High-Performance Liquid Chromatography (HPLC)

  HPLC : Introduction

High performance liquid chromatography (HPLC) is basically a highly improved form of column liquid chromatography. Instead of a solvent being allowed to drip through a column under gravity, it is forced through under high pressures of up to 400 atmospheres.

That makes it much faster. All chromatographic separations, including HPLC operate under the same basic principle; separation of a sample into its constituent parts because of the difference in the relative affinities of different molecules for the mobile phase and the stationary phase used in the separation.

  HPLC : Introduction

GC was discussed in details in the previous tutorial; thus comparing GC with HPLC will clear the understanding about both the chromatography.

High performance liquid chromatography (HPLC) is basically a highly improved form of column liquid chromatography. Instead of a solvent being allowed to drip through a column under gravity, it is forced through under high pressures of up to 400 atmospheres.

That makes it much faster. All chromatographic separations, including HPLC operate under the same basic principle; separation of a sample into its constituent parts because of the difference in the relative affinities of different molecules for the mobile phase and the stationary phase used in the separation.

  HPLC versus GC


The field of HPLC applications overlaps a large section of gas phase chromatography, to which can be added the analyses of many compounds that are thermolabile, or very polar or of high molecular weight.

Using gas as its mobile phase, can analyze compounds which can be “dissolved” in gas. Therefore, only volatile and semi-volatile compounds (generally small molecules) can be analyzed by GC. Derivatization will be helpful to turn non-volatile compounds into volatile ones to be analyze using GC, but it does not always work

Using liquid as mobile phase, can analyze compounds which can be dissolved in liquid. Volatility is not that important in HPLC. Immediately, we see HPLC can be used on much more analytes than GC, because there are a lot of non-volatile compounds can be dissolved (at least a little bit) in water, methanol, acetonitrile, acetone, or hexane, etc. Therefore, currently, HPLC is used more often than GC.


GC typically uses very long columns (commonly 10-30 meter). So it does give better separation result compared with HPLC which use much shorter columns (commonly 5-25 cm).

Operational Temperature

HPLC separations are mostly carried out at ambient temperatures whereas Gas Chromatography separations are carried out at elevated temperatures which can be held at a constant value (isothermal) or variable as decided by the temperature program


Gas chromatography separations are mainly carried out on compounds ranging in molecular weights up to a few hundreds. Such compounds separate on differences in their volatilities and remain stable at high temperatures.

HPLC separation compounds have higher molecular weights ranging from a few hundreds to several millions for large polymers and biomolecules. Such compounds can be analysed at room temperature only because at elevated temperatures they tend to degrade.

Mobile Phase Detection

Liquids used as carrier in HPLC generally have higher viscosity in comparison to gases used in Gas Chromatography. This results in increased column back pressures in HPLC. It is for this reason that HPLC columns are much shorter and have wider diameters in comparison to GC columns which can be much longer and narrower.  Increased column length improves resolution between closely spaced peaks. As the trend is towards faster analysis columns used for HPLC are as short as 1 cm in length.


HPLC detection is commonly based on nondestructive detection such as UV, RI, photodiode array detectors, conductivity and laser detection. On the other hand Gas Chromatography detection is based largely on destructive principles such as a FID, NPD and FPD. Mass spectrometry detectors common to both LC and GC are destructive in nature.


HPLC solvents are costly in comparison to gases used for GC analysis. In addition maintenance cost is also higher due to high pressures developed in pumps and columns

  Stationary and Mobile Phase of HPLC

Stationary phase

For the majority of applications silica gel still represents the basic material used to pack HPLC columns, spherical particles, sometimes porous, with a diameter of between 2 to 5µm assures a compact and homogeneous packing of the column

Common silica gels used in HPLC contain around five Silanol groups Si-OH per m2.

