Lecture 1,2, Introduction To Chromatography-1

.
5/8/2021 Prepared By Asmare T.

Chapter Four chromatography
.
⸎ Introduction to chromatography
⸎ Common terms
⸎ Basic parameters
⸎ Phases of chromatography
⸎ Principle of chromatography
⸎ Limitations of chromatography
⸎ Advantages
⸎ Classification of chromatography
5/8/2021 ⸎ Application of chromatography..
Prepared By Asmare T.
Introduction to Chromatography
.
Amobile Astationary
KD=CS/CM
⸎ KD=Distribution coefficient
⸎ CS= Molar concentration of solute in stationary phase
⸎ CM= Molar concentration of solute in mobile phase

.
Three components thus form the basis of the chromatography technique.
⸎ Stationary phase: This phase is always composed of a “solid” phase or “a

layer of a liquid adsorbed on the surface solid support”.
⸎ Mobile phase: This phase is always composed of “liquid” or a “gaseous

component.”
⸎ Separated molecules: The type of interaction between the stationary phase,

mobile phase, and substances contained in the mixture is the basic
component
5/8/2021
effective on the separation of molecules from each other.
.
Mobile phase
⸎ It has been rightly termed as the lifeline of the HPLC system.
⸎ The mobile phase is a chemically inert gas that serves to carry the
molecules of the analyte through the heated column.
⸎ It plays the important role of transport of the sample through the

separation column and subsequently to the detector for the separation,
purification, analysis and identification of the separated components.

Generally solvents having low viscosities are employed in chromatography. This
is due to the fact that the rate of flow of a solvent varies inversely as its viscosity.
In normal phase chromatography, the stationary phase is polar, polar molecules
will spend more time adsorbed on the stationary phase, while less polar ones will
be carried more quickly by the non-polar mobile phase.

.
Polar and non-polar in chromatography
⸎ In normal-phase chromatography, the stationary phase is polar and the

mobile phase is nonpolar. ... Retention decreases as the amount of
polar solvent in the mobile phase increases. In reversed phase
chromatography, the most polar compounds elute first with the most
nonpolar compounds eluting last.

Introduction to chromatography
 Chromatography is essentially a physical method of

separation in which the components of a mixture are
separated by their distribution between two phases; one
of these phases in the form of immobile (stationary
phase), while the other is a fluid (mobile phase) that

 A separation results from repeated sorption/desorption

events during the movement of the sample components
along the stationary phase in the direction of mobile-phase
migration.
 Useful separations require an adequate difference in the

strength of the physical interactions for the sample
components in the two phases.

Chromatography
 It is based on the Greek word chroma, for colour. graphy mean

writing. This technique is extensively used to separate mixtures
into their components, purify compounds and also to test its
purity.
 Is a process which is based on multiple repetition of sorption and

desorption of substances. It involves passing a mixture dissolved
in a "mobile phase" through a stationary phase.
Common mobile phases used include any miscible combination of water with various organic
solvents (the most common are acetonitrile and methanol). The analyte can be identified by
measuring the sample's absorption of light at different wavelengths.

Separation parameters of chromatography
tRB
tRA
Detector Signal
A B
tM
𝑊𝑎 𝑊𝑏
2 2
tM tR1 tR2 Time

tRC
Rs = 2(tRC-tRA)
tRB 𝑊𝑎+ 𝑊𝑏 + 𝑊c
tRA
Detector Signal
A B C
tM
𝑊𝑎 𝑊𝑏 𝑊𝑐
2 2 2
tM tR1 tR2 Time

.
 Chromatography is an important biophysical technique
that enables the
 Separation
of the components of a mixture for
 Identification
qualitative and quantitative analysis.
 Purification

.  In chromatography solutes are separated by dynamic
differential migration process in a system consisting of two or

more phases, one of which moves continuously in a given
direction and in which the individual substances exhibit different
mobilities by reason of differences in adsorption, partition,
solubility, vapor pressure, molecular size, or ionic charge density

. Chromatography is:
 A method of physically separating mixtures of gases, liquids, or

dissolved substances.
 Used to identify drugs, poisons and many other substances
 Separation is determined by the molecular size and/or charge.

