Kiran Ukey*
Rugved Sathawane
Jayashri Dandale
Indrajeet Gonjari
Pratiksha Rajguru
Kajal Bansode
- High Performance Liquid Chromatography
HPLC is powerful and widely used approach for isolating, determining, and measuring particular components in a liquid mixture. [1] When assessing new formulations, monitoring reaction changes throughout synthesis processes or scale up, verifying the peak purity of novel chemical entitis, and performing quality control and assurance on the finished drug products, HPLC is the preferred technique. [2] It is a more advanced form of a liquid chromatography that uses high pressure to move a solvent (the mobile phase) through a stationary phase packed column. HPLC has isolated each individual chemical component from the sample mixture based on its unique affinities for the mobile phase or adsorbent substance in the column, causing various constituents to separate in the column, causing various constituents to separate as they travel at different velocities. [3]
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- Principle
HPLC works on the basis of separating components interactions with a stationary phase as they get carried by a mobile phase. Small particle size of stationary phase which gives high surface area to make separation more specific and precise. The use of micro syringes allows samples to be injected into pumps that provide high pressure flow of the mobile phase. [4] When small volume of analyte is injected into column the components will move with different affinity in column and separate out with different retention time and on recorder will give distinct and resolved peaks which is used in analysis of analyte. [5]
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- Classification of HPLC can be done as [6]
HPLC is often divided into two subclasses according on the mode of operation:
- NP-HPLC
The term “Normal Phase High Performance Liquid Chromatography” (NP-HPLC) refers to methods where the mobile phase is less polar than the stationary phase. In NP- HPLC SiO2, NH2, -CN, NO2, ALO3 and diol are used as stationary phase and cyclohexane [7-8]
- RP-HPLC
In RP-HPLC mobile phase used is polar or slightly polar, but the SP is non poIar. Separation is primarily based on hydrophobic interactions. [9] Non-polar analytes in the polar mobile phase are attracted to and interact effectively with non-polar SP, leading to longer retention. Polar analytes have weak interactions with the SP and elute quickly as they are more soluble in the polar MP. [10]
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- Instrumentation
The common parts of HPLC instrumentation are
- Solvent reservoir
- Pumps
- Sample injector
- Column
- Detector
Figure 1: Schematic diagram of HPLC
- Solvent reservoir
A reservoir made of glass holds the contents of the mobile phase. The polar and non- polar liquid components that make up the mobile phase, or solvent, in HPLC are often mixed together, and the amounts of these components vary based on the sample’s makeup. Usually polar and non-polar solvents are stored separately in different container and used in proper ratio in isocratic and gradient elution. [11-12]
- Pumps
Pumps are designed to prevent pulsation during the change in composition of mobile phase. HPLC pump continuously pump the mobile phase towards the column with constant pressure and constant flow rate of pump may up to 200 bar depending upon flow rate. Depending on needs of the analysis, the operational pressure limits can vary greatly, ranging from 2000 to 5000 psi in normal analytical operation. [13-14] Three commonly used pump types are
- Constant pressure pump: This uses pressure from a gas cylinder to generate a steady, continuous flow rate across the column. The solvent chamber can be quickly refilled due to the valve configuration. To produce high liquid pressures, a low-pressure gas source is required.
- Syringe type pump: The column receives the steady flow rate through a controlled anchor mechanism. Adjusting the motor’s voltage determines the solvent supply rate. Significant drawbacks include restricted solvent storage and restricted gradient operation.
- Resiprocating piston pump: These uses the rotating action of a piston in a hydraulic chamber to deliver the solvents. Because one pump is in the delivery cycle and the other is in the filling cycle, reciprocating pump systems provide smooth solvent supply. Both gradient operation and high pressure output at a consistent flow rate are achievable.
- Sample Injector
Sample injector used to add the sample to the active mobile phase. Liquid samples with a volume of 0.1-100 mL can be loaded into an HPLC injector with excessive pressure (in the range of to 4,000 psi) and satisfactory reproducibility. When a user has to detect a more number of samples, an auto sampler is an automatic version. Samples are injected into the mobile phase stream at a fixed volume using injectors. To maintain a high degree of accuracy, injection must be inert and repeatable. [15-16] The sample can be introduced into the injection port in three crucial ways.
- Loop Injection: In this type, fixed volume loop injector is used to introduce a fixed quantity of volume.
- Valve Injection: This makes an use of an injection valve to introduce an alterable volume.
- On column Injection: In which, a syringe is used to insert a varied volume through a septum.
