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

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...

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...

Protocol: Sample preparation for Cell Cycle Analysis by Flow Cytometry

Protocol: Preparation of Samples for Cell Cycle Analysis by Flow Cytometry: 1) Suspend approximately million cells (10^6) in 1mL of phosphate-buffered saline solution (1x). And with the help of pipette, gently aspirate about 7-8 times, to make the single cell suspension. 2) Take a centrifuge tube and add 4mL of 70% ethanol. (70% ethanol should be one ice before using). and add 1mL of cells in PBS to this tube. Keep the tubes on ice for minimum 2 hours at 4℃.. This step will fix the cells. ( cells can kept at 4 ℃ for a week). 3) Centrifuge the ethanol fixed cells for 5 minutes at 300g. Carefully take out the supernatant. Do not disturb the pellet. 4) Add 5mL of cold 1x PBS to this pellet and suspend the cells using pipette. 5) Centrifuge the tubes at 300g for 5 minutes. and discard the supernatant. Again do not disturb the pellet. 6) if required, step 5 can be repeated for one more time. 7) Add 1mL of Propidium Iodide (PI...

BIOMAG

Search This Blog