv­ery, that they may agree like broth­ers, and wor­ship me their lord.

Dick: The first thing we do, let’s kill all the lawyers.

Cade: Nay, that I mean to do.

Henry The Sixth, Part 2 Act 4, scene 2, 71–78

Shakespeare’s char­ac­ter Dick the Butcher’s idea of a per­fect soci­ety was one where jus­tice pre­vailed because there were no lawyers. The trai­tor­ous Jack Cade had not so noble a rea­son for want­ing to get rid of all of the lawyers. He wanted to become the auto­crat in a quasi-communistic social rev­o­lu­tion. Cade alleges that all lawyers do is use laws and lan­guage set up by fel­low lawyers to oppress and ruin the life of every day man. There­fore, in his esti­ma­tion, no jus­tice results.

That is an extreme view.

I sug­gest that per­haps jus­tice best results when we have an edu­cated and orga­nized defense bar who is sci­en­tif­i­cally edu­cated in foren­sic sci­ence. As I have blogged before, the foren­sic sci­ence com­mu­nity as cur­rently prac­ticed in the United States today is very flawed. Extremely flawed. Fun­da­men­tally flawed. In some cases, if not the major­ity of cases, it is utterly unsci­en­tific. I agree with the sen­ti­ments that the crim­i­nal defense com­mu­nity shares a large por­tion of the blame. Many lawyers well before our times, let come into evi­dence prac­tices, tech­niques, and “the­o­ries” that had just but the very veneer of sci­ence and were, how­ever, any­thing but sci­en­tific and far from valid. This insti­tu­tional prop­a­ga­tion of error is a large hur­dle for many of us to over­come now where busi­ness as usual or sim­ple rep­e­ti­tion is some­how equated with validity.

So what are we to do?

Set up pro­grams where we edu­cate the defense bar.

I would like to high­light one of these: The Amer­i­can Chem­i­cal Soci­ety Hands-on Foren­sic Chro­matog­ra­phy course.

It is a five-days hands-on class con­ducted at Axion Ana­lyt­i­cal Lab­o­ra­to­ries, Inc. in Chicago, Illi­nois. This hands-on course is taught by three icons of chro­matog­ra­phy (Dr. Harold McNair, PhD, Dr. Lee Polite, PhD and Mr. Lew Fox) and two attor­neys who spe­cial­ize in eval­u­at­ing chro­matog­ra­phy and foren­sic sci­ence related cases (Justin J. McShane and Josh D. Lee).

 

The agenda includes:

Day 1

8:15am Reg­is­tra­tion and Snacks
8:30am Intro­duc­tion to Gas Chromatography
9:30am Inlet Sys­tems for Liq­uid Injections
10:30am Lab 1: GC Famil­iar­iza­tion and Parameters
11:45 Lab Review
12:00pm Lunch
1:00pm Fun­da­men­tals of Sep­a­ra­tion — Resolution
2:30pm GC The­ory
3:30pm Cap­il­lary Columns
4:30pm Lab 2: Col­umn Installation
6:00pm End of Session
7:30pm Group Din­ner

Day 2

8:30am Quan­ti­ta­tive Analysis
9:30am Dis­cus­sion of QC in the Foren­sic World
10:30am Lab 3: GC Quantitation
12:00pm Lunch
1:00pm Lab Review
2:00pm Head­space GC
4:00pm Lab 4: Head­space Demo
5:00pm End of Session
6:30pm Axion Labs spon­sored Group Dinner

Day 3

8:30am GC-MS The­ory
10:30am LC-MS The­ory
12:00pm Lunch
1:00pm Labs 5–7: Wet Lab –Sam­ple Preparation/ Inte­gra­tion / GC-MS Instrumentation
3:00pm FID Detec­tor
4:00pm Dis­cov­ery
5:30pm End of Session
7:05pm Sport­ing event

Day 4

8:30am Ethics
9:30am Lab 8: Walk­ing Down a Case-How to Sort, Iden­tify and Exam­ine Data (Con­fronta­tion Clause)
11:00 am Gen­eral Review of Major Concepts
12:00pm Lunch
1:00pm Defenses That Work
3:00pm Lab 9: Trou­bleshoot­ing GC Problems
4:30pm Lab 10: Beers and Data Round­table (Bring your own data set eval­u­ate with your small group)
8:30pm End

