Beginner's Guide to Liquid Chromatography

Alumina

A porous, particulate form of aluminum oxide [Al₂0₃] used as a stationary phase in normal-phase adsorption chromatography. Alumina has a highly active basic surface; the pH of a 10% aqueous slurry is about 10. It is successively washed with strong acid to make neutral and acidic grades [slurry pH 7.5 and 4, resp.]. Alumina is more hygroscopic than silica. Its activity is measured according to the Brockmann† scale for water content; e.g., Activity Grade I contains 1% H₂O.

†H. Brockmann and H. Schodder, Ber. 74: 73 (1941).

Baseline*

The portion of the chromatogram recording the detector response when only the mobile phase emerges from the column.

Cartridge

A type of column, without endfittings, that consists simply of an open tube wherein the packing material is retained by a frit at either end. SPE cartridges may be operated in parallel on a vacuum-manifold. HPLC cartridges are placed into a cartridge holder that has fluid connections built into each end. Cartridge columns are easy to change, less expensive, and more convenient than conventional columns with integral endfittings.

Chromatogram*

A graphical or other presentation of detector response or other quantity used as a measure of the concentration of the analyte in the effluent versus effluent volume or time. In planar chromatography [e.g., thin-layer chromatography or paper chromatography], chromatogram may refer to the paper or layer containing the separated zones.

Chromatography*

A dynamic physicochemical method of separation in which the components to be separated are distributed between two phases, one of which is stationary [the stationary phase] while the other [the mobile phase] moves relative to the stationary phase.

Column Volume* [Vc]

The geometric volume of the part of the tube that contains the packing [internal cross-sectional area of the tube multiplied by the packed bed length, L]. The interparticle volume of the column, also called the interstitial volume, is the volume occupied by the mobile phase between the particles in the packed bed. The void volume [V₀] is the total volume occupied by the mobile phase, i.e. the sum of the interstitial volume and the intraparticle volume [also called pore volume].

Detector* [see Sensitivity]

A device that indicates a change in the composition of the eluent by measuring physical or chemical properties [e.g., UV/visible light absorbance, differential refractive index, fluorescence, or conductivity]. If the detector’s response is linear with respect to sample concentration, then, by suitable calibration with standards, the amount of a component may be quantitated. Often, it may be beneficial to use two different types of detectors in series. In this way, more corroboratory or specific information may be obtained about the sample analytes. Some detectors [e.g., electrochemical, mass spectrometric] are destructive; i.e., they effect a chemical change in the sample components. If a detector of this type is paired with a non-destructive detector, it is usually placed second in the flow path.

Display

A device that records the electrical response of a detector on a computer screen in the form of a chromatogram. Advanced data recording systems also perform calculations using sophisticated algorithms, e.g., to integrate peak areas, subtract baselines, match spectra, quantitate components, and identify unknowns by comparison to standard libraries.

Efficiency [H, see Plate Number, Resolution, Sensitivity, Speed]

A measure of a column’s ability to resist the dispersion of a sample band as it passes through the packed bed. An efficient column minimizes band dispersion or bandspreading. Higher efficiency is important for effective separation, greater sensitivity, and/or identification of similar components in a complex sample mixture.

Nobelists Martin and Synge, by analogy to distillation, introduced the concept of plate height [H, or H.E.T.P., height equivalent to a theoretical plate] as a measure of chromatographic efficiency and as a means to compare column performance.† Presaging HPLC and UPLC technology, they recognized that a homogeneous bed packed with the smallest possible particle size [requiring higher pressure] was key to maximum efficiency. The relation between column and separation system parameters that affect bandspreading was later described in an equation by van Deemter.††

Chromatographers often refer to a quantity that they can calculate easily and directly from measurements made on a chromatogram, namely plate number [N], as efficiency. Plate height is then determined from the ratio of the length of the column bed to N [H = L/N; methods of calculating N from a chromatogram are shown in Figure U]. It is important to note that calculation of N or H using these methods is correct only for isocratic conditions and cannot be used for gradient separations.

†A.J.P. Martin and R.M. Synge, Biochem. J. 35: 1358–1368 [1941]
††J.J. van Deemter, F. J. Zuiderweg and A. Klinkenberg, Chem. Eng. Sci. 5: 271–289 [1956]

Eluate

The portion of the eluent that emerges from the column outlet containing analytes in solution. In analytical HPLC, the eluate is examined by the detector for the concentration or mass of analytes therein. In preparative HPLC, the eluate is collected continuously in aliquots at uniform time or volume intervals, or discontinuously only when a detector indicates the presence of a peak of interest. These fractions are subsequently processed to obtain purified compounds.

