EXP # 1
Object:
To selection of the proper mobile phase
Apparatus:
TLC plate, TLC tank, graduated cylinder, jet
Reagents:
Hexane
Chloroform
Observation:
Hexane
|
Chloroform
|
Separation from UV light
|
||
1
|
1
|
|
||
0.8
|
1.2
|
|
||
0.6
|
1.4
|
|
||
0.4
|
1.6
|
|
||
0.2
|
1.8
|
|
||
0.0
|
2.0
|
|
Result:
It was observed that as soon Hexane concentration was decrease and Chloroform concentration was increase there was maximum separation.
It was observed that as soon Hexane concentration was decrease and Chloroform concentration was increase there was maximum separation.
EXPERIMENT # 02
Object:
To examine dyes in inks by ascending paper chromatography
Apparatus:
Chromatography paper, 9 samples of ink, hanger, paper pine, tile, dryer,
Capillary
Chemicals:
N-butanol (40ml)
Glacial Acetic acid (10ml)
Water (50ml)
Observation:
Pelikan (Black)
|
Dollar (Blue)
|
Quick (Red)
|
Pelikan (Red)
|
Dollar (Black)
|
Quick (Blue)
|
Pelikan (Blue)
|
Dollar (Red)
|
Quick (Black)
|
|
|
|
|
|
|
|
|
|
Calculation:
Rf = Distance cover by Component
Distance cover by Solvent
For Pelikan (Black):
Rf1 = 0.3 / 4.9
= 0.061
Rf2 = 2.3 / 4.9
= 0.469
For Dollar (Blue):
Rf = 0.9 / 4.9
= 0.183
For Quick (Red):
Rf = 1.5 / 4.9
= 0.306
For Pelikan (Blue):
Rf1 = 0.8 / 4.9
= 0.163
Rf2 = 2.0 / 4.9
= 0.408
For Dollar (Black):
Rf1 = 0.7 / 4.9
= 0.142
Rf2 = 1.5 / 4.9
= 0.306
For Quick (Blue):
Rf = 0.9 / 4.9
= 0.183
For Pelikan (Black):
Rf = 1.9 / 4.9
= 0.387
For Dollar (Red):
Rf1 = 0.6 / 4.9
= 0.122
Rf2 = 2 / 4.9
= 0.408
For Quick (Black):
Rf1 = 0.4 / 4.9
= 0.0816
Rf2 = 0.7 / 4.9 = 0.142
Result:
The
dyes in ink is examined by paper chromatography and it is found by various Rf
values which are shown in calculation
EXPERIMENT # 03
Object:
To examine the dyes in inks by radial chromatography
Apparatus:
Chromatography paper, 2 samples of ink, 2 Petri dish of different sizes,
Chemicals:
N-butanol
(4ml)
Glacial Acetic acid (1ml)
Water (5ml)
Observation:
Sample 1
|
Sample 2
|
|
No separation
|
Calculation:
Rf = Distance cover by Component
Distance cover by Solvent
For Sample 1:
Rf1 = 0.3 / 0.7
= 0.428
Rf2 = 0.5/ 0.7
= 0.714
Result:
The Rf values of the dye in the Ink by Radial Chromatography is
found to be 0.428 & 0.714
EXPERIMENT NO 4
Object:
Conditioning and Capacity
Chemicals:
· 6 %
Hcl solution
· 6
%Nacl solution
· 0.1 N
NaOH
Observation:
Volume of NaOH
|
||
Initial Reading
|
Final Reading
|
Difference
|
0
|
13.8
|
13.8
|
13.8
|
27.6
|
13.8
|
27.6
|
41.4
|
13.8
|
Calculation:
The wet volume capacity = ( ml. of Effluent × Concentration
of effluent ) / ml of Resin
Concentration of effluent:
N1V1=N2V2
(0.1)(13.8) = N2(25)
N2 = 2.89x10 equivalent
The wet volume capacity = (25)( 2.89x10) / (20)
The wet volume capacity = 3.6125 x10 equivalent
Result:
The wet volume Capacity is found to be 3.6125 x10 equivalent
EXP # 5
Object: Preparation of Column
Materials:
- Glass
column
- Activated
absorbent
- Solvent(Mobile phase)
Observations:
Method 1: column filed with adsorbent and then with
