A purity percentage on a peptide certificate is the output of one separation under one set of conditions: the share of the integrated ultraviolet signal the largest peak accounts for. This covers what reversed-phase HPLC and electrospray mass spectrometry each measure, how the percentage is computed from a chromatogram, why two vendors’ figures do not line up, and what it leaves out.
What the two methods answer
Reversed-phase high-performance liquid chromatography (RP-HPLC) answers how much of the material is one species: it separates the sample by hydrophobicity and reports the largest peak’s area as a percentage of total integrated peak area. Electrospray-ionization mass spectrometry (ESI-MS) answers whether that species is the intended molecule: it measures mass-to-charge and returns a molecular mass to set against the one calculated from the sequence.
Neither answers the other’s question. A clean preparation of the wrong compound passes on purity and fails on identity; the right compound at 90 % does the reverse. Vitro’s certificates carry them as two rows, Identity by ESI-MS and Purity by RP-HPLC at 220 nm, because they are two measurements.
| Method | Question | Reported as | Cannot answer |
|---|---|---|---|
| RP-HPLC, 220 nm | How much of what eluted is a single species? | Area percent of total integrated peak area | What any peak is |
| ESI-MS | What is the molecular mass? | Mass-to-charge peaks deconvoluted to one mass | How much of the sample that mass accounts for |
Chromatography quantifies peaks without naming them, and mass spectrometry names a mass without quantifying it.
Reading a chromatogram
A chromatogram plots detector response against time. The figure below is the reversed-phase HPLC trace a purity percentage is read off: absorbance at 220 nm up the vertical axis, retention time in minutes along the horizontal, ticked at 0, 5, 10, 15 and 20 for a twenty-minute run.
Retention time is the interval between the sample entering the system and the apex of a peak, and it records how strongly that species was held. The mobile phase strengthens through the run, 5 to 65 % acetonitrile in the method Vitro’s certificate states, so hydrophilic species leave early and hydrophobic ones late. A retention time is a property of the method, not a constant of the molecule.
The flat line the trace sits on is the baseline, the detector’s reading with nothing absorbing in the flow cell. Integration measures the area between the curve and that baseline, from where a peak lifts off to where it returns. Area rather than height counts: a short broad peak can carry more signal than a tall narrow one.
Three peaks are drawn. The tall one near the middle of the run is the main peak, shaded, with a marker at its apex and a leader line reading area ÷ total area = purity %. The two smaller ones, one eluting before it and one after, each rise from the baseline and return to it before anything else starts. That return is what fully resolved means: the integrator can put a boundary between peaks and assign the area either side without guessing.
The percentage is that division: sum the areas of all integrated peaks, divide the main peak’s area by that sum, multiply by 100. Vitro’s certificate reports 99.2 % against a specification of ≥ 98.0 % of area, beside a single max impurity row capping the largest peak other than the main one at ≤ 1.0 % of area; that row reads 0.4 %. On the figure, the two flanking peaks are the candidates that row would rank.
The figure draws those two larger than that arithmetic would put them: at 0.4 % of the area they would be ripples just above the baseline, drawn here tall enough to be seen.
Why two vendors’ purity figures are not comparable
An area percent, the main peak’s area over the total integrated peak area, is a property of the separation as much as of the material. Two laboratories running the same lot on different columns, gradients or detection wavelengths report different percentages, none of them wrong. A purity figure quoted without the conditions that produced it cannot be set against another.
Vitro’s certificate prints the conditions beside the result: reversed-phase C18, 4.6 × 150 mm, 5 µm particles; a gradient from 5 to 65 % acetonitrile in 0.1 % trifluoroacetic acid over 20 minutes; 1.0 mL/min; detection at 220 nm. Each of those moves the number.
| Condition | Change | Effect |
|---|---|---|
| Gradient slope | Same solvent range over a longer run | Pulls close-eluting species out of the main peak; the percentage usually falls |
| Column length, particle size | Longer column, smaller particles | More separating power, more impurities resolved out |
| Stationary phase | C18 to C8 or C4 | Different selectivity; a pair split on one phase can merge on another |
| Detection wavelength | 220 nm to 280 nm | Changes every species’ response, so changes every peak’s share |
| Run length | Shorter run | Late species never elute and are counted nowhere |
Wavelength is the condition most often left off a quoted figure. 214 nm and 220 nm both sit on the absorbance of the backbone amide bond, which every residue of every peptide contributes to, so almost any peptide-like species shows up. 280 nm sits on the aromatic side chains, tryptophan and tyrosine, where a sequence with no aromatic residue is close to invisible and an impurity carrying one absorbs out of proportion to its mass. The two wavelengths count different populations, so their percentages are not two versions of one number.
No wavelength fixes co-elution. A species leaving the column at the same time as the main peak is integrated inside it, and changing the detector changes the response, not the separation. Resolving it takes a different separation: a shallower gradient, another stationary phase, or another mobile-phase pH. That is why a certificate prints the conditions with the number, walked field by field in how to read a Certificate of Analysis.