Eluent

The mobile phase [see Elution Chromatography].

Eluotropic Series

A list of solvents ordered by elution strength with reference to specified analytes on a standard sorbent. Such a series is useful when developing both isocratic and gradient elution methods. Trappe coined this term after showing that a sequence of solvents of increasing polarity could separate lipid fractions on alumina.† Later, Snyder measured and tabulated solvent strength parameters for a large list of solvents on several normal-phase LC sorbents.†† Neher created a very useful nomogram by which equi-eluotropic [constant elution strength] mixtures of normal-phase solvents could be chosen to optimize the selectivity of TLC separations.†††

A typical normal-phase eluotropic series would start at the weak end with non-polar aliphatic hydrocarbons, e.g., pentane or hexane, then progress successively to benzene [an aromatic hydrocarbon], dichloromethane [a chlorinated hydrocarbon], diethyl ether, ethyl acetate [an ester], acetone [a ketone], and, finally, methanol [an alcohol] at the strong end [see Figure R-1].

†W. Trappe, Biochem. Z. 305: 150 [1940]
††L. R. Snyder, Principles of Adsorption Chromatography, Marcel Dekker [1968], pp. 192–197
†††R. Neher in G.B. Marini-Bettòlo, ed., Thin-Layer Chromatography, Elsevier [1964] pp. 75–86.

Elute* [verb]

To chromatograph by elution chromatography. The process of elution may be stopped while all the sample components are still on the chromatographic bed [planar thin-layer or paper chromatography] or continued until the components have left the chromatographic bed [column chromatography].

Note: The term elute is preferred to develop [a term used in planar chromatography], to avoid confusion with the practice of method development, whereby a separation system [the combination of mobile and stationary phases] is optimized for a particular separation.

Elution Chromatography*

A procedure for chromatographic separation in which the mobile phase is continuously passed through the chromatographic bed. In HPLC, once the detector baseline has stabilized and the separation system has reached equilibrium, a finite slug of sample is introduced into the flowing mobile phase stream. Elution continues until all analytes of interest have passed through the detector.

Elution Strength

A measure of the affinity of a solvent relative to that of the analyte for the stationary phase. A weak solvent cannot displace the analyte, causing it to be strongly retained on the stationary phase. A strong solvent may totally displace all the analyte molecules and carry them through the column unretained. To achieve a proper balance of effective separation and reasonable elution volume, solvents are often blended to set up an appropriate competition between the phases, thereby optimizing both selectivity and separation time for a given set of analytes [see Selectivity].

Dipole moment, dielectric constant, hydrogen bonding, molecular size and shape, and surface tension may give some indication of elution strength. Elution strength is also determined by the separation mode. An eluotropic series of solvents may be ordered by increasing strength in one direction under adsorption or normal-phase conditions; that order may be nearly opposite under reversed-phase partition conditions [see Figure R-1].

Fluorescence Detector

Fluorescence detectors excite a sample with a specified wavelength of light. This causes certain compounds to fluoresce and emit light at a higher wavelength. A sensor, set to a specific emission wavelength and masked so as not to be blinded by the excitation source, collects only the emitted light. Often analytes that do not natively fluoresce may be derivatized to take advantage of the high sensitivity and selectivity of this form of detection, e.g., AccQ•Tag derivatization of amino acids.

Flow Rate*

The volume of mobile phase passing through the column in unit time. In HPLC systems, the flow rate is set by the controller for the solvent delivery system [pump]. Flow rate accuracy can be checked by timed collection and measurement of the effluent at the column outlet. Since a solvent’s density varies with temperature, any calibration or flow rate measurement must take this variable into account. Most accurate determinations are made, when possible, by weight, not volume.

Uniformity [precision] and reproducibility of flow rate is important to many LC techniques, especially in separations where retention times are key to analyte identification, or in gel-permeation chromatography where calibration and correlation of retention times are critical to accurate molecular-weight-distribution measurements of polymers.

Often, separation conditions are compared by means of linear velocity, not flow rate. The linear velocity is calculated by dividing the flow rate by the cross-sectional area of the column. While flow rate is expressed in volume/time [e.g., mL/min], linear velocity is measured in length/time [e.g., mm/sec].