solvent
Method 2: column filed with slurry of solvent and
Absorbent
Method 3: column filed with solvent and then with
absorbent
Time(mint)
|
Volume(ml)
|
||
Method 1
|
Method2
|
Method3
|
|
5
|
2
|
2.1
|
2
|
10
|
8
|
6.5
|
7
|
15
|
8.6
|
8.8
|
7.2
|
Calculation:
Flow rate= Volume/Time
Flow rate
|
Method 1
|
Method 2
|
Method 3
|
1
|
0.40ml/min
|
0.42 ml/min
|
0.40 ml/min
|
2
|
0.80 ml/min
|
0.65 ml/min
|
0.70 ml/min
|
3
|
0.57 ml/min
|
0.58 ml/min
|
0.48 ml/min
|
EXP # 6
Separation of dyes by adsorption Chromatography
Materials:
· Glass
column
· Alumina
· Ethanol
· Water
Observation:
Dye
|
Retention time
|
Retention volume
|
Content
|
TR (min)
|
VR (ml)
|
Methyl blue
|
31.56
|
2.8
|
Methyl yellow
|
16.40
|
2.1
|
Calculation:
· Column
resolution Rs:
Rs = Az/w
Where Az =15.16 min (dif of time of solution
arrival)
W = 23.98 min (average time of solute arrival)
So
Rs = 15.16 / 23.98
Rs = 0.632
Average rate of migration v(cm/mint):
U
= L/tR
L = 20 cm (length of column)
Dye content
|
Rate of migration U(cm/min)
|
Methyl blue
|
0.633
|
Methyl red
|
1.219
|
EXP # 7
Object:
Analysis of Dye mixture by TLC
Chemicals:
· Alcohol
40ml
· Water
10 ml
Observation:
Dye Content
|
Height in cm
|
Methyl Blue
|
0.5
|
Methyl Yellow
|
7.5
|
Graph has been taken out from the Spectroscopy which is
attached with the practical
Calculation:
Rf =
Distance cover by Component
Distance cover by Solvent
Methyl Blue
Rf1 = 0.5 / 9.0
= 0.055
Methyl Yellow
Rf2 = 7.5 / 9.0
= 0.833
Result:
Dye Content
|
Rf Value
|
Methyl Blue
|
0.055
|
Methyl Yellow
|
0.833
|
Ion-exchange chromatography
Ion-exchange chromatography (or ion
chromatography) is a process that allows the separation of ions and polar
molecules based on their charge. It can be used for almost any kind of
charged molecule including large proteins, small nucleotides and amino
acids. The solution to be injected is usually called a sample, and
the individually separated components are called analytes. It is
often used in protein purification, water analysis, and quality control.
Principle:
Ion exchange chromatography retains analyte molecules
on the column based on coulombic (ionic) interactions. The stationary
phase surface displays ionic functional groups (R-X) that interact with analyte
ions of opposite charge. This type of chromatography is further subdivided into
cation exchange chromatography and anion exchange chromatography. The ionic
compound consisting of the cationic species M+ and the anionic species B- can
be retained by the stationary phase.
Cation exchange chromatography retains positively charged cations because
the stationary phase displays a negatively charged functional group:
Anion exchange chromatography retains anions using
positively charged functional group:
Note that the ion strength of either C+ or A- in the mobile
phase can be adjusted to shift the equilibrium position and thus retention
time.
The ion chromatogram shows a typical chromatogram obtained
with an anion exchange column.
