Beginners Guide to Liquid Chromatography

Joseph C. Arsenault, Patrick D. McDonald, Ph.D.

[ Table of Contents ]

Table of ContentsWhat Is Liquid Chromatography? .................................................................................................................... 6 - Brief History and Definition ................................................................................................................. 6 - Liquid Chromatography [LC] Techniques ............................................................................................... 7 - What Is High-performance Liquid Chromatography [HPLC]? ..............................................................10 - What Is Ultra-performance Liquid Chromatography [UPLC Technology]? .......................................11How Does a High-performance Liquid Chromatograph Work? .....................................................................11 - HPLC System Diagram .........................................................................................................................11 - HPLC Operation ...................................................................................................................................13 - What Is a Detector? ..............................................................................................................................15 - What Is a Chromatogram? ...................................................................................................................15 - Identifying and Quantitating Compounds..........................................................................................16 - Isocratic and Gradient LC System Operation .....................................................................................19 - HPLC Scale [Analytical, Preparative, and Process] ............................................................................21HPLC Column Hardware.................................................................................................................................25 - Design ..................................................................................................................................................25 - Separation Performance Resolution................................................................................................26 - Mechanical Separation Power Efficiency .........................................................................................28 - Chemical Separation Power Selectivity ..........................................................................................28HPLC Separation Modes ................................................................................................................................28 - Separations Based on Polarity: ..........................................................................................................28 - Normal-phase HPLC ..................................................................................................................32 - Reversed-phase HPLC ...............................................................................................................32 - Hydrophilic-interaction Chromatography [HILIC] .....................................................................33 - Hydrophobic-interaction Chromatography [HIC] .....................................................................34 - Separations Based on Charge: Ion-exchange Chromatography [IEC] .................................................35 - Separations Based on Size: Size-exclusion Chromatography [SEC] Gel-permeation Chromatography [GPC] ..............................................................................................37Conclusion .....................................................................................................................................................38References for Further Reading .....................................................................................................................39Appendix: HPLC Nomenclature .....................................................................................................................40 Note: Additional information on words in red throughout text can be found in this section.

List of FiguresFigure A Tswetts Experiment ...................................................................................................................6Figure B Thin-layer Chromatography [TLC] ...............................................................................................7Figure C Paper Chromatography ...............................................................................................................8Figure D-1 Column Chromatography Solid-phase Extraction [SPE] .........................................................9Figure D-2 HPLC Column ........................................................................................................................... 10Figure E High-performance Liquid Chromatography [HPLC] System .................................................... 12Figure F A Typical HPLC [Waters Alliance] System ........................................................................... 13Figure G Understanding How a Chromatographic Column Works Bands ......................................... 14Figure H How Peaks Are Created ........................................................................................................... 15Figure I-1 Identification ........................................................................................................................... 17Figure I-2 Identification and Quantitation .............................................................................................. 18Figure J-1 Isocratic LC System .................................................................................................................. 19Figure J-2 High-pressure-gradient System .............................................................................................. 20Figure J-3 Low-pressure-gradient System ............................................................................................... 21Figure K HPLC System for Purification Preparative Chromatography ............................................... 22Figure L HPLC Column Dimensions ....................................................................................................... 23Figure M-1 Column Hardware Examples ................................................................................................... 25Figure M-2 A Look Inside a Column .......................................................................................................... 26Figure N Column Length and Mechanical Separating Power ................................................................ 27Figure O Particle Size and Mechanical Separating Power .................................................................... 27Figure P Chromatographic Polarity Spectrum by Analyte Functional Group ...................................... 29Figure Q Proper Combination of Mobile and Stationary Phases Effects Separation Based on Polarity .... 29Figure R-1 Mobile-phase Chromatographic Polarity Spectrum ................................................................ 30Figure R-2 Stationary-phase Particle Chromatographic Polarity Spectrum ............................................ 31Figure R-3 Compound/Analyte Chromatographic Polarity Spectrum ...................................................... 31Figure S-1 Normal-phase Chromatography............................................................................................... 32Figure S-2 Reversed-phase Chromatography ............................................................................................ 33Figure T Ion-exchange Chromatography ................................................................................................ 35Figure U Methods for Calculating Plate Number [N] ............................................................................. 47

List of TablesTable A Chromatography Scale............................................................................................................. 23Table B Chromatography Scale vs. Column Diameter and Particle Size ............................................ 24Table C Phase Characteristics for Separations Based on Polarity ...................................................... 34Table D Ion-exchange Guidelines ......................................................................................................... 36

[ List of Figures and Tables ]

6[ What is Liquid Chromatography? ]

What Is Liquid Chromatography?Brief History and Definition

Liquid chromatography was defined in the early 1900s by the work of the Russian botanist, Mikhail S. Tswett. His pioneering studies focused on separating compounds [leaf pigments], extracted from plants using a solvent, in a column packed with particles.

Tswett filled an open glass column with particles. Two specific materials that he found useful were powdered chalk [calcium carbonate] and alumina. He poured his sample [solvent extract of homogenized plant leaves] into the column and allowed it to pass into the par-ticle bed. This was followed by pure solvent. As the sample passed down through the column by gravity, different colored bands could be seen separating because some components were moving faster than others. He related these separated, different-colored bands to the different compounds that were originally contained in the sample. He had created an analytical separation of these compounds based on the differing strength of each compounds chemical attraction to the particles. The compounds that were more strongly attracted to the particles slowed down, while other compounds more strongly attracted to the solvent moved faster. This process can be described as fol-lows: the compounds contained in the sample distrib-ute, or partition differently between the moving solvent, called the mobile phase, and the particles, called the stationary phase. This causes each compound to move at a different speed, thus creating a separation of the compounds. Tswett coined the name chromatography [from the Greek words chroma, meaning color, and graph, meaning writingliterally, color writing] to describe his colorful experiment. [Curiously, the Russian name Tswett means color.] Today, liquid chromatography, in its various forms, has become one of the most powerful tools in analytical chemistry.

