SRIJAN LAHIRI

A91704117082
BTech BT
SEM: 7, SEC: ‘B’

EXPERIMENT TITLE PAGE NO.

NO.

Experiment 1 Solvent-solvent extraction for downstream processing. 2

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Experiment 2 Filtration unit for downstream processing of wine.

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Experiment 3 Solvent extraction of Caffeine

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Experiment 4 Ni-NTA column for protein separation using
chromatography
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 Experiment 1: Solvent-solvent extraction for downstream processing

PRINCIPLE:
Solvent-solvent extraction is the removal of a soluble constituent from one liquid by means of
transferring it to another. Solvent-solvent extraction is also known as liquid-liquid extraction
/separation.
METHOD:
Centrifugal force is used to contact and separate phases of liquid of different specific gravities in
liquid-liquid systems. Inside the liquid-liquid extraction equipment, the spinning rotor contains
concentric perforated sheet metal elements called elements. The elements provide coalescing surface
as liquid flows through the perforation ensuring intimate contact generating gravity forces upto 3,000.
The pod is capable of separating liquids with differentials as low as 0.001 and handling the
continuous flow equivalent of a gravity sieve column with thousands of times its volume. There is a
rotor and shaft assembly have radial tubes which can be removed for periodic cleaning. Each row of
tubes distributes one of the feed liquids or collects an effluent.
Liquids flow in and out through distribution tubes. Heavy liquid is introduced inboard and flows
outward. Light-liquid is introduced outboard and flows inward. The intimate contact that occurs as
these liquids make their journey counter-currently results in the transfer of mass and/or components
from one liquid to the other. This is the general method for all extractors used in liquid-liquid
extraction. A single united can provide 3-5 theoretical stages of extraction.
DISCUSSION:
Solvent-solvent extraction is used in industries of Food & Beverage - mainly for flavouring,
Pharmaceuticals, Specialty Chemicals, Mining, Hydrometallurgy and more.
The reasons behind using an extraction process such as liquid-liquid separation vary, but this process
is considered when: The material is heat sensitive or non-volatile Distillation isn't feasible due to the
materials having similar boiling points, poor relative volatilities, or the formation of azetropes.

BASIC MECHANISM

Red Light liquid Blue Heavy liquid

flows INWARDS. flows OUTWARDS.

Light Liquid
Two Phase Clarification Zone
contact Zone

High Liquid
Clarification Zone

Experiment 2: Filtration unit for downstream processing of wine.

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PRINCIPLE:
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 Basic principle in filtration is to separate the suspended particles or larger molecular mass components
from bulk liquid with porous medium. Driving force can be pressure difference, vacuum,
concentration gradient, valence or electrochemical affinity. Filtration in winemaking is used to
achieve two objectives, clarification and microbial stabilization. In clarification, large particles that
affect the visual appearance of the wine are removed. In microbial stabilization, organisms that affect
the stability of the wine are removed therefore reducing the likelihood of re-fermentation or spoilage.
METHOD:
1. The filtrate side of the first filter sheet is taken and put inside the filter.The other plate is placed
against it and the other side of the second filter sheet is put in the filter. This is done to ensure the
wine goes through the hood and the plate is over the filter sheet.
2. On completion of arranging the filter sheets in the compact plate, the last filter sheet is inserted
and the end plate is placed allowing the filter to shut down.
3. Before closing the filter, it must be ensured that the rough side of the filter sheets are placed
together and filtrate side towards the same. By using a spindle to rotate, the filter is closed.
4. After closure of the compact plate with slight pressure, rinsing of the filter sheet is done from
outside by allowing water to flow, after 5 minutes the cellulose filter sheets start to swell up.
5.The valves on the upper part is opened slightly to get the air bubbles out during rinsing process and
the bottom valve is half closed to fill the water in the compact plate through the inlet.
6. The rinsing process starts by allowing the water to flow, the water takes some time to get in and
we could see the wine water coming out of the outlet as a yellow colour signifying it to be natural; the
flow rate measurement is done.The valves are opened slowly after rinsing to let the water flow out of
the compact plate.
7. The compact plate is tightened by a spindle to have the final press in it; after that it is steamed,
cooled down and finally filtered.
DISCUSSION: One of the

reasons to filter a wine is to improve its appearance.

Allowing the wine to settle in barrel or tank is a great way
to clarify it, but sometimes the spent yeast and other
particles simply won’t settle to the bottom in the alloted
time. Filtration can remove those particles and leave the
wine limpid and bright. Another reason to filter is for
microbial stability. Filtration is used
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for microbial stabilization of wine by removing, or

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 controlling the proliferation of foreign and cultured
organisms.