Capacity
The capacity of an ion exchanger is a quantitative measure
of its ability to take up exchangeable counter-ions and is therefore of major
importance. The capacity may be expressed as total ionic capacity, available
capacity or dynamic capacity. The total ionic capacity is the number of charged
substituent groups per gram dry ion exchanger or per ml swollen gel. Total
capacity can be measured by titration with a strong acid or base.
The actual amount of protein which can be bound to an ion
exchanger , under defined experimental conditions, is referred to as the
available capacity for the gel. If the defined conditions include the flow rate
at which the gel was operated, the amount bound is referred to as the dynamic
capacity for the ion exchanger .
Available and dynamic capacities depend upon:
The properties of the protein.
The properties of the ion exchanger .
The chosen experimental conditions.
The properties of the protein which determine the available
or dynamic capacity on a particular ion exchange matrix are its molecular size
and its charge/pH rela- tionship. The capacity of an ion exchanger is thus
different for different proteins.
On a porous matrix used for ion exchange chromatography,
molecules which are small enough to enter the pores will exhibit a higher
available capacity than those molecules which are restricted to the charged
substituent’s on the surface of the gel.
Similarly, since the interaction is ionic, the protein’s
charge/pH relationship must be such that the protein carries the correct net
charge, at a sufficiently high surface charge density, to be bound to a
particular ion exchanger under the chosen buffer conditions.
The properties of the ion exchange matrix which determine
its available capacity for a particular protein are the exclusion limit of the
matrix, and the type and number of the charged substituents. High available
capacity is obtained by having a matrix which is macro porous and highly
substituted with ionic groups which maintain their charge over a wide range of
experimental conditions. Non-porous matrices have considerably lower capacity
than porous matrices, but higher efficiency due to shorter diffusion distances.
The experimental conditions which affect the observed
capacity are pH, the ionic strength of the buffer, the nature of the
counter-ion, the flow rate and the temperature. The flow rate is of particular
importance with respect to dynamic capacity, which decreases as the flow rate
is increased. These conditions should always be taken into consideration when
comparing available capacities for different ion exchangers.
Column Chromatography
Column chromatography in chemistry is a method used to
purify individual chemical compounds from mixtures of compounds. It is often
used for preparative applications on scales from micrograms up to kilograms.
Column preparation:
Column is a glass tube with a diameter from 50 mm and a
height of 50 cm to 1 m with a tap at the bottom. Two methods are generally
used to prepare a column
· The
dry method and the wet method. For the dry method, the column is first filled
with dry stationary phase powder, followed by the addition of mobile phase,
which is flushed through the column until it is completely wet, and from this
point is never allowed to run dry.
· For
the wet method, a slurry is prepared of the eluent with the stationary phase
powder and then carefully poured into the column. Care must be taken to avoid
air bubbles.
Stationary phase (adsorbent)
The stationary phase or adsorbent in
column chromatography is a solid. The most common stationary phase for column
chromatography is silica gel, followed by alumina. Cellulose powder has often
been used in the past. Also possible are ion exchange chromatography,
reversed-phase chromatography (RP), affinity chromatography or expanded bed
adsorption (EBA). The stationary phases are usually finely ground powders or
gels and/or are micro porous for an increased surface; though in EBA a
fluidized bed is used.
Mobile phase (eluent)
The mobile phase or eluent is
either a pure solvent or a mixture of different solvents. It is chosen so that
the retention factor value of the compound of interest is roughly around 0.2 -
0.3 in order to minimize the time and the amount of eluent to run the
chromatography. The eluent has also been chosen so that the different compounds
can be separated effectively. The eluent is optimized in small scale pretests,
often using thin layer chromatography (TLC) with the same stationary phase.
A faster flow rate of the eluent minimizes the time required
to run a column and thereby minimizes diffusion, resulting in a better
separation, see Van Deemter's equation. A simple laboratory column runs by
gravity flow. The flow rate of such a column can be increased by extending the
fresh eluent filled column above the top of the stationary phase or decreased
by the tap controls. Better flow rates can be achieved by using a pump or by
using compressed gas (e.g. air, nitrogen, or argon) to push the solvent through
the column (flash column chromatography).
