High Performance Liquid Chromatography

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3.1.1: Introduction:

Liquid chromatography is a physical separation technique conducted in liquid phase. A sample is separated into its constituent (or analytes) by distributing it between the stationary phase and mobile phase. Mobile phase can be organic solvent like hexane and stationary phase can be porous silica particle packed in column. High Performance Liquid Chromatography (HPLC) is an advance form of liquid chromatography which uses small column through which the mobile phase is pumped at high pressure.[1]

3.1.2: Theory/Principle:

3.1.3: Mobile Phase:

Most commonly used mobile phase for HPLC analysis are petroleum ether, cyclohexane, toluene, benzene, esters, chloroform, ethyl ether, methyl ethyl ketone, alcohols, water, organic acids. These mobile phases along with their polarity and retention time are shown in table below:[2]

Table 1: Solvents used as mobile phase in HPLC

Low Polarity

High polarity


Petroleum ether

Carbon tetrachloride






Ethyl ether


Methyl ethyl ketone



Organic acids

Long Retention Time

Short Retention Time

Normally, the mobile phase consist of mixture of polar and non- polar solvents such as water and ethanol.[3]

In gradient elution HPLC, the polarity of the mobile phase is continuously changed by changing the ratio of the solvents in the mobile phase. It is most useful way to control the retention volume or retention time of the sample component.[2]

Mobile phase must be chosen that would not interfere with the measurements by the detector. For example, in case of ultraviolet absorption detector, the solvent cannot absorb ultraviolet radiations. Mixture of methanol, ethanol and propanol with heptanes and chloroform with heptanes are popular choices as HPLC mobile phases. Mixture of methanol with water is used in reverse phase HPLC as mobile phase.[2]

Solvent Strength and Selectivity:

Solvent strength is the ability of the solvent to elute the solute from a column. The strength of the solvent is depending on its polarity. In case of normal phase liquid chromatography, non- polar solvent like hexane is weak solvent while polar solvent like water is strong solvent and in reverse phase chromatography, non- polar solvent like organic solvents are strong solvents and polar solvents like water is a weak solvent.[1]

3.1.4: Stationary Phase:

The stationary phase is a liquid film coated on a packing material consisting of 3 - 10µm porous silica particles in liquid chromatography.[4]

Some of the stationary phases used in HPLC are listed in table below: [2]

Table 2: Stationary phases used in HPLC

High polarity

Low polarity


Magnesium oxide


Silica gel

Calcium oxide

Magnesium carbonate

Potassium carbonate

Sodium carbonate



High adsorption

Least adsorption

Mostly used solid adsorbent is silica gel. The SiOH groups on silica gel make the gel weakly acidic and attract basic compounds. Generally silica gel attracts bases in order of their strength; i.e; strong bases are retained on silica gel columns longer than weak bases. Silica gel is used as packing material in the form of pure particles and as a pellicle on the solid support. A pellicle is a thin layer of coating on a surface. The pellicles used in HPLC are chemically bonded to the surface of the support material. HPLC pellicular materials are larger and more easily packed into the column than the micro particulate pure adsorbents and the resulting columns are more efficient.[2]

Alumina is another solid adsorbent which is widely used in column packing material. Alumina is basic, it retains acidic compounds. Alumina is mostly used in pellicular form.[2]

3.1.5: Instrumentation:

The basic components of HPLC are:

Solvent/ mobile supply system

Sample injection system



Recording system

a: Solvent/Mobile phase supply system:

The basic function of solvent supply system is to deliver reproducible, precise and constant flow of mobile phase.[8] the solvent supply system have high pressure pumps and provide gradient elution. Solvent reservoirs are filled with solvents of different polarities, which are miscible. The solvents used are must be pure and degassed.[7]

b: Sample injection system:

Sampling loop is widely used for the introduction of sample in liquid chromatography.[4] sampling loops are easily changed. Loops having volume ranging from 0.5 µL to 2mL are available.[6]

c: Columns:

The high performance liquid chromatography has two types of columns.

