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From simple separations to detailed wavelength analysis, we examine the important concepts and techniques needed to make liquid chromatography work its best in the laboratory.
The science of chromatography has been around for well over 100 years when plant pigments were first separated by a scientist named Mikhail Tsvet. Since 1900, chromatography has evolved into one of a laboratory’s primary sources of information. In this column, we will examine an important branch of chromatography essential to the cannabis industry—liquid chromatography. From simple separations to detailed wavelength analysis, we examine the important concepts and techniques needed to make liquid chromatography work its best in the laboratory.
The science of chromatography originates from the Greek words to write with color. The basic concept started with the separation of plant pigments into bands of color early in the 20th century. Mikahil Tsvet injected plant extracts mixed with petroleum ether into a glass column containing calcium carbonate. As the liquid progressed through the column over several hours, bands of color were observed that were later identified as chlorophyll a, chlorophyll b, xanthine, and carotene.
Over the subsequent decades this procedure was expanded as a separation technique by the Nobel Prize winning work of Archer J.P. Martin and Richard L.M. Synge in the 1940s and 1950s. The pair had worked together at the Wool Industries Research Association in Leeds. During their work they had created columns packed with silica and water as a stationary phase and then used chloroform as a mobile phase to separate N-acetamino acids from protein hydrolysates. This new development worked with the affinity of the target compounds with the different phases allowing for separation and collection of individual compounds. Their work established a form of chromatography called partition chemistry and was the basis for methods we now know as paper, gas, and liquid chromatography. They also attempted to explain the theory of solute concentration, resolution, and theoretical plates. The team went on to publish their work; “A New Form of Chromatogram Employing Two Liquid Phases” in the Biochemical Journal in 1941 and was awarded a Nobel Prize in 1952.
Since the initial discoveries of the early to mid-20th century the science of chromatography has seen rapid growth and expansion into an important and fundamental tool of many analytical laboratories. The techniques have involved from classical wet chemistry techniques to more instrument driven measurements facilitating an explosion of instrumentation and methodologies.
Chromatography allows for the separation of a mixture of compounds by diluting the mixture into a mobile phase which then passes through or over a stationary phase to create separation by the interaction and affinity of individual compounds for one of the phases of the chromatography system (Figure 1a). Mobile phase is a solvent or gas that flows through a chromatography system and is also referred to as an eluant. Stationary phase is a liquid or solid material that is fixed in a chromatography system. The properties of the mobile phase and the stationary phase are opposite of each other and allow for the partitioning of analytes as the mobile phase and test mixture flow through or over the stationary phase (Figure 1b).
The basic function of chromatography procedures is to separate a mixture of components into their constituent compounds. The purpose for separation falls into two categories: preparative chromatography or analytical chromatography. Preparative chromatography is used to separate compounds for later isolation or purification which then can be used in other processes. Analytical chromatography is used in quantitative and qualitative analysis of complex mixtures.
Chromatography processes are further divided into two major categories (liquid or gas chromatography) based on the composition of their mobile phase component. Gas chromatography (GC) uses a carrier gas, such as hydrogen or helium, to separate compounds based on their interaction with a stationary phase or based on their boiling points. Mixtures are heated and vaporized in an injection port and are moved through or across a stationary phase to separate compounds before reaching a detector that provides data on concentration or identity of components. Gas chromatography is predominately used for analytical methods, rather than preparatory methods.
In liquid chromatography (LC), compounds are separated by dissolving a sample into a liquid mobile phase which is then passed over a solid stationary phase. Individual compounds transition in and out of each of the phases depending on their affinity with each phase. LC can be further divided into the type of solid phase or media that is used in the technique. Planar chromatography techniques use a flat single dimension matrix or solid phase. These techniques include paper and thin layer chromatography (TLC). TLC is a common planar chromatography technique used in qualitative analysis to separate non-volatile and semi-volatile compounds. Mixtures are applied to a plate layered with stationary phase and then submerged at the end into a developing chamber of mobile phase solvent. As time progresses the spots of a mixture travel up through capillary action through the thin layer of solid phase, which separates into bands of compounds (Figure 2).
Column chromatography uses columns coated or packed with stationary phase. Sample mixture and mobile phase flows through the column and over the stationary phase to elute compounds over time. Early chromatography consisted of filling a glass column with a stationary phase, which allows mobile phase to gravity feed through the column and elute out at the bottom of the column. More modern techniques of liquid chromatography use pumps to increase pressure and flow of mobile phase through the column to force eluent through the chromatographic system, such as high performance liquid chromatography (HPLC). HPLC can be used for preparation, separation, and analytical processes depending upon the type of system.
