IZMIR INSTITUTE OF TECHNOLOGY

DEPARTMENT OF CHEMICAL ENGINEERING

CHE 310
CHEMICAL ENGINEERING LABORATORY I

COLUMN CHROMATOGRAPHY

Submitted to:
Prof. Dr. Aslı YÜKSEL ÖZŞEN

Res. Asst. Tuğçe TUNÇ GULİYEV

Submitted by:
Group B2

Tuğçenur KIRILMAZ 260208022C

Sabahattin ÖNAL 260202043

Görkem HEPDÜZYOL 270202042

Spring

May 13, 2022

ABSTRACT

The aim of this experiment is to separate methylene blue and methyl orange of different
polarities using column chromatography and then examine the separated chemicals with a
visual spectrophotometer. A column filled with activated alumina was used for
chromatography. This was a separation experiment performed with a mixture of methylene blue
and methyl orange as the mobile phase and a mixture of ethanol and water as the stationary
phase. Separation of the mixture was visualized using a chromatogram. Since methyl orange is
more polar than methylene blue, the stationary phase is highly polar. As a result of the polarity
difference, the flow rate between the compounds of the mixture changed, as the methyl orange
interacted more with Alumina. Ethyl alcohol and water had dead times of 144 seconds and 58
seconds, respectively. According to these findings, water is more polar than ethyl alcohol.
Methylene blue and methyl orange retention times were measured to be 597 seconds and 445
seconds, respectively. The output concentrations of methylene blue and methyl orange were
determined using calibration curve data, and they were 0.01126 mg/mL and 0.00995 mg/mL,
respectively. The capacitance factor of methyl orange was discovered to be 6.67 and that of
methylene blue to be 3.146. In addition, the mole and partition coefficients of methyl orange
and methylene blue in the mobile phase were calculated. The selectivity factor was found to be
1.54. There were some mistakes or errors in laboratory measurements because capacity factors
of each solution and selectivity factor could not be obtained as expected.
TABLE OF CONTENTS

ABSTRACT ...........................................................................................................................2
TABLE OF CONTENTS........................................................................................................3
LIST OF FIGURES ................................................................................................................4
LIST OF TABLES .................................................................................................................5
1.INTRODUCTION ...............................................................................................................6
2.THEORY AND PRINCIPLES.............................................................................................7
3. EXPERIMENTAL............................................................................................................ 10
4.RESULTS AND DISCUSSION ........................................................................................ 12
4.1. Calculation of Average Linear Rate of Movement for Mobil Phase ............................ 12
4.2. Average Linear Rate of Analyte Migration................................................................. 12
4.3. Moles of Analyte in Mobile and Stationary Phase ...................................................... 13
4.4. Calculation of Partition Coefficient ............................................................................ 19
4.5. Calculation of Capacity (Retention) Factors of Dye Solutions .................................... 19
4.6. Selecivity Factor ........................................................................................................ 20
5. Question for Considerations .......................................................................................... 21
5. CONCLUSION ................................................................................................................ 23
6. NOMENCLATURE ......................................................................................................... 24
7. REFERENCES ................................................................................................................. 25
8. APPENDIX ...................................................................................................................... 26
LIST OF FIGURES

Figure 1. Column chromatography technique [3] ....................................................................6

Figure 2. Separation of a mixture into its components column chromatography [4] .................7
Figure 3. Column chromatography .........................................................................................8
Figure 4. A sample chromatogram ..........................................................................................9
Figure 5. Experimental set-up ............................................................................................... 11
Figure 6. Calibration Curve for Methylene Blue ................................................................... 15
Figure 7. Calibration Curve for Methyl Orange..................................................................... 18
Figure 8. Structures of Dyes ................................................................................................. 21
Figure 9. Data for Column Chromatography ......................................................................... 26
Figure 10. Excel Data ...........................................................................................................28
LIST OF TABLES

Table 1. Calibration Data for Methylene Blue ....................................................................... 14

Table 2. Calibration Data for Methyl Orange ........................................................................ 17
1.INTRODUCTION

Chromatography is an analytical technique, however, it is widely used to separate a

mixture of chemicals into its components. The purpose of this experiment is to understand the
principles of column chromatography used to separate a mixture, to separate methylene blue
and methyl orange using an alumina packed column. Thus, the separated substances are
determined using a visible spectrophotometer. To achieve these goals, this technique makes use
of the difference in distribution between mobile and stationary phases. A vertical glass column
is filled with solid stationary phase, liquid mobile phase, and sample in column
chromatography. Gravity aids the movement of the sample along the column.

There are different variations for this technique such as paper, thin layer and column
chromatography [1]. Paper chromatography is a technique used in analytical chemistry to
separate dissolved chemicals between paper layers according to different migration rates. It is
a low-cost yet powerful analytical tool that requires only a small amount of material [2]. Thin
layer chromatography (TLC) is a technique that uses a thin stationary phase by an inert support
to separate the components of a mixture. It can be done on an analytical scale to monitor the
progress of a reaction, or it can be done on a preparative scale to purify small amounts of a
compound. A stationary solid phase adsorbs and separates the compounds passing through it
with the help of a liquid mobile phase in column chromatography. Column chromatography
gives a chance to separate both liquid/solid (adsorption) and liquid/liquid (partition) mixtures.
Stationary phase and mobile phase are the two main elements in the column
chromatography process mentioned above. Immobilized stationary phase on the support
particles or the inner wall of the column tube. The mobile phase is the phase that is moving in
one direction. Elution is the process by which the mobile phase passes through the column and
consists of the separated/analyzed sample and the solvent that moves the sample along the
column.

