There is a large dif­fer­ence between a sin­gle col­umn analy­sis and a dual col­umn analy­sis when it comes to the abil­ity to most cor­rectly iden­tify and quan­ti­tate an unknown in the sci­en­tific world.

In foren­sic sci­ence, we are con­stantly test­ing unknowns. What is meant by this is that we have a sam­ple that is seized from a crime scene or from a per­son, but we don’t know what it con­tains. For exam­ple, in blood analy­sis for EtOH in an alleged DUI case, we have a sam­ple of blood that is taken from the accused, but just by look­ing at it, we can­not know if there is even ethanol in it, and even if present, how much there is. We need to ana­lyze it using instru­men­ta­tion in a sci­en­tific manner.

When we are look­ing to be sci­en­tific about our analy­sis we are look­ing to be as spe­cific as pos­si­ble, and try­ing not to be merely selec­tive. There is a large and impor­tant sci­en­tific dif­fer­ence between being selec­tive and spe­cific. As we wrote before on this blog: Metrol­ogy in Quan­ta­tive Mea­sure: Is it Spe­cific or Selec­tive or Neither…

The Inter­na­tional Union of Pure and Applied Chem­istry (IUPAC), which is the world author­ity on chem­i­cal nomen­cla­ture, ter­mi­nol­ogy, stan­dard­ized meth­ods for mea­sure­ment, atomic weights and other crit­i­cally eval­u­ated data and oth­ers have defined the dif­fer­ence between these often con­fused terms as follows:

A spe­cific reac­tion or test is one that occurs only with the sub­stance of inter­est, while a selec­tive reac­tion or test is one that can occur with other sub­stances but exhibits a degree of pref­er­ence for the sub­stance of inter­est.  Few reac­tions are spe­cific, but many “exhibit selectivity”.

Other com­mon def­i­n­i­tions include:

Selec­tiv­ity gives an indi­ca­tion of how strongly the result is affected by other com­po­nents in the sample.

and also

Selec­tiv­ity refers to the extent to which the method can be used to deter­mine par­tic­u­lar ana­lytes in mix­tures or matri­ces with­out inter­fer­ences from other com­po­nents of sim­i­lar behavior.

A selec­tive test may be not a spe­cific test due to cross-reactivity, inter­fer­ence, or codetermination.

So, we search for he most spe­cific form of analy­sis. In the world of DUI for EtOH, the gov­ern­ment typ­i­cally set­tles for Head­space gas Chro­matog­ra­phy with Flame Ion­iza­tion Detec­tor (HS-GC-FID). GC-FID is not the most spe­cific test avail­able for EtOH exam­i­na­tion as there is Gas Chro­matog­ra­phy with Mass Spec­trom­e­try (GC-MS) for exam­ple which is much more selec­tive and bor­ders on spe­cific when it comes to EtOH analy­sis, but for what­ever pol­icy rea­son, the gov­ern­ment chooses not to do the most scein­tific thing which is to use the most spe­cific assay avail­able. There is no sci­en­tific rea­son not to test for EtOH on the most spe­cific assay avail­able. In fact, it could be legit­i­mately argued that rely­ing on GC-FID instead of GC-MS for EtOH deter­mi­na­tion and quan­tifi­ca­tion is not sci­en­tific as GC-MS exists and is read­ily avail­able. How­ever, that is a post for another day.

As we are seemly inex­plic­a­bly stuck with the sci­en­tific step-sister of analy­sis in GC-FID as opposed to GC-MS, we must look at ways that the gov­ern­ment chooses to employ GC-FID to see whether or not as an assay it is valid. As our last series of posts “Method Val­i­da­tion for Lawyers” revealed, there is power in the words “valid” and “valid­ity.” With­out hav­ing a truly valid method that has been proven to be suit­able for its intended pur­pose, we can­not have a valid result.

Some foren­sic lab­o­ra­to­ries choose to use a con­fig­u­ra­tion in GC-FID that is known as a sin­gle col­umn, sin­gle injec­tion setup. In this set up there is one installed col­umn and the ana­lyst makes one injec­tion (or the autosam­pler does) to test the sample.

With­out any sci­en­tific doubt, a sin­gle col­umn method of analy­sis is not foren­si­cally or sci­en­tif­i­cally defen­si­ble or acceptable.

Remem­ber that when we use GC-FID, we can never achieve true speci­ficity, the most we can hope for is the pos­si­bil­ity of being merely selec­tive as demon­strated and proven through the res­o­lu­tion stan­dard (sep­a­ra­tion matrix/standard mix). The qual­i­ta­tive result is only based upon one cri­te­ria which is the reten­tion time. Reten­tion times through any given col­umn are not unique to one spe­cific volatile organic com­pound (VOC) to the exclu­sion of every thing else in the uni­verse. Hence, we have the often repeated phrase that all legit­i­mate tech­ni­cally trained chro­matog­ra­phers know and can recite in their sleep—the lim­i­ta­tion of GC-FID is that the reten­tion time is merely char­ac­ter­is­tic of a com­pound and cer­tainly not adju­dica­tive or con­fir­ma­tory of the speci­ficity of that com­pound— mean­ing a peak at a given reten­tion time is not a unique qual­i­ta­tive mea­sure (to the exclu­sion of every other com­pound in the universe).

Don’t take just my word for it con­sider the following:

As ven­er­ated Pro­fes­sor Harold McNair, PhD writes in his book, Basic Gas Chro­matog­ra­phy,

Reten­tion times are char­ac­ter­is­tic of a GC sys­tem, but they are not unique, so GC reten­tion times can­not be used for qual­i­ta­tive confirmation.

He fur­ther writes:

Iden­ti­fi­ca­tion of an unknown by com­par­i­son to reten­tion times using stan­dards that forms the basis of the qual­i­ta­tive analy­sis [in GC-FID analysis].