Commercially available HPLC systems have different features and selling points, but all systems have some basic modules or components. The key to liquid chromatography is the mobile phase, thus all HPLC systems have one or more mobile phase pumps. Mobile phase is stored in reservoirs that are plumbed into a pumping module. Binary pumps can pump two separate channels or mobile phases at the same time. Quaternary pumps can pump up to four different channels or mobile phases at the same time to the system.
Modern HPLC systems contain solvent degassing modules that remove bubbles and dissolved gases in the mobile phases, which can cause baseline noise or hinder the efficiency of the chromatography system. Solvent degassing can be accomplished by helium purging, vacuum degassing, sonication, or a combination of these methods. Older HPLC systems often lacked an online degassing module and relied on external helium sparging to dissolved air in the mobile phase while in the reservoir bottles.
Another more modern module to HPLC systems is an autosampler compartment instead of single manual injection port. Autosamplers allow for loading multiple samples that are injected without human involvement and therefore reduce sampling errors. Prior to widespread use of autosamplers, sample injections were made manually and were subject to higher error and variability.
The final two components of a liquid chromatography system are: the column compartment (or column oven) and the detector. The column compartment is a temperature-controlled compartment that contains the chromatography column. Many modern systems have the ability for a wide range of heating and cooling functions.
The next and sometimes final component is a detector module that records the separated analytes that elute from the column and produce a signal that a chromatograph records on a chromatogram. A chromatogram is the visual data record of the analyte responses recorded as they elute from the column while a chromatograph is the instrumentation that records the data (Figure 3). After the data is recorded the mobile phase and eluent usually ends up as waste for an analytical system or can be separately collected for use in a prep chromatography system.
The data produced is often represented as peaks or patterns that correspond to separate components in a mixture expressed over time. The time which a selected analyte elutes is called its retention time. The retention time is the amount of time it takes for an analyte to pass through the system from injection to detection. Retention times change with different conditions such as pH, temperature and stationary phase type, column dimensions, and mobile phase or solvent compositions.
The mobile phase or solvent composition is an important element of chromatography. The type and characteristics of the selected solute and mobile phase influence the affinity of the analytes with the different chromatography phases. An important concept of liquid chromatography is polarity.
Polarity occurs in molecules and solutions when there is either a significant difference in charge, electronegativity, or ionic bonds leading to high dipole moments (large differences in charge). Molecules or solvents with high dipole moments are polar whereas molecules or solutions with equal sharing of bonds (covalent or polar covalent) have little to no charge and are nonpolar. Polarity can be ranked using a polarity index (P’), which is a relative measure of a solvent or solution with various polar matrices. The higher the polarity index, the more polar the solvent (Table I).
Early chromatography was dominated by what is now called normal phase liquid chromatography (NPLC) or adsorption chromatography where the mobile phase consisted of a nonpolar solvent such as hexane while the stationary phase was composed of polar materials such as silica. Modern NPLC columns include amino, cyano, silica and supercritical fluid chromatography columns (SFC). The analytes analyzed by NPLC are more hydrophobic and nonpolar in nature.
The most commonly used mode of liquid chromatography is reversed-phase chromatography or partition chromatography in which the mobile phase is polar while the stationary phase is composed of nonpolar materials such as n-octyl (C8) or n-octyldecyl (C18) hydrocarbon chains. The selection of a reversed-phase column depends on the type of analytes and mobile phase composition. Analytes for reversed-phase LC tend to be more hydrophilic and polar than analytes studied by NPLC.
The ability of a solvent or mobile phase to pull analytes from the stationary phase or adsorbent is called its eluent strength, elution power or eluotropic value (ε0) and is dependent upon the polarity of the mobile phase and the stationary phase. Eluotropic series rank solvents by their ability to displace an analyte or solute from the stationary phase. The eluotropic value (ε0 ) expresses the measurement of the solvent or mobile phases absorptive energy based on a particular substrate or stationary phase such as aluminum oxide. The greater the ε0 , the more polar the solvent and the greater its ability to elute analytes from the column (Table II).
Solvents or mobile phases that are part of a chromatographic method often work together to create separation of analytes. In general, the solvents are similar in their polarity with differences in potential elution strength. In all cases the solvents must be compatible and miscible. Miscibility is the ability of solutions to mix together in all proportions to form a homogenous solution. In many instances, polar and nonpolar solvents are immiscible or unable to form a homogenous solution (Table III).
