Liquid Chromatography

The defining feature of liquid chromatography is a liquid mobile phase. The stationary phases are typically liquids immobilized on solids, either through physical adsorption or chemical bonding. Chromatography performed with two liquid phases is known as partition chromatography. In partition chromatography, interactions between the analytes and both the mobile and stationary phases play important roles in determining the effectiveness of a separation, and the elution order. The mobile phase composition can be adjusted to produce the desired results.

Normal-phase and reversed-phase chromatography

Partition chromatography is divided into two types, based on the relative polarities of the mobile and stationary phases. Early versions of partition chromatography featured highly polar stationary phases and relatively non-polar mobile phases, and so this type of chromatography is referred to as normal-phase. Reversed-phase is when the stationary phase is non-polar and the mobile phase is relatively polar. For normal-phase separations, the least polar analytes elute first, and elution times can be decreased by increasing the mobile phase polarity. For reversed-phase separations, the most polar analytes elute first, and elution times can be decreased by decreasing the mobile phase polarity. The thyme leaf analysis employs a reversed-phase separation.

High-Performance Liquid Chromatography

High-performance liquid chromatography (HPLC) utilizes stationary phases with very small packing particles (≤ 10 µm) to achieve highly efficient and well resolved separations. Moving the mobile phase through these densely packed, small particles at a sufficient rate requires columns and instruments that can operate at high pressures. HPLC is sometimes referred to as high-pressure liquid chromatography.

HPLC Instrumentation

The first reliable commercially available instruments for HPLC were developed in the 1970s. Since its inception, HPLC has evolved into a highly sophisticated instrumental technique. HPLC instruments are currently used across a variety of chemical and biological research disciplines for analytical and preparative purposes. Modern HPLC instruments typically share the following features:

• Automated solvent handling and proportioning - A programmable multi-channel pump controls the flow rate and composition of mobile phase solvent.

• Automated sample injection systems - Programmable robots handle and inject tens to hundreds of samples for automated analysis.

• Capillary tubing - The instrument is plumbed with narrow tubing that minimizes the volume of the system and prevents turbulent flow.

• Temperature controlled column compartment - The column is stored in insulated compartment that can be held at a constant temperature to ensure analysis takes place under consistent conditions.

• Packed columns - Columns for liquid chromatography are typically filled with particles that are coated with the stationary phase.

• Electronic detector - A device sensitive to the presence of analytes eluting off the column generates an electronic signal.

• Computer and software control - The instrument is controlled by a computer through a software graphic user interface. Data from the instrument is digitized and saved on the computer for data analysis and reporting.

Mobile Phase Pumping System

The requirements for HPLC pumps are demanding. They must be able to generate relatively pulse-free outputs with pressures up to several thousand pounds per square inch. They must also operate reliably and reproducibly over a broad range of flow rates (0.1 to 10 mL/min) and they must be compatible with a variety of solvents. They also typically handle a very heavy workload, running 10 hours a day or more when used for high-throughput analysis. These pumps also commonly feature on-board filtering and degassing apparatus, to remove particles and dissolved gasses from mobile phase solvents.

Reciprocating pumps

Most HPLC systems are equipped with reciprocating pumps. These pumps use the back-and-forth motion of motor driven pistons to move the solvent through the system. One-way check valves ensure that solvent flows in one direction through the pump. By their nature, reciprocating pumps produce a pulsed flow, which will cause baseline noise if not properly dampened. They offer several advantages, however, including adaptability to gradient elutions, high output pressures (up to 10,000 psi), constant flow rates, and small internal volumes (< 500 µL). While these pumps are capable of generating very high output pressures, it should be noted that every column has a pressure limit that should not be exceeded.

Flow control and Programming

Many HPLC systems feature multi-channel, programmable pumps that are capable of adjusting the mobile phase composition continuously throughout the course of a separation. The instruments used for the thymol separation are equipped with 4-channel pumps (quaternary pump) that allow 4 different solvents, or solutions, to be mixed on-board the instrument in any proportion. This mixing is performed by a proportioning valve that rapidly switches between the solvent channels at intervals varied to achieve the specified solvent composition.

Automated Sample Injection System

Most modern HPLC instruments are equipped with an autosampler/injector module that automates the sample introduction process. The auto-sampler robot injects liquid samples into the instrument for analysis. Trays on the autosampler can typically store around 50 to 100 samples that can be run on the instrument in any sequence and analyzed with any parameters the user desires to program. Samples are typically stored in standardized autosampler vials capped with pierce-able septa (rubber top).

To run a sample, the robot moves the vial to the injector, which removes the specified amount of sample through the septum using a needle. The sample is sucked into a loop of capillary tubing attached to a valve. The valve position can change to insert the sampling loop between the HPLC pump and the column, allowing mobile phase to sweep the sample onto the column. Once the sample has been injected, the robot retrieves the vial and returns it to its position in the storage tray.

HPLC Columns

HPLC columns are typically constructed of stainless-steel tubing that can withstand the high pressures applied by the instrument. These tubes are packed with the stationary phase and sealed on the ends with frits to hold the packing in, and connectors to interface the column with the instrument tubing. Typical HPLC columns are around 10 to 30 cm long, with internal diameters of around 4 to 10 mm. The size of the stationary phase packing particles is commonly 3 to 10 µm. However, columns with dimensions outside of these ranges, both smaller and larger, are available for specialized applications.

HPLC stationary phase

Stationary phases for partition chromatography generally consist of liquids chemically bonded to solid support structures. For reversed-phase HPLC the stationary phase is usually a siloxane with a hydrocarbon chain, typically 8 carbons (C8) or 18 carbons (C18) long. The support structure is typically porous silica, to which the siloxane is chemically bonded.

Guard columns

It is typical to place a short, cheap, sacrificial column with the same stationary phase before the analytical column. This lengthens the life of the analytical column by filtering and capturing any molecules that might get permanently retained in the column.

Temperature Controlled Column Compartment

More consistent separations and retention times can be obtained by precisely controlling the column temperature. Running the column at elevated temperatures has the added advantages of decreasing solvent viscosities, resulting in lower pumping pressures, and increasing analyte solubility. It is generally a good practice to hold the column several degrees above typical ambient temperatures (around 30 º C) when a thermostatted compartment is available. However, one should not exceed the maximum operating temperature of the column. While temperature gradients are commonplace in gas chromatography, long equilibration times make them impractical for liquid chromatography, and they are seldom, if ever, used.

Detectors

Detectors for liquid chromatography either respond to a bulk property of the mobile phase that changes in the presence of dissolved analyte (e.g., refractive index and density), or they respond to a property of the analyte not shared by the mobile phase (e.g., absorbance or fluorescence).

Diode Array Detector

For the thyme extract analysis, you will use an absorbance detector that will respond to the UV absorbance from thymol and other extract components. This system measures the absorbance by flowing the mobile phase through an optical cell after it exits the column. A UVvis light source is shone through the cell and is dispersed onto a diode array detector for absorbance measurements. As absorbing analytes elute from the column, the measured absorbance increases with the concentration of analyte present. Thymol and carvacrol both have absorbance peaks in the UV near 275 nm and can be detected by monitoring this wavelength.