Practical Approaches to - Peptide Isolation - Glossary

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 composition3. 4
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 system.
Deprotection – the removal of amino acid side chain protecting groups. Also the removal of N-terminal peptide protecting groups in solid phase peptide synthesis.
Depyrogenation – column cleaning procedure which uses 1 M sodium hydroxide to remove compounds (i.e., polysaccharides) that could contaminate the final peptide product and induce an allergy-like reaction.
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.16
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 – the 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.

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