CHEN90009 Fermentation Processes Practical Laboratory Experiment 2

Separation of Biomolecules by Size Exclusion Chromatography

Important Notes Work in GROUPS of THREE or FOUR for this laboratory session You must wear your LABCOAT and SAFETY GLASSES at all times The 2 laboratory sessions in this subject count 10% towards your final grade

Laboratory Objectives By the end of this session you should: Have gained experience in bioseparation techniques including: standard curve determination, sample injection and fraction sampling, and biomolecule concentration determination. Have developed a basic understanding of size-exclusion chromatography. Have been introduced to the concept of separation yield and recovery. Laboratory Outline This laboratory is unstructured and will require good time management and coordination between and within groups. During the experiment you will be required to perform the listed tasks in the listed order. Note that part a and b may be done simultaneously by different members of the group. 1. Laboratory Introduction and Planning 20 mins 2. Downstream Separation Experiment 3.5 hrs a. Create at least two calibration curves (one without and one with Bradford assay) b. Pack and prepare gel filtration column c. Start sample injection d. Examine the separation of biomolecules through the column e. Take samples and analyse for the various biomolecule concentrations f. Determine yield and recovery (can be done after the lab) 3. Clean up 10 mins

Two groups, A and B, will work together to create the four required standard curves. Each group will prepare two sets of standard solutions, one for the coloured biomolecule and one of protein with Bradford assay, and obtain their calibration curves. These four sets of calibration curves will then be shared between the two groups for the concentration determination of biomolecules in the samples. (See your demonstrator to discuss the detailed arrangements.)

Separation of Biomolecules by Size-Exclusion Chromatography Many different bioseparation methods are in use today for the downstream processing of biological products, predominantly to isolate and recover mixtures of biological product into pure components. Chromatography is one of the most commonly used methods due to their high selectivity for separating components of similar properties. It includes size exclusion or gel permeation chromatography, ion-exchange chromatography, affinity chromatography. During the chromatographic process, the components in a mixture are separated as they are carried through the column by a gas or liquid stream known as mobile phase or eluent, with the column being packed with a stationary phase of solid particles. In this experiment, size exclusion chromatography (SEC) will be used for the separation into pure components of a mixture containing biomolecules of different molecular weights. This process involves the use of stable, physically inert spherical particles of porous matrix, packed in a column, to separate the components of the mixture based on their molecular size differences. When the sample is applied to the top of the column and a mobile phase is allowed to elute through the column, the molecules or solutes partition themselves between the stationary and mobile phases. The smallest molecules diffuse furthest into the matrix of the stationary phase and take longest to elute compared to the larger molecules. Thus depending on the fractionation range of the gel matrix, the largest molecules will elute through the column first, followed by the next largest molecules, with the smallest molecules eluting last. As the molecules of differing sizes repeatedly migrate in and out of the pores of the matrix as they move along the column, sample injected to the top of the column is separated into solute bands or peaks as the components exit the column, as shown in Figure 1 (for the elution of a two-component mixture). The time from the injection of sample to the time of the peak for each component is termed the retention time, tR. Note that the volume of eluent, or the retention volume, of each component can be determined for a given flowrate of the mobile phase through the column.

Sample injection

t R2 t R1
Concentration

Elution of component 1

Elution of component 2

t w1

T ime

t w2

Figure 1: Chromatogram of the elution of a mixture of two solutes. tR is the retention time and tw the band width of each solute eluted. The column efficiency for separating solutes or components can be determined from the extent 2

of broadening of the solute bands during elution. Band broadening occurs as the initially narrow band of solutes applied to the top of the packed column disperses as the solutes migrate through the column. It is caused by several factors such as non-uniformity in the flow patterns in the column, and slow mass transfer of solutes to and from interface with the stationary phase. The degree of band broadening is a function of the column inefficiency, which can be defined by the height equivalent of a theoretical plate (HETP). The HETP, H of a column of length L is given by (Richardson et al., 2005):

