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chromatography and HPLC principles

Technical Report · January 2018

DOI: 10.13140/RG.2.2.33635.25126


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Hayder Obayes Hashim

University of Babylon


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Chromatography and HPLC principles
By : Dr Hayder Obayes Hashim
Chromatography : The word “chromatography” means
The term chromatography is employed to describe a wide verity “writing with colour” and refers to the
of separation techniques but all of these techniques share the early observations on the separation of
same principle , this principle can be described as the difference dyes by paper chromatography.
in interaction properties of the analyte to two phases .
One of these phases is relatively static and called stationary Depended on the kind of phases the
phase and the other is relatively mobile and called mobile phase chromatography can be classified in to
. several types
All the chromatography type that have a liquid mobile phase are Mobile Stationary Type of
called liquid chromatography or LC . and due to the popularity ph. ph chromatography
and versatility of LC chromatography , we will discuss this type
gas gas Gas- gas
and for more specific term column LC .
gas liqued Gas- liquid
Conventional LC system
The simplest method to perform column chromatography is
called low pressure chromatography in this method the gas Solid Gas- solid
movement of the mobile phase occur solely by gravity or by aid chromatograpy
of low pressure pump . Liquid liqued Liquid – liquid
The simplest system is composed from chromatography
1- Mobile phase reserver : which is a container to reserve Liquid solid Liquid- solid
the mobile phase . this container is fixed directly or by chromatography
tube to the column Solid Solid Solid – solid
2- The column : it is a relatively wide hollow tube packed chromatography
with a specific kind of materials , this material represent
the stationary phase of the system .
3- A suitable detection method to monitor the content of
the post column mobile phase .(usually it is UV/vis
The general view of method :
Assume that we have a mixture of three compound as the
a- Compound A which has no interaction with the stationary
b- Compound B which has a little interaction with stationary
c- Compound C has a higher degree of interaction with the
stationary phase
When this mixture is applied to the top of the column and
permitting the mobile phase to flow with specific flow rate,
a- the compound A which has no interaction with the
stationary phase will move through the column with a
rate similar to the mobile phase rate
b- compound B which has a little interaction with stationary
phase will be slowed by the stationary phase and will
move through the column with a rate lesser than the
mobile phase movement rate
c- compound C which has more interaction with stationary
phase will be slowed more than compound B and due to
this its movement rate through the column will be lesser
than compound B .
in this case we now separate the three compounds . and for
detect the passage of three compounds from the column outlet
we have to monitor the absorbance change in the outlet mobile
phase . the change in absorbance in all over the procedure is
drawing on a paper or by computer software and called
chromatogram and then analyzed to study the separation
Chromatography and HPLC principles
By : Dr Hayder Obayes Hashim
parameters .

stationary phase

The action moods of stationary phase

stationary phase interact with analytes and retained its
movement through the column , by mean of several ways
depending on the stationary phase kind , this ways or moods
are such as :
1- Adsorption Chromatography
The principle of adsorption chromatography is known from
classical column. A relatively polar material with a high
specific surface area is used as the stationary phase, silica
being the most popular. The mobile phase is relatively non-
polar (heptane to tetrahydrofuran). The different extents to
which the various types of molecules in the mixture are
adsorbed on the stationary phase provide the separation
effect. In this kind the polar compounds are eluted later than In ion exchange chromatography
non-polar compounds. This kind of chromatography is also 1- The negative stationary phase is
called normal phase chromatography . called cations exchanger
In the last example if the stationary phase which used has 2- The positive stationary phase is
this kind of separation then the compounds have to be as called anions exchanger
the following :
A: non polar , B: has a little polarity , C has more polarity
than C
Reversed-Phase Chromatography The reverse of the
above applies: (a) The stationary phase is very non-polar.
(b) The mobile phase is relatively polar (water to
tetrahydrofuran). In this kind the non-polar compounds are
eluted later than polar compounds. In the last example if the
stationary phase which used has this kind of separation then
the compounds have to be as the following : A : polar , B: a
little non polar , C: more non polar than B
2- Ion-exchange chromatography (IEC)
is based on the principle that opposites attract. Ion-
exchange chromatography is used to separate charged
analytes and therefore occurs as a result of interaction
between a charged solute and an oppositely charged,
solid stationary phase. Ion-exchange chromatography

