High Pressure Liquid Chromatgraphy (HPLC).

The previous post was on the chromatography in general. We discussed about the basics and different types of the chromatography. I recommend you read that post before you start this one. In this post, we discuss about one of the important type of chromatography, HPLC, in details.

High ‘performance’ liquid chromatography or high ‘pressure’ liquid chromatography is a type of column chromatography. In the conventional column chromatography, the column is packed with solid absorbent particles of average sizes from 30 to 90 μm, into a cylindrical glass or plastic tube. The solid adsorbent packed in the column makes the stationary phase and the liquid solvent(s) make the mobile phase, which drips through the column under the influence of gravity.

Conventional Column chromatography — Fig 1: Basic Column Chromatography (Edvotek)

In HPLC also, the stationary phase is the solid material filling up the column and the mobile phase are the liquid solvents. However, in the HPLC, the column is much more tightly packed with material having very small diameter (2–50 μm in average particle size). The advantage of using small packing materials is increase in the surface area, which in turn increases the sensitivity and the resolution of the process, hence called the high performance.

As the HPLC column is narrower and more tightly packed, the liquid mobile phase cannot simply pass through the column due to the increased resistance. Even if the mobile phase did pass through the column, it would require a lot of time (maybe days) and even effect the resolution. Hence the mobile phase has to be pushed through the column, which is done by applying high pressure using a pump. The application of pressure leads to increase in both; the speed and the resolution of the separation.

The choice of the mobile phase and stationary phase depends on the properties of the analyte. HPLC is of two different types, normal phase and reverse phase, depending on the polarity of the mobile phase and the stationary phase.

• Normal-phase HPLC

In the normal phase HPLC, the stationary phase is hydrophilic and the mobile phase is hydrophobic. The stationary phase is made of hydrophilic material like silica or other materials linked with polar groups like cyano or amino groups. Common nonpolar solvents used in HPLC are hexane and methylene chloride. Due to the hydrophobic mobile phase, the hydrophobic (non polar) compounds elute first and the hydrophilic (polar) compounds are eluted later.

• Reverse-Phase HPLC

In the reverse-phase chromatography, the stationary phase is non-polar while the mobile phase is polar. The stationary phase is made non polar by linking long hydrophobic carbon chains to the silica particles (C-8 and C-18 columns). Commonly used polar solvents are acetonitrile, methanol and tetrahydrofuran (THF).

– HPLC can be used for both analytical chromatography or preparative purposes

• Analytical HPLC

In this type, the molecules are separated for analysis of the component, i.e. for identification or quantification purposes. Usually the analytical HPLC are carried out in narrower columns like around 250 mm long x 4.6 mm inner diameter. The flow rate is maintained at around 0.5 to 1 ml/ min

• Preparative HPLC

Preparative chromatography involves the separation and isolation of one or more components of the sample. The components isolated can be used for some other analytical or experimental studies. Hence this is used as preparatory step for the next experiment. preparative HPLC are carried out in columns with bigger inner diameter and bigger particle size (10 microns in some cases). Standard column dimensions range from 50 to 200 mm inner diameter and 250 to 1000 mm in length. The flow rate of the mobile phase is kept high to around 4-5 ml/min.

Instrumentation:

Shown in the fig 3 is the flow chart of the HPLC instrumentation. The basic HPLC system includes reservoir(s) for solvent(s), a high-pressure pump, sample injector, the column, a detection system, an outlet for waste (and/or fraction collector) and computational system.

1. Solvent reservoir:

The solvent reservoir contains the mobile phase or the solvent. The mobile phase may be a single solvent or a mix of solvents. Some of the instrument have provision for upto 4 solvents. Having more than one solvent reservoir options is useful if using gradient mode. The solvent has to be degassed before pumping it into the column, to remove any air bubble which could hinder the flow of mobile phase through the column. The solvents (mobile phase) can be degassed manually or some instruments do have inbuilt degassing system.

If the mobile phase is kept same throughout the run, it is known as isocratic elution. Whereas in the process where the mobile phase is changed at different time is known as gradient elution. Eg. if the mobile phase is 10% methanol and 90% water throughout the run, it is isocratic elution. If the solvent is changed from 10% methanol (90% water) to 30% methanol (70% water) to 50% methanol (50% water) at the end, the elution is called gradient elution.

