22 High pressure liquid chromatography

Dr. Varinder Kaur

Objectives: To study the basics of high performance liquid chromatography and know the following questions.

What is high performance liquid chromatography?

What are the main components of HPLC?

How is it better over GC?

What are the main sample preparation methods used with HPLC?

1. Description

High performance liquid chromatography is advanced version of column chromatography. The standard column chromatography was inefficient due to slow separation, gravity dependent flow of solvent and manual operation. Therefore, on the basis of exhaustive efforts of various scientists, HPLC hardware and instrumentation developed in 1970s. In this technique, the mobile phase was operated under high pressures of up to 400 atmospheres with the help of pumps instead of dripping through a column under gravity and sample is handled by injection valves. This development solved many problems associated with the classical liquid chromatographic techniques as well as gas chromatography.

Principle

The basic principle of HPLC is same as column chromatography i.e. separation of components between stationary and mobile phase on the basis of their relative affinities for the mobile phase and the stationary phase.

2. Types of HPLC

The HPLC is divided into following categories depending upon the phase system (stationary) used. The normal phase and reverse phase chromatography are discussed below, however, others are discussed in respective modules.

2.1 Normal phase HPLC

In normal phase HPLC, separation occurs due to the distribution of components in a non-polar mobile phase and polar stationary phase. The polar components interact with the polar stationary phase and elute slowly with the non-polar mobile phase whereas less polar components come out very rapidly. The stationary phase is usually silica and solvents used for mobile phase are generally hexane, methylene chloride, chloroform, diethyl ether, and mixtures of these.

2.2 Reverse Phase HPLC (RP-HPLC)

In reverse phase HPLC, a polar mobile phase and nonpolar (hydrophobic) stationary phase is selected for the separation of components. The less polar components are retained on the stationary phase while polar components elute with the mobile phase. For RP-HPLC, mixture of polar solvents like water-methanol, water-acetonitrile, methanol-acetonitrile etc are used for the separation of components.

3. Instrumentation

The instrumentation of HPLC system involves various components like solvent reservoir, pumps, sample injection system, column and detector (Fig. 1). Each component is discussed below.

3.1 Solvent reservoir

The reservoir is the part where solvent is stored for the system. It consists of an inert container with inert lines and reservoir filters. The containers are generally air tight glass containers with outlets for the delivery of solvent. The lines are the inert tubings used to deliver the solvent from the container to the pump. At the end of the tubing, reservoir filters are fixed to remove the impurities or gas bubbles if any. The solvent in the reservoir is filtered and degassed prior to use either by filtration or ultrasonication. In most of the systems, different containers are used for different solvents and the composition of mobile phase is monitored by the software as per the requirement.

3.2 Pump

A pump is used to deliver the mobile phase from solvent resorvoir to the whole system. The systems usually consist of two pumps, which work at a high pressure (up to 42000 kPa or 6000 psi) to push the mobile phase via column to detector. The material used for the construction of pump is usually inert. Nowadays quality of pumps is improved and a pump can deliver high volumes of mobile phase at precise and accurate pulse free flow. Depending upon the composition of the solvent during a run, solvent system is termed as isocratic and gradient system. In isocratic system, a single solvent or solvent mixture of constant composition is employed for the separation of a sample into its components. In gradient elution, mixture of two or more solvents is employed and the composition is monitored in between the run. The variation of solvent composition can be varied by programming continuously or in a series of steps. This enhances the efficiency of the separation.

3.3 Sample Injector or injection system

The sample injector is used to introduce the sample under investigation into the HPLC system. The sample injection system consists of a Rheodyne sampler, which is easy to use, reliable, capable of reproducing injection volumes, and can have variable loop size. It consists of two modes; 1) Load, 2) inject. In the ‘load’ mode solvent delivered by the pump flows directly to the column and then detector and does not cross through the injection port. In this way the sample injected into the valve is filled in the sample loop as per its capacity and extra sample goes to the drain. In ‘inject’ mode, the solvent delivered by the pump passes through the sample loop before entering the column and then delivered to the detector. This delivers the sample form the sample loop into the column where they get separated.