Figure 1. Column chromatography technique [3]

Chromatography is a powerful proposition that exploits the polarity difference between

separated components. Polarity is caused by an unequal distribution of partial charge among
atoms in a compound. Electronegative atoms have a higher probability of having a partially
negative charge, whereas electropositive atoms have a higher probability of having a partially
positive charge. The separation of partial charges results in the formation of a dipole.
 Column chromatography is performed in a series of steps. To begin with, the upper level
of the mobile phase should be the same as the stationary phase. That is, the stationary phase
should be soaked in the solvent. The compound mixture that needs to be separated is added
from the top of the column at this stage. It is allowed to adsorb on the surface of the silica by
turning on the tap below. The solvent or a suitable solvent mixture is then added slowly and
carefully, first touching the side of the glass column. Throughout the process, the solvent is
added as many times as necessary. A schematic diagram of the column chromatography is
shown in Figure 2 below. Compounds in the compound mixture move with the eluent when the
tap is turned on, depending on the polarity of the sample molecule. Non-polar components move
faster than polar components. The higher polarity compound will have a lower tendency to
move with the mobile phase. The less polar compound moves faster. When the column reaches
the end, a clean test tube is used to collect the less polar sample. The sample is then polarized
and the most polar compound is collected in separate test tubes. Therefore, column
chromatography is used to separate or purify a mixture of compounds.

Figure 2. Separation of a mixture into its components column chromatography [4]

2.THEORY AND PRINCIPLES

Chromatography is the technique that differentially disperses the compound to be

separated between the mobile phase and the stationary phase. Some of the types of
chromatography are paper, thin layer and column chromatography. Column chromatography
has an advantage over other types of chromatography because it can identify and purify all
components of mixtures. It is also frequently used because it has both analytical and preparatory
applications.
The main working principle of column chromatography is to separate mixtures into
components as liquid-solid (adsorption) or liquid-liquid (partition) using a solid adsorbent
stationary phase and solvents as mobile phase. The different components in the mixture and the
stationary and mobile phases will interact to different degrees, resulting in a separation. The
number of components of a mixture can be determined by this technique. It consists of column,
tube and stationary phase. The stationary phase, the adsorbent, is placed on a vertical glass
column and the mobile phase is added to it. Separation into its components occurs due to
polarity differences of the compounds in a column, under the influence of gravity and with the
help of external forces, as in Figure 3.

Figure 3. Column chromatography

Partition Coefficient (Equilibrium Constant) (K)

It refers to the equilibrium between the adsorbed component and the solvent in the
stationary phase as the mobile phase moves down the column onto the mobile phase.
𝐴𝑚𝑜𝑏𝑖𝑙𝑒 ↔ 𝐴𝑠𝑡𝑎𝑡𝑖𝑜𝑛𝑎𝑟𝑦
𝐶𝑠
𝐾= 𝐸𝑞𝑢𝑎𝑡𝑖𝑜𝑛 1
𝐶𝑚
• 𝐾: 𝐸𝑞𝑢𝑖𝑙𝑖𝑏𝑟𝑖𝑢𝑚 𝑐𝑜𝑛𝑠𝑡𝑎𝑛𝑡 (𝑝𝑎𝑟𝑡𝑖𝑡𝑖𝑜𝑛 𝑐𝑜𝑒𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑡 )𝑓𝑜𝑟 𝑠𝑜𝑙𝑢𝑡𝑒 𝐴
• 𝐶𝑠 : 𝑀𝑜𝑙𝑎𝑟 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑎𝑛𝑎𝑙𝑦𝑡𝑒 𝐴 𝑖𝑛 𝑡ℎ𝑒 𝑠𝑡𝑎𝑡𝑖𝑜𝑛𝑎𝑟𝑦 𝑝ℎ𝑎𝑠𝑒
• 𝐶𝑚 : 𝑀𝑜𝑙𝑎𝑟 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑎𝑛𝑎𝑙𝑦𝑡𝑒 𝐴 𝑖𝑛 𝑡ℎ𝑒 𝑚𝑜𝑏𝑖𝑙𝑒 𝑝ℎ𝑎𝑠𝑒

Retention Time (tR)

The retention time is the time between when the sample is introduced into the column
and when the analyte reaches the end of the column.
𝐿
𝑡𝑅 = 𝐸𝑞𝑢𝑎𝑡𝑖𝑜𝑛 2
𝑣
• 𝑡𝑅 : 𝑅𝑒𝑡𝑒𝑛𝑡𝑖𝑜𝑛 𝑡𝑖𝑚𝑒
• 𝐿: 𝐿𝑒𝑛𝑔ℎ𝑡 𝑜𝑓 𝑝𝑎𝑐𝑘𝑖𝑛𝑔
• 𝑣: 𝐴𝑣𝑒𝑟𝑎𝑔𝑒 𝑙𝑖𝑛𝑒𝑎𝑟 𝑟𝑎𝑡𝑒 𝑜𝑓 𝑎𝑛𝑎𝑙𝑦𝑡𝑒 𝑚𝑖𝑔𝑟𝑎𝑡𝑖𝑜𝑛
This time was measured for each sample individually.
Dead Time (tm)
Dead time is the time period for the mobile phase to flow through the column
𝐿
𝑡𝑚 = 𝐸𝑞𝑢𝑎𝑡𝑖𝑜𝑛 3
𝑢
 • 𝑡𝑚 : 𝐷𝑒𝑎𝑑 𝑡𝑖𝑚𝑒
• 𝐿: 𝐿𝑒𝑛𝑔ℎ𝑡 𝑜𝑓 𝑝𝑎𝑐𝑘𝑖𝑛𝑔
• 𝑢: 𝐴𝑣𝑒𝑟𝑎𝑔𝑒 𝑙𝑖𝑛𝑒𝑎𝑟 𝑟𝑎𝑡𝑒 𝑜𝑓 𝑚𝑜𝑣𝑒𝑚𝑒𝑛𝑡 𝑜𝑓 𝑚𝑜𝑏𝑖𝑙𝑒 𝑝ℎ𝑎𝑠𝑒
A simple chromatogram is shown below as in figure 4.

