High-ph Reversed Phase
High-ph Reversed Phase - Related Videos
03:31
Reversed-Phase High-Performance Liquid Chromatography: A Robust Method for Quantitation of Fluorescently Labeled and Derivatized Sialic Acids Isolated from Mouse Liver
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2025
In this video, we demonstrate the detection of sialic acids, N-acetylneuraminic acid and N-glycolylneuraminic acid, using high-performance liquid chromatography, HPLC, following derivatization with a suitable fluorescent labeling reagent to aid in fluorescence detection.
12:22
The Use of Reverse Phase Protein Arrays (RPPA) to Explore Protein Expression Variation within Individual Renal Cell Cancers
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Cited by 12 •
2013
RPPA enables the protein expression of hundreds of samples, printed on nitrocellulose slides to be interrogated simultaneously, using fluorescently labelled antibodies. This technique has been applied to study the effect of drug treatment heterogeneity within clear cell renal carcinoma.
06:32
Reverse Phase Protein Arrays Based Protein Expression Analysis: A Procedure for Simultaneous Quantification of Expression of Multiple Proteins from Cell Lysate
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2023
This video demonstrates the methodology for performing Reverse Phase Protein Arrays (RPPA) to study protein expression patterns from cell lysate. In RPPA, the lysate containing a mixture of proteins is printed on nitrocellulose slides and proteins of interest are analyzed using fluorescently labeled antibodies.
02:33
Phase Transitions: Sublimation and Deposition
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2020
Some solids can transition directly into the gaseous state, bypassing the liquid state, via a process known as sublimation. At room temperature and standard pressure, a piece of dry ice (solid CO2) sublimes, appearing to gradually disappear without ever forming any liquid. Snow and ice sublimate at temperatures below the melting point of water, a slow process that may be accelerated by winds and the reduced atmospheric pressures at high altitudes. When solid iodine is warmed, the solid sublimes...
02:39
Phase Diagrams
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2020
A phase diagram combines plots of pressure versus temperature for the liquid-gas, solid-liquid, and solid-gas phase-transition equilibria of a substance. These diagrams indicate the physical states that exist under specific conditions of pressure and temperature and also provide the pressure dependence of the phase-transition temperatures (melting points, sublimation points, boiling points). Regions or areas labeled solid, liquid, and gas represent single phases, while lines or curves represent...
FAQs
Alkaline mobile-phase conditions change the ionization state of analytes, including peptides and proteins. Because ionization affects how molecules interact with the nonpolar stationary material, compounds can show different retention and separation behavior than they would under other pH conditions. This pH-dependent change in selectivity is the central reason the method can resolve components that separate differently at low pH.
The two approaches can provide orthogonal selectivity, meaning they distinguish components through different separation behavior rather than reproducing the same pattern. Applying both conditions can therefore separate complementary subsets of peptides or proteins. In biochemical workflows, this multidimensional strategy helps address the complexity of biological samples and supports more comprehensive molecular characterization.
The aqueous-organic gradient determines when compounds elute from the stationary material. As solvent composition changes, analytes with different hydrophobic properties experience different interactions with that material and therefore separate across the run. This ordered elution enables a complex biochemical sample to be divided into fractions for subsequent analysis.
The workflow begins by passing the biochemical sample through a nonpolar stationary phase under alkaline mobile-phase conditions. An aqueous-organic gradient then changes the elution environment, allowing components to emerge according to their interactions with the stationary material. The resulting separation can be used to fractionate peptides or proteins before downstream biochemical or mass spectrometric analysis.
High-ph Reversed Phase fractionation separates a complex peptide or protein mixture into less complex portions before mass spectrometric measurement. Lowering the complexity of each portion can improve peptide identification and makes it possible to examine more components than a single undivided sample might reveal. This supports broader characterization of biological samples.
It is most useful when a biological sample contains many peptides or proteins whose analysis would benefit from prior separation. Fractionation can organize the mixture into simpler portions, while the alkaline selectivity adds information that low-pH reversed-phase separation may not provide. These features support biochemical analysis, proteomics, and more complete characterization of complex samples.