 

Day 5

8:30am Trou­bleshoot­ing Lab Review
9:00am Cross exam­i­na­tion of an Analyst
10:30am Direct Exam­i­na­tion of an Expert
12:00pm Trou­ble Shoot­ing Lab Review
12:30pm Soft stop of the course with gen­eral discussion
2:00pm Hard Stop-End of Course

This class attracts full of attor­neys from all across the United States. In this class atten­dees not only are instructed in the class­room the­ory that under­lies chro­matog­ra­phy (both liq­uid and gas chro­matog­ra­phy) in gen­eral and the spe­cific the­o­ries that allow for head­space analy­sis and how Flame Ion­iza­tion Detec­tor, UV-DAD and Mass Spec­trom­e­try (EI and EC based) works, and get to see the instru­ments, but they also get to do the fol­low­ing with their own two hands and more:

Hands-on Lab 1

Instru­ment Famil­iar­iza­tion where the atten­dees pre­form direct injec­tions into a Gas Chromatograph-Flame Ion­iza­tion Detec­tor (GC-FID) using EtOH sam­ples, and learn to inter­pret GC-FID chro­matograms, import and manip­u­late a method, change the dif­fer­ent vari­ables on the GC-FID and elu­ci­date the results.

Hands-on Lab 2

The atten­dees on their own GC machine remove and install cap­il­lary columns and all of the com­po­nents of the injec­tor (septa, liner, gold seal, etc.), check for effi­cien­cies, res­o­lu­tion, the­o­ret­i­cal plates, and learn about split ver­sus split­less injec­tor settings.

Hands-on Lab 3

The atten­dees on their own GC machine estab­lish a cal­i­bra­tion curve from CRMs for EtOH. The atten­dees on their own GC machine ana­lyze the response and pro­gram a cal­i­bra­tion curve (exter­nal stan­dard) and also use the Inter­nal Stan­dard method to assure qual­ity. The atten­dees on their own GC machine estab­lish meth­ods and report­ing of this cru­cial part of testing.

Hands-on Lab 4

The atten­dees con­tinue to use Head­space Gas Chromatograph-Flame Ion­iza­tion Detec­tor and also use Gas Chro­matog­ra­phy Mass Spec­trom­e­try (GC-MS) sys­tem, and use an High Per­for­mance Liq­uid Chro­matog­ra­phy (HPLC) system.

Hands-on Lab 5

Sam­ple Preparation-sampling ver­sus sam­ple selec­tion is demon­strated. The atten­dees do their own Pipet­ting. The atten­dees do their own con­trol chart­ing. The atten­dees use vol­u­met­ric flasks. The atten­dees learn about pre-analysis error hands-on and how it affects quan­tifi­ca­tion. The atten­dees are intro­duced to issues of metrol­ogy and Uncer­tainty Measurement.

Hands-on Lab 6

Inte­gra­tion. The atten­dees on their own Chem­Sta­tion work­sta­tion learn about inte­gra­tion and how easy it is to manip­u­late the data. The atten­dees manip­u­late their own data. The atten­dees learn what to look for that shows that the data was manip­u­lated, and the atten­dees learn how to manip­u­late data so that it is not dis­cov­er­able with­out the raw com­puter data in the soft­ware files.

Hands-on Lab 7

GC-MS Instru­men­ta­tion. The atten­dees see how easy life is for an ana­lyst. The atten­dees get to see the analy­sis of the raw data on a GC-MS and dis­cover how a true novice can turn into an “expert” with a sim­ple push of a but­ton. The atten­dees see with their own two eyes the “hid­den” data that the Gov­ern­ment and its lab­o­ra­tory doesn’t want any­one to see that will reveal the truth that the sup­posed “gold stan­dard” that pro­vides for the alleged “unequiv­o­cal iden­ti­fi­ca­tion” of test­ing of unknowns that is GC-MS is not perfect.