If more than one solvent is used simultaneously during a method or mobile phases are mixed, the polarity index changes. To determine the new polarity of a solution the composition of the mixture is calculated with the known polarity indices for the solvents to obtain a new polarity index using the following equation:
P’AB = ΦA P′A + ΦB P′B
For example, if you are mixing two solvents such as acetonitrile and water in a mixture or ratio of 70:30, your first solvent P’ is 5.8 (ACN) multiplied by concentration 0.7. Your second solvent water’s P’ of 10.2 is multiplied by 0.3 and added to your first result to give a mixture with the polarity index of 7.1.
0.7 (5.8) + (0.3) (10.2) = 7.12
The process of mixing of mobile phase can happen by either creating premixed mobile phases in a single mobile phase reservoir or by combining pumping channels with mixing occurring in a mixing cell within the HPLC system. The mixing of channels allows for either control of a steady mix or different ratios of mobile phase over a course of a method run. Isocratic elution is a method based on the principle of a steady composition of mobile phase that may be composed of a single or multiple solvent. In these methods, the proportion of the mobile phase components do not change over the course of the sample run. Gradient elution methods allow the concentration of mobile phase components to change over the course of a method, usually to increases separation or improve resolution of analytes in the chromatogram. Generally, in a reversed-phase separation the initial mobile phase is more polar (aqueous) then changes composition of mobile phase so that it becomes less polar (organic) to the final time. Gradient methods require a time built into the method to allow flushing of retained analytes off the column and then a return or reequilbration of the system to the starting conditions.
The resolution and peak shape of gradient methods overall tend to be higher and sharper than in isocratic methods. Gradient methods also tend to allow for faster analysis. But there are types of analysis detectors and columns that do not function for gradient methods.
A second important variable is chromatography is the selection of a column. Columns are grouped by the mode of analysis and analytical targets. As was stated previously, nonpolar analytes are more commonly examined using normal phase liquid chromatography while polar analytes tend to be examined using reversed-phase liquid chromatography.
HPLC columns all have some basic structural features in common. The starting point of the column is the outer casing or shell, which is commonly composed of either stainless steel or a rigid polymer that can withstand the pressure created by the flow of mobile phase in the system. The length and diameter of these columns vary greatly depending upon the intended analysis and system limitations. The most common type of HPLC columns are called packed columns measuring between 30 to 300 mm compared to capillary columns used in gas chromatography. Most HPLC columns are packed with porous silica particles (3–10 µm), which are bonded with stationary phase.
The silica particles can vary in size and shape (symmetrical or asymmetrical) according to brand and size specifications. The particles have pores that are the largest source of surface area in the column (Figure 5). The particles pack together and create interstitial spaces which contributes to the void volume of the column. Void volume is the total volume of mobile phase in the interstitial space (space between particles) and the pores. Sometimes void volume is called dead volume or column volume and is generally the minimal amount of mobile phase that needs to be pumped through the column before analytes can reach a detector.
One way to determine void volume requires the column dimensions and pore volume (which is usually a maximum of 70% for a fully porous column). The void is calculated in microliters (µL) by the equation:
VV (µl) = (d2 * π * L * VP) / 4
Where d is the column diameter (mm), L is the column length (mm), and VP is the pore volume.
Most columns can be roughly calculated for an approximate void volume and dead or dwell time using this calculation or by a simpler method where an unretained analyte or standard is injected and the time the peak appears is recorded. The noted time and the system flow are then multiplied to determine the volume of the column and connecting tubing. Generally, a typical 4.6 x 100 mm column will have void volume of approximately 1 mL, which means if you are running at 0.5 mL/1 min that you will have a dwell time or dead time of just over 2 min (Table IV).
The silica particles are the substrate for the bonding of the stationary phase, but not all of silica molecules will bond with the stationary phase. The silica layer is composed of silanols and bare silica (Figure 5a), which are all potential sites for bonding with other silica sites or the stationary phase. The stationary phase bonds to some but not all of the silica particles (Figure 5b). Some columns are treated with endcapping compounds such as trimethylsilyl group (TMS), which bind many of the remaining unbound sites. This endcapping makes the silica nonpolar where bare silica and free silanols are slightly acidic and polar. Endcapping is also a means of protecting the column from the harsh polar solvents that otherwise could attack the polar sites.
Columns are known by the type of molecule that is used as the stationary phase. Some of the most commonly used reversed-phase LC columns use a nonpolar alkane stationary phase such as n-octyl (C8) or n-octyldecyl (C18) (also known as ODS) hydrocarbon chains. The type of stationary phase depends on the type of mobile phases being used and the characteristics of the target analytes (Figure 6). Samples that are soluble in water or polar solvents are either separated by reversed-phase or ion exchange chromatography. The target analytes are then examined to determine if they are ionic, polar, or neutral, which then further dictates the column stationary phases that will be most efficient at peak separation.