. Advantages of chromatography
 Can separate complex mixtures with great precision. Even very similar
components, such as proteins that may only vary by a single amino acid.
 Can purify basically any soluble or volatile substance if the right
adsorbent material, carrier fluid, and operating conditions are employed.

. Advantages of chromatography
 Exact quantitative analysis is done even from trace compounds.

 Small material consumption.
 The quantization has a broad linearity range.
 Analyses of several compound can be done during one run.
 Chromatography is a fast analysis method.

Stationary Phase
Separation
Mobile
Mixture Phase Components
Affinity to Stationary Affinity to Mobile
Components
Phase Phase • Analyze
Blue Insoluble in Mobile Phase
----------------
• Identify
Black  
Red  
• Purify
Yellow  
Prepared By     T.
Asmare 
•Quantify 5/8/2021
.
Purpose of Chromatography
Analytical - determine chemical composition of a sample
Preparative - purify and collect one or more components

of a sample

. Importants of chromatography:
 Creating vaccinations. Chromatography is useful in determining

which antibodies fight various diseases and viruses. ...
 Food testing. ...
 Beverage testing. ...
 Drug testing. ...
 Forensic testing.

.
The purpose of chromatography is to separate a complex

mixture into individual component exploiting the partition
effect which distribute the molecules into the different
phases.

.
A distribution of a molecule between two phases A and B is
given by a distribution coefficient, Kd. In most of the
chromatography techniques, phase A is stationary phase or
matrix and phase B is mobile phase or buffer.
Concentration in Phase A
Kd =
Concentration in Phase B

Chromatography
Chromatogram - Detector signal vs.

retention time or volume
Detector Signal
1 2
time or volume

Classification of Chromatography
Chromatographic system consists of two phases
Mobile Phase (a moving solvent)
The Phase that travels or moving through the column (gas or liquid) –
transport sample through the column.
Stationary Phase (immobile matrix)
Immiscible solid or liquid phase that fixed in place in the column or on a

solid support – retain analytes within the column.

.  Fluid entering the column is the eluent, while fluid
leaving the column is the eluate.
 Elution is the process of passing liquid or gas through the

column

Theoretical bases of chromatography
Purpose of the mobile phase in HPLC
 It plays the important role of transport of the sample through the

separation column and subsequently to the detector for identification of
the separated components. The mobile phase moves through the
stationary phase picking up the compounds to be tested.

A good mobile phase has the following characteristics.
 Compatible with the column packing/stationary phase and the

sample.
 Gives good separation of the analytes of interest.
 Is of the highest purity.

Requirements to a mobile phase:
 Should dissolve investigated sample;
 Small viscosity
 Possible excretion from it divided components;
 It must be inertness in relation to materials of all parts of chro.
 Meets the requirements of the chosen detector;
 The safe and cheap

 Stationary phases are usually very polar, while mobile phases vary
widely in polarity, but are less polar than the stationary phase. This is
called normal phase (NP).
 Since the stationary phase is hydrophilic in this technique, molecules

with hydrophilic properties contained within the mobile phase will have
a high affinity for the stationary phase, and therefore will adsorb to the
column packing.

.Principles of chromatography:
 A substance placed in contact with two immiscible phases, one

moving and one stationary, will equilibrate between them. A
reproducible fraction will partition into each phase, depending on
the relative affinity of the substance for each phase.
 A substance which has affinity for the moving or mobile phase

will be moved rapidly through the system.

.Principles of chromatography:
 A material which has a stronger affinity for the stationary phase,

on the other hand, will spend more time immobilized in that
phase, and will take a longer time to pass through the system.
Therefore, it will be separated from the first substance.

Principle of Chromatography (how does chromatography work)
.
⸎ Chromatography is based on the
principle where molecules in mixture
applied onto the surface or into the
solid, and fluid stationary phase (stable
phase) is separating from each other
while moving with the aid of a mobile
phase.