Hands-on Lab 8

How to Obtain Discovery/Walking Down a Case/Defenses that Win-Advanced Issue Spot­ting: In this lab, the best prac­tices in how to obtain dis­cov­ery with a spe­cial empha­sis on how to strate­gi­cally and prac­ti­cally build a record so that the atten­dees can get the raw data in its un-manipulated raw com­puter form and also in its print form. The atten­dees go through an actual case that was lit­i­gated that fea­tured experts on both sides, and issue spot all of the prob­lems with the dis­cov­ery and the data as it was presented.

Hands-on Lab 9

Trou­bleshoot­ing. The atten­dees on their own GC machine run an unknown sam­ple which may result in some sort of “prob­lem” in the chro­matogram. The atten­dees issue spot the prob­lem and ratio­nal­ize what is wrong, and fix it.

Hands-on Lab 10

The atten­dees with their lab part­ners go over their own data set from real cases to fig­ure out what is wrong with the atten­dees’ local laboratory.

Grad­u­ates of the group include:

STATE LAST NAME FIRST NAME   STATE LAST NAME FIRST NAME
Alaska Slone Fred New Jer­sey Her­nan­dez Steven
Ari­zona St. Louis Joe New Jer­sey Levow Evan
Cal­i­for­nia Barba Manny New Mex­ico Frechette Rod­er­ick
Cal­i­for­nia Brehmer Jeremy Okla­homa Edge Bruce
Cal­i­for­nia Ganci Eric Okla­homa Fabian Stephen
Cal­i­for­nia Gore­lick Lynn Okla­homa Hosty Tom
Cal­i­for­nia Laun­dry Vir­ginia Okla­homa Lee (x5)
Josh D.
Cal­i­for­nia Mid­dle­brook Richard Okla­homa Pat­ter­son Clint
Cal­i­for­nia Moore Ron Okla­homa Sifers Jeff
Cal­i­for­nia Sturm Craig Ore­gon Carini, Jr. Peter
Cal­i­for­nia Tie­mann Roland Penn­syl­va­nia Bar­rouk Tim
Cal­i­for­nia Was­son James Penn­syl­va­nia Man­ches­ter Brian
Cal­i­for­nia Wap­ner Terry Penn­syl­va­nia McShane (x6)
Justin
Col­orado Bussey Tim Penn­syl­va­nia Sher­man Mike
Col­orado Cessna Christo­pher Ten­nessee Garza (x2)
Mar­cos
Col­orado Her­ringer William Ten­nessee May Roger
Col­orado Savela Jason Ten­nessee McK­in­ney Rob
Col­orado Orr Rhid­ian Ten­nessee Ryan Edward
Florida Kessler Mike Texas Bal­a­gia Jaime
Florida McIn­tosh Brett Texas But­ler Jim
Geor­gia Adams Clark Texas Boatwright Nicky
Geor­gia Bab­son Rocky Texas Case Kelly
Geor­gia Caron Brian Texas Cof­fey Mimi
Geor­gia Frye Kim Texas de la Paz Brent
Geor­gia Par­man Ann Texas del Cueto Andrew
Geor­gia Stein George Texas DeLuca Matt
Illi­nois Ram­sell Don­ald Texas Flood Tyler
Illi­nois Toney Sarah Texas Grant Dean­dra
Kansas Hul­nick Les Texas Hamil­ton Stephen
Louisiana Delatte (x2)
Glynn Texas Hunter David
Louisiana Bates, Jr. James Texas McK­in­ney Troy
Mary­land Alpert Andrew Texas Mur­phy Doug
Mary­land Bruck­heim Michael Texas Ray Ben­nie
Mary­land Stamm Lenny Texas Segura Anthony
Mass­a­chu­setts Ober­hauser Gre­gory Texas Stauf­fer Phil
Michi­gan Boyle Michael Texas Trichter (x2)
Gary
Min­nesota Ram­say Charles Texas Wilder Dou­glas
Mis­souri East­man Jef­frey Utah Schatz Jason
Mis­souri Holling­shead Jeremy Vir­ginia Keefer Bob
Mis­souri Ward Carl Vir­ginia Solak– (x2)
Michael
Nebraska Dowd­ing Steve Wash­ing­ton Calla­han Linda
Nebraska Island Bell Wash­ing­ton DeBray Ted
Nevada Hayes Dale West Vir­ginia Wag­ner Harley
New Hamp­shire Russ­man Ryan Wis­con­sin Stuck­ert Lau­ren
New Hamp­shire Tenn John

There have been 89 grad­u­ates to date.

