Topic: Generic GC Cross (Isothermal Static Headspace Gas Chromatography with Flame Ionization Detector)

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1 Texas Criminal Defense Lawyers Association 9 th Annual Top Gun DWI Blood Breath & Beyond August 11-12, 2011 Crowne Plaza Houston, Texas Topic: Generic GC Cross (Isothermal Static Headspace Gas Chromatography with Flame Ionization Detector) Speaker: Deandra Grant 6808 Hill Meadow Dr :: Austin, Texas :: p :: f ::

2 GENERIC GC CROSS (Isothermal Static Headspace Gas Chromatography with Flame Ionization Detector) By Deandra Grant Q: You don't hold yourself out as an expert in all forensic science? Q: It would be fair to limit your expertise to be in procedures and protocol and its implementation with respect to alcohol analysis? Q: The testing in this case was done using headspace gas chromatography with flame ionization detector? Q: I want to ask you some questions about the education you have listed on your cv. Do you understand? Q: You list here your education. That would be traditional brick and mortar style education? Q: You received a bachelors degree in Microbiology from Texas A&M in Q: Did you run a headspace gas chromatograph with flame ionization detector while employed by the Charles County Government in Maryland from ? Q: You were not doing headspace gas chromatography with flame ionization detector using a capillary column for purposes of determining ethanol? Q: The actual title that you were conferred at that point in time was bachelor of science? Q: In college did you take any sort of dedicated course in gas chromatography? Q: You took no dedicated course for a year or even a semester in headspace gas chromatography with flame ionization detector? Q: You did not receive any instruction in college on specifically how to use a headspace configured gas chromatograph with flame ionization detector. Q: Your only exposure was part of some sort of survey course on analytical devices. Q: Next in your education you have the University of Houston. That is also a brick and mortar education? Q: You did some graduate work in chemistry.

3 Q: You did not take any sort of dedicated course in gas chromatography with flame ionization detector? Q: You do not have a Master s degree. Q: You do not have a PhD. Q: I want to ask you some questions about the professional qualifications you have listed on your cv. Do you understand? Q: Did you run a headspace gas chromatograph with flame ionization detector while employed at Day and Zimmerman ammunition plant from ? Q: You were not doing headspace gas chromatography with flame ionization detector using a capillary column for purposes of determining ethanol? Q: Did you run a headspace gas chromatograph with flame ionization detector while employed by the Charles County Government in Maryland from ? Q: You were not doing headspace gas chromatography with flame ionization detector using a capillary column for purposes of determining ethanol? Q: Did you run a headspace gas chromatograph with flame ionization detector while employed by Edna Woods Laboratories from ? Q: You were not doing headspace gas chromatography with flame ionization detector using a capillary column for purposes of determining ethanol? Q: You do not hold yourself out as an expert in the theories and the principles of chromatography? Q: You do not hold yourself to be an expert in analytical chemistry, specifically the underlying principles of chromatography, do you? Q: How many analytical chemistry courses have you taken? Q: Titles? Q: In 1991 you went to work for the Houston Crime Lab. Q: You list experience on various instruments within that lab. Q: Did you ever participate in a training course in headspace gas chromatography with flame ionization detector that was put on by the manufacturer? Q: Your supervisors taught you how to use the gas chromatograph?

4 Q: You were taught to follow a certain protocol? Q: Who is the supervisor that taught you? Q: Can you share with us her particular technical scientific background? Q: You have not completed any training by the machine s manufacturer? Q: You have not completed any formal training put on by the American Chemical Society in headspace gas chromatography with flame ionization detector? Q: Are you a member of SOFT, the Society of Forensic Toxicologists? Q: Are you a member of the IACT, the International Association of Chemical Testers? Q: Have you taken any courses with the ACS or the American Chemical Society? Q: You have no publications listed. Q: You have not published anything having to do with the science of chromatography. Q: You have not published anything having to do with flame ionization detection. Q: You have not published anything having to do with blood alcohol analysis using gas chromatography flame ionization detection. Q: I want to ask you questions about your work in this case. Do you understand? Q: You're interested in reporting what we call in science a true result? Q: You would agree with me that in science, there is no such thing as trust me. Q: It s more like trust in God, all others bring data. Q: You received a subpoena from the District Attorney's Office to be here today. Q: Prior to today's testimony you took the time to gather what you believed to be relevant information with respect to this particular test. Q: You knew that you were going to come into court today subject to that subpoena specifically to testify much in the way that you have done in the past? Q: You would be subject to questions by the Prosecution? Q: And by the Defense?