H=

L L = N 16 (t R / tw )2

(1)

where N is the number of theoretical plates in the column, and tR and tw are retention time and band width of the component, as shown in Figure 1. To achieve high separation efficiency, it is therefore desirable to have low H and high N. In this experiment, a mixture containing three biomolecules: Haemoglobin (Mw=67,000), Trypsin (Mw=23,400) and Vitamin B12 (Mw=1,355), will be employed to investigate the use size exclusion chromatography for bioseparation based on molecular size differences. By measuring the absorbance of the sample fractions as they elute from the column packed with a chosen SEC gel, the concentration variation of the components as a function of eluent volume or time can be obtained, and the efficiency of the column determined. In addition, the effect of column dimension in terms of the packing height will be investigated. Concentration Determination Beer-Lambert Law The Beer-Lambert law (or Beer's law) is the linear relationship between absorbance, or sometimes referred to as optical density, OD, and concentration C of an absorbing species. It I can be written as: OD = log( ) = l C (2) I0 where I0 and I are the intensities of light entering and leaving the sample of concentration C and path length l , and is the wavelength-dependent molar absorptivity coefficient. OD is

the optical density of the solution at wavelength . (Note that the units of will depend on the units of C used.) The Beer-Lambert law is usually not obeyed at high concentrations, and the relationship can be used only in the linear concentration region. Use of Spectrophotometer for Concentration Analysis

Since Haemoglobin and Vitamin B12 are coloured (reddish brown and pink, respectively), they can be detected by visible spectrophotometry. Specifically, the concentrations of Vitamin B12 and Haemoglobin can be determined by measuring the absorbance at their respective maximum absorbance wavelength of 362 and 406nm. However, in addition to the maximum absorbance at 362nm for Vitamin B12 and at 406nm for Haemoglobin, Vitamin B12 also shows significant absorbance at 406nm, and likewise for Haemoglobin at 362nm. Hence in order to determine the correct concentrations of these two biomolecule components in a solution, it is necessary to measure the absorbances at the two wavelengths, i.e. at 362 and 406nm, and use two simultaneous Beer-Lambert equations to solve for the unknown concentrations, C B12 and C Hb , as follow:

OD362 nm = B12 / 362 nm l CB12 + Hb / 362 nm l CHb

OD406 nm = B12 / 406 nm l CB12 + Hb / 406 nm l CHb

3

(3)

where O D is the total absorbance at wavelength , X / is the molar absorptivity coefficient of species X at wavelength , and l is the path length (i.e. 1cm). Each of the four X / values can be determined from the slope of the appropriate calibration curve of absorbance of component X at wavelength versus concentration. In using the above equations the assumptions are that the absorbances of the components at each wavelength are additive and there is no interaction between the components. Trypsin, on the other hand, is a colourless protein, but its concentration can still be determined by visible spectrophotometry by reacting with a protein-binding dye. There are several protein assays which are available for the quantification of proteins; in this experiment the Bradford protein assay will be used as it proves to be sufficiently sensitive, reproducible and simple to carry out (see Bradford, 1976). For this assay Coomassie Blue dye is used; it binds with certain proteins and changes colour (from red) to blue, with a maximum absorbance at 595nm. Therefore the intensity of colour, i.e. the absorbance, at 595nm is directly related to the concentration of the bound proteins. However Haemoglobin will also bind with the Coomassie Blue dye to form protein-dye complex. So for solutions containing both these proteins, the total absorbance is the sum of the contributions from each component, i.e. OD595nm = Tryp /595nm l CTryp + Hb /595nm l CHb (4) where Tryp / 595nm can be determined from the calibration curve for the absorbance of Trypsindye complex at 595nm, and likewise for Hb / 595 nm , which can be obtained from the calibration curve for Haemoglobin-dye complex. In order to determine the concentrations of the individual proteins or biomolecules in solution containing all three components, one needs to first determine the concentrations of the Haemoglobin and Vitamin B12 by solving the simultaneous equations given in (2) (for known molar absorptivity coefficients at the appropriate wavelength). Then using the Bradford assay one can further determine the concentration of Trypsin using equation (3) for a known concentration of Haemoglobin and given molar absorptivity coefficients of the two protein-dye complexes at 595nm.
Experimental Procedure General equipments and reagents

The following items are required for all parts of this experiment: 100-1000l pipetter and rack of pipette tips (blue coloured tips) 500-5000l pipetter and rack of pipette tips (large, colourless tips) Access to a vortex machine and spectrophotometer PBS buffer