Chromatography and HPLC principles
By : Dr Hayder Obayes Hashim
can be applied to any solute that can acquire a charge in
solution. If the stationary phase has a negative charge
this charge will interact with positive analytes and cause
its retardation ( slowed its movement )
In the last example if the stationary phase which used
has this kind of separation with a negative charge then
the compounds have to be as the following : A :
negative charged , B: a little positive , C: more positive
than B

3- Affinity chromatography
is based on the lock-and-key mechanism prevalent in
biological systems. The retention mechanism is very
specific, but the technique is more time-consuming and
more expensive than those employing other retention
mechanisms. In this kind of chromatography the
stationary phase can contain :
a- Antibody to interact with its specific antigen
b- Antigen to interact with its specific antibody
c- Enzyme to interact with its specific coenzyme or
substrate analogue
d- ssDNA to interact with its complementary ssDNA
e- other
4- Size-exclusion chromatography (SEC)
is based on the sieving principle. In SEC, the stationary
phase particles are manufactured with a wide range of
pore sizes, causing the stationary phase to behave like a
molecular sieve. As a result of the sieving action, the
analytes are separated on the basis of size, with the
larger ones eluting first (BOCOF, big ones come out


The eluted compounds are transported by the mobile phase to
the detector and recorded as Gaussian (bell-shaped) curves.
The signals are known as peaks and the whole entity is the
chromatogram. The peaks give qualitative and quantitative
information on the mixture in question:
(a) Qualitative: the retention time of a component is always
constant under identical chromatographic conditions. The
retention time is the period that elapses between sample
injection and the recording of the signal maximum.
(b) Quantitative: both the area and height of a peak are
proportional to the amount of a compound injected. A calibration
graph can be derived from peak areas or heights obtained for
various solutions of precisely known concentration and a peak-
size comparison can then be used to determine the
concentration of an unknown sample.

Also The chromatogram can be used to provide information on

separation efficiency . Here w is the peak width at the baseline,
t0 is the dead time or retention time of an unretained solute
(compound A in the last example ) , i.e. the time required by the
mobile phase to pass through the column . Hence the linear flow
velocity, u, can be calculated as

Chromatography and HPLC principles
By : Dr Hayder Obayes Hashim

where L is the column length. A non-retained compound, i.e.

one that is not retained by the stationary phase, appears at the
end of the column at t0.
tR is the retention time ; this is the period between sample
injection and recording of the peak maximum. Two compounds
can be separated if they have different retention times .

Calculate the k and α values of compounds 1
and 2 in the Fig. if the
t 0 = 12:5 sec ; tR1= 33:1 sec; tR2 = 70:5 sec.

Then α = K2/K1 = 4.6/1.6 = 2.875

. t’R is the net retention time or adjusted retention time.and it is

equal to
t’R = tR – t0

t0 is identical for all eluted substances and represents the

mobile-phase residence time.
t’R is the stationary phase residence time and is different for
each separated compound. The longer a compound remains in
the stationary phase, the later it becomes eluted.
Retention time is a function of mobile phase flow velocity and
column length. If the mobile phase is flowing slowly or if the
column is long, then t0 is large and hence so is tR;
tR is therefore not suitable for characterizing a compound.
Therefore the retention factor or k value (formerly known as the
capacity factor ) is preferred:

k is independent of the column length and mobile phase flow-

rate and represents the molar ratio of the compound in the
Chromatography and HPLC principles
By : Dr Hayder Obayes Hashim
stationary and the mobile phase .
Two components in a mixture cannot be separated unless they
have different k values, the means of assessment being
provided by the separation factor, α, formerly known as the
relative retention

If α =1, then no separation takes place as the retention times

are identical. The separation factor is a measure of the
chromatographic system’s potential for separating two
compounds, i.e. its selectivity. Selection of the
stationary and mobile phases can affect the value of α.

The resolution, R, of two neighboring peaks is defined by the Example . How many theoretical plates
ratio of the distance between the two peak maxima, i.e. the distance emerge from calculations based on the
between the two retention times, tR, and the arithmetic mean of the last peak in the chromatogram
two peak widths, w . Resolution is a term used to describe the Solution :
degree of separation between neighboring solute bands or peaks.