Figure 4. A sample chromatogram

Relation between Retention Time and Partition Coefficient

The following equation shows the average linear rate analyte migration expressed by
the mobile phase velocity.
𝑚𝑜𝑙𝑒𝑠 𝑜𝑓 𝑎𝑛𝑎𝑙𝑦𝑡𝑒 𝑖𝑛 𝑚𝑜𝑏𝑖𝑙𝑒 𝑝ℎ𝑎𝑠𝑒
𝑣 = 𝑢( )
𝑡𝑜𝑡𝑎𝑙 𝑚𝑜𝑙𝑒𝑠 𝑜𝑓 𝑎𝑛𝑎𝑙𝑦𝑡𝑒
𝐶𝑚 𝑉𝑚 𝑢
𝑣=𝑢 = 𝐸𝑞𝑢𝑎𝑡𝑖𝑜𝑛 4
𝐶𝑚 𝑉𝑚 + 𝐶𝑠 𝑉𝑠 𝐶𝑉
1+ 𝑠 𝑠
𝐶𝑚 𝑉𝑚
𝐶𝑠
Substituting K for ⁄𝐶 ;
𝑚
𝑢
𝑣= 𝐸𝑞𝑢𝑎𝑡𝑖𝑜𝑛 5
𝐾𝑉
1+ 𝑉𝑠
𝑚

• 𝑉𝑚 : 𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑡ℎ𝑒 𝑚𝑜𝑏𝑖𝑙𝑒 𝑝ℎ𝑎𝑠𝑒

• 𝑉𝑠 : 𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑡ℎ𝑒 𝑠𝑡𝑎𝑡𝑖𝑜𝑛𝑎𝑟𝑦 𝑝ℎ𝑎𝑠𝑒
• 𝐾: 𝑃𝑎𝑟𝑡𝑖𝑡𝑖𝑜𝑛 𝑐𝑜𝑒𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑡
• 𝑣: 𝐴𝑣𝑒𝑟𝑎𝑔𝑒 𝑙𝑖𝑛𝑒𝑎𝑟 𝑟𝑎𝑡𝑒 𝑜𝑓 𝑎𝑛𝑎𝑙𝑦𝑡𝑒 𝑚𝑖𝑔𝑟𝑎𝑡𝑖𝑜𝑛
• 𝑢: 𝐴𝑣𝑒𝑟𝑎𝑔𝑒 𝑙𝑖𝑛𝑒𝑎𝑟 𝑟𝑎𝑡𝑒 𝑜𝑓 𝑚𝑜𝑣𝑒𝑚𝑒𝑛𝑡 𝑜𝑓 𝑚𝑜𝑏𝑖𝑙𝑒 𝑝ℎ𝑎𝑠𝑒
Capacity (Retention) Factor (𝒌′𝑨 )
The capacitance factor is the ratio of the time spent in the stationary phase to the time
spent by the sample component in the mobile phase. This ratio is dimensionless and is not
affected by the geometric properties of the column.
 𝑇𝑖𝑚𝑒 𝑠𝑜𝑙𝑢𝑡𝑒 𝑠𝑝𝑒𝑛𝑑𝑠 𝑖𝑛 𝑠𝑡𝑎𝑡𝑖𝑜𝑛𝑎𝑟𝑦 𝑝ℎ𝑎𝑠𝑒
𝒌′𝑨 =
𝑇𝑖𝑚𝑒 𝑠𝑜𝑙𝑢𝑡𝑒 𝑠𝑝𝑒𝑛𝑑𝑠 𝑖𝑛 𝑚𝑜𝑏𝑖𝑙𝑒 𝑝ℎ𝑎𝑠𝑒
𝑡𝑅 − 𝑡𝑀
𝑘𝐴′ = 𝐸𝑞𝑢𝑎𝑡𝑖𝑜𝑛 6
𝑡𝑀
𝐶𝑠 𝑉𝑠 𝑉𝑠
𝑘𝐴′ = =𝐾 𝐸𝑞𝑢𝑎𝑡𝑖𝑜𝑛 7
𝐶𝑚 𝑉𝑚 𝑉𝑚
If equation 7 is substituted for equation 5.
𝑢
𝑣= 𝐸𝑞𝑢𝑎𝑡𝑖𝑜𝑛 8
1 + 𝑘𝐴′
Following equation may be used to derive 𝑘𝐴′ from chromatogram,
𝐿 𝐿
= 𝐸𝑞𝑢𝑎𝑡𝑖𝑜𝑛 9
𝑡𝑅 𝑡𝑀 ∗ (1 + 𝑘𝐴′ )
Selectivity (Separation) Factor (α)
The selectivity factor (α) describes the separation of two species on the column;
𝐾𝑠𝑝𝑒𝑐𝑖𝑒𝑠 𝐵
𝑎= 𝐸𝑞𝑢𝑎𝑡𝑖𝑜𝑛 10
𝐾𝑠𝑝𝑒𝑐𝑖𝑒𝑠 𝐴

• 𝐾𝑏 : 𝑃𝑎𝑟𝑡𝑖𝑡𝑖𝑜𝑛 𝑐𝑜𝑒𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑡 𝑓𝑜𝑟 𝑡ℎ𝑒 𝑚𝑜𝑟𝑒 𝑠𝑡𝑟𝑜𝑛𝑔𝑙𝑦 𝑟𝑒𝑡𝑎𝑖𝑛𝑒𝑑 𝑠𝑝𝑒𝑐𝑖𝑒𝑠 𝐵

• 𝐾𝑎 : 𝑃𝑎𝑟𝑡𝑖𝑡𝑖𝑜𝑛 𝑐𝑜𝑒𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑡 𝑓𝑜𝑟 𝑡ℎ𝑒 𝑙𝑒𝑠 𝑠𝑡𝑟𝑜𝑛𝑔𝑙𝑦 ℎ𝑒𝑙𝑑 𝑠𝑝𝑒𝑐𝑖𝑒𝑠 𝐴
Selectivity factor must greater than one.
Factors Effecting the Column Efficiency
• Linear velocity of mobile phase
• Diffusion coefficient of mobile phase
• Diffusion coefficient of stationary phase
• Capacity factor
• Diameter of packing particle
• Diameter and length of the column