Calibration Curves for Haemoglobin and Vitamin B12

In order to quantitatively determine the concentrations of Haemoglobin and Vitamin B12 in solutions, four standard curves must be obtained: one for each component at each of the two wavelengths of 362 and 406nm. From these curves, the molar absorptivity coefficients for the Haemoglobin and Vitamin B12 at 362 and 406nm can be determined. For this part of the experiment, groups A and B will work together to produce the four standard curves. Group A will prepare the standard solutions containing Haemoglobin and obtain the standard curves and molar absorptivity coefficients at the two wavelengths of 362 4

and 406nm, and group B will do the same for standard solutions containing Vitamin B12. These results will then be shared between the two groups. Equipment and Reagents Ten clean and dry test tubes placed in a test-tube rack Ten spectrophotometer cuvettes One of two stock solutions: Haemoglobin (containing 1.0mg/ml Hb in PBS buffer) or Vitamin B12 (containing 1.0mg/ml B12 in PBS buffer)

Procedure 1. Label the 10 test tubes from 0 to 9. In each batch of test-tubes, make standard solutions of different concentrations of haemoglobin(Group A) or Vitamin B12 (Group B) from the stock solutions and PBS buffer, according to the Table 1 or Table 2 below:
Table 1 (Group A) Absorbance of Haemoglobin solutions at 406 and 362nm

Standard Haemoglobin No. conc. (mg/ml) 0 0 1 0.04 2 0.08 3 0.12 4 0.16 5 0.2 6 0.4 7 0.6 8 0.8 9 1.0

Haemoglobin Stock solution (ml) 0 0.2 0.4 0.6 0.8 1 2 3 4 5

PBS buffer (ml) 5 4.8 4.6 4.4 4.2 4 3 2 1 0

Absorbance (OD 406nm)

Absorbance (OD 362nm)

Table 2 (Group B) Absorbance of Vitamin B12 solutions at 406 and 362nm

Standard No. 0 1 2 3 4 5 6 7 8 9

Vit. B12 conc. (mg/ml) 0 0.02 0.04 0.06 0.08 0.1 0.2 0.4 0.6 0.8

Vitamin B12 Stock solution (ml) 0 0.1 0.2 0.3 0.4 0.5 1 2 3 4

PBS buffer (ml) 5 4.9 4.8 4.7 4.6 4.5 4 3 2 1

Absorbance (OD 406nm)

Absorbance (OD 362nm)

2. Set the spectrophotometer to measure at 362nm and zero using the blank solution, i.e. Standard No. 0. Then measure and record the absorbance of each solution. Note: the absorbance obtained for the solutions should not exceed a maximum OD of about 2. 5

Also the absorbance of the grey-shaded cells may be omitted as they will probably give either too high or too low optical density reading. 3. Repeat step 2 to measure absorbance at 406nm. 4. Enter the absorbance values into the spreadsheet to create the biomolecule calibration curves. Record the slope of these calibration curves for your subsequent calculations.
Calibration Curves with Bradford Protein Assay

The two proteins used in this experiment, i.e. Haemoglobin and Trypsin, both bind to Coomassie Blue dye to form blue protein-dye complex in solution. The total concentration of these two proteins, which is equivalent to the concentration of the protein-dye complex, can be determined by measuring the absorbance at 595nm. However, a calibration curve for each of the two protein-dye complexes must be obtained individually in order to determine the molar absorptivity coefficient for each. Equipment and Reagents 12 clean and dry test tubes placed in a test-tube rack Six spectrophotometer cuvettes One of two stock solutions: Haemoglobin (containing 1.0mg/ml Hb in PBS buffer) or Trypsin (containing 1.0mg/ml Trypsin in PBS buffer) Bradford Protein Assay Reagent

Procedure 1. Separate the test-tubes into 2 batches of six each. Label the two batches of test-tubes from 0 to 5. 2. In the first batch of test-tubes, make standard solutions of different concentrations of haemoglobin (Group A) or Trypsin (Group B) from the stock solutions and PBS buffer, according to the Table 3 or Table 4 below:
Table 3 (Group A) Concentration of Haemoglobin-dye complex standard solutions