The particle size of the stationary phase is generally finer
in flash column chromatography than in gravity column chromatography. For
example, one of the most widely used silica gel grades in the former technique
is mesh 230 – 400 (40 – 63 µm), while the latter technique typically requires
mesh 70 – 230 (63 – 200 µm) silica gel.
A spreadsheet that assists in the successful development of
flash columns has been developed. The spreadsheet estimates the retention
volume and band volume of analytes, the fraction numbers expected to contain
each analyte, and the resolution between adjacent peaks. This information
allows users to select optimal parameters for preparative-scale separations
before the flash column itself is attempted.
Calculation:-
The higher the resolution of the chromatogram, the better
the extent of separation of the samples the column gives. From Chromatogram,
i.e. conc. of eluent vs. retention time the retention time and curve width are
taken to calculate resolution.
Resolution (Rs):
Rs = 2(tRB – tRA)/(wB + wA)
Where:
tRB = retention time of solute B
tRA = retention time of solute A
wB = Gaussian curve width of solute B
wA = Gaussian curve width of solute A
Plate Number (N):
N = (tR2)/(w/4)2
Plate Height (H):
H = L/N
Where L is the length of the column
tRB = retention time of solute B
tRA = retention time of solute A
wB = Gaussian curve width of solute B
wA = Gaussian curve width of solute A
Plate Number (N):
N = (tR2)/(w/4)2
Plate Height (H):
H = L/N
Where L is the length of the column
Retention Time: The time from the start of
signal detection by the detector to the peak height of the elution
concentration profile of each different sample.
Curve Width: The width of the concentration
profile curve of the different samples in the chromatogram in units of time.
PREPARATION OF COLUMN
MATERIALS
Glass Column
Activated Adsorbent Solvent (Mobile Phase)
n-Hexane
METHOD 1
Close the tip of the column with the thin film of column plug
Mark the column up to 20 cm high
Fill the column with dry activated adsorbent up to 20 cm mar
Introduce the n-Hexane & collect it after each 5 minute & take 3 readings
OBSERVATION
Activated Adsorbent Solvent (Mobile Phase)
n-Hexane
METHOD 1
Close the tip of the column with the thin film of column plug
Mark the column up to 20 cm high
Fill the column with dry activated adsorbent up to 20 cm mar
Introduce the n-Hexane & collect it after each 5 minute & take 3 readings
OBSERVATION
Serial No
|
TIME (minutes)
|
VOLUME (ml)
|
1
|
05:00:00
|
1.6
|
2
|
05:00:00
|
1.7
|
3
|
05:00:00
|
1.6
|
Mean Volume = (1.6 + 1.7 + 1.6)
/ 3 = 1.633 ml
RESULT
The volumetric flow rate = volume/time = 1.633
/ 5 = 0.3266 ml/min
PREPARATION OF COLUMN
MATERIALS
- Glass
Column
- Activated
Adsorbent Solvent (SILICA) (Mobile Phase)
- n-Hexane
METHOD 2
- Empty
the previous column, wash with n-hexane & close its tip with cotton.
- From
previously available adsorbent & solvent prepare the slurry.
- Fill
the column with prepared slurry up to 20 cm mark.
- Introduce
the n-Hexane & collect it after each 5 minute & take 3 readings
OBSERVATION
Serial No
|
TIME (minutes)
|
VOLUME (ml)
|
1
|
05:00:00
|
3.5
|
2
|
05:00:00
|
3.5
|
3
|
05:00:00
|
3.5
|
Mean Volume = (3.5 + 3.5 + 3.5)
/ 3 = 3.5 ml
RESULT
The volumetric flow rate = volume/time = 3.5
/ 5 = 0.700 ml/min
PREPARATION OF COLUMN
MATERIALS
- Glass
Column
- Activated
Adsorbent Solvent (Mobile Phase)
- n-Hexane
METHOD 3
- Empty
the column & dry it with Acetone.