He con­cludes:

Unfor­tu­nately, GC sys­tems can­not con­firm the iden­tity or struc­ture of any peak. Reten­tion times are related to par­ti­tion coef­fi­cients (Chap­ter 3); and while they are char­ac­ter­is­tic of a well-defined sys­tem, they are not unique.

How do we acknowl­edge this lim­i­ta­tion in the lack of speci­ficity and try to mit­i­gate it?

We can add a dif­fer­ent col­umn and ana­lyze the sam­ple con­cur­rently on both columns. As we learned in our post What is a Gas Chro­matog­ra­phy col­umn and why should I care?, the col­umn, if prop­erly selected and prop­erly installed, is what pri­mar­ily causes the sep­a­ra­tion of the var­i­ous VOCs to occur. What we do is select two dif­fer­ent columns with two dif­fer­ent sta­tion­ary phases. We attach a y-splitter that will take the sin­gle injec­tion made into the injec­tor port and divide the sam­ple into two dif­fer­ent path­ways with one part of the sam­ple going to one col­umn for analy­sis and the sec­ond part going to another col­umn for analysis.

The strength of a well-designed dual col­umn analy­sis method where the sta­tion­ary phase is dif­fer­ent between the two columns is that this dif­fer­ence in the sta­tion­ary phase will cause dif­fer­ent sep­a­ra­tion of the ana­lytes (both in terms of reten­tion time and pos­si­bly even elut­ing order) as proven by the chro­matograms of the analy­sis of the res­o­lu­tion stan­dard (sep­a­ra­tion matrix/standard mix). This dif­fer­ent elut­ing order and dif­fer­ent reten­tion times only min­i­mizes, but does not entirely elim­i­nate the pos­si­bil­ity of co-elution as again, the resolv­ing (sep­a­rat­ing) power of the method is only deter­mined by one non-unique mea­sure which is the two reten­tion times. Even though there is a change in the elut­ing order poten­tially and the reten­tion times are dif­fer­ent based upon the sta­tion­ary phase com­po­si­tion, again, it must be empha­sized that the basic lim­i­ta­tion of GC-FID remains in that a reten­tion time is merely char­ac­ter­is­tic of the ana­lyte, but is cer­tainly not confirmatory.

This is why dual col­umn is referred to as the poor man’s Mass Spec as it has a more orthog­o­nal approach towards the qual­i­ta­tive mea­sure than does a sin­gle column.

How the cur­rent trend of the crime lab­o­ra­tory using a dual col­umn GC-FID is alarm­ingly unscientific.

What is most alarm­ing to me is the trend that is devel­op­ing across the United States where the sec­ond col­umn is not being used for quan­tifi­ca­tion at all. In this trend that I see sweep­ing all across the US, the sec­ond col­umn is merely being used as a “con­fir­ma­tory” col­umn in that if the reten­tion time matches with the standards/controls in the Qual­ity Con­trol sam­ples, then it is pre­sented as “ver­i­fied” in terms of the qual­i­ta­tive mea­sure by sim­ply that sec­ond col­umn match­ing reten­tion time with the knowns that act as the standards/controls. As we explain above, that is a dan­ger­ous and unsci­en­tific approach.

The rea­son that this is so impor­tant given the above con­text (there are other rea­sons that it is alarm­ing, but I want to stick to this ref­er­ence) is that with­out the sec­ond col­umn giv­ing a quan­ti­ta­tive mea­sure, we can­not fairly elim­i­nate co-elution (where two com­pounds elute at the same time, but only get iden­ti­fied and quan­ti­tated as one com­pound) because if the sec­ond col­umn is used to also quan­tify it will serve as an indi­rect deter­mi­na­tion of whether or not there is co-elution. If the sec­ond col­umn also pro­vides a quan­tifi­ca­tion of the unknown, we would exam­ine the pre­ci­sion of the quan­ti­ta­tive results (how closely the num­bers agree among those given by col­umn A and those of col­umn B– do the A’s match the B’s?). If there is co-elution that was “dis­cov­ered” by the dual col­umn approach, then we would expect to see impre­ci­sion between these num­bers (the A’s don’t match the B’s).

Now that they have decided as an orga­ni­za­tion to not quan­ti­tate on the sec­ond col­umn, we have lost a vital part of qual­ity con­trol and the lab super­vi­sor has lost a pow­er­ful tool of qual­ity assur­ance. It is just bad science.

Fur­ther, there is no legit­i­mate sci­en­tific rea­son for quan­ti­at­ing on the sec­ond col­umn. If you are doing good qual­ity work ad your meth­ods and instru­ments are in con­trol, then your num­bers should agree.

It’s not a time thing.

As it is a sin­gle injec­tion y-splitter dual col­umn analy­sis any­way, the amount of time it takes to make a cal­i­bra­tion curve, eval­u­ate it and then incor­po­rate it into the soft­ware for one col­umn is vir­tu­ally the same amount of time to do the same on col­umn 2. There is vir­tu­ally no added time. It makes no sense from a sci­ence point-of-view.

So in con­clu­sion, we can fairly con­clude the following:

• Sci­en­tif­i­cally, we always want to test our unknowns on the most spe­cific assay available.
• GC-FID is not the most spe­cific assay available.
• Lab­o­ra­to­ries that use sin­gle col­umn GC-FID as their method of analy­sis do not pro­duce foren­si­cally or sci­en­tif­i­cally defen­si­ble or accept­able results.
• Lab­o­ra­to­ries who use dual col­umn GC-FID that do not quan­ti­tate on both their columns do not pro­duce foren­si­cally or sci­en­tif­i­cally defen­si­ble or accept­able results.
• Good sci­ence is not always prac­ticed in the mod­ern state crime laboratory.