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Chromatography

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Chromatography

We throw a lot of things down the plughole, from food scraps tipped down the sink to wipes and tissue flushed down the toilet. Between them, they contain all manner of substances - preservatives, disinfectants, stabilisers, bleaching agents, and more. If the levels of any of these chemicals get too high, they can damage the fragile ecosystems of our rivers, lakes, and other waterways. So how can we accurately measure the concentration of these potentially harmful substances in wastewater? One such method is chromatography.

Chromatography is a separation and analytical technique used to split a soluble mixture into its component parts.

  • This article is about chromatography in chemistry.
  • We'll start by giving a broad overview of chromatography, including a deep dive into its history, before exploring its underlying principles.
  • We'll then look more closely at different types of chromatography.
  • Finally, we'll discuss the uses of chromatography.

What is chromatography?

Let's say that we have a deep purple ink. Although it may look like it contains just one colour, with chromatography we can split it up into all its separate pigments - blues, reds, perhaps some yellow and green. This is just one example of chromatography. In this case, it is used to separate a mixture. However chromatography can also be used to analyse mixtures - for example, identifying active ingredients in a drug, or finding out about the products of a reaction.

The method of chromatography

There are a few different types of chromatography and each has a slightly different process. However, the overall method stays the same. Chromatography involves the following steps:

  • Take a soluble mixture, known as the solute.
  • Add a small amount of the mixture to a static solid, liquid, or gas. This static medium is known as the stationary phase.
  • Add some sort of solvent. This is known as the mobile phase.
  • The solvent dissolves the mixture and carries it through the stationary phase.
  • Different components of the mixture travel through the stationary phase at different speeds. Because of this, they separate out into clear, distinct spots or bands, that we can view on a chromatogram.

One of the simplest forms of chromatography is paper chromatography. It's quite likely that you have carried it out at school before. Here's what the typical setup for paper chromatography would look like:

Chromatography paper chromatography StudySmarterThe typical setup for paper chromatography. Anna Brewer, StudySmarter Originals

Chromatography was invented in 1900 by the Italian-Russian botanist Mikhail Tsvet, who wanted a way of separating pigments from plant extracts. The word chromatography comes from the Greek words chroma, meaning 'colour', and graphein, meaning 'to write'. Interestingly enough, Tsvet's surname is Russian for 'colour' as well.

Chromatography achieved most of its major breakthroughs in the 1940s and 1950s, thanks to Archer Martin and Richard Synge. In fact, they won the 1952 Nobel Prize in Chemistry for their invention of a particular type of chromatography, known as partition chromatography.

Principles of chromatography

We introduced you to some key words up above, in particular stationary phase, mobile phase, and chromatogram. These are some of the basic principles of chromatography. Let's now explore exactly what they mean.

Stationary phase

The stationary phase is a static solid, liquid, or gel. The solvent carries the soluble mixture up the stationary phase.

The stationary phase is - as the name suggests - well, stationary. It doesn't move. Examples include paper and silica powder.

Mobile phase

The mobile phase is the solvent used to carry the mixture analysed through the stationary phase.

In contrast to the stationary phase, the mobile phase moves. It is a solvent that dissolves the solute you want to analyse or separate, and carries it through the stationary phase.

Chromatograms

Once the chromatography has finished, you'll be left with some evidence of the process. The mixture will have separated out on the stationary phase into different spots or bands. The leftover stationary phase, complete with all of its spots, is called the chromatogram.

A chromatogram is a column or strip of material containing components separated from a mixture by chromatography. It is essentially the output of a chromatography experiment.

For example, in paper chromatography, the stationary phase is a sheet of paper. Once you've finished the experiment, the chromatogram is the paper with its final arrangement of different spots.

Chromatography paper chromatography StudySmarterA chromatogram for paper chromatography. Anna Brewer, StudySmarter Originals

We'll briefly explore paper chromatography in a bit. However, if you can't wait, check out Paper Chromatography for a more detailed look.

Let's focus on two new terms: relative affinity, and retention factor.

Relative affinity

Components within the solute mixture move at different speeds through the stationary phase. This is all to do with their relative affinities to the two phases.

In chromatography, relative affinity describes how well a component is attracted to either the stationary or mobile phase. It determines how quickly the component moves through the stationary phase.

Components that experience a stronger attraction to the stationary phase are said to have a stronger affinity to the stationary phase. They are less soluble in the solvent and are more attracted to the static medium. The mobile phase can't carry them as easily - this means that the components move more slowly through the stationary phase.

