Glossary

Short, plain definitions of the terms used across Lab Notes. Where a page covers a term in depth, the entry links to it.

A

Accreditation

Formal recognition by an accreditation body that a lab is competent to carry out specific tests. For testing labs the standard is ISO/IEC 17025, and accreditation covers only the tests listed in the lab’s scope (see Scope of accreditation).

Amino acid

A small molecule carrying both an amino group and a carboxyl group; amino acids are the building blocks of peptides and proteins. Twenty amino acids make up most proteins, and synthetic peptides can include others.

Area percent

The area of one peak in a chromatogram as a percentage of the total area of all integrated peaks. It is a ratio of detector signals, not of masses (see HPLC and how to read a chromatogram).

Assay

In analytical chemistry, a test that measures how much of a named substance a sample contains, usually against a reference standard. The word is also used loosely for any test, so check the method and the unit.

C

Cake

The porous solid left after freeze-drying. It keeps roughly the shape of the frozen fill, and its pores are where the ice crystals were (see Why research peptides ship as a powder).

Certificate of analysis (COA)

A lab’s report on one sample from one lot, listing each test with its method, specification and the value found. It describes that sample on the day it was measured (see How to read a certificate of analysis).

Chromatogram

The plot an HPLC instrument produces: detector signal against time, with a peak for each separated component the detector can see.

Cleavage

In peptide synthesis, the final step in which strong acid releases the finished chain from the resin and removes its side-chain protecting groups (see How peptides are made).

Counterion

The negatively charged partner that pairs with each positive charge on a peptide, making the dried peptide a salt. Trifluoroacetate, acetate and chloride are common, and the counterion counts toward the powder’s mass (see Purity, net content, net peptide content and salt forms).

Coupling

The step in solid-phase synthesis that joins the next amino acid to the growing chain by forming a peptide bond. Where a coupling fails on some chains, it leaves deletion sequences: chains missing one residue.

C-terminus

The end of a peptide chain with a free carboxyl group, written on the right by convention. In solid-phase synthesis the chain is anchored to the resin by this end.

D

Dalton (Da)

The unit of molecular mass used in mass spectrometry, one twelfth of the mass of a carbon-12 atom. A peptide near 1,800 Da has about 1,800 times that mass.

Deamidation

A reaction in which an asparagine or glutamine side chain loses ammonia, raising the mass by about 1 Da. Asparagine followed by glycine deamidates fastest (see Storage science for lyophilized material).

Deconvolution

Turning the family of multiply charged peaks in an electrospray mass spectrum back into a single neutral mass (see Mass spectrometry: identity, not purity).

E

Electrospray ionization (ESI)

A gentle way to turn molecules into ions for mass spectrometry, by spraying the liquid that carries them from a fine capillary held at high voltage. Peptides usually come out carrying several extra protons.

Endotoxin

A class of molecules (lipopolysaccharides) from the outer membrane of Gram-negative bacteria. It is a contaminant that HPLC purity does not measure; it is detected with the LAL test and can interfere with laboratory assays.

F

Fmoc

9-Fluorenylmethoxycarbonyl, the protecting group that blocks the amino end of each incoming amino acid in the most common form of solid-phase synthesis. A mild base removes it before the next coupling.

G

Gradient

In reversed-phase HPLC, the planned rise in the share of organic solvent in the mobile phase during a run. It is part of the method, and a retention time means nothing without it.

H

HPLC

High-performance liquid chromatography: a technique that separates the components of a sample by pumping it through a packed column, and records them with a detector as they leave. Peptide purity is usually measured by reversed-phase HPLC with UV detection.

Hygroscopic

Tending to absorb water from the air. Freeze-dried peptides are hygroscopic, so keeping moisture out matters in storage.

I

Identity

Whether the main substance in a sample is the intended molecule. For peptides it is usually tested by mass spectrometry, comparing the measured mass with the mass calculated from the sequence.

ISO/IEC 17025

The international standard for the competence of testing and calibration laboratories; the current edition dates from 2017. Labs are accredited to it, for a defined scope, by an accreditation body.

K

Karl Fischer titration

A chemical method that reacts specifically with water, used to measure the water content of a powder.

L

LAL

Limulus amebocyte lysate, a reagent prepared from the amebocytes of the horseshoe crab (Limulus) that reacts with bacterial endotoxin. It is used in the laboratory test for endotoxin.

LC-MS

Liquid chromatography coupled to mass spectrometry: an HPLC separation whose output flows straight into a mass spectrometer, so each peak can be given a mass.

Lot

A quantity of material made in one production run and given one identifying number; also called a batch. A certificate describes only the lot it names.

Lyophilization

Freeze-drying: freezing a material, removing the ice by sublimation under vacuum, then driving off the remaining bound water by gentle warming. It leaves a dry, porous solid.

M

Mass spectrometry (MS)

A technique that measures the mass-to-charge ratio of ions. For peptides it is used mainly to confirm identity; it does not measure purity.

Mass-to-charge ratio (m/z)

An ion’s mass in daltons divided by the number of charges it carries: the quantity a mass spectrometer actually measures.

Monoisotopic mass

The mass of a molecule calculated with every atom as its most abundant isotope. The average mass, based on each element’s average atomic mass, is about one dalton higher for a peptide near 1,800 Da.

N

Net peptide content

The share of a powder’s mass that is peptide, as opposed to counterion, water and residual solvent. It is usually measured by amino acid analysis and commonly falls somewhere around 70 to 90 percent for freeze-dried peptide.

N-terminus

The end of a peptide chain with a free amino group, written on the left by convention.

P

Peptide bond

The amide bond joining the carboxyl group of one amino acid to the amino group of the next. Peptide bonds absorb far-ultraviolet light, which is what HPLC detection at 210 to 220 nm relies on.

Purity

For peptides, usually the area percent of the main peak in an HPLC-UV chromatogram: the share of the separated, UV-visible material that is the main component. It is not the peptide content of the powder.

R

Residual moisture

The water left in a material after freeze-drying, usually reported as percent by mass and measured by Karl Fischer titration.

Residue

One amino acid unit within a peptide chain. A 16-residue peptide is built from 16 amino acids.

Retention time

The time from loading a sample to the top of a component’s peak in a chromatogram. It is reproducible under a fixed method, but on its own it does not prove identity.

Reversed-phase HPLC

The usual HPLC mode for peptides: a column packed with water-repelling particles, commonly C18, and a mobile phase whose share of organic solvent rises during the run, so peptides leave in order of increasing hydrophobicity.

S

Salt form

The counterion a peptide is paired with, such as trifluoroacetate, acetate or chloride. The same peptide in different salt forms gives powders with different peptide shares by mass.

Scope of accreditation

The specific tests, methods and materials for which a lab holds accreditation. A test outside the scope is not accredited, whatever the lab’s letterhead says.

Sequence

The order of amino acids in a peptide chain, written from the N-terminus to the C-terminus.

Solid-phase peptide synthesis (SPPS)

Building a peptide one amino acid at a time on insoluble resin beads, washing away excess reagents after each step. Most synthetic peptides are made this way.

Specification

A limit set before a test is run, such as “not less than 98.0%”. “Conforms” means the value met it; read the value as well as the verdict.

Sublimation

A direct change from solid to vapor without melting. Freeze-drying uses it to remove ice below the pressure of water’s triple point, about 611 Pa.

T

Trifluoroacetic acid (TFA)

A strong acid used to cleave peptides from the resin and as an additive in HPLC mobile phases. Because of this, many synthetic peptides are supplied as TFA salts.