1. Paper Chromatography

In this chromatography technique, the substance is analyzed by allowing the flow of solvents on a a specially designed filter paper. Due to capillary action, the solvent rises up on the paper and due to differential migration of the substances of a mixture, the separation is achieved.

In this type of chromatographic technique, Whatman Paper I is extensively used and this paper is available in different grades. The sample is prepared by using proper solvent and is applied on the paper as bands.

The sample is applied in drops on the spots with the help of a capillary tube. The spotting is done slowly and carefully in such a way that spots occupy only small areas.

Selection of Solvents:

Depending on the nature of the substances, the number of solvents are used for the separation. Using minimum number of solvents is preferable. Solvents used should be shaken well when more than one components of solvents is mixed. and by keeping in mind that, solvents should be used in specific rations to separate different substances.

Chromatogram Development
Image Source: Wikipedia

Location of Spots:

The developed paper is kept on a hot plate for drying for few minutes. There are two types of methods that can be used to locate the spots.

1) Physical Methods, where ultraviolet light and radioactive elements are used.

2) Chemical Methods, where locating reagents are used which may be liquids, solids or gases, here, the colorless spots are made colored by reagents to locate them.

Note: The method described above is ascending paper chromatography, where the solvent flows upwards on the paper by capillary action.
In descending paper chromatography, the solvent is allowed to flow down by gravitational pull on a paper by keeping the solvent at the top of the chromatographic chamber and by capillary action.

Next: 2. Thin Layer Chromatography (TLC)

Comments

Nanodrop

Thermofisher, Nanodrop 1000 Nanodrop is a spectrophotometer which is designed to measure the concentration of nucleic acids at 260nm and Proteins at 280nm present in a very small minute volume. It is also capable of measuring the concentrations of dsDNA, RNA, ssRNA and purified protein. Required sample size is usually 1µL. Nanodrop instruments are usually full spectrum spectrophotometer, i.e., Ultra violet and visible range wavelengths (250nm to 750nm). Nanodrop is commonly used for the quantification of the DNA concentration and RNA concentration in a given sample. However there are many other applications which can be performed on this instrument. It works on the principle of Spectrophotometer or Sample Retention System.... Principle of Nanodrop: "The Nanodrop works on the principle of sample retention system.. When the small volume of sample is introduced, the arm and pedestal will work together and make the sample in the form of colum...

Statistics in Flow Cytometry Data and "MFI" values

The speed of the flow cytometry offers wide range of data points and data plots. Due to its sensitivity and versatility, it has been used extensively. Flow cytometer can detect up to 1000s of cells per second.. But, it should be noted that the flow cytometry also involves the statistics, its significance, calculating the fluorescence intensity.. Statistics in flow cytometry involves, total number of data points acquired, percentage population, most importantly mean and median fluorescence intensity, and others. Most of us get confused about the term "MFI", whether it means mean fluorescence intensity or median fluorescence intensity.. Truth is, both.. It means it can be described as either mean or median fluorescence intensity. Some researchers use Mean for MFI and some use Median for MFI values. (Keep...

Compensation in Flow Cytometry (Fluorescence Spill Over Correction).

The term "Compensation" in flow cytometers refers to the process of correcting fluorescence spill over. The compensation matrix ensures that the fluorescence emitted is detected in a particular detector which is from the fluorescence being measured. As we all know that most of the fluorescence molecules have broad emission range. if more than one one fluorescence is selected, their emission spectra may overlap. To correct this spectral overlap in cytometry data, a mathematical matrix called compensation is used. The Intention of this article is to teach you the compensation from basic theory to practical. This example for FITC and PE fluorescence molecules shows that, Some of the FITC fluorescence is being detected in PE Detector. and Some of the PE fluorescence is being detected in FITC Detectors. Task here is to make sure that in cytometry data, FITC is in FITC detector only and PE is in PE Detector only....

BIOMAG

Search This Blog