3. How does chromatography work?
A mixture is introduced onto a
“column” that contains an adsorbent
Solvent
(mobile phase)
Plant pigments
Packing material
(Stationary
phase)
4. How does chromatography work?
A mixture is introduced onto a
“column” that contains an adsorbent
Solvent flows through the column and
components move at different rates,
resulting in separations
Solvent
(mobile phase)
Plant pigments
Packing material
(Stationary
phase)
5. How does chromatography work?
A mixture is introduced onto a
“column” that contains an adsorbent
Solvent flows through the column and
components move at different rates,
resulting in separations
The components flow out of the
column and can be collected
individually or measured
Chromatography is used to:
Identify components
Determine how much
Purify substances
Solvent
(mobile phase)
Plant pigments
Packing material
(Stationary
phase)
6.
7.
8.
9.
10.
11.
12.
13.
14. Pump Operation: Filling Stroke
outlet
check
valve
pump
head
high pressure solvent out
low pressure solvent in
inlet
check
valve
piston
15. Pump Operation: Delivery Stroke
outlet
check
valve
pump
head
high pressure solvent out
low pressure solvent in
inlet
check
valve
piston
16. Pump Operation: Filling Stroke
outlet
check
valve
pump
head
high pressure solvent out
low pressure solvent in
inlet
check
valve
piston
17. Pump Operation: Delivery Stroke
outlet
check
valve
pump
head
high pressure solvent out
low pressure solvent in
inlet
check
valve
piston
This presentation will provide a brief introduction to the theory and practice of liquid chromatography. Aspects of method development will be covered in a separate presentation. Instrumentation often differs significantly among manufacturers, but the principles of operation are the same. Analytical methods usually can be transferred from one instrument to another, as long as the basic requirements are maintained. Some variability is to be expected based on differences in instrumentation, but the changes in performance can often be compensated by adjusting operational parameters.
Chromatography is an example of a process by which a mixture is separated into at least two fractions with different compositions. Other separation methods exist, such as distillation, recrystallization, and extraction.
Chromatography was first described over 100 years ago by a Russian botanist named Mikail Tswett. His research focused on the separation and purification of chlorophyll pigments from plant extracts. Plant extracts were applied at the top of a tube containing a powdered chalky adsorbent, and a solvent was allowed to flow though the tube. Bands appeared over time along the column that were created by the different plant pigments. For the development of this technique, Tswett is widely regarded as the father of chromatography.
Separations serve three purposes: identification of the components in a mixture, measurement of how much of each component is present, and purification.
Although the original research was carried out with colored pigments, and the demonstration that will be shown in this presentation utilizes food dyes, the chromatographic process does not depend on color, but instead is based on chemical properties of the components and how they interact with the adsorbent.
This figure shows a few basic aspects of chromatography.
A chromatographic column consists of a tube that contains an adsorbent.
1) A mixture is introduced onto the “column” to begin the separation.
Solvent flows though the tube either by gravity, or with the action of a pump, and the components move through the column at different rates, resulting in separations.
When a component flows out of the bottom of the column, it can be detected, measured, and collected.
Thus, chromatography is used to analyze mixtures by identifying the components, determining the amount of each present, and in some cases, purifying the separate parts.
This figure illustrates the basic components of a liquid chromatograph. Different types of tubing are used for low and high pressure components, and different sized internal diameters is appropriate between various components.
Solvent reservoirs are connected to the liquid chromatography with low pressure, 1/8” polyethylene tubing (lines a,b,c) with either 1/8” Swagelok ferrules, or flangeless fittings, depending on the instrument
Lines indicated in red operate at high pressure. For pressures less than about 7000 psi, PEEK polymeric tubing can be used. For ultra high pressure liquid chromatographs, stainless steel tubing must be used. 0.010” tubing is typically used for these applications, although 0.007” or 0.005” tubing may occasionally be required to achieve low dead volume and highest system efficiency.
