Detergents used in biomedical laboratories are mild surfactants (=surface acting agents), used for the disruption of cell

membranes (cell lysis) and the release of intracellular materials in a soluble form. Detergents break the protein-protein,
protein-lipid and lipid-lipid associations, denature proteins and other macromolecules, prevent unspecific binding in
immunochemical assays and protein crystallization.
There are many types of detergents. New detergents, usually for specific applications, are continually being developed
[1]. This article reviews the characteristics and applications of most common detergents.
Type Chemicals
ionic sodium dodecyl sulfate (SDS), deoxycholate, cholate, sarkosyl
non-ionic triton X-100, DDM, digitonin, tween 20, tween 80
zwitterionic CHAPS
chaotropic urea
Table 1. Classification of detergents.

Detergents are organic compounds comprised of a hydrophobic hydrocarbon moiety and a hydrophilic charged
headgroup (Fig. 1A). When dissolved in water at a given concentration and temperature, detergent molecules will form
micelles, with the hydrophobic part in the interior of the micelle and the headgroup at the exterior (Fig. 1B). The
hydrophobic core of the micelle binds to the hydrophobic regions of proteins. The number of detergent molecules in a
micelle is the aggregation number, an important parameter used to assess membrane protein solubility [2]. The length
of the hydrophobic region is directly proportional to the degree of hydrophobicity and it is quite constant among
detergents, while the charged headgroup is variable. Based on its characteristics, the common detergents are
categorized into three types: ionic (anionic or cationic), zwitterionic and non-ionic. The minimal detergent concentration
at which micelles are observed at a specific temperature is called Critical Micelle Concentration (CMC). At any
concentrations lower than the CMC, only monomers are observed; at concentrations higher than CMC both micelles
and monomers co-exist, along with other non-micellar phases that are not dissolved in water. Likewise, the lowest
temperature at which micelles are formed is called Critical Micelle Temperature (CMT). Both temperature and
concentration are important parameters of phase separation and solubility of a detergent. CMC is also affected by the
degree of lipophilicity of the headgroup. Generally, a low lipophilic or lipophobic character results in high CMC.
[enlarge]
Figure 1. General structure of detergent monomers. Adapted from Anatrace.

