Chromatography Basics
The science of chromatography has been around for well over a hundred 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. From simple separations to detailed wavelength analysis we will examine the important concepts and techniques needed to make chromatography work its best in the laboratory.
History of Chromatography
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 twentieth 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 which 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 1940’s 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 proteinhydrolysates. 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-twentieth 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.
The basics of chromatography
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 which flows through a chromatography system and is also referred to as an eluant. Stationary phase is a liquid or solid material which 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 ABOVE).
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, helium etc., 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 and/or identity of components. Gas chromatography are predominately 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 which 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 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 to elute out 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 like high-performance liquid chromatography (HPLC). HPLC can be used for preparation, separation, and analytical processes depending upon the type of system.
HPLC System Components
All HPLC systems commercially available have different features and selling points, but all systems have some basic modules or components. The key to liquid chromatography is the mobile phase so all HPLC systems have one or more mobile phase pumps. Mobile phase is stored in reservoirs which 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 which 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 which are injected without human involvement and 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.
GC System Configurations
The general configuration of a gas chromatography system starts with the carrier gas which is either contained in a cylinder or produced using a gas generator depending on the type of gas. Cylinders containing flammable gases such as hydrogen are commonly threaded in the opposite direction of nonflammable gas cylinders to prevent common mix-ups.
The gas is controlled by a flow controller or a regulator and is plumbed into the GC system usually near the injection port. The injection port is the entry into the system for samples and connects to the column. Gas flow through the injection port and then the column allowing for the partitioning of analytes between phases. The final components are the detectors which output an electronic signal to the chromatograph and the waste for the system (FIGURE 4).
Gas chromatography systems are plumbed to a variety of gases depending on the detectors and type of analysis. A carrier gas is common to all the most commonly used configurations. This carrier gas is the mobile phase which carries analytes to the column for partition chromatography. Most gas lines are plumbed from either a cylinder, Dewar or generator with a series of filters or traps intended to trap any contamination or potential compounds which could interfere with analysis.
The most common traps for GC systems include an oxygen trap, a moisture trap, and a hydrocarbon trap. The oxygen trap removes oxygen from the purified gas which could damage the GC column. Oxygen traps are often composed of metal and an inert reagent. The goal of the trap is to reduce oxygen concentrations to below 20 ppb. Many oxygen traps can remove small organic molecules and sulfur from the gases.
Figuring out the phases
The mobile phase composition is an important element of liquid and gas 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 where molecules or solutions with equal sharing of bonds (covalent or polar covalent) 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 1).
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 like hexane while the stationary phase was composed of polar materials such as silica. Modern NPLC columns include amino, cyano, silica and super-critical fluid chromatography columns (SFC). The analytes analyzed by NPLC are more hydrophobic and non-polar in nature.
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 2).
Solvents or mobile phases which 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 3).
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:
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 (I) 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 which 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 (FIGURE 5).
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 which do not function for gradient methods.
Gas Mobile Phases
Gas chromatography, as the name implies use gas as the mobile phase. These gases are often referred to as the ‘carrier’ gas and include helium, hydrogen, nitrogen, methane, or argon. There are four main considerations in the choice of carrier gas:
- Application
- Efficiency
- Availability
- Cost
The application, type of column and analysis can each dictate the type and grade of the required carrier or make-up gas. A make-up gas is an additional gas plumbed into the system to produce a specific effect on the detector or system. The lower in concentration of the potential target analyte, the higher the purity of carrier and make-up gas are needed for analyses. The higher the purity of gases, the higher the cost of the gas.
The next consideration of efficiency deals with the chromatographic efficiency of the system and theoretical plates as was discussed in our previous chromatography columns. Efficiency is a measure of theoretical plates in a chromatographic system. As has been discussed previously, chromatography columns do not have physical plates which can be measured so the plates are theoretical to describe the efficiency of the column and is called height equivalent to a theoretical plate (HETP) and is measured by the van Deemter equation in gas chromatography analyses:
A is the Eddy Diffusion Parameter, B is the longitudinal diffusion coefficient of eluting particles resulting in dispersion [m2 s−1], C is the mass transfer coefficient of resistance of analyte between mobile (m) and stationary phase (s) and u is linear velocity or speed [m s−1]
The first term of the equation is the eddy diffusion parameter and refers to the diffusion or mixing of substances by a turbulent, swirling motion around objects. In certain types of columns where the stationary phase is particulate in nature, currents or eddies can form and create forces which effect the HETP. These types of columns include packed GC columns (FIGURE 6A). Columns which are tubular in nature (i.e., capillary columns) do not have the same forces so the term A of the equation is zero (FIGURE 6B).
