HPLC Machine
HPLC analysis or High Performance Liquid Chromatography is an important method in chemistry used to detect, separate, and quantify a compound. HPLC (High Pressure Liquid Chromatography or High Performance Liquid Chromatography) is one of the most widely used chromatography methods.
Chromatography methods (chroma meaning color and graph meaning drawing) were first recognized by Tsvet (Mikhail Tsvet), a Russian botanist who worked on separating green leaf pigments.
The results from the HPLC machine are called chromatograms.
How HPLC Works:
HPLC consists of two phases: stationary and mobile. The stationary phase is solid or liquid (dense materials inside the column), and the mobile phase is liquid. The basis of separation in chromatography methods is the relative affinity of each component to the stationary phase as the mobile phase passes over the stationary phase. In this way, the species that has more affinity to the stationary phase moves along the column at a slower speed, and the species that has more affinity to the mobile phase passes through the column at a faster speed.
Initially, the compounds are dissolved in a solvent and then flow under high pressure inside a chromatography column, thus separating the mixed compounds in the column. The degree of separation depends on the amount of interaction between the dissolved compounds and the stationary phase.
Types of HPLC Machines:
- HPLC
- FPLC
- UHPLC
- HPLC PREPARATIVE
Applications of HPLC Machines:
HPLC is a common and very powerful chromatography technique with applications in areas such as pharmaceuticals, bioanalysis, food and beverage, clinical, forensic, environmental, and drug development laboratories.
- Pharmaceutical Applications
- Environmental Applications
- Forensic Applications
- Food and Flavor
- Clinical Applications
Main Parts of the Machine and Schematic of How It Works:
- Solvent Delivery System (Pump)
- Injector
- Column
- Detector
- Computer for Data Evaluation
Advantages:
- HPLC is very easy to use.
- The analysis time is very short.
- In the HPLC system, the two stationary and mobile phases act with a competitive mechanism, which leads to accurate analyzes.
- In this method, for each group of specific compounds, there are special selective detectors that increase the quantity and quality of the analysis in the best possible way.
- Unstable and non-volatile organic compounds are easily analyzed.
- Separation, purification, and identification of proteins and organic compounds, especially pharmaceutical compounds, are well done with this method.
- Quantitative analysis is possible with high sensitivity and accuracy.
- Determining the structure of polymers
- Comparing the structure of different proteins
- Any impurity with different polarities is detectable.
- The presence of impurities with very high molecular weight is detectable.
- The presence of water in the test samples does not cause any problems during the analysis.
- Ease of reproducibility is one of the major advantages of the HPLC system.
Disadvantages:
- Problem in electronic lines
- Problem in the quality of the chromatography column
- Problem in the mobile phase flow
- Problem in the chromatography temperature
- Problem in the pH of the mobile phase flow
- Problem in how the sample is injected
- Problem in the type of detector
- Problem in quality control of the analysis
- Problem in analyzing the results
Conclusion:
High-performance liquid chromatography is a suitable method for separating, measuring, and determining the type of materials. This technique, in combination with other methods and advanced detectors such as mass spectrometry, has many applications in various sciences. Choosing the mobile and stationary phases, choosing the detector, and determining the flow rate of the mobile phase are among the basic issues in setting up a suitable HPLC method.