Ionic detergents
Ionic detergents are comprised of a hydrophobic chain and a charged headgroup which can be either anion or cation.
They generally have higher CMC value than nonionic detergents. These detergents are harsh.
Due to their charged headgroups, ionic detergents cannot be removed by ion exchange chromatography.
Sodium dodecyl sulfate (SDS)
The anionic SDS is a very effective surfactant in solubilizing almost all proteins. It disrupts non-covalent bonds within
and between proteins; thus it denatures them, resulting in the loss of their native conformation and function. SDS binds
to a protein with a ratio 1.4:1 w/w (or one SDS anion per two amino acids) and therefore will mask the charge of the
protein. SDS adds a negative charge to all proteins in the sample regardless of their isoelectric point (pI). That is the
main reason for its wide use in SDS polyacrylamide gel electrophoresis (SDS-PAGE). Usually, for a complete cell lysis
in the presence of SDS, a sample must be sonicated or passed through a needle (19G) several times to ensure DNA
degradation. SDS should never be used when active proteins are required or when protein-protein interactions are
studied. Additional precautions should be taken when ionic detergents are used because some of their properties may
be altered in buffers with variable ionic strength (e.g. CMC falls down to 0.5 from 8 mM when the NaCl concentration
increases from 0 to 500 mM). Furthermore, SDS precipitates at low temperatures, and this effect is enhanced in the
presence of potassium salts. This phenomenon can be exploited to remove SDS from a protein sample [3].
Deoxycholate and cholate
Deoxycholate and cholate fall in this category even though they do not contain a charged headgroup. Their polar groups
are distributed in different parts of the chains. They are used to solubilize membranes.
Sarkosyl
Sarkosyl, also known as sarcosyl or sodium lauroyl sarcosinate, is an anionic surfactant. It is amphiphilic due to the
hydrophobic 14-carbon chain (lauroyl) and the hydrophilic carboxylate. The nitrogen atom in the amide linkage is not pH
active and remains neutral regardless of pH. The carboxylate with a pKa value of 3.6 is negatively charged in any
physiological solution. In addition, sarkosyl has a very high surface activity throughout a wide pH range, and its surface
tension is about 3.4 102 to 2.9 102N/m.
Sarkosyl is prepared from lauroyl chloride and sarcosine in the presence of sodium hydroxide, and is purified by
recrystallization from alcohol, or by acidification with mineral acid, separation of the free acid, and neutralization of the
free acid. Sarkosyl reported in literature is usually from Sigma. The most widely used commercial form of acyl sarcosine
is a 30% aqueous solution.
Sarkosyl is widely used in cosmetic formulations as shampoos, body washes, facial washes, and surfactant-cleansing
agents. Sarkosyl was used at concentrations of 2.78-12.9% in soaps, based on gas chromatographic analysis of the
products [4]. Sarkosyl is used in the metal finishing end processing industries for their crystal modifying, anti-rust, and
anti-corrosion properties. Sarkosyl has been also used to improve wetting and penetration of topical pharmaceutical
products. In food industry, sarkosyl is approved for use in processing, packaging, and transporting food for human
consumption, and in adhesives used in food storage or transportation.
Sarkosyl is also widely utilized in laboratory experiments due to its good water solubility, high foam stability, and strong
sorption capacity to proteins. It can inhibit the initiation of DNA transcription. Sarkosyl serves as a detergent to
permeabilize cells and extract proteins in isolation and purification techniques such as western blot and indirect ELISA.
One major application of sarkosyl is for solubilizing and refolding proteins from inclusion bodies. Eukaryotic recombinant
proteins overexpressed in Escherichia coli tend to form inclusion bodies. Sarkosyl can extract natively folded proteins
from inclusion bodies. Usually an inclusion body pellet is solubilized in 0.3% sarkosyl. Incubation of inclusion bodies
with 10% sarkosyl effectively solubilized > 95% of proteins, while high-yield recovery of sarkosyl-solubilized fusion
proteins was obtained with a specific ratio of Triton X-100 and CHAPS [5]. Earlier work involved solubilizing inclusion
bodies with denaturants such as urea or guanidinium hydrochloride and refolding by slow dilution. However, most of the
solubilized proteins aggregate and precipitate upon removal of the strong detergents. Sarkosyl has been found to be an
effective solubilizing agent that allows refolding at higher protein concentrations (as much as 10-fold higher when
compared to using guanidinium hydrochloride [6] ). Moreover, sarkosyl allows refolding of solubilized protein with less
aggregation than urea or guanidinium hydrochloride. Proteins in the soluble extract with sarkosyl can be stored at 4C
for a week before affinity purification. However, sarkosyl interferes with the subsequent chromatographic process and
must be removed from the solution by dilution, or dialysis.
Non-ionic detergent
Nonionic detergents have uncharged and hydrophilic headgroups. They are considered mild surfactants as they break
protein-lipid, lipid-lipid associations, but not protein-protein interactions, and most of them do not denature proteins.
Therefore, proteins are solubilized and isolated in their native and active form, retaining their protein interactors. They
are preferred for the isolation of membrane proteins.
[enlarge]
Figure 2. Structure and formulas of some common detergents. Adapted from G-Biosciences.

The Triton family

Triton X-100 is a typical nonionic surfactant and the surfactant of choice for most immunoprecipitation experiments. All
members of this family (Triton X100, Triton X114, Nonidet P40 [NP-40], Igepal CA-630) are quite similar and differ only
in their average number (n) of monomers per micelle (9.6, 8.0, 9.0, and 9.5, respectively) and in the size distribution of
the PEG-based headgroup [7]. Triton X100 derives from polyoxyethylene and contains an alkylphenyl hydrophobic
group. The CMC value is low and therefore it can not be easily removed by dialysis. The cloud point is 64 oC and at this
temperature phase separation is observed.
[enlarge]
Figure 3. Stucture and formulas of Tween 20 and Tween 80.