Mechanism of wine filtration using Plate and Frame Filters

Experiment 3: Solvent extraction of Caffeine

PRINCIPLE:
Solvent extraction, also called liquid-liquid extraction (LLE) and partitioning, is a method to separate
compounds based on their relative solubilities in two different immiscible liquids. Immiscible liquids
are ones that cannot get mixed up together and separate into layers when shaken together. These
liquids are usually water and an organic solvent. LLE is an extraction of a substance from one liquid
into another liquid phase. The most common use of the distribution principle is in the extraction of
substances by solvents, which are often employed in a laboratory. Most common use is in the
extraction of caffeine.
PROCEDURE:
a. Weigh the total amount of coffee and add in a beaker.
b. Prepare 25% Hydrochloric acid solution by mixing 25ml HCl in 1L Water.
c. Add the acid solution to the beaker, and stir it thoroughly using HOT-STIRRER (heated till
75℃)
d. Then filter the mixture using a funnel and a cotton plug.
e. Collect the filtrate (liquid filtered) in another 5L beaker by Gravity filtration.
f. Then add 60gm Sodium Carbonate in the beaker containing the filtered liquid (this step
neutralises excess HCl and maintains a basic pH). And mix it thoroughly.
g. Transfer the solution to a funnel attached to the stand.
h. Add 10ml DCM to the solution in the funnel and shake it to mix thoroughly.
i. After 15 minutes, drain the DCM layer into an Erlenmeyer Flask.
j. Thereafter, to recover the DCM, pour the DCM stored in the flask in a fresh funnel.
k. Add saturated Sodium Chloride solution in the funnel containing DCM to wash it and mix
thoroughly, and filtered to a different funnel.
l. Then drain the filtered solution to a flask to e distilled.
m. Place the flask containing the solution to be distilled in a water bath.
n. The dried-out extract (due to distillation and evaporation of DCM) was transferred to a small
beaker by the help of a spatula and add DCM to it.
o. Place the beaker in a Water-bath, and allow the DCM to evaporate.
p. Weigh the extract after the evaporation of DCM on a weighing machine.
q. Add 15 ml of Methanol to the beaker containing the Extract to make a saturated solution, and
mix thoroughly.
r. Place the beaker on a hot plate at low heat.
s. The place it in an ice-box, forcing Crystallisation.
t. Stir the crystals and pour it in a small vacuum filtration apparatus using pipette rear.
u. Collect the clean crystals in a beaker and weigh.
DISCUSSION:
Caffeine is an Alkaloid, having three amines and imine group, giving it a basic character. Though it is
soluble in water, HCl is added which makes it even more soluble, and helps in the conversion of
caffeine into its salt, Caffeine Hydrochloride. This is because, one of the nitrogen in the molecule
(which is basic), traps the proton of the acid forming caffeine cation, while the chlorine holds the
negative charge (Anion), forming an ionic compound, which is more soluble in water, and thereby
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“moves out of” the coffee and into the solvent. The solvent, now containing dissolved caffeine, is then
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 separated from the coffee. If the solvent is allowed to evaporate or is removed in some manner, the
caffeine will be left behind as a white powder.

Experiment 4: Ni-NTA column for protein separation using chromatography

PRINCIPLE:
Adding a N- or C-terminal His6-tag to be expressed using the RTS E. coli Linear Template
Generation Set, His6-tag. These His6-tagged proteins can be purified in one step by immobilized
metal affinity chromatography (IMAC) on a nickel-nitrilotriacetic acid (Ni-NTA) column. In a single
step, this affinity matrix can purify a protein (starting concentration less than 1% of the total protein)
to more than 95% homogeneity.
The green fluorescent protein (GFP) is a protein composed of 238 amino acid residues (26.9 kDa) that
exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range.
Nitrilotriacetic acid (NTA) is a tetradentate chelating adsorbent. NTA occupies four of six ligand
binding sites of the nickel ion, leaving two sites free for interaction with the His6-tag. NTA binds
metal ions tightly, allowing use of stringent washes. Histidine residues on the tag, connected via a
short linker to the C- or N-terminus of the protein, bind to the Ni-ions. The protein can be eluted by
competitive displacement with imidazole.

METHOD:
Firstly, grow single bacterial colonies in a plate in order to express protein. First step is that we must
amplify these bacteria, in order to amplify the bacteria, we will pick a single colony from the plate
and inoculate a small 5 ml primary culture. Fill the column with Ni-NTA resin to create a bed volume
of 0.6 ml. Now, Close the column and mount the lure lock syringe (without plunger) as a buffer
reservoir. Equilibrate the column with 10 to 15 bed volumes (6 – 9 ml) of equilibration buffer. Apply
the sample to the column by gravity flow. Keep a small portion of the sample for assays. (Often, we
can apply the contents of the RTS reaction chamber directly to the column. However, if we see
precipitate in the sample that might clog the column, centrifuge the sample at 10 000 x g for 1 min to
remove the precipitate before applying the sample to the column). Immediately after the sample has
entered the resin, wash the column with 10 bed volumes (6 ml) washing buffer. Beginning with the
first washes, collect 1 ml fractions of effluent from the column throughout the entire purification.
Monitor the progress of the purification by analysing each fraction by SDS-PAGE, Western blotting
and/or activity assay. Use the unpurified sample as a reference in these assays. Elute non-specifically
bound proteins with 10 bed volumes (6 ml) elution buffer 1. (The imidazole concentration in elution
buffer 1 must be optimized for each protein). Elute specifically bound protein with 10 bed volumes (6
ml) elution buffer 2. After all, specifically bound protein has been eluted from the column, wash the
column with 10 bed volumes (6 ml) elution buffer 3.

DISCUSSION:
The gene has a 6X His-Tag to facilitate its purification in sufficient amount. Histidine-tagged protein
purification uses affinity chromatography to capture recombinant proteins with 4–10 histidine
residues. Histidine-tagged proteins are commonly purified using Immobilized Metal Affinity
Chromatography (IMAC). IMAC is based on the interaction between amino acid residues and
divalent metal ions immobilized on resins. Histidine interacts most strongly, and histidine-tagged
proteins have extra high affinity because of the multiple histidine residues. The chemical imidazole is
used to elute histidine-tagged proteins. In general, by increasing the concentration of imidazole in
your binding and wash buffer we decrease unspecific binding. Conversely, by decreasing the
concentration we get a higher capacity due to a stronger affinity interaction. Some target proteins
require more purification steps to reach desired purity levels. His-tagged GFP is an easily inducible
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 protein and the protein was produced in large quantities when induced making the secondary culture
green.
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