Understanding Peptide Purity: HPLC & Mass Spec

When a vial is labeled "BPC-157, 98% purity," what does that actually mean? Where did that number come from? And should you trust it?

These questions matter more than most people realize. A peptide at 98% purity and the same peptide at 85% purity are not the same product. That 13% difference could contain truncated sequences, deletion variants, oxidized forms, or residual synthesis chemicals — any of which can alter experimental results, trigger unexpected biological effects, or simply render the peptide less potent than expected.

The two analytical methods that underpin virtually all peptide quality assessment are high-performance liquid chromatography (HPLC) and mass spectrometry (MS). HPLC answers the question "how pure is it?" MS answers "what is it?" Together, they form the minimum standard for any credible peptide quality evaluation — and understanding what they measure is the first step toward reading a certificate of analysis with confidence.

Why Purity Matters
HPLC: Measuring How Pure a Peptide Is
Mass Spectrometry: Confirming What the Peptide Is
How HPLC and Mass Spec Work Together
What Purity Percentages Actually Mean
Purity vs. Net Peptide Content
Common Impurities in Synthetic Peptides
How to Read a Certificate of Analysis
Red Flags in Purity Documentation
Research Grade vs. GMP Grade
FAQ
The Bottom Line
References

Why Purity Matters {#why-purity-matters}

Peptide purity is not a vanity metric. It directly affects experimental reliability, biological activity, and safety.

Impurities can bind to the same receptors as the target peptide, acting as partial agonists, antagonists, or off-target activators. In cell-based assays, even small amounts of a deletion variant or oxidized peptide can produce confusing dose-response curves. In animal studies, impurities may cause inflammatory reactions or immune responses that get wrongly attributed to the peptide being studied.

A 2025 review in the Journal of Peptide Science found that separation quality during purification has a direct, quantifiable impact on downstream results — and that optimizing analytical and preparative methods can push initial purifications above 90% purity with yields exceeding 30% (JPS, 2025).

For anyone interpreting peptide research, asking "what was the purity?" is as fundamental as asking "what was the dose?" If a study does not report purity data, that is a meaningful omission.

HPLC: Measuring How Pure a Peptide Is {#hplc-measuring-how-pure-a-peptide-is}

High-performance liquid chromatography is the workhorse method for quantifying peptide purity. It separates the components of a sample based on their physical and chemical properties, then measures how much of each component is present.

How RP-HPLC Works

The standard technique for peptides is reversed-phase HPLC (RP-HPLC). Here is the basic setup:

The column contains tiny silica particles coated with hydrophobic alkyl chains — most commonly C18 (18-carbon chains). These chains attract and hold hydrophobic molecules. The column is typically 15-25 cm long with a 4.6 mm internal diameter for analytical work.

The mobile phase is a mixture of water and an organic solvent (usually acetonitrile), with a small amount of acid (trifluoroacetic acid or formic acid) to control pH and improve peak shape. The analysis starts with mostly water and gradually increases the organic solvent concentration — a process called gradient elution.

Separation happens because different peptides have different hydrophobicities. Less hydrophobic peptides interact weakly with the C18 chains and elute (wash off) early. More hydrophobic peptides stick longer and elute later. Through experience, chromatographers have found that about 90% of all peptides elute from a C18 column by 30% acetonitrile concentration.

Detection typically uses UV absorbance at 214-220 nm, where the peptide bond absorbs strongly. The detector produces a chromatogram — a graph of signal intensity versus time — where each peak represents a distinct component in the sample.

Reading the Chromatogram

The target peptide should appear as a single, dominant peak. Its purity is calculated by dividing the area under the main peak by the total area of all peaks, expressed as a percentage. A sample with a main peak comprising 98% of total peak area has 98% HPLC purity.

Small peaks flanking the main peak typically represent closely related impurities — deletion sequences, incompletely deprotected variants, or oxidized forms. Peaks far from the main peak may represent more structurally distinct impurities or synthesis byproducts.

Limitations of HPLC Alone

HPLC tells you how much of the sample is the main component, but it does not tell you what that main component is. A peak with perfect symmetry and 99% area could, in theory, represent the wrong peptide entirely — a synthesis error that produced a different sequence with similar chromatographic properties. This is why mass spectrometry is always needed alongside HPLC.

Some impurities also co-elute with the target peptide (they come off the column at the same time), hiding within the main peak. Isoaspartate variants and deamidated species, for instance, can share nearly identical retention times with the intended peptide, making them invisible to standard HPLC methods without additional separation optimization.

