What is the mobile phase in gel filtration chromatography?
In a gel filtration chromatography column, the stationary phase is composed of a porous matrix, and the mobile phase is the buffer that flows in between the matrix beads. The beads have a defined pore size range, known as the fractionation range.
What is a reverse phase chromatography?
Reversed-phase chromatography employs a polar (aqueous) mobile phase. As a result, hydrophobic molecules in the polar mobile phase tend to adsorb to the hydrophobic stationary phase, and hydrophilic molecules in the mobile phase will pass through the column and are eluted first.
What does Blue Dextran do?
Blue Dextran is a high-molecular-weight glucose polymer (original mol wt 2 x 10(6) g/mol) containing covalently bonded Reactive Blue 2 dye (approximately mmol/g dextran). This blue dye is known for its high binding affinity to a wide variety of proteins, with a particularly high affinity for serum albumin.
Is Blue Dextran a protein?
Abstract. A procedure is described to utilize blue dextran-Sepharose as an affinity chromatographic column specific for the super-secondary structure called the dinucleotide fold, which forms the binding sites for substrates and effectors on a wide range of proteins.
What is the difference between reverse phase and normal phase chromatography?
It is typical for normal-phase chromatography on silica that the mobile phase is 100% organic; no water is used. Reversed-Phase HPLC. The term reversed-phase describes the chromatography mode that is just the opposite of normal phase, namely the use of a polar mobile phase and a non-polar [hydrophobic] stationary phase
What is meant by c18?
C18 columns are HPLC (high performance liquid chromatography) columns that use a C18 substance as the stationary phase. C18 HPLC columns are used in environmental sciences and chemical analysis, as well as industries such as pharmaceutical and environmental sciences, to analyze individual parts of chemical mixtures.
What is peak in HPLC?
A chromatogram is a representation of the separation that has chemically [chromatographically] occurred in the HPLC system. A series of peaks rising from a baseline is drawn on a time axis. Each peak represents the detector response for a different compound.
What is c8 column?
The most popular column is C18 column, this is followed by C8 columns. Both C18 and C8 are dedicated reversed-phase columns. C18 and C8 refer to the alkyl chain length of the bonded phase of the column.
What is the meaning of ODS column?
An ODS column is filled with a packing of octadecylsilyl groups (ODS groups or C18 groups) chemically bonded to a silica gel carrier. ODS columns are used for reverse-phase chromatography.
What is meant by carbon loading in HPLC column?
Carbon load is the percent by. Carbon load is the percent by weight of carbon on the stationary phase. It measures how much organic material has been chemically attached to the surface of silica. For a reversed phase column, more carbon load usually means more retention for nonpolar compounds.
What is Endcapped?
In chromatography, endcapping refers to the replacement of accessible silanol groups in a bonded stationary phase by trimethylsilyl groups.
What is tailing and fronting?
Tailing and Fronting of Chromatographic Peaks. The chromatographic peak in (a) is an example of tailing, which occurs when some sites on the stationary phase retain the solute more strongly than other sites. The peak in (b) is an example of fronting, which most often is the result of overloading the column with sample.
What is peak fronting?
Peaks fronting occurs when the sample capacity of the analytical column is exceeded, which can happen in both GC and HPLC experiments. This overloading effect results from poor sample solubility in the stationary phase, the injection of too much sample, or operating at a “k” value (capacity factor) that is too low.
What is the tailing factor?
Definition: Tailing factor. The tailing factor is a measure of peak tailing. It is defined as the distance from the front slope of the peak to the back slope divided by twice the distance from the center line of the peak to the front slope, with all measurements made at 5% of the maximum peak height.
What is the asymmetry factor?
Definition: Asymmetry factor. The asymmetry factor is a measure of peak tailing. It is defined as the distance from the center line of the peak to the back slope divided by the distance from the center line of the peak to the front slope, with all measurements made at 10% of the maximum peak height.
What is system suitability test in HPLC?
USP states:1. System suitability tests are an integral part of gas and liquid chromatographic methods. They are used to verify that the resolution and reproducibility of the chromatographic system are adequate for the analysis to be done.
After injection, a narrow chromatographic band is broaden during its movement through the column. The higher the column band broadening, the smaller the number of components that can be separated in a given time. In other words, the sharpness of the peak is an indication of how good, or efficient a column is.
What is the meaning of system suitability?
Definition: System suitability. System suitability is defined by ICH as "the checking of a system, before or during analysis of unknowns, to ensure system performance." System suitability criteria may include such factors as plate count, tailing, retention, and/or resolution.
Updated: 2nd October 2019