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Joshua Martinez

English

363, Bruce

Chromatography

Chromatography is a laboratory technique in which a mixture is separated into smaller

components when passed through an inert material using chromatographs. This laboratory

technique measures the retention times for analytes, a chemical substance that is being

identified, to pass through a column of absorbents. There are several types of chromatography

techniques that use different phases. The three major methods of chromatography are Column

Chromatography, Gas Chromatography and Thin-Layer Chromatography. This laboratory

separation method consists of mainly liquid substances, but gases and solids can be used

depending on the technique, which are referred to as the mobile phases when mixed with a

solvent. A solvent is a liquid which has various components dissolve, forming a solution

(Britannica, 2011). Essentially all of the separation techniques utilize a mobile phase that passes

through a stationary phase that sits in the column of the chromatograph. The stationary phase

selectively absorbs material in the mobile phase as it passes through.

History

In the mid 1800’s, a professor and his student by the names of Christian Friedrich

Schonbein and Friedrich Goppelsroeder were the first to publish attempts to study the rate that

different substances travel through a filter paper ("History of chromatography", 2019). Instead
of today’s methods which measure absorption as being responsible for the movement, they

thought capillary action was the reason for the movement of substances. Capillary action is the

movement of liquids through narrow spaces without any external force assisting it (Saig, 2018).

The technique of column chromatography separation was first used in Russia by Mikhail Tsvet

in the early 20th century. He primarily used this technique to separate different plant pigments,

which gave the technique its name. The pigments that Tsvet was separating were chlorophyll

(green pigments), carotenes (orange and yellow pigments), and xanthophylls (yellow pigments)

("Chromatography", 2019).

Later on, in the 20th century new types of this technique were developed. Two scientists

by the name Archer John Port Martin and Richard Laurence Millington Synge further developed

chromatography by establishing principles of partition chromatography. Partition

Chromatography is a type of liquid Chromatography which separates the components based on

solubilities (International Union, 2006). These two scientists went on to win the Nobel prize in

Chemistry in 1952. From these new principles several other chromatographic techniques were

developed ("Chromatography", 2019).

Column Chromatography

Column Chromatography, one of the most common techniques, separates specific compounds

depending on the type of stationary phase is used to pack the column. The column can separate the

eluent, mobile phase consisting of a solvent that dissolves the sample, based on the molecule’s

hydrophobicity (affinity for water substances), polarity (the “pull” atoms have on their electrons), or
electrostatic charge (either a positive or negative charge of the atom) (Torres, 2016). Different sized

columns can be used when packing the stationary phase depending on the amount of substance you are

attempting to separate. After packing the column, the eluent is placed on top of both the sample and

stationary phase. The bottom end nozzle is then opened to allow the sample to flow through the

column. Depending on molecular properties, the analyte which has a weaker interaction with the

stationary phase will pass through it more rapidly while the analytes with a stronger interaction will take

a longer time to pass (Torres, 2016). The time that it took the desired analyte to pass will then aid the

researcher in determining what kind of molecules were compromised of the analyte.

The above diagram is a depiction of column chromatography. The first column on the left is the
beginning of this technique with only the stationary and sample loaded. The mobile phase is then
placed on top on the sample forming the eluent and then as time passes the different analytes pass
through separately depending on their interactions. The first analyte is then collected as it passes
through, then the second is collected as it passes through.
Gas Chromatography (GC)

To the left is a diagram of a basic gas


chromatograph. Starting from the top,
we have the sample gas which is carried
by an inert gas, or unreactive gas, which
forms the mobile phase. The mobile
phase is then carried passed the
stationary phase in the column. The
detector then determines the retention
times of the contents that make it
through which will help the researcher
determine what the substance is.

Gas Chromatography, GC, is a method of separation used in order to separate volatile

compounds. Volatile compounds are organic compounds that, at standard room temperature

of about 23C, have an elevated vapor pressure ("Volatile organic compound", 2019). In this

technique, the sample is mixed with a solvent and then vaporized and passed through the stationary

phase in order to separate. The mobile phase of this technique is compromised of an inert gas, or noble

gas. Unlike the other techniques of separation, the inert gas in the mobile phase does not interact with

the analyte but instead helps carry the molecules of the sample (Libretexts, 2019).

Inlets, also known as injectors, are the entrance attached to the column head for the

sample to be placed into the chromatograph. These inlets introduce the sample into a small

heated chamber from a syringe, the heat breaks down the sample and turns the liquid into a

gas which is then carried to the column by either the entirety, “split-less” injection, or in

portions, “split” injection (Kumar, 2015). Another type of inlet is the “on-column” inlet where the

sample is introduced in its entirety without heat. Programmed Temperature Vaporizing Injector

introduces the sample just below its boiling point through the split line. (Kumar, 2015)
Columns of GC are a long tubing where the stationary phase is held at can come in two

different types: packed columns or capillary columns, which is also known as being open

tubular. Capillary columns come in two different forms usually made of glass (Libretexts, 2019).