n-dodecyl--D-maltoside and other maltosides

The n-dodecyl--D-maltoside (DDM) is a glycosidic surfactant, increasingly used in hydrophobic and membrane protein
isolation, when the protein activity needs to be preserved. It is proved to be more efficient than others, such as CHAPS,
or NP-40 [8]. The glycochain in its lipophilic site, its high CMC of 0.17 mM and the interface of the micelles create an
aqueous-like microenvironment ideal for solubilizing and retaining the stability of membrane and hydrophobic proteins.
Other maltosides have different lengths of the hydrophoic alkyl chains such as beta-decyl-maltoside. Glucoside (octyl-
glucoside) can also be used in the protein experiments [9-11].
Digitonin
Digitonin, from Purple Foxglove (Digitalis purpurea), is used for the solubilization of eukaryotic plasma membranes.
Sarkosyl and Triton X-100 solubilize bacterial inner, but not outer membranes.
The Tween family
Tween-20 and Tween-80 are polysorbate surfactants with a fatty acid ester moiety and a long polyoxyethylene chain.
They have very low CMC and are generally gentle surfactants, they do not affect protein activity and they are effective in
solubilization. They are not common ingredients of cell lysis buffers; they are routinely used as washing agents in
immunoblotting and ELISA in order to minimize unspecific binding of antibodies and to remove unbound moieties.
One common question regarding the Tween family detergents is the difference between Tween 20 and Tween 80, two
most commonly used members. Tween 20 has lauric acid, while Tween 80 has oleic acid, see figure 3. Table 2
summarizes various aspects between them. The difference is sometimes important, especially in an in vivo study, since
Tween 20 and Tween 80 have different levels of hemolytic effect [12].
Synonyms Chemical Molecular Density Appearance Applications
Formula Weight (g/mL)
Tween polysorbate 20, C58H114O26 1228 1.1 Clear, a broad range of
20 polyoxyethylene yellow to applications: as
sorbitan yellow-green a blocking agent
monolaurate, viscous in PBS or TBS
PEG (20) liquid wash buffers for
 ELISA, Western
blotting and
other
immunoassay
methods; for
sorbitan lysing
monolaurate mammalian
cells; and as a
solubilizing
agent for
membrane
proteins.
as a stabilizing
polysorbate 80,
agent for
polyoxyethylene
amber proteins; used in
sorbitan
Tween 1.06- colored tests for the
monooleate, C64H124O26 1310
80 1.09 viscous identification of
PEG (80)
liquid phenotype of
sorbitan
some
monooleate
mycobacteria.
Table 2. Characteristics of Tween 20 and Tween 80

Most non-ionic detergents interfere with ultra-violet (UV) spectrophotometry, especially Triton X100, as they contain a
phenyl ring and they absorb UV light. Therefore, protein determination at 280 nm would be imprecise.
Zwitterionic detergents
The headgroups of zwitterionic (or amphoteric) detergents are hydrophilic and contain both positive and negative
charges in equal numbers, resulting in zero net charge. They are more harsh surfactants than the nonionic ones. A
typical zwitterionic detergent is 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, better known as CHAPS.
Its high CMC (6 mM at room temperature) allows efficient removal by dialysis. It is very common in sample preparation
at concentrations of 2-4% for isoelectric focusing and 2D electrophoresis. CHAPSO differs with CHAPS in that it
contains a more polar headgroup, which makes it more soluble. Thus, CHAPSO is mainly used for solubilization of
integral membrane proteins.
Chaotropes
Chaotropic agents are similar substances to surfactants in that they break non-covalent interactions (hydrogen bonds,
dipole-dipole interactions, hydrophobic interactions) facilitating protein denaturation, which in this case is usually
reversible. Urea alone, or in combination with thiourea or with other detergents, is the most widely used chaotrope with
applications in 2D-gel electrophoresis and in-solution enzymatic digestion of proteins for preparation during proteomic
workflows. When using Urea, extra care must be taken to not heat the sample above 37C as this will lead to
carbamylation of proteins [13].
Detergents for the isolation of membrane proteins
For membrane protein solubility, a detergent with high CMC should be chosen and the volume of the buffer would also
be crucial as enough detergent should be present to solubilize all membrane proteins in the sample. According to Linke
[2], at least one micelle is needed per membrane protein molecule in order to sufficiently mimic the lipid environment of
a membrane (Fig. 1C-D). In principle, by changing the temperature and the concentration of salt in the buffer, an
effective solubilization of membrane proteins can be achieved taking advantage of the phase separation. In this case,
the membrane proteins surrounded by the micelles precipitate with the detergent and the soluble proteins remain in the
supernatant. The temperature at which the detergent solution separates into two phases is called the cloud point. In
addition to the temperature, the cloud point is affected by additives in the buffer such as glycerol or salts (e.g. Triton
X114 has a cloud point of 23C but in the presence of 20% glycerol, the cloud point declines to 4C). This is very
important since the stability of a protein is affected by high temperatures.
Choice of detergents
A good detergent should be able to lyse cells, solubilize proteins and be suitable for downstream application. In addition,
the solublized protein in native or denatured form should be considered. There is no ideal detergent for all applications
and even in the same application the result varies (Table 3). Therefore, trial and error is the best strategy and the use of
a mixture of detergents should also be tested. In addition, fresh preparation of detergent working solution is usually the
best practice to avoid hydrolysis and oxidation.
Deterge MW MW CM Aggregati Clou Avg. Streng Dialyza Application
nt (Da) (Da) C on No. d Micell th ble s
monom micel (m Poin ar
er le M) t Weigh
 25oC (oC) t
Cell lysis,
Electrophore
18,00
SDS 289 7-10 62 >100 18,000 Harsh Yes sis, WB,
0
hybridizatio
n
Enzyme
immunoassa
Triton 90,00 0.2- ys, IP,
625 100-155 65 80,000 Mild No
X-100 0 0.9 Membrane
solubilizatio
n
CHAPS 615 6,150 6 10 >100 6,150 Mild Yes IEF, IP
90,00 0.05 45-
NP-40 680 Mild No IEF
0 9 50
n-
dodecyl Protein
--D- 511 0.15 98 50,000 Crystalizatio
maltosid n
e
WB, ELISA,
Tween- Enzyme
1228 0.06 76 mild No
20 immunoassa
ys
Membrane
Digitoni 70,00
1229 <0.5 60 70,000 Mild No solubilizatio
n 0
n
Table 3. Properties and main applications of common detergents. Information from University of Florida, BCH6206. WB, western
blotting; IP, immunoprecipitation; IEF, isoelectric focusing;