The lines indicated in blue are typically low pressure, perhaps 100 psi or less. Tubing inside diameter should be 0.020” or larger to prevent possible blockage that could result in breakage of the detector cell. In some instances, bubble formation in the cell can be reduced by adding a pressure restrictor to the outlet line.
Most current chromatographic systems utilize one or more reciprocating pistons in conjunction with check valves and other components to provide hydraulic potential. Analytical methods may use isocratic conditions with a constant mobile phase composition, or gradient elution conditions with a changing mobile phase composition as a function of time.
The composition of the mobile phase is controlled by proportioned mixing of solvents at the inlet of the pump during the fill stroke of the pump, or by combining the high pressure flows from of two separate pumps at quantified flow rates.
Fluid flow can be made more uniform by the action of pulse dampner. This component acts to store and then release hydraulic pressure during the fill stroke of the pump. When multiple pumps work in combination, flow can be more uniform since the fill and delivery strokes of each pump are independent.
Conventional LC pumps have an upper pressure limit of approximately 6000 psi. This upper limit has been expanded in more recent instrumentation, and is often identified as ultrahigh pressure liquid chromatography or UHPLC.
Other specialized instrumentation is available for use with microbore columns with internal diameter of approximately 1 mm, or capillary columns with even smaller inside diameters. These instruments are optimized to work with the smaller scale components required for these applications.
This slide shows the principle of operation for low pressure mixing chromatographic pumps. The technology is well established for this type of solvent delivery, and this design approach is very common.
High pressure mixing pumps are less common than pumps that proportion mixing at the inlet. It is sometimes argued that the use of dual pumps can help to provide a more even, pulse-free flow profile, and to reduce system volume.
The precision of solvent delivery systems to deliver individual solvents is reduced at low fractional compositions.
If a method requires a composition that is less than about 1 to 2 %, it is better to prepare the mixed solvent manually, by mass.
At fractions less than a percent, the proportioning valve might only be active for a few milliseconds during each fill stroke of the pump, resulting in poor reproducibility.
Check valves are a key component in most pump designs. These devices are used in pairs to direct solvent flow in a single direction. Operation is dependent on the seal made between a small ball and ring. At high pressure, the force on the seal is significant, and the components are typically made of synthetic sapphire or ruby because of the harness of these materials. Foreign particulate matter and deposited solids from buffers reduce the effectiveness of check valves, and the result is poor pump performance. If the check valve can be disassembled, it may be possible to clean the affected parts and restore function by sonication of the parts in an appropriate solvent. Cartridge style check valves are intended for easy replacement, but it may be worth trying cleaning steps with these parts, as well.
Some manufacturers have designed pumps using active inlet valves rather than the passive ball and seat design. Operation is synchronized to the pump piston. During the fill stroke, the valve is opened to permit flow of the solvent into the pump head. The valve is then closed for the compression stroke. This action provides less resistance to the solvent during the fill stroke, and as a result, there is less tendency for cavitation and less need to degas solvents.
Designs have been based on cam-driven and solenoid-actuated valves.
If check valves are allowed to fully dry, as may occur after long periods of non use, the part must primed for proper function. Isopropanol is the best solvent for this purpose, due to its ability to wet surfaces, its compatibility with other solvents, and its relatively high viscosity. To prime the check valves, initially try pumping isopropanol from one of the solvent reservoirs. If this is not effective, isopropanol can be forced into the pump head using a syringe. Be sure to direct the solvent into the inlet check valve of the pump. When all bubbles are cleared from the pump, reassemble and purge the lines with the pump.
The following animation illustrates the function of the pump mechanism. As the piston is retracted during the fill stroke, solvent flows past the inlet check valve ball-and-seat into the pump head. The outlet check valve prevent any backflow of solvent from the column, which is under pressure.
In the compression stroke of the piston, the inlet check valve is sealed and the solvent is forced past the outlet check valve seat into the high pressure side of the apparatus.
At the limit of this stroke, the ball in the outlet check valve again is seated on the ring, isolating the piston cylinder from the high pressure solvent, and the cycle repeats. When the check valves are functioning optimally, pressure in a closed line can be maintained without pump action. The rate of loss in static pressure is symptomatic of problems with the outlet check valve.