.
⸎ The factors effective on this separation
process include molecular characteristics
related to adsorption (liquid-solid), partition
(liquid-solid), and affinity or differences
among their molecular weights.

.⸎ Because of these differences, some
components of the mixture stay longer in

the stationary phase, and they move
slowly in the chromatography system,
while others pass rapidly into the mobile
phase, and leave the system faster.

. Factors affecting chromatography
 Column temperature
 Intermolecular interaction between the two phases
 Extent of dispersion of solute molecules over the stationary

phase

Common terms of chromatography
.
⸎ Chromatograph - equipment that enables a sophisticated separation
⸎ Eluent - Fluid entering column/ solvent that carries the analyte.
⸎ Eluate - Mobile phase leaving the column.
⸎ Stationary phase - Immobilized phase.
⸎ Chromatogram: Visual output of the chromatograph
⸎ The detector refers to the instrument used for qualitative and quantitative
detection of analytes after separation.
.
⸎ Elution is the process of removing analytes from the adsorbent by running
a solvent, called an "eluent".
⸎ Elute to wash out or to remove (adsorbed material) from an adsorbent by

means of a solvent.
⸎ Eluent is the portion of the mobile phase, which carries the sample
components with it. Eluate is the combination of the mobile phase and the
analytes. Therefore, eluate is what we are interested. We add eluent to the
column, and eluate is what is coming out of the column.
Chromatography terms
.
 Retention time : Time takes for a particular analyte to
pass through the system (from the column inlet to the detector)
under set conditions.
 Sample (Anylate) :Substance analyzed in chromatography.
 Solvent : Any substance capable of solubilizing another

substance.

Typical pathways of several molecules during elution.
.

.
 Theoretical base of chromatography

 Retention time, retention factor
 Factors affecting retention time
 Resolution (peak broadening)
 Selectivity factor, capacity factor
 Column efficiency, plate number
 Principal application chromatography
.
What causes the separation between molecules in chromatography?
 The different components of the mixture travel through the

stationary phase at different speeds, causing them to separate from
one another.
 The nature of the specific mobile and stationary phases determines

which substances travel more quickly or slowly, and is how they are
separated.

. The chromatographic method of separation, in general, involves following steps
1. Adsorption or retention of substances on the stationary phase
2. Separation of the adsorption of substances by the mobile phase
3. Recovery of the separated substances by a continuous flow of

the mobile phase; the method being called elution
4. Qualitative and Quantitative analysis of the eluted substances

.
Differential migration occurs when individual components from a mixture
migrate different distances.
Separation through chromatography takes place due to:
Adsorption of molecules
Differential migration or
Partition of molecules

.
 Components of the mixture with similar properties to the mobile

phase elute earlier while the components of the mixture with
similar properties to the stationary phase elute later.
 Generally, it can be either a liquid or a gas. On the other hand, the

stationary phase is the type of medium of chromatography, stay
fixed to the column.

tRB
tRA
Detector Signal
A B
tM
𝑊𝑎 𝑊𝑏
2 2
tM tR1 tR2 Time

.
Peak broadening and column efficiency
 To obtain optimal separations, sharp, symmetrical

chromatographic peaks must be obtained. This means
that band broadening must be limited. It is also beneficial
to measure the efficiency of the column.

tRC
Rs = 2(tRC-tRA)
tRB 𝑊𝑎+ 𝑊𝑏 + 𝑊c
tRA
Detector Signal
A B C
tM
𝑊𝑎 𝑊𝑏 𝑊𝑐
2 2 2
tM tR1 tR2 Time

 Peak Base: An interpolation of the base line between the
extremities of the peak. .
 Peak Area: The area enclosed by the peak and the peak
base.
 Peak Height: The distance from the peak maximum to the
peak base.
 Peak Width (tW): The magnitude of the peak base
intercepted by the tangents to the inflection points of the
peak.
 Retention time (tR): The time between sample injection
and the appearance of a solute peak at the detector.
 Dead time (t0): The time for mobile phase to pass through
a column.
 Adjusted Retention Time (tR') is the time solute spent in
5/8/2021 the stationary phase andPrepared
equals to T.tR' = [ tR- t0].
By Asmare
 Adjusted Retention Time (tR') tR' = [ tR- t0].
 The Capacity Factor (k') is used to describe the migration rates of
solutes on columns, It is defined as:
For solute A, kA' = (tR,A – t0)/t0 ; = t'R,A/t0
 The Selectivity Factor () of a column for the two solutes A and B
in a mixture is defined as
= k'B/k'A
 Chromatoaraohic column efficiency is determined by

