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Analytical Methods and Purity

An HPLC purity result rests on three silent assumptions. Each of them can fail — and always in the direction of overestimation.

Liquid chromatography is the default method for purity determination, and that is precisely why its assumptions tend to go unnoticed. A result of “99.3% by HPLC” looks like an absolute measurement, yet it is the sum of several silent assumptions: that everything in the sample came off the column; that it came off separately; and that the detector saw it in proportion to its amount. Any of these assumptions can fail to hold, and each fails in the same direction — inflating the result.

This text describes exactly where the limits of the method lie, when an independent technique is needed, and how to read a chromatographic result without attributing to it a meaning it does not have.

The detector sees a chromophore, not a substance

The most common setup is ultraviolet detection. A signal arises when a molecule absorbs radiation at the selected wavelength — that is, when it has a suitable system of conjugated bonds. A substance without a chromophore at that wavelength is transparent to the detector: it will pass through the column, elute from it, and leave no trace on the chromatogram.

Hence the first consequence: area normalization, i.e., calculating the share of the main peak in the sum of all peaks, tacitly assumes that all components of the sample produce a signal. A single impurity without a chromophore is enough to inflate the result — and without any visible symptom on the chromatogram.

The second consequence is more subtle: even when all components absorb, they do so with different intensities. The same mass fraction gives a different peak area for different substances. This is why serious assay determinations are performed against a reference standard rather than by area normalization alone — and why purity determination methods are being developed in variants with internal standard correction.1

There are also detectors that answer different questions. Polarimetric detection, described in the chromatographic literature as early as the late 1980s, responds to optical activity and makes it possible to see what an absorbance detector cannot distinguish.2 The choice of detector is therefore a choice of which impurities stand a chance of being noticed.

Coelution: two compounds, one peak

If an impurity leaves the column at the same time as the main substance, its area is added to the main peak. The purity result goes up, and the chromatogram looks exemplary — a single tall, symmetrical peak.

This is exactly why dedicated procedures for testing peak homogeneity were developed. Principal component analysis of diode array detector data makes it possible to check whether the spectrum recorded on the leading edge, at the apex, and on the trailing edge is the same spectrum.3 If it is not — two substances are sitting under a single apex.

This verification is not a formality. Chromatographic methods for specific substances are sometimes refined over years precisely in order to separate what previously eluted together — this applies both to pharmacopeial methods4 and to determinations developed from scratch for difficult systems.5 Method development is largely a history of successive separations that had not been seen before.

What does not come off the column

The third silent assumption states that everything that went in came out. That is not always the case. Strongly retained substances may leave the column long after data acquisition has ended — and if the run time has been shortened, they simply will not appear in the result. Others bind irreversibly to the packing and do not elute at all.

The symptom may be baseline drift or ghost peaks in the next injection. The symptom may also be silence — and that is the most difficult case, because it looks identical to a clean sample. Work devoted to the purification of compounds with difficult chromatographic behavior shows how much effort must go into simply establishing what elutes from the column and what does not.6

When chromatography alone is not enough

The conclusion from the above is not that the method is unreliable — it is that the method answers a narrower question than is usually attributed to it. This is why the certification of materials relies on orthogonal techniques based on a different physical principle.

Quantitative nuclear magnetic resonance measures the number of nuclei, not absorbance, so an impurity without a chromophore is not invisible to it. The use of proton spectra for determining the purity of pharmaceutical reference materials is described in the metrological literature.7

Mass balance approaches the problem from the side of the impurities: water, residual solvents, and residue on ignition are determined, and the content of the substance is calculated by difference. This is how, among others, amino acid reference materials were certified.8

Combining two independent methods is the strongest solution. In the certification of a caffeine reference material, the spectrophotometric result was verified by quantitative NMR — each method has a different blind spot, so agreement between the results carries information that neither of them would provide on its own.9

How to read a chromatographic result

A few questions worth asking before accepting the number.

  • At what wavelength was the signal recorded? This defines what had a chance of being visible.
  • How long was the run? A short run time carries the risk that something had not yet come off the column.
  • Does the result come from area normalization or from comparison against a reference standard? These are two different quantities.
  • Was the homogeneity of the main peak checked? Without this, the result contains an assumption of complete separation.
  • Has any independent method confirmed the order of magnitude? One technique means one blind spot.