As the name indicates, the piston seal provides a seal against the piston surface. This part is a normal wear item that must be replaced periodically, and it is useful to be familiar with this maintenance procedure.
The design of piston seals is interesting. A circular spring separates the two edges of the seal that contact the piston and the pump head cylinder. The spring helps to apply pressure against the piston at low pressure, but it also holds the seal open to permit solvent entry. Solvent under pressure applies force to these surfaces such that the sealing action is improved with increased pressure.
Some piston seals do not have a flange, and for such parts it may be possible to install replacements backwards. In such cases, the mistake is immediately apparent as leaks will readily occur. When a worn seal is removed for replacement, be sure to note the orientation of the spring relative to the piston.
Several examples of injector bodies are shown in these photographs. Injection valves are either used as a stand alone device in combination with a syringe, or as part of an autosampler.
As with pump seals, the rotor seal in an injector is subject to wear, and replacement may be required. Fortunately, such maintenance is needed only infrequently.
The rotor seal in the photograph is highly worn. The scratch between the machined slots will cause leaks or irreproducible injections.
The design and function of an injector should be understood so that modifications to instrumentation can be made, or custom hardware applications designed.
In the fill position, flow from the pump enters and exits the valve through a single circular groove, shown in red. Also in this position, the sample is introduced into the injection loop shown in blue. Any excess sample is directed to waste. Excellent precision results when the loop is completely filled. It is also possible to partially fill the injection loop, and the resulting injection precision depends on the precision of the syringe metering mechanism. Injection precision for autosamplers is usually less than 1% RSD.
When the injector is switched, the flow from the pump is redirected to through the sample loop onto the column. In this position, the sample introduction line is plumbed to waste, and this function is not used.
This slide illustrates autosamplers from two manufacturers. Autosamplers are available with refrigerated sample trays and can accommodate different sized sample vials or well plates.
When using an autosampler after a period of non use, the system should be flushed to remove any lingering bubbles that might cling to the metering syringe. A demonstration of this procedure will be provided later in this presentation as part of startup procedures.
Column properties influence every aspect of the analytical method. Column selection is probably the single most important aspect of method development that will directly affect measurement accuracy and precision. This topic will be discussed in detail in a separate presentation.
Several important column properties can be highlighted.
The stationary phase is produced by chemical modification of the particulate substrate. Numerous types of columns are available that offer different chromatographic selectivity
The substrate provides a high surface area media to support the stationary phase. Physical properties of the particles directly influence column efficiency, and chemical properties influence selectivity.
Column dimensions affect details of the method, and impact method sensitivity and applicability to specific instrumentation
An understanding of physical and chemical properties of LC columns will greatly assist the development and optimization of robust analytical methods.
1Detectors can be grouped by their response characteristics to different compounds. The designations of specific vs universal is somewhat arbitrary, but the intention is to provide some indication of how the detectors can be used. For applications that require the detection of a few compounds in a complex matrix, a selective detector is appropriate; whereas if all compounds must be characterized, as is the case with purity analyses, the detector should have broad response characteristics. Specific detectors depend on differences in functional groups and so such detectors often are sensitive to spectral differences. Universal detectors depend on bulk property differences between the mobile phase and the solute.
A few examples can be provided for specific and universal detectors.
2Specific detectors include…
Examples of universal detectors include…
In general, specific detectors are usually more sensitive than universal detectors. This table provides a few of the more important properties that must be considered when selecting a detector during method development. Absorbance detectors are perhaps the most common general purpose detector, and they can exhibit high specificity and sensitivity. In contrast, refractive index detectors offer broad response characteristics but with significantly lower sensitivity.
In selecting a detector during method development, consider the requirements of the analysis, and balance the needs for sensitivity, selectivity, and mobile phase conditions.
Early in the development of modern liquid chromatography, fixed wavelength absorbance detectors were common. These detectors utilized a mercury vapor lamp to provide a high intensity UV light source, typically in combination with a 254 nm filter.
