HPLC
High Performance Liquid Chromatography
(Is an advance type of Liquid Chromatography)
(Is an advance type of Liquid Chromatography)
INTRODUCTION
It is a form of column chromatography used frequently in all field of Life science and Analytical
Chemistry. HPLC is used to separate components of a mixture by using a variety
of chemical interactions between the substance being analyzed and the
chromatography column. We can analyzed the Vitamins, API, Enzymes,
Photochemical, Toxins and Microbial toxin content with the help of HPLC.
How Does HPLC Work?
Nomenclature
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.
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.
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.
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.
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.
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].
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.
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.
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.
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.††
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]
††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.
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].
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 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.
††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].
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.
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 appropriatecompetition between the phases, thereby optimizing both selectivity and
separation time for a given set of analytes [see Selectivity].
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 appropriatecompetition 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-phaseconditions;
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.
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.
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. Gelpermeation 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.††
Separation based mainly upon exclusion effects due to differences in molecular size and/or shape. Gelpermeation 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]
††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.
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 programcan 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.
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.
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].
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.
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].
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.
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.
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 thepeak 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.
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 thepeak 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 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. [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, VR, or retention time, tR] and peak width must be expressed in the same units, because N is a dimensionless number. Note that the 5 sigma
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 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. [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, VR, or retention time, tR] and peak width must be expressed in the same units, because N is a dimensionless number. Note that the 5 sigma
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].
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. Rs = 1.25 indicates that two peaks of equal width are just separated at the baseline. When Rs = 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].
The separation of two peaks, expressed as the difference in their corresponding retention times, divided by their average peak width at the baseline. Rs = 1.25 indicates that two peaks of equal width are just separated at the baseline. When Rs = 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 = tR'/tM [see Retention Time and Selectivity].
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 = tR'/tM [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, tR', is calculated by subtracting from tR thehold-up time [tM, the time from injection to the elution of the peak maximum of a totally unretained analyte].
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, tR', is calculated by subtracting from tR thehold-up time [tM, 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.]
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 isseparation factor = 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] is always ≥ 1. If σ [σ]. It equals the ratio of retention factors, k2/k1 [see Retention Factor]; by definition, 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 isseparation factor = 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] is always ≥ 1. If σ [σ]. It equals the ratio of retention factors, k2/k1 [see Retention Factor]; by definition, σ
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 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.
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].†
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.
†See #3 on list of References
for Further Reading above.
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].
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.
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.
The components of a basic high-performance
liquid chromatography [HPLC] system are shown in the simple diagram in Figure
E.
A reservoir holds the solvent [called the
mobile phase, because it moves]. A high-pressure pump [solvent delivery system
or solvent manager] is used to generate and meter a specified flow rate of
mobile phase, typically milliliters per minute. An injector [sample manager or
autosampler] is able to introduce [inject] the sample into the continuously
flowing mobile phase stream that carries the sample into the HPLC column. The
column contains the chromatographic packing material needed to effect the
separation. This packing material is called the stationary phase because it is
held in place by the column hardware. A detector is needed to see the separated compound bands as they elute from the HPLC
column [most compounds have no color, so we cannot see them with our eyes]. The
mobile phase exits the detector and can be sent to waste, or collected, as
desired. When the mobile phase contains a separated compound band, HPLC
provides the ability to collect this fraction of the eluate containing that
purified compound for further study. This is called preparative chromatography
[discussed in the section on HPLC Scale].
Note that high-pressure tubing and fittings
are used to interconnect the pump, injector, column, and detector components to
form the conduit for the mobile phase, sample, and separated compound bands.
Figure
E: High-Performance Liquid Chromatography [HPLC] System
The detector is wired to the computer data
station, the HPLC system component that records the electrical signal needed to
generate the chromatogram on its display and to identify and quantitate the
concentration of the sample constituents (see Figure F). Since sample compound
characteristics can be very different, several types of detectors have been
developed. For example, if a compound can absorb ultraviolet light, a
UV-absorbance detector is used. If the compound fluoresces, a fluorescence
detector is used. If the compound does not have either of these
characteristics, a more universal type of detector is used, such as an
evaporative-light-scattering detector [ELSD]. The most powerful approach is the
use multiple detectors in series. For example, a UV and/or ELSD detector may be
used in combination with a mass spectrometer [MS] to analyze the results of the
chromatographic separation. This provides, from a single injection, more
comprehensive information about an analyte. The practice of coupling a mass
spectrometer to an HPLC system is called LC/MS.