Plant Extractin Solvent

Colored"Bands"

Figure A: Tswetts Experiment

7[ Liquid Chromatography [LC] Techniques ]

Liquid Chromatography [LC] Techniques

Liquid chromatography can be performed using planar [Techniques 1 and 2] or column techniques [Technique 3]. Column liquid chromatography is the most powerful and has the highest capacity for sample. In all cases, the sample first must be dissolved in a liquid that is then transported either onto, or into, the chromato-graphic device.

Technique 1. The sample is spotted onto, and then flows through, a thin layer of chromatographic particles [stationary phase] fixed onto the surface of a glass plate [Figure B]. The bottom edge of the plate is placed in a solvent. Flow is created by capillary action as the solvent [mobile phase] diffuses into the dry particle layer and moves up the glass plate. This technique is called thin-layer chromatography or TLC.

Figure B: Thin-layer Chromatography

Note that the black sample is a mixture of FD&C yellow, red and blue food dyes that has been chromatographically separated.

Time Zero After X Minutes

Solvent Solvent

Solvent Tank

Original Sample

8[ Beginners Guide to Liquid Chromatography ]

Technique 2. In Figure C, samples are spotted onto paper [stationary phase]. Solvent [mobile phase] is then added to the center of the spot to create an outward radial flow. This is a form of paper chromatography. [Classic paper chromatography is performed in a manner similar to that of TLC with linear flow.] In the upper image, the same black FD&C dye sample is applied to the paper.

Figure C: Paper Chromatography

Notice the difference in separation power for this particular paper when compared to the TLC plate. The green ring indicates that the paper cannot separate the yellow and blue dyes from each other, but it could separate those dyes from the red dyes. In the bottom image, a green sample, made up of the same yellow and blue dyes, is applied to the paper. As you would predict, the paper cannot separate the two dyes. In the middle, a purple sample, made up of red and blue dyes, was applied to the paper. They are well separated.

[ Paper Chromatography ]

9[ Column Chromatography - Solid-phase Extraction (SPE) ]

Technique 3. In this, the most powerful approach, the sample passes through a column or a cartridge device containing appropriate particles [stationary phase]. These particles are called the chromatographic packing material. Solvent [mobile phase] flows through the device. In solid-phase extraction [SPE], the sample is loaded onto the cartridge and the solvent stream carries the sample through the device. As in Tswetts experi-ment, the compounds in the sample are then separated by traveling at different individual speeds through the device. Here the black sample is loaded onto a cartridge. Different solvents are used in each step to create the separation.

Load Sample(Black)

StepElute 1

StepElute 2

One cartridge can separate all three dyes

StepElute 3

NOTE: Different strength solvents can be used to separate the dyes.

Stationary Phase

Particles

Figure D-1: Column Chromatography Solid-phase Extraction [SPE]

When the cartridge format is utilized, there are several ways to achieve flow. Gravity or vacuum can be used for columns that are not designed to withstand pressure. Typically, the particles in this case are larger in diameter [> 50 microns] so that there is less resistance to flow. Open glass columns [Tswetts experiment] are an example of this. In addition, small plastic columns, typically in the shape of syringe barrels, can be filled with packing-material particles and used to perform sample preparation. This is called solid-phase extraction [SPE]. Here, the chromatographic device, called a cartridge, is used, usually with vacuum-assisted flow, to clean up a very complex sample before it is analyzed further.

Smaller particle sizes [

11

[ What is Ultra-performance Liquid Chromatography? ]

may easily be identified. HPLC can be, and has been, applied to just about any sample, such as pharmaceuti-cals, food, nutraceuticals, cosmetics, environmental matrices, forensic samples, and industrial chemicals.

What Is Ultra-performance Liquid Chromatography [UPLC Technology]?

In 2004, further advances in instrumentation and column technology were made to achieve very sig-nificant increases in resolution, speed, and sensitivity in liquid chromatography. Columns with smaller particles [1.7 micron] and instrumentation with specialized capabilities designed to deliver mobile phase at 15,000 psi [1,000 bar] were needed to achieve a new level of performance. A new system had to be holistically created to perform ultra-performance liquid chromatography [UPLC technology].

Basic research is being conducted today by scientists working with columns containing even smaller 1-micron-diameter particles and instrumentation capable of performing at 100,000 psi [6,800 bar]. This provides a glimpse of what we may expect in the future.

How Does a High-performance Liquid Chromatograph Work?HPLC System Diagram

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 com-pounds 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.

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 concentra-tion of the sample constituents (see Figure F). Since sample compound characteristics can be very differ-ent, 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.

Solvent(Mobile Phase)

Reservoir

Waste

PumpSolvent Manager

Solvent Delivery System

InjectorAutoSampler

Sample Manager

HPLC ColumnPacking Material Chromatogram

Peaks = Yellow, Red, Blue

Detector

Sample

Computer Data Station

Figure E: High-performance Liquid Chromatography [HPLC] System

12

[ How Does High-performance Liquid Chromatography Work? ]

40

Appendix: HPLC Nomenclature

*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: .

AluminaA porous, particulate form of aluminum oxide [Al203] used as a stationary phase in normal-phase adsorp-tion 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.

CartridgeA 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 col-umns 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.

[ Appendix: HPLC Nomenclature ]

41

[ Appendix: HPLC Nomenclature ]

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 detectors 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 differ-ent 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.

DisplayA device that records the electrical response of a detector on a computer screen in the form of a chro-matogram. 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 columns 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 band-spreading was later described in an equation by van Deemter.

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