Chemistry, General/Organic – Instrumental Analysis Source: Conklin et al., J. Chem. Educ. 2014, 91, 889–891 | Lab Experiment
Difficulty: Introductory to Intermediate Prerequisites: Basic understanding of molecular structure (functional groups, bonds). Familiarity with Beer’s Law and the concept of a calibration curve is helpful but not essential.
This topic sits at the intersection of organic chemistry and instrumental analysis. You are learning how to use infrared light to identify and quantify a specific molecule (ethanol) in a complex mixture (gasoline). The technique, FT-IR spectroscopy, is one of the workhorses of analytical chemistry, and this experiment is a practical, real-world application of it.
If you have covered functional groups and know that different bonds absorb IR light at characteristic frequencies, you have enough background. If you have not, start with a primer on IR spectroscopy before diving in here.
Ethanol in gasoline can be measured by collecting an FT-IR spectrum and comparing the size of the O–H absorption (from ethanol) to the C–H absorption (from alkanes). You build a calibration curve from known ethanol/hexanes mixtures, then use it to calculate the ethanol percentage in a real gasoline sample. The method is straightforward, reasonably accurate, and teaches core quantitative analysis skills.
FT-IR spectroscopy (Fourier-transform infrared spectroscopy)
An analytical technique that passes infrared light through (or bounces it off) a sample and measures which wavelengths the sample absorbs. The pattern of absorptions reveals what functional groups are present, and the size of each absorption can tell you how much of a substance is there.
In simple terms, it is a way to fingerprint molecules using infrared light.
Oxygenate
A compound containing oxygen that is added to gasoline to promote more complete combustion and reduce harmful emissions. Ethanol is the primary oxygenate used in the United States today; MTBE was the main one before it was phased out.
Think of it as an oxygen-delivery additive that helps fuel burn cleaner.
O–H absorption (O–H stretch)
The broad infrared absorption band in the 3500–3000 cm⁻¹ region caused by the stretching vibration of the oxygen–hydrogen bond in alcohols. In this experiment, it is the signal that comes from ethanol.
In simple terms, this is the "ethanol signal" in the IR spectrum.
C–H absorption (C–H stretch)
The infrared absorption band in the 3000–2800 cm⁻¹ region caused by the stretching vibrations of carbon–hydrogen bonds in alkanes. In this experiment, these absorptions come primarily from the hexanes (or gasoline alkanes) and serve as the internal reference signal.
Think of it as the "alkane signal" that stays roughly constant and lets you measure ethanol against it.
Wavenumber (cm⁻¹)
The unit used to describe the position of an absorption in an IR spectrum. It is the reciprocal of the wavelength in centimetres. Higher wavenumbers correspond to higher-energy vibrations.
In simple terms, it is the x-axis label on an IR spectrum, measured in inverse centimetres.
ATR (attenuated total reflectance)
A sampling technique for IR spectroscopy where the sample is pressed against a crystal and the infrared beam bounces internally within the crystal, interacting with the sample at the surface. It requires very little sample and no special preparation.
Think of it as a "press and measure" accessory for the IR spectrometer.
Calibration curve (standard curve)
A graph that plots a known property (here, percent ethanol) against a measured response (here, the ratio of O–H to C–H peak areas). Once built from standards of known concentration, it lets you read off the concentration of an unknown sample from its measured response.
In simple terms, it is the reference chart you build so you can translate a measurement into a concentration.
Internal standard
A component in the sample whose signal is used as a reference point against which the analyte signal is measured. In this experiment, the alkane C–H absorption acts as a quasi-internal standard because the alkane content of gasoline is relatively constant and provides a built-in reference.
E10 / E85
Shorthand for gasoline blended with ethanol. E10 contains 10% ethanol by volume (the most common blend at petrol stations). E85 contains 85% ethanol and is only available in limited areas near ethanol production facilities.
Ethanol is the primary oxygenate blended into US gasoline. It replaced MTBE (methyl tert-butyl ether), which was phased out due to groundwater contamination concerns.
Ethanol is produced from ethene, grain (mainly corn), and as a byproduct of beer production.