Figure
F: A Typical HPLC [Waters Alliance] System
HPLC Operation
A simple way to understand how we achieve the separation of the compounds contained in a sample is to view the diagram in Figure G.
A simple way to understand how we achieve the separation of the compounds contained in a sample is to view the diagram in Figure G.
Mobile phase enters the column from the left,
passes through the particle bed, and exits at the right. Flow direction is
represented by green arrows. First, consider the top image; it represents the
column at time zero [the moment of injection], when the sample enters the
column and begins to form a band. The sample shown here, a mixture of yellow,
red, and blue dyes, appears at the inlet of the column as a single black band.
[In reality, this sample could be anything that can be dissolved in a solvent;
typically the compounds would be colorless and the column wall opaque, so we
would need a detector to see the separated compounds as they elute.]
After a few minutes [lower image], during
which mobile phase flows continuously and steadily past the packing material
particles, we can see that the individual dyes have moved in separate bands at
different speeds. This is because there is a competition between the mobile
phase and the stationary phase for attracting each of the dyes or analytes. Notice
that the yellow dye band moves the fastest and is about to exit the column. The
yellow dye likes [is attracted to] the mobile phase more than the other dyes.
Therefore, it moves at a faster speed, closer to that of the mobile phase. The blue dye
band likes the packing material more than the mobile phase. Its stronger
attraction to the particles causes it to move significantly slower. In other words, it is the most retained compound in this
sample mixture. The red dye band has an intermediate attraction for the mobile
phase and therefore moves at an intermediate speed through the column. Since each dye band moves at
different speed, we are able to separate it chromatographically.
Figure
G: Understanding How a Chromatographic Column Works – Bands
What Is a Detector?
As the separated dye bands leave the column, they pass immediately into the detector. The detector contains a flow cell that sees [detects] each separated compound band against a background of mobile phase [see Figure H]. [In reality, solutions of many compounds at typical HPLC analytical concentrations are colorless.] An appropriate detector has the ability to sense the presence of a compound and send its corresponding electrical signal to a computer data station. A choice is made among many different types of detectors, depending upon the characteristics and concentrations of the compounds that need to be separated and analyzed, as discussed earlier.
As the separated dye bands leave the column, they pass immediately into the detector. The detector contains a flow cell that sees [detects] each separated compound band against a background of mobile phase [see Figure H]. [In reality, solutions of many compounds at typical HPLC analytical concentrations are colorless.] An appropriate detector has the ability to sense the presence of a compound and send its corresponding electrical signal to a computer data station. A choice is made among many different types of detectors, depending upon the characteristics and concentrations of the compounds that need to be separated and analyzed, as discussed earlier.
What Is a Chromatogram?
A chromatogram is a representation of the separation that has chemically [chromatographically] occurred in the HPLC system. A series of peaks rising from a baseline is drawn on a time axis. Each peak represents the detector response for a different compound. The chromatogram is plotted by the computer data station [see Figure H].
A chromatogram is a representation of the separation that has chemically [chromatographically] occurred in the HPLC system. A series of peaks rising from a baseline is drawn on a time axis. Each peak represents the detector response for a different compound. The chromatogram is plotted by the computer data station [see Figure H].
Figure
H: How Peaks Are Created
In Figure H, the yellow band has completely
passed through the detector flow cell; the electrical signal generated has been
sent to the computer data station. The resulting chromatogram has begun to
appear on screen. Note that the chromatogram begins when the sample was first
injected and starts as a straight line set near the bottom of the screen. This
is called the baseline; it represents pure mobile phase passing through the
flow cell over time. As the yellow analyte band passes through the flow cell, a
stronger signal is sent to the computer. The line curves, first upward, and
then downward, in proportion to the concentration of the yellow dye in the
sample band. This creates a peak in the chromatogram. After the yellow band
passes completely out of the detector cell, the signal level returns to the
baseline; the flow cell now has, once again, only pure mobile phase in it.
Since the yellow band moves fastest, eluting first from the column, it is the
first peak drawn.
