Analysis Of Breath Analyzers Biology Essay

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Breath analysis, a promising new field of medicine and medical instrumentation, potentially offers non-invasive, real-time, and point-of-care (POC) disease diagnostics and metabolic status monitoring. Numerous breath biomarkers have been detected and quantified so far by using the GC-MS technique. Recent advances in laser spectroscopic techniques and laser sources have driven breath analysis to new heights, moving from laboratory research to commercial reality. Laser spectroscopic detection techniques not only have high-sensitivity and high-selectivity, as equivalently offered by the MS-based techniques, but also have the advantageous features of near real-time response, low instrument costs, and POC function. Of the approximately 35 established breath biomarkers, such as acetone, ammonia, carbon dioxide, ethane, methane, and nitric oxide, 14 species in exhaled human breath have been analyzed by high-sensitivity laser spectroscopic techniques, namely, tunable diode laser absorption spectroscopy (TDLAS), cavity ring down spectroscopy (CRDS), integrated cavity output spectroscopy (ICOS), cavity enhanced absorption spectroscopy (CEAS), cavity leak-out spectroscopy (CALOS), photo acoustic spectroscopy (PAS), quartz-enhanced photo acoustic spectroscopy (QEPAS), and optical frequency comb cavity-enhanced absorption spectroscopy (OFC-CEAS). Spectral fingerprints of the measured biomarkers span from the UV to the mid-IR spectral regions and the detection limits achieved by the laser techniques range from parts per million to parts per billion levels. Sensors using the laser spectroscopic techniques for a few breath biomarkers, e.g., carbon dioxide, nitric oxide, etc. are commercially available. This review presents an update on the latest developments in laser-based breath analysis.


It is generally accepted that modern breath analysis started with the discovery made by Pauling in 1971 that hundreds of volatile organic compounds (VOCs) are present in normal human breath at the levels of parts per billion (ppb) or lower This discovery, together with other early breath studies, has regenerated research interest in human breath analysis for non-invasive disease diagnosis and metabolic status monitoring. Breath analysis can be classified into two groups: analysis of breath metabolites after administration of a drug or substrate and analysis of breath compounds produced endogenously due to a particular physiological status. Normal human breath contains a few atmospheric molecules, e.g., H2O, CO2, N2, O2, in relatively high concentrations, several VOCs, e.g., acetone, isoprene, propanols, etc., at the parts per million (ppm) or sub ppm levels, and about four hundred major VOCs (of more than 1,000 breath compounds) at the ppb or parts per trillion (ppt) levels. To date, some VOCs have been established as biomarkers for specific diseases or metabolic disorders. For instance, alkanes are present in the case of lung cancer and formaldehyde in the case of breast cancer; the presence of isoprene in human breath is related to blood cholesterol levels; and patients with Type 1 diabetes have excess acetone in their breath. Such knowledge suggests that breath analysis is useful for human disease diagnosis and/or metabolic status monitoring. However, due to the low concentrations and large quantity of trace compounds in exhaled breath, breath analysis requires a highly sensitive and highly selective instrument in order to identify and determine concentrations of specific biomarkers. One major technique primarily employed for breath gas analysis is gas chromatography-mass spectrometry (GC-MS). This method has a routine detection sensitivity of ppb to ppt and can analyze multiple compounds simultaneously and selectively; yet, GC-MS requires complicated procedures for sample collection and pre-concentration and also has high instrument costs. Extensive studies have been conducted to identify and quantify breath biomarkers using GC-MS, to improve the methods of breath sample preparation, and to miniaturize devices. However, current MS-based breath analyses are still limited to laboratory research, beyond the consideration of an affordable, real-time, point-of-care (POC) clinical instrument. In addition to conventional GC-MS methods, a relatively new technique, proton transfer reaction mass spectrometry (PTR-MS), has been used for breath profiling. Vacuum-free ion mobility spectroscopy (IMS) combined with a multi-capillary column has also been used for identification of metabolites and bacteria in human breath. IMS is slightly less sensitive than GC-MS and PTR-MS and shows much potential for the development of a hand-held breath device. By comparison, selected ion flow tube mass spectrometry (SIFT-MS), which also belongs to the MS-based category, performs exceptionally well in clinical breath analysis; on-line breath analysis of many breath compounds under various physiological conditions have been conducted in clinics with actual human breath. Breath analysis is also conducted by using electrical sensors, which are comparatively inexpensive and smaller in size, but they have low detection selectivity and require frequent calibrations.