The mode of action of silica gel is adsorption,  phenomenon that leads to the accumulation of a compound at the interface between the stationary and mobile phases

Bonded Silica Gel : Silica gel is very polar in nature, to reduce excessive gel’s polarity, the silanol groups are exploited in order to provide sites of covalent bonding for organic molecules

Bonded silica gel, modified in this way, behave as a liquid in that the separation mechanism now depends on the partition coefficient instead of adsorption coefficient . Often used in RP-HPLC

  • Aluminium oxide Al2O3
  • Zirconium oxide ZrO2

Mobile Phase

If the stationary phase is polar, then the technique is said to be normal phase chromatography and a less polar mobile phase is used, If the stationary phase is non-polar, or only weakly polar then the technique is called reversed phase chromatography (RP-HPLC)

A polar mobile phase is selected (most commonly water with a modifying solvent such as methanol or acetonitrile).

Four different types of interaction are possible between solvent molecules and

the analyte:

  • Dipolar interactions when analyte and solvent both possess dipole moments
  • Dispersion due to the attraction between molecules in proximity
  • Dielectrics, which favour the solubility of ionic species in polar solvents

  Types of HPLC

  HPLC Type : Mode and Principle of Separation

Mode of Separation

Normal phase chromatography - stationary phase is polar(hydrophilic) and mobile face is non-polar (hydrophobic).

Reverse phase chromatography- stationary face is non-polar(hydrophobic) and mobile face is Polar (hydrophilic).

Polar-Polar bonds and Non Polar-Non Polar bonds have more affinity than Polar-Non Polar bonds

Reverse phase chromatography is more commonly used as drugs are usually hydrophilic

Principle of Separation

Absorption Chromatography solute molecules bond directly to the surface of the stationary phase the component which has more affinity towards mobile phase elutes first & the component which has less affinity towards stationary phase elutes later. No two components have same affinity towards mobile phase &stationary phase

Ion-exchange chromatography Ion exchange chromatography is a process that allows the separation of ions and polar molecules based on their charge. Retention is based on the attraction between solute ions and charged sites bound to the stationary phase. Ions of the same charge are excluded. The use of a resin (the stationary solid phase) is used to covalently attach anions or cations onto it.

Ion-pair chromatography - It is a form of chromatography in which ions in solution can be "paired“ or neutralized and separated as an ion pair on a reversed-phase column.

Ion-pairing agents are usually ionic compounds that contain a hydrocarbon chain that imparts a certain hydrophobacity so that the ionpair can be retained on a reversed-phase column

Gel permeation chromatography - This type of chromatography lacks an attractive interaction between the stationary phase and solute.

The liquid or gaseous phase passes through a porous gel which separates the molecules according to its size. The pores are normally small and exclude the larger solute molecules, but allows smaller molecules to enter the gel, causing them to flow through a larger volume. This causes the larger molecules to pass through the column at a faster rate than the smaller ones.

  HPLC Type : Affinity and Elution


Affinity Chromatography - This is the most selective type of chromatography employed. Example, the immobilized molecule may be an antibody to some specific protein. When solute containing a mixture of proteins are passed by this molecule, only the specific protein is reacted to this antibody binding it to the stationary phase. This protein is later extracted by changing the ionic strength or pH.

Chiral chromatography - involves the separation of stereoisomers. In the case ofenantiomers, these have no chemical or physical differences apart from being three-dimensional mirror images. Conventional chromatography or other separation processes are incapable of separating them. To enable chiral separations to take place, either the mobile phase or the stationary phase must themselves be made chiral, giving differing affinities between the analytes.

A molecular organic compound whose structural formula reveals the presence of an asymmetric centre leads generally to a mixture of two possible enantiomers, R and S in variable quantities

Hydrophobic Interaction Chromatography (HIC), is principally employed for the separation of bio-organic compounds such as water soluble proteins. This separation takes advantage of the hydrophobic differences among the compounds present in the sample.

Compared to reversed phase chromatography, the density of the ligand on the matrix is much lower and allows mild elution conditions, helping to preserve biological activity. HIC is particularly suitable for samples precipitated with ammonium sulfate or eluted in high salt concentrations since high ionic strength buffers enhance the hydrophobic interaction.

Isocratic elution  & Gradient elution

Isocratic elution –

Separation in which the mobile phase composition remains constant throughout the procedure is termed isocratic elution. The resolution of the chromatogram depends on the number of theoretical plates.