A little while later, the red band reaches
the flow cell. The signal rises up from the baseline as the red band first
enters the cell, and the peak representing the red band begins to be drawn. In
this diagram, the red band has not fully passed through the flow cell. The
diagram shows what the red band and red peak would look like if we stopped the
process at this moment. Since most of the red band has passed through the cell,
most of the peak has been drawn, as shown by the solid line. If we could
restart, the red band would completely pass through the flow cell and the red
peak would be completed [dotted line]. The blue band, the most strongly
retained, travels at the slowest rate and elutes after the red band. The dotted
line shows you how the completed chromatogram would appear if we had let the
run continue to its conclusion. It is interesting to note that the width of the
blue peak will be the broadest because the width of the blue analyte band,
while narrowest on the column, becomes the widest as it elutes from the column.
This is because it moves more slowly through the chromatographic packing
material bed and requires more time [and mobile phase volume] to be eluted
completely. Since mobile phase is continuously flowing at a fixed rate, this
means that the blue band widens and is more dilute. Since the detector responds
in proportion to the concentration of the band, the blue peak is lower in
height, but larger in width.
Identifying and Quantitating Compounds
In Figure H, three dye
compounds are represented by three peaks separated in time in the chromatogram.
Each elutes at a specific location, measured by the elapsed time between the
moment of injection [time zero] and the time when the peak maximum elutes. By
comparing each peak’s retention time [tR] with that of injected reference
standards in the same chromatographic system [same mobile and stationary
phase], a chromatographer may be able to identify each compound.
Figure I-1: Identification
In the chromatogram shown in
Figure I-1, the chromatographer knew that, under these LC system conditions,
the analyte, acrylamide, would be separated and elute from the column at 2.85
minutes [retention time]. Whenever a new sample, which happened to contain
acrylamide, was injected into the LC system under the same conditions, a peak
would be present at 2.85 minutes [see Sample B in Figure I-2].
[For a better understanding of
why some compounds move more slowly [are better retained] than others, please
review the HPLC Separation Modes section on page 28].
Once identity is established,
the next piece of important information is how much of each compound was
present in the sample. The chromatogram and the related data from the detector
help us calculate the concentration of each compound. The detector basically
responds to the concentration of the compound band as it passes through the
flow cell. The more concentrated it is, the stronger the signal; this is seen
as a greater peak height above the baseline.
Figure I-2: Identification and Quantitation
In Figure I-2, chromatograms
for Samples A and B, on the same time scale, are stacked one above the other.
The same volume of sample was injected in both runs. Both chromatograms display
a peak at a retention time [tR] of 2.85 minutes, indicating that each sample contains
acrylamide. However, Sample A displays a much bigger peak for acrylamide. The
area under a peak [peak area count] is a measure of the concentration of the
compound it represents. This area value is integrated and calculated
automatically by the computer data station. In this example, the peak for
acrylamide in Sample A has 10 times the area of that for Sample B. Using
reference standards, it can be determined that Sample A contains 10 picograms
of acrylamide, which is ten times the amount in Sample B [1 picogram]. Note
there is another peak [not identified] that elutes at 1.8 minutes in both
samples. Since the area counts for this peak in both samples are about the
same, this unknown compound may have the same concentration in both samples.
Isocratic and Gradient LC
System Operation
Two basic elution modes are used in HPLC. The first is called isocratic elution. In this mode, the mobile phase, either a pure solvent or a mixture, remains the same throughout the run. A typical system is outlined in Figure J-1.
Two basic elution modes are used in HPLC. The first is called isocratic elution. In this mode, the mobile phase, either a pure solvent or a mixture, remains the same throughout the run. A typical system is outlined in Figure J-1.
Figure J-1: Isocratic LC
System
The second type is called
gradient elution, wherein, as its name implies, the
mobile phase composition changes during the separation. This mode is useful for samples
that contain compounds that span a wide range of chromatographic polarity [see
section on HPLC Separation Modes]. As the separation proceeds, the elution
strength of the mobile phase is increased to elute the more strongly retained
sample components.
In the simplest case, shown in
Figure J-2, there are two bottles of solvents and two pumps. The speed of each
pump is managed by the gradient controller to deliver more or less of each
solvent over the course of the separation. The two streams are combined in the
mixer to create the actual mobile phase composition that is delivered to the
column over time. At the beginning, the mobile phase contains a higher
proportion of the weaker solvent [Solvent A]. Over time, the proportion of the
stronger solvent [Solvent B] is increased, according to a predetermined
timetable, the mixer is downstream of the pumps; thus
the gradient is created under high pressure. Other
HPLC systems are designed to mix multiple streams of solvents under low
pressure, ahead of a single pump. A gradient proportioning valve
selects from the four solvent bottles, changing the strength of the mobile
phase over time.
