Purity Calculators

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Purity describes the proportion of a desired substance in a sample relative to the total sample mass or mole content. A 99% pure compound contains 99% of the desired molecule and 1% impurities. Purity assessment is critical in pharmaceutical manufacturing (drug purity requirements), analytical chemistry (reagent purity grade), molecular biology (nucleic acid and protein purity), and food science (ingredient purity). Methods for purity assessment include HPLC (area% for small molecules), SDS-PAGE (protein purity by Coomassie staining), spectrophotometric ratios (A260/A280 for nucleic acids), melting point determination (sharp melting = pure compound), and titration (acid-base purity assay).

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Purity Percentage

Purity % = (mass of pure substance / total sample mass) × 100

Or: purity % = (area of target peak / total peak area) × 100 in HPLC chromatograms.

Purity Grades

  • ACS Reagent Grade: purity defined by American Chemical Society specifications (typically ≥ 95–99%); suitable for analytical work
  • HPLC grade (solvents): very high purity; low UV absorbance; for HPLC mobile phases
  • Pharmaceutical grade (USP/EP): meets United States Pharmacopeia or European Pharmacopoeia; ≥ 98–99.5% for most APIs
  • Research grade: adequate for most laboratory purposes; may be 90–95%

HPLC Purity (Small Molecules)

Reversed-phase HPLC: inject sample; measure peak areas at 220 or 254 nm; purity = (target peak area / sum of all peak areas) × 100%. HPLC purity is the standard for API (active pharmaceutical ingredient) characterization. Requirement: ≥ 95% (research); ≥ 98–99% (regulatory submission).

Nucleic Acid Purity (A260/A280)

Pure DNA: A260/A280 ≈ 1.8–2.0. Pure RNA: ≈ 2.0. Protein contamination: ratio < 1.8. Phenol contamination: ratio < 1.8. A260/A230 also used: pure nucleic acids ≈ 2.0–2.2; < 1.8 suggests organic compound or salt carryover.

Protein Purity (SDS-PAGE)

Run SDS-PAGE; stain with Coomassie; densitometry of target band vs. total protein in lane: purity % = (target band intensity / total intensity) × 100. Typical: > 90% for biochemical assays; > 95% for structural studies.

Glossary

Purity
The proportion of the desired substance in a sample: purity % = (target/total) × 100; measured by HPLC (area%), SDS-PAGE (densitometry), A260/A280 (nucleic acids), or other analytical methods.
HPLC Purity
Reversed-phase HPLC peak area percentage; gold standard for small molecule purity; purity = target peak area/total peak area × 100%; typically ≥ 98–99% required for pharmaceutical regulatory filing.
A260/A280 Ratio
Spectrophotometric purity indicator for nucleic acids; pure dsDNA ≈ 1.8–2.0; pure RNA ≈ 2.0; below expected = protein or phenol contamination; measured by NanoDrop in 1–2 μL sample.

Frequently Asked Questions

HPLC is the gold standard for small molecule API purity. Procedure: dissolve API in mobile phase; inject onto reversed-phase C18 column; elute with gradient; detect at UV (typically 220 nm for general detection or 254 nm for aromatic compounds). Purity = (target peak area / Σ all peak areas) × 100%. Advantages: separates structurally related impurities; identifies specific impurities by retention time comparison to reference standards. Regulatory requirements: ICH Q3A guidelines require identification of impurities ≥ 0.1% in drug substances; qualification ≥ 0.15%; ≥ 99% total purity typically expected for NCE (new chemical entity) regulatory filing. Complementary methods: ¹H-NMR (confirms structure and detects solvent/reagent impurities); mass spectrometry (identifies unknown impurities by molecular mass); elemental analysis (C, H, N, S content).

A260/A280 ratio: pure dsDNA ≈ 1.8–2.0; pure ssRNA ≈ 2.0. Low ratio (< 1.8 for DNA): protein contamination (proteins absorb at 280 nm from Trp/Tyr); phenol carryover from extraction (phenol absorbs 270–280 nm); chloroform or other organic solvents. High ratio (> 2.0 for DNA): RNA contamination; degraded DNA; certain buffer conditions. A260/A230 ratio: an additional purity indicator; pure nucleic acids ≈ 2.0–2.2; low ratio (< 1.8): organic compound contamination (phenol, guanidinium, EDTA absorb at 230 nm); carbohydrate or salt carryover. Both ratios are measured simultaneously on a NanoDrop spectrophotometer in seconds with 1–2 μL sample.

SDS-PAGE + Coomassie staining: the standard visual method. Procedure: run SDS-PAGE; stain; quantify by densitometry (ImageJ or gel analysis software). Purity = target band density / all band densities × 100%. Detection limit: Coomassie ~100–500 ng per band; silver stain ~1–5 ng per band (10–100× more sensitive). Other methods: Western blot: detects specific proteins with antibodies but is not quantitative for total purity. Reverse-phase HPLC (RP-HPLC): high resolution for protein purity; especially useful for antibody characterization. Size exclusion chromatography (SEC): separates by size; quantifies monomer vs. aggregates → important for biopharmaceutical quality. Mass spectrometry: identifies specific impurities by mass; confirms identity and post-translational modifications.

Application-specific purity requirements: Basic biochemical assays (enzyme activity, Western blot): ≥ 80–90% protein purity; functional impurities are the concern, not minor contaminants. Structural biology (X-ray crystallography, cryo-EM): ≥ 95–99%; aggregates, flexible regions, or heterogeneity prevent crystal formation or confound cryo-EM reconstruction. Pharmaceutical APIs: ≥ 98–99.5% by HPLC for regulatory submission; each impurity qualified (ICH Q3A). Oligonucleotides (PCR primers): desalting purification ≈ 80–90% (adequate for PCR); HPLC purification ≥ 95% (required for therapeutic oligonucleotides). NMR spectroscopy: ≥ 95% (impurity signals interfere with spectrum interpretation). Cell culture reagents: ≥ 99% for amino acids, salts; endotoxin content may be more important than chemical purity for cell-based assays.