3. EXPERIMENTAL

Materials

• Methylene blue (MW = 373.90 g/mol)

• Methyl orange (MW = 327.34 g/mol)
• Ethyl alcohol
• Water
• Activated Alumina 90 (Aluminum oxide 90 standardized, Merck 101097, Activity I)

Equipment

• Column (glass burette with a glass stopcock at the bottom)

 • Glass wool
• Ring stand and clamp for holding column
• Tubes for collecting of fractions
• UV-Visible spectrophotometer

Figure 5. Experimental set-up

Procedure

• First, glass wool was placed at the bottom of the chromatography column to which
alumina was added to seal the outlet.
• A slurry was obtained by combining 10 mL of ethanol and 10 g of alumina. This is then
left to feed into the colon and after it has been placed at a height of 4-5 cm.
• A 0.25 𝑚𝑔/𝑚𝐿 methyl orange solution was prepared with water. A 0.25 𝑚𝑔/𝑚𝐿
methylene blue solution was prepared with ethanol.
• The column was filled with 0.6 mL of methylene blue and 0.4 mL of methyl orange.
• The interval between when the dye solution was added to the column and when the first
blue color appeared at the exit of the column was recorded. At this time, the retention
time of methylene blue was recorded.
• To be used in methylene blue calibration, x10, x50, x100, x150, x200 diluted from
reference solutions and stock solutions were used, respectively.
• In ethanol, 0.25 mg/ml methylene blue was used as a stock solution.
• As a final step, samples were determined by measuring and recording the absorbance
values of the samples collected from the column.
• For methyl orange solution, the steps were repeated.
4.RESULTS AND DISCUSSION

Column chromatography was used in this experiment to separate methylene blue and
methyl orange, and the solvents for this technique were determined to be ethyl alcohol and
water. Activated alumina was also used as the stationary phase in this experiment. Ethyl alcohol
and water are used to dissolve methylene blue and methyl orange, respectively. Compounds are
separated based on their polarity because polarity is an important property in column
chromatography. Polar molecules exchange information via dipole-dipole intermolecular forces
and hydrogen bonds. The solvents used in this experiment have varying degrees of polarity.
Water has a higher affinity for hydrogen bonding than ethanol, making it more polar.
Furthermore, the polarities of methyl orange and methylene blue differ. To determine the
concentration of the solution passing through the column, a UV-visible spectrophotometer is
used. All of the calculations are listed below to help you understand the purpose of this
experiment.

4.1. Calculation of Average Linear Rate of Movement for Mobil Phase

The column length was measured as 6.3 cm (0.063 m). The formula required to
calculate the average linear motion velocity of the mobile phase is as follows.
𝐿
𝑡𝑀 =
𝑢

For Ethanol;
𝑡𝑀 = 144 𝑠
𝐿 0.063 𝑚 𝑚
𝑢= = = 0.0004375
𝑇𝑚 144 𝑠 𝑠

For Water;
𝑡𝑀 = 58 𝑠
𝐿 0.063 𝑚 𝑚
𝑢= = = 0.00108
𝑡𝑚 58 𝑠 𝑠
Because a highly polar solvent rapidly transports highly polar molecules through the
column, the more polar eluent is water.

4.2. Average Linear Rate of Analyte Migration

For calculation of average linear rate of analyte migration, required formula as follow.
𝐿
𝑡𝑅 =
𝑣
 For Methylene Blue;
𝑡𝑅 = 597 𝑠
𝐿 0.063 𝑚 𝑚
𝑣= = = 0.000105
𝑡𝑅 597 𝑠 𝑠
For Methyl Orange;
𝑡𝑅 = 445 𝑠
𝐿 0.063 𝑚 𝑚
𝑣= = = 0.0001416
𝑡𝑅 445 𝑠 𝑠

4.3. Moles of Analyte in Mobile and Stationary Phase

Methylene blue and methyl orange were diluted at 10, 50, 100, 150, and 200 times and
calculations were performed by below equation,
𝐶1 ∗ 𝑉1 = 𝐶2 ∗ 𝑉2
For dilution factor 10;
The volume of was taken as 3 mL for 0.25 mg/mL. The required volume for 0.025
mg/mL.
𝑚𝑔 𝑚𝑔
0.25 ∗ 3 𝑚𝐿 = 0.025 ∗𝑉
𝑚𝐿 𝑚𝐿 2
𝑉2 = 30 𝑚𝐿
30 − 3 = 27 𝑚𝐿 needed solvent

For dilution factor 50;

10 times diluted solution is was used to found V2
𝑚𝑔 𝑚𝑔
0.025 ∗ 5 𝑚𝐿 = 0.005 ∗𝑉
𝑚𝐿 𝑚𝐿 2
𝑉2 = 25 𝑚𝐿
25 − 5 = 20 𝑚𝐿 needed solvent

For dilution factor 100;

10 times diluted solution is used to found V2.
𝑚𝑔 𝑚𝑔
0.025 ∗ 2 𝑚𝐿 = 0.0025 ∗𝑉
𝑚𝐿 𝑚𝐿 2
𝑉2 = 20 𝑚𝐿
 20 − 2 = 18 𝑚𝐿 needed solvent

For dilution factor 150;

50 times diluted solution is used to found V2.
𝑚𝑔 𝑚𝑔
0.005 ∗ 6 𝑚𝐿 = 0.00167 ∗𝑉
𝑚𝐿 𝑚𝐿 2
𝑉2 = 18 𝑚𝐿
18 − 6 = 12 𝑚𝐿 needed solvent

For dilution factor 200;

10 times diluted solution is used to found V2.
𝑚𝑔 𝑚𝑔
0.025 ∗ 1 𝑚𝐿 = 0.00125 ∗𝑉
𝑚𝐿 𝑚𝐿 2
𝑉2 = 20 𝑚𝐿
20 − 1 = 19 𝑚𝐿 needed solvent

A calibration curve was first required to determine the concentration. Thus, the number
of moles could be calculated. Dye solutions were diluted 10, 50, 100, 150 and 200 times to
generate standard solutions for the calibration curve. The absorbance value of each solution was
measured as shown in Table 1. Then, the absorbance of the passed solutions was measured by
comparing them with the standard values, and the concentration of the passed solutions was
determined.