Variable wavelength absorbance detectors use broadband sources in combination with a monochrometer to provide detection at specific wavelengths. The practical range for these detectors is approximately 200 nm to 700 nm. Depending on the control software used, the detection wavelength can be changed during the analysis to optimize selectivity and sensitivity for different analytes. Absorbance spectra can be obtained by stopped flow scanning of spectra.
Diode array absorbance detectors are variable wavelength detectors that use an array of light sensitive diodes to simultaneously collect the dispersed light from the monochrometer. Continuous, real time absorbance spectra can be collected that can assist in the identification of unknown compounds and the determination of compound purity. Because full spectral information can be saved, chromatograms can be reprocessed and detection optimized after the analyses are completed.
1Fluorescence detectors provide very high selectivity and sensitivity.
2Because relatively few compounds exhibit native fluorescence, if the analytes of interest do fluoresce, selective analyses are possible.
3Non-fluorescent analytes can sometimes be derivatized using fluorescent derivatizing reagents either before or after the chromatographic separation to permit fluorescence detection.
4Time programed fluorescence detection is particularly useful to set individual excitation & emission wavelengths that are optimized for individual analytes. These steps add complexity to the overall analytical method, but may be warranted to achieve measurement goals. Fluorescence detection has a few notable issues.
5Response is influenced by environmental conditions that include temperature, pH, solvent properties, and dissolved oxygen.
6Some compounds may exhibit photo decomposition due to the high intensity of the light source. In practice, these parameters can be adequately controlled; however, the analyst should be aware of the potential issues.
Refractive index detectors offer broad applicability to many analytes, but with limited sensitivity.
1The principle of operation is based on the change in refractive index of the mobile phase as the solute passes through the flow cell.
2Consequently, Refractive index detectors are used with isocratic elution methods, since the refractive index of the mobile phase changes with gradient elution.
3RI detectors are still commonly used for the analysis of polymers, but the low sensitivity of the technique precludes consideration for use in trace level applications or complex mixtures.
4Refractive index is considered a universal detector since responses are observed for nearly every compound
The evaporative light scattering detector, or ELSD for short, is another example of a universal detector.
The principle of operation is based on the formation of solute particles when a nebulized stream of the mobile phase is evaporated in a heated drift tube. These particles scatter a beam of light, which is then detected. Compounds must be less volatile than the mobile phase so that particles result when the solvents are evaporated.
The detector exhibits low sensitivity with a nonlinear response. Unlike refractive index detection, this detector can be used with gradient elution.
A family of detectors can be categorized under the broad description of electrochemical detectors.
1This approach to detection requires that the analytes are electroactive.
2Electrochemical detectors can utilize measurement of resistance,
3voltage,
4current, or
5capacitance of the species.
6Challenges exist in achieving stable and reproducible results, but difficulties in implementation may be offset by gains in selectivity and sensitivity that are commonly observed.
One of the most powerful analytical techniques results from the coupling of liquid chromatography with mass spectrometry. This topic will be discussed in detail in a separate presentation, but a few aspects of the technology can be summarized.
1 The effluent from the column is introduced into a mass spectrometer through an interface that eliminates most of the solvent.
2 Ions created in the source are separated on the basis of their mass to charge ratio, and the abundance of each can be quantified.
3 LCMS exhibits extremely high selectivity, since species are separated in both chromatographic and mass spectrometric domains.
4 Sensitivity of the approach is exceptional, and quantitation of low nanograms mass levels is now routinely achieved.
5 The resolution of a mass spectrometer depends on the type of mass analyzer employed, which commonly is based on quadrupole, time of flight, or ion trap technology.
6 The use of multiple mass filters, such as triple quadrupole mass spectrometry or MS-MS for short, provides even greater selectivity.
Startup procedures for liquid chromatography mostly involve elimination of air bubbles from the fluid pathways, and wetting of seals and check valves. Most flow reproducibility problems can be traced to these sources, or to worn seals. More effort may be required to resolve these issues if an instrument has not be used for an extended period of time. 1 Begin by turning on the power to all modules.
