Reversed-Phase Chromatography – Principle, Protocol, Applications

Reversed-Phase Chromatography (RPC) is a type of liquid chromatography used for separation and analysis of compounds. It separates molecules mainly according to their hydrophobic nature.

In this method the stationary phase is non-polar. The stationary phase is usually silica gel attached with hydrophobic alkyl chains. Common phases are C18 and C8.

The mobile phase is polar in nature. It is usually water mixed with organic solvent like methanol or acetonitrile. The sample components move between mobile phase and stationary phase.

Polar molecules do not bind strongly with the non-polar stationary phase. So they come out from the column first. Non-polar molecules bind more strongly with the stationary phase and remain in the column for longer time.

This method is called reversed-phase because it is opposite to normal-phase chromatography. In normal phase the stationary phase is polar and mobile phase is non-polar. But in reversed-phase chromatography, the stationary phase is non-polar and mobile phase is polar.

Earlier normal-phase chromatography was used more. But many biological and pharmaceutical compounds were not separated properly by that method. So silica particles were modified with hydrocarbon chains to make hydrophobic surface.

Reversed-Phase Chromatography is now commonly used in HPLC. It is used in pharmaceutical analysis, biochemical analysis, environmental sample analysis and purification of many organic compounds.

Principle of Reversed-Phase Chromatography

Principle of Reversed-Phase Chromatography is based on hydrophobic interaction between sample molecules and non-polar stationary phase. The compounds are separated according to their hydrophobicity.

In this method the stationary phase is non-polar. Common stationary phases are C18 and C8 bonded silica column. The mobile phase is polar and it is usually water mixed with methanol or acetonitrile.

When the sample is passed through the column, different compounds distribute between mobile phase and stationary phase. Polar compounds prefer the polar mobile phase. So they move faster through the column.

Non-polar compounds interact more with the non-polar stationary phase. So they remain in the column for longer time. More hydrophobic compound is retained more strongly.

Thus polar compounds elute first from the column. Less polar and more hydrophobic compounds elute later. In this way the separation occurs in Reversed-Phase Chromatography.

Parts of Reversed-Phase Chromatography

The following are the main parts of Reversed-Phase Chromatography–

1. Mobile phase
   It is the liquid phase which carries the sample through the system. It is usually polar in nature and made up of water or buffer with methanol or acetonitrile.
2. Degasser
   It is used to remove dissolved gases from the mobile phase. If gas remains in solvent, bubbles may form and baseline may become unstable.
3. Pump
   It is used to push the mobile phase through the column. It gives constant flow of solvent under high pressure.
4. Injector
   It is used for adding the sample into the mobile phase. It may be manual injector or automatic autosampler.
5. Column
   It is the main part where separation takes place. In RPC, the column is usually packed with non-polar stationary phase.
6. Stationary phase
   It is present inside the column. It is commonly silica gel bonded with hydrophobic chains like C18 or C8.
7. Column oven
   It is used to maintain fixed temperature of the column. Constant temperature gives more reproducible retention time and better separation.
8. Detector
   It is used to detect the compounds after they come out from the column. Common detectors are UV-Visible detector, fluorescence detector and mass spectrometer (MS).
9. Data system
   It is used to record the detector signal. The signal is converted into chromatogram and the peaks are analysed.
10. Waste container
    It is used to collect the mobile phase and unwanted eluent after detection. Organic solvent waste should be collected safely.

Protocol of Reverse-phase chromatography

The following are the steps of Reverse-Phase Chromatography–

1. At first the column is washed with suitable solvent. This prepares the non-polar stationary phase for separation.
2. The column is then equilibrated with starting mobile phase. The mobile phase is usually water or buffer with little amount of methanol or acetonitrile.
3. Equilibration is done until the baseline becomes stable. Pressure should also remain constant.
4. The sample is dissolved in polar solvent. The sample should be clear and filtered before loading.
5. The sample is injected into the column by injector or autosampler. The mobile phase carries the sample into the column.
6. Inside the column the sample components come in contact with non-polar stationary phase. Different compounds bind with it in different strength.
7. Polar compounds bind weakly with the stationary phase. So these compounds move fast and come out first.
8. Hydrophobic compounds bind strongly with the stationary phase. So these compounds remain in column for more time.
9. The column is washed with starting mobile phase. Weakly bound impurities and polar substances are removed.
10. The retained compounds are eluted by increasing organic solvent in the mobile phase. Methanol or acetonitrile is increased slowly.
11. When organic solvent increases, the hydrophobic binding becomes weak. Then the compounds start to come out from the column.
12. The compounds coming out from the column are detected by detector. UV-visible detector or mass spectrometer (MS) may be used.
13. The detector gives signal to data system. The result is recorded as chromatogram.
14. After the run the column is washed with strong organic solvent. Remaining compounds are removed from the column.
15. The column is again equilibrated with starting mobile phase. Then it is ready for next sample.

