How Liquid Chromatography Works & How Excedr Saves You Time & Money

Classically, chromatography uses a polar stationary phase and a non-polar mobile phase to separate molecules. However, in reversed-phase chromatography, the opposite occurs. Also referred to as RPC, this technique employs a non-polar stationary phase and a polar mobile phase.

When the sample’s hydrophobic molecules, seemingly repelled from water, are in the polar mobile phase, they tend to adsorb to the non-polar stationary phase. In contrast, the hydrophilic molecules present, which are attracted to water and tend to be dissolved by water, will be transported along the chromatography column and elute first.

Mixtures of water or aqueous buffers and organic solvents are used in reversed-phase to elute components from a reversed-phase column. Gradient elution, in which the water-solvent composition changes as a function of time, is often used to separate a sample containing a wide range of components. Reversed-phase chromatography is one of the most widely used types of HPLC.

In normal-phase chromatography, the basis for separation is based on the classic principle of liquid chromatography, where the stationary phase is polar, and the mobile phase is non-polar.

Generally, the stationary phase is most commonly made up of silica gel, while the mobile phase is predominantly an alkane (saturated hydrocarbons) or a mixture containing a high proportion of an alkane. Based on these polarities, the least polar compounds elute first, and the most polar compounds elute last. Normal phase chromatography, the oldest mode of liquid chromatography, is very useful for separating water-sensitive compounds, geometric isomers, cis-trans isomers, and chiral compounds.

Ion chromatography, or ion-exchange chromatography (IEX), is a liquid chromatography technique used to separate charged particles from a liquid and measure their concentration. Ion exchange chromatography systems can analyze particles such as anions, cations, organic salts, and proteins. They are typically used in environmental, manufacturing, food, pharmaceutical, and chemical industries.

In ion-exchange chromatography, the stationary phase is made up of a large number of acid groups attached to a polymeric resin, while the mobile phase is an aqueous buffer, such as an inorganic salt dissolved in a suitable solvent. The molecules separate based on their affinity to the ion exchanger, either an anion exchanger or a cation exchanger. Cation exchange chromatography, where a cation exchanger is used, retains positively charged cations. In contrast, anion exchange chromatography, where an anion exchanger is used, retains negatively charged anions.

Ion exchange chromatography is most commonly used to separate ions and polar molecules such as amino acids, large proteins, peptides, or almost any kind of charged biomolecule.

In size-exclusion chromatography, there is no chemical interaction between the sample molecules and the stationary phase. Instead, molecules are separated depending on their size relative to the pore size of the stationary phase. The largest molecules present elute fastest, as smaller molecules – which can permeate the pores – elute more slowly. This technique, also known as gel permeation chromatography (GPC) or gel filtration chromatography (GF), is widely used to separate polymer and proteins.

Modern HPLC systems have been improved to work at much higher pressures, and therefore can use much smaller particle sizes in the columns (<2 μm).

These systems, referred to as ultra-high-performance liquid chromatography (UHPLC) systems, can work at pressures of 120 MPa (17,405 lbf/in 2 ), or about 1200 atmospheres. The term “UPLC” is a trademark of the Waters Corporation but is sometimes used to refer to the more general technique of UHPLC.

In addition to UHPLC, there is another subset of liquid chromatography, referred to as fast protein liquid chromatography (FPLC).

FPLC is generally applied only to proteins; however, it has broad applications because of the wide choice of resins and buffers. In contrast to HPLC, the buffer pressure used is relatively low, typically less than 5 bar, but the flow rate is relatively high, typically 1-5 ml/min. FPLC can be readily scaled from analysis of milligrams of mixtures in columns with a total volume of 5ml or less to industrial production of kilograms of purified protein in columns with volumes of many liters.

Not to be mistaken with FPLC, there are also flash chromatography systems on the market which minimize human involvement in the purification process. These systems are typically referred to as low-pressure liquid chromatography (LPLC) systems and were designed to address the highly time-consuming column chromatography stage, which oftentimes bottlenecks the productivity of process labs.

Liquid chromatography pumps vary in pressure capacity. Their performance is measured on their ability to yield a consistent and reproducible flow rate. Pressure may reach as high as 60 MPa (6000 lbf/in 2 ), or about 600 atmospheres. In HPLC, the pump is also often referred to as a solvent delivery system. It provides a controlled, precise flow of the mobile phase and eluent to the column.

HPLC pumps can deliver relatively pulseless flows against significant backpressure. Their flow rates are reproducible even when the backpressure changes. Many HPLC pumps are available as standalone models that are easy to operate. It is also possible to generate gradient elutions. This is useful for HPLC as well as other applications.

Some of the critical components of an HPLC pump include the piston, driver, pump head, high-pressure pump unit, check valve, proportioning valve, mixer, pulse damper, and degasser. Many pumps available in LC systems today include:

• Constant pressure pumps
• Constant flow pumps
• Reciprocating pumps
• Syringe pumps
• Dual-piston pumps
• High-pressure/low-pressure mixing pumps

The system and pump most suited to your laboratory’s needs will depend upon several factors, most importantly your flow rate requirements.

The injector can be a single injection or an automated injection system. An injector for an HPLC system should provide injection of the liquid sample, or analyte, within the range of 0.1-100 mL of volume with high reproducibility under high pressure.

