Glossary Of HPLC-LC Separation Terms – S

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.

S

S: The solvent-strength parameter in reversed-phase chromatography. The solute-dependent slope of a plot of log₁₀ k versus volume fraction of organic modifier. S varies with modifier type, stationary phase, and temperature.

σ ² : See peak variance.Salting-out effect: Using a high-concentration salt buffer in the mobile phase to cause a low-polarity analyte to have a decreased solubility in water and therefore precipitate or come out of solution. Most often used for the hydrophobic interaction chromatography of proteins when proteins are precipitated first at high salt concentrations and then eluted by gradual dilution using reversed-gradient elution.

Sample capacity: Refers to the amount of sample that can be injected onto an LC column without overloading. Often expressed as grams of sample per gram of packing. Overloading is defined as the sample mass injected when the column efficiency decreases by 10% from its normal value; sometimes called sample loading.

Sampling rate: See data acquisition rate

Saturator column: See precolumn.

SAX: See strong anion exchanger.

Scaleability: In going from analytical to preparative chromatography, refers to the reproducibility of results on columns of different internal diameters when using the same particle size and bonded phase; normally a larger diameter column is used to increase capacity; a linear scaleup process minimizes time required to optimize preparative separations.

SCX: See strong cation exchanger.

SEC: See size-exclusion chromatography and steric exclusion chromatography.

Sedimentation: A technique used for the sizing of resins for ion-exchange chromatography. A broad distribution of beads are placed in a solvent, often water, in a container that is affixed to a stationary surface. Based on particle size and particle density, the beads will settle at different velocities into a gradient of sizes, and the fraction of interest is removed. Workers can obtain very narrow cuts of particle size by sedimentation.

Selectivity or selectivity factor (α): Old term replaced by the separation factor. Sometimes called relative retention.

Selectivity coefficient (k _A/B ): In ion-exchange chromatography, the equilibrium coefficient obtained by applying the law of mass action to an ion exchanger and characterizing the ability of an ion exchanger to select two ions present in the same solution using electroosmotic flow. For example, the exchange of Na⁺ for H⁺ k _Na/H = ([Na]_S [H]_M)/([Na]_M [H]_S).

Semipreparative chromatography: Refers to preparative LC performed on analytical (4–5 mm i.d.) or slightly larger (6–10 mm i.d.) columns. Normal injection size would be milligram- to low-gram-size samples.

Separation factor (α): A thermodynamic factor that is a measure of relative retention of two substances. Formerly called selectivity or selectivity factor. The relative retention; α = t _R2´/t _R1´ = k ₂/k ₁, where t _R2´ and t _R1´ are the adjusted retention times of peaks 2 and 1, respectively, and k ₂ and k ₁ are the corresponding retention factors.

Separation impedance (E): A figure of merit developed by John Knox to compare the efficiency of two chromatographic systems that normalize for both analysis time and pressure drop; E = t_Rδp/N² ν(1 + k), where t_R is the retention time, δp is the pressure drop, N is the efficiency, ν is the reduced velocity, and k the retention factor. The lower the value of E, the better the system.

SFC: See supercritical fluid chromatography.

Silanol: The Si–OH group found on the surface of silica gel. Silanols vary in strength depending upon their location, relationship to each other, and the metal content of the silica. The strongest silanols are acidic and often lead to undesirable interactions with basic compounds during chromatography.

Silanophile: A compound that has high affinity for active or acidic silanol groups on a silica surface. Usually a strongly basic amine.

Silica gel: The most widely used HPLC packing. It has an amorphous structure, is porous, and is composed of siloxane and silanol groups. It is used in all modes of LC as a bare packing for adsorption, as the support for liquid–liquid chromatography or for chemically bonded phases, and as an SEC packing with various pore sizes. Microparticulate silicas of 3-, 5-, and 10-μm average particle diameter are used in HPLC. Compared with irregular silicas, spherical silicas are preferred in modern analytical HPLC columns because of their packing reproducibility and lower pressure drops. Sometimes called silica.

Siloxane: The Si–O–Si bond. A principal bond found in silica gel or a silylated compound or bonded phase. Stable, except at high pH values. Has little effect on the HPLC separation.

