Chromatography is one of the most powerful tools in analytical chemistry and is one of the most commonly found instruments in the laboratory. In this technique, the mobile phase is a liquid.
High-Performance Liquid Chromatography (HPLC)
High-pressure liquid chromatography, now known as high-performance liquid chromatography (HPLC), is a chromatographic technique used to identify, quantify, separate and purify individual compounds present in a mixture .
How does high-performance liquid chromatography work?
In high-performance liquid chromatography (HPLC) the sample mixture is passed along with a liquid solvent under high pressure through a column filled with a solid adsorbent material. The pressure within the system is built-up with the help of pumps. The working principle is that each compound in the mixture interacts slightly different with the adsorbent material in the column, resulting in varying flow rates for the different components. This leads to separation of the components as they flow out of the column. The adsorbent material used is typically granular, made up of solid particles, such as silica, constituting the ‘stationary phase’. The pressurized liquid is a mixture of solvents, such as water and organic liquids like methanol and acetonitrile, which constitute the ‘mobile phase’.
The detector is connected to a digital microprocessor and user software for data acquisition and analysis. The separated compounds are visualized as peaks with the number of peaks corresponding to the number of separated components in the mixture. The area of the peak is proportional to the concentration of the compound present within the mixture . The resolution between two peaks in chromatographic techniques is the extent to which substances are separated during the experiment. Higher resolution reflects good separation of the compounds.
What is high-performance liquid chromatography used for?
High-performance liquid chromatography has a wide range of applications in the fields of biochemistry, analytical chemistry, pharmaceuticals, forensics, food research, and other areas. For example, HPLC is also used in the testing for banned substances in athletes.
Why would you use high-performance liquid chromatography?
HPLC is both affordable and adaptable. It provides useful benefits such as data management and instrument validation.
Ultra High-Performance Liquid Chromatography (UHPLC)
Ultra-high performance liquid chromatography (UHPLC) is a variant of HPLC which uses smaller particles to enhance chromatographic performance and requires higher pressures. In a similar way to HPLC, the water contaminants can negatively impact on results, however, due to the higher sensitivities of the UHPLC, this can be more extreme.
Both HPLC and UHPLC can be used for liquid chromatography, but the equipment needed to run both differs and each has different benefits. The shorter column length means that Ultra high-performance liquid chromatography provides a better resolution than traditional HPLC.
Ion chromatography allows the separation of ions and polar molecules based on their interaction with a column of ion-exchange media. It can be used for almost any kind of charged molecule including large proteins, small nucleotides, and amino acids. It is very widely used for the determination of inorganic and organic cations and anions.
The quality of reagent water affects nearly every aspect of HPLC analysis, ranging from sample and standard preparation to column rinsing and elution. This also makes water the most consumed reagent in HPLC.
Typically pure water (Type II) is used to prepare blanks, standards, eluents and for sample pre-treatment; however as gradient HPLC is capable of extremely low detection limits, the water requirement is more stringent and the highest quality is required.
A large proportion of HPLC performance problems are due to the poor quality of water used in preparing HPLC eluents, standards and samples. Poor quality or contaminated water affects resolution by introducing ghost peaks, altering stationary phase selectivity and impacting baselines during chromatographic separation.
Moreover, it can lead to the build-up of contaminants in the stationary phase, which can cause blockage of the column. This results in an increase in pressure and a shift in sample running time. Poor quality data such as shifting retention times, loss of resolution or ghost peaks could be an indication of contamination caused by organics, ions, bacteria or particulates.
What are the different types of contaminants in water that affect HPLC results?
Organic contamination of ultrapure water may affect chromatographic separation in different ways:
(i) Reducing column life - Organic molecules that bind to the surface of the column can slow-down access of sample and solvent molecules to the binding sites within the column beads (stationary phase). This results in a decrease in the column’s ability to separate compounds or loss of resolution and a shorter column life.
(ii) Reducing sensitivity - Organic molecules in the eluent water may compete with sample molecules for binding to the column beads (stationary phase). This decreases the number of sample molecules binding to the column, consequently reducing the number of molecules released during the elution process.
(iii) Inaccurate data - Contaminant or ghost peaks can be obtained from organics that may accumulate at the head of the column and later get collected as eluate.