Uses of Reverse-phase chromatography

The following are the uses of Reversed-Phase Chromatography–

• It is used in pharmaceutical industry for drug discovery. New drug molecules are separated and checked by this method.
• It is used for testing purity of active pharmaceutical ingredients (APIs). Finished drug products are also analysed by this method.
• It is used for pharmacokinetic study. Drug concentration can be measured from biological samples like human plasma.
• It is used in biochemical analysis. Proteins, peptides and nucleic acids are separated and purified by this method.
• It is used for peptide mapping. Different peptide fragments are separated and identified.
• It is used for protein sequencing work. It also helps in study of post-translational modification of proteins.
• It is used in environmental monitoring. Trace pollutants, pesticide residues and hydrophobic contaminants are detected from water, soil and air samples.
• It is used for detection of endocrine disruptors. These compounds are present in very low amount and can be analysed by this method.
• It is used in food and beverage quality control. Vitamins, proteins and essential fatty acids can be measured.
• It is used for detection of food spoilage markers. Mycotoxins, contaminants and illegal adulterants can also be detected.
• It is used for analysis of food additives. Artificial colour, preservatives, flavour and aroma compounds are checked by this method.
• It is used for lipid analysis. Different classes of lipids present in biological samples can be separated and estimated.
• It is used in cosmetic industry. Active ingredients like retinol in anti-aging cream can be measured.
• It is used in industrial product analysis. Fine chemicals, synthetic polymers and other hydrophobic compounds are analysed by this method.

Advantages of Reverse-phase chromatography

The following are the advantages of Reversed-Phase Chromatography–

• It is used for many types of compounds. Small organic molecules, drugs, lipids, peptides and proteins can be analysed by this method.
• It is the most commonly used mode of HPLC. Most of the analytical methods are done by reversed-phase system.
• It gives good resolution. Closely related compounds can be separated when proper column and mobile phase is used.
• It has good sensitivity. Very small amount of sample can also be detected by using suitable detector.
• It gives reproducible result. Retention time remains almost same when same condition is maintained.
• It is more stable than normal-phase chromatography. Moisture does not affect it much like normal phase system.
• It gives fast separation. Many samples can be analysed in less time.
• The mobile phase can be changed easily. Water, buffer, methanol and acetonitrile are commonly used.
• pH and gradient elution can be adjusted. This helps in improving separation of difficult mixture.
• It can be used with many detectors. UV-visible detector, fluorescence detector and mass spectrometer (MS) can be connected.
• It is easy to operate. Standard HPLC system and ready-made columns are available for this method.
• It is suitable for automation. Sample injection, solvent gradient and data recording can be controlled by instrument.
• It needs small amount of sample. So sample loss is less and result can be obtained from limited sample.
• It is economical for routine analysis. The method gives accurate result with small sample and common solvents.

Limitations of Reverse-phase chromatography

The following are the limitations of Reversed-Phase Chromatography–

• It is not very suitable for highly polar compounds. Such compounds do not bind properly with non-polar stationary phase and may come out very fast.
• Water insoluble compounds may create problem in this method. The sample must be dissolved properly before injection.
• Some amines may not give good peak shape. They may interact with free silanol groups present on silica surface.
• The column may be damaged at very high or very low pH. Silica based C18 and C8 columns are not stable in extreme pH condition.
• High temperature may also damage the stationary phase. So the column should be used within its recommended temperature range.
• Peak tailing may occur in basic compounds. This happens due to secondary interaction between analyte and unreacted silanol groups.
• Phase collapse may occur when 100% water is used as mobile phase. The hydrophobic chains may collapse and retention becomes poor.
• If phase collapse occurs, peak shape becomes bad. Retention time also becomes non-reproducible.
• Sample recovery is limited in analytical run. Once the sample passes through detector and waste line, it is not usually recovered.
• Large amount of organic solvent may be required. Acetonitrile and methanol are toxic and inflammable solvents.
• Organic solvent waste is produced after the run. This waste needs proper collection and disposal.
• The running cost is high. Pure solvents, expensive column and instrument maintenance are needed.
• High pressure is used in RPC system. This may cause wear of pump, tubing and other instrument parts.
• It needs technical skill for operation. Gradient control, column selection and troubleshooting should be done carefully.
• It cannot confirm compound identity by itself. For exact identification, it is often connected with mass spectrometry (MS) or other detector.