The most common blend is E10 (10% ethanol by volume). Higher blends (E5 through E85) exist, but E85 is largely restricted to areas near ethanol-manufacturing plants.
Environmental, safety, and health concerns around ethanol-gasoline blends make accurate measurement of ethanol content important.
Different chemical bonds absorb infrared light at characteristic frequencies. The O–H bond in ethanol and the C–H bonds in alkanes absorb in distinct, well-separated regions of the IR spectrum.
O–H stretch region: approximately 3500–3000 cm⁻¹. This absorption is present only when ethanol (or another alcohol) is in the mixture. Water is not soluble in the alkane portion of gasoline, so any O–H signal comes from ethanol.
C–H stretch region: approximately 3000–2800 cm⁻¹. This absorption comes from the alkanes that make up the bulk of gasoline (or hexanes in the model system). It serves as the internal reference.
By comparing the area of the O–H peak to the area of the C–H peak, you get a ratio that is directly related to the percentage of ethanol present.
Model system: ethanol mixed with hexanes at known percentages (0%, 1%, 3%, 5%, 10%, 30%, 50%, 85% by volume). Hexanes are used because they are a major component of gasoline and are representative of C5–C12 alkanes.
For each standard, collect an FT-IR spectrum in absorbance mode.
Measure the area under the O–H absorption (about 3660–3045 cm⁻¹) and the area under the C–H absorption (about 3045–2760 cm⁻¹).
Calculate the ratio: (Area of O–H peak) / (Area of C–H peaks).
Plot percent ethanol (x-axis) against the area ratio (y-axis). The resulting calibration curve is linear, with typical R² values of 0.99.
Collect an FT-IR spectrum of the gasoline sample using the same instrument settings and baseline corrections as the standards.
Measure the O–H and C–H peak areas and calculate their ratio.
Use the calibration equation to determine the ethanol percentage:
% Ethanol = [(Area of O–H peak) / (Area of C–H peaks) – (y-intercept)] / Slope
A representative calibration equation from the paper: y = 0.0268x + 0.0039 (R² = 0.99249), where x = % ethanol by volume.
Reagent-grade ethanol is denatured with methanol and 2-propanol (it contains 90.25% ethanol, 4.75% methanol, 5.00% 2-propanol). These other alcohols introduce small systematic errors.
The C–H region also picks up some signal from the alcohol itself, so treating it as a pure alkane reference is an approximation. At low ethanol concentrations (E10 and below), this error is small.
The limit of detection is approximately 3% ethanol. Below that, the O–H signal is indistinguishable from the hexanes-only baseline (the 1% standard is indistinguishable from 0%).
Consistency in defining peak integration regions and baseline correction is critical. Inconsistent settings between standards and samples will degrade accuracy.
Student results in a general-education course averaged 14% (range 12–18%) for gasoline advertised as containing up to 10% ethanol, largely because of difficulties in preparing standard solutions precisely.
Calibration equation (linear fit):
y = 0.0268x + 0.0039
where y = (Area of O–H peak) / (Area of C–H peaks) and x = % ethanol by volume. R² = 0.99.
Rearranged to solve for % ethanol:
% Ethanol = [y – 0.0039] / 0.0268
Or in general form:
% Ethanol = [(Area O–H / Area C–H) – y-intercept] / Slope
Key spectral regions to memorise:
O–H stretch (ethanol signal): 3660–3045 cm⁻¹ (broad band)
C–H stretch (alkane reference): 3045–2760 cm⁻¹ (sharp, strong peaks)
The old MTBE fingerprint region (1000–800 cm⁻¹) is no longer used because MTBE has been phased out of gasoline.
This is how fuel regulators and quality-control labs verify that the ethanol content printed on the pump matches what is actually in the tank. FT-IR is one of several methods (alongside gas chromatography, NMR, and Raman spectroscopy) used for routine fuel analysis.
The same ratio-based IR approach can be adapted to quantify other additives or contaminants in fuel blends, and the calibration-curve workflow you learn here is the backbone of nearly every quantitative instrumental method in chemistry.
Students often assume the O–H signal in gasoline could come from water. It does not. Water is insoluble in the alkane mixture that makes up gasoline, so any O–H absorption is from ethanol.