For Methylene Blue;

Table 1. Calibration Data for Methylene Blue

Concentration
Absorbance 1 Absorbance 2 Absorbance 3 Average
(mg/ml)
0,0000 0,0000 0,0000 0,0000 0,0000
0,025 2,79 2,751 2,747 2,762666667
0,005 2,174 2,148 2,151 2,157666667
0,0025 1,211 1,208 1,208 1,209
0,00167 0,846 0,845 0,846 0,845666667
0,00125 0,601 0,601 0,602 0,601333333
 Figure 6 shows the concentration values in mg/mL on the x-axis and the absorbance
values on the y-axis. As can be seen in the data table above, when the calibration curve was
created with the results obtained experimentally, the graph did not take a linear shape. It was
noticed that the solution that disrupted the linearity was the solution diluted 10 times, and the
absorbance value was lower than the required value. Therefore, in order to ensure linearity, the
solution which diluted 10 times was not taken into account while plotting the graph. R2 = 0.9964
indicates that the curve's linearity is desirable. The slope of the curve in Figure 6 was
determined to be 4448.71.

Calibration Curve for Methylene Blue

2.5000
Average Absorbance

2.0000
y = 448.71x
R² = 0.9964
1.5000

1.0000

0.5000

0.0000
0.0000 0.0010 0.0020 0.0030 0.0040 0.0050
Concentration (mg/ml)

Figure 6. Calibration Curve for Methylene Blue

The collected sample, which had a volume of 20 mL, had an absorbance value of 1.011.
The following equation can be used to calculate the concentration of the standard solution.

𝐴 = 𝑆𝑙𝑜𝑝𝑒 ∗ 𝐶 ∗ 𝑙 and (l ~ 1cm)

𝐴𝑏𝑠𝑜𝑟𝑝𝑡𝑖𝑜𝑛 1.011 𝑚𝑔
𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛𝑒𝑥𝑖𝑡 = ( )= = 0.00225
𝑆𝑙𝑜𝑝𝑒 448.71 𝑚𝐿

Mole of methylene blue in the mobile phase was calculated with the exit concentration
value:
𝑚𝑔 1𝑔
𝐶 ∗ 𝑉 (0.00225 ( 𝑚𝐿 ) ∗ 20𝑚𝐿) ∗ 1000 𝑚𝑔
𝑛𝑒𝑥𝑖𝑡,𝑀𝑒𝑡ℎ𝑦𝑙𝑒𝑛𝑒 𝐵𝑙𝑢𝑒 = = 𝑔 = 1.408 ∗ 10−7 𝑚𝑜𝑙𝑒
𝑀𝑊 319.85 𝑚𝑜𝑙𝑒

To found moles of stationary phase, initial mole of methylene blue is needed. So, to
found initial mole of methylene blue:
 𝑚𝑔 1𝑔
𝐶 ∗ 𝑉 (0.25 (𝑚𝐿 ) ∗ 0.6𝑚𝐿) ∗ 1000 𝑚𝑔
𝑛𝑖𝑛𝑖𝑡𝑖𝑎𝑙,𝑀𝑒𝑡ℎ𝑦𝑙𝑒𝑛𝑒 𝐵𝑙𝑢𝑒 = = 𝑔 = 4.689 ∗ 10−7 𝑚𝑜𝑙𝑒
𝑀𝑊 319.85 𝑚𝑜𝑙𝑒

Since exit (mobile phase) and initial (total) mole of methylene blue known, stationary
mole of methylene blue calculated below:
𝑛𝑠𝑡𝑎𝑡𝑖𝑜𝑛𝑎𝑟𝑦 𝑝ℎ𝑎𝑠𝑒,𝑚𝑒𝑡ℎ𝑦𝑙𝑒𝑛𝑒 𝑏𝑙𝑢𝑒 = 𝑛𝑖𝑛𝑖𝑡𝑖𝑎𝑙,𝑀𝑒𝑡ℎ𝑦𝑙𝑒𝑛𝑒 𝐵𝑙𝑢𝑒 − 𝑛𝑒𝑥𝑖𝑡,𝑀𝑒𝑡ℎ𝑦𝑙𝑒𝑛𝑒 𝐵𝑙𝑢𝑒

𝑛𝑠𝑡𝑎𝑡𝑖𝑜𝑛𝑎𝑟𝑦 𝑝ℎ𝑎𝑠𝑒,𝑚𝑒𝑡ℎ𝑦𝑙𝑒𝑛𝑒 𝑏𝑙𝑢𝑒 = (4.689 ∗ 10−7 − 1.408 ∗ 10−7 ) = 3.2808 ∗ 10−7 𝑚𝑜𝑙𝑒

According to result, 3.2808 ∗ 10−7 𝑚𝑜𝑙𝑒 methylene blue remained in stationary

phase.
For Methyl Orange;
The needed volume calculations were done on the methylene blue, are valid for the also
methylene orange. Same volume calculations and same volume values are shown below.
𝐶1 ∗ 𝑉1 = 𝐶2 ∗ 𝑉2

For dilution factor 10;

The stock solution is was used. The volume of was taken as 3 mL for 0.25 mg/mL.
The required volume for 0.025 mg/mL. to found V2.

𝑚𝑔 𝑚𝑔
0.25 ∗ 3 𝑚𝐿 = 0.025 ∗𝑉
𝑚𝐿 𝑚𝐿 2
𝑉2 = 30 𝑚𝐿
30 − 3 = 27 𝑚𝐿 needed solvent

For dilution factor 50;

10 times diluted solution is used to found V2.

