How purity and identity are established

Two laboratory procedures do most of the work here, and they answer two different questions. Chromatography asks how much of a sample is one thing. Mass spectrometry asks what that thing weighs. A certificate of analysis reports both, for one batch, and this page sets out what those procedures do, what their output looks like, what ends up on the certificate, and how a vial is matched back to the document that describes it.

Chromatography

Reversed-phase HPLC separates a sample into its components and reports the main peak as a proportion of the total integrated area.

Mass spectrometry

Measures molecular mass, so what is in the vial can be compared with the mass calculated from the stated sequence.

Batch records

One batch, one certificate. The number printed on the vial is the number the certificate is issued against.

Two procedures, two different questions

Purity and identity are separate questions and no single measurement answers both. A chromatogram cannot tell you what a peak is. A mass spectrum cannot tell you how much of the sample it accounts for. A certificate carries both results because either on its own is incomplete.

Reversed-phase HPLC

High-performance liquid chromatography separates a mixture by pushing it through a column. The sample is dissolved, loaded onto the column, and carried through a bed of fine non-polar particles by solvent under high pressure, while the composition of that solvent is changed along a gradient. Components leave the column at different times according to how strongly each is held by the packing, and a detector at the far end records ultraviolet absorbance (conventionally at 220 nm, where the peptide bond itself absorbs) against time.

The output is a chromatogram: a flat baseline with peaks rising off it. The time at which a peak appears is its retention time. The area beneath a peak is proportional to how much of that component passed the detector. Purity is reported by area normalisation: the area of the main peak expressed as a percentage of the total area of every peak the software integrated.

That figure is a relative measure, and it is worth being precise about what it does not cover. It compares peaks with peaks. It cannot report anything that does not absorb at the wavelength used, anything that never leaves the column, or anything that leaves it at the same moment as the main peak. Water and counter-ion are determined separately for exactly that reason.

Mass spectrometry

Mass spectrometry measures mass rather than quantity. In electrospray ionisation the sample is sprayed from a fine capillary held at high voltage; the solvent evaporates from the droplets and what is left is a population of gas-phase ions. The analyser separates those ions by their mass-to-charge ratio and a detector counts them.

The output is a spectrum: intensity plotted against mass-to-charge ratio. A peptide does not usually appear as a single line: it appears as a series of ions carrying different numbers of charges, and that series is deconvoluted to one molecular mass.

Identity is then established by comparison. The mass calculated from the stated amino-acid sequence is a fixed arithmetic result. The observed mass is a measurement. Agreement between the two, within the tolerance the instrument and the method allow, is the evidence that the material in the vial is the compound named on the label. Disagreement is a failed result, and a batch that fails is not released.

Supporting determinations

Appearance is recorded by eye against a written specification. For a lyophilised peptide that specification is normally a white to off-white cake or powder, and anything else is a deviation to be investigated rather than a matter of opinion.

Water content is determined by Karl Fischer titration, a reaction that responds to water specifically rather than to everything volatile. Counter-ion content (commonly acetate or trifluoroacetate, depending on how the material was purified) is determined chromatographically, because the counter-ion is present by design and is not something an HPLC purity figure at 220 nm was ever going to report.

Net peptide content states how much of the mass in the vial is peptide rather than water and counter-ion. Where it is determined, the certificate names the method used to determine it.

What a certificate of analysis contains

A certificate of analysis is a record of what was measured, by which method, against which specification, for one batch. It is not a statement about a product line and it does not transfer between batches. A second batch of the same compound gets a second certificate with its own numbers on it.

Product identity
The compound as named on the label, its physical form, and the quantity in the vial.
Batch number
The single number tying the document to one synthesis, one analytical run and one set of filled vials.
Dates
Date of manufacture, date of analysis, and the date by which the batch should be re-analysed if it is still held.
Appearance
The written specification, and what was actually observed.
Purity
The method (reversed-phase HPLC, the detection wavelength, and the integration approach) together with the specification and the result.
Identity
The method (mass spectrometry), the mass calculated from the sequence, and the mass observed.
Water content
The method, the specification and the result.
Counter-ion content
Which counter-ion is present, the method, the specification and the result.
Storage
The conditions the batch was released under, which are the conditions printed on the vial label.
Authorisation
Who issued the certificate, in what capacity, and on what date.