Recent advances in high-sensitivity, high-selectivity laser spectroscopic techniques as well as laser sources make it possible for breath analysis to advance from the MS-based, time-consuming, laboratory studies to laser-based, real-time, clinical testing. Many breath biomarkers have been detected by the laser-based techniques, and detection sensitivities are comparable with those from MS-based measurements, e.g., ranging from the ppm to ppt levels. Several excellent reviews have discussed the current status, trends, and challenges of clinical breath analysis, the MS-based analytical techniques in breath analysis, and advanced laser techniques and laser sources in breath analysis.

Chemistry Involved:

When the user exhales into the breathalyzer, any ethanol present in their breath is oxidized to acetic acid at the anode:

CH3CH2OH(g) + H2O(l) → CH3CO2H(l) + 4H+(aq) + 4e-

At the cathode, atmospheric oxygen is reduced:

O2(g) + 4H+(aq) + 4e- → 2H2O(l)

The overall reaction is the oxidation of ethanol to acetic acid and water.

CH3CH2OH(l) + O2(g) → CH3COOH(l) + H2O(l)

The electrical current produced by this reaction is measured, processed, and displayed as an approximation of overall blood alcohol content by the breathalyzer.

Breath analyzers do not directly measure blood alcohol content or concentration, which requires the analysis of a blood sample. Instead, they estimate BAC indirectly by measuring the amount of alcohol in one's breath. Two breathalyzer technologies are most prevalent. Desktop analyzers generally use infrared spectrophotometer technology, electrochemical fuel cell technology, or a combination of the two. Hand-held field testing devices are generally based on electrochemical platinum fuel cell analysis and, depending upon jurisdiction, may be used by officers in the field as a form of "field sobriety test" commonly called PBT (preliminary breath test) or PAS (preliminary alcohol screening) or as evidential devices in POA (point of arrest) testing.

How does ethanol get to the lungs?

Ethanol must first enter the bloodstream

Ethanol is a water soluble compound-it dissolves readily in water-based solutions and once it is swallowed, it moves into water spaces throughout the body, including the bloodstream. Here is a brief review of what happens:

§ Molecules of ethanol travel across the membranes made of epithelial cells that line the stomach and small intestine.

§ Ethanol molecules move across the membrane via passive diffusion.

§ Once on the other side of the gut cells, these small ethanol molecules then easily pass through the

walls of the tiny capillaries that line the gut.

Absorption of ethanol from the gut into the bloodstream. Ethanol molecules in the gut diffuse passively across epithelial cells, through the interstitial space, and then into nearby capillaries.

Ethanol travels through the bloodstream to the lungs

Ethanol, in its liquid form, travels in the capillaries to the veins, and then heads up to the lungs. It takes the same route that deoxygenated blood takes to become oxygenated.

§ Ethanol is carried by the venous circulation to the right side of the heart.

§ Ethanol enters the right atrium (like a foyer), and then moves into the right ventricle (a pump).

§ The right ventricle pumps the venous blood to the lungs.

§ In the lungs, red blood cells unload carbon dioxide and pick up oxygen brought in by respiration (breathing).

§ Some of the ethanol dissolved in the blood gets eliminated as a gas by the lungs during exhalation.

§ Ethanol that remains in the blood returns to the left side of the heart (the atrium).

§ The left atrium supplies blood to the left ventricle of the heart, which pumps the blood to the rest of the water-containing compartments and tissues of the body via the arteries.

The branch of the circulatory system that moves blood between the heart and the lungs is called the pulmonary circulation, while the branch that moves blood throughout the rest of the body is called the systemic circulation.

