INTRODUCTION

Chromatography is a technique for separating mixtures into their component in order to

analyse, identify, purity, and quantify the mixture or component. Chromatography is define as a
process which is similar to adsorption in the gas or liquid, the mixtures is let to pass through a
bed of porous particles but the feed is introduced in a small amount rather than continuous. The
separated component or individual component will move through the bed at different rates and
are collected at the exits. The bed is continuously regenerated by the passage of gas or liquid and
it can be used for a long period. Only small amounts of the feed mixture are separated at a time.

The components that are strongly retained will moved slowly while the component that
are weakly retained travel more rapidly, a detector is required to be placed at the end of the
chromatograph in order to quantify the analyte. Based on mobile phase, chromatography can be
separated into two major categories which is liquid chromatography and gas chromatography.

General classification Specific Method Stationary Phase

Liquid chromatography Liquid-liquid or partition
Liquid-bonded surface
(LC)
Liquid-solid or adsorption
Ion exchange size exclusion
Gas chromatography (GC) Gas-liquid
Gas-bonded phase
Gas solid
Table 1 : classification of chromatography
Liquid adsorbed on solids
Organic species bonded to a
solid surface
Solid
Ion-exchange resin liquid in
interstices of a polymer
solid
Liquid adsorbed on a solid
Organic species bonded to a
solid surface
Solid

1|Page
 Liquid chromatography is defined as an analytical technique that is useful for separating
ions or molecules. [1] If the sample solution is in contact with a second solid or liquid phase, the
different solutes will interact with the other phase to differing degrees due to differences in
adsorption, ion-exchange, partitioning , or size. The differences will allow the mixture
component to be separated into its individual component. Different component in the mixtures
will pass through a column at different rates between the mobile liquid phase and stationary
phase.

High performance liquid chromatography (HPLC) is one of the powerful tools that
usually being used. HPLC is a column chromatography but it already highly improved. Usually
the liquid is allow to drip under gravity but with HPLC the liquid is forced to operates under high
pressure up to 400 atmosphere. It also allows you to use a very small particle size for the column
packing material which gives a much greater surface area for interactions between the stationary
phase and the molecules flowing past it. This allows a much better separation of the components
of the mixture. [2]

Mobile phase enters the column from the left, and passes through the particle bed, and
exits at the right. Flow direction is shown by the arrow. First, at the moment of the injection, it
represents that the column is at zero. When the sample enters the column and it begins to form a
band. The sample shown here, a mixture of yellow, red, and blue dyes, appears at the inlet of the
column as a single black band but in real life, this sample could be anything that can be dissolved
in a solvent, typically the compounds would be colourless and the column wall opaque, so we
would need a detector to see the separated compounds as they elute. [3]

2|Page
MASS

TRANSFER

In liquid chromatography, the solute or component in the mixture of mobile phase is migrate
through the whole column.[4] If the mixture contained more than one and the components are in
colour, the compound will be separated into distinct band within the column. [4] This is due to
difference in polarity of the component that passed through the column where by one component
will move faster than the other.[4] Each band formed are made up from particular and distinct
component. In short, those components are separated in a column based on the components
affinity of the mobile phase mixture.[4]

The separation process in liquid chromatography of many components in the moving mixture
involving the mass transfer process. There are three types of mass transfer in this liquid
chromatography which are longitudinal diffusion, eddy diffusion (dispersion) and adsorption-
desorption process.[5] The separation technique chosen for the liquid chromatography is high-
performance liquid chromatography (HPLC) where the absorbent material is a solid.[10]

The type of mass transfer involved is taken from the most general and comprehensive theory of
band broadening in chromatographic column made by Giddings in 1960s.[5] He proposed that the
finite width of an eluted peak is the result from the combination of a few independent sources of

3|Page
sample dispersion.[5] The theory introduced by Giddings is still valid up until today, thus it can be
classified as types of mass transfer involved in liquid chromatography.[5]

Figure 2 The mass transfer involved in broadening of the band due to longitudinal diffusion.