In contrast, components that experience a stronger attraction to the mobile phase are said to have a stronger affinity to the mobile phase. They are more soluble in the solvent and less attracted to the static medium. The mobile phase is really good at carrying them about, so these components travel more quickly through the stationary phase.

What causes these differing relative affinities? As we mentioned, it is all to do with attraction to the two phases.

Let's say that the stationary phase consists of a polar molecule. This means it experiences permanent dipole-dipole forces between itself and any other polar components in the starting mixture. On the other hand, we'd typically use a nonpolar solvent. This means there would only be weak van der Waal forces between the solvent and the components. Polar components therefore experience a much stronger attraction between themselves and the stationary phase, than themselves and the mobile phase. They are more attracted to the stationary phase, are less soluble in the solvent, so we can say that they have a greater affinity to the stationary phase.

Retention factors

We now know that different components travel at different speeds through the stationary phase due to their relative affinities to the two stages. This means that in a given time period, different components will travel different distances. We can see this because they show up as clear, distinct spots.

We use the ratio between the distance travelled by each spot and the total distance travelled by the solvent to calculate things known as retention factors, or Rf values.

Rf values are important because they help us identify components. A particular component always produces the same Rf value under a certain set of conditions - i.e., if things like temperature, mobile phase, and stationary phase are exactly the same. If we calculate the Rf value for a particular component, we can compare it to values in a database to find out the identity of this unknown substance.

To find Rf values, divide the distance travelled by each component by the total distance travelled by the solvent.

Chromatography Rf values StudySmarterCalculating Rf values. Anna Brewer, StudySmarter Originals

In the example above, the blue spot has travelled 7.4 cm and the solvent has travelled 9.8 cm. To calculate its Rf value, we use the following equation:

Rf values have no units and are generally given to two decimal places.

Some types of chromatography use retention times instead of retention factors. These measure the time taken for each component to move through the stationary phase.

Now that you know about relative affinities, can you predict how Rf values vary between components?

  • Components with a greater affinity to the stationary phase travel more slowly through the medium. They move less far in a given time period and so have a lower retention factor.
  • Components with a greater affinity to the mobile phase move more quickly through the medium. They move further in a given time period and so have a higher retention factor.

Types of chromatography

There are multiple types of chromatography. They differ in their mobile and stationary phases and their methods, but all follow the principles outlined above. Types include:

  • Thin-layer chromatography (TLC).
  • Paper chromatography.
  • Gas chromatography.
  • Column chromatography.
  • Partition chromatography.
  • High-performance liquid chromatography (HPLC).

Here's a handy table to help you compare them.

NameStationary phaseMobile phaseNotes
TLCPlate covered in thin layer of silica gelLiquid solventAccurate.Uses small samples.
PaperPaperLiquid solventCheap.Uses small samples.
GasTube filled with silica powderGaseous solventRequires heating.Extremely sensitive.
ColumnColumn filled with silica powderLiquid solventMostly used for separation.
PartitionColumn filled with liquid held in place on a solid supportLiquid solventSeparation occurs due to differing solubilities in the two liquids.
HPLCColumn filled with silica powderLiquid solventUses pressure to speed up the process.

If you want to find out more about some of these types of chromatography, we've got you covered - check out Thin-Layer Chromatography, Column Chromatography, Gas Chromatography, Paper Chromatography and Ion Exchange Chromatography.

Uses of chromatography

We've already discussed some of the uses of chromatography - for example, analysing wastewater. But chromatography has a myriad of different applications. These include:

  • Drug detection in urine.
  • Analysing metabolites in bodily fluids.
  • Extracting pigments from plant extracts.
  • Isolating active ingredients in drugs.
  • Purifying compounds.
  • Separating mixtures of proteins, amino acids, or nucleotides.
  • Quality control of alcoholic drinks.

Do you remember the horse meat scandal from 2013? Some meat products sold as beef were actually found to contain horse. But scientists were only able to prove the meat's origins by blending it up and analysing it using chromatography alongside samples of pure horse meat.