HPLC Scale [Analytical,
Preparative, and Process]
We have discussed how HPLC
provides analytical data that can be used both to identify and to quantify
compounds present in a sample. However, HPLC can also be used to purify and
collect desired amounts of each compound, using a fraction collector downstream
of the detector flow cell. This process is called preparative chromatography
[see Figure K].
In preparative chromatography,
the scientist is able to collect the individual analytes as they elute from the
column [e.g.,
in this example: yellow, then red, then blue].
Figure K: HPLC System for Purification: Preparative
Chromatography
The fraction collector
selectively collects the eluate that now contains a purified analyte, for a
specified length of time. The vessels are moved so that each collects only a
single analyte peak.
A scientist determines goals
for purity level and amount. Coupled with knowledge of the complexity of the
sample and the nature and concentration of the desired analytes relative to
that of the matrix constituents, these goals, in turn, determine the amount of
sample that needs to be processed and the required capacity of the HPLC system.
In general, as the sample size increases, the size of the HPLC column will
become larger and the pump will need higher volume-flow-rate capacity.
Determining the capacity of an HPLC system is called selecting the HPLC scale.
Table A lists various HPLC scales and their chromatographic objectives.
The ability to maximize
selectivity with a specific combination of HPLC stationary and mobile
phases—achieving the largest possible separation between two sample components
of interest—is critical in determining the requirements for scaling up a
separation [see discussion on HPLC Separation Modes]. Capacity then becomes a
matter of scaling the column volume [Vc] to the amount of sample to be injected
and choosing an appropriate particle size [determines pressure and efficiency;
see discussion of Separation Power]. Column volume, a function of bed length
[L] and internal diameter [i.d.], determines the amount of packing material
[particles] that can be contained (see Figure L).
In general, HPLC columns range
from 20 mm to 500 mm in length [L] and 1 mm to 100 mm in internal diameter
[i.d.]. As the scale of chromatography increases, so do column dimensions,
especially the cross-sectional area. To optimize throughput, mobile phase flow
rates must increase in proportion to cross-sectional area. If a smaller
particle size is desirable for more separation power, pumps must then be
designed to sustain higher mobile-phase-volume flow rates at high backpressure.
Table B presents some simple guidelines on selecting the column i.d. and
particle size range recommended for each scale of chromatography.
For example, a
semi-preparative-scale application [red X] would use a column with an internal
diameter of 10–40 mm containing 5–15 micron particles. Column length could then
be calculated based on how much purified compound needs to be processed during
each run and on how much separation power is required.
HPLC Column Hardware
A column tube and fittings must
contain the chromatographic packing material [stationary phase] that is used to
effect a separation. It must withstand backpressure created both during
manufacture and in use. Also, it must provide a well-controlled [leak-free,
minimum-volume, and zero-dead-volume] flow path for the sample at its inlet,
and analyte bands at its outlet, and be chemically inert relative to the
separation system [sample, mobile, and stationary phases]. Most columns are
constructed of stainless steel for highest pressure resistance. PEEK™[an engineered plastic]
and glass, while less pressure tolerant, may be used when inert surfaces are
required for special chemical or biological applications. [Figure M-1].
A glass column wall offers a
visual advantage. In the photo in Figure M-2, flow has been stopped while the
sample bands are still in the column. You can see that the three dyes in the
injected sample mixture have already separated in the bed; the yellow analyte,
traveling fastest, is just about to exit the column.
Separation Performance –
Resolution
The degree to which two compounds are separated is called chromatographic resolution [RS]. Two principal factors that determine the overall separation power or resolution that can be achieved by an HPLC column are: mechanical separation power, created by the column length, particle size, and packed-bed uniformity, and chemical separation power, created by the physicochemical competition for compounds between the packing material and the mobile phase. Efficiency is a measure of mechanical separation power, while selectivity is a measure of chemical separation power.
The degree to which two compounds are separated is called chromatographic resolution [RS]. Two principal factors that determine the overall separation power or resolution that can be achieved by an HPLC column are: mechanical separation power, created by the column length, particle size, and packed-bed uniformity, and chemical separation power, created by the physicochemical competition for compounds between the packing material and the mobile phase. Efficiency is a measure of mechanical separation power, while selectivity is a measure of chemical separation power.
