Lab Manual For DSP

Downstream Processing Laboratory 10 BT72
Sl. No Name of the Experiment Page No.

Introduction to downstream processing
1. Tangential Flow Filtration System
2. Centrifugation Studies During settling of yeast cells
3. Yeast cells disruption by mechanical method

4. Design of thickener for batch sedimentation using
Kynch’s theory
5. Estimation of Organic acid by calorimetric method
6. Determination of partition coefficient and yield of

yeast cells using aqueous two phase extraction
7. Extraction Of Pigments From Spinach And
Estimation By Thin Layer Chromatography
8. Ammonium Sulfate Precipitation Of Proteins Using

Yeast Cell Suspension
9. Determination of drying time for a given sample in

Vacuum tray drier.
10. Gas Chromatography analysis for a compound

11. Crystallization of a product
12. Analysis of a compound using HPLC, Gas
Chromatography.
CONTENTS
Department of Biotechnology, R. V .College of Engineering, Bangalore 1

Introduction to downstream processing

Downstream processing refers to the recovery and purification of biologicals,
biosynthetic products, particularly pharmaceuticals, from natural sources such as animal
or plant tissue or fermentation broth, It is an essential step in the manufacture of
pharmaceuticals such as antibiotics, hormones (e.g. insulin and human growth hormone),
antibodies (e.g. infliximab and abciximab) and vaccines; antibodies and enzymes used in
diagnostics; industrial enzymes; and natural fragrance and flavor compounds.
Downstream processing is usually considered a specialized field in biochemical
engineering, itself a specialization within chemical engineering, though many of the key
technologies were developed by chemists and biologists for laboratory-scale separation of
biological products.
Downstream processing and analytical bioseparation both refer to the separation or

purification of biological products, but at different scales of operation and for different
purposes. Downstream processing implies manufacture of a purified product fit for a
specific use, generally in marketable quantities, while analytical bioseparation refers to
purification for the sole purpose of measuring a component or components of a mixture,
and may deal with sample sizes as small as a single cell.
Stages in downstream processing
A widely recognized heuristic for categorizing downstream processing operations divides

them into four groups which are applied in order to bring a product from its natural state
as a component of a tissue, cell or fermentation broth through progressive improvements
in purity and concentration.
Removal of insolubles is the first step and involves the capture of the product as a solute
in a particulate-free liquid, for example the separation of cells, cell debris or other
particulate matter from fermentation broth containing an antibiotic. Typical operations to
achieve this are filtration, centrifugation, sedimentation, flocculation, electro-
precipitation, and gravity settling. Additional operations such as grinding,
homogenization, or leaching, required to recover products from solid sources such as
plant and animal tissues, are usually included in this group.
Product isolation is the removal of those components whose properties vary markedly
from that of the desired product. For most products, water is the chief impurity and
isolation steps are designed to remove most of it, reducing the volume of material to be

handled and concentrating the product. Solvent extraction, adsorption, ultrafiltration, and
precipitation are some of the unit operations involved.
Product purification is done to separate those contaminants that resemble the product
very closely in physical and chemical properties. Consequently steps in this stage are
expensive to carry out and require sensitive and sophisticated equipment. This stage
contributes a significant fraction of the entire downstream processing expenditure.
Examples of operations include affinity, size exclusion, reversed phase chromatography,
crystallization and fractional precipitation.
Product polishing describes the final processing steps which end with packaging of the
product in a form that is stable, easily transportable and convenient. Crystallization,
desiccation, lyophilization and spray drying are typical unit operations. Depending on the
product and its intended use, polishing may also include operations to sterilize the
product and remove or deactivate trace contaminants which might compromise product
safety. Such operations might include the removal of viruses or depyrogenation.
A few product recovery methods may be considered to combine two or more stages. For
example, expanded bed adsorption accomplishes removal of insolubles and product
isolation in a single step. Affinity chromatography often isolates and purifies in a single
step.

YEAST CELLS DISRUPTION BY MECHANICAL METHOD

Objective
To carry out disruption of yeast cells mechanically and to assay the total protein and
enzyme content
Apparatus
Yeast, DNS, sucrose, invertase, BSA, spectrophotometer
Principle
Cell disruption is the process of breaking open the cell membrane and cell wall, to release
the contents of the cell into the surrounding media. The main aim of cell disruption is to
ensure that the labile materials are not denatured by the process or hydrolyzed by the
enzymes. Two methods are employed:-
i) By physical methods like thermolysis, osmotic shock, ultrasonication,
high pressure homogenizer and bead mill homogenizers.
ii) By chemical methods like alkali treatment, detergent solubilization, lipid
permeabilization by organic solvents and enzymatic method.
The method used is here is basic grinding using mortar and pestle. We then study the
action of invertase on sucrose hydrolysis and determine its activity compare it to standard
glucose curve. Also, we estimate the protein content in the cell using the Biuret method
of protein estimation.
Procedure
(a) Disruption
2 gm of dry yeast is taken and placed into 10 ml acetate buffer (pH 4.6) and ground in
with pestle and mortar. This suspension is then centrifuged at 4000 rpm for 5 min and the
supernatant is taken for the enzyme and protein assay.
(b) Invertase activity

A glucose standard graph is prepared by taking 6 test tubes and adding 0, 0.2, 0.4, 0.6,
0.8, 1.0 ml of glucose (stock sol-1 mg/ml made upto mark with pH 4.6 buffer) and
making volume up to 1 ml in all the 6 test tubes. 1 ml DNS is added and boiled in a water
bath for 10 min. 3 ml of distilled water is added and OD is taken at 540 nm. For test
sample, 2 test tubes, 1 test and 1 control tube is taken. 1 ml of sucrose is (stock sol- 0.1%
w/v made upto mark with pH 4.6 buffer) added and volume is made up to 4 ml using pH
4.6 acetate buffer in both tubes. 1 ml invertase (extract) is added and incubated for 20 and
30 mins respectively. 1 ml DNS is added to 6 tubes and OD taken at 540 nm. Standard
graph of OD540 vs glucose concentration (μg/ml) is plotted and concentration of test
determined.
(c) Protein estimation

