Practical Approaches to Peptide Isolation

Active splitter

Device which is used to manage mass spec detector signal by sampling the preparative flow stream with a defined split ratio as programmed by the user. The sample is diluted and transferred to the detectors with a makeup solvent.

At-column dilution

Patented injection technique that increases mass capacity, improves resolution, increases column lifetime and improves LC system ruggedness by diluting sample in strong solvent at the head of the column.

Average mass

 Mass of an ion or molecule weighted for its isotopic composition.

Centroid

Continuum data processed to display a single, centered data point for each distribution of ions in a mass spectrum and are displayed as sticks in the mass spectrum.

Charge state

Mass spectrometers operate on the basis of the mass-to-charge ratio (m/z), where z is defined as the charge state. Calculated by dividing 1 Da, the theoretical mass difference between isotopes, by the molecule’s actual mass difference between isotopes.
single charge m/z = (M+H)/1
double charge m/z = (M+2H)/2
n charge m/z = (M+nH)/n
Isotope peaks of n-charged ions are separated by 1/n Da (e.g., isotope peaks of doubly charged ions are separated by 0.5 Da). Multiple charging extends the mass range of the mass spectrometer.

Chromophore

Molecular group that absorbs light at a particular frequency.

Column volume

Volume inside the column body.

Continuum data

Full profile of the distribution of all of the signals present in a mass spectrum.

Cleavage

The removal of the peptide from the resin in solid phase peptide synthesis.

Delay volume

The volume from the point of gradient mixing to the head of the column in a chromatographic

Deprotection

The removal of amino acid side chain protecting groups. Also the removal of N-terminal peptide protecting groups in solid phase peptide synthesis.

Dwell volume

Same as system volume.

Electrospray

Atmospheric ionization technique which produces very little fragmentation and is useful for analyzing polar compounds and biomolecules.

Focused gradient

Gradients which target only the product by rapidly increasing the solvent strength from the low concentrations used for sample loading to about 5% below the expected elution point for the product peak. For peptides, the shallow segment of the gradient is best run at approximately 0.25–0.33% change per column volume. Once the peptide product is eluted, the column is washed with a high percentage of organic solvent and then reequilibrated at initial conditions.

HPLC index

Predicts the percentage of acetonitrile required to elute the peptide from a C₁₈ µBondapak Column using TFA:water:acetonitrile as the buffer system, as described by Browne, Bennet, and Solomon.

Hydrophobic

Tending not to dissolve in, mix with, or be wetted by water.

Ion-exchange chromatography

Separation of compounds using columns with charged functionality bonded to their surface.

Ionization

Process by which a molecule obtains an electric charge by gaining or losing electrons.

Isocratic

Chromatography in which the mobile-phase composition remains constant over the course of the separation

Linear gradient

Separation method which changes the mobile-phase composition over the course of a defined period of time.

Loading

He amount of compound that can be applied to a column of specific dimensions.

Make-up solvent

Solvent used to dilute and transfer sample split from the prep stream to the detectors in a mass-directed purification system.

Mass capacity

Mass load; directly proportional to the volume of the column.

Mobile-phase

Solvents used to elute compounds from a column.

Mobile-phase modifier

Additives mixed into the chromatographic solvents for improving the separation.

Monoisotopic mass

Mass calculated using the most abundant isotopes of each element in the molecule.

Passive splitter

Device used to continuously sample the prep stream with a defined split ratio; manages the detector signal in mass-directed purification.

Resolution

The width of two peaks relative to the distance between those peaks.

Reversed-phase chromatography

A type of separation method in which the mobile- phase is more polar than the packing material. Compounds are separated based on their interaction with the non-polar stationary phase.

Scaling

Maintaining a separation upon transfer from a small column to a large column or vice versa.

Segmented gradient

Separation method which maintains the slope before and after a shallow, focused portion of the gradient, thereby preserving the chromatographic profile for these parts of the separation.

Shallow gradient

Separation method in which the rate of change of organic solvent concentration per column volume is low.

Side-chain protecting group

Organic molecules attached to amino acid side chains to prevent the side chains from reacting with other molecules during peptide synthesis.

Silanol

Chemical group located on the silica substrate column packing material which is available for interaction with sample.

Size-exclusion chromatography

Separation of compounds based on their size in solution.

Slope

The rate of change in organic solvent composition per column volume during a gradient separation.

Solid-phase peptide synthesis

Process by which a peptide is constructed by sequentially adding amino acids to a growing chain with the first C-terminal amino acid linked to a solid support.

Solution phase peptide synthesis

Process by which peptides are made in solution with organic synthetic reactions. Short peptide segments may be condensed together to form long peptide sequences.

Split ratio

The amount of material continuously “removed” and diluted from the sample stream before transfer to the detectors during mass-directed purification.

System volume

Same as dwell volume and delay volume.