5 Q: You came into this entire event knowing you were going to have to scientifically defend the accuracy of your conclusion in this case. Q: You have reviewed your work before coming here today to testify. Q: This work meets your standards of professionalism? Q: Do you consider yourself a scientist first or an employee of the government first? Q: As a scientist you don't mind the scrutiny that we're going to go through in terms of determining whether or not this is a verified, true, precise accurate? Q: You are familiar with the concept of an uncertainty budget? Q: An uncertainty budget presents a range of possible error. Q: There is no uncertainty budget included with the result in this case. Q: The result is reported as a definitive result. Q: The range of possible error is omitted from the test result. Q: The scientific community does not find that to be an acceptable method of reporting? Q: The reported result in this case is not completely free of error? Q: You find it to be acceptable to report a conclusion without the range of possible analytical error? Q: You are familiar with DNA reports and the uncertainty budgets included with their results. Q: That is a forensic test. Q: The uncertainty, or rate of error, is always reported. Q: You did not report the rate of error in this case. Q: I want to ask you some specific questions about gas chromatography. Do you understand? Q: Gas chromatography is what we call separation science. Q: Separation is the key component of gas chromatography. Q: The first component is what is called the headspace device.

6 Q: The second component is called a gas chromatograph. Q: The third component is called a flame ionization detector. Q: The headspace device is connected by a transfer tube to the injector port of the gas chromatograph. Q: Inside the gas chromatograph is what we call a column. Q: In this case 2 columns were used. Q: The gas chromatograph is heated. Q: It is connected to what we call a flame ionization detector. Q: A complex mixture, such as blood, is injected from the headspace device into the column where it is supposed to separate out the component parts. Q: After separation, the components are burned in the flame ionization detector to determine the amount. Q: You cannot determine the quantity until you have achieved separation. Q: A chromatogram is the recorded representation of this method of testing. Q: I want to ask you about the blood sample in this particular case. Do you understand? Q: Without making a single assumption, can you tell us the true blood alcohol content of my client at the time his blood was drawn? Q: Without making a single assumption, can you tell us that the blood alcohol content, the numeric value as being reported is true, accurate, precise, reliable, verified, and repeatable without making a single assumption along the way? Q: Without making a single assumption you can do that? Q: You are familiar with the term pre-collection error? Q: It is important for the manufacturer specifications be followed? Q: You are aware of what the manufacturer s storage instructions are for their blood tubes? Q: Now, this particular case involves a DPS blood kit.

7 Q: You are aware of an insert that comes with the blood kit that includes directions and proper use such as temperature and tolerances? Q: DPS blood kits contain blood collection tubes. Q: Blood collection tubes have temperature storage requirements. Q: Temperatures that are too hot or too cold can compromise the blood tube. Q: A compromised blood tube could allow contaminants to get inside. Q: You have no knowledge of how this blood kit was stored before being used in this case. Q: You cannot testify that it was not exposed to heat or cold beyond the manufacturer s specifications. Q: You re aware that the blood is supposed to be refrigerated? Q: Blood is immediately put into a refrigerator when it comes into your lab? Q: When a tube comes into the lab for analysis, it comes in one perhaps out of many? Q: You don't have any sort of photographic evidence to demonstrate what the blood tube looked like when it arrived at the lab. Q: There is no photographic evidence that verifies that there were intact integrity seals covering the tube? Q: There is nothing would prohibit you from taking digital photos of the evidence? Q: This tube is designed specifically be properly filled at 10 milliliters? Q: The vacuum inside the blood tube is measured to that specification? Q: Less than 10 ml could be an indicator that the vacuum was compromised. Q: If vacuum was able to escape from a blood tube, then it s possible contaminants could have gotten in. Q: The blood tubes have expiration dates. Q: The expiration dates refer to the seal and vacuum integrity? Q: I want to ask you about the importance of the chemicals inside the blood tube. Do you understand?