Standard Haemoglobin No. conc. (mg/ml) 0 0 1 0.2 2 0.4 3 0.6 4 0.8 5 1.0

Haemoglobin Stock solution (ml) 0 1 2 3 4 5

PBS buffer (ml) 5 4 3 2 1 0

Table 4 (Group B) Concentrations of Trypsin-dye complex standard solutions

Standard No. 0 1 2 3 4 5

Trypsin conc. (mg/ml) 0 0.2 0.4 0.6 0.8 1.0

Trypsin Stock solution (ml) 0 1 2 3 4 5

PBS buffer (ml) 5 4 3 2 1 0

3. Into the second batch of test-tubes marked 1 to 5, pipette 100L of each of the standard solutions and 5ml of dye reagent. In test-tube marked 0, pipette 100L of PBS buffer and 5ml of dye reagent; this is to be used as blank. Vortex and let stand at room temperature for exactly 10 minutes. 4. Set the spectrophotometer to measure at 595nm and zero using the blank solution, i.e. Standard No. 0+ dye. Then measure the absorbance of each solution at 595nm and record the readings in Table 5. Note: the absorbance measured for any solution should not exceed a maximum value of about 2.
Table 5 Absorbance of Hb-dye or Trypsin-dye complex standard solutions at 595nm

Protein-dye complex Standard No. 0 1 2 3 4 5

Absorbance (OD 595nm)

5. Enter the absorbance values into the spreadsheet to create the protein-dye complex calibration curves. Record the slope of these two calibration curves for your subsequent calculations.
SEC Column Packing

Gel permeation or size exclusion chromatographic technique is used for the separation, or commonly referred to as fractionation, of polymers or biomolecules based on differences in the molecular size or weight. The molecular weight range over which the SEC medium can separate molecules is referred to as the selectivity of the matrix or packing medium. In addition to the selectivity of the medium, the efficiency to separate a mixture of polymers or biomolecules into individual components depends on several other factors, such as column dimensions and packing density, flowrate, sample volume, etc. In this experiment, the effect of the column dimension, i.e. packing height, will be investigated while the column diameter is kept constant. Group A will use short columns of between 1214ml of packing, and group B will use long columns of between 18-20ml of packing. The effect of packing efficiency or packing density on the separation efficiency will be investigated by comparing the results from two different groups using the same column heights.

The packing medium to be used in this work is Sepahcryl S-100 gel (from Amersham Biosciences) which is a composite, prepared by covalently cross-linking allyl dextran with N,N-methylene bisacrylamide to from a hydrophilic matrix of high mechanical strength. The Sephacryl S-100 gel consists of particle size ranging from 45-75m, and has a fractionation range for molecular weights between 1103 and 1105. (The gel is supplied swollen in a suspension containing 20% ethanol as a preservative. However, the gel used here have been equilibrated in PBS buffer before use.) In this experiment, a 25ml burette will be used as the filtration column, and the flowrate of the buffer through the column will be controlled manually by maintaining a constant head of liquid (i.e. buffer) above the packed medium. Equipment and Reagents One 25mL burette and burette stand One 25ml measuring cylinder One 100ml beaker Disposable pipettes Glass wool Metal rod Column packing medium (Sephacryl S-100 gel which has been equilibrated with PBS buffer)

Procedure 1. To pack the column for the biomolecules separation experiment, first suspend the packing medium by stirring it gently and then pour into the measuring cylinder a volume of medium approximately 1.2 times that of the required packed column volume. Dilute with PBS buffer to 2 times the required volume. 2. Check that the column is held firmly on the stand. Close the tap of the column and fill with 2 to 3 ml of PBS buffer. 3. Again gently shake or stir the packing medium slurry in the measuring cylinder, and gently but quickly pour some of the slurry into the column. Wait for the medium to begin to settle and keep topping up with the remaining slurry until it has all been transferred. Open the tap to let some buffer elute out if necessary. You must continue to agitate the slurry in the measuring cylinder to prevent it from settling prematurely. 4. Open the tap to let the PBS buffer run off and for the gel to pack under gravity in the column. Meanwhile keep adding PBS buffer to the top of the column to maintain a liquid head of about 5ml above the packing. Keep this going until the packed volume becomes constant. Make sure never to let the column run dry. 5. Close the tap when a stabilised bed has been reached, making sure there is some buffer above the packing.
Sample Application and Elution

