Understanding Flowthrough, Wash and Eluate Fractions

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In summary, chromatography is a process used to separate substances from a mixture. It involves applying a sample to a column and then moving it along the column as different substances adsorb to the column. Eluate fraction is the final product, and it's the one you're most likely wondering about.
  • #1
indoubt
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chromatography:

we have to collect the fractions from the column. i wonder what is the difference between;

flowthrough fraction, wash fraction and eluate fraction?

i think i know what it means with eluate fraction, but what about the other two?

hope for replies!
thanks a bunch!
 
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  • #2
It seems there are a few folks posting here working on similar topics in class (i.e., chromatography). Why don't you try to help each other out a bit? You'll learn it better to try answering each other's questions (it's like practicing for a quiz or test).

Please start out by telling us what you think the eluate fraction is, since that's the one you know.
 
  • #3
I'm not sure quite what you mean, but I know chromatography has to do with the substances in a mixture's affinity to the paper. I hope this helps you out a little.

Paden Roder
 
  • #4
indoubt, just think about the different steps you go through during chromotography when you apply your sample to the column. You apply your sample, you wash it, you elute it. Washing takes away non-binding or low-affinity binding molecules, eluting is a wash step where the high-affinity molecules binding to the column will lower their affinity and wash out.
 
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  • #5
PRodQuanta said:
I'm not sure quite what you mean, but I know chromatography has to do with the substances in a mixture's affinity to the paper. I hope this helps you out a little.

Paden Roder

You're thinking of thin layer chromatography (TLC). Indoubt is asking about column chromatography. The basic principles are similar in terms of adsorption of a compound to a matrix, but column chromatography takes it a further, allowing you to not only separate your sample, but collect it in a more purified form so you can then use it for something else.

Here's a website with some basic column chromatography methods and pictures of the procedure. At the end, it demonstrates using TLC to confirm successful separation of the components of the original mixture. http://www.wfu.edu/academics/chemistry/courses/CC/
 
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FAQ: Understanding Flowthrough, Wash and Eluate Fractions

What is flowthrough, wash, and eluate fractionation?

Flowthrough, wash, and eluate fractionation is a process used in chromatography to separate and purify different components in a mixture. It involves passing a sample through a chromatography column, where the different components will interact differently with the stationary phase and elute at different times. This results in the sample being separated into three fractions: flowthrough, which contains components that did not bind to the stationary phase, wash, which contains weakly bound components, and eluate, which contains strongly bound or captured components.

How is flowthrough, wash, and eluate fractionation used in scientific research?

Flowthrough, wash, and eluate fractionation is commonly used in scientific research for protein purification, isolation of DNA or RNA, and separation of complex mixtures for analysis. It is also used in the production of biopharmaceuticals and in various biotechnology processes.

What factors affect the separation of flowthrough, wash, and eluate fractions?

The type and strength of the stationary phase, the properties of the components in the mixture, the flow rate of the sample, and the composition of the elution buffer are some of the factors that can affect the separation of flowthrough, wash, and eluate fractions. Temperature, pH, and ionic strength can also have an impact on the separation process.

How can flowthrough, wash, and eluate fractions be analyzed?

The fractions can be analyzed using various techniques such as gel electrophoresis, mass spectrometry, or spectroscopy. These methods can provide information about the size, shape, and chemical properties of the components in each fraction, helping to identify and characterize them.

What are some limitations of flowthrough, wash, and eluate fractionation?

One limitation is that not all components in a mixture may be separated using this technique, as some may have similar interactions with the stationary phase. Another limitation is that the separation may not be completely pure, and some components may still be present in other fractions. Additionally, the process can be time-consuming and may require specialized equipment and expertise.

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