Mass Spectrometry: Confirming What the Peptide Is {#mass-spectrometry-confirming-what-the-peptide-is}

If HPLC is the scale, mass spectrometry is the fingerprint. It confirms the molecular identity of a peptide by measuring its exact mass.

How It Works

Mass spectrometry works by converting peptide molecules into gas-phase ions, then measuring their mass-to-charge ratio (m/z). The two primary ionization methods used for peptides are:

Electrospray ionization (ESI) sprays the peptide solution through a charged needle, producing multiply charged ions. ESI is gentle enough to preserve peptide structure and is easily coupled directly to HPLC systems (creating LC-MS), making it ideal for routine quality control.

Matrix-assisted laser desorption ionization (MALDI) mixes the peptide with a crystalline matrix and uses a laser to ionize the sample. MALDI typically produces singly charged ions and is often used for quick identity checks and higher molecular weight peptides.

What the Mass Spectrum Shows

The resulting spectrum shows peaks at specific m/z values. For a peptide of known sequence, you can calculate the expected molecular weight. If the observed mass matches the theoretical mass (typically within 0.1% or better), the identity is confirmed.

Mass differences reveal specific problems:

Mass +16 Da: Likely an oxidation event (addition of one oxygen atom, often at methionine)
Mass -18 Da: Possible dehydration or cyclization
Mass differences of ~100-200 Da: Missing or extra amino acid residues
Mass +80 Da: Phosphorylation
Shifted mass patterns: Possible sequence errors, incomplete cleavage, or modification failures

Tandem Mass Spectrometry (MS/MS)

For more detailed structural confirmation, tandem MS (MS/MS) fragments the peptide and analyzes the fragments. By comparing the fragmentation pattern to the predicted pattern for the expected sequence, researchers can verify not just the molecular weight but the actual amino acid order. This is the most definitive confirmation of peptide identity available.

How HPLC and Mass Spec Work Together {#how-hplc-and-mass-spec-work-together}

The most powerful approach is LC-MS — liquid chromatography directly coupled to mass spectrometry. In this setup, the HPLC separates components in real time, and the mass spectrometer identifies each component as it elutes from the column.

This combination solves the limitations of either technique alone:

HPLC separates co-eluting impurities that mass spec alone might miss
Mass spec identifies each separated peak, catching cases where the wrong peptide has the "right" HPLC profile
Together, they can detect and identify impurities that represent less than 0.1% of the total sample

For routine quality control, most laboratories run HPLC and MS as separate analyses. For detailed characterization or troubleshooting, LC-MS provides the most complete picture in a single run.

What Purity Percentages Actually Mean {#what-purity-percentages-actually-mean}

When a supplier reports "purity: 98%," they are saying that 98% of the peptide-related material in the sample, as measured by HPLC peak area integration, corresponds to the target peptide. The remaining 2% is other peptide-related species — deletion sequences, truncated forms, oxidized variants, and other synthesis byproducts.

This number has important nuances:

Purity is method-dependent. Different HPLC conditions (column type, gradient, mobile phase) can produce different purity values for the same sample. Two laboratories might report 97% and 95% purity for identical material simply because their methods resolve impurities differently. This is why reputable certificates of analysis include method details.

Purity targets depend on application. General guidance:

>95%: Suitable for most research applications
>98%: Recommended for quantitative biological assays, structural studies, and sensitive in vivo work
>99%: Required for clinical-grade applications and some pharmaceutical studies

Purity does not equal potency. A 98%-pure peptide is not necessarily 98% potent. Biological activity depends on correct folding, disulfide bond formation, and post-translational modifications — factors that purity measurements do not capture.

Purity vs. Net Peptide Content {#purity-vs-net-peptide-content}

This distinction trips up many researchers and is worth understanding clearly.

HPLC purity measures the percentage of the target peptide relative to other peptide-related impurities. It answers: "Of the peptide material in this vial, how much is the correct peptide?"

Net peptide content measures the percentage of the vial's total weight that is actual peptide (as opposed to water, salts, counterions like trifluoroacetate, and residual solvents). It answers: "Of the total material in this vial, how much is peptide of any kind?"

A vial might contain a peptide at 98% HPLC purity but only 65% net peptide content. This means:

65% of the weight is peptide material
Of that 65%, 98% is the correct target peptide
The remaining 35% of total weight is water, TFA counterions, and salts

Net peptide content typically falls between 60% and 90%, even for high-purity peptides. This matters for accurate dosing calculations — if you weigh out 5 mg of powder but the net peptide content is 70%, you actually have 3.5 mg of peptide. For more on this topic, see our guide on how to calculate peptide dosages.