1- Plate height (Height Equivarent to Theoretical Plate (HETP)= L/N
2- Number of Theoretical Plates(N) =16 (tR/tW)2
L = length of column
 Resolution of the column (Rs) = (tR,B – tR,A)/0.5 (tw,A+tW,B)
5/8/2021 Rs = √N/4 [α-1]/ByαAsmare T. x

x Prepared [k'B/(k'B+1)]
Column efficiency Selectivity Capacity
.
Types of Phase
⸸ A non-polar solvent such as hexane is used in the normal phase

while a polar solvent such as methanol is used in the reverse
phase in HPLC while
⸸ A polar stationary phase is used in the forward phase

chromatography while a non-polar stationary phase is used in
the reverse phase.

Solvents (eluents) divide on weak and strong.
 Weak eluents are a little adsorbed by stationary phase therefore

distribution factors D of sorbate is high.
⸎ Strong eluents are strongly adsorbed by stationary phase, therefore

distribution factors D of sorbed substances (sorbate) is low.
⸎ The eluent strength (ε°) is a measure of the. solvent adsorption

energy, •The more polar the solvent, the greater is its eluent.

Methods of elution
 When a separation uses a single mobile phase of fixed composition

it is called an isocratic elution. It is often difficult, however, to find
a single mobile-phase composition that is suitable for all solutes.
 Gradient elution is the process of changing the mobile phase’s

solvent strength to enhance the separation of both early and late
eluting solutes.

 All chromatographic systems have a mobile phase that transports the

analytes through the column and a stationary phase coated onto the
column or on the resin beads in the column.
 The stationary phase loosely interacts with each analyte based on its
chemical structure, resulting in the separation of each analyte as a
function of time spent in the separation column.

 The less analytes interact with the stationary phase, the faster they are
transported through the system.
 The reverse is true for less mobile analytes that have stronger
interactions. Thus, the many analytes in a sample are identified by
retention time in the system for a given set of conditions..

 In GC, these conditions include the gas (mobile phase) pressure, flow
rate, linear velocity, and temperature of the separation column.
 In HPLC, the mobile phase (liquid) pressure, flow rate, linear velocity,
and the polarity of the mobile phase all affect a compounds’ retention
time.

Applications of Chromatography
1.Pharmaceutical sector
 To identify and analyze samples for the presence of trace

elements or chemicals.
 Separation of compounds based on their molecular weight and

element composition.
 Detects the unknown compounds and purity of mixture.
 In drug development.

2. In Molecular Biology Studies
 Various hyphenated techniques in chromatography applied in the

study of metabolomics and proteomics along with nucleic acid
research.
 HPLC is used in Protein Separation like Insulin Purification, Plasma

Fractionation, and Enzyme Purification and also in various
departments like Fuel Industry, biotechnology, and biochemical
processes.
.
3.Food Analysis
 Analysis of foods is concerned with confirm the presence and

determination the quantities of the analytes (lipids, proteins,
carbohydrates, preservatives, flavours, colorants, and also
vitamins, steroids, and pesticide residues).
 In food spoilage and additive detection
 Determining the nutritional quality of food

. 4. Drug Analysis
 Chromatography (gas) is widely applied to identification of the active

components, possible impurities as well as the metabolites
Forensic Analysis
 In forensic cases, very little sample is available, and the concentration

of the sample components may be very low.
 GC is a useful due to its high sensitivity and separation efficiency. In

forensic pathology and crime scene testing like analyzing blood and
hair samples of crime place.
5.Chemical industry
 In testing water samples and also checks air quality.
 HPLC and GC are very much used for detecting various

contaminants such as polychlorinated biphenyl (PCBs) in
pesticides and oils.
 In various life sciences applications