Gel-Permeation Chromatography*

Separation based mainly upon exclusion effects due to differences in molecular size and/or shape. Gel-permeation chromatography and gel filtration chromatography describe the process when the stationary phase is a swollen gel. Both are forms of size-exclusion chromatography. Porath and Flodin first described gel-filtration using dextran gels and aqueous mobile phases for the size-based separation of biomolecules.† Moore applied similar principles to the separation of organic polymers by size in solution using organic-solvent mobile phases on porous polystyrene-divinylbenzene polymer gels.††

†J. Porath, P. Flodin, Nature 183: 1657–1659 [1959]
††J.C. Moore, U.S. Patent 3,326,875 [filed Jan. 1963; issued June 1967]

Gradient

The change over time in the relative concentrations of two [or more] miscible solvent components that form a mobile phase of increasing elution strength. A step gradient is typically used in solid-phase extraction; in each step, the eluent composition is changed abruptly from a weaker mobile phase to a stronger mobile phase. It is even possible, by drying the SPE sorbent bed in between steps, to change from one solvent to another immiscible solvent.

A continuous gradient is typically generated by a low- or high-pressure mixing system [see Figures J-2 and J-3] according to a pre-determined curve [linear or non-linear] representing the concentration of the stronger solvent B in the initial solvent A over a fixed time period. A hold at a fixed isocratic solvent composition can be programmed at any time point within a continuous gradient. At the end of a separation, the gradient program can also be set to return to the initial mobile phase composition to re-equilibrate the column in preparation for the injection of the next sample. Sophisticated HPLC systems can blend as many as four or more solvents [or solvent mixtures] into a continuous gradient.

Injector [Autosampler, Sample Manager]

A mechanism for accurately and precisely introducing [injecting] a discrete, predetermined volume of a sample solution into the flowing mobile phase stream. The injector can be a simple manual device, or a sophisticated autosampler that can be programmed for unattended injections of many samples from an array of individual vials or wells in a predetermined sequence. Sample compartments in these systems may even be temperature controlled to maintain sample integrity over many hours of operation.

Most modern injectors incorporate some form of syringe-filled sample loop that can be switched on- or offline by means of a multi-port valve. A well-designed, minimal-internal-volume injection system is situated as close to the column inlet as possible and minimizes the spreading of the sample band. Between sample injections, it is also capable of being flushed to waste by mobile phase, or a wash solvent, to prevent carryover [contamination of the present sample by a previous one].

Samples are best prepared for injection, if possible, by dissolving them in the mobile phase into which they will be injected; this may prevent issues with separation and/or detection. If another solvent must be used, it is desirable that its elution strength be equal to or less than that of the mobile phase. It is often wise to mix a bit of a sample solution with the mobile phase offline to test for precipitation or miscibility issues that might compromise a successful separation.

Inlet

The end of the column bed where the mobile phase stream and sample enter. A porous, inert frit retains the packing material and protects the sorbent bed inlet from particulate contamination. Good HPLC practice dictates that samples and mobile phases should be particulate-free; this becomes imperative for small-particle columns whose inlets are much more easily plugged. If the column bed inlet becomes clogged and exhibits higher-than-normal backpressure, sometimes, reversing the flow direction while directing the effluent to waste may dislodge and flush out sample debris that sits atop the frit. If the debris has penetrated the frit and is lodged in the inlet end of the bed itself, then the column has most likely reached the end of its useful life.

Ion-Exchange Chromatography* [see section: Separations Based on Charge]

This separation mode is based mainly on differences in the ion-exchange affinities of the sample components. Separation of primarily inorganic ionic species in water or buffered aqueous mobile phases on small particle, superficially porous, high-efficiency, ion-exchange columns followed by conductometric or electrochemical detection is referred to as ion chromatography [IC].

Isocratic Elution*

A procedure in which the composition of the mobile phase remains constant during the elution process.

Liquid Chromatography* [LC]

A separation technique in which the mobile phase is a liquid. Liquid chromatography can be carried out either in a column or on a plane [TLC or paper chromatography]. Modern liquid chromatography utilizing smaller particles and higher inlet pressure was termed high-performance (or high-pressure) liquid chromatography [HPLC] in 1970. In 2004, ultra-performance liquid chromatography dramatically raised the performance of LC to a new plateau [see UPLC Technology].

Mobile Phase* [see Eluate, Eluent]

A fluid that percolates, in a definite direction, through the length of the stationary-phase sorbent bed. The mobile phase may be a liquid [liquid chromatography] or a gas [gas chromatography] or a supercritical fluid [supercritical-fluid chromatography]. In gas chromatography the expression carrier gas may be used for the mobile phase. In elution chromatography, the mobile phase may also be called the eluent, while the word eluate is defined as the portion of the mobile phase that has passed through the sorbent bed and contains the compounds of interest in solution.