Initially 6 tubes of increasing standard Bovine Serum Albumin (BSA) (stock sol- 10
mg/ml made upto mark with distilled water) of 0.2, 0.4, 0.6, 0.8, and 1 ml are taken and
volume is made up to 1 ml using water. To this, 9 ml Biuret solution is added and
incubated for 20 min at 37C. This is followed by taking the OD at 540 nm. Plot a
standard graph of OD540 Vs BSA concentration. 2 test tubes are taken and 0.5 ml and 1 ml
of supernatant are added. Volume made up to 1 ml using water. 9 ml of Biuret solution is
added and incubated for 2 min at room temperature and their OD is taken. These values
are compared with the concentration of standard graph and protein is estimated.
Observations
Protein Estimation (standard BSA = 10 mg/ml)
Standard Distilled Biuret Incubation O.D at 540 nm
BSA water (ml) time (min)
0 1.0 9 20
0.2 0.8 9 20
0.4 0.6 9 20
0.6 0.4 9 20
0.8 0.2 9 20
1.0 0.0 9 20

Supernatant of Biuret Incubation time O.D at

protein extract (min) 540nm
(ml)
0.5 9 20
1 9 20
Activity = Micromoles of product formed x Total volume

Incubation time x ml of protein
Result
Activity of enzyme invertase was estimated and protein concentration was calculated as
_______________________________ respectively.

DESIGN OF THICKENER FOR BATCH SEDIMENTATION USING

KYNCH’S THEORY
Objective
To carry out a batch sedimentation test and to design a thickener required to handle 5
tons per day of slurry with an initial concentration of 15 g/l to an overflow concentration
of 150 g/l in the presence of 10 ml/L of supersaturated ferric alum solution.
Apparatus
1 liter beaker, yeast cells, stop watch
Principle
Separation of fine solids from slurry in large tonnage is carried out in a thickener. The
process is best described by batch sedimentation and from this, the area of the thickener
can be calculated. In the operation of thickener a basic assumption is that the material to
be settled contains flocks of uniform size and shape and they settle under
hinderedsettling.
The capacity of a thickener is determined by the fact that the solids initially present in the
feed must be able to settle through all zones of slurry concentrations from that of the
initial feed, when introduced into the thickener. If the area provided is not sufficient the
solids will build up through the settling zone until finally some solids are discharged in
the overflow. The method based on mathematical analysis proposed by Kynch, propounds
that settling time and concentration of the zone that limits capacity can be determined
through batch sedimentation test. From the graph of Z versus 0, settling velocity as a
function of concentration, it is possible to calculate the solids handling capacity per unit
area. The lowest value of the solid handling capacity is used to determine the thickener
area.
Formulae used:
C0 Z0
Concentration of solids on limiting layer, C L  ---------------------------- (1)
Zi
Zi - Z L
Velocity of solid particles in the limiting layer, VL  --------------- (2)
L

The minimum cross sectional area of the thickener can be determined by the following
equation:
VL
L LCL 
1 1 ------------------------- (3)

C L Cu
The minimum cross-sectional area of the thickener can be determined by rearranging eq.
(3)
L LCL
A
VL
m2 ------------------------------ (4)
1 1

C L Cu
where,
CL = concentration of the solids in the limiting layer, Kg/m3
C0 = initial concentration of solids, Kg/m3
Cu = underflow (final solids) concentration, Kg/m3
Z0 = initial height of the interface, m
ZL = height of the interface at time θ
VL = velocity of solid particle sin the limiting layer, m/s
A = minimum cross-sectional area of the thickener, m2
Procedure
The graduated glass cylinder should be cleaned thoroughly before starting the experiment
and the experiment need to be carried out in clear light.
1. Prepare yeast solution by grinding 15 g of yeast and making upto 1000ml mark
with water in 1 l measuring jar.
2. Add 10 ml of supersaturated ferric alum solution and stir to get uniform
concentration.
3. Note down the initial height Z0, simultaneously start a stop watch to note down
the time taken for each cm height of fall of the interface.
4. Carry on the experiment till the height of the interface remains almost constant.

5. Plot interface height (Z) versus time θ.

6. Calculate the slope (VL) using eq. (1) and concentration using eq. (2).
7. Draw a graph of the solid handling capacity and the concentration of the limiting
VL
layer i.e. 1 1 versus VL. The minimum value from the graph is used in eq.

C L Cu
(4) to determine the area of the thickener.
Observation and calculations

Table 1.
Sl No. Interface height, Zx 102, m Time, θ, s
1.
2.
Table 2.
Sl Zi x 102, ZL x 102, θL, s CL, VL x x104, 1 1 VL A,

C L Cu 1 1 ,
No. m m kg/m3 m/s  m2
C L Cu
, m3/kg
m3/kg

Downstream Processing Laboratory Manual
Nature of graph
Z x 102, m
Result
The minimum area of the thickener needed to settle a fermentation broth with an initial
concentration of 15 g/l in the presence of 10 ml/L of supersaturated ferric alum solution
is_______________m2. θ, s
Department of Biotechnology, RVCE, Bangalore 10