Chromatography - Key takeaways

  • Chromatography is a separation and analytical technique used to split a soluble mixture into its component parts.
  • Chromatography involves a static medium, known as the stationary phase, a solvent, known as the mobile phase, and a mixture that you want to analyse. The mobile phase carries the mixture through the stationary phase. Different components within the mixture have different affinities to each of the phases and so move at different speeds through the stationary phase. This causes them to separate.
  • A chromatogram is a column or strip of material containing components separated from a mixture by chromatography. It is essentially the output of a chromatography experiment.
  • Relative affinity describes how well a component is attracted to either the stationary or mobile phase. It determines how quickly the component moves through the stationary phase.
  • Retention factors (Rf values) show the ratio between the distance travelled by each component and the distance travelled by the solvent.
  • Under the same conditions, the same component always produces the same Rf value.
  • Types of chromatography include TLC, paper chromatography, column chromatography and HPLC.
  • Uses of chromatography include separating mixtures, wastewater analysis, and isolating active ingredients in drugs.

Frequently Asked Questions about Chromatography

Chromatography is a separation and analytical technique used to split a soluble mixture into its component parts.

Chromatography has a variety of uses. These include analysing waste water, separating mixtures, purifying compounds and extracting active ingredients from drugs.

Chromatography has a few basic principles. In chromatography, a solvent, known as the mobile phase, carries a soluble mixture up a static medium, known as the stationary phase. Different components within the mixture have different relative affinities to each of the phases. This causes them to travel through the stationary phase at different speeds and separate out into distinct spots or layers.

In chromatography, different components within the mixture have different affinities to the mobile and stationary phases. This means that some components move faster through the stationary phase than others and causes the components to separate out into distinct spots or layers.

There are multiple different types of chromatography. These include TLC, paper chromatography, ion exchange chromatography and column chromatography.

Final Chromatography Quiz

Question

What is the stationary phase in TLC?

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Answer

A thin metal or plastic plate covered in a layer of silica gel or alumina.

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Question

What is the mobile phase in TLC?

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Answer

A solvent.

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Question

How do you view colourless components on TLC chromatograms? Give two methods.

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Answer

  • Add a substance that fluoresces under UV light to the solvent and view the chromatogram under UV light.
  • Spray the chromatogram with a substance that forms coloured compounds, such as ninhydrin.

Show question

Question

Give three advantages of TLC over paper chromatography.

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Answer

  • It is faster.
  • It can use smaller samples.
  • It is more reliable.
  • It is easier to handle as the plates are more sturdy than paper.

Show question

Question

A mixture contains an alcohol and an alkene. Predict which molecule will travel further up the plate in TLC.

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Answer

The alkene

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Question

Substance A has an Rf value of 0.8. Substance B has an Rf value of 0.4. Predict which substance has a higher affinity to the mobile phase.

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Answer

Substance A

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Question

In TLC, substance A travels 12.3 cm up the plate. The total distance travelled by the solvent is 15.8 cm. Calculate substance A's Rf value. 

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Answer

0.78

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Question

What is column chromatography?

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Answer

A separation technique used to separate single components from a mixture dissolved in a fluid. 

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Question

Give two uses of column chromatography.

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Answer

(E.g.)

  • Isolating active ingredients.
  • Separating mixtures such as a mix of amino acids.
  • Isolating metabolites from biological samples.
  • Removing impurities.

Show question

Question

What is the stationary phase in column chromatography?

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Answer

A thin plate covered in silica gel.

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Question

What's the mobile phase in column chromatography?

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Answer

A solvent.

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Question

Which of the following do you need for column chromatography?

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Answer

A glass column.

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Question

What's an eluent?

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Answer

Another term for the solvent, also known as the mobile phase.

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Question

Describe the steps involved in column chromatography.

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Answer

  1. Place a layer of mineral wool at the bottom of the glass tube.
  2. Fill the tube with a solid such as silica powder, which is your stationary phase.
  3. Saturate the solid with a solvent, your mobile phase.
  4. Pour your sample mixture into the top of the column.
  5. Pour the solvent in a continuous stream down the column and collect the effluent that flows out of the bottom.

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Question

What is retention time?

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Answer

Retention time is the time taken for a particular component within the sample mixture to travel through the column. In other words, it is the time from sample injection to component detection.

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Question

A sample contains a carboxylic acid and an alkane. Which has a shorter retention time? (In this experiment, the stationary phase is silica powder and the mobile phase is a nonpolar solvent.) 

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Answer

The alkane.

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Question

Which of the following has a greater affinity to silica powder?

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Answer

An amine.

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Question

Give two advantages of column chromatography.