For the first type of mass transfer involve which is longitudinal diffusion, by definition the
longitudinal diffusion is the diffusion of solute molecules in the direction of flow of the mobile
phase.[6] By referring to Figure 2, the band is broadening as the time increasing. [7] This is because
the more solute from the mobile phase mixture will transfer from the moving mixture into the
band produced. The flow rate of the mobile phase also give the effect to the longitudinal
diffusion where the flow rate of mixture decrease, the degree of band-broadening will increase.[7]

The degree of band broadening of any chromatographic peak can be related to the column plate
height as the column plate height is the ratio of length of column to a theoretical plate. [8] The
theoretical plate is dependent on the degree of band-broadening. As the band-broadening
increase, the theoretical plate will decrease.[8] The lower in theoretical plate will eventually give
large number of the column plate height. But when referring to the goal of any chromatographic
separation, the desired column plate height should be the lowest possible value.[8]

Thus, the degree of band-broadening must be remained at minimum in order to achieve the target
of chromatographic separation which is the lowest possible value of column plate height. There

4|Page
are three ways to minimize the longitudinal diffusion which are using high flow rate of mobile
phase, using short and as narrow as possible of the tubing system.[9]

Figure 3 The mass transfer involved due to eddy dispersion (diffusion).

The definition of the eddy dispersion of diffusion is a diffusion process by which substances are
mixed in the atmosphere or in any fluid system due to eddy motion. [11] The effect of eddy motion
can be taken as the solute travel around the support particles inside the column where the support
particles usually in sphere shaped.[12]

The travel path of the solutes may differ between each other since the distribution of the support
particles in the column is random between one place to another. [12] By referring to the Figure 3
above, the upper side of the column has much more support particles compared to the lower side
of the column. Thus, the solute that passes the column at the bottom will arrive earlier than the
solute travelled at the top of the column. The time taken for the solute to travel at different flow
path inside the column may vary along with the distribution of the support particles in the
column.

5|Page
Figure 4 The mass transfer involved due to adsorption.

By definition, the adsorption-desorption is the interaction between the solute molecules and
active sites on the stationary phase.[13] By referring to Figure 4 above, the mass transfer involved
due to adsorption is clearly shown. The interaction involve between the solute and active sites is
dependable on the polarity of solute as well as the polarity of the active site. [13] This interaction is
one of the example for the statement of polar like polar.[13]

This type of mass transfer in liquid chromatography process occur as the solute bind directly to
the surface of the stationary phase where the common stationary phase is solid. [14] Furthermore,
the stationary phase may have variety of adsorption sites which are different in terms of tenacity
with which molecule they bind and the relative abundance of the molecule.[14] The desorption is a
process which is a reverse process of an adsorption process. In short, the desorption is the
process where the solute detached from the active site of the stationary phase.

6|Page
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)

High performance liquid chromatography is basically a highly improved form of column

chromatography. Solvent is forced through column under high pressures of up to 400 atm which
makes it much faster instead being allowed to drip through a column under gravity force.

It allows the use of column packing material of very much smaller particle size which gives
much greater surface area for interactions between the stationary phase and the molecules
flowing past it. This allows a much better separation of the components of the mixture. [15]

Figure 5 A flow scheme of HPLC.

There are two types of high performance liquid chromatography, namely:

1. Normal phase HPLC

7|Page
 2. Reversed phase HPLC

Normal phase HPLC

The stationary phase is polar and the mobile phase is nonpolar in normal-phase chromatography.
Silica are typically used as stationary phases for normal-phase chromatography. There are also
bonded normal phase material. They have organic moieties with cyano and amino functional
groups. [16]

In normal-phase chromatography, polar compounds in the mixture being passed through the
column will stick longer to the polar silica than non-polar compounds will. The non-polar ones
will therefore pass more quickly through the column. As the amount of non-polar solvent in the
mobile phase increases, retention also increases.