ESTIMATION OF CITRIC ACID IN UNKNOWN SAMPLE BY

CALORIMETRIC METHOD
Objective
To estimate citric acid in unknown sample by calorimetric method.
Apparatus
citric acid, H2SO4, metaphosphoric acid, KMNO4
Theory
Citric acid is an important chemical used in medicines, flavoring extract, food and
candies, manufacture of ink, dyeing and engraving. Several different species of moulds
have the ability to convert sugar to citric acid but Aspergillus niger is most widely used
for its commercial production.
Estimation
The reaction involves oxidation of citric acid by potassium permanganate in the presence
of bromine. The pentabromoacetone produced is extracted from the oxidation mixture
with petroleum ether and treated with thiourea borax solution. A yellow color complex is
formed. This is proportional to intensity of pentabromoacetone (PBA) in solution. The
quantity of citric acid in the solution is derived from the calibration curve of the standard
citric acid solution (concentration versus Optical density at 440 nm).
Procedure
1) Take 0, 0.1, 0.2, 0.4, 0.6, 0.8 and 1 ml of standard citric acid (2 mg/ml) in test tube B,
1, 2, 3, 4, 5 and 6.
2) Add distilled water to make up to l ml.
3) Add 1 ml of 9N H2S04 followed by 0.3 ml of 40 % metaphosphoric acid and mix after
addition.
4) Incubate in ice bath for 10 min.
5) Add 0.5 ml of 2M KBr followed by 1.5 ml of 6.5% KMNO4.
6) Mix thoroughly and incubate in ice bath for 10 mm.
7) Add 6% H202 dropwise and shake until KMNO4 is decolorized.

8) Transfer into iodine flask and add 5 ml petroleum ether. Shake for 2 min to extract.
PBA into petroleum ether. The 2 layers separate after sometime.
9) 2 ml from upper layer of petroleum ether layer is pipetted into the test tube.
10) Add 6 ml of thiourea borax solution (pH 9.2) to each test tube and shake well for 2
min.
11) A golden yellow color is developed because complex formed between thiourea and
pentabromoacetone. Take OD at 440 nm.

Observations and Calculations
Keep in ice water bath for 10 min

Sl. Standard Distilled 9N 40 % 2M 6.5% 6% Petrole Thio OD
No. citric water H2SO4 Metaphos KBr KMNO4 H2O2 um borax at
acid (ml) (ml) (ml) phoric (ml) ether solution 440
acid (ml) (ml) (ml) nm
B 0 1 1 0.3 0.5 1.5 5 6
1 0.2 0.8 1 0.3 0.5 1.5 5 6
2 0.4 0.6 1 0.3 0.5 1.5 5 6
3 0.6 0.4 1 0.3 0.5 1.5 5 6
4 0.8 0.2 1 0.3 0.5 1.5 5 6
5 1 0 1 0.3 0.5 1.5 5 6
6 unknown 1 0.3 0.5 1.5 5 6

7
unknown
1
0.3
0.5
Department of Biotechnology, R. V .College of Engineering, Bangalore

1.5
Mix and keep in ice water bath for 10 min
Add drop wise until color decolorizes

5
Shake the mixture for 2 min and leave for separation. Two layers are formed. Take 2 ml
from upper (petroleum ether) layer
6
14

Nature of graph
OD
Concentration of citric acid, μg/ml
Result
The concentration of citric acid in test sample 1 was ______________ μg /ml
The concentration of citric acid in test sample 2 was ______________ μg/ml

DETERMINATON OF PARTITION COEFFICIENT & YIELD OF

YEAST CELLS USING AQUEOUS TWO PHASE EXTRACTION
Objective
To determine the partition coefficient and yield of total protein present in yeast cells or
yeast extract powder (bacteriology grade) using Polyethylene Glycol (PEG) 4000 and
ammonium sulfate or potassium phosphate system.
Apparatus
Beakers, separating funnel, PEG 4000, ammonium sulfate, potassium dihydrogen
phosphate (KH2PO4), dipottasium hydrogen phosphate (K2HPO4), beaker’s yeast or yeast
powder (bacteriology grade).
Theory
Aqueous two phase system is formed by dissolving a polymer and a salt or a pair of
polymers in water above a certain critical concentration. Major advantages of aqueous
two phase extraction (ATPE) include high extraction capacity, biocompatible
environment, low interfacial tension, high throughput, low energy requirements.
A phase diagram for a typical PEG/Salt ATPS is shown in the figure below. Both
polymer-salt are separately miscible with water in all proportions with each other. As the
concentration increases above a critical value, phase separation occurs with the formation
of Polymer (PEG) rich upper phase and salt (KH2PO4) rich lower phase, each containing
more than 70-80% water. The curve formed by joining these points of critical
concentrations, which separates the homogeneous region and the two-phase region, is
known as a binodal. If a mixture of total composition represented by a point A 2 (7.93%
PEG and 15.3% salt) is taken, it will separate into two phases. The composition of the top
and bottom phase will be T2 (28.15% PEG and 4.55%salt) and B2 (1.01% PEG and
19.41%salt), respectively. Pairs of points, like T2 and B2 are called nodes and the straight
line joining the nodes T2B2 is called the tie line. Point O represents the critical point
having a small vanishing tie line that just separates into two phases of equal volume and
composition.

Estimation of volume ratio

The volume ratio of the top and bottom phases can be obtained by the inverse lever arm
rule, as given by:
Vt volume of top phase A B ……….. (1)

Vr    1 1
VB volume of bottom phase A 1T1
Estimation of partition coefficient

The partition coefficient (K) is calculated as the ratio of the equilibrium concentration of
the protein or enzyme in top phase (CT ) to that in bottom phase (CB) .
CT
K ……….. (2)
CB
Estimation of yield
Yield (%) of protein or enzyme in top phase is calculated by using the following
equation:
KVr
Yield   100 ……….. (3)
1  KVr
where,
Vr = volume ratio
K = partition coefficient of protein or enzyme
Yield (%) of protein or enzyme in bottom phase is calculated by using the following
equation:
1
Yield   100 ……….. (4)
1  KVr

40
T1 Binodal Curve
35 Two Phase Region
30 T2
PEG low tie line high tie Line
4000,
%w/w 25
medium tie line
20
15
10 Homogeneous region
A2
O A1
5
B2 B1
0
0 5 10 15 20 25 30
Potassium Phosphate, % w/w
Fig. Phase diagram for PEG 4000/Potassium phosphate at pH=7 at 20C