Normal-Phase Chromatography*

An elution procedure in which the stationary phase is more polar than the mobile phase. This term is used in liquid chromatography to emphasize the contrast to reversed-phase chromatography.

Peak* [see Plate Number]

The portion of a differential chromatogram recording the detector response while a single component is eluted from the column. If separation is incomplete, two or more components may be eluted as one unresolved peak. Peaks eluted under optimal conditions from a well-packed, efficient column, operated in a system that minimizes bandspreading, approach the shape of a Gaussian distribution. Quantitation is usually done by measuring the peak area [enclosed by the baseline and the peak curve]. Less often, peak height [the distance measured from the peak apex to the baseline] may be used for quantitation. This procedure requires that both the peak width and the peak shape remain constant.

Plate Number* [N, see Efficiency]

A number indicative of column performance [mechanical separation power or efficiency, also called plate count, number of theoretical plates, or theoretical plate number]. It relates the magnitude of a peak’s retention to its width [variance or bandspread]. In order to calculate a plate count, it is assumed that a peak can be represented by a Gaussian distribution [a statistical bell curve]. At the inflection points [60.7% of peak height], the width of a Gaussian curve is twice the standard deviation [σ] about its mean [located at the peak apex]. As shown in Figure U, a Gaussian curve’s peak width measured at other fractions of peak height can be expressed in precisely defined multiples of σ. Peak retention [retention volume, V_R, or retention time, t_R] and peak width must be expressed in the same units, because N is a dimensionless number. Note that the 5 sigma method of calculating N is a more stringent measure of column homogeneity and performance, as it is more severely affected by peak asymmetry. Computer data stations can automatically delineate each resolved peak and calculate its corresponding plate number.

Preparative Chromatography

The process of using liquid chromatography to isolate a compound in a quantity and at a purity level sufficient for further experiments or uses. For pharmaceutical or biotechnological purification processes, columns several feet in diameter can be used for multiple kilograms of material. For isolating just a few micrograms of a valuable natural product, an analytical HPLC column is sufficient. Both are preparative chromatographic approaches, differing only in scale [see section on HPLC Scale and Table A].

Resolution* [Rs, see Selectivity]

The separation of two peaks, expressed as the difference in their corresponding retention times, divided by their average peak width at the baseline. R_s = 1.25 indicates that two peaks of equal width are just separated at the baseline. When R_s = 0.6, the only visual indication of the presence of two peaks on a chromatogram is a small notch near the peak apex. Higher efficiency columns produce narrower peaks and improve resolution for difficult separations; however, resolution increases by only the square root of N. The most powerful method of increasing resolution is to increase selectivity by altering the mobile/stationary phase combination used for the chromatographic separation [see section on Chemical Separation Power].

Retention Factor* [k]

A measure of the time the sample component resides in the stationary phase relative to the time it resides in the mobile phase; it expresses how much longer a sample component is retarded by the stationary phase than it would take to travel through the column with the velocity of the mobile phase. Mathematically, it is the ratio of the adjusted retention time [volume] and the hold-up time [volume]: k = t_R'/t_M [see Retention Time and Selectivity].

Note: In the past, this term has also been expressed as partition ratio, capacity ratio, capacity factor, or mass distribution ratio and symbolized by k'.

Retention Time* [tR]

The time between the start of elution [typically, in HPLC, the moment of injection or sample introduction] and the emergence of the peak maximum. The adjusted retention time, t_R', is calculated by subtracting from t_R the hold-up time [t_M, the time from injection to the elution of the peak maximum of a totally unretained analyte].

Reversed-Phase Chromatography*

An elution procedure used in liquid chromatography in which the mobile phase is significantly more polar than the stationary phase, e.g. a microporous silica-based material with alkyl chains chemically bonded to its accessible surface. Note: Avoid the incorrect term reverse phase. [See Reference 4 for some novel ideas on the mechanism of reversed-phase separations.]

Selectivity [Separation Factor, σ]

A term used to describe the magnitude of the difference between the relative thermodynamic affinities of a pair of analytes for the specified mobile and stationary phases that comprise the separation system. The proper term is separation factor [σ]. It equals the ratio of retention factors, k2/k1 [see Retention Factor]; by definition, σ is always ≥ 1. If σ = 1, then both peaks co-elute, and no separation is obtained. It is important in preparative chromatography to maximize α for highest sample loadability and throughput. [see section on Chemical Separation Power]

Sensitivity* [S]

The signal output per unit concentration or unit mass of a substance in the mobile phase entering the detector, e.g., the slope of a linear calibration curve [see Detector]. For concentration-sensitive detectors [e.g., UV/VIS absorbance], sensitivity is the ratio of peak height to analyte concentration in the peak. For mass-flow-sensitive detectors, it is the ratio of peak height to unit mass. If sensitivity is to be a unique performance characteristic, it must depend only on the chemical measurement process, not upon scale factors.