Common Impurities in Synthetic Peptides {#common-impurities-in-synthetic-peptides}

Solid-phase peptide synthesis (SPPS), the standard method for producing research peptides, generates characteristic impurity profiles. Understanding these impurities helps interpret both quality data and experimental results. For background on how peptides are made, see our peptide synthesis methods guide.

Deletion sequences. During SPPS, each amino acid is added one at a time. If a coupling step fails or is incomplete, the next amino acid is added to a chain that is one residue short. The result is a peptide missing a single amino acid — often difficult to separate from the full-length product because its chromatographic properties may be very similar.

Truncated sequences. If the synthesis terminates prematurely, shorter fragments accumulate. These are usually easier to detect and remove during purification because their retention times differ more from the target.

Incomplete deprotection. During synthesis, reactive amino acid side chains are protected with temporary chemical groups. If deprotection is incomplete during the final cleavage step, the peptide retains one or more protecting groups, adding mass and potentially blocking biological activity.

Oxidized variants. Methionine and cysteine residues are particularly susceptible to oxidation during synthesis, cleavage, purification, and storage. Oxidized methionine (+16 Da) is one of the most common modifications seen in peptide quality control.

Racemization products. Under certain synthesis conditions, L-amino acids can convert to D-amino acids (racemize). These epimerized peptides have the same molecular weight as the target but may have reduced or altered biological activity.

Residual solvents and reagents. Trace amounts of TFA, DMF, piperidine, and other synthesis chemicals can persist after purification. While typically present at very low levels, they should be monitored, especially for peptides intended for cell culture or in vivo work.

How to Read a Certificate of Analysis {#how-to-read-a-certificate-of-analysis}

A certificate of analysis (CoA) is the quality passport for a peptide batch. It summarizes all testing performed and should provide enough information to assess whether the peptide meets your needs. For a step-by-step walkthrough, see our detailed guide on how to read a peptide CoA.

A credible CoA should include:

Peptide identity. Name, sequence, molecular formula, and theoretical molecular weight.

HPLC purity data. The reported purity percentage, the chromatogram itself (or at least a description of the method), the column type, mobile phase composition, gradient conditions, and detection wavelength. Without method details, the purity number lacks context.

Mass spectrometry data. The observed molecular weight and the mass spectrum (or at least the key m/z values). The observed mass should match the theoretical mass within an acceptable tolerance — typically within 0.1% or 1 Da for ESI-MS.

Batch and lot information. A unique lot number tied to a specific synthesis run. This allows traceability and comparison between batches.

Physical appearance. Color and form (typically white to off-white powder for lyophilized peptides).

Storage conditions. Recommended temperature and handling instructions.

Date of analysis. When the testing was performed.

For more on verifying quality through independent testing, see our guide on third-party peptide purity testing.

Red Flags in Purity Documentation {#red-flags-in-purity-documentation}

Not all CoAs are created equal. Here is what should raise concerns:

No mass spectrometry data. An HPLC purity number without MS confirmation is incomplete. HPLC alone cannot confirm identity — the main peak could be the wrong peptide. Any credible supplier includes MS data.

Purity reported as 100% or 99.99%. Analytical instruments have measurement uncertainty. A reported purity of 100.0% suggests either rounding, a poorly calibrated instrument, or fabrication. Real analytical results show realistic values like 98.3% or 96.7%.

No chromatogram or method details. A purity number without the supporting chromatogram and analytical conditions is unverifiable. You cannot assess peak shape, baseline quality, or whether impurity peaks are adequately resolved.

Generic or template-looking CoAs. If every peptide from a supplier has an identical-looking CoA with suspiciously similar purity values, the data may be templated rather than batch-specific.

Missing lot numbers. Without a unique batch identifier, there is no way to trace the data back to a specific synthesis run or compare results to independent testing.

Inconsistency between HPLC and MS. If the HPLC shows a single clean peak but the MS reveals multiple molecular species, something is wrong — likely co-eluting impurities that the HPLC method did not resolve.

Research Grade vs. GMP Grade {#research-grade-vs-gmp-grade}

The terms "research grade" and "GMP grade" describe fundamentally different quality systems, not just different purity levels.

Research grade peptides are produced and tested under basic quality control. They typically include HPLC and MS analysis, are labeled "for research use only," and are suitable for in vitro studies, cell assays, and preclinical research. Quality documentation is a standard CoA.