Mechanical Separation Power –
Efficiency
If a column bed is stable and uniformly packed, its mechanical separation power is determined by the column length and the particle size. Mechanical separation power, also called efficiency, is often measured and compared by a plate number [symbol = N]. Smaller-particle chromatographic beds have higher efficiency and higher backpressure. For a given particle size, more mechanical separation power is gained by increasing column length. However, the trade-offs are longer chromatographic run times, greater solvent consumption, and higher backpressure. Shorter column lengths minimize all these variables but also reduce mechanical separation power, as shown in Figure N.
If a column bed is stable and uniformly packed, its mechanical separation power is determined by the column length and the particle size. Mechanical separation power, also called efficiency, is often measured and compared by a plate number [symbol = N]. Smaller-particle chromatographic beds have higher efficiency and higher backpressure. For a given particle size, more mechanical separation power is gained by increasing column length. However, the trade-offs are longer chromatographic run times, greater solvent consumption, and higher backpressure. Shorter column lengths minimize all these variables but also reduce mechanical separation power, as shown in Figure N.
Figure N: Column Length and Mechanical Separating Power [Same
Particle Size]
Figure O: Particle Size and
Mechanical Separating Power [Same Column Length]
For a given particle chemistry,
mobile phase, and flow rate, as shown in Figure O, a column of the same length
and i.d., but with a smaller particle size, will deliver more mechanical
separation power in the same time. However, its backpressure will be much
higher.
Chemical Separation Power –
Selectivity
The choice of a combination of particle chemistry [stationary phase] and mobile-phase composition—the separation system—will determine the degree of chemical separation power [how we change the speed of each analyte]. Optimizing selectivity is the most powerful means of creating a separation; this may obviate the need for the brute force of the highest possible mechanical efficiency. To create a separation of any two specified compounds, a scientist may choose among a multiplicity of phase combinations [stationary phase and mobile phase] and retention mechanisms [modes of chromatography]. These are discussed in the next section.
The choice of a combination of particle chemistry [stationary phase] and mobile-phase composition—the separation system—will determine the degree of chemical separation power [how we change the speed of each analyte]. Optimizing selectivity is the most powerful means of creating a separation; this may obviate the need for the brute force of the highest possible mechanical efficiency. To create a separation of any two specified compounds, a scientist may choose among a multiplicity of phase combinations [stationary phase and mobile phase] and retention mechanisms [modes of chromatography]. These are discussed in the next section.
HPLC Separation Modes
In general, three primary
characteristics of chemical compounds can be used to create HPLC separations.
They are:
• Polarity
• Electrical Charge
• Molecular Size
• Electrical Charge
• Molecular Size
First, let’s consider polarity
and the two primary separation modes that exploit this characteristic: normal
phase and reversed-phase chromatography.
Separations Based on Polarity
A molecule’s structure, activity, and physicochemical characteristics are determined by the arrangement of its constituent atoms and the bonds between them. Within a molecule, a specific arrangement of certain atoms that is responsible for special properties and predictable chemical reactions is called a functional group. This structure often determines whether the molecule is polar or non-polar. Organic molecules are sorted into classes according to the principal functional group(s) each contains. Using a separation mode based on polarity, the relative chromatographic retention of different kinds of molecules is largely determined by the nature and location of these functional groups. As shown in Figure P, classes of molecules can be ordered by their relative retention into a range or spectrum of chromatographic polarity from highly polar to highly non-polar.
A molecule’s structure, activity, and physicochemical characteristics are determined by the arrangement of its constituent atoms and the bonds between them. Within a molecule, a specific arrangement of certain atoms that is responsible for special properties and predictable chemical reactions is called a functional group. This structure often determines whether the molecule is polar or non-polar. Organic molecules are sorted into classes according to the principal functional group(s) each contains. Using a separation mode based on polarity, the relative chromatographic retention of different kinds of molecules is largely determined by the nature and location of these functional groups. As shown in Figure P, classes of molecules can be ordered by their relative retention into a range or spectrum of chromatographic polarity from highly polar to highly non-polar.
Water [a small molecule with a
high dipole moment] is a polar compound. Benzene [an aromatic hydrocarbon] is a
non-polar compound. Molecules with similar chromatographic polarity tend to be
attracted to each other; those with dissimilar polarity exhibit much weaker
attraction, if any, and may even repel one another. This becomes the basis for
chromatographic separation modes based on polarity.
Another way to think of this is
by the familiar analogy: oil [non-polar] and water [polar] don’t mix. Unlike in
magnetism where opposite poles attract each other, chromatographic separations
based on polarity depend upon the stronger attraction between likes and the
weaker attraction between opposites. Remember, “Like
attracts like” in
polarity-based chromatography.
