High performance liquid chromatography, also known as high-pressure liquid chromatography, is a chromatographic technique for separating a mixture of substances that is used in biochemistry and analytical chemistry to identify, measure, and purify the mixture's constituent components. A liquid sample is injected into a solvent stream (mobile phase) that flows through a column containing a separation medium (stationary phase). As samples travel through the column, they separate from one another through a process known as differential migration. The HPLC system consists primarily of an infusion pump, a sampler, a chromatographic column, a detector, and a data recording and processing device. Key components include the infusion pump, chromatographic column, and detector.
Martin and Synge described the discovery of liquid-liquid partition chromatography in 1941, laying the groundwork for gas liquid chromatography and high performance liquid chromatography as well. They also proposed the idea of the Height Equivalent to Theoretical Plates, which has since been accepted as a measure of chromatographic efficiency.
TYPES OF HPLC TECHNIQUES:
1. Normal phase Chromatography
2. Reverse phase Chromatography
3. Ion exchange/ Ion chromatography
4. Size exclusion chromatography
5. Bio-Affinity chromatography
6. Partition Chromatography
TYPES OF ELUTION TECHNIQUES:
1. Isocratic separation
2. Gradient separation
An HPLC separation is considered isocratic elution if the mobile phase's composition doesn't change during the process. Changing the ratio of polar to non-polar compounds in the mobile phase during the sample run is frequently the only way to elute every chemical in the sample in a reasonable length of time while retaining peak resolution. This method, called gradient chromatography, is preferred when a sample has constituents with a broad range of polarity. In the case of a reverse phase gradient, the solvent gradually becomes more non-polar after beginning comparatively polar. The most thorough peak separation is provided by the gradient elution, which doesn't take too long.
A gradient elution can separate a sample comprising chemicals with a wide range of polarities in a shorter time period without sacrificing resolution in earlier peaks or excessive widening of later peaks. Gradient elution, on the other hand, necessitates more complex and expensive equipment, as well as greater difficulty in maintaining a consistent flow rate as the mobile phase composition changes constantly. Gradient elution, particularly at high speeds, highlights the limitations of lower-quality experimental apparatus, making results less reproducible in equipment that is already prone to variance. If the flow rate or composition of the mobile phase changes, the results will not be replicable.
The HPLC detector, placed at the end of the column, must register the presence of diverse sample components while excluding the solvent. As a result, there is no universal detector that is effective for all separations. A UV absorption detector is a popular HPLC detector since most medium to large molecules absorb UV radiation. Fluorescence and refractive index detectors are also employed in specialized applications. The combination of an HPLC separation and an NMR detector is a relatively recent invention. This permits the sample's pure components to be identified and measured using nuclear magnetic resonance after being separated by HPLC in a single integrated process.
If the stationary phase is more polar than the mobile phase, the separation is considered normal phase. If the stationary phase is less polar than the mobile phase, the separation occurs in reverse phase. In reverse phase HPLC, a compound's retention period increases as the polarity of the species decreases. The key to effective and efficient separation is to find the right balance of polar and non-polar components in the mobile phase. The goal is for all of the chemicals to elute in as little time as feasible while allowing for the resolution of individual peaks. Typical columns for normal phase separation are filled with alumina or silica. Reverse phase separation is commonly achieved using alkyl, aliphatic, or phenyl-bonded phases. HPLC is useful for both quantitative and qualitative applications, or the identification and quantification of compounds. Nowadays, reverse phase HPLC may be utilized for practically all HPLC separations, with normal phase HPLC being employed extremely seldom. Ion exchange chromatography is a more effective method for separating inorganic ions than reverse phase HPLC (RPLC), which is ineffective for all but a few types of separations. It is unable to separate polynucleotides (which adsorb irreversibly to the reverse phase packing) or polysaccharides (which are too hydrophilic for any solid phase adsorption to occur). Finally, RPLC has limited selectivity and cannot efficiently separate extremely hydrophobic molecules. With a few notable exceptions, nearly all additional compound types can be separated using RPLC.
Comparable simple and aromatic hydrocarbons, even those with only one methylene group separating them, can be successfully separated using RPLC. Simple amines, lipids, carbohydrates, and even pharmaceutically active compounds can all be successfully separated with RPLC. Proteins, peptides, and amino acids can all be separated using RPLC. Lastly, biologically derived compounds are separated using RPLC. In commercial applications, RPLC is frequently used to evaluate the caffeine concentration in coffee products in order to guarantee the quality and purity of ground coffee. HPLC is a useful instrument in an analyst's toolbox, especially when it comes to isolating a substance before additional analysis. The separation is said to be in the normal phase if the stationary phase is more polar than the mobile phase. The separation is reverse phase if the stationary phase is less polar than the mobile phase. A compound's retention period in reverse phase HPLC increases as the species' polarity decreases. Finding the right proportion of polar to non-polar components in the mobile phase is essential for a successful and efficient separation. The objective is for all of the chemicals to elute as quickly as feasible while preserving the ability to resolve specific peaks. Typically, silica or alumina are packed into columns for normal phase separation. For reverse phase separation, alkyl, aliphatic, or phenyl linked phases are usually utilized.