Difficulty: Intermediate | Prerequisites: Amino acid biosynthesis and transamination theory (see companion notes). Basic understanding of electromagnetic radiation and chromatography principles.
Once you have run a transamination reaction, you need to confirm that it worked and identify the product. This topic covers two analytical techniques used in tandem: UV/Vis spectroscopy to monitor the reaction's progress in real time, and thin-layer chromatography (TLC) to identify the amino acid product after the reaction is complete. Both techniques appear across organic and biochemistry labs, and understanding what the data means (and what can go wrong) is as important as running the instruments.
UV/Vis spectroscopy tracks transamination by measuring absorbance at two wavelengths: 300 nm (PMP absorbs here) and 385 nm (PLP absorbs here). As the reaction proceeds, the 300 nm signal should fall and the 385 nm signal should rise. TLC then identifies the amino acid product by comparing Rf values and spot characteristics (colour, UV activity) against known standards.
UV/Vis spectroscopy
A technique that measures how much ultraviolet or visible light a sample absorbs at each wavelength. The absorbance at a given wavelength is proportional to the concentration of the absorbing species (Beer-Lambert law). In simple terms, you shine light through the solution and measure what comes out the other side to see how much of a particular molecule is present.
Absorbance
A unitless quantity equal to log₁₀(I₀/I), where I₀ is the incident light intensity and I is the transmitted intensity. Higher absorbance means the sample is absorbing more light at that wavelength, which means more of the absorbing molecule is present.
Beer-Lambert law
A = εlc, where A is absorbance, ε is the molar absorptivity (a constant for each molecule at each wavelength), l is the path length of the cuvette, and c is the concentration. This is the equation that lets you convert absorbance readings into concentration data.
Thin-layer chromatography (TLC)
A chromatographic technique in which compounds are separated on a thin layer of adsorbent (usually silica gel) on a glass or plastic plate. The sample is spotted near the bottom, and a solvent (mobile phase) travels up by capillary action, carrying different compounds to different heights depending on their polarity.
Rf value (retention factor)
The ratio of the distance a compound travels to the distance the solvent front travels, measured from the origin. Rf = distance of spot / distance of solvent front. Values range from 0 to 1. Each compound has a characteristic Rf under given conditions, which is how you identify unknowns by comparison with standards.
Ninhydrin stain
A chemical stain that reacts with free amino groups to produce a coloured spot (typically purple or reddish). Used to visualise amino acids on a TLC plate, since most amino acids do not absorb UV light and would otherwise be invisible.
Rotary evaporation (rotovap)
A technique for removing solvent under reduced pressure. In this experiment, the reaction mixture is concentrated by rotovap before TLC analysis so that the amino acid product is present at high enough concentration to show up as a visible spot.
Two wavelengths are monitored simultaneously:
300 nm: PMP (the starting amine donor) absorbs here. As PMP is consumed, absorbance at 300 nm should decrease over time.
385 nm: PLP (the aldehyde product) absorbs here. As PLP is formed, absorbance at 385 nm should increase over time.
Readings are taken at regular intervals (every 20 minutes in this experiment) to build an absorbance-versus-time plot.
A linear trendline fitted to each dataset gives the rate of PMP consumption and PLP formation. The slope at 300 nm should be negative; the slope at 385 nm should be positive.
Expected result: negative slope at 300 nm, positive slope at 385 nm, confirming conversion of PMP to PLP.
What went wrong in this lab: most groups obtained inconsistent slopes. Possible sources of error include:
Contamination of the aliquots extracted at each time point.
Inaccurate timing of the 20-minute intervals.
Inaccurate volumes of reagents (methanol, PMP solution, zinc perchlorate).
PMP or the keto acid may have degraded or not been at the expected concentration.
The R² values reported (0.68 at 300 nm, 0.94 at 385 nm) indicate a poor linear fit for the 300 nm data and a reasonable fit for the 385 nm data. When R² is low, the trendline does not explain much of the variation, and drawing conclusions from the slope alone is unreliable.