In Isocratic elution late-eluting peaks get very flat and broad. Best for the analysis of simple samples

Gradient elution - separation in which the mobile phase composition is changed during the separation process is described as a gradient elution (composition is altered during the analysis – normally by increasing the amount of organic modifier)

  • Gradient elution decreases the retention of the later-eluting components so that they elute faster, giving narrower peaks . This also improves the peak shape and the peak height
  • Best for the analysis of complex samples
  • Often used in method development for unknown mixtures
  • Linear gradients are most popular - The elution strength usually increases with time, as shown above, where the gradient starts at 20% B and ends at 60% B over 30 minutes. The gradient shown is a linear gradient that changes at a rate of 1.33% solvent B per min
  • The initial composition is chosen so that the strength is appropriate to retain and resolve early eluting analytes
  • The elution strength is then increased in a pre determined way to elute compounds with optimum resolution
  • The final mobile phase composition is chosen to ensure elution of all compounds of interest from the column within a reasonable time
  • It is possible to increase the organic modifier concentration to wash strongly retained, potentially contaminating components from the column

Gradient elution is best suited to analyses carried out using reversed phase, normal phase separations using bonded stationary phases, and for ion exchange chromatography

Particular pumps are required to carry out gradient HPLC analysis, which allow on-line mixing of the mobile phase components

Benefits of Gradient Elution-

Improved resolution · Increased sensitivity · Ability to separate complex samples · Shorter analysis times · Decrease in column deterioration due to strongly retained components

  HPLC Terminology

Theoretical Plate

A theoretical plate is a hypothetical zone or stage in which two phases, such as the liquid and vapor phases of a substance, establish an equilibrium with each other. Such equilibrium stages may also be referred to as an equilibrium stage, ideal stage, or a theoretical tray. 

Having more theoretical plates increases the efficiency of the separation process be it either a distillationabsorptionchromatographicadsorption or similar process.

Potential problems are associated with isocratic analysis

range of analyte polarities is broad, some analytes may be poorly retained and resolution is lost with peaks eluting

It is possible that some components will be irreversibly adsorbed on the column and cause contamination

Column Efficiency

Peak capacity will increase proportionally to the square root of column efficiency therefore, doubling column efficiency will increase peak capacity, but only by 40%

N = L / H

N = no of plate , H height of plate and L length of plate (theoretical Plates)

Void Volume

The void volume is the volume of mobile phase (Vm or V0) in a column. For example, if the stationary phase occupies 40% of the total column volume, the void volume would be 60% of the total column volume. Consider a column that is 25 cm long with an inner diameter of 1 cm. The total column volume is 19.6 mL (V = ∏ r2L = 3.14 * 0.52 * 25 cm). If the mobile phase occupies 60% of the column volume, the void volume is 11.8 mL.

Dead Volume

The dead volume is the volume of an HPLC system between the point of injection to the point of detection, excluding the column.


End Capped Column - To prevent unwanted interactions between the solutes and any unreacted –SiOH groups, the silica frequently is “capped” by reacting it with  Si(CH3)3Cl; such columns are designated as end-capped. The stationary phase may be partially soluble in the mobile phase, causing it to “bleed” from the column over time. To prevent this loss of stationary phase, it is covalently bound to the silica Particles

Degassing - mobile-phase solvents must be treated to remove dissolved gases, such as N2 and O2, and small particulate matter, such as dust. Dissolved gases often lead to the formation of gas bubbles when the mobile phase enters the detector, resulting in a distortion of the detector’s signal. Degassing is accomplished in several ways, but the most common are the use of a vacuum pump or sparging with an inert gas, such as He, which has a low solubility in the mobile phase

  Guard Column - HPLC

Column - Considered the “heart of the chromatograph” the column’s stationary phase separates the sample components of interest using various physical and chemical parameters.