Ethanol vaporizes to a gas in the lungs

In the lungs, ethanol moves by passive diffusion from the capillaries into the surrounding air sacs called alveoli. Unlike the process of active transport, passive diffusion requires no additional energy to move molecules. The energy comes from the difference in the concentration of ethanol between the two compartments: blood and lung alveoli (substances move from areas of high concentration to areas of low concentration).

Let's look more closely at the process of ethanol vaporization.

At room and body temperatures, ethanol can exist as a liquid or a gas.

§ Liquid - individual ethanol molecules are tethered to one another by two types of chemical forces: hydrogen bonds and Van der Waals forces.

§ Gas - when liquid ethanol molecules have enough energy to break the hydrogen bonds connecting them together, they escape into the gas state.

Compounds, like ethanol, that can easily change from a liquid to gas are called volatile compounds. Volatility is related to the number of hydrogen bonds. Let's compare ethanol and water. Individual ethanol molecules can only form three hydrogen bonds with neighbouring ethanol molecules, while water molecules can form as many as four hydrogen bonds with neighbouring water molecules. So, ethanol requires less energy to break three hydrogen bonds (ethanol) than four hydrogen bonds (water). Therefore, ethanol is more volatile than water.

How does the Breath analyzer work?

The original version of the Breath Analyzer included a mouthpiece and two chambers containing liquid connected to a meter that detects a change in colour. To use the Breath Analyzer, the subject exhales through the mouthpiece into a test chamber filled with a reddish-orange solution of potassium dichromate (K2Cr2O7).

In the Breath Analyzer, alcohol reacts with the reddish-orange potassium dichromate solution and turns green. The degree of the color change is directly related to the level of alcohol in the expelled air.

A photocell compares the difference in colors between the reacted mixture in the test chamber and a reference chamber containing unreacted mixture. The difference in colors produces an electrical current, which can be converted into a quantitative value for the BAC. Read on to understand how the chemical reaction produces the color change.

Redox chemistry inside the Breath Analyzer.

The chemical reaction inside the Breathalyzerâ„¢ includes both oxidation and reduction. The Breath Analyzer contains a chamber with several compounds to support these reactions. They include:

§ Potassium dichromate

§ Sulphuric acid

§ Silver nitrate

When the potassium dichromate solution in the Breath Analyzer reacts with ethanol, the potassium dichromate loses an oxygen atom.

§ This process is called reduction when a compound loses oxygen, gains hydrogen, or gains (partially gains) electrons.

§ The reduction converts orange potassium dichromate into a green solution containing chromium sulphate.

At the same time dichromate ion gets reduced to chromium ion, ethanol gets oxidized to acetic acid.

Oxidation reactions often occur simultaneously with reduction reactions and are commonly abbreviated as redox reactions.

§ Oxidation occurs when an element combines with oxygen to give an oxide. For example, the oxide of hydrogen is water.

§ Oxidation is the gain of oxygen, the loss of hydrogen, or the loss (or partial loss) of electrons

Silver nitrate serves as a catalyst for the reaction to increase the rate at which the dichromate gets reduced.

Sulphuric acid in the test chamber helps to remove the alcohol from the exhaled air into the test solution and to provide the necessary acidic conditions.

Oxidation and reduction (redox) reactions are opposing reactions that occur simultaneously.

