Experiment

0: Lab safety & statistics

1. Accuracy: how close you are to the true value
2. Precision: closeness of how the measurements are
3. MSDS: provides information about chemical product, company identification,
composition, hazards, first aid measures, handling and storage, physical and
chemical properties, stability, etc. to ensure it can be used properly in the lab
4. Average (Mean)
$
#
a. 𝑥 = $%&
'
5. Standard Deviation
$ ,
$%&(#*#)
a. 𝜎 =
'*-
6. Absolute error: magnitude of the difference between the exact value and the
approximation
a. |𝑥 − 𝑥|
7. Relative error: absolute error divided by the value obtained
|#*#|
a.
#
|#*#|
b. Percent relative error: ×100%
#

8. Systematic error: instrumental errors, data is off by the same amount every
instance (calibration problem)
a. Can be corrected, avoidable
9. Random error: due to human error; data is always slightly off no matter how
close it is to the theoretical value
a. Unavoidable
10. Confidence intervals: stating the true mean μ is likely to lie within a certain
distance from the measure of the mean
6∙8
a. 𝜇 = 𝑥 +
'
11. Student’s t and use of t-Table
|#*='>?' A;<BC|
a. 𝑡:;<: = 𝑛
8
b. if 𝑡:;<: > 𝑡86BEC'6 , then there is a significant difference
12. Significance Testing


Experiment 1: extracting ethanol from alcoholic beverages and the subsequent
analysis of this extract by gas-liquid chromatography for qualitative analysis
1. Perform solvent-solvent extraction in order to remove a chemical compound
from a complex mixture
a. Remove/isolate the compound of interest (analyte) from rest of the
mixture of chemical compounds (sample matrix)
b. Can be done by solvent-solvent extraction and it’s based on
differences in solubility of the analyte in different liquids
i. Two solvents must not be miscible (mix freely) with one
another and thus form two separate and distinct layers or
phases (one layer is aqueous phase and the second layer is
organic/non-aqueous phase)
1. In this experiment, ethanol is the solute (S) from a
series of beer samples
2. Pentanol (Vpentanol) will be added to a given volume (Vaq)
of beer sample (beer sample is treated as an aqueous
phase because there is a large amount of water present)
ii. Solute (S) will distribute itself between two immiscible liquid
phases: 𝑆;GCB>B8 ⇌ 𝑆IC'6;'><
1. 𝐶𝐻L 𝐶𝐻M 𝑂𝐻;GBC>B8 ⇌ 𝐶𝐻L 𝐶𝐻M 𝑂𝐻IC'6;'><
c. Partition coefficient: ratio of concentrations of solute in each layer
defines the K
[Q]STUV$&W YZ[ YZ, \Z]^$_V$S`
i. 𝐾 = =
[Q]VX YZ[ YZ, \ZVXa^Sab
d. Fraction of ethanol remaining
'VX YVX eVX eVX
i. 𝑞 = = = fSTUV$&W =
'VX d'STUV$&W YVX eVX dYSTUV$&W eSTUV$&W eVX d eSTUV$&W
fVX
eVX

eVX dgeSTUV$&W
ii. Multiple extractions (n=number of extractions)
eVX
1. 𝑞 = ( )'
eVX dgeSTUV$&W
2. Gas Chromatography
a. Based on differential migration of chemical species through some
medium
b. A syringe is used to inject a small quantity (in microliters) of a volatile
liquid sample into the gas chromatograph through a heated injection
port
c. Heated injection port vaporizes the liquid sample and mixes it with
the He carrier gas, creating a gaseous mixture (carrier gas serves as
mobile phase)
i. Carrier gas will carry gaseous mixture through the column
1. Unlike other chromatographic methods uses, where
mobile phase is a liquid
 d. Either be an open tubular column, where stationary phase is coated
on the inside walls, or packed column, containing particles coated
with a high boiling point liquid stationary phase
e. Partitioning process involves volatile organic molecules moving
between mobile gas phase and liquid stationary phase
i. Molecules will dissolve into the liquid stationary phase from
the gas phase and diffuse back out of the liquid stationary
phase and back into the gas phase (equilibrium); occurs along
the column length
1. Method is also called gas-liquid chromatography
because particles distribute themselves between the gas
phase and liquid stationary phase
ii. Longer the phase stays in the column, the longer it will take to
elute
f. Thermal conductivity detector: measures differences in thermal
conductivities between carrier gas and the gaseous sample
components
i. Voltage values will change as particles pass by
3. Stationary phase
a. Packed column; in this experiment there is crushed firebrick
(common supporting material)
b. Thin film of non-volatile liquid is coated on to make solid support
stay; in this experiment Carbowax is used which interacts favorably
with polar molecules (ex: alcohols)
i. Nonpolar molecules will be able to pass by more quickly than
polar molecules
c. Elutions based on vapor pressure (boiling points) for nonpolar
compounds of hydrocarbons (lower boiling points are eluted first)
d. In a mixture of polar and nonpolar compounds, nonpolar would be
eluted first against the polar stationary phase, as polar compounds
will interact strongly with the phase