The next class (which is full) will be in April is sched­uled to have the fol­low­ing folks:

Patrick Maher

MD

Hunter Bie­der­man

TX

Wayne R. Foote

ME

John Hun­sucker

OK

Andrew Mishlove

WI

Michael J Snure

FL

Clark Adams (2nd time through)

GA

Andrew Bucher

OH

N. Cole Williams

NC

Jay M. Tiftickjian

CO

Bruce Edge (2nd time through)

OK

Brent de la Paz (2nd time through)

TX

Paul Liam McGlone

VA

Kevin Leck­er­man

PA

Jon W Woolsey

CA

Gor­don Senerius

SC

Nico La Hood

TX

Bryan E DePowell

PA

Joseph Cit­ron

GA

John j Eastland

TX

Jonathon Rands

WA

Shawn Dor­ward

PA

Jared Bartell

CA

To insure jus­tice, we need to have an edu­cated defense bar. We need more sci­en­tific pro­grams like this one.

Hemol­y­sis is a par­tic­u­lar prob­lem in the proper quan­tifi­ca­tion of EtOH in a blood sample.

First let’s define what it is:

What does it look like?

Red blood cells with­out (left and mid­dle) and with (right) hemol­y­sis. If as lit­tle as 0.5% of the red blood cells are hemolyzed, the released hemo­glo­bin will cause the serum or plasma to appear pale red or cherry red in color. Note that the hemolyzed sam­ple is trans­par­ent, because there are no cells to scat­ter light.

If there is hemol­y­sis, it can be gross and very notice­able as it pro­duces a light pink hue to the blood instead of the cus­tom­ary red blown that we have all seen as it is taken from the arm or it can be slight. Most fail­ures in life and in processes are not typ­i­cally gross in nature.

When run­ning head­space gas chro­matog­ra­phy with flame ion­iza­tion detec­tor (HS-GC-FID), hemol­y­sis will always pro­duce a higher BAC result than if there was no hemol­y­sis. It has to do not so much with the sam­ple but rather the tech­nique of sam­ple intro­duc­tion mean­ing the head­space tech­nique itself.

Using the head­space tech­nique, we are not directly sam­pling the blood (the liq­uid phase) but instead the head­space above the liq­uid phase (the gaseous phase). As such, it is sub­ject and depen­dent to the prin­ci­ples of Henry’s Law. Henry’s Law states that at equi­lib­rium at a con­stant pres­sure, tem­per­a­ture and flow in a closed sys­tem, the amount in the head­space is pro­por­tional to the amount liq­uid phase. It is sta­tic and pre­dictable at equi­lib­rium. This rela­tion­ship is called the par­ti­tion ratio or K.

For exam­ple, based upon empir­i­cal and valid research we know that the par­ti­tion ratio of ethanol in a true aque­ous (water) sam­ple at 50 degrees is 1220:1.

Some folks mis­take the con­cept of par­ti­tion ratio. A par­ti­tion ratio can be thought of as the mea­sure­ment of resis­tance of the volatile organic com­pound to elute (escape) to the gaseous phase. The higher the par­ti­tion ratio the more resis­tance. The higher the par­ti­tion ratio (K), the more resis­tance there is for the VOC to escape into the gaseous phase and there­fore there will be less VOC present in the gaseous phase to examine.

What effects par­ti­tion ratio are a few fac­tors includ­ing but not lim­ited to Beta which for our pur­poses can be sim­pli­fied to the matrix that the liq­uid phase is. In short, the more stuff in the matrix, the higher the par­ti­tion ratio because there is more “stuff” that the VOC has to travel through to escape the gaseous phase. In inverse is also true. The less stuff, in the matrix, the lower the par­ti­tion ratio because there is less “stuff” that the VOC has to inter­act with and travel through to escape to the gaseous phase.