To design a chromatographic
separation system [see Figure Q], we create competition for the various
compounds contained in the sample by choosing a mobile phase and a stationary
phase with different polarities. Then, compounds in the sample that are similar
in polarity to the stationary phase [column packing material] will be delayed
because they are more strongly attracted to the particles. Compounds whose
polarity is similar to that of the mobile phase will be preferentially
attracted to it and move faster.
In this way, based upon
differences in the relative attraction of each compound for each phase, a
separation is created by changing the speeds of the analytes.
Figures R-1, R-2, and R-3
display typical chromatographic polarity ranges for mobile phases, stationary
phases, and sample analytes, respectively. Let’s consider each in turn to see
how a chromatographer chooses the appropriate phases to develop the attraction
competition needed to achieve a polarity-based HPLC separation.
Figure R-1: Mobile Phase Chromatographic Polarity Spectrum
A scale, such as that shown in
Figure R-1, upon which some common solvents are placed in order of relative
chromatographic polarity is called an eluotropic series. Mobile phase molecules
that compete effectively with analyte molecules for the attractive stationary
phase sites displace these analytes, causing them to move faster through the column
[weakly retained]. Water is at the polar end of mobile-phase-solvent scale,
while hexane, an aliphatic hydrocarbon, is at the non-polar end. In between,
single solvents, as well as miscible-solvent mixtures [blended in proportions
appropriate to meet specific separation requirements], can be placed in order
of elution strength. Which end of the scale represents the ‘strongest’ mobile
phase depends upon the nature of the stationary phase surface where the
competition for the analyte molecules occurs.
Silica has an active,
hydrophilic [water-loving] surface containing acidic silanol
[silicon-containing analog of alcohol] functional groups. Consequently, it
falls at the polar end of the stationary-phase scale shown in Figure R-2. The
activity or polarity of the silica surface may be modified selectively by
chemically bonding to it less polar functional groups [bonded phase]. Examples
shown here include, in order of decreasing polarity, cyanopropylsilyl- [CN],
n-octylsilyl- [C8], and n-octadecylsilyl- [C18, ODS] moieties on silica. The
latter is a hydrophobic [water-hating], very non-polar packing. repeats the chromatographic polarity spectrum of our sample [shown
in Figure P]. After considering the polarity of both phases, then, for a given
stationary phase, a chromatographer must choose a mobile phase in which the
analytes of interest are retained, but not so strongly that they cannot be
eluted. Among solvents of similar strength, the chromatographer considers which
phase combination may best exploit the more subtle differences in analyte
polarity and solubility to maximize the selectivity of the chromatographic
system. Like attracts like, but, as you probably can imagine from the
discussion so far, creating a separation based upon polarity involves knowledge
of the sample and experience with various kinds of analytes and retention
modes. To summarize, the chromatographer will choose the best combination of a
mobile phase and particle stationary phase with appropriately opposite
polarities. Then, as the sample analytes move through the column, the rule like
attracts like will
determine which analytes slow down and which proceed at a faster speed.
Normal-Phase HPLC
In his separations of plant extracts, Tswett was successful using a polar stationary phase [chalk in a glass column; see Figure A] with a much less polar [non-polar] mobile phase. This classical mode of chromatography became known as normal phase.
In his separations of plant extracts, Tswett was successful using a polar stationary phase [chalk in a glass column; see Figure A] with a much less polar [non-polar] mobile phase. This classical mode of chromatography became known as normal phase.
Figure S-1 represents a
normal-phase chromatographic separation of our three-dye test mixture. The
stationary phase is polar and retains the polar yellow dye most strongly. The
relatively non-polar blue dye is won in the retention competition by the mobile
phase, a non-polar solvent, and elutes quickly. Since the blue dye is most like
the mobile phase [both are non-polar], it moves faster. It is typical for
normal-phase chromatography on silica that the mobile phase is 100% organic; no
water is used.
Reversed-Phase HPLC
The term reversed-phase describes the chromatography mode that is just the opposite of normal phase, namely the use of a polar mobile phase and a non-polar [hydrophobic] stationary phase. Figure S-2 illustrates the black three-dye mixture being separated using such a protocol.
The term reversed-phase describes the chromatography mode that is just the opposite of normal phase, namely the use of a polar mobile phase and a non-polar [hydrophobic] stationary phase. Figure S-2 illustrates the black three-dye mixture being separated using such a protocol.