The Sephacryl S-200 gel used in this work has a fractionation range for molecular weights between 1103 and 1105 dalton. It is thus suitable for separation of the ternary biomolecules mixture consisting of Vitamin B12 (Mw=1,355), Trypsin (Mw=23,400) and Haemoglobin (Mw=67,000). As the three biomolecules are considerably different in their molecular weights, but are all within the fractionation range of the S-200 gel, they are expected to elute from the column at different times and eluent volumes, and can thus be separated into individual 8

components. After the application of the mixture to the packed column, the various components will elute from the column in order of decreasing molecular size. The entire separation process should be complete, with all the components having eluted, after one packed bed volume of buffer having passed through the column. By considering the molecular weight of the various biomolecules in the mixture, you should be able to identify the order, as well as estimate the eluent volume, and eluent time for a given flowrate, that each component will exit the column. As the components elute from the column, the aim is to measure the concentration of the components as a function of eluent volume. In order to obtain a true representation of the variation in concentration of each component at different eluent volumes, especially the peak concentrations for each component, a large number of small eluent volumes (i.e. samples) should be collected. If the volume of the eluent samples collected is too large, especially near the peak, the true peak concentration will not be detected as the value will be diluted by the lower concentrations of the eluent on either side of the peak. However, the number of samples collected must be balanced against the time and effort required to analyse these samples. A total of 20 samples are recommended, using eluent volumes ranging from 0.5 to 1.5 ml. You need to plan this part of the experiment carefully in order to obtain meaningful plots of concentration of the biomolecules against eluent volume. Record the eluent volumes that you plan to collect during the separation process in Table 6 (at the end of the notes), and check with your demonstrator before commencing your sample application. Several methods of sample application can be used, but for small column and sample size used in this experiment, the sample will be applied directly to the top of the column via a pipette. The recommended volume of sample is between 1 to 4% of the column volume, with the lower the sample volume applied the higher the resolution that can be achieved. After the injection of sample, the entire separation takes place as one packed bed volume of buffer elutes through the column. As Vitamin B12 will elute through the column last, it is also possible to determine when all the components have been collected and the separation process is complete. This will be marked by the disappearance of the last pinkish colour of the eluent through the column. Equipment and Reagents At least twenty 1.5ml micro-centrifuge tubes for sample collection Sample of ternary-biomolecule mixture, containing Haemoglobin (8mg/ml), Trypsin (10mg/ml), Vitamin B12 (4mg/ml).

Procedure 1. 2. 3. Label the micro-centrifuge tubes from 1 to 20. Prepare a volume of the ternary-biomolecule mixture equivalent to 1% of the packed bed volume, ready for pipette transfer to the column. Open the tap of the column to let the buffer elute through the packing until the level of liquid is just above the column packing, then gently apply the ternary-biomolecule mixture (from the pipetter) to the top of the column packing. Let some more buffer flow out of the column until the whole volume of the injected 9

4.

mixture has entered the top of the column packing. Do not add buffer before this time, or you will dilute the sample (i.e. by increasing the sample volume) and the elution peaks will spread out. 5. As soon as the last trace of the ternary-biomolecule mixture has entered the column packing, gently add buffer to the top of the column to maintain a constant head of about 5ml buffer above the packing. Collect the first few ml of eluent (which should not contain any biomolecule) in a test-tube. When the first hint of reddish brown Haemoglobin fraction is noticeable near (i.e. 2-3 ml above) the bottom of the packed column, start your sample collection using the labelled micro-centrifuge tubes. Collect small fractions of the eluent as planned in Table 6, using the markings on the burette to note the eluent volume and ensuring that none of the eluent is lost during sample collection. Keep topping up the column with buffer so that there is always a head of about 5ml buffer above the packing. There is no need to close the tap between each sample collection, as this will slow down the elution unnecessarily. Note: If you have collected larger or smaller volume of samples than planned, make sure to record the correct sample volume in Table 6. 8. Continue the sample collection until all the biomolecule fractions have been collected, i.e. when the last trace of pink Vitamin B12 fraction have eluted through. The total volume of all the eluent fractions collected should be approximately equivalent to one packed bed volume. Continue adding extra buffer to the top of the column and allow the buffer to elute through the column for as long as time allows, to equilibrate and clean the packing medium in the column. Again do not let the column run dry. At the end of the experiment, close the tap and seal the top of the column with a small piece of Parafilm.

6. 7.

9.