2 Fill the solvent reservoirs and [3] eliminate air bubbles from the supply lines either manually with a syringe, or by running the pump at a high flow rate with the purge value open.
4 If the pump appears to operate normally and provide normal flow, assume that the check valves are operating properly. This can be revisited later if there are indications of flow irreproducibility
5 If applicable, assess if bubbles are present in the autosampler metering syringe. Some autosamplers use a different approach to sampling, so this may not apply. Eliminate bubbles through priming or manual purging.
6 Replace inline filter frits and precolumns as warranted.
7 With the purge valve closed, apply flow at 1 mL/min of methanol or acetonitrile, and monitor the baseline and system pressure. If a stable baseline and constant pressure is achieved, the flow system components are probably functioning OK.
8 Before attempting quantitative measurements, injection reproducibility of the autosampler should be assessed.
Prepare a vial containing methanol or acetonitrile with a drop of acetone. Inject the sample repeatedly, perhaps 10 to 20 times to determine injection reproducibility. The nominal specification for most autosamplers is 1% relative standard deviation, or less.
9 If periodic fluctuations occur in the baseline, the problem may result from a bubble in the detector flow cell. Flush the detector with isopropanol to eliminate bubbles.
1 No special precautions are needed for instrument shutdown.
2 To extend the life of detector lamps, efforts should be made to turn off detectors when they are not being used.
3 Also, if buffers are employed, all flow paths should be flushed with a compatible solvent if the system will not be used for an extended period of time.
The following video illustrates some of the steps involved in the startup of a liquid chromatograph, particularly after an extended period of nonuse. Begin by filling the solvent reservoirs with the appropriate solvents.
If the inlet tubing is fitted with a filter, it may be a good idea to replace the filter, especially if the existing filter shows any signs of discoloration that might indicate contamination with solids.
To clear the solvent lines of air, open the purge valve and activate the pump with the selection of the appropriate inlet. Select a moderately high flow rate of 5 to 10 mL/min, and purge each line separately until all air bubbles are cleared. The purge valve waste line should also indicate the absence of air bubbles.
If the autosampler has an accessible metering syringe, either inject solvent from a sample vial or activate the priming routine from the control software and observe the plunger. No air bubbles should be visible as the plunger is retracted. If bubbles are visible, the software priming routine may act to dislodge the bubbles.
If this is not effective, carefully remove the syringe and fill it with methanol or acetonitrile. Replace the syringe in the autosampler and attach the plunger with the retaining screw. Test the syringe to confirm that the bubble has been cleared.
Solvent lines can also be cleared manually by the use of a syringe fitted with a disposable pipet tip. Trim the pipette tip with a razor blade so that a seal is made with the syringe. To use, simply insert the pipette tip into the end of the solvent line, and withdraw the syringe plunger.
Check valves are integral components for most pump designs. The ball and seat parts are highly susceptible to fouling by particulate matter, and when their operation is impeded, pressure delivered by the pump is erratic. To determine proper function, collect the flow pumped through the column over at least a one minute period and compare this volume with the volume based on the specified flow rate. If the collected volume is significantly less than the flowrate would indicate, then the check valves or pump seals are not fully functioning.
To evaluate check valves for proper function, the flow path can be blocked after the pressure transducer, shown as point A in the figure. Under normal operating conditions the pump will shut off as the pressure limit is exceeded. This pressure should be maintained and only gradually decrease with time, if the check valves are functioning properly. A rapid decrease in pressure indicates likely check valve malfunction.
Under isocratic conditions, system pressure should be constant within a few psi. If wide variations in pressure are evident, particularly timed with pump action, then this also indicates pump problems. When irregular pressure is noted, the cause of the problem should be identified and remedied. Pressure fluctuations invariably result in retention irreproducibility, and poor measurement precision. The likely sources of the problems are the wear points of the check values and piston seals. These should be replaced as needed.
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