One form of capillary columns contains finely divided, inert, solid support material that is

coated on the wall of the column with the stationary phase, also known as wall-coated open

tubular (Libretexts, 2019). The second form is also known as a support-coated open tubular

column. In these the columns are first coated with extremely small layer of adsorbent solid that

is treated with the stationary phase. Of these two capillary columns the, the support-coated

can hold a greater volume of stationary phase while the wall-coated is more efficient in

separation (Libretexts, 2019). Packed columns are usually made of glass or metal with a larger

diameter but do have a shorter length than capillary columns. Due to the greater diameter and

shorter length, the packed columns are way less efficient than wall-coated open tubular

columns (Libretexts, 2019).

Detectors are used at the end of the chromatogram to give a quantitative (being able to

measure) measurement of the analytes as they pass through with the inert gas (Libretexts,

2019). Regardless of the type of detector, there are two key parts that work together to

transduce (converts a physical amount into an electrical signal) (Libretexts, 2019). The first part is

a sensor at the end of the column and the second is an “electrical equipment used to digitize

the analog signal” for a computer to analyze the collected chromatogram (Libretexts, 2019).

Depending on the type of sample need to be analyzed there are several types of detectors.

Some types of detectors are Mass Spectrometer, Flame Ionization, Thermal Conductivity,

Electron-Capture, Atomic Emission, and Chemiluminescence detectors (Libretexts, 2019).


Thin-layer chromatography (TLC)

The image to the left is a TLC plate at


the end of an experiment. The
bottom of the plate is where the
sample is placed prior to the
experiment. Blotches directly above
each sample starting position is the
end distance that the analyte
traveled due to capillary action.

TLC is a common technique of chromatography in order to identify non-volatile organic

compounds and determine their purity. Just like the other techniques, a stationary phase is used but

composed of a finely ground material such as silica gel. Silica gel is used due its ability to fluoresce (shine

or glow) in Ultra-Violet light. The stationary phase is thinly layered as a “thick slurry” onto a glass place

or a metal film ("Thin-layer chromatography", 2019).

When the plate is ready, it is then placed into a small amount of the mobile phase in a beaker.

The sample is then elevated up the plate by capillary action. Capillary action is the ability of a liquid to

become elevated depending on its own adhesiveness properties (Dictionary.com, 2019). The After the

experiment, the analyte spots are visualized when a UV light is projected onto the plate ("Thin-layer

chromatography", 2019). The distance traveled by the sample is then measured and compared to the

mobile phase, Retardation factor. If the substance being measured has a similar structure to that of the

mobile phase then the retardation factor will be higher. To keep a consistent measurement for the
substance that is being measured, a known compound is applied to the plate ("Thin-layer

chromatography", 2019).

References

1. Britannica, T. E. (2011, February 07). Solvent. Retrieved from

https://www.britannica.com/science/solvent-chemistry

2. Capillary action. (n.d.). Retrieved from

https://www.dictionary.com/browse/capillary--action

3. Chromatography. (2019, February 15). Retrieved from

https://en.wikipedia.org/wiki/Chromatography

4. History of chromatography. (2019, February 23). Retrieved from

https://en.wikipedia.org/wiki/History_of_chromatography

5. International Union. (2006). Partition chromatography. Retrieved from

https://goldbook.iupac.org/html/P/P04436.html

6. Kumar, P. (2015, October 16). Top 12 Types of Chromatographic Techniques |

Biochemistry. Retrieved from

http://www.biologydiscussion.com/biochemistry/chromatography-techniques/top-

12-types-of-chromatographic-techniques-biochemistry/12730

7. Libretexts. (2019, February 23). Gas Chromatography. Retrieved from

https://chem.libretexts.org/Bookshelves/Analytical_Chemistry/Supplemental_Modules

_(Analytical_Chemistry)/Instrumental_Analysis/Chromatography/Gas_Chromatography

8. Saig, A. (2018, December 10). Retrieved from

https://davidson.weizmann.ac.il/en/online/askexpert/chemistry/what-capillary-action-

and-how-it-affected-gravity-ariel-michal
9. Thin-layer chromatography. (2019, February 08). Retrieved from

https://en.wikipedia.org/wiki/Thin-layer_chromatography

10. Torres, J. (2016, August 07). The Basics of Running a Chromatography Column. Retrieved

from https://bitesizebio.com/29947/basics-chromatography-column/

11. Volatile organic compound. (2019, February 05). Retrieved from

https://en.wikipedia.org/wiki/Volatile_organic_compound

Post Write

In this assignment I utilized a few different definition strategies throughout my writing.

There are some examples of parenthetical definitions, sentence definitions, graphics, partition,

and principles of operation implemented into this extended definition.

Acknowledgment

I would like to thank my table partner, Gerardo Sandoval, for being specific on what I

should improve on and suggesting what I should and/or remove.

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