The ability to detect [qualify] or measure [quantify] an analyte is governed by many instrumental and chemical factors. Well-resolved peaks [maximum selectivity] eluting from high efficiency columns [narrow peak width with good symmetry for maximum peak height] as well as good detector sensitivity and specificity are ideal. Both the separation system interference and electronic component noise should also be minimized to achieve maximum sensitivity.

Solid-Phase Extraction [SPE]

A sample preparation technique that uses LC principles to isolate, enrich, and/or purify analytes from a complex matrix applied to a miniature chromatographic bed. Offline SPE is done [manually or via automation] with larger particles in individual plastic cartridges or in micro-elution plate wells, using low positive pressure or vacuum to assist flow. Online SPE is done with smaller particles in miniature HPLC columns using higher pressures and a valve to switch the SPE column online with the primary HPLC column, or offline to waste, as appropriate.

SPE methods use step gradients [see Gradient] to accomplish bed conditioning, sample loading, washing, and elution steps. Samples are loaded typically under conditions where the k of important analytes is as high as possible, so that they are fully retained during loading and washing steps. Elution is then done by switching to a much stronger solvent mixture [see Elution Strength]. The goal is to remove matrix interferences and to isolate the analyte in a solution, and at a concentration, suitable for subsequent analysis.

Speed [see Efficiency, Flow Rate, Resolution]

A benefit of operating LC separations at higher linear velocities using smaller-volume, smaller-particle analytical columns, or larger-volume, larger-particle preparative columns. Order-of-magnitude advances in LC speed came in 1972 [with the use of 10 μm particles and pumps capable of delivering accurate mobile-phase flow at 6000 psi], in 1976 [with 75-μm preparative columns operated at a flow rate of 500 mL/min], and in 2004 [with the introduction of UPLC technology—1.7 μm-particle columns operated at 15,000 psi].†

High-speed analytical LC systems must not only accommodate higher pressures throughout the fluidics; injector cycle time must be short; gradient mixers must be capable of rapid turnaround between samples; detector sensors must rapidly respond to tiny changes in eluate composition; and data systems must collect the dozens of points each second required to plot and to quantitate narrow peaks accurately.

Together, higher resolution, higher speed, and higher efficiency typically deliver higher throughput. More samples can be analyzed in a workday. Larger quantities of compound can be purified per run or per process period.

Stationary Phase*

One of the two phases forming a chromatographic system. It may be a solid, a gel, or a liquid. If a liquid, it may be distributed on a solid. This solid may or may not contribute to the separation process. The liquid may also be chemically bonded to the solid [bonded phase] or immobilized onto it [immobilized phase].

The expression chromatographic bed or sorbent may be used as a general term to denote any of the different forms in which the stationary phase is used.

The use of the term liquid phase to denote the mobile phase in LC is discouraged. This avoids confusion with gas chromatography where the stationary phase is called a liquid phase [most often a liquid coated on a solid support].

Open-column liquid-liquid partition chromatography [LLC] did not translate well to HPLC. It was supplanted by the use of bonded-phase packings. LLC proved incompatible with modern detectors because of problems with bleed of the stationary-phase-liquid coating off its solid support, thereby contaminating the immiscible liquid mobile phase.

UPLC Technology

The use of a high-efficiency LC system holistically designed to accommodate sub-2 μm particles and very high operating pressure is termed ultra-performance liquid chromatography [UPLC technology].† The major benefits of this technology are significant improvements in resolution over HPLC, and/or faster run times while maintaining the resolution seen in an existing HPLC separation.

†For more information, visit: https://www.waters.com/uplc

*Indicates a definition adapted from: L.S. Ettre, Nomenclature for Chromatography, Pure Appl. Chem. 65: 819–872 [1993], © 1993 IUPAC; an updated version of this comprehensive report is available in the Orange Book, Chapter 9: Separations [1997] at: <https://media.iupac.org/publications/analytical_compendium/>.

In this Primer

What is HPLC (High Performance Liquid Chromatography)

How Does High Performance Liquid Chromatography Work?

Identifying and Quantitating Compounds

HPLC Column Hardware

HPLC Separation Modes

Appendix: HPLC Nomenclature