Breathe Biomarkers

Acetaldehyde (C2H4O) Acetaldehyde has been identified as a biomarker for alcoholism and lung cancer [18,112]. Normal human breath acetaldehyde ranges from 0 to 140 ppb [113]. Only one publication has reported the investigation of acetaldehyde in exhaled human breath [75], in which measurements of acetaldehyde in exhaled breath following the ingestion of 375 mL of wine (12.5% alcohol) were conducted by using the TDLAS technique. Acetaldehyde exhibits a strong absorption band in the spectral range of 1,680 to 1,820 cm-1), which is attributed to the υ4 band, and the strongest absorption is at 1,764 cm-1. In that study, however, the absorption of acetaldehyde near 1,727.1 cm-1) was measured at 26 Torr in an astigmatic Herriott cell with an effective path-length of 100 m. The detection limit for the acetaldehyde was reported to be 80 ppb and 30 ppb for an integration time of 5 and 45 s, respectively. The authors noted that if the molecular fingerprint at (1,764 cm-1) is used, the detection limits can be improved by a factor of 2, which will be close to the required detection limit, 10 ppb, for analysis of acetaldehyde in human breath. Measurements of acetaldehyde in non-breath gas using the laser spectroscopic techniques have been reported and can be read elsewhere. Acetone ((CH3)2CO).The mean acetone concentration in healthy breath reported in the literature varies from 0.39 to 0.85 ppm, and the overall mean value is ~0.49 ± 0.20 ppm. Elevated acetone concentrations in the exhaled breath of diabetic subjects have been reported. Elevated breath acetone also exists in exhaled breath of children who are on a high-fat diet for the treatment of epilepsy. Acetone concentrations in human breath can also be an indicator of congestive heart failure and cardiac index. CRDS of acetone in the UV and NIR spectral regions have been reported. Wang et al. demonstrated a portable acetone detection device using CRDS at a single wavelength at 266 nm and evaluated the instrument performance using both standard acetone solution samples and actual human breath under various situations.

Ammonia (NH3) Ammonia concentration in normal human breath is in the range of 0.25-2.9 ppm. Ammonia is a biomarker of renal failure, Helicobacter pyroli and oral cavity disease. One of the earliest measurements of ammonia in human breath was reported by Lachish et al. They used the TDLAS technique combined with a high resolution MIR lead-salt light source. In the near-real time measurement of ammonia, the laser was locked on the peak of the absorption line of ammonia at 930.76 cm-1. The absorption of ammonia was observed in a sample cell with a path-length of 50 cm. The detection limit was 1 ppm with an integration time of 10 s. Manne et al reported a prototype breath gas analyzer based on the CRDS technique for quantification of ammonia in human breath. A thermo-electrically cooled distributed feedback (DFB) quantum cascade diode laser (QCDL) was used as the MIR laser source operating at 10.3 μm (970 cm-1). The system showed a detection limit of 50 ppb for ammonia with a response time of 20 s. Recently, the same group has developed a sensor for measurements of breath ammonia with a pulsed quantum cascade laser using intra- and inter-pulse spectroscopic techniques. The system utilized an astigmatic Herriott gas cell with a 150 m effective path-length and a pulsed DFB-QCDL operating at 10.6 μm. The detection limits for breath ammonia of the system were 3 ppb when the system was operated in the intra-pulse method with an integration time of less than 10 s and 4 ppb in the inter-pulse method with an integration time of ~5 s. Simultaneous measurements of multiple breath species, including ammonia, were demonstrated by the Ye group using the OFC-CEAS technique. In that study, an NIR spectral window centered around 1.51 μm (6,623 cm-1) was used, where absorptions of ammonia and water were relatively overlapped. Using calibrated samples with 4.4 ppm ammonia in nitrogen as a reference, they obtained a detection limit of 18 ppb. Moskalenko et al. reported an automated TDLAS system operating in the MIR and measured multiple compounds in smokers' and non-smokers' exhaled breath, including ammonia, carbon monoxide, methane, and carbon dioxide. They reported ammonia concentrations in the smokers and non-smokers' breath had no significant difference and they were all in the range of 130-200 ppb. The detection limit for ammonia measured at 10.3 mm was 5 ppb for a response time of 30 sec.

Carbon dioxide (CO2) and 13C-isotope Carbon dioxide and carbon isotopes have been established as a biomarker of Helicobacter pyroli infections, liver malfunction, and excessive growth of bacteria in body etc. CO2 has a strong absorption around 4.3 μm (2,326 cm-1) which allows highly sensitive detection of CO2 and its 13C isotope using direct laser absorption spectroscopy with MIR laser sources, such as lead-salt lasers as shown by Becker et al. [128] and DFG light sources as shown by Erdelyi et al. Crosson et al conducted measurements of CO2 in breath samples using cw-CRDS with an external cavity diode laser (ECDL) at 1.597 μm (6,262 cm-1) in a laboratory system at 100 Torr, a detection limit of 3 ppm for CO2 was demonstrated.