- Fill
the column with n-hexane up to 20 cm mark.
- Introduce
the adsorbent slowly up to the 20 cm mark.
- Collect
the sample after each 5 minute & take 3 readings.
OBSERVATION
Serial No
|
TIME (minutes)
|
VOLUME (ml)
|
1
|
05:00:00
|
4.5
|
2
|
05:00:00
|
4.5
|
3
|
05:00:00
|
4.6
|
Mean Volume = (4.5 + 4.5 + 4.6)
/ 3 = 4.533 ml
RESULT
The volumetric flow rate = volume/time = 4.533
/ 5 = 0.906 ml/min
DISCUSSION
COLUMN CHROMATOGRAPHY
It is a method used to purify individual chemical compounds
from mixtures of compounds. It is often used for preparative applications on
scales from micrograms up to kilograms.
The individual components are retained by the stationary
phase differently & separate from each other while they are running at
different speeds through the column with the eluant. At the end of the column
they elute one at a time. During the entire chromatography process the eluant
is collected in a series of fractions. The composition of the eluant flow can
be monitored & each fraction is analyzed for dissolved compounds.
For Example: By Analytical Chromatography, UV
Absorption or Fluorescence.
Colored compounds (or fluorescent compounds with the aid of
an UV lamp) can be seen through the glass wall as moving bands.
THE SOLVENT (MOBILE PHASE)
The polarity of the solvent which is passed through the
column affects the relative rates at which compounds move through the column.
Polar solvents can more effectively compete with the polar molecules of a
mixture for the polar sites on the adsorbent surface & will also better
solvate the polar constituents. Consequently, a high polar solvent will move
even high polar molecules rapidly through the column. If a solvent is too
polar, movement becomes too rapid and little or no separation of the components
of a mixture will result, if a solvent is not polar enough, no compounds will
elute from the column. Proper choice of an eluting solvent is thus crucial to
the successful application of column chromatography as a separation technique.
PACKING THE COLUMN
In order to obtain maximum efficiency the column must be
evenly packed. While open tabular chromatography using gravity feed of solvent
is restricted to packing particles>150 μm (in order to obtain acceptable
flow rates), the column must be packed as uniformly as possible to minimize
distortion of the chromatographic boundaries. Channeling is usually caused by
the inclusion of air bubbles during packing.
PROCEDURE FOR GRAVITY COLUMN CHROMATOGRAPHY
There are 2 ways to pack a gravity column:
§ THE SLURRY METHOD FOR PACKING
§ DRY-PACK METHOD FOR PACKING
THE SLURRY METHOD FOR PACKING
In the slurry method of column packing, mix the adsorbent
with the solvent & then pour this slurry into the prepared column. The
advantage of slurry method is that they eliminate air bubbles from forming in
the column as it packs.
When finished packing, drain the excess solvent until it
just reaches the top level of the alumina. Close the screw clamp. Your column
is now “packed”
Sometimes a small amount of sand is added to the top of the
column to prevent it from being disturbed when fresh solvent is added.
THE DRY PACK METHOD FOR PACKING
This method is easier, but can lead to bubbles in the
column. Obtain as empty column filling it with a small piece of glass wool,
& affix a pinch to the bottom of the column. Place the column in the
vertical position, close the pinch clamp, and fill the column with the solvent.
Allow solvent to drain from the column to prevent overflowing. Let the alumina
settle & gently tap the column with a pencil so that the alumina will pack
tightly into the column. Drain the solvent until the solvent is just even with
the surface of the alumina.