Normal Phase HPLC are used when the compounds are [17]

o too hydrophobic or too hydrophilic for separation using RPLC

o not soluble in water

o may decompose in water

o isomers that require separation

o for Prep and Process scale chromatography

Although it is described as "normal", it isn't the most commonly used form of HPLC.

8|Page
Figure 6 Chemistry of stationary phase.

Reversed phase HPLC

Any chromatographic method that uses a hydrophobic stationary phase is considered as reversed-
phase chromatography (also known as RPC, reverse-phase chromatography, or hydrophobic
chromatography) includes. RPC refers to liquid (rather than gas) chromatography.

A hydrophobic stationary phase, which has a stronger affinity for hydrophobic compounds is
created from a technique using alkyl chains covalently bonded to the solid. In reversed phase
HPLC, the use of a hydrophobic stationary phase can be considered the reverse of normal phase
chromatography. Reversed-phase chromatography employs a polar (aqueous) mobile phase. As a
result, hydrophobic molecules in the polar mobile phase tend to adsorb to the hydrophobic
stationary phase, and hydrophilic molecules in the mobile phase will pass through the column
and are eluted first.

By decreasing the polarity of the mobile phase using an organic (non-polar) solvent, which
reduces hydrophobic interactions, hydrophobic molecules can be eluted from the column. The
more hydrophobic the molecule, the more strongly it will bind to the stationary phase, and the
higher the concentration of organic solvent that will be required to elute the molecule.

In this case, there will be a strong attraction between the polar solvent and polar molecules in the
mixture being passed through the column. There won't be as much attraction between the

9|Page
hydrocarbon chains attached to the silica (the stationary phase) and the polar molecules in the
solution. Polar molecules in the mixture will therefore spend most of their time moving with the
solvent.

Non-polar compounds in the mixture will tend to form attractions with the hydrocarbon groups
because of van der Waals dispersion forces. They will also be less soluble in the solvent because
of the need to break hydrogen bonds as they squeeze in between the water or methanol
molecules, for example. They therefore spend less time in solution in the solvent and this will
slow them down on their way through the column which means that now the polar molecules
will travel through the column more quickly. [18]

Reversed phase HPLC is the most commonly used form of HPLC.

THE ISSUE THAT FACED IN HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

There are many technique can be used in the chromatography such as Liquid
Chromatography, Gas Chromatography, Ion Chromatography and also High Performance Liquid
Chromatography. One of the issue/problem that we can discuss more is High Performance Liquid
Chromatography also known as HPLC.

I found one case that related to this technique. [19] It is about a laboratory director at the
analytical laboratories. He shared the experience with his team member during their work using
chromatography. He said that there are different ways in which his service laboratory uses
chromatography techniques to analyze complex mixtures for various clients. They are often
asked to analyze trace component in samples such as nutraceuticals, botanicals, cosmetics and
others.

Besides, the chemist at the analytical laboratory also develop new chromatography
method and modify existing ones to get the best possible results in the least amount of time.
From the problem statement, it is stated that they faced a challenge with High Performance
Liquid Chromatography. They use HPLC to do a lot of separation work since their sample matrix
tends to be very complex.

10 | P a g e
 For their analysis, they have to separate individual components. Traditional HPLC can do
the job, but it takes a long time for achieving good separation. They have to slow down the
gradient and that consumes a lot of solvent.

HOW TO OVERCOME THE ISSUE IN THE HIGH PERFORMANCE LIQUID

CHROMATOGRAPHY?

To overcome this challenge they have now shifted to using Ultra Performance Liquid
Chromatography (UPLC), where the packing particles in the column have much smaller size and
that increase column efficiency. The column efficiencies increase three to five times compared to
traditional HPLC, with the larger particle size in a traditional HPLC we cannot increase the
solvent velocity too much, as the back pressure in the column can increase too.