GMP (Good Manufacturing Practice) grade peptides are produced under a fully validated quality system per ICH Q7 guidelines. This includes validated analytical methods, documented manufacturing procedures, batch records, stability testing, endotoxin testing, sterility testing (for injectable products), and quality oversight by trained personnel. GMP grade is required for human clinical trials and approved drugs.

The practical difference: a research-grade peptide might be 98% pure and perfectly suitable for a cell culture experiment. But it would not have the documentation, traceability, or validated testing needed for clinical use. GMP compliance adds substantial cost — often 10-fold or more — but provides the regulatory-grade assurance needed for human administration.

FAQ {#faq}

What purity level do I need for my research?

For most in vitro studies and general research, purity above 95% is standard. For quantitative biological assays, receptor binding studies, or in vivo work, aim for 98% or higher. For NMR structural studies or clinical applications, 99%+ is typical. The key is matching purity to the sensitivity of your application — a screening assay is more tolerant of impurities than a dose-response curve determination.

Can HPLC alone confirm peptide identity?

No. HPLC measures the relative amount of each component but does not identify what those components are. A peak with the expected retention time could theoretically be a different molecule with similar hydrophobicity. Mass spectrometry is required to confirm that the main peak is actually the intended peptide.

What is the difference between HPLC purity and net peptide content?

HPLC purity measures the target peptide as a percentage of all peptide-related material. Net peptide content measures total peptide as a percentage of the vial's weight (including water, salts, and counterions). A peptide can be 98% pure by HPLC but have only 70% net peptide content — the remaining weight is non-peptide material like TFA salts and absorbed moisture.

Why do some impurities have the same mass as the target peptide?

Certain modifications, like isoaspartate formation (isomerization of aspartate) or racemization (conversion from L- to D-amino acid), do not change molecular mass. These isobaric impurities require chromatographic separation rather than mass-based detection, which is one reason why HPLC and MS must be used together rather than relying on either alone.

How should I store peptides to maintain purity?

Lyophilized (freeze-dried) peptides are most stable stored at -20 degrees C or below, protected from moisture and light. Once reconstituted in solution, peptides degrade faster and should be stored at -20 degrees C in single-use aliquots to avoid repeated freeze-thaw cycles. Some peptides, particularly those containing methionine or cysteine, are susceptible to oxidation and benefit from storage under inert gas (nitrogen or argon). See our peptide storage guide for detailed recommendations.

What does "endotoxin tested" mean on a CoA?

Endotoxin testing (usually by LAL assay) measures bacterial lipopolysaccharide contamination. This is important for peptides used in cell culture or in vivo research because endotoxins can activate immune responses and confound experimental results. Endotoxin levels below 0.1 EU/mg are generally considered acceptable for most research applications. This test is standard for GMP-grade peptides but optional for research-grade material.

The Bottom Line {#the-bottom-line}

HPLC and mass spectrometry are not interchangeable — they answer different questions. HPLC tells you how pure the peptide is. Mass spectrometry tells you what the peptide is. Neither alone gives the full picture. Any peptide used in serious research should have both measurements, documented in a batch-specific certificate of analysis with method details and supporting data.

The good news is that reading a CoA is not complicated once you know what to look for: an HPLC purity above 95% (for most applications), a mass spectrum confirming the expected molecular weight, a unique lot number, and enough method detail to make the results interpretable. Red flags — missing MS data, suspiciously perfect purity values, template-looking documents — are straightforward to spot once you know the patterns.

Peptide quality assessment is not about blind trust in a number on a label. It is about understanding where that number came from, what it does and does not tell you, and whether the supporting evidence holds up. For a deeper dive into evaluating peptide quality, start with our guide on reading a certificate of analysis and third-party verification methods.

References {#references}

Biosynth. (2024). Analytical methods and quality control for peptide products. Biosynth
Keil, F., et al. (2025). Improvement of analysis and transferability in peptide purification: From HPLC to FPLC and back again. Journal of Peptide Science. PMC
VYDAC. The handbook of analysis and purification of peptides and proteins by reversed-phase HPLC. Wolfson Centre PDF
Creative Proteomics. Peptide purity analysis service — methods overview. Creative Proteomics
Verified Peptides. (2024). Decoding lab reports: Peptide verification report metrics. Verified Peptides
Biovera. (2024). Quality control standards for research peptides. Biovera
Waters Corporation. (2017). Selecting a reversed-phase column for the peptide mapping analysis of a biotherapeutic protein. Waters
Spartan Peptides. (2025). Quality control in peptide research — Interpreting HPLC & mass spec purity. Spartan Peptides

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