So, in incor­po­rat­ing the above we arrive at the fol­low­ing conclusions:

  1. The low­est par­ti­tion ratio pos­si­ble is in the case of aque­ous matrix that con­tains EtOH.
  2. The high­est par­ti­tion pos­si­ble is if the EtOH is a matrix with a lot of “stuff” (think blood).

There­fore, between the two, the higher EtOH con­cen­tra­tion reported will be in the one that is clos­est to the one that is most aque­ous like as the par­ti­tion ratio low­est in the aque­ous matrix.

Hemol­y­sis or the red blood cell burstis will change the par­ti­tion ratio to be closer to that of an aque­ous sam­ple. The result is lower par­ti­tion ratio and more EtOH in the head­space and more EtOH reported.

So if you had two aliquots from the same homog­e­nized blood sam­ple and there­fore would objec­tively have the same EtOH in aliquot one and aliquot 2, but before you ana­lyzed them you took aliquot 2 and hemolyzed the sam­ple, the hemolyzed aliquot 2 would report a higher BAC than that of aliquot 1. If there was direct liq­uid injec­tion, then we would expect they would actu­ally have the same BAC reported.

GC consumables and preventive maintenance

Lots of pros­e­cu­tors and police sci­en­tists like to present the Gas Chro­mato­graph (GC) and its results as totally infal­li­ble, not sub­ject to inter­pre­ta­tion, and never wrong.

While GC tech­nol­ogy is good (in fact if the analy­sis is con­ducted on a sta­ble and prop­erly main­tained and installed instru­ment using a val­i­dated method with a well-trained and well-educated true sci­en­tist oper­at­ing it is per­haps the best qual­i­ta­tive device avail­able to us now), it is not any­thing more than a machine. A fancy machine, but still a machine.

Like all machines, it breaks down.

Like all machines, it requires pre­ven­tive main­te­nance periodically.

Like all machines, it should be very strictly mon­i­tored to insure that it is pro­duc­ing cor­rect (valid) results.

There is a myth that I see in court and in tran­script after tran­script that this machine, the GC, reg­u­lates and mon­i­tors itself and if any­thing with this machine “goes wrong,” it will not pro­duce a result.

Horse hockey.

Pure horse hockey.

Just as your car will keep dri­ving along even if you ignore the need for oil changes, this machine, the GC, will keep on spit­ting out results even if not prop­erly main­tained. Notice I wrote that it would pro­duce results, but not that it would pro­duce the cor­rect (valid) results. There is a big dif­fer­ence between the two.

Just as your car will keep dri­ving on bald tires but it may not get you safely where you want to go on a rainy day, this machine, the GC will keep on spit­ting out results even if the direc­tions for best use are not fol­lowed. Notice I wrote that it would pro­duce results, but not that it would pro­duce the cor­rect (valid) results. There is a big dif­fer­ence between the two.

There is a dif­fer­ence between what could be done, what should be done and what is done dur­ing pre­ven­tive main­te­nance of a GC. The bot­tom line is that your lab­o­ra­tory should have a writ­ten pol­icy pro­ce­dures and instruc­tions as far as what must be done and what trig­gers it. It should always be a data dri­ven decision.

The best way to look at it is from a sys­tem point-of-view. Remem­ber this whole device is a won­der­fully com­pli­cated inter­ac­tion of var­i­ous parts that must come together per­fectly to get a valid result.

  1. I would start by look­ing at the Gas Cylin­ders. It is some­thing that is taken for granted, and by no means is a given. I took a tour of Air­Gas and it was fas­ci­nat­ing. An impor­tant con­cept is not sim­ply the gas purity as deliv­ered to the cylin­der, but also the “born on date” of the cylin­der itself. You can read about this impor­tant con­cept and poten­tial source of con­t­a­m­i­na­tion here. Born on Date– Not Just for Beer But Gas Cylin­ders Too
  2. Next I would look at whether or not there is a gas purifi­ca­tion sys­tem in place that is between the cylin­der and the instrument.
An example of an installed gas purification system

An exam­ple of an installed gas purifi­ca­tion system

There are dif­fer­ent types and per­for­mance char­ac­ter­is­tics of these gas puri­fiers (e.g., mois­ture puri­fiers to remove H2O, hydro­car­bon traps that remove volatile organic com­pounds, and oxy­gen scrub­bers to remove oxy­gen). The four shown con­sist of four gas puri­fiers to remove mois­ture, con­t­a­m­i­nat­ing organic sub­stances (VOCs) and oxy­gen. All of those can con­tribute to a “bad result.”