Here some differences between Traditional HPLC and Ultra Performance Liquid
Chromatography (UPLC):

HPLC UPLC

The similarities:
HPLC and UPLC are both Liquid Chromatography used in separating the components
of a compound
The differences:

HPLC stands for High Particle sizes less than 2um can be
Performance Liquid used.
allows for better separation that
Chromatography is a
particle size of 5um that are used
technique used to separate
in HPLC
different constituents of a
Also allows for a very fast
compound.
It is the most widely used analysis.
in UPLC, this pressure can go up
technique to identify,
to 100MPa, which is what makes
quantify and separate
this technique so very exciting and

11 | P a g e
 components of a mixture efficient.
It uses high pressure to push UPLC is a trademark technology
solvents through the of Waters Corporation, people use
column. it as a general term for a separate
HPLC is widely used in technique.
biochemistry for the Ultra performance liquid
analysis of constituents of a chromatography (UPLC) makes it
compound possible to perform very high-
It is an ideal way for resolution separations in short
separation and identification periods of time.
of amino acids, nucleic Ultra performance liquid
acids, proteins, chromatography (UPLC) employs
hydrocarbons, pesticides, particles smaller than 2 m in
carbohydrates, antibiotics, diameter to achieve superior
steroids and countless other resolution, speed, and sensitivity
inorganic substances. compared with high-performance
pump pressure in HPLC is liquid chromatography (HPLC).[21]
40MPa
It allows for higher
resolution and speed of
analysis, HPLC columns
can be reused without
repacking or regeneration,
better control of parameters
affecting efficiency of
separation, easy automation
of instrument operation and
data analysis, and
adaptability to large scale
procedures.
it supports very small
particle sizes and large
surface areas and allows for

12 | P a g e
 application of high pressure
for solvent to flow.[20]

The summary:

1. HPLC is high performance liquid chromatography, whereas UPLC is ultra high

performance liquid chromatography

2. In essence, UPLC is just a special variant of HPLC

3. The pump pressure, which is 40Mpa (400 atmospheres) in HPLC, can go up to 100MPa
in UPLC. UPLC also allows for much smaller particle size, normally less than 2um
whereas it is normally 5um in HPLC

CONCLUSION

As we know High-performance liquid chromatography or high-pressure liquid

chromatography (HPLC) is a chromatographic method that is used to separate a mixture of
compounds in analytical chemistry and biochemistry so as to identify, quantify or purify the
individual components of the mixture. There are some benefit that we can get from using HPLC
such as

Controls and automates chromatography instrumentation

Provides data management, security features, and reporting and instrument validation.
Powerful and adaptable
Increases productivity by managing all the areas of analysis - from sample to instrument, and

13 | P a g e
 from separation to reporting results.
Affordable

Because of the benefits that has been listed above HPLCs can be used in the following
applications:

Water purification
Preconcentration of trace components
Ligand-exchange chromatography
Ion-exchange chromatography of proteins
High-pH anion-exchange chromatography of carbohydrates and oligosaccharides

Other than HPLC we also have another method to separate a mixture compound in liquid
which is Ultra-Performance Liquid Chromatography (UPLC) . UPLC Is an analysis method
which is commonly used in analytical chemistry (and in the pharmaceutical industry). Its
operation is based on column chromatography, which is often used to investigate mixtures. The
different analytes (the substances to be tested) can be separated (and identified) on the basis of
their polarity, and the interaction between the analyte itself, the stationary phase and the mobile
phase. From using UPLC we can get the benefit such as

Improved chromatographic performance

More accurate, better visualized results that are available earlier .
increased productivity for each individual in your decision process
More sustainable use of lab resources through reduced costs in energy and solvent
Faster time to market

Because of the benefits that has been listed above UPLCs can be used in the following
applications:

Determination of Pesticides in Groundwater

Improved Resolving Power in Peptide Maps

14 | P a g e
 Rapid Dose Formulation Analysis
Analysis of Traditional Chinese Medicines (TCM)
Identification of Metabolites

Therefore using UPLC is more efficient than using HLPC in separation of components in a
mixture. Contrasting between HPLC and UPLC. Using UPLC is better than HPLC because:

UPLC gives faster results with better resolution than HLPC.