Class-wide data showed large variation between groups (slopes ranging from negative hundreds to positive thousands), which means no single group's absorbance data was reliable enough to identify the unknown on its own.
After rotovap concentration and acidification with HCl (2 to 3 drops), the sample is spotted on a TLC plate alongside known standards.
Four lanes were spotted in this experiment:
Unknown sample
Pyridoxamine dihydrochloride (a reference for the cofactor)
Alanine (one possible amino acid product)
Phenylalanine (the other possible amino acid product)
Rf values observed:
Unknown: 0.40
Pyridoxamine dihydrochloride: 0.20
Alanine: 0.55
Phenylalanine: 0.50
The unknown's Rf (0.40) is closer to phenylalanine (0.50) than to alanine (0.55), providing initial evidence for identification.
UV activity: the unknown spot, pyridoxamine dihydrochloride, and phenylalanine all showed up under UV light. The alanine spot did not appear until after ninhydrin staining. This is a useful distinguishing feature because phenylalanine's aromatic ring absorbs UV, while alanine has no chromophore.
Spot colour after staining: the unknown and phenylalanine both had reddish tints; alanine was yellowish. Colour match supports the identification.
Conclusion from TLC: the unknown is phenylalanine, identified by the convergence of three lines of evidence: Rf value, UV activity, and spot colour.
UV/Vis relies on quantitative measurement of small absorbance changes over time. Small errors in technique compound across multiple readings.
TLC is a qualitative comparison. You are matching an unknown against standards run under identical conditions on the same plate. It is more robust against the kind of systematic errors that plagued the absorbance data in this lab.
Beer-Lambert law:
A = εlc
Where: A = absorbance (unitless), ε = molar absorptivity (L mol⁻¹ cm⁻¹), l = path length (cm), c = concentration (mol L⁻¹)
Rf calculation:
Rf = distance travelled by compound / distance travelled by solvent front
Example from the lab: unknown spot travelled 0.8 cm, solvent front travelled 2.0 cm → Rf = 0.8 / 2.0 = 0.40
Linear regression equations from the lab data:
At 385 nm: f(x) = 3.758x + 0.091, R² = 0.943
At 300 nm: f(x) = 2.722x + 2.364, R² = 0.678
(Note: both slopes were positive, which is unexpected for the 300 nm data. This inconsistency is discussed above.)
UV/Vis spectroscopy is one of the most widely used analytical techniques in chemistry and biology. It is the basis of clinical assays (measuring enzyme activity, drug concentrations in blood), quality control in manufacturing, and environmental monitoring (measuring water pollutant levels). TLC remains a standard quick-check method in pharmaceutical labs for confirming the identity and purity of drug compounds before committing to more expensive analyses like HPLC or mass spectrometry.
Students often assume a higher Rf value means a "better" match. It does not. The unknown's Rf should be compared to each standard, and the closest match is the identification. In this experiment, the unknown (0.40) is closer to phenylalanine (0.50) than to alanine (0.55), but none of the values are identical. Small differences are normal because Rf depends on exact conditions (temperature, solvent composition, plate preparation).
Another error is treating UV/Vis absorbance data as automatically reliable. Absorbance readings are only meaningful when the instrument is properly blanked, the cuvettes are clean, and the concentrations fall within the linear range of Beer-Lambert. Values above approximately 2.0 often indicate the detector is saturated, and the 300 nm readings in this lab (around 2.3 to 2.5) may have been affected by this.
Students sometimes conclude that if the UV/Vis data is inconsistent, the entire experiment failed. TLC provides an independent line of evidence, and in this lab it gave a clear identification even though the spectroscopy data did not.
Confusing UV activity with ninhydrin staining is a common mix-up. UV activity (glowing or quenching under a UV lamp) depends on the molecule having a chromophore (like an aromatic ring). Ninhydrin staining depends on a free amino group. A molecule can show one, both, or neither.
⚠️ Be able to explain what absorbance at 300 nm vs. 385 nm tells you about PMP and PLP concentrations, and why the slopes should go in opposite directions.