𝑚𝑔 𝑚𝑔
0.025 ∗ 5 𝑚𝐿 = 0.005 ∗𝑉
𝑚𝐿 𝑚𝐿 2
𝑉2 = 25 𝑚𝐿
25 − 5 = 20 𝑚𝐿 needed solvent

For dilution factor 100;

10 times diluted solution is used to found V2.
 𝑚𝑔 𝑚𝑔
0.025 ∗ 2 𝑚𝐿 = 0.0025 ∗𝑉
𝑚𝐿 𝑚𝐿 2
𝑉2 = 20 𝑚𝐿
20 − 2 = 18 𝑚𝐿 needed solvent

For dilution factor 150;

50 times diluted solution is used to found V2.

𝑚𝑔 𝑚𝑔
0.005 ∗ 6 𝑚𝐿 = 0.00167 ∗𝑉
𝑚𝐿 𝑚𝐿 2
𝑉2 = 18 𝑚𝐿
18 − 6 = 12 𝑚𝐿 needed solvent

For dilution factor 200;

10 times diluted solution is used to found V2.

𝑚𝑔 𝑚𝑔
0.025 ∗ 1 𝑚𝐿 = 0.00125 ∗𝑉
𝑚𝐿 𝑚𝐿 2
𝑉2 = 20 𝑚𝐿
20 − 1 = 19 𝑚𝐿 needed solvent

As a result of the calculations seen above, the volumes needed for the solution were
determined and methyl orange solutions were prepared according to these values. Then,
absorbance values of solutions are measured by visible spectrophotometer as shown below.

Table 2. Calibration Data for Methyl Orange

Concentration
Absorbance 1 Absorbance 2 Absorbance 3 Average
(mg/ml)
0.0000 0.0000 0.0000 0.0000 0.0000
0.025 3.443 3.261 3.159 3.287667
0.005 0.858 0.859 0.858 0.858333
0.0025 0.423 0.424 0.424 0.423667
0.00167 0.278 0.279 0.279 0.278667
0.00125 0.207 0.207 0.207 0.207
 According to obtained concentration and absorbance data, a calibration curve is plotted.
It was concluded that the desired linearity was achieved because the R value was close to 1.
The R value was equal to 0.9956 and the slope of the calibration curve was equal to 133.62.
The plot is shown below as Figure 7.

Calibration Curve for Methyl Orange

4.0000

3.5000
Average Absorbance

3.0000
y = 133.62x
2.5000 R² = 0.9956
2.0000

1.5000

1.0000

0.5000

0.0000
0.0000 0.0050 0.0100 0.0150 0.0200 0.0250 0.0300
Concentration (mg/ml)

Figure 7. Calibration Curve for Methyl Orange

The collected sample, which had a volume of 20 mL, had an absorbance value of
0.266. The following equation can be used to calculate the concentration of the standard
solution.
𝐴 = 𝑆𝑙𝑜𝑝𝑒 ∗ 𝐶 ∗ 𝑙 and (l ~ 1cm)
𝐴𝑏𝑠𝑜𝑟𝑝𝑡𝑖𝑜𝑛 0.266 𝑚𝑔
𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛𝑒𝑥𝑖𝑡 = ( ) ∗ 𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝐹𝑎𝑐𝑡𝑜𝑟 = ∗ 1 = 0.00199
𝑆𝑙𝑜𝑝𝑒 133.62 𝑚𝐿
Mole of methyl orange in the mobile phase was calculated with the exit concentration
value:
𝑚𝑔 1𝑔
𝐶 ∗ 𝑉 (0.00199 (𝑚𝐿 ) ∗ 20𝑚𝐿) ∗ 1000 𝑚𝑔
𝑛𝑒𝑥𝑖𝑡,𝑀𝑒𝑡ℎ𝑦𝑙 𝑂𝑟𝑎𝑛𝑔𝑒 = = 𝑔 = 1.216 ∗ 10−7 𝑚𝑜𝑙𝑒
𝑀𝑊 327.33 𝑚𝑜𝑙𝑒

To found moles of stationary phase, initial mole of methyl orange is needed. So, to
found initial mole of methylene blue:
𝑚𝑔 1𝑔
𝐶 ∗ 𝑉 (0.25 (𝑚𝐿 ) ∗ 0.4𝑚𝐿) ∗ 1000 𝑚𝑔
𝑛𝑖𝑛𝑖𝑡𝑖𝑎𝑙,𝑀𝑒𝑡ℎ𝑦𝑙 𝑂𝑟𝑎𝑛𝑔𝑒 = = 𝑔 = 3.055 ∗ 10−7 𝑚𝑜𝑙𝑒
𝑀𝑊 327.33 𝑚𝑜𝑙𝑒

Since exit (mobile phase) and initial (total) mole of methyl orange are known, stationary
mole of methyl orange calculated below:
 𝑛𝑠𝑡𝑎𝑡𝑖𝑜𝑛𝑎𝑟𝑦 𝑝ℎ𝑎𝑠𝑒,𝑚𝑒𝑡ℎ𝑦𝑙 𝑜𝑟𝑎𝑛𝑔𝑒 = 𝑛𝑖𝑛𝑖𝑡𝑖𝑎𝑙,𝑀𝑒𝑡ℎ𝑦𝑙 𝑂𝑟𝑎𝑛𝑔𝑒 − 𝑛𝑒𝑥𝑖𝑡,𝑀𝑒𝑡ℎ𝑦𝑙 𝑂𝑟𝑎𝑛𝑔𝑒

𝑛𝑠𝑡𝑎𝑡𝑖𝑜𝑛𝑎𝑟𝑦 𝑝ℎ𝑎𝑠𝑒,𝑚𝑒𝑡ℎ𝑦𝑙 𝑂𝑟𝑎𝑛𝑔𝑒 = (3.055 ∗ 10−7 − 1.216 ∗ 10−7 ) = 1.839 𝑚𝑜𝑙𝑒

According to result, 3.2808 ∗ 10−7 𝑚𝑜𝑙𝑒 methyl orange remained in stationary phase.