.
The most important separation parameters are:
 Retention time: tR
 Retention factor: k
 Selectivity: α, tells us about the difference in retention between the
individual components (calculated by the ratio of their retentions).
 Efficiency: N, also referred to as the plate number, shows the relation
between retention time and peak width and defines column quality and
separation power.
. Retention time
 Retention time (RT) is a measure of the time taken for a solute to pass
through a column
 Every sample injected onto a column will take a finite time to travel
through the column, even if there is no affinity of the sample components
for the stationary phase.
 In this case, the time between applying the sample and its elution from the
column will only depend on the speed at which the mobile phase is flowing
through the column.
.
Retention time (R.t) is the time that a solute spends in a column or it can be
defined as the time spent in the stationary and mobile phases. The stronger the
interaction, the more will be the interaction time.
 Column type, dimension & temp.

 The Mobile Phase Flow-rate
 The mobile phase composition
 Integration of the phases
 The presence of active components
The time taken for the mobile phase to pass through the column is called
tM. Or the time for an unretained solute to reach the detector from the point
of injection is called the column dead time or the hold up time(tM). The
time between injection and elution of a retained sample component is called
the retention time (tR)
 The retention time is the sum of time a sample component spends in the
mobile phase and the amount of time it spends in the stationary phase..

Retention factor (K) or capacity factor
 It is a variable indicating how much time a component spends in the

stationary phase compared to a non-retained inert component.
 It is capacity of a stationary phase to attract a component,
 The (mobile and stationary phase) can be regarded as a system of two

immiscible phases.
 When there is sufficient interaction, a sample component will be

distributed between the two phases.
Retention factor (K) or capacity factor
K= tR - to
Detector Signal to
to: Non-retention time
t'R1 = tR1-tM tR: Retention time
t'R2 = tR2-tM
tM K: Retention factor
tM tR1 tR2 Time
Adjueted (corrected) retention time: t'R = tR-tM

.
Capacity/retention factor (K)
 Shows how long a sample can be retained by the stationary phase.
 Calculated as, k = (Tr - To)/To, where Tr is the retention time of the target
and To is the unretained peak time.
 If Tr = To, then K=0, then the sample is not retained by the stationary
phase. Which means co-elution (no interaction with the stationary phase).

.
Capacity /retention factor (K)
 k = 0 , Component does not spend any time in the stationary phase & therefore
it is not retained.
 k = 1 , Component spends just as much time in the stationary phase than in the
mobile phase.
 k < 1, Elution is so fast, accurate determination of the R.t is very difficult
 k >1, Elution takes a very long time. There is strong interactions of the analyte
with the surface, Ideally, the retention factor for an analyte is between one and
five.
5/8/2021 To analyze an ingredient, we
Prepared By want
Asmare T. this type of interaction.
.
 The distribution Coefficient, KD describes the way in which the

mixture (sample) distributes itself between two immiscible
phases (stationary and mobile) ·
Concentration component in stationary phase

KD =
Concentration component in mobile phase

. If the distribution coefficient is 1:
 The mixture is distributed throughout the whole column but is maximally

concentrated at the center of the column.
If the distribution coefficient is <1:
 More than 50% of the mixture would be left on solid stationary phase after
each equilibration and the concentration peak is above the center of the
column and vice versa.
If the distribution coefficient is >1:
 More than 50% of the mixture retained on the solid stationary phase, the
concentration peak is below the center of the column.
.
Resolution
 Scientists consider a resolution of 1.0 or higher to represent an

adequate separation. It measures the widths of two adjacent peaks
in the chromatogram by noting where the x-axis values are at the
base of each peak. The x-axis represents retention time, usually
measured in seconds.

.
Resolution ( Rs)
 A resolution value of 1.5 or greater between two peaks will ensure that
the sample components are well ('baseline') separated - to a degree at
which the area or height of each peak may be accurately measured.
 Resolution is calculated using the separation of two peaks in terms of

their average peak width at the base (t R2 > t Rl).