Preparation of yeast extract

20 gm of Baker’s yeast is taken and placed into 100 ml acetate buffer (pH 4-6) and
homogenized with pestle and mortar. This suspension is then centrifuged at 4000 rpm for
10 min and the supernatant taken for further experiments.
Yeast extract (bacteriology grade) can also be used (which is cell debris free) in the same
composition [40 gm of yeast extract and 100 ml acetate buffer (pH 4-6)] for preparation
of stock solution of extract.
Procedure
1) Add the appropriate quantity of PEG 4000 and ammonium sulfate or potassium
phosphate and the remaining water/ crude extract according to the phase diagram at
medium tie line length so as to prepare 100% w/w composition (total quantity 50 ml).
2) In case of ammonium sulfate lower the pH down to 4.6 with HCl (for phase system
containing crude extract) in order to prepare a total of 50 ml phase system. In case of
potassium phosphate calculate the compositions of the salts (mono and di-pottasium
phosphate) taking the ratio of K2HPO4: KH2PO4 = 1.82:1
2) Mix the contents thoroughly using a magnetic stirrer for about 15 min (until all phase
components dissolve).
4) Leave for phase separation in a separating funnel or beaker (about 30 min) or if desired
for faster separation, centrifuge to obtain the clear separation of top and bottom phases in
a glass centrifuge.
4) Note the volumes of top and bottom phases (for phase system containing crude extract)
and calculate the volume ratio.
5) Estimate the protein content in both phases (Biuret method) and calculate partition
coefficient K.
6) Calculate the yield of the protein in both the phases.
Protein estimation
Initially 6 tubes of increasing standard Bovine Serum Albumin (BSA) of 0.2, 0.4, 0.6,
0.8, and 1 ml are taken and volume is made up to 1 ml using water. To this, 9 ml Biuret
solution is added and incubated for 20 min at 37°C. This is followed by taking the OD at
540 nm. We then plot the standard graph of OD Vs BSA concentration. 2 test tubes are
taken and 0.5 ml of supernatant (crude extract) and suitable volumes (approx. 0.2-0.5 ml
of top and bottom phases) are added. Volume is made up to 1 ml using water, 9 ml of
Biuret solution is added and incubated for 20 min at 37C and OD taken at 540 nm.
These values are compared with the concentration of standard graph and protein is
estimated.
Observation and Calculation
Sl % tie Concentration Concentration Volume Volume Volume Partition Yield
No. line of protein in of protein in of top of ratio coefficient of
length top phase bottom phase phase bottom (Vr) (K) protein
(TLL) phase (%)
1
2
3
Result
The partition coefficient (K) of the protein = ____________
The yield of the protein = ____________ %.

AMMONIUM SULFATE PRECIPITATION OF PROTEINS USING

YEAST CELL SUSPENSION
Objective
To carry out bulk precipitation of protein from yeast cell suspension using of ammonium
sulfate salt.
Apparatus
Centrifuge, Spectrophotometer, Beakers/Test tubes, Ammonium Sulfate, Yeast cells, ice
bath, magnetic stirrer.
Principle
The solubility of proteins is markedly affected by the ionic strength of the
medium. As the ionic strength is increased, protein solubility at first increases and this is
called “salting in”. However beyond a certain point the solubility begins to decrease and
this is known as “salting out”. At low ionic strengths the activity coefficients of the
ionizable groups of the proteins are decreased so that their effective concentration is
decreased. This is because the ionizable groups become surrounded by counter ions
which prevent interaction between ionizable groups. Thus protein-protein interactions are
decreased and the solubility is increased. At high ionic strengths much water becomes
bounded by the added ions that not enough remains to properly hydrate the proteins. As a
result protein- protein interactions exceed protein-water interaction and the solubility
decreases. Because of differences in structure and amino-acid sequence, proteins differ
in their salting in and salting out behavior. This forms the basis for the fractional
precipitation of proteins by salt.
Ammonium sulfate is a particularly useful salt for the precipitation of protein. It is
available in highly purified form, has great solubility allowing for significant changes in
the ionic strength and is inexpensive. Changes in ammonium sulfate concentration of
solution can be brought about either by adding solid ammonium sulfate or by adding a
solution of known saturation, generally a fully saturated solution (100%) solution.

Procedure
A. Preparation of Homogenate
1) Weigh 20 g of crude yeast cells and homogenize by using mortar and pestle
with precooled 100 ml phosphate buffer (pH 7.5). Separate the cell debris and
extract by centrifuging at 4000 rpm for 10 min. Re-centrifuge the supernatant
to get the clear supernatant .The supernatant (crude extract) recovered is used
for further experiments. Retain 1 ml sample from supernatant for protein
analysis.
B. Ammonium sulfate precipitation
1) Divide the supernant obtained into 4 equal volumes for precipitation at different
saturation percentages of ammonium sulphate.
2) Weigh 40%, 50%, 60% and 70 % saturation equivalent of ammonium sulfate for
the precipitation process( 238 g for 40%, 313g for 50%, 390g for 60%, 516g for
70% for one litre of the supernatant)
3) Calculate the weight of ammonium sulphate as per the volume of the supernatant
and weigh separately.
4) Take one volume of the supernatant add the ammonium sulphate ( 40 %
equivalent) salt under stirring cold conditions in an ice-bath.
5) During the addition of the ammonium sulphate salt,The protein gets precipitated
leave the solution for 15-20 mins in cold conditions.
6) Centrifuge the mixture and separate the precipitate from supernatant. Measure the
volume of supernatant. Take one ml of supernatant and check for protein
estimation.
7) Dissolve the pellet in 2ml of phosphate buffer pH 7.
8) Repeat steps 4,5 and 6 using 50%, 60%, 70% saturated solution.
9) Protein estimation to be done with Bradford reagent.
10) Prepare standard plots of BSA.
11) Estimate the α protein content in precipitate and supernatant of 40%, 50% 60%
and 70% ammonium sulfate.

