How to Read an HPLC Chromatogram and a Purity Value

What the plot shows

High-performance liquid chromatography (HPLC) separates the components of a sample so that each can be seen on its own. For peptides the usual form is reversed-phase HPLC: the sample is carried through a column packed with a hydrophobic material, and the more hydrophobic a molecule is, the longer it is held back.^{[1, 2]}

A chromatogram plots time along the horizontal axis against detector signal on the vertical axis. For peptides the detector is almost always ultraviolet absorbance, commonly read near 214 nm where the peptide bond absorbs, sometimes alongside 280 nm where tryptophan and tyrosine absorb. Each peak is a group of molecules that left the column together. The height of a peak shows how much signal arrived at that moment; its area is the signal added up over the whole peak.

How the separation is run

The liquid pumped through the column is the mobile phase. It usually starts water-rich and becomes steadily richer in an organic solvent such as acetonitrile, which is called a gradient. As the organic fraction rises, peptides let go of the column in order of increasing hydrophobicity.

Most peptide methods add a small amount of an acid such as trifluoroacetic acid to both solvents. It acts as an ion-pairing reagent: its anion pairs with the positive charges on the peptide and sharpens the peaks. Published work shows that the choice and concentration of this reagent changes which peptides separate from which, so two methods can give two different pictures of the same sample.^[3]

Parts of a chromatogram

• Baseline. The flat line where nothing is leaving the column. A baseline that drifts upward during a gradient is common, because the mobile phase itself absorbs more or less light as its composition changes.
• Solvent front. A disturbance near the start where unretained material passes straight through. It is normally left out of purity calculations.
• Main peak. The largest peak, which is assumed to be the peptide of interest. Assumed is the important word: a peak is not named by its size.
• Minor peaks and shoulders. Smaller peaks before or after the main peak, or bumps on its side, are other substances that are partly or fully separated from it.
• Blank peaks. Peaks that appear when no sample is loaded come from the solvents, the system or earlier runs. A blank run, with the same gradient, shows which peaks to ignore.

Retention time is not identity

Retention time is the time from the start of the run to the top of a peak. It depends on the column, the gradient, the temperature, the flow rate and the ion-pairing reagent, so it only means something when compared with a reference run made on the same system under the same conditions. Even then it is a hint, not proof: two different peptides can leave the column at nearly the same time. Identity is normally confirmed with a second technique; see mass spectrometry for peptide identity.

How a purity percentage is calculated

Chromatographic purity is usually reported as area percent: the area of the main peak divided by the total area of all integrated peaks, multiplied by 100.

Here is a worked example with made-up numbers, only to show the arithmetic. Suppose the integrated areas are 12.5, 1450.0, 8.5 and 29.0 in arbitrary units. The total is 1500.0, and the main peak is 1450.0. Purity = 1450.0 ÷ 1500.0 × 100 = 96.67 %. The same chromatogram would give a different number if the small peak at 8.5 were not integrated or if a shoulder were counted as part of the main peak. That is why a purity value should always come with the integration settings and the chromatogram itself.

What area percent leaves out

• Anything the detector cannot see. Water, salts and counter-ions give no signal at 214 nm, so they are not in the denominator. This is why a high purity does not mean the powder is nearly all peptide; see purity versus net peptide content.
• Unequal response. Different peptides absorb light differently. At 214 nm the signal depends on the number of peptide bonds and on certain side chains, with tryptophan, phenylalanine, tyrosine and histidine adding noticeably more than most residues.^[4] An impurity with a different composition does not have to give the same signal per gram as the main peptide.
• Hidden co-eluting species. A single sharp peak can still contain two substances with the same hydrophobicity. A second method, such as a different column, a different pH or mass spectrometry, is how this is checked. Impurity work on a model peptide hormone has shown how mass spectrometry finds and identifies related impurities that matter for an accurate value.^[5]
• Function. Purity says nothing about what a peptide does in any system. It is a statement about how much of the detected signal belongs to one peak.

A short checklist for reading a report

1. Look at the chromatogram, not only the number. Is the baseline flat? Are there peaks the number does not mention?
2. Find the method details: column type, gradient, wavelength and mobile-phase additive. A purity value without them cannot be reproduced.
3. Check that the sample name and the date match the material you hold.
4. Look for a blank run, so you know which peaks belong to the system.
5. Look for a mass-spectrometry result for the main peak. Purity and identity are separate questions.
6. Remember that a purity number is not a content number: weigh with the purity and content calculator if you know both.

A note on stated values

Sealed Peptides product pages list purity as stated by the supplier. A stated value is a starting point; confirming it for your own material needs your own analysis, with a method you can describe and a chromatogram you can show.