2. Pump:

The column in the HPLC is narrow and tightly packed. The mobile phase cannot simply pass through due to gravity. Hence a pump is placed between the solvent reservoir and the column. The pump pushes the solvent through the column continuously at a constant volume or pressure. The peristaltic pump is commonly used.

3. Sample injector:

Sample injector is an automated system fixed within the modern instruments which draws the sample from the sample tube and injects it into the HPLC system just before the column, which is then carried through the column by the mobile phase.

4. Column:

As previously mentioned the HPLC columns are tightly packed with small beads, having average size in the range of 2–50 μm. Common packing materials in HPLC columns include silica or hydroxyapatite media and polymeric resins such as polystyrene divinylbenzene.

A range of choice of column materials are available to choose from depending on the properties of the sample. The properties that can be exploited are polarity, ionic exchange, ionic interaction, size exclusion and so on.

HPLC column different sizes — Fig 4: Different options in sizes in HPLC columns (Orochem Technologies)

The HPLC columns also come in range of sizes depending on the purpose of the study, that is preparative or analytical. The internal diameter (ID) and volume of a column determine both how much sample can be loaded onto a column and the sensitivity of separation. The lesser the inner diameter of the column better is the smaller separation and sensitivity.

A much smaller column similar to HPLC column, called as guard column, is usually placed between the sample injector and the analytical column. The guard column retains the contaminants, if any, in the sample and protects (guards) the main column. Hence it helps increase the lifetime and maintain the resolution of the analytical column.

5. Detector:

As the sample components pass through the stationary phase carried by the mobile phase, they are separated. These separated components then are passed through a detector . The detector detects the presence of any compound carried by the mobile phase. Different types of detectors used in the HPLC are

a. UV, VIS, and PDA Detectors

These detectors detect the changes in the absorbance of the eluent. By measuring the difference between the absorbance in the eluent flowing through a flow cell with and without the sample, the amount of sample can be determined. These detectors can detect the compounds at ~pg level.

UV detector is commonly used detector for HPLC. UV detector may have wavelength between 195 to 370 nm whereas the VIS detector uses longer wavelength (400 to 700 nm).

PDA Detector also known as Diode array detectors (DAD detector) do not a single wavelength but a spectrum of light. In this, the light from the lamps is passed directly through the flow cell. The light is then dispersed using diffraction grating, and the amount of the dispersed light is estimated for different wavelength in the photodiode arrays. Hence entire spectrum is obtained. The difference in the spectrum (any absorbed wavelength) is detected in presence and absence of the analyte.

PDA detector DAD detector — Fig 5: Representation of PDA detector

b. Reflex Index Detector

RI detector measures change in reflex index between the sample cell containing the effluent and the reference cell containing only the mobile phase. When any compound is present in the effluent, there is a change in the reflex index and change in the angle of the incident light beam. This change can be measured and detected. However RI detector has lower sensitivity compared to UV detector. Nonetheless it is useful when the samples do not absorb UV, such as sugar, alcohol, or inorganic ions.

c. Evaporative Light Scattering Detector

ELSD can detect non-volatile to semi-volatile compounds with a good sensitivity (at ng level). The effluent is converted to fine spray by a nebulizer and mobile phase is heated and evaporated. The light (laser beam) is passed through the evaporated compound and scattered light is detected. This is useful in detection of lipids, sugar, and high molecular weight analytes.

d. Mass Spectrometer

The HPLC can be connected to a Mass spectrometer for further detection and analysis. The analyse can be identified by their. masses. The mass spectrometer can even identify the structure of the analytes, in case it’s unknown.

6. Waste collector or/ and Fraction collector:

The effluent is either discarded (in analytical HPLC) or collected ( preparative) after flowing through the detector. The fraction collector is connected next to the detector if the effluent containing certain compound has to be collected if the analyte has to be used for further studies.

7. Computation and Data Analysis:

Finally the detectors are connected to the computer, which records the data from the detector in form of chromatogram. The different data can be obtained with help of the chromatogram like the retention time and amount of the components of the sample.