A chromatographic separation involves a stationary phase and a mobile phase. The mobile phase moves over the stationary phase to separate the analyte components on the basis of their relative interaction/affinity with the two distinct phases. High-performance liquid chromatography is a type of liquid column chromatography. The stationary phase in this case is compactly packed into a column while the mobile phase is a liquid that sweeps pass the stationary phase.
The small particle size of the stationary phase packing in HPLC allows a high surface area for analyte separation thus the name high-”performance” liquid chromatography. But as a consequence of this compactly packed stationary phase, a large amount of pressure needs to be applied in order to push the mobile phase through it. Thus, HPLC is also sometimes known as high-”pressure” liquid chromatography.
To fully understand how to perform an HPLC experiment, we should be aware of the basic terminology associated with the topic.
Chromatographic terms related to high-performance liquid chromatography
|Eluent||The mobile phase travelling through the column|
|Eluate||Mobile phase emerging from the column that consists of analyte components in it|
|Eluite||The analyte components eluting from the column except the mobile phase
i.e., eluite=eluate-mobile phase
|Effluent||Wastage in result of a chromatographic separation|
|Elution||The overall chromatographic process|
|Isocratic elution||Maintaining a single mobile phase composition throughout the chromatographic separation|
|Gradient elution||Varying the mobile phase composition amid the chromatographic separation|
|Retention time||Time taken for the analyte components to pass
through the chromatographic column and to get eluted out of it
For more basic terms related to chromatography in general, refer to our article what is chromatography.
HPLC mobile phase
An HPLC mobile phase is an organic solvent or a combination of solvents that must have the following characteristics.
- Ultra-high purity: Impurities present in the mobile phase can cause fluctuations in its flow through the column thus negatively impacting the analytical separation.
- An HPLC grade mobile phase should be used.
- The solvents used in the mobile phase should be non-reactive.
- The mobile phase should be de-gassed. Degassing means removing any air bubbles present in the solvent which can otherwise develop back pressure thus degrading the stationary phase packing.
- The mobile phase could either be used for isocratic elution and/or for gradient elution.
- In case of gradient elution, a binary or a quaternary solvent system is usually used for high-performance liquid chromatography i.e., a mobile phase composition consisting of two or four organic solvents respectively.
- Popularly used organic solvent choices for an HPLC experiment includes alcohols (such as ethanol and methanol), acetic acid, acetonitrile and different pH buffers prepared in high purity, HPLC grade water. The solvents are filtered using an extremely fine filter paper with pore size 0.25-0.45 µm prior to use.
Stationary phase packing in HPLC
High-performance liquid chromatography can be performed in both normal phase or reverse phase modes as per requirement however the latter is preferred in most of the analyses. The stationary phase could be a solid or a liquid coated onto a solid support. Silica (SiO2) gel is the most commonly used stationary phase packing for performing an HPLC experiment. Bare silica is highly polar in nature and retains strongly polar components from the analyte mixture.
The silanol activity of silica however can be masked with hydrophobic, long chain alkyl groups to prepare a non-polar stationary phase for performing HPLC in reverse phase mode. The alkyl group could be a propyl (C3H7), a butyl (C4H9), an octyl (C8H17) or an octadecyl (C18H37) group depending on the hydrophobicity required.C8 and C18 columns are the most favourable such as an octadecylsilane (ODS) column commercially available in the market.
For a more detailed understanding of stationary phase packing in HPLC columns and their different modes of interaction with analyte components, refer to our article: what is stationary phase in chromatography.
High-performance liquid chromatography (HPLC) is a state-of-the-art analytic separation and identification technique that requires an integrated infrastructure. All the major components of a complex HPLC operation are as discussed in this section.
1. Solvent Reservoir
Highly pure, filtered solvents are initially placed in the HPLC solvent reservoirs. The solvents are degassed using various mechanisms: ultrasonication, magnetic stirring, heating, nebulization with helium gas and/or using a vacuum pump.
2. HPLC Pumps
The degassed solvents are pumped into the solvent mixture using high quality HPLC pumps where the mobile phase composition required for a particular analysis is set. The mobile phase so prepared is then pumped into the guard column.
Two different types of pumps can be used for HPLC namely constant pressure pumps and constant flow pumps. The constant pressure pump maintains a constant pressure while varying the mobile phase flow rate. The constant flow pump on the other hand, maintains a constant flow rate while varying the pressure. The constant flow pump overall is a better choice for HPLC to ensure a steady flow of the mobile phase through the column. A pressure as high as 6000 Psi is maintained in an HPLC column with the aid of these mechanical pumps.
3. Guard Column/Pre-Column
The guard column is a mini version of the main analytical column. It protects the main column from any possible impurities present in the mobile phase. The guard column is packed in a similar manner as the main column but it has a shorter length and a smaller internal diameter in comparison to the former. The mobile phase is first passed through this column so that any trace impurities present in it can get retained onto the stationary phase packing. This inevitably highlights the importance of maintaining a high purity in an HPLC analysis.
