High-performance liquid Chromatography is now an extremely versatile and effective separation and analytical method through the years. It is a sophisticated form of liquid chromatography LC. Rather than introducing the solvent to the pillar and letting it drip down under the influence of gravity, in HPLC, the sample is forced through the column under high pressures of almost 400 atm, leading to quicker and more efficient separation. This technique is also known as high-pressure liquid chromatography.
- The Principle of HPLC
HPLC follows the same basic principle as chromatography. Various elements in the sample have varying affinities to the adsorbent material. This causes a difference in the flow rate for each element which leads to their separation because they come from the column. The only difference is the speed and sensitivity of HPLC are much greater than that of LC because of the use of high pressure. The size of pressure applied depends upon several factors like the length and width of the column, flow rate, size of particles at the stationary phase, and mobile phase composition.
- Columns
HPLC columns are Normally made from stainless steel and are 50 – 300mm long with an inner diameter of 2 – 5mm. They are full of all the adsorbents stationary phase of particle size 3 — 10µm.
- Sample Injector
The sample is injected Into the column via an injector that is capable of handling sample volumes in the range of 0.1 – 100mL under high pressures of up to 4000psi.
- Reservoir
The solvent or the Mobile phase is put in a glass reservoir. It is generally a combination of polar and non-polar liquids whose concentrations are determined by the sample composition.
- Pump
The solvent in the Mobile phase is aspirated by a pump from the reservoir and forced through the HPLC column and then the sensor.
- Detector
what is hplc The sensor in an HPLC System can be found at the end of the column and it finds the parts of the sample which elute from the column. Various kinds of detectors like fluorescence, mass-spectrometric, UV-spectroscopic, and electrochemical sensors are used.
- Data collection systems
The signal from the Sensor is received by recorders that are utilized to process, store, and reproduce chromatographic data. The information is interpreted and integrated by means of a computer that produces a user friendly chromatograph.
High Performance liquid chromatography has been extensively employed for the analysis of antibiotics since it is superior to traditional microbiological assays concerning specificity, sensitivity and evaluation period. In this guide, HPLC conditions for the analysis of a number of antibiotics are outlined. For analysis of biological samples, not extraction procedures but also derivatization methods are clarified, if needed. Since it is not feasible to record HPLC methods for many antibiotics in clinical use, only a few have been selected from each class. Where a stereoisomer is present for the antibiotic of curiosity, the HPLC conditions that have the ability to resolve stereoisomers are clarified.