Glossary Of HPLC-LC Separation Terms – P

The listing should be helpful to those just starting in HPLC but it also can serve as a refresher for long-time users in the field.

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P

Δp: See head pressure.

Pa: See pascal.

Packing: The adsorbent, gel, or solid support used in an HPLC column. Most modern analytical HPLC packings are less than 10 μm in average diameter, and 5 μm is the current favorite.

Paired-ion chromatography: Same as ion-pair chromatography.

Particle size (d p ): The average particle size of the packing in the LC column. A 5-μm d p column would be packed with particles with a definite particle-size distribution because packings are never monodisperse. See also monodisperse particles, particle size distribution, and polydisperse particles.

Particle-size distribution: A measure of the distribution of the particles used to pack the LC column. In HPLC, a narrow particle-size distribution is desirable. A particle-size distribution of d p ± 10% would mean that 90% of the particles fall between 9 and 11 μm for an average 10-μm d p packing.

Particulates: Generally refers to a small particles found in the mobile phase that can cause back pressure problems by lodging in frits; it can also refer to the small particles packed into HPLC columns

Partition chromatography: Separation process in which one of two liquid phases is held stationary on a solid support (stationary phase) while the other is allowed to flow freely down the column (mobile phase). Solutes partition themselves between the two phases based on their individual partition coefficients. Liquid–liquid chromatography is an example; modern bonded-phase chromatography can be considered to be a form of partition chromatography in which one of the liquid phases is actually bonded to the solid support. Mechanistically partition chromatography implies that the solute becomes at least partially embedded within the stationary phase, which is impregnated, coated, or bonded to the substrate. In contrast to an adsorption process in which the solute does not penetrate into the retentive surface or interphase.Partition coefficient (K): The ratio of the equilibrium concentration of solute in the stationary phase relative to the equilibrium concentration of solute in the mobile phase. Also called distribution coefficient, KD, and distribution constant (Kc).

Pascal (Pa): A unit of pressure. 1 MPa is approximately 10 bar (atm) or 150 psi.

Peak: The profile of an analyte compound as it elutes from a column through a detector; usually depicted on a visual output on a recorder or printer based on the detector's electrical response.

Peak area: The area measured under a chromatographic peak; usually measured by an integrator or data system; the peak area is related to the amount of substance eluted in a peak.

Peak capacity (n): The number of equally well-resolved peaks (n) that can be fit in a chromatogram between the holdup volume and some upper limit in retention. For R = 1, n is given by the approximation 1 + 0.25[(N)¼ ln(1 + k n)], where R is the resolution, N is the number of theoretical plates, and k n is the retention factor for peak n.

Peak dispersion: See band broadening.

Peak doublet: A split peak generally caused by a column void. Could be closely eluted compounds.

Peak height: The height of a chromatographic peak as measured from the baseline to the peak apex; the peak height is related to the amount of substance eluted in a peak.

Peak shape: Describes the profile of a chromatography peak. Theory assumes a Gaussian peak shape (perfectly symmetrical). Peak asymmetry factor describes shape as a ratio. See Figures 1 and 2. See also asymmetry.

Peak tracking: A way of matching peaks that contain the same compound between different experimental runs during method development. Relies upon detection parameters of each pure analyte. Diode-array detectors and mass spectrometers are among the best detectors for peak tracking because of their specificity.

Peak variance (σ 2 ): The second central moment of the peak about the retention time. For a Gaussian peak, the variance is the fundamental parameter controlling peak width. See Figure 2. See also Gaussian peak.

Peak volume: The total volume occupied by a chromatographic peak as it passes through the detector; VR= F x wb (see Figure 2).

Peak width (w b ): Same as bandwidth. See Figure 2.Pellicular packing: See porous-layer bead.

Percent B solvent (% B solvent): Refers to the stronger solvent in a binary solvent mixture. % A solvent would be the weaker solvent analog.

Perfusion chromatography: Refers to chromatography performed using particles with very large pores (4000–8000 Å) called throughpores (megapores or gigapores). Eluent flows between the large pores and through the particles' 300–1000 Å interconnecting pores, called diffusive pores. Best suited for the preparative separation of macromolecules.

Permeability (B o ): Also called column permeability and specific permeability. A term expressing the resistance of the packed column to the flow of mobile phase. For a packed column, B o ≈ d p 2 ´3 /[180(1 – ´)2 ] = d p 2 /1000. A column with high permeability gives a low pressure drop.

Permeation: Refers to the SEC process in which a solute can enter a mobile-phase-filled pore of the packing.

Phase ratio (β): The relative amount of stationary to mobile phase in the column. In partition chromatography, β = V S/V M, where V S and V M are the volume, of stationary and mobile phase in the column, respectively. The retention factor is the product of the phase ratio and the partition coefficient.

Phenyl phase: A popular nonpolar bonded phase prepared by the reaction of dimethylphenylchloro- or alkoxysilane with silica gel. Reportedly has affinity for aromatic-containing compounds and does impart a different selectivity compared with alkyl-bonded phases.

Pirkle column: Chiral, brush-type stationary phases based on 3,5-dinitrobenzoylphenylglycine silica used in the separation of a wide variety of enantiomers. Named after its developer, William Pirkle of the University of Illinois.

Planar chromatography: A separation technique in which the stationary phase is present as or on a plane (IUPAC). Typical forms are paper and thin-layer chromatography.

Plate height (H): See HETP.

