Glossary Of HPLC-LC Separation Terms – R

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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r: See relative retention.

Radial compression: Using radial pressure applied to a flexible wall column to reduce wall effects.

Radial diffusion–dispersion: Diffusion–dispersion across the LC column in a radial direction. If the sample is injected into the exact center of a column, it will spread not only in a longitudinal direction as it moves down the column but also radially, which allows the solute to reach the wall region where the eluent velocity is different than in the center of the column.

Re: See Reynolds number.

Recovery: The amount of solute or sample that is eluted from a column relative to the amount injected. Excellent recovery is important for good quantitation, preparative separations, especially biomolecules, and good peak shape and resolution. Reasons for inadequate recovery can be solute interaction with active sites on the packing, column frits, and column tubing. Compound decomposition during the separation process also can affect recovery.

Recycling chromatography: A technique in which the column effluent is recirculated onto the head of the column to take advantage of extended column length. Can be performed on a single column by passing the effluent through the pump again. An alternative technique uses two columns connected by a switching valve where the effluent of one column is directed onto the head of the other column. Very seldom used in HPLC and then only in exclusion chromatography.

Reduced plate height (h): Used to compare efficiencies of different columns; h = H/d p, where H is the height equivalent to a theoretical plate and d p is the particle diameter. An h value of 2 or less at the optimum velocity is considered to be a well-packed HPLC column.

Reduced velocity (ν): Used with the reduced plate height to compare different packed chromatographic columns. It relates the solute diffusion coefficient (D M) in the mobile phase to the particle size of the column packing (d p); ν = ud p/D M, where u is the average interstitial mobile-phase linear velocity. See also Knox equation.

Refractive index peak: A pseudo-peak normally found near the dead volume that results from the refractive index sensitivity of absorbance and other detectors. See also vacancy peak.

Regeneration: Regenerating the packing in the column to its initial state after a gradient elution. Mobile phase is passed through the column stepwise or in a gradient. The stationary phase is restored or solvated to its initial condition. In ion exchange, regeneration involves replacing ions taken up in the exchange process with the original ions, which occupied the exchange sites. Regeneration also can refer to bringing any column back to its original state; for example, removing impurities with a strong solvent.

Relative retention (r): Retention relative to a standard; r = t R´/t R(st)´ = k/k st, where t R´ is the adjusted retention time of the component of interest, t R(st)´ is the adjusted retention time of the standard, k and k st are the corresponding retention factors. For two adjacent peaks, α expresses the relative retention and is called separation factor (formerly called selectivity or selectivity factor); calculated as 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.

Residual silanols: The silanol (–Si–OH) groups that remain on the surface of a packing after chemically bonding a phase onto its surface. These silanol groups, which may be present in very small pores, may be inaccessible to a reacting bulky organosilane such as octadecydimethyl-chlorosilane) but may be accessible to small polar compounds. Often they are removed by endcapping with a small organosilane such as trimethylchlorosilane. See also endcapping.

Resin: A solid polymeric packing used in ion-exchange separations. The most popular resins are PS–DVB copolymers with particle sizes less than 10 μm. Ionic functionality is incorporated into the resin.

Resolution (R s ): Ability of a column to separate chromatographic peaks; Rs ≡ (tR2 – tR1)/[(wb1 + wb2)/2], where tR2 and tR1 are the retention times of the two peaks and wb is the baseline width of the peaks. It usually is expressed in terms of the separation of two peaks. A value of 1 is considered to be the minimum for a measurable separation to occur and to allow good quantitation. A value of 0.6 is required to discern a valley between two equal-height peaks. A value of 1.5 is considered sufficient for baseline resolution for two peaks of equal height. Values of 1.7 or greater generally are desirable for rugged methods. See Figure 2.

Resolution equation: Also called the general resolution equation and the Purnell equation; R = 4N¼ [(α – 1)/α][k/(1 + k)], where N is the efficiency, α is the separation factor, and k is the retention factor.

Retention factor (k): The period of time that the sample component resides in the stationary phase relative to the time it resides in the mobile phase. It is calculated from the adjusted retention time divided by the holdup time; k = (t R – t M)/t M, where t R is retention time for the sample peak and t M is the retention time for an unretained peak. (Formerly, k´ was used, and it was called the capacity factor or the capacity ratio.)

Retention time (t R ): Also called the total retention time. The time between injection and the appearance of the peak maximum. The total retention volume (V R) is determined by multiplying the retention time by the flow rate. The adjusted retention time (t R´) adjusts for the column void volume; t R´ = t R – t M. It usually is measured from the point of injection to the apex of the peak, but it should be measured to the center of gravity of the peak for asymmetric peaks.

Retention volume (V R ): The volume of mobile phase required to elute a substance from the column; V R = F t R or V R = V M + K D V S, where V M is the void volume, K D is the distribution coefficient, and V S is the stationary-phase volume. See also retention time.

Reversed-phase chromatography: The most frequently used mode in HPLC. Uses low-polarity packings such as octadecyl- or octylsilane phases bonded to silica or neutral polymeric beads. The mobile phase usually is water or water-miscible organic solvents such as methanol or acetonitrile. Elution usually occurs based on the relative hydrophobicity or lipophilicity of the solutes. The more hydrophobic, the stronger the retention. The greater the water solubility of the analyte, the less it is retained. The technique has many variations in which various mobile-phase additives impart a different selectivity. For example, adding a buffer and a tetraalkylammonium salt to an anion analysis would allow ion-pairing to occur and generate separations that rival those of ion-exchange chromatography. More than 90% of HPLC analysts use reversed-phase chromatography.

Reynolds number (Re): The ratio of viscous to inertial energy of the moving fluid. A measurement of flow in a smooth unpacked pipe; Re = ud/(η/ρ), where u is the average velocity (in centimenters per second), d is the pipe diameter, η is the viscosity (in grams per centimeter seconds), and ρ is the density (in grams per cubic centimeters). At low Re, viscous friction dominates and controls fluid motion, making it slow and steady. In an unpacked tube, flow becomes fully turbulent when Re exceeds 4200. In a packed bed, u is replaced with the average interstitial velocity and d with the average particle diameter. Flow becomes turbulent in a packed bed at Re values greater than approximately 10 but is not fully turbulent until Re exceeds 100–200.

R s See resolution.

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