You see a vial labeled "≥98% purity" and think, great, that's good stuff. But what does that number actually mean? How was it measured? And—here's the important part—how do you know it's real?
Understanding peptide purity isn't just about reading a label. It's about knowing whether your research is built on solid ground or quicksand. The difference between 95% and 98% purity isn't just 3%—it's the difference between reproducible experiments and data you can't trust.
This guide breaks down how peptide testing actually works, teaches you to read a Certificate of Analysis like a pro, and shows you the red flags that scream "low-quality supplier."
What "Purity" Actually Means (And Doesn't)
When we say a peptide is 98% pure, we mean 98% of the peptide content is the correct amino acid sequence. The other 2%? That's synthesis byproducts—deletion sequences where an amino acid got skipped, truncations, or other molecular near-misses.
But here's what purity doesn't include:
- Counterions: Salts like trifluoroacetate (TFA) or acetate from the purification process. They're stuck to your peptide and they're not going anywhere.
- Residual Water: Even "dry" lyophilized powder contains 5-10% water by weight.
- Stabilizers: Things like mannitol or trehalose, if your peptide needs them.
This is why a "5mg vial" doesn't contain exactly 5mg of active peptide. It contains 5mg of peptide plus all that other stuff. The actual peptide content by weight is usually 75-85%. Good suppliers list this separately on the COA so you know what you're really working with.
HPLC: The Gold Standard (and What It's Actually Doing)
How HPLC Works
High-Performance Liquid Chromatography is the industry standard for measuring peptide purity. Here's the concept: you dissolve your peptide and pump it through a column packed with a hydrophobic material. Then you run a gradient of increasing organic solvent (usually acetonitrile) to wash molecules out.
Different molecules stick to that hydrophobic column differently. Your target peptide might elute at 12 minutes, while an impurity comes out at 13 minutes. A UV detector watches everything exit and records peaks—each peak represents a different compound.
For peptides, reversed-phase HPLC (RP-HPLC) is the go-to method because peptide hydrophobicity tends to separate sequences nicely.
Reading an HPLC Chromatogram
A chromatogram is just a graph:
- X-axis: Time (how long it took for something to come out of the column)
- Y-axis: UV absorbance (how much of it there is, basically)
The main peak—usually the tallest one—is your target peptide. Smaller peaks are impurities. Purity gets calculated like this:
Purity (%) = (Area of Main Peak ÷ Total Area of All Peaks) × 100
A high-quality peptide shows one dominant peak taking up >98% of the total area, with minimal noise from other peaks. Multiple significant peaks? That's a sign of poor synthesis or inadequate purification, and your experiments are going to suffer for it.
Peak Integration: Where the Cheating Happens
The way you draw baselines and integrate peak areas can dramatically change the purity number. Some suppliers play games here—manipulating baselines to inflate purity percentages. This is why third-party testing matters. Independent labs follow standardized methods.
Mass Spectrometry: Confirming You Got What You Paid For
HPLC tells you how pure your sample is. Mass spectrometry (MS) tells you what's actually in the vial.
Here's the thing: HPLC can't distinguish between your target peptide and a closely-related impurity that happens to elute at the same time. Mass spec measures the actual molecular weight—so you know if you're holding the peptide you ordered or something that just looks similar on a chromatogram.
How Mass Spec Works
The two common methods for peptides are:
- ESI-MS (Electrospray Ionization): Standard for most peptides. Gives you an accurate molecular weight.
- MALDI-TOF: Better for larger peptides and proteins, but more expensive.
A proper mass spec report shows:
- Expected mass: The calculated molecular weight of your target peptide
- Observed mass: What the instrument actually measured
- Delta: The difference (should be within ±1-2 Da for small peptides)
If the observed mass doesn't match the expected mass, you don't have the right peptide. Full stop.
What a Real COA Looks Like
A Certificate of Analysis is your peptide's report card. Here's what should be on it:
The Non-Negotiables
- Peptide Information: Name, sequence, CAS number, molecular weight
- Batch/Lot Number: So you can trace which specific batch you got
- HPLC Purity: The percentage and the actual chromatogram image
- Mass Spectrometry Data: Expected vs. observed molecular weight
- Peptide Content: The actual peptide percentage by weight (usually 75-85%)
- Appearance: What it should look like (white to off-white powder, etc.)