4.4. Calculation of Partition Coefficient

The equilibrium between the component adsorbed on the stationary phase and in the
eluent is established while the eluent flows down the column. Between the stationary and
mobile phases, an analyte (component) (A) is in equilibrium. The partition coefficient is the
ratio of the concentration of a analyte when the two concentrations are at this equilibrium.
𝐴𝑚𝑜𝑏𝑖𝑙𝑒 ↔ 𝐴𝑠𝑡𝑎𝑡𝑖𝑜𝑛𝑎𝑟𝑦
𝐶𝑠
𝐾=
𝐶𝑚
K: equilibrium constant (partition coefficient) for solute A.
Cs: molar concentration of analyte A in the stationary phase.
Cm: molar concentration of analyte A in the mobile phase.
As can be seen from the partition coefficient equation, it is the ratio of molar
concentration of analyte A in stationary phase and molar concentration of a analyte A in the
mobile phase. The volumes of both phases were assumed to be equal in this calculation.
Therefore, the mole ratio can be directly obtained.

𝐹𝑜𝑟 𝑀𝑒𝑡ℎ𝑦𝑙𝑒𝑛𝑒 𝐵𝑙𝑢𝑒;

𝐶𝑠 𝑛𝑠 3.2808 ∗ 10−7
𝐾𝑀𝐵 = = = = 2.329
𝐶𝑚 𝑛𝑚 1.408 ∗ 10−7
𝐹𝑜𝑟 𝑀𝑒𝑡ℎ𝑦𝑙 𝑂𝑟𝑎𝑛𝑔𝑒;
𝐶𝑠 𝑛𝑠 1.838 ∗ 10−7
𝐾𝑀𝑂 = = = = 1.512
𝐶𝑚 𝑛𝑚 1.216 ∗ 10−7
As seen in the calculations above, the partition coefficients were 2.329 and 1.512 for
methylene blue and methyl orange, respectively. That is, the partition coefficient of methylene
blue was found to be larger. This result was as expected because methyl orange has a greater
polarity due to its molecular structure.

4.5. Calculation of Capacity (Retention) Factors of Dye Solutions

The capacity (retention) factor (kA) is the ratio of time spent in the stationary phase to
time spent in the mobile phase for a sample component. It shows how much longer the
stationary phase delays a sample component compared to how long it would take for it to travel
through the column at the mobile phase's velocity. The capacity factor calculation formula is:
𝑢 𝑢
𝑣= => 𝑘 ′ = − 1
1 + 𝑘′ 𝑣
𝑘 ′ = 𝑐𝑎𝑝𝑎𝑐𝑖𝑡𝑦 𝑓𝑎𝑐𝑡𝑜𝑟
𝑢 = 𝑎𝑣𝑒𝑟𝑎𝑔𝑒 𝑙𝑖𝑛𝑒𝑎𝑟 𝑟𝑎𝑡𝑒 𝑜𝑓 𝑡ℎ𝑒 𝑚𝑜𝑣𝑒𝑚𝑒𝑛𝑡 𝑜𝑓 𝑚𝑜𝑏𝑖𝑙𝑒 𝑝ℎ𝑎𝑠𝑒
𝑣 = 𝑎𝑣𝑒𝑟𝑎𝑔𝑒 𝑙𝑖𝑛𝑒𝑎𝑟 𝑟𝑎𝑡𝑒 𝑜𝑓 𝑎𝑛𝑎𝑙𝑦𝑡𝑒 𝑚𝑖𝑔𝑟𝑎𝑡𝑖𝑜𝑛

Capacity Factor of Methylene Blue:

𝑚
𝑢 4.375 ∗ 10−4 𝑠
′
𝑘 = −1 = 𝑚 − 1 = 3.146
𝑣 1.055 ∗ 10−4 𝑠

Capacity Factor of Methyl Orange:

𝑚
𝑢 1.086 ∗ 10−3 𝑠
𝑘′ = − 1 = 𝑚 − 1 = 6.672
𝑣 1.416 ∗ 10−4 𝑠

When the results obtained were examined, it was observed that the capacity factor of
methyl orange had a higher value compared to methylene blue. In other words, it was concluded
that methyl orange spent more time in the stationary phase. This is because the methyl orange
and stationary phase are polar. Thus, since both states are polar, polar molecules of methyl
orange were kept in stationary phase.

4.6. Selecivity Factor

Selectivity factor (α) describes the separation of two species on the column. In this
experiment these species are methylene blue and methyl orange. By its definition, the selectivity
factor must be greater than 1. The equation of selectivity factor is below:
𝐾𝐵 𝐾𝑀𝑂
∝= =
𝐾𝐴 𝐾𝑀𝐵
𝐾𝐵 = 𝑝𝑎𝑟𝑡𝑖𝑡𝑖𝑜𝑛 𝑐𝑜𝑒𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑡 𝑓𝑜𝑟 𝑡ℎ𝑒 𝑚𝑜𝑟𝑒 𝑠𝑡𝑟𝑜𝑛𝑔𝑙𝑦 𝑟𝑒𝑡𝑎𝑖𝑛𝑒𝑑 𝑠𝑝𝑒𝑐𝑖𝑒𝑠 𝐵
𝐾𝐴 = 𝑝𝑎𝑟𝑡𝑖𝑡𝑖𝑜𝑛 𝑐𝑜𝑒𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑡 𝑓𝑜𝑟 𝑡ℎ𝑒 𝑙𝑒𝑠𝑠 𝑠𝑡𝑟𝑜𝑛𝑔𝑙𝑦 ℎ𝑒𝑙𝑑 (𝑚𝑜𝑟𝑒 𝑟𝑎𝑝𝑖𝑑𝑙𝑦 𝑒𝑙𝑢𝑡𝑒𝑑) 𝑠𝑝𝑒𝑐𝑖𝑒𝑠 𝐴.