Precautions of Reversed-Phase Chromatography

The following are the precautions of Reversed-Phase Chromatography–

• Personal protective equipment (PPE) should be used during the work. Lab coat, gloves and safety glasses are used to protect skin and eyes from harmful chemicals.
• Toxic and volatile solvents should be handled in proper ventilation. Solvents like acetonitrile and methanol may produce harmful vapour.
• Organic solvents should be kept away from flame and ignition source. These solvents are highly inflammable in nature.
• Mobile phase should be filtered before use. Solid particles present in solvent may block the column and increase pressure.
• Mobile phase should be degassed before running. It removes dissolved gases and prevents bubble formation in the detector and tubing.
• Degassing should not be done for very long time. Organic solvents may evaporate and change the mobile phase composition.
• Silica based C18 and C8 columns should not be used at extreme pH. High pH or very low pH may damage the silica surface.
• High pH buffer should be protected from air. Carbon dioxide from air may enter into buffer and change the pH.
• Column should not be stored in pure water for long time. It may cause microbial growth and sometimes collapse of stationary phase.
• Column should be stored in suitable organic solvent mixture. Generally 50-80% organic solvent is used for long term storage.
• Eluent bottles should be kept closed. This prevents evaporation of solvent and change in solvent strength.
• Neutral aqueous solution should not be stored for many days. It may support microbial growth and contaminate the system.
• Solvent change should be done carefully. When changing from one solvent system to another, intermediate solvent like isopropanol may be passed.
• Salt containing mobile phase should be flushed properly from the system. Salt precipitation may block tubing, column or detector cell.
• Flow restrictor may be used after detector. It prevents air accumulation inside the detector cell.
• Waste solvent should be collected in labelled container. Strong acid, organic solvent and other waste should be disposed safely.

Troubleshoot for Reversed-Phase Chromatography

The following are the troubleshoot for Reversed-Phase Chromatography–

• Baseline noise occurs due to dissolved gas or air bubble in the mobile phase. The solvent should be degassed properly, system should be purged and leakage should be checked.
• Unstable baseline may occur due to air in detector cell or fluid leak in the system. The mobile phase line, detector flow cell and fittings should be checked properly.
• Peak tailing occurs mostly in basic compounds. These compounds may interact with free silanol groups present on silica surface, so pH may be lowered or endcapped column may be used.
• Poor peak shape may occur due to dead volume in capillary connection. Ferrule and tubing connection should be fitted properly without extra space.
• Loss of retention may occur in highly aqueous mobile phase. If organic solvent is less than 5%, C18 phase may collapse and retention becomes poor.
• Phase collapse can be reduced by using special aqueous compatible column. Polar endcapped, polar embedded or short chain alkyl column may be used.
• Column degradation occurs when silica column is used at extreme pH. At high pH silica may dissolve and at very low pH bonded phase may be removed.
• Extreme pH problem can be solved by using suitable special column. Polymeric column like PLRP-S can be used when very low or very high pH is required.
• Column damage may occur due to high temperature. The column should be used below the temperature limit given by manufacturer.
• High backpressure occurs due to blockage by particles or salt precipitation. Sample and mobile phase should be filtered through 0.22 µm or 0.45 µm membrane filter.
• Pressure change may occur due to change in temperature. Solvent viscosity changes with temperature, so column oven or constant temperature should be maintained.
• Buffer precipitation occurs when high organic solvent is mixed with some buffer salts. Buffer solubility should be checked before mixing with acetonitrile or methanol.
• Retention time shifting occurs due to poor column equilibration. The column should be equilibrated with more than 10 column volumes of starting mobile phase.
• Different retention time in different instrument may occur due to dwell volume difference. The gradient delay volume of the instrument should be considered during method transfer.
• Change in elution order may occur during method transfer. Mobile phase mixing, gradient program and instrument dwell volume should be checked.

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