ESI-MS and charge-state envelopes
Electrospray ionization sprays the sample from an acidified solution through a charged needle, and the molecules reaching the analyzer carry one or more added protons. A mass analyzer measures mass-to-charge (m/z) rather than mass, so a molecule of neutral mass M carrying n protons appears at (M + n × 1.008) ÷ n. One compound therefore gives a series of peaks, one per charge state, rather than a single peak.
That series is the charge-state envelope. Peptides take protons at the N-terminus and at the basic side chains, arginine, lysine and histidine, so the number of sites rises with chain length and the population spreads over several charge states. A short peptide appears essentially as its singly protonated ion; a long one distributes across four, five or more, each at a lower m/z than the last.
| Ion | Charges (n) | m/z |
|---|---|---|
| [M+H]⁺ | 1 | 4001.0 |
| [M+2H]²⁺ | 2 | 2001.0 |
| [M+3H]³⁺ | 3 | 1334.3 |
| [M+4H]⁴⁺ | 4 | 1001.0 |
| [M+5H]⁵⁺ | 5 | 801.0 |
Multiple charging is also what brings a large molecule inside a modest scan range. Vitro’s certificate states ESI in positive mode, direct infusion, scanning 100 to 1500 m/z, and a 4,000 Da peptide falls inside it from the third charge state upwards, though its neutral mass is more than twice the ceiling. A tripeptide of a few hundred daltons needs no such help and reads as the single [M+H]⁺ ion that certificate’s identity row quotes.
Deconvolution runs that arithmetic backwards. Adjacent peaks differ by one charge, enough to solve for both n and M, and the answer is checked against the rest of the series; a peak fitting no charge state of M belongs to something else in the vial.
Intensity within an envelope is not abundance. Ionization efficiency varies by species, and on direct infusion the whole sample enters unseparated, so a minor component that ionizes readily can out-signal a major one. Quantity comes from the chromatogram.
A matching mass is also weaker evidence than it looks. It does not distinguish sequence permutations, which have identical composition, nor residues of equal mass: leucine and isoleucine are exactly isobaric at a residue mass of 113.084 Da, and glutamine and lysine differ by 0.036 Da, which a high-resolution analyzer separates and a unit-resolution one does not. Telling these apart takes fragmenting the ion and reading the fragment masses, or a separation that resolves them.
What area percent cannot see
Area percent is the share of one detector’s integrated signal. It counts a species only if that species absorbs at 220 nm, comes off the column, and comes off resolved from the main peak inside the integration window, the stretch of the run the software integrates. That window normally opens after the solvent front, the unretained material leaving at the void volume, the volume of mobile phase the column holds. Anything failing one of those conditions contributes no area and shifts no percentage, whatever share of the vial’s mass it is.
That is why a lot can read 99.2 % by area and still be several percent water and salt by weight. Vitro’s specimen certificate shows that same lot at 5.1 % water and 6.8 % counter-ion, the anion of the acid the peptide is isolated as a salt of. The three figures do not contradict each other: one is a fraction of ultraviolet peak area, the other two fractions of the weighed solid.
| Missed | Why | Covered by |
|---|---|---|
| Counter-ion | Acetate and trifluoroacetate absorb little at 220 nm and elute near the void volume | Counter-ion (acetate) row, method HPLC, specification Report |
| Residual water | No chromophore at all; leaves with the solvent front | Water content, Karl Fischer |
| Inorganic salts and non-absorbing scavengers such as triisopropylsilane or ethanedithiol | No absorbance at 220 nm, so no peak to integrate; aromatic cleavage scavengers such as thioanisole and phenol do absorb, and are counted | Not quantified individually on Vitro’s certificate |
| Co-eluting related species | Same retention as the main peak; integrated inside it | Identity by ESI-MS, and only for a gross mass difference |
| Strongly retained species | Not eluted by the specimen method’s 65 % acetonitrile ceiling; stay on the column | Nothing; the denominator is only what came off |
| Unretained material | Elutes in the void volume, usually excluded from integration | Nothing |
Area percent is not mass percent either. Response at 220 nm scales with the number of backbone amide bonds, which a peptide carries at roughly one per 110 Da, so a truncated fragment understates its own share of the solid only slightly: dropping one residue from a ten-residue sequence shifts response per milligram by about 1 %. The gap is larger for a species that gains or loses a chromophore, or carries no amide bond at all.
Co-elution is the limit that matters most, because the species most likely to be present most resemble the peptide. An oxidized methionine at +16 Da, a deamidated asparagine at +0.98 Da, a single-residue deletion, or a D-epimer can sit inside the main peak under one gradient and resolve away under a shallower one. The chemistry that generates them is covered in storing research peptides.
Read the percentage beside the rows covering what it omits: water content, counter-ion content, gravimetric net content, and identity. How those rows net into a peptide mass is in how to read a Certificate of Analysis; what the salt form means for a diluent is in reconstituting lyophilized peptides. No certificate row covers a species that both co-elutes and matches the mass, which is what an orthogonal separation is for. To pull a lot’s certificate, enter the lot number at verify.
For laboratory research use only. Not for human or veterinary use, and not intended to diagnose, treat, cure, or prevent any disease.