.

.
Resolution ( Rs)
 The degree of separation between any two peaks, A and B, can be

expressed as the resolution of the peaks. This is defined as the difference
between the two retention times divided by their average peak width.

. Resolution
A resolution of 1.5 is considered a complete separation. LC-MS may allow for
lower resolution (and shorter columns) yet still achieve accurate quantitation.
2.3% mutual overlap 1.5 % mutual overlap
Resolution = 1.0 Resolution = 1.5

Fig Resolution and peak separation
.

.
What causes peak broadening?
 The sample injection volume is related to broadening of the sample

zone in the first stage of column separation. Therefore, increasing the
injection volume can result in peak broadening.
 In particular, the higher the ratio of strong solvent in the sample

solvent, the greater the effects.

.

.
 The constant KD is a physico chemical constant that only depends on the
type of analyte, the type of stationary phase and the temperature. It does not
depend on the volumes of the phases. The retention factor k describes the
distribution of sample components in terms of true mass amounts in both
phases, rather than in terms of their concentrations. The retention factor can
be written as
where,
Vs = volume of stationary phase in the column
Vm = volume of mobile phase
5/8/2021 Prepared in the column
By Asmare T.
.  Concentration profiles for the bands containing solutes A and B in
Figure at time tl and a later time t2
 B is more strongly retained by the stationary phase than is A, B lags

during the migration and elutes last.
Detector
A B A B
5/8/2021 Distance migrated

. tR
tM
Detector Signal
Retention factor or capacity factor (k)
is defined as the ratio of time an analyte
is retained in the stationary phase to the
time it is retained in the mobile phase,
Time
Retention factor, K tR-tM)

K= t
tR = Retention time M
tM = Non-retention time
.
The distribution constant is the concentration of a component in or on the stationary
phase divided by the concentration of the component in the mobile phase in
equilibrium conditions. Amobile phase Bstationary phase
The equilibrium constant, Kc for this reaction is called the distribution constant, the
partition ratio, or the partition coefficient,
KC = CS/CM = nS/vs
Where; nM/vM
CS and CM are the molar conc. of the solute;
nS and nM are the number of moles of analyte;
Vs and vM are the volumes of the stationary and mobile phases
.
 Retention time is the time that a solute spends in a column or
it can be defined as the time spent in the stationary and
mobile phases. It is the function of Kc.
 Retention time, tR, The time it takes after sample injection

for the analyte peak to reach the detector
 Dead time, tm or void time, the time for the unretained

species to reach the detector

.
Factors affect retention time
 The retention time depends on many factors: analysis conditions, type

of column, column dimension, degradation of column, existence of
active points such as contamination. and so on.
 If citing a familiar example, all peaks appear at shorter times when

you cut off part of column. The dead time is the time after each event
during which the system is not able to record another event.

The analyte has been retained because it spends
a time , tR, in the stationary phase. The retention
time is then tR = ts + tm
The average linear rate of solute migration through the column
where, L is the length of the column packing
The average linear rate of movement of the M.P molecules,
where tm, the dead time.

Volumetric Flow Rate and Linear Flow Velocity the mobile-phase flow is usually
characterized by the volumetric flow rate, F (cm3 /min) at the column outlet.

Column resolution
Resolution (zone broadening) depends on;
 u (linear flow rate) - low flow favors increased resolution
 H (plate height) (or N number of plates) - use smaller particles, lengthen

column, viscosity of mobile phase (diffusion)
 (selectivity factor) ,a - vary temperature, composition of column/mobile

phase
 kA (capacity factor) -vary temperature, composition of column/mobile

phase
Column resolution
Resolution (zone broadening)
The resolution of a chromatographic column is a quantitative

measure of its ability to separate analytes A and B .
So, separation of mixtures depends on:
 Width of solute peaks (want narrow) efficiency
 Spacing between peaks (want large spacing) selectivity

Column resolution
Factors influencing resolution
 Capacity factor K
 Selectivity or separation factor α (alpha)
 Column efficiency or
 Number of theoretical plates N.