Silylation: The reaction process of an organochloro- or organoalkoxysilane with a compound that contains an reactive group. In LC, it refers to the process of derivatizing the solute before chromatography to make it detectable or to prevent unwanted stationary-phase interactions. It also can refer to the process of adding a chemically bonded phase to a solid support or deactivating the packing to reduce surface activity.

Simulated moving bed: A chromatographic system involving a series of columns and valves set up to simulate the countercurrent movement of the mobile and stationary phases and enable the continuous removal of product and reapplication of sample. A complex form of recycle chromatography used in preparative-scale chromatography.

Size-exclusion chromatography (SEC): Same as steric exclusion chromatography.

Slurry packing: The technique most often used to pack HPLC columns with microparticles. The packing is suspended in a slurry of approximately 10% (w/v) and rapidly pumped into the empty column using special high-pressure pumps.

Snyder ´ ^o : Solvent-strength parameter in adsorption chromatography. The energy of solvent adsorption per unit surface area occupied by the solvent.

Soap chromatography: The earlier name for ion-pair chromatography. Long-chain soaps or detergents were used as the mobile-phase additives.

Sol gel: Silica gel formed by the aggregation of silica sol. Generates Type B silica gel with lower surface acidity, lower trace metal, lower surface area and porosity, and greater high-pH stability than older Type A silica gels.

Solid-phase extraction (SPE): A technique for sample preparation using a 20–40 μm d _p solid-phase packing contained in a small plastic cartridge, disk, or in the wells of a 96-well flowthrough plate. The solid stationary phases used are identical to HPLC packings. Although related to chromatography, the principle of SPE is different and is sometimes called digital chromatography. The process as most often practiced requires four steps: conditioning the sorbent, adding the sample, washing away the impurities, and eluting the sample in as small a volume as possible with a strong solvent.

Solid support: Same as support.

Solute: See also analyte.

Solvent: The liquid used to dissolve a sample for injection into an HPLC column or CE capillary. Sometimes refers to the mobile phase used. See also eluent.

Solvent demixing: Occurs when two solvents with very different strengths — A is the weak solvent, and B is the strong solvent — are used with unmodified silica or alumina. The strong solvent (B) will be adsorbed preferentially by the active surface of the stationary phase until it is saturated; until this occurs, the weak solvent (A) will be enriched or demixed as it travels down the column. Eventually, when the entire column is saturated with solvent B, this solvent will be eluted, mixed with solvent A at the initial strength, and sample components will be eluted with the sudden change in solvent strength.

Solvent selectivity: Ability of a solvent to influence selectivity. For example, a change in solvent strength from 5% to 10% solvent B or a change from methanol to acetonitrile as the reversed-phase organic modifier will affect band spacing.

Solvent-selectivity triangle: A useful guide for choosing among different solvents for changing band spacing. Solvent selectivity is dependent on dipole moment, acidity, and basicity of the solvent molecule. See reference 4 for details.

Solvent strength: Refers to the ability of a solvent to elute a particular solute or compound from a column. Snyder described this quality for linear elution adsorption chromatography (liquid–solid chromatography) on alumina and quantitatively rated solvents in an eluotropic series. Less-extensive data are available for silica and carbon adsorbents. See also Snyder ´^o .

Sorb: The process of being retained by a stationary phase when the retention mechanism — adsorption, absorption, or partitioning — is unclear.

Sorbent: Refers to a packing used in LC. Common sorbents are polymers, silica gel, alumina, titania, zirconia, and chemically modified materials.SPE: See solid-phase extraction.

Specific surface area: The surface area of an LC packing based on measurement by an accepted technique such as the BET method using nitrogen adsorption.

Spherical packing: Refers to spherical, solid packing materials. In analytical HPLC, spherical packings generally are preferred over irregular particles, but irregular particles often are used in preparative work because of their lower cost.

Standards: A sample which contains known quantities of the compounds of interest. Standards are used to help identify sample peaks by comparing the time in which they elute to the retention times obtained through the injection of the sample under the same conditions. For quantitation, external standards are compounds that are used to construct calibration curves of detector output (peak area or peak height) vs. concentration; the concentration of unknowns are determined by fitting the detector output to the calibration curve. Internal standards are compounds of known concentration with different retention times that are added to the sample and relative detector responses between the internal standard and the unknown are compared in order to quantitatively measure unknown compounds.