What one looks like

The document below shows the shape of a certificate rather than the contents of one. Nothing in it is a result. The batch number is deliberately not a real batch number and every value is a placeholder in square brackets. To obtain the certificate for a batch you are holding, ask for it by its number on check a batch.

Appearance, identity and purity are on every certificate. The determinations below them are reported where the laboratory made them, so a certificate for a batch you hold may carry some of them and not others.

PXP Peptides

Certificate of analysis

Example: specimen layout

Product
[Compound name], lyophilised powder
Batch number
PXP-EXAMPLE-0000
Quantity per vial
[n] mg
Date of manufacture
[YYYY-MM-DD]
Date of analysis
[YYYY-MM-DD]
Re-analysis date
[YYYY-MM-DD]
Storage
[Storage condition this batch was released under]
Analysis by
[Name of the testing laboratory]
Results
Test Method Specification Result
Appearance Visual White to off-white lyophilised powder Conforms
Identity ESI mass spectrometry Observed mass agrees with [theoretical mass] Da [observed] Da
Purity RP-HPLC with UV detection ≥ [nn.n] % [nn.n] %
Reported where determined
Single impurity RP-HPLC with UV detection Largest single peak ≤ [n.n] % [n.n] %
Water content Karl Fischer titration ≤ [n.n] % [n.n] %
Counter-ion Ion chromatography [counter-ion], [n.n] to [n.n] % [n.n] %
Net peptide content [Method] Reported [nn.n] %

Authorised by: [Name], [position]

Date of issue: [YYYY-MM-DD]

Example only. This is a specimen layout, not a certificate. No value on it is a result for any batch of any material, and it must not be copied, circulated or presented as though it were one.

Batch numbering, and what it is for

A batch is one synthesis, purified and analysed together. Everything downstream of that is tied to its number, which is what makes it possible to take a vial off a shelf and find the document that describes it.

Assigned at manufacture

A batch number is assigned when the material is made and does not change afterwards. It identifies one synthesis and the vials filled from it.

Carried on the analysis

The certificate of analysis is issued against that number. Results belong to the batch and not to the product line, so a repeat batch of the same compound has its own certificate with its own figures on it.

Printed on what arrives

The number is on the vial label, repeated on the outer packaging and recorded on the despatch note. Quoting it is enough for us to retrieve the certificate.

What arrives, and how it is packed

Nothing below describes what any material is for or what should be done with it. This is supply and handling information: what is in the box, how it travels, and the conditions each batch was released under.

In the box

Each vial is glass, closed with a stopper and a crimped seal, and labelled with the compound, the quantity, the batch number and the storage condition for that batch.

The despatch note lists every line with its quantity and its batch number, so the contents of a box can be checked against the paperwork before anything is opened. A certificate of analysis exists for each batch supplied and is issued on request, by batch number.

Packaging

Outer packaging is plain. There is no branding on it, no description of what is inside and no indication of value. Vials are packed so that they cannot move against one another in transit.

Consignments are despatched from the UK by Royal Mail Tracked 24, with a tracking number sent by email when the order leaves. Despatch and delivery times are on the FAQ page.

Storage and cold chain

Peptides are supplied lyophilised, a dry solid rather than a solution. Storage conditions differ by line: the condition for a given batch is printed on the vial label, repeated on its certificate of analysis and shown on the product page. Where those disagree, the label on the vial is the one that governs.

Material should be brought to its stated storage condition on arrival rather than left in the packaging it travelled in.

Request a certificate of analysis

Certificates are issued by batch. Give us the number printed on the vial or on the despatch note and we will send the certificate for that batch. If the number is not to hand, name the compound and roughly when it was ordered and we will find it.

Check a batch

Research use only

Research use only. Not for human or veterinary consumption, not for diagnostic or therapeutic use, and not a medicine or a food.

  • Everything listed on this site is supplied for laboratory research, to laboratories, universities and research organisations.
  • Purchasers must be 18 or over and must be buying for research or institutional use.
  • Nothing on this site describes how any material should be used, and PXP does not supply protocols, quantities or methods of administration on request.
  • The buyer is responsible for safe handling, for storage after delivery, and for compliance with the law in their own jurisdiction.

The full position is set out in our research-use policy. If something on this page is not clear, ask us.