Many handheld breathalyzers sold to consumers use a silicon oxide sensor (also called a semiconductor sensor) to determine the blood alcohol concentration. These sensors are far more prone to contamination and interference from other substances besides breath alcohol. The sensors require recalibration or replacement every six months. Higher end personal breathalyzers and professional-use breath alcohol testers use platinum fuel cell sensors.These too require recalibration but at less frequent intervals than semiconductor devices, usually once a year.

Calibration is the process of checking and adjusting the internal settings of a breathalyzer by comparing and adjusting its test results to a known alcohol standard. Law enforcement breathalyzers are meticulously maintained and re-calibrated frequently to ensure accuracy.

There are two methods of calibrating a precision fuel cell breathalyzer, the Wet Bath and the Dry Gas method. Each method requires specialized equipment and factory trained technicians. It is not a procedure that can be conducted by untrained users or without the proper equipment.

The Dry-Gas Method utilizes a portable calibration standard which is a precise mixture of alcohol and inert nitrogen available in a pressurized canister. Initial equipment costs are less than alternative methods and the steps required are fewer. The equipment is also portable allowing calibrations to be done when and where required.

The Wet Bath Method utilizes an alcohol/water standard in a precise specialized alcohol concentration, contained and delivered in specialized simulator equipment. Wet bath apparatus has a higher initial cost and is not intended to be portable. The standard must be fresh and replaced regularly.

Some semiconductor models are designed to allow the sensor module to be replaced without the need to send the unit to a calibration lab.

Breathalyzer myths

There are a number of substances or techniques that can supposedly "fool" a breathalyzer (i.e., generate a lower blood alcohol content).

A 2003 episode of the popular science television show MythBusters tested a number of methods that supposedly allow a person to fool a breathalyzer test. The methods tested includedbreath mints, onions, denture cream, mouthwash, pennies and batteries; all of these methods proved ineffective. The show noted that using items such as breath mints, onions, denturecream and mouthwash to cover the smell of alcohol may fool a person, but, since they will not actually reduce a person's BAC, there will be no effect on a breathalyzer test regardless of the quantity used. Pennies supposedly produce a chemical reaction, while batteries supposedly create an electrical charge, yet neither of these methods affected the breathalyzer results.

The Mythbusters episode also pointed out another complication: It would be necessary to insert the item into one's mouth (e.g. eat an onion, rinse with mouthwash, conceal a battery), take the breath test, and then possibly remove the item - all of which would have to be accomplished discreetly enough to avoid alerting the police officers administering the test (who would obviously become very suspicious if they noticed that a person was inserting items into their mouth prior to taking a breath test). It would likely be very difficult, especially for someone in an intoxicated state, to be able to accomplish such a feat.

In addition, the show noted that breath tests are often verified with blood tests (which are more accurate) and that even if a person somehow managed to fool a breath test, a blood test would certainly confirm a person's guilt.

Other substances that might reduce the BAC reading include a bag of activated charcoal concealed in the mouth (to absorb alcohol vapor), an oxidizing gas (such as N2O, Cl2, O3, etc.) that would fool a fuel cell type detector, or an organic interferent to fool an infrared absorption detector. The infrared absorption detector is more vulnerable to interference than a laboratory instrument measuring a continuous absorption spectrum since it only makes measurements at particular discrete wavelengths. However, due to the fact that any interference can only cause higher absorption, not lower, the estimated blood alcohol content will be overestimate. Additionally, Cl2 is rather toxic and corrosive.

A 2007 episode of the Spike network's show Manswers showed some of the more common and not-so-common ways of attempts to beat the breathalyzer, none of which work. Test 1 was to suck on a copper coin. (Actually, copper coins are now generally often only copper-coated and mostly zinc or steel.Test 2 was to hold a battery on the tongue. Test 3 was to chew gum. None of these tests showed a "pass" reading if the subject had consumed alcohol.