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Answer

(E.g.)

  • You can analyse large amounts of a sample.
  • The stationary phase is generally low cost and easy to dispose of.
  • It has a wide variety of potential applications.

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Question

What is the mobile phase in paper chromatography?

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Answer

A solvent.

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What is the stationary phase in paper chromatography?

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Answer

Chromatography paper.

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Question

What do you use to draw the base line in paper chromatography?

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Answer

Pencil.

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Question

In paper chromatography, the starting solvent level should be _____.

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Answer

Below the pencil line containing the spot of mixture.

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Question

In paper chromatography, components that travel faster up the paper have a greater affinity to the ______.

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Answer

Mobile phase.

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Question

In paper chromatography, more soluble components have a _____ affinity to the mobile phase.

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Answer

Stronger.

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Question

What are the units for Rf values?

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Answer

cm

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Question

What are the advantages of paper chromatography?

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Answer

  • It is fast.
  • It is cheap.
  • It is simple.
  • It uses small amounts of the mixture.
  • It can analyse organic and inorganic compounds.

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Question

What are the uses of paper chromatography?

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Answer

  • Separating mixtures.
  • Obtaining pure compounds.
  • Removing impurities.
  • Analysing drugs.

Show question

Question

What is chromatography?

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Answer

A separation and analytical technique used to split a soluble mixture into its component parts. 

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Question

What are the two phases in chromatography?

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Answer

Mobile phase.

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Question

What is relative affinity?

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Answer

Relative affinity describes the strength of the attraction between the component, the stationary phase, and the mobile phase.

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Question

In paper chromatography, the stationary phase is polar and the mobile phase is nonpolar. Which of the following statements are correct?

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Answer

Polar components will have a greater affinity to the stationary phase.

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Question

Give three uses of chromatography.

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Answer

Separating mixtures, drug detection, analysing wastewater, purifying compounds, isolating active ingredients.

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Question

Which of the following are types of chromatography?

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Answer

Gas

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Question

What is ion exchange chromatography most commonly used for?

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Answer

Separating charged molecules

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What is the stationary phase in ion exchange chromatography?

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Answer

An ionically charged matrix.

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Question

How many different mobile phases are used in ion exchange chromatography?

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Answer

Multiple

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Question

A typical mobile phase used in ion exchange chromatography consists of _____.

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Answer

A buffer solution.

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Question

The mobile phases used in ion exchange chromatography vary in _____.

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Answer

Ionic strength

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Question

What is gas chromatography?

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Answer

An analytical technique that analyses components of a sample in the gas phase.

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What is a phase?

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Answer

It is a state such as gas, liquid, or solid.

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Question

What is the mobile phase in GC?

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Answer

The mobile phase is an inert gas which carries the analyte components along the column towards the detector.

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Question

What is the stationary phase in GC?


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Answer

This is the phase which doesn't move since it is attached to the column.

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Question

What is the retention time?

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Answer

The time taken by a compound in a mixture to pass through the chromatography column and reach the detector.

Show question

Question

What are the key steps involved in a GC-MS system?

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Answer

The sample is injected.


The chemicals in the mixture separate at the GC column and exit it one by one. 


The separated components are sent into a mass spectrometer which acts as a detector. 


A detailed mass spectrum is produced by the spectrometer for each component and this can be used to identify the components, as well as showing what the original sample consisted of.

Show question

Question

What are three advantages of gas chromatography?


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Answer

-  It has a high level of sensitivity, meaning a wider range of compounds can be detected.

- Gas chromatography is an easy technique that gives accurate data and results.

-  It has a high resolution which allows for different types of compounds to be separated.

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Question


Describe the capillary column

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Answer

A wire-like object on which the stationary phase is coated. It's usually made up of materials such as stainless steel, borosilicate glass, or fused silica. The column is coated with a polyamide on the outer surface. Most capillary columns are approximately 1.5 - 10 m long and 2-4 mm thick.

Show question

Question

What acts as the mobile phase in gas chromatography?


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Answer

 An unreactive gas such as helium

Show question

Question

What is a chromatogram?

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Answer

A chromatogram is the result of a chromatography run. It shows the results after the components of a gaseous mixture are separated. 

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Question

Where is the sample injected in GC?

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Answer

Sample injection port

Show question

Question

What does the area under each peak in a chromatogram represent?

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Answer

The area under each peak on the chromatogram represents the relative amounts of each component.    

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