As shown above this sys­tem is con­fig­ured as con­tain­ing:
High Capac­ity Mois­ture trap
High Capac­ity Hydro­car­bon Trap
High Capac­ity Oxy­gen Trap
Back-up Indi­cat­ing Oxy­gen Trap

     3. Next, I would move on and look at the injec­tor port.

There are many dif­fer­ent parts of the injec­tor port.  It is the sep­tum, the inlet liner, the O-ring, and the golden seal that make up the injec­tor port con­sum­ables.  These are the con­sum­able por­tions (just in the injec­tor port) that must be replaced peri­od­i­cally to insure repro­ducibil­ity and robust­ness in the GC.  There is no hard and fast “must replace by” time for these items as deter­mined by the man­u­fac­turer or val­i­dated in a sci­en­tific sense.  Agi­lent rec­om­mends (key word) monthly replace­ment of these items for Volatile Organic Com­pound (VOCs) sam­pled by head­space, which is what we usu­ally do.  If a lab has such a pro­ce­dure it is out of the abun­dance of cau­tion not because the man­u­fac­turer says you must to do so.

The wear on these con­sum­ables depends upon the use of the GC (vol­ume of through­put) and the matrix influ­enced. If it is direct liq­uid injec­tion, then these con­sum­ables will have to be replaced a lot more fre­quently than with head­space. If it is not direct injec­tion and if the instru­ment, it is seek­ing to sam­ple only VOCs and the method always calls for head­space analy­sis (i.e., it is a ded­i­cated machine) then these items do not need to be replaced as fre­quently.  They do need to be replaced.  Gen­er­ally in indus­try there is a Good Lab­o­ra­tory Prac­tice instruc­tion that is set up with some sort of replace­ment sched­ule.  That being said we, in the foren­sic sci­ence arena, are in the non-regulated world (as opposed to EPA or FDA) and there­fore what I most see with labs all across the US they either have some sort of replace­ment sched­ule (which is very rare) or when they see abnor­mal results, then they con­sult with the man­u­fac­turer who teaches them the impor­tance of these consumables.

The func­tion of the inlet liner is to act as a last place of resort to trap any “junk” that would be intro­duced onto the col­umn and also to allow for homog­e­nized dis­tri­b­u­tion of the vapor to reduce ran­dom sam­pling error.  The pri­mary pur­pose of the golden seal is to seal the inlet to help main­tain the chro­mato­graphic con­di­tions and also pro­vide for uni­for­mity in the dis­tri­b­u­tion of the vapor in the inlet.  It is housed within the injec­tor por­tion of the instrument.

 

Fig­ure 1 above:  a label­ing of the injec­tor port housing

 

Fig­ure 2 above:  The new inlet liner with O-ring  (N.B., there are also many dif­fer­ent types and con­fig­u­ra­tions of inlet lin­ers that can affect the per­for­mance of the instru­ment and the valid­ity of the result both in terms of the qual­i­ta­tive mea­sure and the quan­ti­ta­tive mea­sure of that qual­i­ta­tive measure.)

 

Fig­ure 3 above:  Exploded view of the reduc­ing nut hous­ing at the bot­tom of the injec­tor port hous­ing and a close up of the gold seal

 

Fig­ure 4 above:  a graphite ferral.

 

Fig­ure 5 above:  a pic­ture of an installed inlet liner

 

Fig­ure 6 above: 2 used septa

 

Fig­ure 7 above: an easy way to check the integrity of a septum

Video 1 above: Video that I took while dis­as­sem­bling an GC injec­tor port

Video 2 above: A video I took of dis­as­sem­bling a FID

Video 3:  A good video of the process of remov­ing and installing var­i­ous con­sum­ables from the oven side.