4. Sample Injector
Three types of injectors can be used in high-performance liquid chromatography to inject the sample components into the main analytical column:
i) Stop Flow Injection
ii) Loop Injection
iii) Micro Volume Injection
In all types of injections, a sharp syringe needle is used to inject the analyte mixture. The blunt needle protects the injection port from any suspected damage.
5. Analytical Column
The analytical column is the main component of an HPLC system. It is where the ‘actual separation’’ occurs. The column is usually made of stainless steel and commercially packed using different types of stationary phase material as we have discussed earlier. A long column (3-25) cm with a narrow diameter usually 4.6 mm is used for the sake of high-performance liquid chromatography. A longer column allows greater separation efficiency which leads to good peak separation and a high resolution of the chromatographic process overall.
Another important component of a high-performance liquid chromatographic system are the detectors used for identifying the analyte components. An ideal HPLC detector should be sensitive enough to respond to even a low analyte concentration and to provide a linear outcome. The detector should also be insensitive to minor fluctuations in temperature and solvent composition.
Different types of detectors can be used for an HPLC experiment. These include; ultra-violet (UV) detector, a photodiode array detector, a refractive index detector, evaporative light-scattering detector, charged-aerosol detector, electrochemical detector and/or a conductivity detector. All these different types of detectors operate on differing principles with a common goal i.e., analyte identification. The choice of detector depends on the type of sample and the purpose of analysis. The detector response is finally fed to a computer which then illustrates it in the form of a chromatogram.
7. HPLC Chromatogram
The chromatogram obtained from a high-performance liquid chromatography is plotted using detector response versus retention time. The chromatogram displays peaks where each chromatographic peak is indicative of a single analyte component. The component can then be identified using the data present in an HPLC library by matching the peak shape, retention time and retention volume with the standards.
A single, sharp peak, adequately separated from adjacent peaks represents a good analytical separation. Peak broadening, overlapping of adjacent peaks and an unstable base line refers to a poor HPLC separation as shown in the Figure 1 below.
The area under the peak can be used to determine the concentration of the targeted analyte. In this way, high-performance liquid chromatography aids in both a quantitative as well as a qualitative analysis.
Interpreting HPLC results
For this study, we take the example of a reverse phase high-performance liquid chromatographic (RP-HPLC) experiment for separating and identifying components A, B, C and D from a complex mixture M. A C-18 packed HPLC column having dimensions 150 mm x 4.6 mm was used. Methanol containing 1% acetic acid: acetonitrile (70:30) was used as the mobile phase in an isocratic mode of elution, maintaining the flow rate at 1mL/min. A UV detector was used at λmax 254 nm. The chromatogram obtained from the analysis is as shown in Figure 2.
The standard solutions of A, B, C and D prepared using the same solvent were also passed through the HPLC column under similar conditions. The peak as well as the retention time obtained for standard A if matches exactly with that obtained for any component from the series of peaks shown on Figure 2, then it allows qualitative identification for that component.
The calibration curve, plotting peak area versus analyte concentration at this particular λmax can then be obtained for standard A in order to measure the amount of A present in the mixture quantitatively, using the steps given below:
Step I: Treat the peak A as a triangle, find the area of the triangle by multiplying the height of the peak with its width.
Step II: Repeat step I for determining the area under the peak for standard A at different known concentrations.
Step III: Plot and use the standard calibration curve to determine the unknown analyte concentration.
Adding a known amount of standard and recording the detector response against it for quantitative HPLC analysis is called an ‘internal standard method’’.
Method precision, reproducibility, LOD, LOQ, and standard deviation are other parameters related to the results obtained from an HPLC analysis that fall under the scope of HPLC method development and validation.
Limitations of HPLC
HPLC is a complex technique, fascinated with detectors. In addition to that, it comes with high demands of purity, sensitivity and efficiency which makes high-performance liquid chromatography an expensive protocol. Maintaining the column in a good health requires frequent inspection by trained technicians.
Applications of HPLC
High-performance liquid chromatography is important for a diversity of analysis from everyday laboratory usage to complex industrial applications. It is one of the most versatile chromatographic techniques thus can never be ignored in the modern scientific world whether it be in the pharmaceutical sector, environmental analysis, food industry, forensic industry and/or clinical sector. Read more specific uses of different types of chromatography in a subsequent article in our series.
HPLC is fast, effective and efficient and is here to stay for many years with frequent advancements in its infrastructure and method development as and when required.
You may also find the following information sources on different aspects of HPLC useful:
1.B, P., D. Akalanka, S. r. G H, J. P and A. P (2013). “A Review – Importance of RP-HPLC in Analytical Method Development.” International Journal of Novel Trends in Pharmaceutical Sciences 3(1): 15-23.
2. Borman, S. (1987). “Eluent, Effluent, Eluate, and Eluite.” Analytical Chemistry 59(2):
3. C.Harris, D. (2010). Quantitative Chemical Analysis.
4. Snyder, L. R., J. J. Kirkland and J. L. Glajch (2016). Practical HPLC Method Development.