Stagnant mobile phase: The fraction of the mobile phase contained within the pores of the particle.

Stationary phase: The chromatographically retentive immobile phase involved in the chromatographic process. The stationary phase in LC can be a solid, a bonded, an immobilized or a coated phase on a solid support or a wall-coated phase. The stationary phase often characterizes the LC mode. For example, silica gel is used in adsorption chromatography and octadecylsilane bonded phase is used in reversed-phase chromatography.

Stationary zone: To be distinguished from the stationary phase. The stationary zone includes the stagnant mobile phase and the chromatographically active stationary phase.

Stepwise elution: Using eluents of different compositions during a chromatographic run. These eluents are added in a stepwise manner with a pump or a selector valve. Gradient elution is the continuous version of changing solvent composition.

Steric exclusion chromatography: A major mode of LC in which samples are separated by virtue of their size in solution. Also known as size-exclusion chromatography, gel-permeation chromatography, gel-filtration chromatography, and gel chromatography. Steric exclusion chromatography is used most often for polymer separation and characterization.

Sterically protected bonded phase: Bonded phase that has sterically protecting bulky functional groups such as isopropyl and isobutyl surrounding a siloxane covalent surface bond. Prevents attacks on siloxane bond, catalyzed hydrolysis, and loss of bonded phase at pH levels less than 3.

Straight-phase chromatography: Same as normal-phase chromatography.

Strong anion exchanger: Anion-exchange packing with strongly basic ionogenic groups such as tetraalkylammonium groups.

Strong cation exchanger: Cation-exchange packing with strongly acidic ionogenic groups such as sulfonate groups.

Strong solvent: In general, refers to a solvent which is a good solvent for a chemical compound; in chromatography, refers to the mobile phase constituent that provides a higher solvent strength that causes an analyte to elute more quickly from the column; in a water-acetonitrile binary solvent system for reversed-phase LC, acetonitrile would be considered to be the strong solvent.

Sub-2- μ m: A term that refers to the use of porous packings below 2-μm average particle diameter; current products vary from 1.5 to 2.0 μm

Sulfonyl cation exchanger: A strong cation-exchange functionality found in resin-based packings, usually propyl-SO₃H. May come in cationic forms such as sodium, ammonium, silver, and calcium.

Supercritical fluid chromatography (SFC): A technique that uses a supercritical fluid as the mobile phase. The technique has been applied to the separation of substances that cannot be handled effectively by LC (because of detection problems) or GC (because of the lack of volatility). Examples include separations of triglycerides, hydrocarbons, and fatty acids. GC detectors and HPLC pumps have been used together in SFC.

Superficial velocity (u _s ): The hypothetical velocity that a mobile phase would have if the same column were operated unpacked but with the same flow rate; u _s = F/A _c, where F is the flow rate and A _c is the cross-sectional area of the tube.

Superficially porous packing: Same as porous-layer bead.

Support: Refers to solid particles. A support can be naked, coated, or have a chemically bonded phase in HPLC. Normally the solid support doesn't contribute to the chromatographic process.

Suppressor column: Refers to the column placed after the ion-exchange column. Its purpose is to remove or suppress the ionization of buffer ions so that sample ions can be observed in a weakly conducting background with a conductivity detector. Sometimes membrane suppressors are used rather than a column.

Surface area: Refers to the total area of the solid surface in an adsorbent as determined by an accepted measurement technique such as the BET method, which uses nitrogen adsorption. The surface area of a typical porous adsorbent such as silica gel can vary from less than 100 to 600 m²/g.

Surface coverage: Usually refers to the mass of stationary phase per unit area bonded to an LC support. Often expressed in micromoles per square meter of surface. Sometimes the percentage of carbon is given as an indicator of surface coverage.

Swelling–shrinking: Process in which resins and gels increase or decrease their volume because of their solvent environment. Swelling is dependent upon the degree of cross-linking; low-cross-linking resins will swell and shrink more than highly cross-linked resins. If swelling occurs in a packed column blockage, increased back pressure can occur, and column efficiency can be affected.

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