Video 4:  A good video of the process of remov­ing and installing var­i­ous con­sum­ables on the injec­tor side.

 

Video 5:  Two good videoes of the process of remov­ing and installing a column.

As an aside, in my opin­ion hav­ing done it about 60 times, prop­erly installing a col­umn and replac­ing all con­sum­ables prop­erly where you get desired high effi­ciency is actu­ally pretty tough to do.  A lot of labs out­source the instal­la­tion and chang­ing of their GC columns and con­sum­ables to a third party ser­vice provider for this very reason.

A fre­quently asked ques­tion on the list­servs that I belong to basi­cally asks the fol­low­ing question:

In GC-FID use to quan­tify Blood Alco­hol Con­tent where EtOH is the tar­get ana­lyte, how does the machine arrive at the reported number?

The machine is called a Gas Chro­mato­graph with a Flame Ion­iza­tion Detec­tor (GC-FID). Typ­i­cally the sam­ple is intro­duced by way of head­space. The most fre­quently used tech­nique is tech­ni­cally called Sta­tic Head­space Isother­mal Wall Coated Open Tubu­lar Gas Chro­matog­ra­phy with Flame Ion­iza­tion Detector.

First a crash course on the Flame Ion­iza­tion Detec­tor (FID).

The FID is the part of the appa­ra­tus that quan­ti­tates the result of the chro­mato­graphic efflu­ent. Remem­ber that the num­ber one rule of GC-FID is that you must demon­strate with data (actu­ally prove) proper res­o­lu­tion first. The mea­sure of qual­i­ta­tive selec­tiv­ity (sep­a­ra­tion) must be proven before we can validly quantitate.

The FID actu­ally is a destruc­tive, mass count­ing device. It lit­er­ally burns all carbon-hydogen bonds (C-H bonds) from what comes off the col­umn. The cur­rent is sensed by an elec­trom­e­ter, con­verted to a dig­i­tal form and sent to an out­put device that gives us the peak. It counts the increase in the num­ber of ions between a cath­ode and a diode. A polar­iz­ing volt­age attracts these ions to a col­lec­tor located near the flame. This cur­rent is mea­sured with a high-impedance picoam­me­ter. An elec­trom­e­ter is an elec­tri­cal instru­ment for mea­sur­ing elec­tric charge or elec­tri­cal poten­tial dif­fer­ence. The response is pro­por­tional to the num­ber of C-H bonds. So, there­fore, iden­ti­cal amounts of methanol, ethanol, butanol and hexa­nol would not give equal area count responses. The FID itself emits an ana­log (con­stant) sig­nal; yet, it is reported through a com­puter sys­tem that is dig­i­tal in nature.

There are two main meth­ods of deter­min­ing quan­ti­ta­tion when it comes to GC-FID: (1) Peak height, and (2)Peak area.

Peak Height versus Peak Area

Peak Height ver­sus Peak Area

Peak height is an anti­quated method of quan­tifi­ca­tion that is not used a lot. It harkens back to a time when there were no com­puter pro­grams. Lab­Corp and cer­tain labs in Cal­i­for­nia still use the peak height method. Both are based upon the stan­dard dose response cal­i­bra­tion curve.

In order to deter­mine either peak height or peak area, the cru­cial bound­ary is the baseline.

The baseline and its determination is a crucial bound

The base­line and its deter­mi­na­tion is a cru­cial bound

It is essen­tial to remem­ber that all detec­tors have ana­lyt­i­cal noise. There is no way of record­ing zero. No machine can mea­sure true zero. There is always going to be an off­set. As an aside, this is why on a cal­i­bra­tion curve zero (the ori­gin) can­not be used as a legit­i­mate data point in deter­min­ing the slope which gives us the quantitation.

While there is a peak, there is, of course, sig­nal. As a result dur­ing the act of detec­tion there is no base­line per se. Instead, the com­puter uses an algo­rithm based upon prior input before the peak and “guesses” what the base­line would be if there was no sig­nal. As this is the computer’s guess, is it is sub­ject to inter­pre­ta­tion and error.