Students sometimes think you can read the ethanol percentage directly from the height of the O–H peak. You cannot. You need the ratio of O–H area to C–H area because the absolute peak size depends on how much total sample the beam interacts with, which varies from run to run.
There is a common mix-up between percent by volume and percent by mass. This experiment measures percent by volume. A 10.5% ethanol-in-hexanes solution by mass corresponds to only about 8.9% by volume because ethanol is less dense than hexanes.
Students sometimes believe the calibration curve should pass through the origin. It may have a small positive y-intercept (0.0039 in the representative data), which is normal and accounted for in the equation.
Be able to identify the O–H and C–H stretch regions on an IR spectrum and explain what each represents in this experiment.
Know how to construct and use a calibration curve: plot the data, find the equation of the line, and use it to determine an unknown concentration.
Understand why a ratio (O–H area / C–H area) is used rather than the raw O–H area alone. This is the concept of an internal standard.
Be prepared to explain why hexanes are a suitable model for gasoline (C5–C12 alkane mixture, readily available, safe).
Know the limit of detection for this method (approximately 3% ethanol) and what that means practically.
True or false: The O–H absorption in gasoline’s IR spectrum could come from dissolved water. (False – water is insoluble in the alkane mixture.)
Fill in the blank: The C–H absorption in this experiment acts as a quasi-_______ standard. (internal)
True or false: A calibration curve with R² = 0.99 means every data point falls exactly on the line. (False – it means the fit is very good, but not perfect.)
The O–H stretch appears in the _______ to _______ cm⁻¹ region. (3500 to 3000, or more precisely 3660 to 3045 for integration purposes.)
True or false: This method can reliably detect 1% ethanol in gasoline. (False – the limit of detection is approximately 3%.)
Q: Why is the ratio of O–H peak area to C–H peak area used, rather than the O–H peak area alone?
A: Using the ratio normalises for differences in how much sample the IR beam interacts with between runs. The C–H signal from the alkanes acts as an internal reference, so variations in path length or sample amount cancel out. This makes the measurement more reproducible.
Q: A gasoline sample gives an O–H/C–H area ratio of 0.272. Using the calibration equation y = 0.0268x + 0.0039, calculate the ethanol percentage.
A: x = (0.272 – 0.0039) / 0.0268 = 0.2681 / 0.0268 = 10.0% ethanol by volume.
Q: Explain why hexanes are chosen as the solvent for preparing calibration standards instead of using ethanol-free gasoline.
A: Hexanes are a commercially available mixture of C6 alkanes that are representative of the C5–C12 alkanes found in gasoline. Ethanol-free gasoline is difficult to obtain in many areas, and removing ethanol from gasoline by water extraction introduces additional hazards (aromatic hydrocarbons) and takes extra time. Hexanes are cheap, safe, and consistently available.
Q: A student obtains a calibration curve but finds that several data points fall well off the linear fit. What is the most likely source of this error?
A: The most likely cause is inaccurate preparation of the standard solutions (imprecise measurement of ethanol and hexanes volumes). Other possible causes include inconsistent peak integration regions or baseline correction settings between measurements.
Q: Why does this method have a detection limit of approximately 3% ethanol?
A: At very low ethanol concentrations, the O–H absorption is too small to distinguish from the baseline noise. The 1% standard produces an area ratio that is indistinguishable from that of pure hexanes (0% ethanol), so concentrations below about 3% cannot be reliably measured.
This material connects directly to Beer’s Law and quantitative spectroscopy: the idea that absorbance is proportional to concentration is what makes the calibration curve linear. If your course covers UV-Vis spectroscopy, the workflow (prepare standards, measure absorbance, plot calibration curve, determine unknown) is identical.
The experiment also ties into organic chemistry functional group identification. Recognising O–H and C–H stretches on an IR spectrum is a core skill in any organic chemistry lab, and this experiment gives you a quantitative reason to care about peak areas, not just peak positions.
For environmental chemistry, the ethanol-in-gasoline question connects to biofuels policy, emissions regulations, and the broader shift from petroleum-only fuels to blended fuels.
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