8 Q: The tubes have powder inside of them. Q: The powder contains an anti-coagulant to prevent the blood from clotting. Q: The powder also contains a preservative to prevent fermentation. Q: It is important for the person drawing the blood to properly mix the chemicals with the blood. Q: The manufacturer s suggest a certain number of times the tube should be inverted in order to ensure proper mixing. Q: You have no personal knowledge that the proper number of inversions were done in this case. Q: The integrity of the result in part depends upon the proportion of the additives in the tube to the blood that is collected. Q: It is important for the integrity of the result that ultimately comes as your basically one sentence report with no uncertainty budget that the additives are properly present inside the tube so as to prevent coagulation and fermentation? Q: You have no personal knowledge that the proper amount of preservative and anti coagulant were present in this tube before blood was drawn. Q: There is a process that's called ion scanning that can be done to figure out whether or not sodium chloride or potassium oxalate were present in the tube. Q: No such testing was done on this tube. Q: The anti coagulant is called potassium oxalate. Q: It stops the blood from clotting. Q: Without an anti coagulant, blood starts to clot immediately. Q: To perform a whole blood analysis on a gas chromatograph, clotting of the blood needs to be prevented. Q: Clotted blood could potentially invalidate the sample for this type of testing. Q: Failure to properly mix the anti coagulant and the blood sample could lead to clotting. Q: The clotting problem can also be spotted by an analyst such as you if it is a large clot. Q: Not all clots are detectible to the visible eye.

9 Q: Very small clots, known as micro clots, are not automatically caught at all times by analysts? Q: You cannot say that this particular sample was free of micro clots. Q: The impact of such clotting, if present, would be unknown. Q: The second chemical found in the tube is a preservative. Q: It s what we call an anti-fermenting agent. Q: Fermentation would be the production of alcohol within the sample. Q: The gas chromatograph cannot tell the difference between alcohol that one drinks and alcohol that is created as the blood breaks down. Q: If the blood sample was not properly mixed with the anti fermenting agent, under the right circumstances, alcohol could be produced within the sample. Q: There has been no ion test to confirm the presence of either of the two additives? Q: Without either of the two additives being in the proper ratio or even being in the tube, you can't tell us what the impact would be on the results in this case. Q: The test result is only as good as the specimen given to you. Q: If there is error in the tube itself or in the collection of the sample, all the great lab work in the world can t fix it. Q: Garbage in, garbage out? Q: You are aware of a yeast called Candida Albicans. Q: It is a very plentiful yeast that is all around us on our skin and in the air? Q: When taking a sample of blood for alcohol testing, a providone-iodine pad is used. Q: The purpose of using the providone-iodine pad is to come as close as possible to creating a sterile venipuncture site? Q: In terms of the use of the providone-iodine, it is an important step in this analysis to eliminate as much as we can Candida Albicans? Q: When cleaning the draw site it is proper procedure to move in concentric circles away from the venipuncture site as opposed to up and down because up and down would just move the contaminants back and forth.

10 Q: If proper procedures were not followed with this blood draw, then it is possible that Candida Albicans might have contaminated the sample. Q: If Candida Albicans were present in this sample, then they could essentially eat the sugar in the blood. Q: The byproduct of yeast and sugar is ethyl alcohol. Q: Ethyl alcohol, or ethanol, is what the gas chromatograph looks for? Q: The gas chromatograph cannot tell the difference between that which ethanol was produced because he drank versus that which is produced as a reaction to Candida Albicans infiltration. Q: A bacterial culture can be run to determine whether fermentation has occurred. Q: A simple blood glucose test could also alert you about possible fermentation. Q: Neither type of test was run in this case. Q: We can t scientifically eliminate that as a possibility in this case. Q: In terms of a possible Candida Albicans issue, you are unable to quantify the impact on a test result. Q: One of the things that s very important when dealing with a blood sample is proper refrigeration. Q: Blood tube manufacturers publish specifications as far as the zone of tolerance of the sample itself? Q: One of the reasons why it's important for the sample to be at particular temperature is to inhibit fermentation? Q: You need both the antifermenting agent and proper temperature control to ensure there is no alcohol production in the sample. Q: From December 8 to December 10 this sample was not within your lab's control? Q: So between the dates of December 8 and December 10 you have no verifiable, traceable information that the tubes in this particular case were refrigerated within the specified zone of tolerance per the manufacturer's specifications. Q: If it were not properly refrigerated, if there is no evidence of that in the record or according to your notes, you cannot tell us what if any impact that lack or potential lack of refrigeration has on the ultimate result as reported by you.