Resolution
Resolution is a measure of the separation of two peaks of

different retention time t in a chromatogram.
where tR is the retention time and wb is the peak width at baseline.

Here compound 1 elutes before compound 2. If the peaks have the
same width

tRB
tRA
Detector Signal
A B
tM
A
𝑊𝑎 𝑊𝑏
2 2
tM tR1 tR2 Time

Selectivity
 Selectivity is a relative measure of the retention of two solutes,

which we define using a selectivity factor, a where solute A has
the smaller retention time. When two solutes elute with identical
retention time, a=1.00; for all other conditions a>1.00

Column Efficiency
Suppose we inject a sample that has a single component. At the

moment we inject the sample it is a narrow band of finite width. As
the sample passes through the column, the width of this band
continually increases in a process we call band broadening. Column
efficiency is a quantitative measure of the extent of bandbroadening.

Column Efficiency
They described column efficiency in terms of the number of

theoretical plates, N, where L is the column’s length and H is the
height of a theoretical plate

.

Lecture 1,2, Introduction To Chromatography-1

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Lecture 1,2, Introduction To Chromatography-1

Uploaded by

Copyright:

Available Formats

.

5/8/2021 Prepared By Asmare T.

⸎ CS= Molar concentration of solute in stationary phase

⸎ CM= Molar concentration of solute in mobile phase

⸎ Stationary phase: This phase is always composed of a “solid” phase or “a

⸎ Mobile phase: This phase is always composed of “liquid” or a “gaseous

⸎ Separated molecules: The type of interaction between the stationary phase,

⸎ It has been rightly termed as the lifeline of the HPLC system.

⸎ It plays the important role of transport of the sample through the

5/8/2021 Prepared By Asmare T.

5/8/2021 Prepared By Asmare T.

⸎ In normal-phase chromatography, the stationary phase is polar and the

5/8/2021 Prepared By Asmare T.

 Chromatography is essentially a physical method of

5/8/2021 Prepared By Asmare T.

 A separation results from repeated sorption/desorption

 Useful separations require an adequate difference in the

5/8/2021 Prepared By Asmare T.

 It is based on the Greek word chroma, for colour. graphy mean

 Is a process which is based on multiple repetition of sorption and

5/8/2021 Prepared By Asmare T.

tM tR1 tR2 Time

5/8/2021 Prepared By Asmare T.

tM tR1 tR2 Time

5/8/2021 Prepared By Asmare T.

differential migration process in a system consisting of two or

5/8/2021 Prepared By Asmare T.

 A method of physically separating mixtures of gases, liquids, or

5/8/2021 Prepared By Asmare T.

5/8/2021 Prepared By Asmare T.

 Exact quantitative analysis is done even from trace compounds.

5/8/2021 Prepared By Asmare T.

Analytical - determine chemical composition of a sample

Preparative - purify and collect one or more components

5/8/2021 Prepared By Asmare T.

 Creating vaccinations. Chromatography is useful in determining

 Food testing. ...

 Beverage testing. ...

 Drug testing. ...

5/8/2021 Prepared By Asmare T.

The purpose of chromatography is to separate a complex

5/8/2021 Prepared By Asmare T.

5/8/2021 Prepared By Asmare T.

Chromatogram - Detector signal vs.

5/8/2021 Prepared By Asmare T.

Mobile Phase (a moving solvent)

Stationary Phase (immobile matrix)

Immiscible solid or liquid phase that fixed in place in the column or on a

5/8/2021 Prepared By Asmare T.

leaving the column is the eluate.

 Elution is the process of passing liquid or gas through the

5/8/2021 Prepared By Asmare T.

Purpose of the mobile phase in HPLC

 It plays the important role of transport of the sample through the

5/8/2021 Prepared By Asmare T.

A good mobile phase has the following characteristics.

 Compatible with the column packing/stationary phase and the

 Gives good separation of the analytes of interest.

 Is of the highest purity.

5/8/2021 Prepared By Asmare T.

 Should dissolve investigated sample;

 Possible excretion from it divided components;

 It must be inertness in relation to materials of all parts of chro.