HPLC columns are generally grouped into one of four categories: ion exchange (IEC), size exclusion (SEC), normal phase (NP), and reversed-phase columns (RP). Ion exchange chromatography (IEC) columns are used to analyze highly polar compounds. The IE columns have an acidic stationary phase such as a strong anion exchange (SAX) or a basic stationary phase such as strong cation exchange columns (SCX). The mobile phase for these columns is polar and may contain salts. Separation occurs by strong ionic exchange between the highly charged phases and the analytes.
Size exclusion chromatography (SEC) (also known as molecular sieve chromatography) separates analytes by size with large molecules being inhibited by the pore size of the stationary phase and therefore elute first before the smaller molecules. This type of chromatography is most often used to separate large biological molecules such as polymers, peptides, and proteins. The most common mobile phase for this technique is water sometimes with added buffers such as an acid or base.
Normal phase columns, as has been discussed earlier, have a polar stationary phase and a nonpolar mobile phase, and are used for the separation of nonpolar analytes. There are new generations of columns such as mixed mode or hydrophilic interaction liquid chromatography (HILIC) columns that have features and stationary phases that cross the lines between all the different types of chromatography. HILIC columns are sometimes referred to as reverse reverse-phase columns since they are meant to be used with extremely polar analytes and employ highly aqueous mobile phase.
Finally, reversed-phase columns which have a nonpolar stationary phase (such as C8 and C18) and polar mobile phases are the most popular columns in analytical use. All manufactures of HPLC columns have their own version of C8 and C18 columns with different features such as high aqueous mobile phase tolerance, special end capping, and increased robustness, to name a few. These types of columns are considered the starting point for most method development and testing since these phases can work with many varied types of analytes and instrument detectors.
Once separation of different analytes has been achieved by the column, the analyte then must be recorded or measured by some type of detector. The most commonly used HPLC detectors are ultraviolet and visible light (UV-vis) detectors, mass spectrometers (MS), refractive index detectors (RID), fluorescence detectors, and evaporative light scattering detectors (ELSD). `
The simplest detectors are refractive index detectors (RID) that measure the refractive index of an analyte against the refractive index of the mobile phase. RIDs are thought be universal detectors capable of detecting any type of analyte since the technique is basically a differential measurement. The draw backs to RIDs are that have low sensitivity and cannot employ gradient methods since the reference cell containing the mobile phase is often a closed system once the cell has been filled. In RID analysis, the work of the separation must be accomplished by the column phase and an isocratic mobile phase. Compounds that elute together (coelute) can not be distinguished from one another.
Fluorescence detectors are sensitive detectors for a select group of compounds that can be excited and emit a light signal (fluoresce). Lamps produce excitation energy to the analyte molecules and the light that is emitted from the analytes is measured by the detector. Evaporative light scattering detectors (ELSD) are used for compounds that neither contain chromophores nor can fluoresce such as sugars and fatty acids. In ELSD, mobile phase is nebulized creating droplets that pass into heated tubes, which evaporate the solvent and leave analyte aerosols that then are passed through a region of light beams where the scattering of the beam is measured by a photodiode or multiplier. Unlike refractive index detectors, fluorescence detectors can use gradient methods to achieve separation.
Ultraviolet and visible light detectors (UV-vis) are absorbance detectors that measure absorbance of light from analytes with a chromophore. A chromophore is a molecule or region of a molecule that is responsible for color. This chromophore region has an energy difference between molecular orbitals that falls within the light spectrum. A molecule’s chromophore absorbs visible light exciting its electrons which allows the detector to measure absorbance in the ultraviolet through visible light range from about 190 to 900 nm with most organic compounds and chromophores falling in the range of 190 to 350 nm (Figure 7 and Table V).
UV-vis detectors are stable and reliable detectors that are easy to operate and a common tool in analytical laboratories. There are several types of UV-vis detectors based on the number of wavelengths monitored and flexibility in data handling. Diode array detectors (DAD) or photo array detectors (PDA) can monitor many different wavelengths up to the entire spectrum while fixed wavelength detectors (FWD) and some variable wavelength detectors are more limited to set wavelengths or bands. UV-vis detectors consist of a deuterium lamp as a light source with wavelengths ranging from 190 to 380 nm and a tungsten lamp to detect longer wavelengths.