11 Q: Between the dates of December 8 and December 10, you cannot verify that the blood sample was not exposed to extreme temperatures. Q: If during this period of time something happened to alter the specimen, no amount of perfect analysis on your part will cure that bad sample. Q: Before it becomes into your lab and becomes basically your responsibility, you can't control what other people do before it gets in the lab? Q: One of the particular issues and problems that may come up is what's called hemolysis? Q: Hemolysis is the rupturing of the red blood cells that create a pinkish type of hue and can cause an abnormal result at the end of the analysis. Q: Hemolysis can be created by excessive jarring events. Shaking the specimen, having it jarred around in the car, that can potentially cause hemolysis? Q: Do you have any evidence as to whether or not this particular sample was subject to any of those but for your visual inspection of the tube itself? Q: You'll concede the fact that hemolysis can come in a spectrum. There can be gross hemolysis which is obvious to slight hemolysis which is maybe not as detectible. Q: So simple visual detection may not catch hemolysis even if one were looking for it. Q: You can t quantify for us the impact hemolysis might ultimately have on the reported result in this case? Q: Between December 10 and December 27 it was in your lab. Q: The protocol at your lab regarding new samples is that they are logged in and then refrigerated. Q: Samples are to be kept at a constant temperature within a certain range. Q: Do we have the temperature logs for the refrigerator where this particular specimen was stored? Q: These refrigerators are laboratory grade refrigerators? Q: Something that, for example, we could get at Best Buy? Q: I want to ask you about errors that can occur during the testing itself. Do you understand? Q: Chromatography is a separation science.

12 Q: By separating, we mean that we take a a mixture of some sort that has multiple volatiles or compounds within it and that through the science of chromatography we can separate and distinguish between those component parts. Q: If a device, an analytical device such as a gas chromatograph even with a flame ionization detector cannot successfully separate between potential volatile samples and cannot distinguish between different compounds, then it is not a useful analytical device. Q: So the number one principle we have before us is the need to prove separation? Q: If we can't prove and verify separation, we're kind of stuck. Q: The gas chromatograph looks like a box. Q: The analytical mechanism that's within it is called a column. Q: There are two different types of columns. There are packed columns and capillary columns. Q: They have different purposes and different uses but both of them can be used for alcohol analysis Q: Your lab uses a capillary column. Q: A capillary column actually looks like a coil. Q: Maybe 30 meters long. Q: It's tightly wound around multiple, multiple times; and inside is packed silica. Q: You know through your past courses and experience that packed silica is used in chromatographic analysis to cause separation. Q: So we have the loopy type of device that sits inside of the box that's 30 meters long, correct? Q: When you take a look at it, the inside of the capillary column, it might be a hair or so in terms of diameter outside of the packed silica or whatever is on the inside, correct? Q: More or less a hair. Q: The overall capillary column is about maybe the size of a basketball in circumference. Q: The capillary column, just like all things over time, it becomes less sensitive the more it's used.

13 Q: And in fact, to go with an acceptable practice that occurs that's called crimping where you cut off the end because it becomes -- I don't know -- gunked. Q: Capillary columns are not cheap. Q: In this particular case, a dual column analysis was performed which means there were 2 columns. Q: Samples are prepared by mixed a tiny amount of the blood with what s called an internal standard. Q: To mix the samples you use what s called a pipette. Q: It is essential that you make precise measurements when preparing samples. Q: You have to go through proficiency training in order to learn how to use a pipette. Q: In fact, pipettes themselves have to be calibrated. Q: Prior to the testing in this case, when was the last time the pipettes used here were calibrated? Q: Uncalibrated pipettes can lead to pipetting errors. Q: The mixed samples are the put into what are called headspace vials and sealed. Q: It is essential that the vials are tightly sealed. Q: Many different samples are prepared at once. Q: In this case, it appears 47 samples were being run in this particular batch. Q: It is essential that the Jonny s sample not get mixed up with Paul s. Q: All of the prepared vials are placed in what s called an auto sampler. Q: The auto sampler is like a carousel. Q: The vials are heated. Q: It is essential that the auto sampler maintain the vials at a constant temperature and pressure. Q: What happens is the piercing mechanism comes in, takes a sample of the headspace gas above the prepared sample and pushes it through the injection port down into the capillary column. Q: The headspace gas is what is actually being tested, not the liquid blood itself.