Sample Analysis Measurement of Biomolecule Concentrations

For the coloured components, i.e. Haemoglobin and Vitamin B12, the concentrations in any sample fraction can be determined by measuring absorbances at 362 and 406nm, and solving the simultaneous equations given in Eq. (3). Thereafter the concentration of the remaining component, i.e. Trypsin, can be determined by the Bradford assay method, by measuring of absorbance at 595nm and solving the equation given in Eq. (4). It is also important to note that in order to obtain a correct absorbance measurement at any given wavelength, the sample volume in the cuvette must be at least 2 ml. This is so that the entire width of the spectrophotometer light beam passes through the sample being measured, and not through air above the sample if the sample volume is insufficient. Equipment and Reagents 21 clean and dry test-tubes At least 20 cuvettes Bradford assay dye reagent

10

Procedure To determine the concentrations of Haemoglobin and Vitamin B12 in the samples: 1. Label the cuvettes (on the opaque side) from 1 to 20. 2. Into each labelled cuvette, pipette 0.3ml of the sample and 2.7ml of buffer. This results in a dilution factor of 10. Record this value in Table 6. 3. Set the spectrophotometer to measure at 362nm and zero using a blank solution containing only the PBS buffer. Measure the absorbance of the samples at 362nm, and record the results in Table 6 4. Set the spectrophotometer to measure at 406nm and zero using a blank solution containing only the PBS buffer. Measure the absorbance of the samples at 406nm, and record the results in Table 6. To determine the concentration of Trypsin using the Bradford protein assay: 1. Label the test-tubes from 1 to 20. 2. Into each labelled test-tubes, pipette 100l of the appropriate sample and 5ml of the Bradford dye reagent. Vortex and let stand at room temperature for exactly 10 minutes. In the last test-tube, pipette 100L PBS buffer and 5ml Bradford dye reagent, to be used as blank. 3. Set the spectrophotometer to measure at 595nm and zero using the blank solution. 4. Measure the absorbance of each sample (from the test-tubes) at 595nm. Record the results in Table 6. Note: If any of the measured absorbance readings exceeds a value of 2, dilute the sample with PBS buffer by a known factor and remeasure the absorbance. 5. Enter all the results in the spreadsheet and determine the concentration of each component in the samples.
Clean-Up

Before you leave the laboratory, please ensure that: 1. All equipment are left clean and electrical appliances are turned off 2. Any spills have been cleaned up, waste and rubbish put away appropriately 3. Test-tubes and other glasswares are rinsed and left to dry 4. All chemicals and equipment are accounted for

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Data analysis

From the absorbance results measured for the various sample fractions at the three different wavelengths, you are now ready to calculate the concentration of the biomolecules using Eqs. (3) and (4). From these results you can obtain a plot showing the concentrations of each of the three biomolecules versus eluent volume. From these plots you should also be able to determine the total amount of each biomolecule recovered in the size-exclusion chromatographic separation process by determining the area under the curve for each component. You can do this by counting the squares under the graph, or by numerically summing the rectangular areas of width (Vn Vn 1 ) and height 1 (Cn + C n 1 ) , where Vn are
2

the cumulative eluent volume and Cn and concentration of the nth sample, respectively. You can also evaluate the column efficiency by comparing the degree of band broadening which occurs as the solutes or biomolecules disperse as they elute through the packed column. That is, the column efficiency for each component can be evaluated in terms of the number of theoretical plates which can be determined using Eq. (1).
Questions

1. For one of the samples collected, show a sample calculation to determine the concentration of the three biomolecule components from the measured absorbances, i.e. O.D. at the three different wavelengths. 2. What are the underlying assumptions in the determination of concentrations of Haemoglobin and Vitamin B12 from the measured absorbance values at 362 and 406nm, using Eq. (3)? 3. Comment on the percentage recoveries that you obtained for the components, and give some reasons why the values may be lower (or higher) than 100%. 4. Comment on the efficiency of the columns for separating the three biomolecules. Suggest ways of improving the efficiency for bioseparation by SEC or gel permeation chromatography.
References and Further reading

1. 2.

Bailey, J. E. and Ollis, D. F. (1986) Biochemical Engineering Fundamentals, 2nd Ed., McGraw-Hill, New York. Bradford, M. M. (1976), A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72, 255. Richardson, J.F. and Harker, J.H., with Backhurst, J.R. (2002), Chemical Engineering, Volume 2, 5th Ed., Butterworth-Heinemann, Amsterdam.

3.

12

Table 6 Sample volume and absorbances of eluents.

Sample No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Sample Volume (ml)

Volume collected (ml)

Dilution factor

Absorbance (OD 362nm)

Absorbance (OD 406nm)

Absorbance (OD 595nm)

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