HPLC Testing Explained: What Peptide Purity Numbers Actually Mean

When a peptide supplier states that a compound is \"98% pure by HPLC,\" what does that number actually mean? For many researchers, the purity percentage is taken at face value — a higher number is better, and that is the extent of the analysis. But HPLC purity is not a simple, absolute measure. It depends on the method used, the conditions chosen, the detection wavelength, and the way the data is processed. Two suppliers could test the same peptide and report different purity values, and both could be technically correct.

This guide explains the fundamentals of HPLC purity testing for peptides, walks through how to read a chromatogram, and identifies the critical factors that affect purity numbers — empowering you to evaluate supplier data with an informed, critical eye.

How HPLC Works: The Basics

High-Performance Liquid Chromatography separates the components of a mixture based on their differential interactions with a stationary phase (the column packing material) and a mobile phase (the solvent flowing through the column). For peptides, the most common approach is reversed-phase HPLC (RP-HPLC).

Reversed-Phase HPLC for Peptides

In RP-HPLC, the stationary phase is hydrophobic (typically C18 or C8 alkyl chains bonded to silica particles), and the mobile phase is a mixture of water and an organic solvent (typically acetonitrile). Peptides are separated based on their hydrophobicity:

• More hydrophilic peptides elute earlier (less interaction with the hydrophobic stationary phase)
• More hydrophobic peptides elute later (stronger interaction, requiring more organic solvent to elute)
• The organic solvent concentration is gradually increased over time (gradient elution) to progressively elute all components

Detection

As separated components elute from the column, they pass through a detector. For peptides, UV detection is standard:

• 214–220 nm: Detects the peptide bond (amide bond absorption). This is the most universal detection wavelength for peptides and is the standard for purity measurement. Every peptide bond absorbs at this wavelength, making it the most equitable basis for comparing different impurities.
• 254 nm: Detects aromatic residues (Trp, Phe, Tyr). More selective but not universal — peptides without aromatic residues will have weak or no signal.
• 280 nm: Primarily detects tryptophan and tyrosine. Even more selective.

The choice of detection wavelength matters. A purity measured at 220 nm may differ from one measured at 254 nm because different impurities absorb differently at each wavelength.

Reading a Chromatogram

A chromatogram is a plot of detector response (y-axis) versus time (x-axis). Here is what to look for:

The Main Peak

This is the tallest peak in the chromatogram and represents your target peptide. Key features to evaluate:

• Retention time: The time at which the peak elutes. This should be consistent between analyses of the same peptide under the same conditions.
• Peak shape: Should be symmetrical and sharp. A tailing peak (asymmetric, with a drawn-out tail on the right side) may indicate column degradation, secondary interactions, or overloading. A fronting peak (tail on the left side) is less common but can indicate sample overloading.
• Peak width: A narrow, sharp peak indicates good chromatographic resolution. A broad peak may indicate poor column performance or the co-elution of multiple species.

Impurity Peaks

Smaller peaks before and after the main peak represent impurities. Common impurities in synthetic peptides include:

• Deletion sequences: Peptides missing one or more amino acids. These typically elute at different retention times from the full-length peptide.
• Truncated sequences: Incomplete synthesis products. Usually more hydrophilic (elute earlier) than the full-length peptide.
• Deamidation products: May elute very close to the main peak, sometimes appearing as a shoulder rather than a resolved peak.
• Oxidized forms: Typically more hydrophilic than the parent peptide and elute earlier.
• Diastereomers: Result from racemization during synthesis. May co-elute with the main peak or appear as closely spaced peaks.

Baseline

The baseline is the detector signal in the absence of eluting compounds. A good chromatogram has a flat, stable baseline. Baseline drift (gradual upward or downward trend) can be normal during gradient elution but should not be excessive. Noisy baseline may indicate detector issues or mobile phase contamination.

How Purity Is Calculated

Area Normalization Method

The standard method for calculating HPLC purity of peptides is area normalization (also called area percent). The formula is:

Purity (%) = (Area of main peak / Total area of all peaks) x 100

This method makes two important assumptions:

• All components have the same detector response factor (extinction coefficient) at the detection wavelength
• All components in the sample are detected (nothing elutes undetected)

Neither assumption is perfectly true, which is why HPLC purity is a relative measurement, not an absolute one.

Integration Parameters

Integration is the process of defining where peaks begin and end and calculating their areas. This is where subjectivity can enter purity determination:

• Threshold sensitivity: How small must a peak be before it is ignored? Setting a high threshold will exclude small impurity peaks and inflate the apparent purity.
• Baseline construction: How the baseline is drawn under peaks affects the calculated area. Tangent-skim versus valley-to-valley integration can yield different results for partially resolved peaks.
• Integration window: Which time range is included in the calculation? Excluding early-eluting or late-eluting peaks reduces the denominator and increases the purity percentage.

This is why requesting the actual chromatogram — not just the purity number — is so important. It allows you to evaluate whether the integration was performed reasonably.