(iv) Shift in retention time - High levels of organics can create a new stationary phase in the column, which can cause a shift in retention time and peak tailing. It can also lead to back pressure increase.
It is, therefore, critical to accurately monitor the level of organics in water used for HPLC applications. Total organic carbon (TOC) is a measure of the total organic species present in water. TOC is measured in units of parts-per-million (ppm) or parts-per-billion (ppb). Organics, present in high ppb, can alter spectral identification of trace components within the mixture and influence peak quantification.
An HPLC system can be contaminated by a large variety of TOC sources. These include water, leaching from purification media, tubing and containers, bacterial contamination and potentially, absorption from the atmosphere. In fact, it is now recommended to avoid using high-purity bottled water that is not freshly purified. This is because HPLC bottled water that has been standing in a laboratory environment for more than 8 h (exposure to atmospheric organics) or distilled water is susceptible to an increase in TOC levels.
Figure 1 compares the chromatogram output from HPLC-grade water and ultrapure water measured at wavelengths of 254 nm and 214 nm. HPLC-grade water has higher TOC levels, which elute out from the column causing baseline shifts, with an increased size and number of peaks, compared to ultrapure water .
Therefore, the use of pure water is critical for HPLC analysis, and it is important to keep water free from all sorts of contaminants. Chromatographers, in addition to ensuring the purity of organic solvents, standards and other HPLC mobile phase components, must also ensure that the reagent water is of high quality and devoid of any contaminants.
2. Particles and Colloids
The presence of particles and colloids in the water sample can cause damage to the pump and injector in addition to physically blocking the column. These can also behave as a solid phase within the column binding to the sample constituents. Colloids can also irreversibly adsorb to the column packing material thereby preventing the sample constituents from binding to the column.
The presence of ions in the solvent can also affect chromatographic separations. The presence of any UV-absorbing ions, such as nitrates, nitrites, sulfates, bromides, chlorides, and fluorides, can pass through the column and appear as a peak in the chromatogram making the data difficult to analyze.
Among the different water contaminants that affect HPLC analysis, organics are by far the most important determinants for water purity. Experimental evidence has strongly suggested that freshly prepared ultrapure water should be the choice for any HPLC as other sources of water, namely distilled water or even HPLC-grade bottled water still contain relatively high amounts of organics, which can compromise the quality of the chromatograms and the performance of the apparatus (see, Figure 1).
It is, therefore, imperative to ensure that high standard water purification systems are used and the system itself is properly maintained.
Make sure that you are using the right water type for your application. Here are the requirements for liquid chromatography applications:
|Sensitivity required||Resistivity |
|Bacteria (CFU/ml)||Endotoxins (EU/ml)||Nucleases||Water grade|
|Ion chromatography||General High|
General lab grade water
Ultrapure water (Type 1)
General lab grade water
Ultrapure water (Type 1)
How does ELGA Veolia solve water purity problems for HPLC?
There are different purification systems currently available with each method having its own set of advantages and limitations. ELGA’s expertise in water purification systems has a long-standing reputation. ELGA has classified water purity into different grades, which can help determine the level of water purity required for a given application. For example, our PURELAB® Chorus 1 Analytical Research system delivers type I ultrapure water with TOC levels as low as 2ppb, that is highly suitable for HPLC experiments. ELGA provides water purification systems that are user-friendly, cost-effective and are low maintenance systems.
The purity of water as a laboratory reagent is crucial for successful experiments. Highly sensitive technologies, such as HPLC requires water of very high purity, which means that the water used should have minimal TOC levels and be devoid of any other contaminants. Freshly prepared ultrapure water is the choice for HPLC experiments and ELGA’s broad range of water purification systems helps researchers globally to ensure that the water used in their experiments are of desired purity.
 Jena A Kumar. HPLC: Highly Accessible Instrument in Pharmaceutical Industry for Effective Method Development. Pharm Anal Acta 2012;3. doi:10.4172/2153-2435.1000147.
 Malviya R, Bansal V, Prakash Pal O, Kumar Sharma P. High performance liquid chromatography: A short review. J Glob Pharma Technol 2010;2:22–6.