C. Estimation of Protein by Bradford method or Lowry method.
Bradfor d method:
1) Take 6 tubes of increasing standard Bovine Serum Albumin (BSA) (stock sol-
1 mg/ml in distilled water) of 0, 0.2, 0.4, 0.6, 0.8, and 1 ml
2) To this, add distilled water upto 2 ml.
3) Add 1 ml Bradford reagent , wait for 5 mins take OD reading at 595.
4) Plot a standard graph of OD595 Vs BSA concentration.
5) Take 2.0 ml buffer add to the pellet after centrifugation obtained from 40% ,
50%. 60% and 70% ammonium sulfate precipitated sample.
6) add 1ml of Bradford reagent wait for 5 mins take OD readings at 595.
7) These values are extrapolated in the standard graph and protein conc was
calculated.
Observation
Protein Estimation (standard BSA = 1 mg/ml)
Standard Distilled Bradford Incubation O.D at 595
BSA water (ml) reagent time (min) nm
0 2.0 1 5
0.2 1.8 1 5
0.4 1.6 1 5
0.6 1.4 1 5
0.8 1.2 1 5
1.0 1.0 1 5
Lowry’s method:
1. Dissolve the pellet from each fraction in 1 ml buffer
2. Add 5 ml reagent C and incubate at RT for 10 min
3. Add 1.0 ml of FC Reagent and incubate in dark for 20 min
4. Check OD at 660nm against blank

5. For blank same as above except pellet.
Pellet of Bradford Incubation time O.D at

protein extract (min) 595nm
(ml)
40% Plus 2 ml buffer 1.0 5
50%
60%
70%
6. Extrapolate from protein content from standard graph
Standard curve for Lowry’s method
Use BSA 1 mg/ml prepared in water

Tube BSA(1mg/ D/W REAGENT INCUBATION FC- INCUB OD
no ml) -C AT RT REAGENT ATION
IN
DARK
1 0 1ml
Blank
2 0.2ml 0.8 660n
3 0.4 0.6 5ml 10 min 0.5 ml 20 min
m
4 0.6 0.4
5 0.8 0.2
6 1.0 0
Step Total protein Yield (%)

content in
precipitate

Crude - 100%
40 % saturated ammonium
sulfate
sulfate
sulfate
sulfate
Result
The proteins content was found highest in ________________ % saturated ammonium
sulfate solution with protein recovery of ________________%.
TANGENTIAL FLOW FILTRATION SYSTEM
AIM: To determine the clean water flux (CWF) and to calculate the membrane required
for the clarification of 1000 litres yeast cell broth in one hour.
APPARATUS: Tangential flow filtration system, yeast cell suspension or broth, double
distilled water, membrane 0.2 micron for microfiltration, measuring cylinders, stop
watch.
Theory: Membrane filtration is a separation technique widely used in the life science
laboratory. Depending on membrane porosity, it can be classified as a microfiltration or

ultrafiltration process. Microfiltration membranes, with pore sizes typically between 0.1
µm and 10 µm, are generally used for clarification and removal of microparticulates or
for cell harvesting. Ultrafiltration membranes, with much smaller pore sizes between
0.001 and 0.1 µm, are used for concentrating and desalting dissolved molecules (proteins,
peptides, nucleic acids, carbohydrates, and other biomolecules), exchanging buffers, and
gross fractionation. Ultrafiltration membranes are typically classified by molecular
weight cutoff (MWCO) rather than pore size.
Tangential Flow Filtration (TFF), also known as crossflow filtration, where the feed
stream passes parallel to the membrane face as one portion passes through the membrane
(permeate) while the remainder (retentate) is recirculated back to the feed reservoir.
Applications:
The primary applications for TFF are concentration, diafiltration (desalting and buffer
exchange), and fractionation of large from small biomolecules. In addition, it can be used
for clarification and removal of cells as well as cellular debris from fermentation or cell
culture broths.
 TFF is easy to set up and use – Simply connect the TFF device to a pump and
pressure gauge(s) with tubing and fittings, add your sample to the reservoir and
you’re ready to go. An example set-up is shown in Figure 1.
 TFF is fast and efficient – It is easier to set up and much faster than dialysis. You
can achieve higher concentrations in less time than with centrifugal devices or
stirred cells.
 Perform two steps with one system – You can concentrate and diafilter a sample
on the same system, saving time and avoiding product loss.
 TFF can be scaled-up or scaled-down – Materials of construction and cassette
path length allow conditions established during pilot-scale trials to be applied to
process-scale applications. TFF devices are available that can process sample
volumes as small as 10 mL or as large as thousands of liters.
 TFF is economical – TFF devices and cassettes can be cleaned and reused, or
disposed of after single use.

Cassette description:
Pore size / MWCO: Lot No.: Membrane area:
TFF System set up:

 Connect the feed line to the peristaltic pump
 Place the cassette in the holder by correctly aligning the permeate and retentate
lines of cassette and the holder correctly.
 Tighten the cassette with a torque wrench at 20 N-m.
 Open all the inlet, permeate valves, check any loose connections etc
 Now the system is ready for flushing the preservative already present in the
membrane.
Determination of clean water flux:
1. First flush the membrane with double distilled water about 1 litre. With inlet
pressure 1.0 bar or 2.0 bar check the permeate flow rate. Discard the water
collected at the feed and permeate side. Drain the system.
2. Use fresh double distilled water run the system at inlet pressure of 1.0 bar,
retentate pressure 0.0 bar and the permeate pressure 0.0 bar. Note down the
value of permeate water flow per minute
Formula:
NWP 25 = CWFt x Kt
A x TMP
NWP 25 = clean water flux at 25 degrees ( l/h x m2 x bar)

CWFt = clean water flow at X degree centigrade ( l/h)
Kt = temperature correction factor ( usually 1.0)
A = Membrane area (m2) ( 0.1 m2)