In the manual sample injection, sample is injected with the help of syringes by the operator. However, in automated sample injection, samples are injected automatically as per the programming commands given to the software. In this case, injections are continuous, operator free, precise, accurate and can run upto 100 samples continuously. The HPLC systems with automated sample injection are expensive as compared to systems with manual sampling.

3.4 Column

The columns are categorized into analytical and guard columns on the basis of their role; analytical and guard column. Analytical Columns is the most important component in HPLC systems because separation of components occurs in this part. The columns are usually made up of polished stainless steel (sometimes, heavy walled glass tubing), with dimensions between 50-300 mm (length) and 2-5 mm   (internal diameter; Microbore columns consists of internal diameter less than 2 mm). These are filled with stationary phase with particle size of 3–10 µm. In some cases, columns are placed in a chamber to control the temperature of the column. Guard Column is also important component. It increases the life of the analytical column because it is fixed in front of the column and used to remove impurities (if any) delivered with the mobile phase. It also helps to saturate the mobile phase with the stationary phase that solvent loss from the analytical column is minimized. The nature of guard-column packing is usually similar to the analytical column, however particle size is larger. This column may be discarded after the contamination and replaced with a new guard column.

3.5 Detector

A detector is used to analyze the mobile phase coming out of the column for the presence of components. It is located at the end of the column and connected via inert tubing for the entry of solvent with components eluted from the column. In HPLC, various detectors are used depending upon the physical property of the sample components. Some of the common detectors used with HPLC are summarized below.

3.5.1 UV-Vis spectrophotometric detectors

It detects the components of a sample by measuring the ability of a component to absorb UV or visible light of particular wavelength. When the mobile phase with eluted component is exposed to the light of certain wavelength, the component absorbs the light and hence, the intensity of the light coming out of the sample decreases. This change in the intensity of the light is measured in terms of the absorbance. Therefore, the separation of the components is observed as sharp peaks in the chromatogram depending upon the retention time of the components. The UV-Visible detectors used with HPLC are further of three types; 1) Fixed wavelength, 2) Variable wavelength, and 3) Diode array detector. In fixed wavelength UV-Vis detector, the components are analyzed only on a fixed wavelength (usually 254 nm). In variable wavelength detectors, absorbance is monitored at a wavelength by choosing from range of wavelengths i.e. 190-900 nm. However, in photodiode array, different wavelengths can be monitors simultaneously according to the physical property of the sample components.

3.5.2 Fluorescence detector

This detector is used to detect the components which are fluorescent in nature. In this case, principle is same as the fluorometers and measure the light emitted by the components upon excitation. This detector has limited use and its sensitivity depends upon the fluorescence properties of the components.

3.5.3 Refractive index detector

It measures the change in the overall of ability of mobile phase to deflect light in a flow cell. The refractive index of mobile phase is considered as reference and the relative change in the property is observed when a component is present in the mobile phase. The amount of deflection is proportional to the concentration of the component.

3.5.4 Electrochemical detector

It measures the current resulted from the oxidation/reduction of the components at a suitable electrode. The detector operates on the principles of amperometry, polarography, coulometry, and conductometry.

4. Data Collection Devices

The data collection devices are used to convert the signals from the detector with the help of electronic integrators, which process, store and reprocess the chromatographic data. These devices deliver the data in terms of chromatograms, which is easy to read and interpret.

5. Sample preparation in HPLC

The sample preparation involves treatment of the real/field samples before analysis. The method of sample preparation varies according to the nature of the sample. In case of solids, the sample must be converted to its soluble/liquid form before the introduction of sample into injection port of HPLC. If the sample is soluble in a solvent, then, it can be injected directly into the system however the analytes must be extracted from the sample in case matrix is insoluble in an appropriate solvent. Some of the common extraction techniques used for the sample preparation of solids and liquids are summarized below.

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