When we use the peak area method of quan­ti­ta­tion, then the ques­tion becomes not only where is the base­line, but where does the com­puter begin to cal­cu­late the begin­ning and end­ing of a peak. This is so very important.

As you can see in the above there are ver­ti­cal hash marks that are labeled “start” and “stop,” these are called “tick marks.” This is the other perime­ter that deter­mines peak area.

So in sum the deter­mi­na­tion of the peak area is a func­tion of 3 bounds: (1) the base­line and (2) start­ing tick mark, and (3) end­ing tick mark. Think of them as bound­ary marks.

There are two types of inte­gra­tion: (1) auto-integration and (2) man­ual integration.

If the but­ton pusher uses auto-integration, then the deter­mi­na­tion of the base­line will be cal­cu­lated the same through­out the run. This is what should be done (pro­vided that the use of the auto-integration events is sta­tic and equally applies to the cal­i­bra­tors and the unknowns that are tested and is part of a truly val­i­dated method).

On the other hand, one can use man­ual inte­gra­tion events to manip­u­late the data. This is a func­tion of the end user. This is typ­i­cally a one-time event in that only one chro­matogram is manip­u­lated. It may be some­thing that can be dis­cov­ered by look­ing at the chro­matogram or it may be utterly undis­cov­er­able unless you have the raw data. (Here is a video that shows this con­cept: The case for raw data: “Inte­gra­tion” in Gas Chro­matog­ra­phy: How to make an inno­cent per­son guilty in a DUI case by manip­u­lat­ing the soft­ware Actu­ally at Axion, we dis­cov­ered another 3 ways of manip­u­lat­ing the raw data so it would be undetectable.)

The key func­tion of all of this is that every­thing remain the same in order for the result to be valid. The inte­gra­tion events must be sta­tic in order for the quan­tifi­ca­tion to be valid.

When look­ing at the EtOH peak:

The unknown that is your client’s sam­ple must use the same inte­gra­tion perime­ters as the cal­i­bra­tors. The bound­ary marks used to deter­mine the peak area for the cal­i­bra­tors in their indi­vid­ual chro­matograms must match that of the unknown. If the base­line remains the same, but the tick marks are wider for the cal­cu­la­tion of the unknown than the cal­i­bra­tors, then this will over-report the Blood Alco­hol Con­tent (BAC). The con­verse to this is true as well. If the tick marks for the cal­i­bra­tor and the unknown remain the same, but the base­line for the unknown is lower than the cal­i­bra­tor, it will over-report the BAC. The con­verse is true as well.

Auto-integration versus manual integration

Auto-integration ver­sus man­ual integration

Above is an exam­ple of how the auto-integration leads to less area and how the man­ual inte­gra­tion leads to more area. More area equates a higher BAC if this is per­formed on the EtOH peak.

What fur­ther com­pli­cates this is the use of the Inter­nal Stan­dard (ISTD). The inte­gra­tion events for the ISTD must also remain sta­tic. A peak is a peak is a peak. The same bound­ary marks apply. How­ever, with respect to the ISTD, the ISTD amount is inversely related to the EtOH. If there is too much ISTD cal­cu­lated, then the BAC will be ratio reduced. The con­verse is true as well.

How can you tell if any of this is done? Well, maybe you can and maybe you can’t as dis­cussed above and in the video. If you trust the lab­o­ra­tory, and if they report the base­line and the tick marks on all of the chro­matograms, you can sim­ply use a light­box or even hold it up to a light with the cal­i­bra­tors and the unknowns on top of one another and see if the bound­ary marks are all treated the same.

Bot­tom line take away: Con­sis­tent and appro­pri­ate inte­gra­tions are sci­en­tif­i­cally defen­si­ble. Incon­sis­tent or inap­pro­pri­ate inte­gra­tions are dif­fi­cult to defend. All QC, Ver­i­fiers and Unknowns must be treated in a con­sis­tent (same) man­ner and per a val­i­dated method. Oth­er­wise, you have a non-validated result.

The take away ques­tion: What does your lab­o­ra­tory do when it reports out it’s BAC?