Plate number: See column plate number.

Plate or plate number: Refers to theoretical plates in a packed column (IUPAC). See also theoretical plate.

Polar: Chemical polarity refers to the dipole-dipole intermolecular forces between the slightly positively-charged end of one molecule to the negative end of another or the same molecule. Molecular polarity is dependent on the difference in electronegativity between atoms in a compound and the asymmetry of the compound's structure. For example, water is polar because of the unequal sharing of its electrons. However, methane is considered non-polar because the carbon shares the hydrogen molecules uniformally.

Polyacrylamide gel: Neutral hydrophilic polymeric packings used in aqueous SEC. Prepared by the copolymerization of acryl-amide with N,N9-methylenebisacrylamide.

Polydisperse particles: Particles that have a substantial range of diameters (>10%).

Polyethyleneimine: An anionic polymeric phase used to coat or bond onto silica or a polymeric packing. Most often used for separating proteins and peptides.

Polymeric packings: Packings based on polymeric materials, usually in the form of spherical beads. Typical polymers used in LC are polystyrene–divinylbenzene (PS–DVB), polydivinylbenzene, polyacryl-amide, polymethylacrylate, polyethylene-oxide, polydextran, and polysaccharide.

Polymeric phase: Refers to a chemically bonded phase in which a polymer species is bonded to silica-based particles.

Polystyrene–divinylbenzene resin (PS–DVB): The most common base polymer for ion-exchange chromatography. Ionic groups are incorporated by various chemical reactions. Neutral PS–DVB beads are used in reversed-phase chromatography. Porosity and mechanical stability can be altered by varying the cross-linking through the DVB content.

Poppe plot: A kinetic plot named after Prof. Hans Poppe [J. Chromatogr. A 778, 3 (1997)], University of Amsterdam, the Netherlands, where the plate time (log [t 0/N]) is depicted as a function of the number of theoretical plates (N) to assess the limits of column performances as a function of particle size, column pressure drop, and so forth.

Pore diameter: Same as mean pore diameter.

Pore size: The average size of a pore in a porous packing. Its value is expressed in angstroms or in nanometers. The pore size determines whether a molecule can diffuse into and out of the packing. See also mean pore diameter.

Pore volume: The total volume of the pores in a porous packing, usually expressed in milliliters per gram. More appropriately called the specific pore volume. It is measured by the BET method of nitrogen adsorption or by mercury-intrusion porosimetry in which mercury is pumped into the pores under high pressure.

Porosity: For a porous substrate, the ratio of the volume of the pores in a particle to volume occupied by the particle. The pore volume is a measure of the porosity and is expressed in milliliters per gram.

Porous-layer bead: A small glass bead coated with a thin layer of stationary phase. The thin layer can be an adsorbent, resin, or a phase chemically bonded onto the adsorbent. These packings were among the first to be used in HPLC. They had 20–40 μm particle sizes, which were larger than the microparticulate packings of today, but were easy to pack and provided adequate efficiency. Also called controlled surface-porosity supports and pellicular materials.

Porous particle: Refers to column packing particles that possess interconnecting pores of specified diameter and pore volume. For HPLC applications, analysts generally use porous particles with diameters less than 10 μm. Larger particles are used in preparative-scale chromatography because of lower cost and higher column permeability.

Porous polymer: A packing material, generally spherical, that is based on organic polymers or copolymers. Popular examples include PS–DVB, polyacrylates, polydextrans, polyacrylamides, and polybutadienes.

Precolumn: A small column placed between the pump and the injector. It removes particulate matter that may be present in the mobile phase, presaturates the mobile phase with stationary phase or with dissolved substrate to prevent a loss of stationary phase or dissolution of the analytical column, and chemically absorbs substances that might interfere with the separation. Its volume has little effect on isocratic elution but contributes a delay to the gradient in gradient elution.

Precolumn Filter: A filter used between the injector and the column (or guard column) to keep unwanted sample components from reaching the column; sometimes called in-line filter, occasionally inlet filter.

Preconcentration: See trace enrichment.

Preparative chromatography: Refers to the process of using LC as a technique for the isolation of a sufficient amount of material for other experimental or functional purposes. For pharmaceutical or biotechnological purifications, large columns of several feet in diameter can be used for multiple grams of material. For isolating a few micrograms of valuable natural product an analytical column with a 4.6-mm i.d. can be used. Based on the intended need of the chromatographer, both size of columns are preparative chromatographic approaches.

Pressure (pressure drop) (Δp): See head pressure.

Pressure injection: Pressure-induced injection in CE. Using pressure or vacuum to inject nanoliter-level volumes of sample into a capillary column. Best for narrow-bore capillaries that have inner diameters less than 10 μm. A version of hydrostatic injection.

Process-scale chromatography: Refers to the use of LC at the industrial-scale level outside of laboratories. Generally requires specially designed columns (usually with diameters > 5 cm), recoverable solvents, low-cost packings (larger and irregular-shaped particles), and overloaded operating conditions compared with laboratory-scale HPLC.

Programmed-temperature chromatography: Varying temperature during a chromatographic run. Seldom used in LC.

PS–DVB: See polystyrene–divinylbenzene resin.

Pulsating flow: Flow originating from a reciprocating pump. Normally, the pulses are dampened by a pulse damper, an electronic pressure feedback circuit, or an active damper pump head. Detectors such as electrochemical and refractive index detectors are greatly affected by flow pulsations.

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