- Dates: Manufacturing and expiration
- Storage Conditions: Recommended temperature
Nice to Have (But Increasingly Expected)
- Endotoxin Testing: Critical for cell culture or animal work (should be <1.0 EU/mg)
- Water Content: From Karl Fischer titration
- Counter-Ion Identity: Which salt form you're actually getting (TFA, acetate, etc.)
- Amino Acid Analysis: Confirms sequence composition, though it's expensive and not always done
Why Third-Party Testing Matters
Manufacturer COAs can be... optimistic. Not always intentionally, but there's an inherent conflict of interest. Third-party labs don't have skin in the game—they just report what they find. At PRC Peptides, every batch gets independent third-party verification before it goes in the catalog.
Red Flags: Spotting Bad Peptides Before You Waste Time
In the COA Itself
- No Chromatogram: Just a purity number with no supporting data? Walk away.
- Multiple Big Impurity Peaks: If you're seeing secondary peaks at >1-2% each, that's not 98% purity no matter what they claim.
- Mass Spec Mismatch: Observed mass differs from expected by more than 2 Da? That's not your peptide.
- Generic Template COA: Looks copy-pasted, no specific batch number or date. Probably fake.
- Suspiciously Perfect Numbers: Exactly 99.5% or 99.9% every time? Real analytical data has variation—98.2%, 98.7%, etc.
- Missing Peptide Content: If they don't distinguish between purity and actual peptide weight percentage, they're hiding something.
From the Supplier
- COA Not Available: You have to beg for it, or they're "working on it." Nope.
- Different COA Every Time: Each order shows wildly varying purity. That's batch-to-batch inconsistency you don't want in your research.
- No Testing Details: Doesn't specify HPLC method, column type, or MS instrument. How do you know they actually tested it?
- Price Too Good to Be True: Significantly cheaper than everyone else? There's a reason. You get what you pay for.
Fake COAs Are More Common Than You Think
Some suppliers provide fabricated or altered COAs. Red flags include: the same generic chromatogram for multiple different peptides, batch numbers that don't match the vial, or data that looks too clean. Always request batch-specific COAs and cross-check that the testing lab is real.
Other Purity Methods (and Why HPLC Wins)
Thin-Layer Chromatography (TLC)
Pros: Cheap and fast
Cons: Can't detect impurities below ~5%. Basically useless for modern peptide QC. If a supplier only provides TLC data, run.
Capillary Electrophoresis (CE)
Pros: High resolution, uses less sample
Cons: Less robust than HPLC for routine testing. Not as widely used in the peptide industry.
UPLC (Ultra-Performance Liquid Chromatography)
Pros: Higher resolution and faster than regular HPLC. Better at separating closely-related impurities.
Cons: More expensive equipment. For research peptides, standard HPLC is usually plenty.
Bottom line: HPLC + mass spectrometry is the gold standard. Purity from HPLC, identity from mass spec. That combination gives you both the quality and confidence you need.
Why This Matters for Your Research
Reproducibility
Lower purity means more batch-to-batch variability. A peptide that's 92% one batch and 96% the next introduces variables you can't control. Your experiments become irreproducible, and you'll never figure out why.
Impurities Aren't Inert
Those impurities aren't just random junk—they're structurally similar peptides. A deletion sequence missing one amino acid might still bind your target receptor, creating off-target effects you can't account for.
Dosing Accuracy
Think you're dosing 1mg but the peptide is only 85% pure? You're actually delivering 0.85mg. Over a whole study, that discrepancy compounds and can completely throw off dose-response relationships.
Publication Standards
High-impact journals increasingly require documentation of peptide purity. Studies using poorly characterized compounds face rejection or demands for re-validation with better material. Save yourself the headache.
Bottom Line
Peptide purity isn't just a marketing claim—it's the foundation your research is built on. Understanding HPLC, knowing what a COA should contain, and spotting red flags are essential skills for anyone working with synthetic peptides.
Always request the COA. Always look at the chromatogram and mass spec data, not just the purity number. Be skeptical of claims without supporting evidence. And remember: the most expensive part of research isn't the cost of the peptide—it's the time and money wasted on experiments with compromised materials.
Quality testing isn't optional. It's what separates publishable science from a month wasted in the lab chasing artifacts.