N, the number of theoretical plates, is one index used to determine the

performance and effectiveness of columns, and is calculated using equation

The value of N reflect the efficiency of the column to separate a

certain analyte and it is used to compare between the efficiency of
two or more columns to separate a certain analyte .
As we see from the equation of N we can recognize that the good
column have to retain the analyte for longest time but in addition
have to keep the peak width at the minimum .
Both of these parameters ( the long retention time and narrow peak
width ) can be enhanced by increasing the surface area of the
stationary phase the best way to increase the surface area of the
stationary phase is by using a stationary phase composed of small A column side view B column side view
particles . but the usage of small particles stationary phase develop a
very high resistance to the movement of the mobile phase and to
overcome this resistance a high pressure pump is used to force the
mobile phase through this small particles stationary phase column .
this kind of chromatography is called high pressure liquid
chromatography or high performance liquid chromatography and
abbreviated to HPLC . A column tope view B column top view

Isocratic and gradient mobile phase: A column :

Isocratic mobile phase: mean that the composition of the mobile larger stationary phase particles
Chromatography and HPLC principles
By : Dr Hayder Obayes Hashim
phase ( type , concentration ) is unchanged in all over the lesser surface area
chromatographic procedure . lesser efficiency (N)
Gradient mobile phase : mean that the mobile phase composition ( lesser resistance to mobile phase
type , concentration ) is changed during the chromatographic movement
procedure . B column :
How to write and understand the mobile phase composition : Smaller stationary phase particles
In isocratic mood ( elution ) is simply represented by writing : the More surface area
procedure or the elution is done by isocratic of ( mobile phase More efficiency (N )
composition and concentration ) for example : More resistance to mobile phase
1- The elution is done by isocratic 70% acetonitrile : this mean movement
the composition of the mobile phase is 70% acetonitrile and
30% water in all over the procedure .
2- The elution is done by isocratic 40% PH 7 , 0.2 M phosphate
buffer and 60% methanol . this mean the composition of the
mobile phase is a mixture of 40% from( PH7 ,0.2M
phosphate buffer) and 60% methanol in all over the Note : in gradient , the summation of
procedure . the concentrations of the all solvents
While in the gradient mood the mobile phase is represented by two at any time are equal to 100% . for
or more solvents type which are changed during the time of the this the references books or the
elution ( procedure ) and always it represented by a table . researchers always mention the
Example 1 : the elution is done by a gradient of two solvents , change in the concentration of one
As in the table mobile phase because the other
time mobile mobile mobile phase concentration can be
min A B easily deduced .
0 10 90
20 100 0 Question :
To Determination of Paracetamol
This mean that the elution started with 10% of the solvent A and and its by-products by HPLC a
then the concentration (V/V) of the A increased gradually through gradient of two mobile phases are
the period 0-20 minutes to 100% , while the solvent B started with used ; A: Acetonitrile , B: Water (pH
90% and gradually decreased through the period 0-20 minutes to 2.75 with H3PO4)
As in the table
0% Time min A% B%
0 10
0.65 10
5.4 60
10.4 60
12 10

1- Deduce the B concentrations

2- Explain the data in this table
3- Draw a diagram represent
the change in the A and B
during the time of the
procedure ?

Chromatography and HPLC principles
By : Dr Hayder Obayes Hashim
Example2 : the elution is done by a gradient of two solvents , A:
acetonitrile and B: ethanol as the following table

time mobile mobile

min A B
0 30 70
2 30 70
5 80 20
15 80 20
This mean that the A concentration (V/V%) started with 30% and
continuo until the 2 minutes at the same value then the
concentration of A increased to 80% gradually through the period
between 2 - 5 minutes then it continuo at the same value along the
period from 5-15 minutes . while the B is started with 70% and
continuo to the 2 minutes at the same value then the concentration
of B decreased gradually to 20% through the period between 2-5
minutes then it continuo at the same value along the period 5-15
minutes .

High pressure liquid chromatography Or high performance liquid
chromatography . it is the most popular technique in pharmaceutical
preparations and measurement due to its simplicity and versatility .
indeed HPLC represent a technique which include complete procedure
(protocol ) . this procedure is performed by the aid of specific device
which called HPLC system or device .
HPLC system competent:
There are many essential and optional components of HPLC
system , here we will discuss the essential components only .

