chromatography scale up

8/8/2019 Chromatography Scale Up

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INTRODUCTION

Chromatography is the science which is studies the separation of molecules based on

differences in their structure or composition. In general, chromatography involves moving a

preparation of the materials to be separated over a stationary support. The molecules in the test

preparation will have different interactions with the stationary support leading to separation of

similar molecules. Test molecules which display tighter interactions with the support will tend to

move more slowly through the support than those molecules with weaker interactions. In this

way, different types of molecules can be separated from each other as they move over the

support. Chromatographic separations can be carried out using a variety of supports, including

immobilized silica on glass plates (thin layer chromatography), volatile gases (gas

chromatography), paper (paper chromatography), and liquids which may incorporate

hydrophilic, insoluble molecules (liquid chromatography).

Chromatographers use many different types of chromatographic techniques in

biotechnology as they bring a molecule from the initial identification stage to the stage of a

becoming a marketed product. The most commonly used of these techniques is liquid

chromatography, which is used to separate the target molecule from undesired contaminants

(usually host-related), as well as to analyze the final product for the requisite purity established

with governmental regulatory groups (such as the FDA).

The biggest changes in chromatography have been associated with the types of

chromatographic supports used in protein purification. Many years ago, cellulose-based

chromatography media were exclusively used for purification of proteins. These media were

eventually replaced with carbohydrate-based supports, which offered better flow properties flow

is the term used to describe the progress of the test solution as it passes over the support.

Carbohydrate based supports could also be more easily cleaned to allow for repeated use. The

current generation of chromatographic supports incorporates synthetic, polymeric beads which

have even higher flow rate capabilities, as well as new approaches in design of the support

particle, or bead.


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METHODOLOGY

1. Firstly, there were 5 concentrations that prepared with different weight which were 0.03g,

0.06g, 0.09g, 0.12g, and 0.15g.

2. All the concentrations were diluted with distilled water.

3. There were 2 columns were used in this experiment which were in big and short size and

long length and small size.

4. The Blue Dextran was used in this experiment and it passed through the column after it

was injected to Buchi Pump Manager equipment.

5. When the level for each vial reached until 10 ml, the time was taken.

6. Then, the absorbance was checked.


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RESULT

A. Bigger column (column diameter = 40x75mm)

Table 1: Result for 0.6% concentration

TUBE TIME(sec) ABSORBANCE

1 42 0.014

2 83 0.016

3 131 0.015

4 178 0.017

5 223 0.050

6 270 0.116

7 315 0.1498 362 0.098

9 408 0.035

10 486 0.008

11 576 0.007

12 669 0.007

Figure 1: Graph of 0.6% Concentration

Absorbance vs Time

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

0 200 400 600 800

Time

Absorba


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Table 2: Result for1.2 % concentration of blue dextran

TUBE TIME (sec) ABSORBANCE

1 67 0.008

2

155 0.0

12

3 244 0.015

4 332 0.010

5 420 0.035

6 508 0.390

7 594 0.398

8 654 0.300

9 699 0.183

10 743 0.094

11 788 0.031

12 833 0.010

Figure 2: Graph of1.2% Concentration

Absorbance vs Time

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0 200 400 600 800 1000Time

Absorban


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1 43 0.011

2 91 0.005

3 136 0.005

4 183 0.003

5 230 0.047

6 275 0.246

7 320 0.309

8 365 0.258

9 410 0.166

10 456 0.078

11 502 0.040

12 548 0.014


Absorbance vs Time

-0.05

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0 100 200 300 400 500 600

Time

Absorban


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1 44 0.008

2 89 0.004

3 136 0.005

4 182 0.006

5 226 0.072

6 272 0.389

7 316 0.509

8 361 0.452

9 405 0.398

10 449 0.323

11 494 0.220

12 537 0.153

13 583 0.086

14 628 0.035

15 673 0.019

16 720 0.014

17 764 0.008


Absorbance vs Time

0

0.1

0.2

0.3

0.4

0.5

0.6

0 200 400 600 800 1000

Time

Absorba


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Table 5: Result for 3.0 % concentration of blue dextran


1 49 0.008

2 99 0.005

3 143 0.004

4 190 0.004

5 235 0.087

6 280 0.391

7 323 0.605

8 367 0.508

9 413 0.361

10 457 0.240

11 503 0.168

12 548 0.121

13 595 0.070

14 638 0.025

15 685 0.013

16 732 0.010

17 776 0.008

Figure 5: Graph of 3.0% Concentration

Absorbance vs Time

-0.1

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 200 400 600 800 1000

Time

Absorba


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B. Hollow Tube (column diameter = 12x150mm)



1 41 0.010

2 88 0.533

3 133 0.035

4 180 0.018

5 227 0.011

6 272 0.009

Figure 1: Graph 0.6 % concentration.

