If you are bored and want to have a little fun, call up your local crime laboratory, ask for someone who preforms their chromatography (it doesn’t matter if it is liquid chromatography or gas chromatography). When you get that person ask them a simple question:

What’s the t0 (pronounced “t subzero”) for your method that you are running?

If you could see them (remember you are over the phone), they would probably look like this:

There will likely be a pause. Then perhaps some confusing babble from the person that is scientific gibberish that means absolutely nothing as it was unresponsive. Or perhaps the person might actually confess that he/she doesn’t even know what t0 is or what you are talking about or why it would be important.

The reason that I am gambling that this will be the likely response at your local crime laboratory is the way that crime laboratories hire and train these analysts. It is a sad fact that there are very few undergraduate institutions that actually meaningfully teach chromatography practice or theory. Even fewer undergraduate institutions actually have a GC or LC. The few that do usually have 1. Usually, it is broken or abused. Even if it is running, the use of it is reserved to graduate students or are dedicated to the professor’s totally esoteric research project, and therefore is a hands-off experience for the poor undergraduate. As a byproduct of this sad reality, a generation of state scientists have intellectually grown up without any of the fundamentals or meaningful practical experience in chromatography as we would hope that they would gain in undergraduate study. What further compounds the issue is the pay scale (which is appalling low) cannot possibly be designed to attract and maintain in the crime laboratory analyst position people who have any real experience and any real theoretical training in chromatography. In fact, when I have looked at the CVs of crime laboratory analysts from my colleagues across the nation, it is highly unusual to find any one who has actually used a GC or LC before they were hired by the crime laboratory. So when they show up for work, they are literally tabula rasa. They know nothing more about the theory and the process than perhaps a 3 year old child. I have seen some truly weird backgrounds. I have seen a French Literature major who had a minor in biology (consisted almost entirely of mammalian biology classes) hired to conduct LC-MS work which requires a lot of understanding in acid-base chemistry to interpret the spectra correctly. I have seen folks who are physics majors who never took a single chemistry class hired to do this work. I have also frequently seen AA graduates with only 2 hard science classes (introduction to biology and introduction to chemistry) with Forensic Science/Police Studies AA degrees. Then there is the internet degree places where the person may be a chemistry major, but never stepped foot in a laboratory at all and never seen a GC or LC in person let alone use it. In sum, very few have even an introductory instrumentation class. They certainly have no exposure to metrology or in quality control or quality assurance procedures. It is a very sad state of affairs.

The crime laboratory, concerned with throughput, wants to get this person who is drawing a paycheck and taking up space in the laboratory to testing samples as soon as humanly possible. The push to “get testing” leads to a totally incomplete education that amounts to little more than teaching someone to be a button pusher doing a mechanical process that requires no thought or inspection.

But back to our main topic—t0.

What is t0?

t0, T0, T sub zero, T naught, T zero, air peak, dead time, tM, hold time, or void time all refer to the same concept. It is the time that it takes for an unretained (meaning no interaction with the stationary phase) molecule to make it from injection to detector. The significance of it cannot be underscored. Every laboratory should have in their validation study for the method the t0 time.

Why it is so important for us as external quality assurance officers for the crime laboratory?

If there is any peak before t0, then it must be from carry-over and not from the injection that we are supposed to be analyzing.

Carryover is the nice scientific name. The reality is that it is unplanned contamination that calls into question the validity of the reported results.

Here is data from a validation study from a forensic laboratory where they proved t0. Remember that t0 will not be at the same time for every GC-FID in the world. It totally depends on the chromatographic column and the chromatographic conditions (primarily temperature and pressure/flow).

There is actually a theoretical manner to measure and accurately predict t0 based upon your conditions through physical chemistry. You then have to experimentally confirm the math on your instrument and test for robustness per the Huber/USP protocol for method validation (more on method validation can be found here Method Validation for Lawyers: A Six Part Series).

Here is a simple exemplar chromatogram using the same method (same column and conditions) where there is a peak detected before t0. The only way that happens is if there is carryover from the last samples that preceded it. There is an old saying in chromatography which says “just because you are done with chromatography doesn’t mean it is done with you” which means that just because you think that everything has eluted from the column doesn’t make it so. This is a very basic reason why there must be blanks between unknowns.

Here is a particularly dangerous chromatogram that demonstrates carryover in the most important chromatogram in the QC for the method which is the proof of separation/resolution:

Here is the other column from the same batch. Note t0 is different as the column is different, but same chromatographic conditions. There is demonstrated carryover and likely/possible carryover demonstrated.

So ask your crime laboratory: “what’s your t0?”