High Performance Liquid Chromatography (HPLC) utilizes a mobile liquid phase with a dissolved analyte and a packed colum n stationary phase, for which analyte molecules have different degrees of affinity. The chromatographic separation operation is performed at high pressure. Infinita Lab, USA offers this test through its laboratory network, to clients in the USA and across the world.... Read More
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High Performance Liquid Chromatography (HPLC)
High Performance Liquid Chromatography (HPLC) utilizes a high-pressure chromatographic column to separate components of liquids or solids dissolved in liquids, followed by detection and analysis. The molecular species of interest (termed analyte) together with the solvent and other components (termed matrix) form the ‘mobile phase’ that flows through the column.The Stationary phase in the column can be a porous solid, stagnant liquid, a liquid layer on the solid phase, or an interfacial layer between liquid and solid.
In HPLC, the analyte molecules are partitioned between mobile and stationary phases, depending on their relative affinities for the stationary phase. The extent of separation is influenced by the molecular structure of the analyte and properties of stationary and mobile phases. Analyte molecules with greater affinity to the stationary phase are held back for longer durations within the column, while the other molecules are eluted earlier. All eluted molecules are detected when they exit the column, with the signal intensity (peak) characterizing the nature and amount of each molecule. Considerations for mobile phase selection include composition, solubility, UV transparency, viscosity, and miscibility with other solvents. Stationary phases are typically porous particles made of inorganic silica compounds, Aluminum oxide or polymeric beads packed into a column.
The HPLC equipment includes injector arrangement, high pressure pump, column, detector and computer based data acquisition, control and analysis. The major types of liquid chromatography include normal phase chromatography, reverse phase chromatography, ion exchange, size exclusion chromatography, and affinity chromatography. Various types of detectors can be used, such as differential refractive index (DRI), UV, fluorescence, radiochemical and electrochemical. HPLC can also be coupled to Mass spectrometer or NMR for further analysis.
The HPLC technique is widely used for separation, purification and analytical applications in academic and industrial research, especially for mixtures of complex synthetic and natural organic molecules.
Common Uses of High Performance Liquid Chromatography (HPLC)
Molecular weight determination of complex organics
Molecular weight distribution determination
Separation and purification of organic compound
Forensics
Pathology testing
Drug evaluation
Proteins fractionation
Environmental testing
Advantages of High Performance Liquid Chromatography (HPLC)
High sensitivity
Identifies complex molecules
Well defined separation.
Wide range of solvents and column packings available
Small quantity of sample.
Limitations of High Performance Liquid Chromatography (HPLC)
Structural information on substances separated by HPLC is limited by the detector system employed.
Compounds with similar structure and polarity can elute simultaneously making identification difficult
In normal phase HPLC, silica packing can be affected by water or protic organic solvents which change the hydration state of the silica.
Industrial Applications of Gel Permeation Chromatography (GPC)
ASTM E572 test method covers the analysis of stainless and alloy steels by Wavelength Dispersive X-ray Fluorescence Spectrometry (WDXRF). It provides rapid, multi-element determinations with sufficient accuracy to assure product quality.
The ASTM D2674 is a standard test method for the analysis of sulfochromate etches solution used in the surface preparation of aluminum. The ASTM D2674 standard specifies a method for determining the efficacy of an etchant used to prepare the surface of aluminum alloys for subsequent adhesive bonding.
An immunological method for quantization of Hevea Natural Rubber (HNRL) proteins using rabbit anti-HNRL serum. Rabbits immunized with HNRL proteins react to the majority of the proteins present, and their sera have the capability to detect most if not all the proteins in HNRL.
ASTM G65 measures the resistance of metallic materials to abrasion using the dry sand/rubber wheel apparatus. The quality, durability, and toughness of the sample are determined using this test. Metallic materials are ranked in their resistance to scratching abrasion under a controlled environment.
ASTM E2141 test methods provide accelerated aging and monitoring of the performance of time-dependent electrochromic devices (ECD) integrated in insulating glass units (IGU). This test helps to understand the relative serviceability of electrochromic glazings applied on ECD.
ASTM C724 test method is used in analyzing the quality and ease of maintenance of a ceramic decoration on architectural-type glass. This test method is useful in the acknowledgment of technical standards.
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