An analytical column which is responsible for separation

Guard column which protect the analytical column from contamination

i: Analytical column:

Analytical columns used in HPLC are constructed from stainless steel material. There length ranging from 30 mm to 300 mm and diameter between 2.1 mm to 4.6 mm. porous silica particles are used to pack the column with the thickness of 3 - 10 µm. The efficiencies of these columns are 40,000 - 60,000 theoretical plates/m.[4]

ii: Guard column:[4]

Analytical columns have face two types of problems which decrease their life time:

Solutes binding to the stationary phase irreversibly which degrade the column efficiency by decreasing the available stationary phase

With the sample, particulate material is injected which clog the analytical column

Before the analytical column, guard column is placed to overcome these problems.

Guard columns are less expensive; however, these are packed with the same packing material and stationary phase as analytical column. Guard columns are replaced regularly because they are intended to be sacrificial.

d: Detectors:

The detectors used in HPLC must have low dead volume to minimize extra column band broadening. The detector should be small and suitable with liquid flow. The detector used in HPLC depend on the nature of the sample.[4] [6]

The most widely used detectors in HPLC are: [4]

Spectroscopic detector

Fluorescence detector

Refractive index detector

Mass spectrometer detector

i: Spectroscopic detector:

The most widely used HPLC detectors are based on ultraviolet - visible absorption and fluorescence.[4]

The sensitivity of ultraviolet - visible (UV) detector is about 10-8 g/ml (0.01 ppm). UV detectors are not temperature sensitive. They are less expensive and can be used with gradient elution.[7]

The most of the UV detectors have a simple interference filter which measures the absorbance at few selected wavelengths. The expensive UV detectors have monochromators that allows absorbance at particular wavelength.[6]

ii: Fluorescence detectors:

Fluorescence detectors are more sensitive as compared to UV detectors. They give improved selectivity.[7]

iii: Refractive Index detectors:

Refractive index detectors are useful for almost all types of compounds but it has poor detection limit. For gradient elution RI detectors are not useful. They are useful only when mobile phase components have same refractive indexes.[4] they are less sensitive as compared to UV detectors.[8]

iv: Mass Spectrometer detector:

Among all detectors mass spectrometric detectors are most useful detectors. It also give structural information to identify the analyte.[4]

e: Recording system:

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3.1.6: Applications:

Purity of Samples:

Purity of samples can be determined by high performance liquid chromatography.[9]

Analysis of drugs:

HPLC is used for the analysis of drugs in biological fluids[9].

Analysis of Polyaromatic Hydrocarbons:

The concentration of polyaromatic hydrocarbons by using HPLC.[4]

Identification of unknown compounds:

Unknown compounds can be identified by comparing them with standards using their retention times.[9]

Analysis of Biological Compounds:

HPLC is very important and useful technique for biotechnology and pharmaceutical industries. The variety of biological compounds like DNA, carbohydrates, proteins, lipids and amino acids are analyzed by HPLC.[8]


3.2: Gas Chromatography and Mass Spectrometry: (GC-MS)


In gas chromatography and mass spectrometric technique (GC-MS) the components of mixture are separated by the GC and these components are identified at molecular level by MS.[7]

3.2.1: Principle:

In gas chromatography, when sample mixture is heated it separates into individual components and converted into vapour states. These vapours are moved through column along with carrier gas such as helium. The separated components emerge from the column and enter into the ionization chamber of mass spectrometer.[7]

In mass spectrometer, molecules are ionized by using beam of highly energetic electrons. The ionization of molecules depends upon the nature of the bonds in the molecules. The molecular ions are broken up into many fragments. There are particular mass to charge value for every ion. In most cases, ions have only one charge so that m/e value is the molecular mass of the ion.[10] [11]

3.2.2: Mobile Phase:

Mobile phase or carrier gas is the important component of gas chromatography. The gas used in GC should be inert toward stationary and mobile phase. Most commonly used carrier gases are Helium, Argon and nitrogen. The purpose of the carrier gas is to carry the sample through the system. Flow rate of the mobile is measured with the help of flow meter placed at the outlet of column.[4] [8]

3.2.3: Stationary Phase:

The stationary phases used in GC should have following characteristics:[4]

It should be chemically inert

It should be thermally stable

It should be of low volatility

The most commonly used stationary phases are squalane, polydimethyl siloxane, 50% methyl - 50 % phenyl polysiloxane, 50% cyanopropyl - 50% phenyl methyl polysiloxane, polyethylene glycol.[4]

3.2.4: Instrumentation:

The instrument used for GC - MS consists of following basic components:

Gas chromatograph:

Inlet system



Mass spectrometer

d: Ion source

e: Mass analyzer (filter)

f: Detector

g: Read out system

Injection port or Inlet system:

The function of the inlet system is to introduce the sample into the column. When the sample is introduced it is vaporized and enters into the column along with the carrier gas. The temperature of heated gas is maintained above the boiling point of highest boiling component.[3] [8]


In order to control the column temperature, column is placed inside the thermostated over.[4]

Oven is operated on two types of analysis:[8]

Isothermal analysis.

Temperature programming.

i: Isothermal analysis:

Isothermal analysis is used when constant temperature throughout the analysis is required i.e. there is 100°C or less difference in the boiling point of low and high boiling components.[8]

ii: Temperature programming:

When the components of a mixture have wide range of boiling point then temperature programming occurs throughout the analysis. It gives better separation.[8]


The gas chromatographic columns are of many types such as U - shaped columns and coiled tube columns.[7]

There are two types of columns used in GC:[7]

Packed columns

Capillary columns

i: Packed columns:

Packed columns are made of stainless steel, glass, aluminium or copper. The length of packed column is 2 - 6 meters. Internal diameter is 2 - 4 mm.[4] The stationary phase is packed in a column.[3] the solid support used in GC columns is diatomaceous earth. It consist of hydrated silica gel.[4]

ii: Capillary columns:

Capillary columns are open tubular columns. Its length is about 100 meters and internal diameter is about 0.1 - 0.5 mm. A very thin film of liquid stationary phase is coated on a metal or glass walls of the tube. [7] Capillary column gives high separation efficiency.[4] Capillary columns gives better resolution as compared to packed column.[3]


The molecules form GC column enter into ion source of MS and these sample molecules are ionized. Ionization potential is the minimum energy required to ionize the sample molecules.[10]

Electron Ionization

Chemical Ionization

i: Electron Ionization:

Electron ionization is the most common ionization technique used in MS. Molecules after entering into MS bombarded with highly energetic electrons emitted from the hot filament. After bombardment, fragments of molecules are produced. A very low pressure is required for electron ionization process.[6] [8]

ii: Chemical Ionization:

In chemical ionization, reagent gas like methane is used. The reagent gas interacts with the sample molecule and produce new ions. The reagent gas methane transfer the charge to the sample molecule.[12]

Fragment ions produced in chemical ionization are much less than electron ionization because of this reason chemical ionization is called "soft ionization technique".[13]

e: Mass Analyzer:

The ions from the ionization chamber enter into mass analyzer. The function of mass analyzers is to resolve the different mass fragments.[6] [10]

Quadrupole mass analyzer:

Quadrupole mass analyzer is the most efficient mass analyzer used in MS. It analyzed the analytes in less than 100 ns time scales. Du to this reason, quadrupole mass analyzer detectors is used in GC - MS coupled technique.[14]

The quadruple mass analyzer consists of four metal electrodes. Each electrode is connected to its opposite electrode electrically. These electrodes are 10 - 15 cm long and 5 - 6 mm in diameter. When these electrodes are polarized, ions of particular mass to charge ratio are allowed to pass through the space between electrodes.[14]

f: Detectors:

g: Read out system