Chromatography and HPLC principles
By : Dr Hayder Obayes Hashim

1- solvent reservoirs
a container always made of glass or plastic . they have to be secure
to prevent evaporation of solvents
2- degasser
A tube made from a porous material which allow gas exchange
under vacuum of the vacuum pump . the degasser function to
remove the gases from the solvents ( the mobile phase) . the gases
vacuoles in the solvent can cause bad separation conditions and lead
to bad signals in the detector ..
3- low pressure gradient Pumps (LPG)
A solvent low pressure pumps which control the amount of solvent
from each reservoir . it is very important to be very precise to pump
the right amount of solvent at a right time . any change in the
amount and the time can change the retention time and decrease the
reproducibility of the procedure . this component found only in the
system which support the gradient and isocratic moods of mobile
phase and omitted in the system which support the isocratic mood
only .
4- High pressure pump
This is the main pump that force the mobile phase to flow through
the column and should have the following properties : # psi : pound per square inch
Constructed of materials inert toward solvents to be used 1bar ≈ 1 atmosphere ≈ 14.5 psi
Deliver high volumes (flow rates) of solvent (to 10 mL/min in Analytical HPLC : this kind of
systems used to detect the quantity
analytical HPLC and more in preparative HPLC)
and the quality of the compounds
Deliver precise and accurate flow (<0.5% variation)
but it is difficult to be used in
Deliver high pressure (up to 6000 psi) purification, i.e. this system do not
Chromatography and HPLC principles
By : Dr Hayder Obayes Hashim
5- Pre-column or saturation column or guard column used in the procedures that aim to
A guard column is introduced before the analytical column to purify a certain compound from a
increase the life of the analytical column by removing not only mixture . i.e. the output of this
particulate matter and contaminants from the solvents but also system is data only .
sample components that bind irreversibly to the stationary phase. Preparative HPLC : this kind of
6- sampling port : systems used mainly in the
It is a tool for introducing the sample (which have to be analyzed ) purification of compound from a
prior to the column front . Due to the high pressure in this place mixture and also to detect the
this port is designed in sophisticated way to fulfill this mission . quantity and the quality of the
7- the main column compounds ( but lesser accuracy
It is always made from a tube of stainless steel packed with the than analytical systems ) . i.e. the
stationary phase . Different type of stationary phase can be used in output of this kind are data and
HPLC and employ any chromatography methods for separation purified compound . this kind is the
such as ion exchange , size exclusion , affinity , and the adsorption main tool in the drugs purification .
chromatography which is the most used method . Column parameters :
8- - the detector 1- Stationary phase type
The detector is a device used to monitor the change in the mobile 2- Stationary phase particles
phase content of analytes after the passage through the column size
There are several types of detectors used in HPLC each one has its 3- Column length
advantages and disadvantages 4- Column internal diameter
 UV-VIS Detector :UV-VIS Detector is the most commonly
used detector. Its response is specific to a particular
compound or class of compounds depending on the presence
of light absorbing functional groups of eluting molecules. It
is cheap and versatile but unspecific .
 Fluorescence Detector :Fluorescence detection offers
greater sensitivity than a UV-VIS detector. However, the
number of naturally fluorescent compounds is smaller in
comparison to light absorbing compounds. This limitation is
overcome by post or pre column derivatization.
 Mass Spectroscopic Detector : Mass spectroscopy offers
very high sensitivity and selectivity. Detection is based on
fragmentation of molecules by electric fields and separation
on basis of mass to charge ratios of fragmented molecules.
LC –MS technique has opened up new application areas due
to advantages of resolution and sensitivity.
 Other detectors : such as refractive index detector , light
scattering detector , biosensor detector ,etc
9- data presentation and analysis method : the data that collected
by the detector is transmitted to a printer or computer software to
draw the chromatogram . then this chromatogram can be analyzed
manually or by specialized software .