Removal of Detergents
The downstream application controls not only the type of detergent but also its concentration, which usually should be
lowered or completely removed. For such purposes, size exclusion chromatography or dialysis can be used but this
requires that the micelle size is different compared to the protein of interest or small enough micelles (i.e high CMC) to
pass the dialysis tubing [2]. Other methods employ the use of non-polar beads or resins that bind detergents,
cyclodextrin inclusion compounds [14], ion-exchange chromatography or protein precipitation. However, the end buffer
after detergent removal should be also chosen with extreme care to avoid precipitation and protein aggregation.
Detergents in Labome Survey
Labome surveys literature for the application of detergents. The following table lists the main suppliers, and number of
the articles, indicating most of the detergents are supplied by Sigma Aldrich as of August 1, 2014.
detergent num suppliers
Triton X-100 34 Thermo Fisher, Amresco, JT Baker, Sigma
Tween-20 19 Thermo Fisher, Amersham Pharmacia, Bio-Rad
SDS 13 Amresco, Bio-Rad, Q.BIOgene, Sigma
NP-40 6 Roche, Sigma
CHAPS 6 EMD Millipore/Merck/Calbiochem, Sigma, JT Baker
digitonin 8 EMD Millipore/Merck, Sigma, Wako
Table 4. Detergents in Labome literature survey. num: number of publications.
Triton X-100
Thermo Fisher Pierce Triton X-100 was used to lyse cell and tissue samples for western blots [15, 16] and
immunocytochemistry [17]. Amresco 30% Triton X-100 was used to homogenize the spinal cords of rats [ 18]. JT Baker
Triton X-100 was used to fix and permeabilize cells for visualizing the actin cytoskeleton [ 19]. Packard Triton X-100 was
used to dissolve primary antibodies for immunocytochemistry [20]. Sigma Triton X-100 was used to permeabilize cells in
immunocytochemistry [21-29], or in blocking buffer for immunohistochemistry [30-33], to solublize samples in western
blots [34, 35], to lyze cells and tissue samples [36-42], in PCR experiments [43], liposome fusion experiments [10],
proteinase K protection assay [44], and in whole mount staining [45].
Tween-20 and Tween-80
Tween-20 is commonly used in washing buffers, such as TBS-T, PBS-T, in various immuno assays.
Fisher 0.05% Tween was used in ELISA [46]. Amersham Pharmacia Tween 20 was used in western blot [47]. Bio-Rad
Tween 20 was used in PBS for immunoblot analysis [36] and for washing tissue sections in immunohistochemistry [48].
Sigma Tween-20 was used in washing blots [23, 35], [49], [50], [51-54], in ELISA [55,56], in immunoprecipitation [57],
in in situ hybridization [58], and in microfluidic array multiplex PCR [59] and others [60, 61].
Sigma Tween-80 was used to dissolve erlotinib [62] and as a supplement to grow M. tuberculosis strains [63].
SDS
Amresco SDS was used in SDS-PAGE [64]. Bio-Rad sodium dodecyl sulfate was used to prepare
radioimmunoprecipitation assay buffer [65]. Q.BIOgene SDS was used to prepare buffer for homogenizing pellets in
western blots [66]. SIGMA SDS was used to prepare buffers for, among others, in vitro octanoylation assays, Laemmli
sample buffer, 2D-DIGE experiments [35, 37, 38, 60, 67-72]. Sigma 10% SDS was used to homogenize the spinal cords
of rats [18].
NP-40
Roche NP-40 was used in cell lysis [73, 74], Sigma NP-40 was used to prepare radioimmunoprecipitation assay buffer
[65], cell lysis buffer [75], homogenization buffer [76] and immunoprecipitation assay RIPA buffer [29].
CHAPS
Calbiochem CHAPS (2% w/v) was used in order to identify circulating protein markers of ovarian cancer [46]. Sigma
CHAPS was used in buffers for purification of recombinant mouse proghrelin [68], immunoprecipitation assays [29],
tissue lysis [77], and protein crystallization [78]. JT Baker CHAPS was used to lyse cells to study viral interaction with
human ASF1 protein [79].
Digitonin
MERCK digitonin was used in immunocytochemistry experiment to study the role of PI4P in plasma membrane identity
[80]. Sigma digitonin was used to permeabilize cells to study the epithelial and mesenchymal cell responses towards
Escherichia coli Shiga toxin 1 [81], to study autophagy [82], prepare extraction buffer for the study of TNFalpha effect on
macrophages [83], perform proteinase K protection assay [44], and to extract RNA [84]. Wako digitonin was used to lyze
cells [85] and perform immunoprecipitaion experiments [86].
Maltosides and glucosides
Anatrace n-decyl-beta-D-maltopyranoside was used for protein purification [87] and at 5 mM [88] ; so were its n-
dodecyl-beta-D-maltoside [89-93] and n-undecyl-beta-D-maltoside [92, 94]. Glycon beta-dodecyl-maltoside and beta-
decyl-maltoside were used in protein purification [11]. Sigma n-dodecyl-beta-maltoside was used in 1% to perform
trypsin cleavage inhibition assays [95] and decyl maltoside was used to perform protein isolation [96].
Octyl-glycoside from Glycon was used in protein purification [10] and protein labeling [11], and Anatrace n-octyl-beta-
glucoside was used in pulse-chase analysis [9].
Others
For protein purifications, Affymetrix octyl glucose neopentyl glycol (OGNPG) at 1% [97], and Sigma cholesteryl
hemisuccinate at 0.1% (w/v) [98] were used.
 Characteristics of Colloidal Dispersions
Colloids, emulsions, and suspensions are polyphasic or heterogenious systems in
which the dispersed phase is found in discrete particles many times the size of most
molecules and ions. The degree of subdivision and the forces associated with large
surface area distinguish the colloid from the other dispersions. The characteristics
of a colloid are a result of its enormous surface per unit weight. Colloids may be
classified as lyophobic or lyophilic.

