Sunday, January 27, 2013

Analytical Chemistry (Practicals) by SAAD ABDUL WAHAB


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EXP # 1

Object:
To selection of the proper mobile phase

Apparatus:
TLC plate, TLC tank, graduated cylinder, jet

Reagents:
Hexane
Chloroform
Observation:
Ratios

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. 

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

                        Rf= 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

                        Rf= 2.0 / 4.9
                              = 0.408

For Dollar (Black):
                       
                        Rf1 = 0.7 / 4.9
                              = 0.142

                        Rf= 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

                        Rf= 2 / 4.9
                              = 0.408

For Quick (Black):

                        Rf1 = 0.4 / 4.9
                               = 0.0816

                        Rf= 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

                        Rf= 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
T(min)
V(ml)
Methyl blue
31.56
2.8
Methyl yellow
16.40
2.1


Calculation:

·         Column resolution Rs:
R= Az/w
Where Az =15.16 min (dif of time of solution arrival)
                W = ­­­23.98 min (average time of solute arrival)
So          
                R= 15.16 / 23.98
R= 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
                       
Rf= 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

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 

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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