Now the most strongly retained
compound is the more non-polar blue dye, as its attraction to the non-polar
stationary phase is greatest. The polar yellow dye, being weakly retained, is
won in competition by the polar, aqueous mobile phase, moves the fastest
through the bed, and elutes earliest like attracts like.
Today, because it is more
reproducible and has broad applicability, reversed-phase chromatography is used
for approximately 75% of all HPLC methods. Most of these protocols use as the
mobile phase an aqueous blend of water with a miscible, polar organic solvent,
such as acetonitrile or methanol. This typically ensures the proper interaction
of analytes with the non-polar, hydrophobic particle surface. A C18–bonded
silica [sometimes called ODS] is the most popular type of reversed-phase HPLC
packing.
Table C presents a summary of
the phase characteristics for the two principal HPLC separation modes based
upon polarity. Remember, for these polarity-based modes, like
attracts like.
Hydrophilic-Interaction
Chromatography [HILIC]
HILIC may be viewed as a variant of normal-phase chromatography. In normal-phase chromatography, the mobile phase is 100% organic. Only traces of water are present in the mobile phase and in the pores of the polar packing particles. Polar analytes bind strongly to the polar stationary phase and may not elute.
HILIC may be viewed as a variant of normal-phase chromatography. In normal-phase chromatography, the mobile phase is 100% organic. Only traces of water are present in the mobile phase and in the pores of the polar packing particles. Polar analytes bind strongly to the polar stationary phase and may not elute.
Adding some water [< 20%] to
the organic mobile phase [typically an aprotic solvent like acetonitrile] makes
it possible to separate and elute polar compounds that are strongly retained in
the normal-phase mode [or weakly retained in the reversed-phase mode]. Water, a
very polar solvent, competes effectively with polar analytes for the stationary
phase. HILIC may be run in either isocratic or gradient elution modes. Polar
compounds that are initially attracted to the polar packing material particles
can be eluted as the polarity [strength] of the mobile phase is increased [by
adding more water]. Analytes are eluted in order of increasing hydrophilicity[chromatographic
polarity relative to water]. Buffers or salts may be added to the mobile phase
to keep ionizable analytes in a single form.
Hydrophobic-Interaction
Chromatography [HIC]
HIC is a type of reversed-phase chromatography that is used to separate large biomolecules, such as proteins. It is usually desirable to maintain these molecules intact in an aqueous solution, avoiding contact with organic solvents or surfaces that might denature them. HIC takes advantage of the hydrophobic interaction of large molecules with a moderately hydrophobic stationary phase, e.g., butyl-bonded [C4], rather than octadecyl-bonded [C18], silica. Initially, higher salt concentrations in water will encourage the proteins to be retained [salted out] on the packing. Gradient separations are typically run by decreasing salt concentration. In this way, biomolecules are eluted in order of increasing hydrophobicity.
HIC is a type of reversed-phase chromatography that is used to separate large biomolecules, such as proteins. It is usually desirable to maintain these molecules intact in an aqueous solution, avoiding contact with organic solvents or surfaces that might denature them. HIC takes advantage of the hydrophobic interaction of large molecules with a moderately hydrophobic stationary phase, e.g., butyl-bonded [C4], rather than octadecyl-bonded [C18], silica. Initially, higher salt concentrations in water will encourage the proteins to be retained [salted out] on the packing. Gradient separations are typically run by decreasing salt concentration. In this way, biomolecules are eluted in order of increasing hydrophobicity.
Separations Based on Charge:
Ion-Exchange Chromatography [IEC]
For separations based on polarity, like is attracted to like and opposites may be repelled. In ion-exchange chromatography and other separations based upon electrical charge, the rule is reversed. Likes may repel, while opposites are attracted to each other. Stationary phases for ion-exchange separations are characterized by the nature and strength of the acidic or basic functions on their surfaces and the types of ions that they attract and retain. Cation exchange is used to retain and separate positively charged ions on a negative surface. Conversely,anion exchange is used to retain and separate negatively charged ions on a positive surface [see Figure T]. With each type of ion exchange, there are at least two general approaches for separation and elution.
For separations based on polarity, like is attracted to like and opposites may be repelled. In ion-exchange chromatography and other separations based upon electrical charge, the rule is reversed. Likes may repel, while opposites are attracted to each other. Stationary phases for ion-exchange separations are characterized by the nature and strength of the acidic or basic functions on their surfaces and the types of ions that they attract and retain. Cation exchange is used to retain and separate positively charged ions on a negative surface. Conversely,anion exchange is used to retain and separate negatively charged ions on a positive surface [see Figure T]. With each type of ion exchange, there are at least two general approaches for separation and elution.