The next most popular type of detector is a mass spectrometer (MS). A mass spectrometer detects the mass of analytes and can be used for identification and quantification of compounds. LC–MS is often a multidetector technique that involves the combination of the mass spectrometer and another detector such as a UV-vis detector. Like the UV-vis detectors, LC–MS techniques are amiable to both isocratic and gradient mobile phase methods.
LC–MS often requires that the analyte compounds have the ability to ionize to be detected. LC–MS systems have one of several ionization methods or sources the most popular include: electrospray ionization (ESI), atmospheric pressures chemical ionization (APCI), and atmospheric pressure photoionization (APPI). ESI utilizes a nebulizer probe or needle and a nebulizer gas. A mist of charged ions is created by the desolvation of mobile phase droplets and application of heat and charge. Polar molecules with their lower ionization energies are easily ionized by ESI. ESI with its lower energy requirements allows for rearrangement of ions, double charging, or additional ions being added to the analyte ions. Additional ions which occur by adding fragments of mobile phase, other analytes, components of glassware or tubing are called adducts. The most common adducts come from sodium in the glassware that can result in M + 23 molecules. Other adducts can be from mobile phase additive such as NH4, or acids. Another type of molecule created in the ESI source are dimers, which is a doubling of the analyte ion. There are many free programs and adduct calculators available online including Excel calculators. The calculator I use most in my laboratory can be downloaded at http://www.lc-ms.nl/ and used in Excel.
APCI is similar to ESI but has the ability to ionize less polar molecules. The solvent evaporation and nebulizer spray is similar to ESI, but the molecules are ionized by the addition of energy from a corona needle. Since higher energy levels are applied to this ionization source, there is a tendency to form less adducts than found in the ESI source.
APPI utilizes a Krypton lamp to ionize analytes with very high ionization energies such as low or nonpolar compounds. The lamp produces photons that ionize the nonpolar compounds by overcoming their ionization energy. Sometimes a highly ionizable dopant or solvent is used to lower the ionization energy by complexing with the analyte and adding the effect of both the photoionization and solvent evaporation in the heated zone.
Once ions are created in the LC–MS, the analyte ions are carried to a detector where they can be fragmented to various degrees with application of charge. Detectors are grouped into three overall technologies: quadrupole, ion traps, and time of flight (ToF). Some newer technologies combine multiple techniques such as "triple quads" (the combination of multiple quadrupoles), Q-ToF (quadrupole and time of flight), and ion trap ToF (ion trap with time of flight). The mostly widely used LC–MS detector is the quadrupole detector (either a single quad or triple quad). In a quadrupole detector, two pair of oppositely charged rods are arrange opposite each other to create a charged tunnel through which analyte ions pass and are fragmented. In a single quadrupole system, there is only one pathway for detection, which often creates the parent ion plus either an adduct or a hydrogen (M+1) or a simple fragment. In a triple quadrupole system, the three sets of quadrupoles can each be set to different fragmentation energy to create an additional set of smaller fragments along with the parent ion which can aid in identification (MS/MS). The first and the third quadrupoles act as mass filters and the second quadrupole becomes a collision cell to create fragments.
Ion trap detectors have a charged reservoir or trap into which the analyte ions are deposited. Fragmentation energy is applied to the trap which fragments the analyte ions and ejects them from the trap. The ion trap can apply multiple different ionization energies to the trap and can created multiple fragments similar to passing through multiple triple quads and is often referred to as MSn due to its ability to produce a large number of fragmentations.
Time of flight detectors measure extremely accurate mass for analyte ions determined by a time-of-flight measurement. Ions enter a charged field and are accelerated through the field with larger ions having slower speed than smaller ions. The resulting measurement produces an extremely accurate mass calculation which can be used as a method of identification.
Chromatography is an important tool in the modern analytical laboratory. The technology has evolved quickly and steadily over the first 100 years of its existence. Most analytical scientists consider their HPLC systems to be essential instruments in the laboratory. It would be impossible to cover all the variations and intricacies of liquid chromatography in one article, especially since volumes and careers have been built around the subject. But my hope is that by examining the history, functionality, and operation of these essential instruments that it makes the task of operation and method development easier to understand. The next step is to dive a bit deeper into the weeds of theory and chemistry of liquid chromatography to understand separation, resolution, efficiency, and so on to allow for better method development and analysis.
Patricia Atkins is a Senior Applications Scientist with SPEX CertiPrep and a member of both the AOAC and ASTM committees for cannabis.
P. Atkins, Cannabis Science and Technology 4(1), 20-30 (2021).