Lyophobic colloids have no affinity for the dispersing medium and are not
solvated. They are stabilized by charge acquired by preferential adsorption of
ions. If water is the dispersing medium, they are known as hydrophobic colloids.

Lyophilic colloids are highly solvated as well as charged. The charge usually is a
result of ionization. If water is the dispersing phase, they are called hydrophilic
colloids. Hydrophobic colloids will aggregate and precipitate if their electrical
charge is removed; however hydrophilic colloids often remain dispersed and do not
precipitate even after the charge is removed or neutralized. Fortunately most
pharmaceutical colloids are of the hydrophilic type and are stabilized by a dual
mechanism.

The large surface of a lyophobic colloid preferentially adsorbs ions. Trace ions
will induce an opposit charge in a neighboring molecule and will then be held to
the surface of the colloid by an ion-induced dipole attraction. It is the adsorption
of trace ions which gives a lyophobic colloid its charge; the charged particles repel
each other and prevent aggregation and precipitation. The adsorbed ions on the
surface of the particle tend to attract oppositely charged ions, and two layers of
oppositely charged electricity results. The thickness of this double layer is small
compared to the diameter of the colloidal particle, and the repuslive force of the
charged colloid does not extend beyond the thickness of the double layer.

This double layer consists of two shells of ions of opposite charge. The inner shell
is narrow and compact, adhering tightly to the colloidal particle. The outer shell is
wide and diffused, with a high concentration of ions near the inner shell and a
progressively lower concentration of ions as the distance from the surface of the
particle to the bulk of the dispersing medium is increased. The outer shell can be
removed and reformed as the particle moves in the dispersion.