14 Q: The flame ionization detector is at the end of the columns and it is literally a flame. Q: The flame ionization detector burns whatever comes off the column. Q: The ultimate result is a graphical representations which is called a chromatogram. Q: Everything that comes off the column and is burned up by the detector produces a peak on the chromatogram. Q: I want to ask you about the batch run in this case. Do you understand? Q: Human blood can contain numerous volatile compounds. Q: There are alcohols that are very similar in structure to ethyl alcohol. Q: It is essential that the gas chromatograph be able to distinguish between alcohols such as methanol, ethanol, isopropanol, etc. Q: Do you have any documentation with you that demonstrates that this particular gas chromatograph was capable of separating out ethanol to the exclusion of all other substances. Q: Are you familiar with the terms standard mix or separation matrix. Q: Running a standard mix a providing the chromatogram would show that this gas chromatograph could tell the difference between ethanol and other substances. Q: Did you bring that with you today. Q: When was the last time something like that was run on this gas chromatograph before this testing was done. Q: You ve had a chance to review the chromatograms associated with this case. Q: You would agree that they contain a lot of unidentified peaks. Q: That means there were other substances coming off the columns that this gas chromatograph was not identifying. Q: You cannot conclusively testify that there was not another unknown substance that was burned up in the detector at the same time as the ethanol. Q: If that occurred, then the peak for that substance and the peak for ethanol would be mixed up with each other. Q: That would result in the sum total of both area peaks being added together and calculated at ethanol.

15 Q: The ethanol value in that circumstance would be overstated. Q: You are familiar with the term internal standard. Q: N-propanol is commonly used as an internal standard in gas chromatography. Q: With an internal standard, you add the same amount to every sample and it should report out the same on every chromatogram. Q: You are familiar with the term blank. Q: A good ethanol blank should be a chromatogram that contains no ethanol and only the internal standard of n-propanol. Q: The importance of a blank is to show that there is no analytical carryover. Q: Carryover would mean that each sample is being contaminated by the previous sample. Q: Ideally a lab would have a protocol in place to make sure a blank was placed between each unknown sample to make sure there was no chance of alcohol carrying over. Q: In other words, put a blank between Sally s sample and Johnny s sample to prevent any chance for cross contamination. Q: It would be crucial in running blood samples to test for ethanol that Sally s sample not spillover into Johnny s sample. Q: Your lab does not put blanks between the unknown samples. Q: So no buffer between Sally and Johnny s samples. Q: Blanks are only run at the end. Q: If an ethanol blank shows ethanol, that would prove that carryover is taking place in the particular batch run. Q: If ethanol were being carried over from sample to sample, then you would not have valid results at the end of the batch run. Q: There were 4 ethanol blanks run for this test 2 on each column. Q: All 4 chromatograms for the ethanol blanks measured ethanol. Q: That means alcohol carryover was going on with both columns.

16 Q: Somewhere along the line before the sample made it to the columns, there was a problem with this gas chromatograph. Q: Your own testing method proves that alcohol from each sample tested is carrying over to the next sample. Q: Do you have with you any learned treatises or peer reviewed scientific articles that would support the idea that any sort of carryover is acceptable in forensic work? Q: From a scientific standpoint, this entire batch should have been re-run. Q: Test results from a batch run where alcohol carryover is taking place are not scientifically valid. Q: Do you ever watch any of those Gordon Ramsey cooking shows? Q: Pretend you re a contestant and the challenge is to bake some cookies. Q: You know how you first put the dry ingredients into a bowl and blend them flour, salt and baking powder? Q: Imagine doing that and then being told that the real challenge was to sift out the baking powder and weigh it. Q: Assume you were given a special sifter that would only allow particles the size of baking powder to pass through onto a scale where they would be measured for weight. Q: You would sift your mix into the sifter and then look at the weight calculation, right? Q: Seems simple enough. Q: What if you were then told that Gordon Ramsey snuck some cinnamon into some of the mixes when no one was looking and cinnamon is the exact same size as baking powder. Q: That would call into question your weight total, right? Q: Unless you had some way to show that you had no cinnamon in your mix, you couldn t be sure that your total weight was correct. Q: What if you then found out that the contestant before you didn t clean the device and some of their baking powder had been left on the scale? Q: Another error in your calculation? Q: You have no data, no chromatogram, nothing here with you today that demonstrates with a scientific certainty that nothing else came off that column with the ethanol.