Strong ion exchangers bear functional groups [e.g., quaternary amines
or sulfonic acids] that are always ionized. They are typically used to retain
and separate weak ions. These weak ions may be
eluted by displacement with a mobile phase containing ions that are more
strongly attracted to the stationary phase sites. Alternately, weak ions may be
retained on the column, then neutralized by in
situ changing
the pH of the mobile phase, causing them to lose their attraction and elute.
Weak ion exchangers [e.g., with secondary-amine or
carboxylic-acid functions] may be neutralized above or below a certain pH value
and lose their ability to retain ions by charge. When charged, they are used to
retain and separate strong ions. If these ions cannot be
eluted by displacement, then the stationary phase exchange sites may be
neutralized, shutting off the ionic attraction, and
permitting elution of the charged analytes
When weak ion exchangers are neutralized,
they may retain and separate species by hydrophobic [reversed-phase] or hydrophilic [normal-phase] interactions; in
these cases, elution strength is determined by the polarity of the mobile phase
[Figure R-1]. Thus, weak ion exchangers may be used for mixed-mode separations
[separations based on both polarity and charge].
Table D outlines guidelines for
the principal categories of ion exchange. For example, to retain a strongly
basicanalyte [always positively charged], use a weak-cation-exchange
stationary phase particle at pH > 7; this assures a negatively charged particle surface. To
release or elute the strong base, lower the pH of the mobile phase below 3;
this removes the surface charge and shuts off the ion-exchange retention
mechanism.
Note that a pKa is the pH value
at which 50% of the functional group is ionized and 50% is neutral. To assure
an essentially neutral, or a fully charged, analyte or particle surface, the pH
must be adjusted to a value at least 2 units beyond the pKa, as appropriate
[indicated in Table D].
Do not use a strong-cation
exchanger to retain a strong base; both remain charged and strongly attracted
to each other, making the base nearly impossible to elute. It can only be
removed by swamping the strong cation exchanger with a competing base that
exhibits even stronger retention and displaces the compound of interest by
winning the competition for the active exchange sites. This approach is rarely
practical, or safe, in HPLC and SPE. [Very strong acids and bases are dangerous
to work with, and they may be corrosive to materials of construction used in
HPLC fluidics!]
Separations Based on Size:
Size-Exclusion Chromatography [SEC] –
Gel-Permeation Chromatography [GPC]
Gel-Permeation Chromatography [GPC]
In the 1950s, Porath and Flodin
discovered that biomolecules could be separated based on their size, rather
than on their charge or polarity, by passing, or filtering,
them through a controlled-porosity, hydrophilic dextran polymer. This process
was termed gel filtration. Later,
an analogous scheme was used to separate synthetic oligomers and polymers using
organic-polymer packings with specific pore-size ranges. This process was
called gel-permeation chromatography [GPC]. Similar separations done using
controlled-porosity silica packings were called size-exclusion chromatography
[SEC]. Introduced in 1963, the first commercial HPLC instruments were designed
for GPC applications [see Reference 3].
All of these techniques are
typically done on stationary phases that have been synthesized with a pore-size
distribution over a range that permits the analytes of interest to enter, or to
be excluded from, more or less of the pore volume of the packing. Smaller
molecules penetrate more of the pores on their passage through the bed. Larger
molecules may only penetrate pores above a certain size so they spend less time
in the bed. The biggest molecules may be totally excluded from pores and pass
only between the particles, eluting very quickly in a small volume. Mobile
phases are chosen for two reasons: first, they are good solvents for the
analytes; and, second, they may prevent any interactions [based on polarity or
charge] between the analytes and the stationary phase surface. In this way, the
larger molecules elute first, while the smaller molecules travel slower
[because they move into and out of more of the pores] and elute later, in
decreasing order of their size in solution. Hence the simple rule: Big
ones come out first.
Since it is possible to
correlate the molecular weight of a polymer with its size in solution, GPC
revolutionized measurement of the molecular-weight distribution of polymers
that, in turn, determines the physical characteristics that may enhance, or
detract from, polymer processing, quality, and performance [how to tell goodfrom bad polymer].
Conclusion
We hope you have enjoyed this
brief introduction to HPLC. We encourage you to read the references below and
to study the Appendix on HPLC Nomenclature.