The second term B is the longitudinal diffusion coefficient which is the constant proportion of the diffusion of one component into another divided by the average speed of diffusion. This term is a calculation of the dispersion as a solute travels down the pathway. In this situation the concentration of the solute is highest in the middle of the pathway and more diffuse on the edges with the solute diffusing more as it passes through the column and contributes to band broadening (FIGURE 7).
The diffusion effects are more pronounced at lower flow rates. Gas chromatography is more effected by these forces due to the exponentially higher diffusion coefficients found in gases as opposed to liquids. The third term is the mass transfer coefficient between the mobile and solid phase. Since the mobile phase (gas) is rapidly moving, an equilibrium may not be reached resulting in peaks being either less retained or retained too highly on the solid phase depending on the depth or thickness of the solid phase (film). The mass transfer is most efficient at lower flow rates which is the opposite of the longitudinal diffusion. The most effective set of conditions is a balance between these two coefficients resulting in a minimal value for the HETP and the optimized linear velocity (u) in a Van Deemter curve (FIGURE 8).
The window of velocity for the optimal velocity can change with the type of carrier gas, solid phase (film) thickness and the inner diameter of the column. Film thickness can influence the mass transfer while smaller diameter columns produce flattened curves with optimal velocity at lower velocities.
The B/u diffusion term is greatly influenced by the type of carrier gas. The goal is to optimize the balance between the type of gas, the gas flow, and the column dimensions to produce an area of optimal practical gas velocities (~1.5 to 2x the optimal velocity) in which the system operates.
Carrier gases and their own van Deemter curves will influence the optimization of the GC system. u = B C {\displaystyle u={\sqrt {\ frac {B}{C}}}} Helium is the one of the most commonly used carrier gases for GC due to its safety and good relatively good van Deemter curve with a range of practical optimum gas velocities (OPGV) between about (25-35 cm/sec). The drawback to helium is that is an expensive commodity which is dependent upon the finite reserves of natural gas. There are no easy was to produce helium (especially within the average laboratory) and helium’s available and cost in some cases offset its ease of use and safety.
Nitrogen is another potential GC carrier gas which unlike helium is fairly inexpensive and can be produced in-house with a liquid nitrogen dewar, or a nitrogen generator and purifier. Nitrogen is a fairly safe gas in the laboratory, but it is not the most optimal gas for GC with a smaller lower range OPGV from ~ 10 to 15 cm/sec.
Finally, hydrogen, like nitrogen is cheap, accessible, and easy to produce but can cause some safety concerns (real and exaggerated) in the laboratory. Hydrogen can be produced using a hydrogen generator or from hydrogen cylinders. Hydrogen has the best OPGV range of values with higher velocities from 35 to 60 cm/sec but there can be possible hydrogenation reactions that may affect some target analytes (FIGURE 9).
As mentioned earlier, there safety concerns regarding the use of hydrogen in the laboratory as a carrier gas. Hydrogen can be explosive at above ~4% volume in air but on the positive side, it quickly diffuses, and most modern GC systems regulate flow and have automated safety shutdowns to prevent accidents. Secondly, there is very little hydrogen flowing through any GC system so the amount of possible leakage at the system is minimal compared to other possible leaks in the gas plumbing system upstream of the chromatograph.
Instrument election and dynamic range
No one piece of instrumentation will fill all needs. Some technologies have a wider range target such as LCMS vs GCMS, but each technique has its limitations and uses. Instrument choice is often dependent on chemistry of target analytes and their potential analytical concentration. For instance, in cannabis there are several classes of organic analytes which are routinely examined including cannabinoids for potency; terpenes and flavonoids for identity, flavor, fragrance and chemical fingerprinting; and pesticide residues or mycotoxins as potential contaminants. By their very nature all these compounds occur in vastly different concentrations. Trace analysis (low ppm or ppb) is the range for any methods created to quantify potentially dangerous contaminants like pesticides or mycotoxins and require systems such as GCMS, LCMS and LCMS/MS which have sensitivity in those low ranges.