⚠️ Know how to calculate an Rf value and use it to identify an unknown by comparison with standards.
⚠️ Understand why UV activity distinguishes phenylalanine (aromatic, absorbs UV) from alanine (non-aromatic, invisible under UV).
⚠️ Be prepared to discuss sources of experimental error in absorbance-vs.-time measurements and explain why class data can be inconsistent.
⚠️ Know the Beer-Lambert law and its assumptions (linear range, clean cuvettes, proper blanking).
True or false: At 385 nm, absorbance should decrease over time as the transamination reaction proceeds.
Fill in the blank: The Rf value is calculated as the distance the __________ travels divided by the distance the __________ travels.
True or false: Alanine absorbs UV light because it contains an aromatic ring.
Fill in the blank: In the Beer-Lambert law, ε represents the __________ of the absorbing species.
True or false: An R² value of 0.68 indicates a strong linear correlation.
Answers: 1. False (385 nm absorbance should increase, because PLP, which absorbs at 385 nm, is being formed). 2. Compound (or spot); solvent front. 3. False (alanine has no aromatic ring and does not absorb UV; phenylalanine does). 4. Molar absorptivity (or molar extinction coefficient). 5. False (0.68 is a moderate-to-weak fit; values above 0.95 are typically considered strong).
Q: A student observes that the absorbance at 300 nm increases over the course of the reaction. What does this suggest, and is it consistent with a successful transamination?
A: An increase at 300 nm suggests PMP concentration is rising, not falling. This is inconsistent with a successful transamination, in which PMP should be consumed. Possible explanations include instrument error, contamination, absorbance readings in the non-linear range (above ~2.0), or inaccurate timing of sample extraction.
Q: On a TLC plate, compound X has an Rf of 0.42, standard A has an Rf of 0.55, and standard B has an Rf of 0.43. Which standard does compound X most likely match?
A: Standard B (Rf 0.43), since it is the closest to compound X (Rf 0.42). A small difference in Rf between an unknown and a standard is expected due to minor variations in spotting and plate conditions.
Q: Why is the TLC plate stained with ninhydrin after UV visualisation?
A: Not all compounds are visible under UV light. Ninhydrin reacts with free amino groups to produce coloured spots, allowing detection of amino acids that lack a UV-absorbing chromophore (such as alanine). Running UV first, then ninhydrin, lets you record which spots are UV-active before the stain obscures the plate.
Q: Explain why UV/Vis readings above an absorbance of 2.0 may be unreliable.
A: An absorbance of 2.0 means only 1% of the incident light reaches the detector (10⁻² = 0.01). At such low transmitted intensities, the signal-to-noise ratio drops sharply, and small measurement errors produce large swings in the calculated absorbance. The Beer-Lambert relationship assumes a linear response, which breaks down at high absorbance values.
Q: In this experiment, three pieces of evidence from the TLC plate pointed to phenylalanine as the unknown. Name them.
A: (1) Rf value: the unknown (0.40) was closer to phenylalanine (0.50) than to alanine (0.55). (2) UV activity: the unknown and phenylalanine both appeared under UV light, while alanine did not. (3) Spot colour: the unknown and phenylalanine both showed reddish tints after staining, while alanine was yellowish.
This material connects to analytical chemistry (Beer-Lambert law and spectrophotometer operation), chromatography theory (partitioning, polarity, and mobile vs. stationary phase), and biochemistry lab methods (enzyme assays often rely on UV/Vis absorbance changes to measure reaction rates). If you go on to study HPLC, the principles of separation and Rf are directly analogous to retention time in column chromatography.
UV/Vis spectroscopy, ultraviolet visible spectroscopy, absorbance, Beer-Lambert law, molar absorptivity, extinction coefficient, 300 nm, 385 nm, thin-layer chromatography, TLC, Rf value, retention factor, ninhydrin stain, amino acid identification, phenylalanine, alanine, pyridoxamine dihydrochloride, rotary evaporation, rotovap, chromophore, aromatic UV absorption, organic chemistry lab techniques, analytical chemistry, spectrophotometry