TMP = Trans membrane Pressure = ( P feed + P retentate) - P perm
2
TEMP = Temperature in degees centigrade.
CLARIFICATION OF YEAST CELL BROTH:
1. Prepare 2 litres of yeast cell suspension with 0.5 % w/v in double distilled water
and use for experiment.
2. Assemble the TFF System, connect the tube for the pump, tighten the cassette wit
adequate torque until a click sound comes.
3. Run the system as the sequence in the following table and note down the required
pressure and flow rates of the the permeate.
4. Calculate the TMP, flow rates and the Flux.
TABLE:
DESCRIPTION P P P TM Permeat Permeat Flux
feed rete permeat P e flow e flow Lmh
ntat e rate rate L/H
e ml/min
Flushing with 1 0 0
double distilled
water to remove
the preservative
Run with double 1 0 0
distilled water for
clean water flux
determination
Buffer rinse 1.5 0
Sample ( yeast cell 0.2 0
broth / suspension)

Buffer rinse 1.0 0 0

Water wash 1.0 0
Distilled water for 1.0 0 0
clean water flux
determination
Preservative 1.0 0 0
circulation ( 0.5%
sodium azide)
Calculation & Result:

Average flux = lmh
From the above, data the average flux = lmh
For processing 1000 litres of yeast cell broth in one hour, membrane area required =
sq mt
CENTRIFUGATION STUDIES DURING SETTLING OF YEAST
CELLS
Objective
To study the effect of:
i) Increasing speeds of centrifugation on the settling of the yeast cell
particles.
ii) Increasing centrifugal times on the settling of yeast cell particles.
Apparatus
Yeast, centrifuge, spectrophotometer, centrifuge tubes
Principle
Centrifugation is a basic separation technique used to separate material of different
densities when gravitational force is insufficient for their separation. Normally a
suspension of slowly under the influence of gravity. This process is called sedimentation.
In centrifugation, the process of settling is added by centrifugal forces.
The centrifugal force, F is given by
F = mω2r
Where m = mass of the particle on which it is acting, w = angular velocity, r = radius.

It has been observed that by fixing the type (density and size) of particles, to be
settled, under the given study and also the type of rotor, the settling of a particle subjected
to centrifugation depends on the speed of centrifugation as well as the time spent by the
particles at that speed. Hence, varying these two parameters (keeping either constant) we
can study centrifugation profiles of particles.
Procedure
1. Prepare 100 ml of stock yeast solution by dissolving 2 g of yeast in
10 ml of warm water (distilled) and make it up to 100 ml using normal water
(distilled) (Since yeast is not easily soluble in normal water use warm distilled water).
2. Pipette out 10 ml of the stock solution into 10 centrifuge tubes.
3. The first 5 tubes are centrifuged for 5 minutes each, with varying
centrifugal speeds of 1000, 1500, 2000, 2500, 3000 rpm. The O.D’s of supernatant
are recorded at 540 nm.
4. The next 5 tubes are centrifuged at 1500 rpm with varying times of
centrifugation of 1, 2, 3, 4, 5 min respectively. Here also, supernatants are collected
and optical densities are noted.
5. Graphs of OD540 vs rpm and OD540 vs Centrifugation time are
plotted.
6. Based on these graphs, the perfect speed for centrifugation and
duration for the same for efficient separation of all particles can be measured.
Tabular Column
Concentration Speed (rpm) Time(min) Optical Density
(540 nm)
1000 5
1500 5
2000 5
2 g / 100 ml
2500 5
3000 5
3500 5
Concentration Speed (rpm) Time(min) Optical Density

(540 nm)
1500 1
1500 2
2 g / 100 ml 1500 3
1500 4
1500 5
Result
The profiles of OD vs rpm and OD vs centrifugation time are plotted.
ESTIMATION OF ORGANIC ACID BY CALORIMETRIC METHOD

Objective
To estimate citric acid from fermentation broth by calorimetric method.
Apparatus
citric acid, H2SO4, metaphosphoric acid, KMNO4
Theory
Citric acid is an important chemical used in medicines, flavoring extract, food and
candies, manufacture of ink, dyeing and engraving. Several different species of moulds
have the ability to convert sugar to citric acid but Aspergillus niger is most widely used
for its commercial production.
Principle
The reaction involves oxidation of citric acid by potassium permanganate in the presence
of bromine. The pentabromoacetone produced is extracted from the oxidation mixture
with petroleum ether and treated with thiourea borax solution. A yellow color complex is
formed. This is proportional to intensity of pentabromoacetone (PBA) in solution. The
quantity of citric acid in the solution is derived from the calibration curve of the standard
citric acid solution (concentration versus Optical density at 440 nm).
Procedure
1) Take 0, 0.1, 0.2, 0.4, 0.6, 0.8 and 1 ml of standard citric acid (2 mg/ml) in test tube
label as B, 1, 2, 3, 4, 5 and 6. and take broth 1 ml ( unknown)

2) Add distilled water to make up to l ml.