Between the surface of the colloid and the main body of the dispersion there exists
a potential. The total potential, E, may be divided into two parts. The first is the
potential between the inner shell and the surface of the colloid . The second is
called the "zeta" or electrokinetic potential and is the potential difference through
the outer shell extending from the outer edge of the inner shell to the body of the
solution. The greater the zeta potential of a lyophobic colloid the greater its
stability.

The factors governing the selective adsorption of ions are not completely known.
For example, if dilute solutions of silver nitrate and sodium iodide are mixed, the
colloidal silver iodide may be positive or negative. It depends on the ions that are
in excess. If iodine is in excess, the particle is negatively charged and if the silver
is in excess the particle will be positively charged.

Hydrophilic colloids acquire a charge by ionization. Most of the gums used in

pharmacy to make colloids like acacia are negatively charged due to the
carboxyllic or sulfate groups on the carbohydrate polymer. Proteins(gelatins)
which are made of amino acids which are zwitter ions can acquire either a positive
or negative charge based on the pH of the solution. Pretreatment with acid or base
can change the isoelectric point of the gelatin. This results in acid treated gelatin
being positively charged at pH 4 - 4.5 while basic treated gelatin is negatively
charged at pH 8.

Hydrophobic colloids can be precipitated by small concentration of electrolytes or

oppositely charged colloids.

When surfactants at very low concentrations are dispersed in water, they tend to
become adsorbed as a closely packed monolayer at the air-water interface. If
additional surfactant is added, it cannot be accommodated at the surface and they
agglomerate in the bulk of the solution forming aggregates called micelles.
Micelles of a given surfactant at a given concentration and temperature contain the
same number of molecules ( 25 to 100). The diameter of the micelles range
between 30 and 80 angstroms. This is called an association colloid. In a
hydrophobic sol the polar portions of the surfactant face inward and the non-polar
face out. In a hydrophilic sol the opposit is true. The lowerset concentration at a
given temperature and pressure at which micelles form is call the critical micell
concentration (CMC). These micelles can increase the solubility of poorly
soluably compounds.

PROCEDURE:

Hydrophilic colloids are commonly used in pharmaceuticals. Many have been

suggested as protective colloids for hydrophobic colloidal particles. Gelatin,
acacia, agar, and sodium carboxymethylcellulose have been used in this capacity.
This laboratory consists of three parts. One student will complete parts A, one
student will complete part B, and two students will complete part C. Part A & B
require that you dry a portion of your product. This is best done in a large petri
dish over a steam bath.
 A. Acacia mucilage(35% W/V) may be used as a protective colloid. Dissolve
0.66g of potassium iodide and 0.66 g of silver nitrate in two separate 10 ml
portions of water. Add 15 ml of Acacia Mucilage to the potassium iodide solution
( make this mixture by slowly adding the dry acacia to the water in small portions.
To this mixture with constant stirring slowly add the silver nitrate solution. Take
about half of the colloid you just formed and evaporate it to dryness over a water
bath. Now try to reconstitute the dry colloid with 10 ml of distilled water.
Compare to the sample of the original colloid.

B. Pour one ml of benzoin tincture into 25 ml of water in a beaker. Add the

benzoin tincture very slowly with mixing. Take half of the mixture and evaporate
it to dryness on a water bath. Try to reconstitute the dry colloid. Compare the
original colloid with the recostituted one. Repeat the experiment using 10 ml of a
2% Sodium Carboxymethylcellulose solution and 2.5 ml of benzoin tincture.
Again take half and evaporate to dryness and reconstitute with water. Compare the
results.

C. The entire class will work on the preparation of a three component phase
diagram for peppermint oil-water-Tween 20 system. Each group will be doing
four titrations. Each group will have assigned different mixtures so that in effect
we will be making 24 titrations. You will measure out the amount of peppermint oil
and Tween 80 and place it into a 100 ml beaker. You will then titrate the mixture to
a cloud point by adding water from the burette. It will be your responsibility to
record the amount of water used in each titration on the white board and to obtain
the other groups numbers to be used in your graph in your groups report. Repeat
each titration at least once. The table below lists the number of grams of surfactant
and the number of grams of oil to be used in each titration. Your group is lettered
from the north of the laboratory. If you do not know what group or team you are
ask the instructo