UPLC uses less of valuable solvents like acetonitrile which lowers cost of the
experiment.

The reduction of solvent use is more environmentally friendly

15 | P a g e
REFENCES

1. Retrieved from

http://www.chemicool.com/definition/liquid_chromatography_lc.html (11 December 2016)

2. Retrieved from

http://www.chemguide.co.uk/analysis/chromatography/hplc.html (11 December 2016)

3. Retrieved from

http://www.waters.com/waters/en_MY/How-Does-High-Performance-Liquid-Chromatography-
Work%3F/nav.htm?cid=10049055&locale=en_MY (11 December 2016)

4.Retrieved from
http://chem.libretexts.org/Core/Analytical_Chemistry/Instrumental_Analysis/Chromatography/Li
quid_Chromatography ( 11 December 2016 ).

5. Retrieved from http://www.chromatographyonline.com/quantification-individual-mass-

transfer-phenomena-liquid-chromatography-further-improvement-column-0 ( 11 December 2016
).

6. Retrieved from http://medical-dictionary.thefreedictionary.com/longitudinal+diffusion ( 11

December 2016 ).

7. Retrieved from http://images.slideplayer.com/7/1667936/slides/slide_20.jpg ( 12 December

2016 ).

8. Retrieved from https://books.google.com.my/books?

id=9L2WRWXBauwC&pg=PA1013&lpg=PA1013&dq=Degree%20of%20%20longitudinal
%20diffusion&source=bl&ots=jPdpcQ0R6a&sig=onuTh4b4itnXaQPAjAuyrTyxsPk&hl=en&sa
=X&redir_esc=y#v=onepage&q=Degree%20of%20%20longitudinal%20diffusion&f=false ( 12
December 2016 ).

9. Retrieved from http://image.slidesharecdn.com/11-141101101533-conversion-

gate02/95/recent-advances-in-hplc-and-gc-9-638.jpg?cb=1414837096 ( 12 December 2016 ).

16 | P a g e
10. Retrieved from https://en.m.wikipedia.org/wiki/High-performance_liquid_chromatography
(13 December 2016 ).

11. Retrieved from https://en.m.wikipedia.org/wiki/Eddy_diffusion ( 12 December 2016 ).

12. Retrieved from http://image.slidesharecdn.com/chrom-lect1-141117150321-conversion-

gate02/95/chromatography-lec-1-35-638.jpg?cb=1416236744 ( 13 December 2016 ).

13. Retrieved from http://chromatographyscience.blogspot.my/2012/08/adsorption-

chromatography.html?m=1 ( 13 December 2016 ).

14. Retrieved from https://global.britannica.com/science/chromatography/Elution-

chromatography#ref619755 ( 13 December 2016 ).

15. Retrieved from http://www.chemguide.co.uk/analysis/chromatography/hplc.html ( 11

December 2016 ).

16. Retrieved from http://www.chromatographyshop.com/html/normal_phase_lc.html ( 11

December 2016 ).

17. Retrieved from https://en.wikipedia.org/wiki/Aqueous_normal-phase_chromatography ( 12

December 2016 ).

18. Retrieved from https://en.wikipedia.org/wiki/Reversed-phase_chromatography( 113

December 2016 ).

19. Retrieved from http://www.labmanager.com/ask-the-expert/2010/06/ask-the-expert-how-to-

overcome-challenges-with-chromatography#.WE-15H2vTvs ( 12 December 2016 ).

20. Retrieved from http://www.differencebetween.com/difference-between-hplc-and-vs-uplc/

( 12 December 2016 ).
21. Retrieved from http://www.sciencedirect.com/science/article/pii/S1044030505009116 ( 13
December 2016 ).

17 | P a g e