- Column
Made mostly of clean stainless steel, columns have internal dimensions of 2 to 5 mm in width and 50 to 300 mm in length. Columns are packed with a SP that contains particles between 3 to 10 µm. Microbore columns have internal diameter of less than 2 mm. During the analysis, maintaining a constant temperature for both the column and the MP is adequate. [17] Various column types are- 1) Guard columns and 2) Analytical columns [18]
Guard column: It prolongs the lifespan of analytical column by eliminating impurities and particulate, matter from solvents. It has same composition compared to analytical column, but it has large particle size.
Analytical column: It is referred to as heart of HPLC, as from it mobile phase continuously passes. The length of column can be vary from 10 to 30 cm with diameter of 4 to 10 mm.
- Detector
Every molecule that elutes from the chromatographic column can be identified by the HPLC detector. The detectors include electrochemical, ultraviolet spectroscopy, mass spectrometric, fluorescence, and evaporative light scattering detectors are utilized. Detector supplied an output from the detector to a computer or recorder, which produces the graph, or a liquid chromatogram of the detector output. Both the necessary sensitivity and a particular response are provided by a detector for the components that the column separates. [19]
Table 1: Detector and their applications
|
|
Detector |
Analytes |
Solvent requirement |
|
1 |
UV Visible |
Any with chromophores |
UV grade non-UV absorbing |
|
2 |
Fluorescence |
Fluorescent |
UV grade non-UV absorbing solvents |
|
3 |
Refractive index (RI) |
Compound with a different RI to that of mobile phase |
Cannot run mobile phase gradients |
|
4 |
Conductivity |
Charged/ polar compounds |
Mobile phase must be conducting |
|
5 |
Mass spectrometer (MS) |
Broad range of compounds |
Must use volatile solvent and volatile buffers |
1.4 Application of HPLC [20]
1. Pharmaceutical application
HPLC has a high linear dynamic range and reliable quantitative precision and accuray, it can be used to quantify various substances in a single run. A useful method for preparing samples for solid dosage forms in aqueous solutions that have been altered with acetonitrile or methanol. There are several ways to separate chiral substances into their respective enantiomers using HPLC. Precolumn derivatization is one method of creating diastereomers.
2. Manufacturing
There are several uses for HPLC in experimental and therapeutic science. This technique is commonly used in pharmaceutical manufacturing as it is an accurate way to determine and confirm the purity of the product. HPLC is not often the primary method used in the production of bulk pharmaceutical compounds, although its ability to yield very pure and superior products. Unfortunately, HPLC tends to increase specificity, precision and accuracy at the expense of increased cost.
3. Research [21]
Research may determine the concentration of potential medicinal candidates, such as asthma drugs and antifungal treatments, using similar assays techniques. When attempting to determine the identify of a species, this method necessitates the use of standard solutions, because purity is crucial in research, it is employed as a technique to verify the outcomes of synthesis procedures. It is definitely useful in observing a variety of species in sample collection also.
4. Medical
Drug analysis is one application of HPLC in medicine, but it is more closely related to nutrient analysis. The most common medium for detecting medication concentrations is urine, but usually medical analyses utilizing HPLC use blood serum as the sample. Other techniques, such as immunoassays, for detecting chemicals that are relevant for clinical research has been evaluated against HPLC. In one instance, the sensitivity of HPLC and competitive protein binding assays (CPBA) for vitamin D detection was evaluated.
- Foods
High-performance liquid chromatography has improved food analysis in ways that are desired. In general, food matrices are complicated, and extracting analytes is a challenging process. Trace elements often contain both unwanted and useful components, and conventional separation and evaluation techniques are not accurate or precise enough to make matters more problematic.
1.5 System Suitability Parameters
A crucial component of the liquid chromatographic approach is a system suitability test. They are used in analysis to make sure that the chromatographic system has adequate resolution and reproducibility. The test’s foundation is the idea that the apparatus, electronics, investigative process, and tester under analysis form a single, integrated system that should be assessed as such. [22] System performance before or during analysis is confirm by determining the parameters such as resoIution, pIate count, reproducibility and taiIing factor. [23]
- Retention time (RT)
Retention time is the interval of time between the injection site and peak maximum appearance. Additionally, it can refer to the time it takes for half of a component to come out from a column. The measuring units are minutes and seconds.
- Theoretical plates (N)
An alternative name for it is column efficiency. Wherever the distribution of the tester between liquid-liquid or solid- solid segment takes place, a column can be seen of as consisting of a large no. of theoretical plates. To determine TP formula is given below: N=16RTW2
Where RT= Retention time and w= Width at the peak’s base.
10.5281/zenodo.15606059