High Performance Liquid Chromatography (HPLC)

What is HPLC?

HPLC can be used to separate the constituents of a compound, tell you how much of each compound is found within the mixture and helps to identify what each compound is.

HPLC is the technique of choice when analysing materials for a wide range of organic compounds. Volatile compounds (VOCs and SVOCs) are usually best analysed by GC or GC-MS but HPLC is applicable to a much greater variety of mixtures, including non-volatile or thermally unstable molecules. Its advantages include versatility, sensitivity, and applicability to very complex mixtures.

How does HPLC work?

Separation using High Performance Liquid Chromatography is based on the affinity of the different compounds within the analyte to the mobile phase (eluent) and the stationary phase. The specific intermolecular interactions between the molecules of a component of the sample and the packing material result, in effect, in these molecules being taken up transitorily on to the stationary phase. 

The greater the interaction with the stationary phase compared with the mobile phase, the longer the time spent interacting with the stationary phase, the longer the time spent on the column and the longer the retention time (Rf) for that component. The power of the technique comes from the wide range of mobile and stationary phases that may be used to fine tune separations.

What are the different uses of HPLC?

HPLC is a commonly used and extremely powerful chromatographic technique, with applications in areas such as pharmaceutical, bioanalytical, food and beverage, clinical, forensic, environmental and drug development laboratories. 

For example, in a medical setting HPLC can be used to determine the contents and concentrations of substances in biological materials. This could include drug analysis of urine or detection of vitamin levels in blood serum.

What are the different types of HPLC?

Normal-Phase HPLC
In normal phase chromatography, the stationary phase is non- polar and the mobile phase is polar. This means that any non-polar substances in the sample elute more quickly as they are more similar to the mobile phase and move quickly. 

Reverse Phase
Reverse phase High Performance Liquid chromatography is the opposite of normal phase. Namely, a polar mobile phase, such as water and a polar organic solvent, is used with a non-polar, hydrophobic stationary phase.

Reverse phase HPLC is often preferred over normal phase HPLC as the use of water as the solvent eliminates the danger of analyte retention times being skewed due to absorption of water into the atmosphere. Reverse High Performance Chromatography is also considered to be more flexible as the hydrophobic stationary phase can be used in conjunction with hydrophobic, hydrophilic, ionic and ionisable compounds to separate out their different compounds.

Isocratic vs gradient elution
Isocratic elution means that a constant gradient is maintained in the mobile phase, whereas a gradient elution refers to an experiment where the concentration of the mobile phase varies. 

Gradient elution has several benefits over isocratic elution as it provides a more even spacing of peaks with similar widths throughout the resulting chromatogram. In isocratic elution, peaks often have a decreased resolution and are exceedingly close together at the beginning of the process, becoming much broader towards the end. Gradient elution can also offer an even shorter run time. 

However, isocratic elution is often favoured over gradient elution because the gradient process requires greater care and regulation on the part of the operator. Isocratic elution also requires less specialised chromatographic equipment.

How does pure water affect HPLC?

HPLC is incredibly dependent upon water purity. Using an impure water source to prepare eluents, blanks, samples and standards could introduce contamination into the experiment, degrading the chromatographic performance by impacting resolution, integration and baselines. As water is the reagent used in the largest volume in HPLC, it is vital that the water chosen is of the correct purity required for the sensitivity of the application.

Which water type should you use?
For HPLC experiments where the applications have a general sensitivity, we recommend Type II+ water. Where the sensitivity of the application is high, Ultrapure Type I+ water should be used as it has a resistivity of more than 18 MΩ.cm, a TOC value of less that 2ppb, less than 1 CFU/mL of bacteria and less than 0.03 endotoxins.

Instrumentation

Pumps:
If the HPLC is a gradient system, then either a low-pressure gradient (LPG) or high pressure gradient (HPG) process is available. In HPG, the solvents are mixed discharge side and are inputted from individual pumps. Whereas, in LPG the solvents are mixed suction side.

Column:
The column is the core of any HPLC system as it is responsible for the separation of the sample compounds. A variety of different HPLC columns are available dependent on the needs of your experiment. For example, the column can be filled with a variety of different packing materials to support the various types of HPLC, such as reverse phase or normal phase.

Detector:
The detector measures the time and amount of each substance that is eluted from the column. The difference in composition over the course of the process is registered by the detector and translated into an electrical signal from which a chromatogram is created.

Chromatographic Parameters

Throughout the chromatographic process, the detector produces electronic signals that can be turned into a chromatogram by an accompanying computer. These diagrams can then be used to determine the substances present within the sample and their quantities. Each signal peak represents an analyte that has been transported by a mobile phase through the column.

A variety of qualitative information can be garnered from these peaks, from the time of the peak to the concentration of the substance (as represented by the area under the graph). 

It is also important to take into consideration the resolution of the process. A resolution value of 1.5 or greater between two peaks means that the sample components are separated to a degree that the height and width of the peaks can be accurately measured. The resolution can be calculated using the Fundamental Resolution equation. 

The HPLC parameters that are considered in this equation are the efficiency factor (N), the retention factor (kappa prime), and the separation factor (alpha). Adjustments, such as changing the solvent used in the experiment or altering the temperature can then be put in place to change these parameters and improve the Resolution of the experiment.

Water Purification Systems supplied by ELGA LabWater

If you’re in need of pure water for your HPLC applications, have a look through our Water Purification Systems, designed to provide you with an efficient supply of the water grade you need, whether it be Ultrapure or Type III. Whether you want to request a demo or ask a question, get in contact today.

ELGA’s expertise and long-established reputation ensure that its experienced team can help customers to determine the particular water purity requirements for their applications. The Company offers a number of water purification systems that have been proved to meet the requirements for HPLC. For example, the bench-top PURELAB Chorus 1 Analytical Research  point-of-use system consistently delivers ultrapure water of 18.2 MΩ.cm (Type I/I+) and TOC less than 2ppb suitable for all these applications.