Chromatography and HPLC principles
By : Dr Hayder Obayes Hashim
HPLC procedures UV/HPLC or HPLC/UV : it is the
If we have a sample ( blood , drug ,water ) the aim of or procedure can be one of HPLC system that contain the
the following : UV detector .
1- To detect the presence of certain compound or compounds in the sample
, e.g. we want to detect the presence of diazepam in patient blood sample
and/or estimate its level , or we want to detect the presence of caffeine the eluent fraction containing the
in plant extract and /or estimate its level . this aim can be easily fulfilled peak of interest : this mean
by UV/HPLC or the other kind of HPLC . collecting the mobile phase
2- To detect unknown compound in the sample , e.g. we want to detect the after the column ( post column )
quality of the effected compound in plant extract , or we want to detect and separate the mobile phase
the compound that induce a certain disease in a patient blood sample ,
which contain peaks in fractions
this is very difficult to fulfilled by UV/HPLC . In this case, it may be
each fraction represent a
necessary to employ detectors that can be used to aid in identification,
such as mass spectrometers. It may also be necessary to collect the certain peak , the fraction that
eluent fraction containing the peak of interest for off-line contain the target peak
characterization using Infra-red or Nuclear Magnetic Resonance represent a pure form of the
Spectroscopy (NMR). target analyte . this is done by
Here we will discuss the first aim and how to determine the presence and preparative HPLC .
quantity of certain compound by using UV/HPLC device because it is the
simplest and most popular .
How can we do our detection and separation by HPLC
First we have to answer this questions : do we want to detect our target
compound by using previously validated method ?
If the answer is “yes” then we have to follow this method exactly .
If the answer is “no” then we have to develop and validate our method , the
development of a new method can be very sophisticated and expense along time
and costly . actually , a master degree thesis can be achieved by develop and
validate a new method for a certain compound .
So we will focus on how to use a previously developed and validated method to
do our qualitative and quantitative determination of the target compound .
What are the information that we have to know from the previously
validated method to do our detection and separation by HPLC :
To do our detection or/and separation we have to know the following :
1- What we have to do with the sample before the introducing to the HPLC
device ( the injection ) . some samples need only filtration while the ** all the samples have to be
other sample need a specific extraction or derivatization or other filtrated by 0.45 µm pore size
treatment before the introducing to the HPLC device . filter , for removal of large
2- The chromatography conditions this include : sample particles .
A. The amount of the sample have to be injected to the device
B. The mobile phase type , concentration ,gradient or isocratic , flow Finding a suitable HPLC method
rate : nowadays we can find several
C. The column parameters ; type of stationary phase , the size of method to detect and separate
stationary phase particles , the column length , the internal diameter each compound , in this case
of the column (ID) , the column temperature we have to choose the method
D. The wave length of the detector according to our need ,material
E. The linearity of the detection . each method have a range of a good and devices availability .
quantity detection so we have to adjust our target compound
quantity ( by dilution or concentration ) to be within this range .
After this we can now do our qualitative and /or quantitative measurement of our
target compound .
Qualitative HPLC analysis :
The aim of qualitative analysis is to answer the question , is the target or certain
compound present in the sample .e.g. if we trying to detect the presence of
arginine ( Arg or R ) in urine sample and to establish this first we have to find
valid procedure for our target ( in this case the Arg ) second we should have a
pure form of Arg to use it as a reference standard , then we run the sample

Chromatography and HPLC principles
By : Dr Hayder Obayes Hashim
according to the procedure and run the standard in other run under the same
condition . if we find a peak in the sample have the same retention time ( tR) as
the retention time of the standard , here we can say that the peak in the sample ** in HPLC each sample and
can be Arg in about 90% accuracy . standard have to analyzed at
least three time ( triplicate run )
then the median of these runs
will represent the output data .

For more rigorous peak assignment it is important that the analysis is also carried
out under orthogonal (different) conditions. The analysis is carried using a
different column and mobile phase, and the retention time and response
compared again. If a match is found, then the confidence in the peak
identification is increased.

Sample Spiking
It is another technique for qualitative detection . The technique of „spiking‟ a
sample involves the addition of a known reference material ( the standard ) to the
sample , in order to confirm the identity of one of the sample component peaks.

In past diagram , one of the peaks in the sample is suspected to be X compound .

The sample is spiked with X ( addition of X) at approximately the same

Chromatography and HPLC principles
By : Dr Hayder Obayes Hashim
concentration as the sample components. If any of the peaks within the
chromatogram gets larger, then that peak may be X. If a new chromatographic
peak is seen, or if any of the peaks develops a „shoulder‟, then it is unlikely that
any of the peaks in the chromatogram is due to X within the sample.

