HPLC, GC-MS and NMR Purity Testing Explained

High-performance liquid chromatography (HPLC)

HPLC separates the components of a sample as they pass through a column packed with a stationary phase, carried by a liquid mobile phase under high pressure. Different compounds travel through the column at different rates depending on their chemical properties, emerging at different times and producing distinct peaks on a chromatogram.

A detector, most commonly a UV absorbance detector, measures each peak as it elutes. The area of the peak corresponding to the target compound, expressed as a percentage of the total peak area, is the basis for the purity figure most often seen on a CoA. HPLC is widely used because it works well for compounds that are not easily vaporised and can be run at ambient or near-ambient temperature.

Gas chromatography–mass spectrometry (GC-MS)

GC-MS combines two techniques. Gas chromatography separates volatile compounds as they travel through a column in a gas stream, similarly to HPLC but using a gas rather than a liquid mobile phase. As each separated compound exits the column, it passes into a mass spectrometer, which fragments the molecule and measures the mass-to-charge ratio of the resulting fragments.

The resulting fragmentation pattern acts as a kind of chemical fingerprint, which can be compared against reference spectra to confirm the identity of a compound, not just its relative quantity. This makes GC-MS particularly useful for identity confirmation and for detecting unexpected trace compounds that a simple UV-based method might miss.

Nuclear magnetic resonance spectroscopy (NMR)

NMR works on a different physical principle entirely. It places a sample in a strong magnetic field and measures how atomic nuclei, typically hydrogen or carbon, respond to a radiofrequency pulse. The resulting spectrum reflects the chemical environment of each nucleus, producing a detailed structural map of the molecule.

Because NMR reports on the whole molecular structure rather than relying on separation and detection of individual peaks, it is often used as a structural confirmation and purity cross-check alongside chromatographic methods, particularly when a laboratory wants independent confirmation that is not dependent on the same separation principles as HPLC or GC.

Comparing the three techniques

No single technique captures every aspect of identity and purity, which is why combining methods is common practice among laboratories that produce thorough documentation.

• HPLC: strong for quantitative purity of non-volatile compounds, moderate identity confirmation via retention time and UV spectrum
• GC-MS: strong identity confirmation via mass fragmentation, well suited to volatile or semi-volatile compounds
• NMR: strong structural confirmation independent of chromatographic separation, useful for detecting certain impurity types that chromatography can miss
• Combined methods provide cross-validation: an unexpected difference between two techniques is a signal worth investigating further

Reading method references on a Certificate of Analysis

When a CoA states a method such as 'Purity: 99.1% (HPLC-UV, 254 nm)', each part of that statement is informative: the numeric result, the technique, the detection wavelength. A laboratory reviewing documentation should look for this level of specificity rather than accepting an unqualified purity percentage with no stated method.

If a report references NMR data, check whether the actual spectrum or only a summary interpretation is provided. A summary is easier to produce but a full spectrum allows for independent review by a laboratory with its own spectroscopic expertise.

Practical implications for laboratory procurement

For most research applications, a CoA backed by at least one chromatographic method (HPLC or GC-MS) with a stated identity confirmation step is a reasonable baseline. Laboratories with more demanding internal quality requirements may request NMR data in addition, particularly for novel or less well-characterised materials.

Understanding these methods also helps when comparing documentation between suppliers: a report that names the specific technique, instrument parameters, and reference standard used is generally more transparent than one that states only a bare percentage.