REQUIREMENTS FOR THE MOBILE PHASE
- The
used solvents must have high purity and low viscosity, strength and
volatility
- The
used solvents must be miscible with each other
- The
samples must be soluble in the mobile phase
THE BEST METHOD
FOR THE PREPARATION
OF COLUMN
The best method of column preparation is found to be the 3rd Method
which is:
- Empty
the column & dry it with Acetone.
- Fill
the column with n-hexane up to 20 cm mark.
- Introduce
the adsorbent slowly up to the 20 cm mark.
The flow rate from 3rd method is found to be = 0.906
ml/min
CONDITIONING AND CAPACITY
APPARATUS
- Resin
Column
- Volumetric
Flask 100ml and 250 ml
- Watch
Glass
- Funnel
- Burette
- Pipette
- Beakers
CHEMICALS
- 6.0
% HCl [6 ml HCl dissolved in 100ml of Distilled Water]
- 6.0
% NaCl [15 gm NaCl dissolved in 250ml Dissolved Water]
- 0.1N
NaOH [0.4 gm of NaOH dissolved in 100ml Distilled Water]
PROCEDURE
Fill the Column with Resin up to 20cm Mark.
Wash the column with 150ml Distilled Water. “20
drops/mint”
Wash with 20ml of 6.0 % HCl. “20 drops/mint”
Wash with 100 ml of H2O. “20 drops/mint”
Wash with 150 ml of 6.0% NaCl. “20 drops/mint”
Wash with 50ml of 6.0% NaCl & note down its pH
OBSERVATION
Initial Reading (ml)
|
Final Reading (ml)
|
Difference Reading (ml)
|
0.0
|
14.2
|
14.2
|
14.2
|
28.5
|
14.3
|
28.5
|
42.7
|
14.2
|
Mean Volume of NaOH consumed = 14.233 ml
CALCULATION
The wet volume capacity = (Volume of NaOH
consumed) x (Concentration of NaOH)
The wet volume capacity = (14.233) x (0.1) = 1.4233
ml
SEPRATION OF DYES BY ADSORPTION CHROLATOGRAPHY
CHEMICALS
- Alumina
- Methanol
- Distilled
Water
- Dye
Solution (Blue + Yellow)
PROCEDURE
- Take
a column & clean it completely
- Make
a mark at the height of 20cm on the column from its tip
- Fill
the column with Alumina till the mark (20 cm)
- Pour
Methanol till the Alumina & Methanol conside at mark (20 cm)
- Take
1 ml of dye solution in a beaker & add some amount of Alumina to make
a dry mixture
- Add
some amount of that prepared mixture into the column
- Start
the stop watch when the blue color starts to flow down from the tip of the
column
- Stops
the stop watch when the blue color stops to flow down from the tip of the
column
- Collect
that blue dye solution in a separate beaker & note down its volume
- Now
add some volume of distilled water to separate yellow dye
- Note
down its time & volume in the way as for the blue dye
CALCULATION
- Retention
Time = TR
- Retention
Volume = VR
- Column
Resolution Rs = Az/W
- Average
Rate of Migration U = L/ TR
OBSERAVTION FOR BLUE DYE
- Retention
Time for Blue Dye = (TR) blue = 20:42:22 = 1242.366
seconds
- Retention
Volume for Blue Dye = (VR) blue = 14.0 ml
OBSERAVTION FOR YELLOW DYE
- Retention
Time for Yellow Dye (TR) yellow = 10:58:72 = 659.2
seconds
- Retention
Volume for Yellow Dye (VR) yellow = 6.8 ml
v Az = Difference of time of the
solution interval
v W = Average time of the solution
interval
v L = Length of the column
CALCULATION FOR Rs
Az = (TR) blue - (TR) yellow = 1242.366
seconds - 659.2 seconds = 583.166 seconds
W = [(TR) blue + (TR) yellow] / 2 = [1242.366
seconds - 659.2 seconds]/2 = 950.783 seconds
L = 20 cm
Now Rs = Az / W
Rs = 583.166/950.783
Rs = 0.6133
CALCULATING AVERAGE RATE OF MIGRATION FOR BLUE DYE “U”
U = 20 / 1242.366
U = 0.0160 ml/seconds
CALCULATING AVERAGE RATE OF MIGRATION FOR YELLOW DYE
“U”
U = 20 / 659.2
U = 0.0303 ml/seconds
DISCUSSION
THE SOLVENT (MOBILE PHASE)
The polarity of the solvent which is passed through the
column affects the relative rates at which compounds move through the column.