Some sample injectors available with modern HPLC systems include injection valves, sample pumps, and autosamplers. Precision, accuracy, and carryover are typically the most important factors to consider when deciding which sample injector best fits your laboratory’s needs.

Furthermore, while manual injection valves are the most cost-effective option to introduce samples, automated injection methods are recommended when high throughput is needed.

Deciding which HPLC column to use for a separation depends on the physical properties of the analyte being separated. These properties generally include any of the following:

• Hydrophobicity/hydrophilicity
• Intermolecular forces (particularly dipole-dipole)
• Intramolecular forces (ionic)
• Size

HPLC column separations are also used to make use of specific differences in the molecular properties of the target molecules. Generally, the structure and chemistry of the HPLC column packing—the stationary phase—determines the analyte elution profile.

Column sizes range from capillary to process scale. The internal diameter and volume of a column determine what volume of sample can be loaded onto a column as well as the sensitivity of separation. Moreover, the column’s internal diameter can affect the separation profile, mainly when using gradient elution, while smaller internal diameters yield increased separation and detection sensitivity. For example, in capillary HPLC, the use of small capillaries facilitates taller peaks on the chromatograph. These taller peaks provide better detection limits for mass spectrometry and other concentration-sensitive detectors.

There is often a trade-off between sensitivity and the sample volume loaded onto a column where analytical separations are concerned.

While traditional columns are made of glass, newer HPLC columns are mostly made up of borosilicate glass (a type of glass mainly made from silica and boron trioxide), acrylic glass, or stainless steel. These columns can also be categorized by the separation mechanism. The columns available include, but are not limited to:

• Ion exchange columns
• Ion exclusion columns
• Size exclusion columns
• Reversed-phase columns
• Normal phase columns

For each column type, the chromatography packing material varies. Additionally, column thermostats are usually included in the LC system for applications that require close control of column temperature. The other advantage of using a thermostat is that more reproducible chromatograms are usually obtained by maintaining column temperature. While organic-based chromatography usually involves column heating, the opposite is true for separating proteins.

HPLC detectors are used to detect components of a sample mixture being eluted from the chromatography column. Furthermore, the detector collects the separated compounds and monitors them, producing an electronic signal that identifies the compounds the detector is designed to respond to.

Each detector type varies in detection method and produces variable results for purity screening. There are many detectors, with multiple ways of categorizing each kind. They typically include:

• UV/Vis detectors: A non-destructive detection method, UV/Vis detectors work on either a fixed or variable wavelength. This means the UV absorption of the effluent is continuously measured at single or multiple wavelengths. These devices are, in general, the most popular detectors for use in liquid chromatography. They are considered absorbance detectors since their design measures the amount of ultraviolet or visible light that a component present in the eluent absorbs.
• Photodiode array (PDA) detectors: Considered another type of UV/Vis detection, a PDA detector can detect an entire spectrum simultaneously.
• Refractive index detectors: This detector can also be categorized as non-destructive and continuously measures the refractive index of the effluent. It has the lowest sensitivity of all detectors; however, it offers excellent stability. Most devices are designed to feature a temperature control structure and an improved thermal design, providing better baseline stability and a shorter initial stabilization time.
• Fluorescence detectors: This detector utilizes the principle of fluorescence spectroscopy and irradiates effluent with a light of set wavelength, measuring the fluorescence of the effluent using single or multiple wavelengths. It’s relatively easy to use and offers sufficient stability.
• Evaporative light scattering (ELSD) detectors: A destructive detection method, ELSD is used for its ability to provide sensitive readings of non-volatile analytes. Using a nebulizer, this detector continuously evaporates the effluent and measures the light scattering of the aerosol created. It is considered a universal detector used for gradient analyses of compounds that cannot be analyzed using an absorbance detector.
• Conductivity detectors: When ionic compounds in a solution are being separated and analyzed, a conductivity detector is used because any solution containing ionic components will conduct electricity. These devices measure electronic resistance, and that measured value is directly proportional to the concentration of ions present in the solution. For this reason, conductivity detectors are generally used in ion chromatography (also referred to as ion-exchange chromatography).
• Mass spectrometers: When mass spectrometers are integrated with LC systems, it’s referred to as LC/MS. Mass spectrometers work by analyzing the molecular weight of the compounds present, which is equal to their mass-to-charge ratio (m/z).

Chromatographic analysis, whether it’s HPLC, GC, or IC, requires the use of a chromatography data system (CDS). The CDS plays a pivotal role in instrument control, data processing, archiving, and report generation.

In general, the data handling and instrument control system, in conjunction with its accompanying software, can be set up for use in three ways:

• As a standalone system that controls two or more chromatographs
• As a standalone system that maintains a single chromatograph, including LC/MS instruments
• As a networked system that controls multiple instruments in one or more labs

As for HPLC system prices, simple setups with a basic detector, injector, and pump are relatively affordable. This is especially the case if you opt for HPLC over UHPLC. However, the cost rises significantly if you increase the number of detection modules, add an autosampler, and upgrade to a high-pressure pump.

While purchasing a new or refurbished system outright may not be in your budget, you can lease the equipment at an affordable rate. Our leases are structured to be customizable to your laboratory’s needs and offer an excellent alternative to purchasing using either your capital, a bank loan, or a line of credit.

Furthermore, by leasing an HPLC system, you can significantly reduce the upfront costs of the machinery and simplify cash flow management through affordable, predictable payments.