The selectivity factor of methyl orange and methylene blue:

𝐾𝑀𝑂 2.329
∝= = = 1.54
𝐾𝑀𝐵 1.512

As mentioned above, selectivity factor should be greater then 1. The selectivity

calculation in this experiment was found as 1.54, as expected. It was concluded that the methyl
orange, that is, the more strongly retained species is greater than the more rapidly eluted specie.
Experimentally, this section has been successful.
5. Question for Considerations
Which eluent is more polar?
Water has a higher hydrogen bonding affinity than ethyl alcohol, therefore water is
more polar.
Which dye is more polar? Why? Is this reflected in their structures given below?

Figure 8. Structures of Dyes

The presence of N(CH3) groups on both sides of Methylene Blue stabilizes the main
molecule and ensures less polarity. However, the fact that methyl orange has different
functional groups at both ends and it increases the force in a single direction and upsets the
balance. As a result, the polarity of methyl orange becomes high.
Suggest a better method to control the rate of flow of mobile phase through the column?

In column chromatography, gravity and pressure forces are used to separate and elute
dyes and solvents. Flow rate can be controlled by creating pressure with the help of a
compressor at the inlet of the column. Also, a pump can also be used to control the rate of ethyl
alcohol and water flow.

How can this system be automated?

The automated chromatography system requires a UV detector, a fraction collector,
column valves and a gradient pump to increase the flow rate of ethanol and water. End-of-
column detectors respond to the state of methylene blue when it first drops, and the detector
can open and close the ethanol valve as needed. Then, graphs of volume over time are plotted.
The detector then sees and detects the methyl orange drop for the first time after opening the
water valve. The same procedure is used for both the ethylene blue and ethyl alcohol solution
and the methyl orange and water solution. Detector signals are plotted for volume change over
time. As a result, methylene blue and methyl orange separation can be done with this
automation system.

Discuss any errors in your experiment and what you would do differently if you were to do this
lab again?
The results show that time measurements are not good and the reason for the errors in
this experiment is due to the wrong measurement of time. It indicates that the recorded
absorbance values are not accurate enough to determine the correct concentration. If the
experiment is to be done again, the time measurements should be done more accurately because
many parameters depend on this time measurement. In addition, absorbance values of diluted
samples can be measured more accurately and precisely in order to obtain more accurate and
more precise results. In addition to these, care should be taken that the column does not remain
dry before and after starting the operations. For this, this problem can be solved by wetting the
column with ethanol or water.

,
5. CONCLUSION

The goal of this experiment was to use column chromatography to separate methylene
blue and methyl orange mixtures. Alumina was used as a stationary phase and, ethanol and
water were used as the mobile phase in this experiment. When the chemical structures are
examined of dyes, since methylene blue has a more symmetric chemical structure, methyl
orange was found to be more polar than methylene blue. For this reason, methylene blue
solution is formed with ethanol and methylene blue; and methyl orange solution is formed with
methyl orange and water. At the beginning of the experiment, a small tube was filled with 0.6
mL methylene blue and 0.4 mL methyl orange. The column's height was measured as 6.3 cm.
First, the dead times of ethanol and water were measured and found to be 144 seconds and 58
seconds, respectively. Accordingly, linear movement of mobile phase was obtained, and it was
concluded that water moves faster. Retention times were measured as 597s for methylene blue
and 445s for methyl orange. Thus, linear rate of migration was found, methyl orange was faster.
Then the visible spectrophotometer was used for our solutions diluted at different ratios as 10,
50, 100, 150 and 200 times; and the absorbance values were found. The absorbance values of
methylene Blue were higher than methyl orange, the main reason for this can be said to be its
dark color. The exit methylene blue's absorbance value found as 1.011 and it is concentration
of exit was calculated as 0.00225 mg/mL by using the calibration curve. Methylene blue moles
in the mobile phase were 1.409*107 moles, while in the stationary phase were 3.28*107 moles.
The same calculations were performed for methyl orange and its collected absorbance value
found as 0.266, also the concentration value found as 0.00199mg/mL. Methyl orange’s moles
in mobile phase obtained as 1.216*107 and moles in stationary phase found as 1.838*107. Then
partition coefficients are calculated, and the results are obtained as 2.328 and 1.511 for
methylene blue and methyl orange, respectively. Theoretically the partition coefficients should
be zero but because of the errors such as laboratory equipment error or human error the partition
coefficients are not equal to zero in this experiment. Lastly, selectivity factor calculated, and it
found 1.54 as expected which is a greater than one. According to the selectivity factor,
methylene blue was first handled with the mobile phase of ethanol, and methyl orange remained
in the stationary phase during this elution.
6. NOMENCLATURE

Notation Meaning
tM Death time (s)
L Length of packing (m)
u Average linear rate of movement of mobile phase
tR Retention time (s)
v Average linear rate of analyte migration
K Equilibrium constant for solute
CS Molar concentration of analyte in the stationary phase
CM Molar concentration of analyte in the mobile phase
k’ Capacity factor
α Selectivity factor
7. REFERENCES

[1] "Column Chramotography " in CHE 310 Chemical Engineering Laboratory 1 Course
Manual. 2022, Izmir Institute of Technology. p. 9-21.
[2]Britannica, T. Editors of Encyclopaedia (2020, May 18). Paper
chromatography. Encyclopedia Britannica.
https://www.britannica.com/science/paper-chromatography
[3]Column chromatography.jpg. (2021, January 2). Wikimedia Commons, the free media
repository. Retrieved 12:26, May 17, 2022 from
https://commons.wikimedia.org/w/index.php?title=File:Column_chromatography.jpg&oldid=52349
5151.

[4] Nishi Srivastava, ... Ravindra N. Kharwar, in Natural Bioactive Compounds, 2021
8. APPENDIX

Figure 9. Data for Column Chromatography

 Figure 10. Excel Data

Tuğçenur Kırılmaz: Abstract, Introductıon, Result and Discussion, Excel

Sabahattin Önal: Theory and Principles, Experimental, Result and Discussion, Excel
Görkem Hepdüzyol: Conclusion, Result and Discussion, Excel