Polar solvents can more effectively compete with the polar molecules of a
mixture for the polar sites on the adsorbent surface & will also better
solvate the polar constituents. Consequently, a high polar solvent will move
even high polar molecules rapidly through the column. If a solvent is too
polar, movement becomes too rapid and little or no separation of the components
of a mixture will result, if a solvent is not polar enough, no compounds will
elute from the column. Proper choice of an eluting solvent is thus crucial to
the successful application of column chromatography as a separation technique.
PACKING THE COLUMN
In order to obtain maximum efficiency the column must be
evenly packed. While open tabular chromatography using gravity feed of solvent
is restricted to packing particles>150 μm (in order to obtain acceptable
flow rates), the column must be packed as uniformly as possible to minimize
distortion of the chromatographic boundaries. Channeling is usually caused by
the inclusion of air bubbles during packing.
PROCEDURE FOR GRAVITY COLUMN CHROMATOGRAPHY
There are 2 ways to pack a gravity column:
§ THE SLURRY METHOD FOR PACKING
§ DRY-PACK METHOD FOR PACKING
THE SLURRY METHOD FOR PACKING
In the slurry method of column packing, mix the adsorbent
with the solvent & then pour this slurry into the prepared column. The
advantage of slurry method is that they eliminate air bubbles from forming in
the column as it packs.
When finished packing, drain the excess solvent until it
just reaches the top level of the alumina. Close the screw clamp. Your column
is now “packed”
Sometimes a small amount of sand is added to the top of the
column to prevent it from being disturbed when fresh solvent is added.
THE DRY PACK METHOD FOR PACKING
This method is easier, but can lead to bubbles in the
column. Obtain as empty column filling it with a small piece of glass wool,
& affix a pinch to the bottom of the column. Place the column in the
vertical position, close the pinch clamp, and fill the column with the solvent.
Allow solvent to drain from the column to prevent overflowing. Let the alumina
settle & gently tap the column with a pencil so that the alumina will pack
tightly into the column. Drain the solvent until the solvent is just even with
the surface of the alumina.
SELECTION OF THE MOILE PHASE
Depending on the adsorbent, its activity, and the class of
the solute compounds, a wide range of solvents can be used as the mobile phase,
such as single solvents of elutropic series or solvents mixtures (Organic,
Aqueous, Aqueous-Organic, & Ionic Solvents).
REQUIREMENTS FOR THE MOBILE PHASE
Mobile phases are of a greater variety than the restricted
number of stationary phases. Many solvents and their mixtures are used as a
mobile phase. The possibility of slight modification of solvent proportions in
a mixture permits the increase of mobile phase number and, thus, different
results in the component separation of the analyzed sample. That is why the
optimum mobile phase selection becomes one of the basic operations for success
of the analysis.
In order to have a better selection, there are the following
among the necessary requirements for the mobile phase.
- The
used solvents must have high purity and low viscosity, strength and
volatility
- The
used solvents must be miscible with each other
- The
samples must be soluble in the mobile phase
RETENTION TIME
The retention is a measure of the speed at which a substance
moves in a chromatographic system. In continuous development systems like HPLC
or GC, where the compounds are eluted with the eluent, the retention is usually
measured as the “Retention Time”, the time between injection & detection.
The retention of a compound often differs considerably
between experiments due to variation of the eluent, the stationary phase,
temperature, and the setup. It is therefore important to compare the retention
of the test compound to that of one or more standard compounds under absolutely
identical conditions.
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