Factors That Affect Reported Purity

Method Parameters

Parameter	Effect on Reported Purity	What to Look For
Column type (C18 vs. C8 vs. C4)	Affects resolution between main peak and impurities	C18 is standard for most peptides
Gradient slope	Steeper gradients may co-elute impurities with main peak	Shallow gradients (0.5–1% per minute) give better resolution
Flow rate	Higher flow rates can reduce resolution	0.5–1.0 mL/min is typical for analytical columns
Detection wavelength	Different wavelengths detect different impurity profiles	220 nm is the standard for peptide purity
Column temperature	Affects selectivity and peak shape	Typically 25–40°C; should be stated
Sample load	Overloading broadens peaks and reduces resolution	Analytical loads should be in the low microgram range

The Co-Elution Problem

One of the most significant limitations of HPLC purity is co-elution — when an impurity has the same retention time as the target peptide and is hidden under the main peak. Common co-eluting impurities include:

• Deamidated variants (mass difference of only +1 Da, often very similar hydrophobicity)
• Diastereomers from racemization at a single residue
• TFA adducts or counterion variants

A peptide that appears 99% pure by one HPLC method may show only 95% purity when analyzed by a different method (different column, different gradient) that resolves the co-eluting impurity. This is why orthogonal analytical methods (MS, ion-exchange chromatography, capillary electrophoresis) provide important complementary information.

What \"98% Pure\" Actually Means

When a COA states \"Purity: 98.2% by RP-HPLC,\" it means:

• Under the specific chromatographic conditions used by that laboratory
• At the specific detection wavelength (usually 220 nm)
• With the specific integration parameters chosen by the analyst
• The main peak accounted for 98.2% of the total detected peak area

It does not mean:

• That 98.2% of the material by weight is the target peptide (counterions, water, and salts are not detected by UV)
• That only 1.8% of impurities are present (co-eluting impurities are missed)
• That the purity would be 98.2% by any other analytical method
• That a different laboratory would necessarily report the same number

Purity vs. Peptide Content

A commonly confused distinction is between HPLC purity and peptide content (also called net peptide content):

• HPLC purity: The percentage of the peptide-related material that is the target peptide (as opposed to peptide impurities). Does not account for non-peptide components.
• Peptide content: The percentage of the total sample weight that is peptide material (as opposed to water, counterions, and salts). Typically determined by amino acid analysis, nitrogen analysis, or UV absorbance at a known extinction coefficient.

A peptide might be 98% pure by HPLC but have only 70% peptide content because the remaining 30% is TFA counterion and residual moisture. For accurate dosing in quantitative experiments, peptide content is the more relevant number.

Evaluating Supplier HPLC Data: A Checklist

• Is an actual chromatogram provided, or just a purity number?
• Are the method parameters listed (column, mobile phase, gradient, wavelength, flow rate)?
• Is the detection wavelength 214–220 nm (the standard for peptide purity)?
• Is the main peak well-resolved from impurity peaks?
• Is the peak shape symmetrical, without excessive tailing or fronting?
• Is the baseline stable and flat?
• Are integration parameters reasonable (no obvious exclusion of visible peaks)?
• Does the retention time make sense for the peptide's expected hydrophobicity?
• Is the purity value consistent with the visual appearance of the chromatogram?

Practical advice: If you are comparing purity values across suppliers, recognize that the numbers are not directly comparable unless the same method was used. A supplier reporting 96% purity using a high-resolution method with a shallow gradient may actually be providing a purer product than a supplier reporting 99% using a low-resolution method with a steep gradient that co-elutes impurities. The chromatogram tells you more than the number.

Frequently Asked Questions

What purity level do I need for my peptide research?

It depends on your application. For antibody production or screening assays, 70–85% purity (desalted grade) is often sufficient. For most in vitro research, 95% or higher is recommended. For quantitative studies like dose-response curves, receptor binding assays, or any work where precise concentration matters, 98% or higher is advisable. For structural studies (NMR, X-ray crystallography), the highest available purity (>99%) is typically required.

Why does my peptide show different purity values from two different suppliers?

Different purity values almost certainly reflect different analytical methods rather than different product quality. Variations in column type, gradient conditions, detection wavelength, and integration parameters can all affect the reported purity. To compare products from different suppliers fairly, you would need to test them side by side using the same HPLC method in the same laboratory. This is one reason why looking at the actual chromatogram is more informative than comparing numbers.

Is HPLC purity the same as total purity?

No. HPLC purity measures only the proportion of peptide-related material that is the target compound. It does not account for water content, counterions (such as TFA or acetate), residual solvents, or inorganic salts. The total purity or peptide content — the actual percentage of the sample that is peptide by weight — is typically 60–80% even for HPLC-pure samples, due to the contribution of counterions and moisture. For accurate quantitative work, both HPLC purity and peptide content should be known.

Can I determine peptide purity using a standard laboratory HPLC?

Yes, if your HPLC system has a UV detector and a reversed-phase C18 column, you can perform a basic purity analysis. Use a water/acetonitrile gradient with 0.1% TFA as the ion-pairing agent, detect at 220 nm, and run a gradient from approximately 5% to 65% acetonitrile over 30–60 minutes. Compare your chromatogram to the supplier's COA chromatogram. While your absolute purity values may differ slightly due to system differences, the overall peak profile should be comparable.