The accuracy and ability to quantify analyte concentrations depends on the instruments analytical specification levels and dynamic range often bracketed by the level of detection (LOD) and level of linearity (LOL). The lowest limit of an analytical system is the limit of detection (LOD), this is the point where a target can be differentiated from a blank or noise with a high degree of confidence (usually over three standard deviations from the noise or blank response). The highest level of accurate quantitation ends with the limit of linearity (LOL) where the linearity of the system starts to skew often due to detector saturation. Peaks which reach LOL appear broad, flatten at their apex, or are cut off before their apex.
The range of the most accuracy (dynamic range) is between the LOD and LOL starting at the limit of quantitation (LOQ). The limit of quantitation (LOQ) is the lower limit of a method or system which the target analyte can be reasonably calculated (over ten standard deviations from the blank or baseline response).
A simple method to determine if a response peak reaches the cutoff for LOD or LOQ is to look at the ratio of signal-to-noise (S/N). A blank baseline in chromatographic systems is rarely flat and straight. The lowest points of the chromatogram are a combination of the true baseline and system noise. Baseline noise are the sum of all the random variations (electrical, temperature, etc.) and contamination or interference from the chemical components.
To determine a peak can be quantified one can either compare relative heights or relative areas. In comparing relative height, the analyst averages the mean height of the noise and compare it to the height of the target peak from the noise mean height. To compare areas, one or more ‘peaks’ in the noise are integrated with similar width to the target peak and the areas are compared. If the ratio is greater than three then it qualifies as within LOD and if the ratio is greater than ten, then that peak can be used for quantitation (LOQ).
The best practice is to integrate the noise at the baseline at several points and average the baseline noise responses then compare to the integrated peak of interest (FIGURE 10). For example, if the chromatographer is interested in peak A, they should integrate areas of baseline near the peak of similar peak widths to the target or measure its height compared to the average range of peak heights found in the noise. If the average of those baseline noise peaks is 100 units (height or area) then peak A must be at least 300 units to meet the LOD criteria of 3X. If peak A is only 200 units, then it fails and cannot be used for either identification or quantification. If peak B is the peak of interest and has a S/N >3 but <10, then it can be used for detection (LOD) and possibly identification but should not be used for quantification (LOQ).
Finally, a peak such as C can be used for both identification and quantification because its S/N value is higher than 10. Instrument sensitivity (represented as S/N) can be increased by decreasing baseline noise without increasing the target response. Noise in system can be created by matrix from the extracted sample, contamination of the sample, and contamination of either the stationary phase or mobile phase.
Proper sample clean-up and processing can sometimes reduce baseline noise, so the target peak is not ‘lost in the weeds’ of the baseline. Often it is believed that one can get a better response by injecting a larger sample aliquot, but, if the sample matrix is a contributor to the noise, then a larger sample means more matrix as well and will not necessarily help with the issues of S/N.
As for the mobile phases, impurities in mobile phase can directly affect baseline noise. The wrong grade of gases in GC can create high baselines while HPLC mobile phases can accumulate contamination by exposure to the laboratory environment. Replacing old solvents with fresh solvent can dramatically lower HPLC baseline noise. In some cases, especially in LCMS and ppb analysis, the use of highly filtered LCMS solvents can also play a role in reducing baseline noise.
Solid phase contamination and build up can play a role in baseline noise. As columns age the backbone materials silanes, siloxanes etc. can break down or lose protective end capping which increases noise. Harsh or acidic HPLC mobile phases can strip column phases and promote column breakdown. By examining the chromatographic baseline and cleaning up the contributing factors it can ensure that more target peaks.
Chromatography is a powerful analytical tool that needs extensive adjustments to maximize its accuracy and efficiency. All the adjustments and fine tuning of parameters ultimately is based on the targets of interest. Generally, chromatographers group targets as either polar or nonpolar analytes, or group the analytes in respects to volatility (nonvolatile, semivolitile and volatile) with some fluctuating between the classifications. Nonpolar analytes such as alkanes and alike often are targets for normal phase chromatographic methods (NPLC) or gas chromatography (GC) while polar analytes such as carboxylic acids, will be examined with reverse phase liquid chromatographic methods (RPLC). The majority of modern HPLC analysis falls under the classification of RPLC.
The chemical properties (in addition to the concentration) of your targets will dictate to a degree the type of instrumentation that will be needed for analysis. As was stated previously, the concentration of analytes can dictate instrumentation. But the chemical nature of the compounds will play an important role not only in selecting the right column but the right detector. Once the chromatographer understands their target analyte’s range requirements, selected instruments, detectors, and targeted chemistries it is time to start building or refining an analysis method.