It is usually made of stainless steel to withstand high pressure caused by the pump to move the mobile phase through the column packing other material include PEEK( Poly Ethyl Ethyl Ketone) and glass

The small particles inside the column are called the “packing” what cause the high back pressure at normal flow rates

Dimensions of the analytical column are usually-straight, Length(5 ~ 25 cm), diameter of column(3 ~ 5 mm), diameter of particle(35μm). Number (40 k ~ 70 k plates/m) (5-25cm)

Guard column is used to remove particular matter and contamination, it protect the analytical column and contains similar packing its temperature is controlled at < 150 °C , 0.1 °C

  Detectors : HPLC

The detector can detect the individual molecules that elute from the column and convert the data into an electrical signal. The detector provides an output to a recorder or computer that results in the liquid chromatogram. Detector is selected based on the analyte or the sample under detection

The most widely used detection methods are based upon the optical properties of the analytes:

  • Absorption,
  • Fluorescence and
  • Refractive index
  • Spectro-photometric Detector

Detection is based upon the Lambert–Beer Law. The absorbance A of the mobile phase is measured at the outlet of the column, at one or several wavelengths  in the UV or visible spectrum

Its is a selective detection. For compounds that do not possess a significant absorption spectrum it is possible to perform derivatization of the analytes prior to detection.

  • Monochromatic detection – single wavelength e.g. 254nm if the source is a mercury lamp
  • Polychromatic detection - several wavelengths (diode array detector)

Ultraviolet (UV) Detectors are more common - This type of detector responds to substances that absorb light. UV detectors cannot be used for testing substances that are low in chromophores (colorless or virtually colorless) as they cannot absorb light at low range. They are cost-effective and popular and are widely used in industry

Fluorescence Detector

About 10 per cent of organic compounds are fluorescent, in that they have the ability to re-emit part of the light absorbed from the excitation source, The intensity of this fluorescence is proportional to the concentration of the analyte, as long as this concentration is kept low. There are a number of reagents that have been developed specifically to synthesized fluorescent derivatives

Specific detector that senses only those substances that emit light. This detector is popular for trace analysis in environmental science, PAH. As it is very sensitive, its response is only linear over a relatively limited concentration range. As there are not many elements that fluoresce , samples must be synthesized to make them detectable.

Mass Spectrometry Detector(HPLC-MS)

The mass spectrometry detector coupled with HPLC is called HPLC-MS. HPLC-MS is the most powerful detector,widely used in pharmaceutical laboratories and research and development.

The principal benefit of HPLC-MS is that it is capable of analyzing and providing molecular identity of a wide range of components.

Refractive Index Detector

This type of detector relies on the Fresnel principle of light transmission through a transparent medium of refractive index . It is designed to measure continuously the difference in the refractive index between the mobile phase ahead of and following the column. The change in refractive index between the two liquids, which appears when a compound is eluting the column, is visualized as an angular displacement of the refracted beam.

Used only in the isocratic conditions

Electrochemical Detector

Another common group of HPLC detectors are those based on electrochemical measurements such as

  • amperometry,
  • voltammetry,
  • coulometry, and
  • conductivity

  Chromatograph Interpretation


Standard Addition HPLC Calibration Method is called Spiking.

 It is used to determine the concentration of an analyte which is in a sample matrix, commonly found when working with clinical, biological or food samples.

Chromatograph Interpretation

The chromatogram is a graph that monitors the signal in the detector over time. As chemicals are detected by the instrument, the signal increases, and the chromatogram displays a "peak." Each peak in the chromatogram indicates the presence of a chemical in the sample

Each peak is labeled with retention time. Retention time indicates how long it takes for a compound to come out of the HPLC column

The first few small peaks in the chromatogram (around 2-3 minutes) are "noise" from the injection we made. We ignore those. The larger peaks represent chemicals in the sample.

Illustration is given below to explain and interpret Chromatogram

Bad Chromatograph

Retention Factor (k)

  Environmental Application of HPLC

  • Phenols in Drinking Water
  • Identification of diphenhydramine in sediment samples
  • Bio-monitering of PAH pollution
  • Toxicity of tetracyclines and tetracycline degradation products to environmentally relevant bacteria.
  • Assessment of TNT toxicity in sediment

The term 'mycotoxin' is usually reserved for the toxic chemical products produced by fungi that readily colonize crops.