3) Add 1 ml of 9N H2S04 followed by 0.3 ml of 40 % metaphosphoric acid and mix after
addition.
4) Incubate in ice bath for 10 min.
5) Add 0.5 ml of 2M KBr followed by 1.5 ml of 6.5% KMNO4.
6) Mix thoroughly and incubate in ice bath for 10 mm.
7) Add 6% H202 dropwise and shake until KMNO4 is decolorized.
8) Transfer into iodine flask and add 5 ml petroleum ether. Shake for 2 min to
extract. PBA into petroleum ether. The 2 layers separate after sometime.
9) 2 ml from upper layer of petroleum ether layer is pipetted into the test tube.
10) Add 6 ml of thiourea borax solution (pH 9.2) to each test tube and shake well
for 2 min.
11) A golden yellow color is developed because complex formed between thiourea
and pentabromoacetone( lower layer).
12) Separate lower layer carefully and use for OD
13) Take OD at 440 nm against Blank.
14) Plot the graph between standard citric acid concentration and the od values
15) Extrapolate the values of the unknown saples from the graph

Table:
Keep in ice water bath for 10 min
Mix and keep in ice water bath for 10 min

Sl. Standard Distill 9N 40 % 2M KBr 6.5% 6% Petrole Thio OD at
No. citric ed H2SO4 Metaphos (ml) KMN H2O2 um borax 440
acid (ml) water (ml) phoric O4 ether solution nm
(ml) acid (ml) (ml) (ml)
B 0 1 1 0.3 0.5 1.5 5 6 Use
1 0.2 0.8 1 0.3 0.5 1.5 5 6
lower
2 0.4 0.6 1 0.3 0.5 1.5 5 6
3 0.6 0.4 1 0.3 0.5 1.5 5 6 layer
4 0.8 0.2 1 0.3 0.5 1.5 5 6 for OD
5 1 0 1 0.3 0.5 1.5 5 6
6 1.0 1 0.3 0.5 1.5 5 6
ml(unkn
own)

7
unknown
1
0.3
0.5
Department of Biotechnology, R. V .College of Engineering, Bangalore

1.5
Add drop wise until color decolorizes

5
Shake the mixture for 2 min and leave for separation. Two layers are formed. Take 2 ml
from upper (petroleum ether) layer
6
35

Nature of graph
OD
Concentration of citric acid, μg/ml
Result
The concentration of citric acid in test sample 1 was ______________ μg /ml
The concentration of citric acid in test sample 2 was ______________ μg/ml
Pecautions for the experiment:
 Use fresh hydrogen peroxide solution

 Mix thoroughly after addition at each step
 Use boiling tubes instead of test tubes

EXTRACTION OF PIGMENTS FROM SPINACH AND

ESTIMATION BY THIN LAYER CHROMATOGRAPHY
Objective
To identify the unknown pigments by comparing its R f value with Rf value of the
standards.
Apparatus
Chromatography chamber with airtight lid, TLC plates, capillary or micropipettes
Principle
The general principle involved in TLC is similar to that of column chromatography i.e.
Adsorption chromatography. Adsorption chromatography is a phenomenon whereby a
substance gets attracted by electrostatic forces to the surface of unit particle. If the
substance is in the solution and the particles insoluble is the solvents, then part of the
substance is adsorbed and part remains in the solution. The ratio between the amount
adsorbed and part remains in the solution, called the adsorption coefficient. So
compounds with different partition coefficient can be separated. In the TLC adsorbent
normally used contains a binding agent such as calcium sulphate, which facilitates the
adherence of the adsorbent to the glass plate. Solvent is allowed to ascend along the
plates by capillary action. During the slow percolation, the solute molecules gets
adsorbed, released and again reabsorbed on the surface of the adsorbent and the molecule
with the least adsorptive goes along with the solvent and reaches first at opposite end.
TLC is useful for both qualitative and quantitative analysis as it yields results of greater
reproducibility when compared with Paper Chromatography.
Procedure
1. Cut a TLC plate into small strips so that they fit the tubes. Do not touch the surface of
the plates.

2. Prepare 150 ml of mobile phase containing the following composition:

 Petroleum ether- 90ml
 Hexane- 24ml
 Ethyl acetate- 15ml
 Acetone- 15ml
 Methanol- 6ml
3.Prepare mobile phase and put into the chamber and allow to equilibrate for 30 mins,
cover the tank with the lid.
4. Take fresh leaves of spinach 10 g in a morter and pestle
5. Add 5 gms of MgSO4 and 10 gm of glass beads
6. Triturate or grind thoroughly for 5-10 minutes until smooth paste is formwed
7. Transfer into Seperating funnel and add 20 ml of acetone shake thoroughly for
10 minutes and keep aside for seperation of layer after 10 minutes take the upper
layer and spot on the TLC plate.
8. Allow to dry for a minute, then carefully dip the plate in the mobile phase
containing chamber in up right position.
9. Allow the mobile phase to run till the mobile phase reaches 90% distance of the plate.
10. Remove the plate from the chamber and make a mark of the mobile phase front.
11. Observe for the movement of the spots and make a note of the distance of the
sample front
12. Measure the distance run by the solvent front and by each of the pigments.
All measurements should be made from the centre of the original spot to the front
of each pigment spot.
13. Calculate how far the pigment has gone relative to the solvent front. This is the
Rf value. (Rf = the distance run by the pigment divided by the distance run by
the solvent or the mobile phase.)
14. Draw a suitable results table. For each pigment record: the distance run, its colour,
Rf value and possible identity b matching with standard Rf values.

Observation and Calculation

Sl. No. Distance run by Distance run by Rf value identity
the solvent the sample
1
2
3
Result: The Rf value for the pigments are found as ____________ after comparing with
standards.
Contents

CYSTALLIZATION OF A PHARMA PRODUCT
Aim
To synthesize , crystallize, dry aspirin and to find the percentage yield.
Background
A component of willow tree bark called salicin has long been recognized as an analgesic
(pain reliever) and antipyretic. A powdered derivative of salicin, acetyl salicylic acid, is
sold as aspirin. Aspirin is the product of the reaction of the acetic anhydride with salicylic
acid. The reaction is acid catalyzed by H 3PO4. If are added the reactants in the molar
ration indicated by stoichiometry of the reaction and the reaction proceeds to completion,
only product will be present at the end. If we add on excess of any of the reactants, the
maximum amount of aspirin that we can produce will be determined by the molar amount
of the reactant not present in excess. This reactant is called limiting reactant.
Purpose Synthesize aspirin and determine feasibility of the synthesize methodology

using % yield. Purity of product is confirmed by measuring its melting point range.
Safety Considerations
This experiment uses salicylic acid, acetic anhydride and phosphoric acid. The salicylic
acid and aspirin may cause irritation to your skin and eyes but not hazardous. An excess
of these can be disposed of in the sink or if packed, in the trash. If you spill some, wipe it
up with a wet paper towel and throw and throw the towel in the trash. Acetic anhydride
and phosphoric acid can cause bad burns. Used in the hood, gloves and safety goggles
should be used.
HOOC – C6H4 – OH + CH3COOC – CH3  HOOC – C6H4 – OCOCH3

Salicylic acid acetic anhydride aspirin
+ CH3COOH
Acetic acid.