Quantitative Analysis :
After the peaks have been identified, the next step in the analysis is
quantification. Quantification uses peak areas or heights to determine the
concentration of a compound in the sample.
Although there are many different types of detector used for quantitative HPLC
analysis, but the quantitative principle remains constant. Each detector will
produce a response that depends upon the AMOUNT of analyte to which it is
responding. The scale of the response may be dependent on the analyte itself as
well as the matrix from which it comes - and therefore standards are usually
employed to „calibrate‟ the instrument response.
There are some general requirements that must be met before a quantitative
analysis can be undertaken, and these include:
• The identity of the component to be analyzed should be known
• The best possible separation of the component should be achieved
• Standards of known purity should be available

Calibration of an HPLC method is necessary to give quantitative results.

However there is more than type of calibration available, and each has its virtues
and limitations. The aim is to select the most appropriate one .
Here we will shortly explain a two type of calibration .
1. External Standard Calibration
This involves analysing a series of standards covering the concentration range of
interest. For example:
Level 1 - 40mg/l
Level 2 - 60mg/l
Level 3 - 80mg/l
Level 4 - 100mg/l
The peak for each component is integrated and identified and the peak area is ** peak integration : it is the
plotted against concentration to give a calibration curve ,When an unknown calculation of peak parameters
sample is run, the peaks are integrated and identified, and the peak areas are such as area , height , width
related to a concentration from the calibration graph.

The simplest method to

calculate peak area ( but less
accurate ) = height * ½ width
** the modern chromatography
software calculate the peak
parameters automatically .
External standards calibration

Chromatography and HPLC principles
By : Dr Hayder Obayes Hashim
If the calibration curve is a straight line, and it goes through zero, it is valid to
use a single point calibration. For example, only the highest standard is run,
and the line passes through this point and zero. If the points do not give a straight
line, or the curve does not pass through zero, a multi-point calibration should be
used, and normally five concentration levels are required.

2-Internal standard calibration

In this method an foreign compound is added to the sample and standards
solution . this foreign compound is called internal standard (IS) . Internal
standards are usually well characterized compounds which are not present in the
sample and is added in a known (constant) concentration to standard and sample
solutions . usually at the beginning of the analytical process to compensate
for losses and variability throughout the sample preparation and analytical
The Good internal standards compound have to be :
 elute near to, but are well resolved from, the analyte of interest
 are chemically and physically similar to the analyte
 are not present in the original sample mixture
 are unreactive towards any of the sample components
 are available in highly pure form
 are added in the concentration range 0.3 - 0.5 of the expected
MAXIMUM analyte concentration .
The calibration curve is constructed by plotting the
(The area of external standard / the area of the internal standard )on the Y axis .
and the (concentration of external standard /concentration of internal standard) on
the X axis .
Then to estimate the concentration of the target analyte in the sample , we do as
in the external standard method but instead we put the value of : ( sample peak
area/internal standard peak area ) on the Y axis to find (the concentration of
sample/concentration of the internal standard) on the X axis . as in the example
below :
To estimate the lysine concentration in plasma . by using 2ng/ml of norvaline as
internal standard the peaks area of the sample and standards as the following

run Concentration of Lys Peak area of Lys Peak area of

ng/ml norvaline (IS)
Ex St1 1 4 2
Ex St2 3 10 1.8
Ex St3 5 22 2.1
Sample ??? 18 3

Standard curve construction

X axis values :
Concentration of Ex st/concentration of (IS)
1 / 2 = 0.5
3 / 2 = 1.5
5 / 2 = 2.5
Y axis values :
The peak area of Ex st / peak area of IS
Ex st 1 : 4 /2 = 2
Ex st 2; 10 /1.8 = 5.5
Ex st3 : 22 / 2.1 = 10.5
Plot each value of X to its corresponding value of Y on the chart
Chromatography and HPLC principles
By : Dr Hayder Obayes Hashim
As the points : (0.5 ,2 ) , (1.5 , 5.5 ) , (2.5 , 10.5 ) and draw a line pass through or
near to all of the points

After this then we calculate the sample value of Y axis as :=

The peak area of the sample /the peak area of the IS
= 18/ 3 = 6
Then find the corresponding value on the X axis as the following

The sample X value represent :

The concentration of the Lys / concentration of IS =1.5
So the concentration of Lys = 1.5 * 2 ( IS. con. ) = 3 ng

Chromatography and HPLC principles
By : Dr Hayder Obayes Hashim


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