Even after multiple rounds of purification, small amounts of
impurities may still exist in the final peptide product.
Therefore, GenScript uses reversed-phase high performance liquid
chromatography (RP-HPLC) to analyze the purity of each custom
What is RP-HPLC?
RP-HPLC is a widely used analytical tool, in which the
components of a complex mixture can be efficiently separated.
The analyte mixture is usually dissolved in water, which is
sometimes also mixed with an organic solvent or an acid to
assist dissolution prior to RP-HPLC analysis. The analyte is
carried by a mobile phase consisting of water and an organic
modifier, which is pumped through a column packed with a
stationary phase which usually is a number of small diameter
particles consisting of carbon chains of a specified length on
the surface. As the mixture is pumped through the column, the
analytes (in this case your custom peptide and small impurities)
adsorb to the hydrophobic surface of the stationary phase. As
the percentage of organic modifier in the mobile phase is
gradually increased, the analytes desorb into the mobile phase.
Desorption of a specific analyte is based on its intrinsic
properties, thus each analyte will remain in the column for a
specific amount of time, called the retention time. Since
peptide bonds maximally absorb UV light at the wavelength of 220
nm, a UV spectrometer is commonly used in RP-HPLC to detect a
peptide as it elutes from the column. The detection signal is
converted to a visual graph called a chromatogram, which is the
plot of UV absorbance vs. elution time.
High resolution of eluted peptides is mainly based on the
selection of columns and the organic modifier, especially its
elution gradient. The height of a theoretical plate of each
column used in the manufacture is regularly checked, and
different gradients are established to separate varying
peptides. Both processes are aimed at maintaining high
resolution of every custom peptide. GenScript takes special care
in developing RP-HPLC protocols to ensure the most effective
separation and the accurate purity determination of your custom
Reading Your Custom Peptide HPLC report
HPLC mobile phase and its elution gradient: The
peptide separation is highly sensitive to the selection of
organic modifier and its elution gradient.
Absorption wavelength: The wavelength
of 220 nm is chosen for UV detection since peptide bonds
(amide bonds) maximally absorb UV at this wavelength.
Column type and dimensions: The
selection of the column plays an important role in RP-HPLC
analysis. A C18 column is usually used to separate peptides.
Dead time: Indicates the amount of time
required for an unretained solute such as the organic solvent
or the acid from the column. Thus, the organic solvent nor the
acid contributes to the area of the target peptide peak.
Major component elution peak (the target peptide):
Shows the retention time of the target peptide as well as the
impurities. The integrated peak area is used for the purity
calculation. The baseline should be smooth, indicating that
the system is stable. The vertical lines separate the
partially overlaid peaks and denote the peak area of the
Peak table: All peaks shown in the
chromatogram are listed according to their retention time. The
Area % of the target peptide is used to calculate the peptide
We use electrospray ionization mass spectrometry (ESI-MS)
analysis to confirm the molecular weight of the target peptide.
What is ESI-MS?
ESI-MS is an analytical tool used to study macromolecules, where
the mass-to-charge ratios (m/z) of all ionized analytes are
respectively determined. As one of the widely used soft
ionization techniques, electrospray ionization produces a whole
charged ion from the solution of analytes without fragmentation
of the macromolecule, which provides a reliable and accurate way
to measure the molecular weight of the macromolecule. Generally
peptides can be ionized into multiple-charged states, either
positively or negatively, depending on the number of basic and
acidic residues in the sequence.
The ionized peptides are then transferred into a mass analyzer
such as a quadruple or a time-of-flight (TOF), which can
separate all charged ions according to their m/z. All these
isolated ions are respectively detected by an ion detector such
as an electron multiplier and finally are converted into a
visual graph called a mass spectrum, which is the plot of ion
abundance vs. m/z value of all detected ions. The mass spectrum
displays all detected m/z signals and the relative intensity of
the ions, which is relative to the highest m/z peak. The highest
peak is set as 100%. For most peptides, the mass spectrum
displays more than one m/z peak representing a series of charge
states. Theoretically the m/z values of these peaks should all
result in the same calculated molecular mass, however,
calculations from these peaks actually vary with each other
slightly due to limited resolution of the instrument. In our MS
report, the calculated molecular weight from the most abundant
m/z peak of the analyte is utilized to largely eliminate
variations from the instrument. GenScript performs regular
cleaning and calibration of mass spectrometers to ensure the
mass accuracy and reproducibility of MS analyses.
Reading Your Custom Peptide MS report
Ionization mode: Either positive or
negative ionization mode can be used for the peptide
Note: In positive ionization mode, the m/z value of the
target peptide can be used to calculate the measured molecular
weight (MW) by using the formula MW=(m/z)*IzI-z
z: +2, the measured
z: +3, the measured
z: +4, the measured
Instrument settings: The optimized
parameters for ESI ion source.
Measured MW by mass spectrometer: The
MW calculated from the most abundant m/z peak of the analyte.