Procedure
1. Weigh about 2 g of salicylic acid and place in a 125 ml Erlenmeyer flask.
2. Measure 4 ml of acetic anhydride and add this to flask containing salicylic
acid. (hood and goggles to be used)
3. Carefully add 5 to 10 drops of 85% H3PO4. , a catalyst to the flask and
swirl to mix everything.
4. Still in the hood, heat the mixture for 10 min in a beaker of warm water
(70 - 80 oC )
5. After heating continuously add 20 ml of distilled water and place it in ice
bath.
6. Filter the solid aspirin using ordinary filter paper.
7. Wash the crystals with 2-3 ml of chilled water.
8. Place washed crystals in a pre weighed watch glass and measure the wet
weight of the aspirin crystals, followed by drying in oven (80°C).
9. For every 5 min measure the weight of the watch glass containing aspirin
crystals.
10. Plot a graph of % moisture content versus time
Nature of graph
% Moisture Content Vs. Time

% Moisture Content
Time

Observations
Sl. Time Wt. of wet solid (gm) Wt. of wet Wt. of moisture Moisture
No (min) + solid retained Content
Wt. of watch glass (gm) (gm) (gm) (%)
1 0
2 5
3 10
4 15
5 20
6 25
7 30
8 35
9 40
10 45
11 ∞
Tabular Column
Calculations
Weight of empty glass =…………… gm
Weight of wet solid = Wt. of wet solid + watch glass – Wt. of watch glass.
Weight of moisture retained = Wt. of wet solid – Wt. of dry solid.
Moisture content = Wt. of moisture retained

Wt. of wet solid at t = 0

Dry Wt . of Aspirin
Moles Aspirin Mol.Wt. of Aspirin
Yield (%)   =
Moles of Salicylic Acid Wt. of Salicylic Acid
Mol .Wt. of Salicylic Acid
Result
The experiment was conducted and the yield of aspirin was calculated to be ……. %.

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Downstream Processing Laboratory 10 BT72

Sl. No Name of the Experiment Page No.

2. Centrifugation Studies During settling of yeast cells

3. Yeast cells disruption by mechanical method

6. Determination of partition coefficient and yield of

8. Ammonium Sulfate Precipitation Of Proteins Using

9. Determination of drying time for a given sample in

10. Gas Chromatography analysis for a compound

Department of Biotechnology, R. V .College of Engineering, Bangalore 1

Introduction to downstream processing

Downstream processing and analytical bioseparation both refer to the separation or

Stages in downstream processing

A widely recognized heuristic for categorizing downstream processing operations divides

Department of Biotechnology, R. V .College of Engineering, Bangalore 2

Department of Biotechnology, R. V .College of Engineering, Bangalore 3

YEAST CELLS DISRUPTION BY MECHANICAL METHOD

Department of Biotechnology, R. V .College of Engineering, Bangalore 4

(c) Protein estimation

Department of Biotechnology, R. V .College of Engineering, Bangalore 5

Supernatant of Biuret Incubation time O.D at

Activity = Micromoles of product formed x Total volume

Department of Biotechnology, R. V .College of Engineering, Bangalore 6

DESIGN OF THICKENER FOR BATCH SEDIMENTATION USING

Department of Biotechnology, R. V .College of Engineering, Bangalore 7

Department of Biotechnology, R. V .College of Engineering, Bangalore 8

5. Plot interface height (Z) versus time θ.

(4) to determine the area of the thickener.

Observation and calculations

Department of Biotechnology, R. V .College of Engineering, Bangalore 9

Department of Biotechnology, RVCE, Bangalore 10

ESTIMATION OF CITRIC ACID IN UNKNOWN SAMPLE BY

Department of Biotechnology, RVCE, Bangalore 11

Department of Biotechnology, R. V .College of Engineering, Bangalore 12

Observations and Calculations

Keep in ice water bath for 10 min

Department of Biotechnology, RVCE, Bangalore 13

Department of Biotechnology, R. V .College of Engineering, Bangalore

Mix and keep in ice water bath for 10 min

Add drop wise until color decolorizes

Department of Biotechnology, R. V .College of Engineering, Bangalore 15

Concentration of citric acid, μg/ml

Department of Biotechnology, RVCE, Bangalore 16

DETERMINATON OF PARTITION COEFFICIENT & YIELD OF

Department of Biotechnology, RVCE, Bangalore 17

Estimation of volume ratio

Vt volume of top phase A B ……….. (1)

Estimation of partition coefficient

Department of Biotechnology, R. V .College of Engineering, Bangalore 18

Fig. Phase diagram for PEG 4000/Potassium phosphate at pH=7 at 20C

Department of Biotechnology, RVCE, Bangalore 19

Preparation of yeast extract

Department of Biotechnology, R. V .College of Engineering, Bangalore 21

AMMONIUM SULFATE PRECIPITATION OF PROTEINS USING

Department of Biotechnology, R. V .College of Engineering, Bangalore 22

Department of Biotechnology, R. V .College of Engineering, Bangalore 23

C. Estimation of Protein by Bradford method or Lowry method.

Department of Biotechnology, R. V .College of Engineering, Bangalore 24

5. For blank same as above except pellet.

Pellet of Bradford Incubation time O.D at

Standard curve for Lowry’s method

Use BSA 1 mg/ml prepared in water

Observations and Calculations

Step Total protein Yield (%)