17 Q: You do have data in the form of chromatograms which demonstrates that alcohol was carrying over from one sample to another. Q: So without a doubt baking powder had been left on the scale. Q: I want to ask you some questions about the calculation you did regarding alcohol concentration at the time of driving. Do you understand? Q: This calculation you just did for the jury, isn t it true that you had already performed the calculation for the DA prior to trial? Q: So when she just gave you that hypothetical information and you punched some numbers into your calculator, you both already knew the answer to her question, right? Q: What is the exact calculation you are making? Q: Have you heard of a scientist by the name of Widmark? Q: Can you tell the jury who Dr. Widmark was? Q: Are you using Dr. Widmark s formula to compute the numbers in this case? Q: You would agree that performing a back extrapolation requires you to use several averages, correct? Q: And you would agree that you have no way of knowing whether my client actually falls into the category of average for any of the factors you are using in your equation? Q: Would you agree that the scientific community frowns on the use of back extrapolation testimony due to the large number of uncertainties involved? Q: In fact, both Dr. Dubowski and Dr. Jones, the two most well-known and well-published experts in this field, disagree with the use of back extrapolation, right? Q: Are you aware that Dr. Jones has referred to the practice as dubious? Q: What elimination rate are you using? Q: Isn t it true that the scientific literature reports a wide range of elimination rates found in test subjects and that.015 (or 0.20 or whatever the witness used) is merely a commonly used AVERAGE?

18 Q: As you sit here today, can you tell this jury what my client s elimination rate was on the night he was arrested? Q: So if it was not (or whatever rate was used), then your calculation would be off, wouldn t it? Q: Another important variable in retrograde extrapolation is the rate of absorption, correct? Q: Would you agree that the calculation itself is meaningless if the person is still absorbing alcohol? Q: In order to perform this calculation, you made the assumption that all of the alcohol in my client s stomach had been fully absorbed and that he was in what is commonly referred to as the elimination phase? Q: Would you agree that the scientific literature also reports a wide range of absorption rates? Q: In fact, Dr. Dubowski and Dr. Jones, as well as many others, have experimented and written extensively on all of the many variables that can impact absorption rates, correct? Q: Do you agree with Dr. Jones that the rate of absorption of alcohol can be impacted by the type of alcohol a person drinks? (ie. beer vs. whiskey) Q: Time of day? (for this factor and the ones that follow, see Garriott s at pg 57) Q: Trauma? Q: Cigarette smoking? Q: Carbonated drinks? Q: Prescription medications? Q: Low blood sugar? Q: Those are lots of variables, aren t they? Q: Food in the stomach?

19 Q: Do you know how much food was in my client s stomach that evening? Q: You re really just making assumptions with regard to whether or not food might have slowed down the alcohol absorption process, right? Q: Body fat vs. muscle mass is another variable that needs to be taken into consideration, wouldn t you agree? Q: Dr. Widmark used Swedish subjects in the 1930 s for his study, correct? Q: In fact, researchers after Widmark have recommended using body mass index numbers to more accurately reflect a population that perhaps is not quite the same as the 1930 s Swedes that Dr. Widmark tested, correct? Q: After all, people today tend to carry more body fat, don t they? Q: When taking into account all of the uncertainty involved in attempting to back extrapolate, the calculated range you just gave the jury of what my client s alcohol concentration MIGHT HAVE BEEN when he was driving amounts to nothing more than a guess, right? (witness might say it s an educated guess but emphasize that it s still a guess)..

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