It is also worth remembering that chromatographic determinations in complex matrices have their own well-documented pitfalls — whether the task is assessing the quality of a plant raw material,10 determining highly polar compounds,11 or analyzing groups of compounds that require specially selected conditions.12 Purity determination methods for specific classes of substances are developed and published separately precisely because universal conditions do not exist.13

What does this mean for someone buying a reagent? Simply that an HPLC result is a good starting point and a weak end point. It says that within the recorded time window, at the selected wavelength, the main peak accounted for a certain share of the signal. That is useful information — provided it is not read as “the container holds 99.3% of this substance”. Where the differences between methods come from is described in our text on what the number on a certificate really means, and the document itself is broken down into its parts in our text on the certificate of analysis.

Sources

Each item below was retrieved from the PubMed database via the E-utilities interface and is not reproduced from memory. PMID numbers link to the source records.

  1. Sun X, Zhang W, Huang T et al. Purity determination of pyributicarb by internal standard correction-high-performance liquid chromatography-quantitative nuclear magnetic resonance. Anal Bioanal Chem. 2020;412:6983-6993. PMID: 32754793. DOI: 10.1007/s00216-020-02832-0.
  2. Lloyd DK, Goodall DM. Polarimetric detection in high-performance liquid chromatography. Chirality. 1989;1:251-64. PMID: 2701852. DOI: 10.1002/chir.530010403.
  3. Wiberg K, Andersson M, Hagman A et al. Peak purity determination with principal component analysis of high-performance liquid chromatography-diode array detection data. J Chromatogr A. 2004;1029:13-20. PMID: 15032344. DOI: 10.1016/j.chroma.2003.12.052.
  4. Salkić A, Otašević B, Zečević M. Improvement of Pharmacopeial High-Performance Liquid Chromatography Method for Determination of Enantiomeric Purity of Moxifloxacin Drug Substance. J Sep Sci. 2025;48:e70190. PMID: 40485329. DOI: 10.1002/jssc.70190.
  5. Samoilov M, Zubareva E, Degterev M. Development and Validation of an Ion-Pair Reverse-Phase High-Performance Liquid Chromatography-Electrospray Ionization Mass Spectrometry Method for Determination of Purity of Nusinersen for Quality Control of Drug Substance or Drug Product. Int J Mol Sci. 2026;27. PMID: 41977479. DOI: 10.3390/ijms27073301.
  6. Lawton LA, Edwards C. Purification of microcystins. J Chromatogr A. 2001;912:191-209. PMID: 11330790. DOI: 10.1016/s0021-9673(01)00592-1.
  7. Monakhova YB, Kohl-Himmelseher M, Kuballa T et al. Determination of the purity of pharmaceutical reference materials by 1H NMR using the standardless PULCON methodology. J Pharm Biomed Anal. 2014;100:381-386. PMID: 25215441. DOI: 10.1016/j.jpba.2014.08.024.
  8. Liu H, Cheow PS, Yong S et al. Determination of purity values of amino acid reference materials by mass balance method: an approach to the quantification of related structure impurities. Anal Bioanal Chem. 2020;412:8023-8037. PMID: 32914399. DOI: 10.1007/s00216-020-02936-7.
  9. Shehata AB, Rizk MS, Rend EA. Certification of caffeine reference material purity by ultraviolet/visible spectrophotometry and high-performance liquid chromatography with diode-array detection as two independent analytical methods. J Food Drug Anal. 2016;24:703-715. PMID: 28911607. DOI: 10.1016/j.jfda.2016.06.009.
  10. Valle García-Rodríguez M, Serrano-Díaz J, Tarantilis PA et al. Determination of saffron quality by high-performance liquid chromatography. J Agric Food Chem. 2014;62:8068-74. PMID: 25075549. DOI: 10.1021/jf5019356.
  11. Liu Y, Xing JL, Shen J et al. [Simultaneous determination of six rare sugars in solid foods by high performance liquid chromatography-evaporative light-scattering detection]. Se Pu. 2023;41:781-788. PMID: 37712542. DOI: 10.3724/SP.J.1123.2023.02014.
  12. Rubert-Nason KF, Hedman CJ, Holeski LM et al. Determination of salicinoids by micro-high-performance liquid chromatography and photodiode array detection. Phytochem Anal. 2014;25:185-91. PMID: 24847528. DOI: 10.1002/pca.2485.
  13. Sobolewska E, Biesaga M. High-Performance Liquid Chromatography Methods for Determining the Purity of Drugs with Weak UV Chromophores – A Review. Crit Rev Anal Chem. 2025;55:419-433. PMID: 38180794. DOI: 10.1080/10408347.2023.2291815.

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