Absorbance s.Time

0

0.1

0.2

0.3

0.4

0.5

0.6

40 90 140 190 240 290

Time (sec)

Absorbance


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1 49 0.010

2 92 0.718

3 137 0.086

4 182 0.012

5 229 0.006

6 275 0.006


Abso bance s.Ti e

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

49 79 109 139 169 199 229

Ti e (sec)

Abso

bance


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1 42 0.005

2 88 0.868

3 133 0.110

4 182 0.017

5 227 0.013

6 270 0.013


Absorbance s. i e

0

0.

0.

0.

0.0.

0.

0.

0.

0.

0 70 100 130 160 190 220 250 280

Ti e sec)

Absor

bance


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1 48 0.013

2 98 1.426

3 145 0.659

4 193 0.012

5 241 0.009

6 288 0.009

Figure 4: Graph 2.4 % concentration

Abs rba ce sTi e

-0.1

0.05

0.2

0.35

0.5

0.65

0.8

0.95

1.1

1.25

1.4

48 78 108 138 168 198 228 258 288

Ti e (sec)

A

bs

rba

ce


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TUBE TIME(sec) ABSORBANCE

1 44 0.015

2 92 1.533

3 138 0.739

4 185 0.038

5 231 0.040

6 282 0.025

7 331 0.022

8 377 0.018

9 421 0.017

Figure 5: Graph 3.0 % concentration

Abs rba ce s.Ti e

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

0 50 100 150 200 250 300 350 400 450

Ti e (sec)

Abs

rb

a

ce


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DISCUSSION

The experiment is run in order to determine the operation and function of

chromatography scale-up. In this experiment, blue dextran was used as a sample that needs to

test. The blue dextran was diluted with difference concentrations which are 0.6%, 1.2%, 1.8%,

2.4% and 3.0%. The difference of concentration is depends on the weight of blue dextran which

is 0.03g, 0.06g, 0.09g, 0.12g and 0.15g. The purpose of the difference weight is to make a

comparison of the absorbance over time on graph. Other than that, the two types of column

which is difference in terms of diameter and length was used. The first column which is bigger

column used has 40 mm of diameter while the second column which is hollow has 12 mm of

diameter.

The blue dextran is a polysaccharide composed of glucose residues and produced by the

fermentation of sucrose by the microorganism Leuconostoc mesenteroides. It is prepared with

various degrees of cross linking to control pore size and is supplied in the form of dry beads of

various degrees of fineness that swell when water is added. Swelling is the process by which the

pores become filled with the liquid to be used as eluant. It is commercially available under the

trade name Sephadex. During this experiment, the dextran that used is difference in terms of

weight so the difference concentration can be produced and make a comparison.

Based on the graph, the comparison can make by looking at the difference concentration.

The higher concentration caused the separation of the sample takes longer compared to the lower

concentration. This was proven by looking at the table which is at the concentration 3.0% with

weight 0.15 g of blue dextran for bigger column; it has 17 tubes to finish collecting the sample

until no blue produce. In average reading for bigger column, the chromatography was started to

produce a higher absorbance reading at 300 to 400 seconds. For example, at 3.0% concentration,

the higher absorbance was detected at 323 seconds. The blue sample started to separate and

insert into the tube at tube 5 but the times recorded is different due to the difference of sample

concentration. When the sample starting come out by passing through the column about two

tubes, the third tube collect the higher absorbance by observe the blue color during the

experiment. It means the more concentrated color the higher the absorbance reading. Then, the


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sample was separated nicely, so the graph was reduced slowly until no more blue sample

produced.

Hollow column actually is difference compared to the bigger column in terms of

diameter. So, the tubes that used to collect the sample separation are little about 6 tubes except

for 3.0% concentration which is used 9 tubes. For this column type, the higher absorbance

actually was detected in the second tube which is faster separation compared to the bigger

column. This occurred faster due to the smaller diameter of column which makes the separation

is faster but unfortunately it is not accurate separate. All tables was shown that second tube

collect higher of concentration readings means it has more diluted sample which has higher

concentrated blue color. When the second tube already collected higher concentrated sample, the

third tube automatically collect less concentrated sample and the difference of absorbance

reading is huge between second and third tube.

Column size was played main role during the separation of sample. By comparison on

graph for both columns, the bigger column has more tubes that collected the separated sample.

This means the diluted blue dextran will passing slowly in the bigger diameter to separate

efficiency and the particle that passed through is a desired product. The bigger diameter of

column chromatography, the efficient of separation occurs although it will take longer to

separate. The particle size of sample also influences the flow down of sample through the

column for separation. The smaller particle of sample, the faster sample flows down through the

column and easily to separate. In fact, the highest peak in graph shown the higher absorbance

produced.

In addition, the polarity of the solvent which is passed through the column also 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 and

will also better solvate the polar constituents. Consequently, a highly polar solvent will move

even highly 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.


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CONCLUSION

The chromatography process was run smoothly and the absorbance readings were

recorded. The purpose of the chromatography is to purify the protein and separate the particles.

The difference of column and concentration of blue dextran actually influence the separation

process. The higher concentration of sample will caused the slower process of separation. Other

than that, the larger diameter of column, the efficient separation of sample even though it

actually takes longer to separate.


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REFERENCE

chromatography scale up

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