Experiment 2: the determination of the isoelectric point of the protein bovine
serum albumin
1. Electrophoresis involves a charged molecule migrating in an electric field
2. Slab electrophoresis: carried out on thin flat layer or slab of some porous
semisolid gel or membrane contained in an aqueous buffer solution
a. Paper electrophoretic strips are immersed in aqueous buffer and near
an electrode
b. Voltage is supplied and electric field is established, charged sample
will begin to migrate
i. E = V/d
3. Capillary electrophoresis: separation occurs in submillimeter capillary tubes;
separates ions based on their electrophoretic mobility with the use of applied
voltage
4. Sample origin: spot or line across the center of the strip
5. Direction of migration: determined by the net sign of its charge at a given pH
a. Positive molecules migrate towards cathode (negative electrode)
b. Negative molecules migrate towards the anode (positive electrode)
c. Molecules with no net charge will not migrate
6. Rate (𝜇CI )
a. 𝜇CI = 𝑒𝑙𝑒𝑐𝑡𝑟𝑜𝑝ℎ𝑜𝑟𝑒𝑡𝑖𝑐 𝑚𝑜𝑏𝑖𝑙𝑖𝑡𝑦 × 𝑚𝑎𝑔𝑛𝑖𝑡𝑢𝑑𝑒 𝑜𝑓 𝑒𝑙𝑒𝑐𝑡𝑟𝑖𝑐 𝑓𝑖𝑒𝑙𝑑 =
:x;yzC × {;z'|6BEC >} C<C:6y|: }|C<E

}y|:6|>';< :>C}}|:|C'6
b. Frictional coefficient can be a result of shape, radius, and viscosity
7. Isoelectric point: pH that leads to electrical neutrality (average charge is 0)
and thus no observed migration, also known as isoelectric pH
I=;-dI=;M
a. pH =
M
b. Only affected by equilibrium reactions that involve species with no
net charge
c. Average charge of molecule is zero
8. Degree to which an acid dissociates depends on the pH and the acid
dissociate constant(s)
a. pH<pka1, positive net charge so molecule migrates towards cathode
b. pH>pka2, negative net charge so molecule migrates towards anode












Experiment 3: using spectrophotometry to determine the pKa of the acid-base
indicator bromothymol blue
•
1. Absorbance= log S
•
•S
a. = 𝑖𝑟𝑟𝑖𝑑𝑖𝑎𝑛𝑐𝑒 𝑏𝑒𝑓𝑜𝑟𝑒, 𝑖𝑟𝑟𝑎𝑑𝑖𝑎𝑛𝑐𝑒 𝑎𝑓𝑡𝑒𝑟
•
b. Irradiance before is measured in the absence of an absorbing solution,
irradiance after is measured when light has passed through the
solution of interest
•
c. Percent transmittance: %T= S ×100%
•
•
d. 𝐴 = log S = − log(𝑇)
•
2. 𝐵𝑒𝑒𝑟 † 𝑠 𝐿𝑎𝑤 = 𝐴Š = 𝜀Š 𝑏𝐶
3. HIn appears as yellow
4. In- appears as blue
5. Color change is an indication of pH change
a. Top benzene ring with the hydroxyl group coming off of the acidic
group is known as a phenol; replaced by double bonded oxygen in
basic form
b. When bromothymol blue loses its proton, the negative charge does
not reside on the oxygen atom in the basic form of bromothymol blue;
the bonds in the top benzene ring structure have been reorganized in
order to delocalize the negative charge
c. In addition to the change in bond structure, the five-member ring that
contains the sulfur atom is broken in this structural rearrangement
process





















Experiment 4: preparation and investigation of buffers
1. Buffered solutions are solutions that show very small changes in pH when
small amounts of strong acid are added, when small amounts of strong base
are added, or when the solution is diluted
2. Contains appreciable amounts of both a weak acid and its conjugate based
a. There is enough weak acid and conjugate base to be capable of equally
reacting with either added acid or base
b. Add too much acid: acid will dissociate to a small extent to produce
small amount of conjugate base
c. Add too much base: base will hydrolyze to produce a small amount of
conjugate acid
3. Henderson Hasselbach equation
•*
a. 𝑝𝐻 = 𝑝𝑘𝑎 + log ( )
Z•
b. Can also use equation to determine the ratio of conjugate base to
weak acid ([A]:[HA]) that would be required to obtain a given buffer
solution pH
•*
c. = 10(IZ*I=;)
Z•
4. Ways to prepare a buffer
a. Using an acid and its conjugate base to prepare the solution
b. Adding small amount of strong acid to a weak base solution; partial
neutralization of weak bases
c. Adding a small amount of strong base to a weak acid solution; partial
neutralization of a weak acid





















Experiment 5: using size exclusion chromatography as a separation technique
1. Also known as gel permeation chromatography when organic solvent is used
2. Known as gel filtration chromatography when aqueous solvent is used
a. Vertical column packed with gel stationary phase and solvent (mobile
phase) is allowed to continually flow through the column by the force
of gravity or a pump
b. Gel particles are porous, semi-rigid, cross-linked polymer (dextran,
polystyrene, polyacrylamide) networks
i. Assume gel particles are spherical to help explain separation
process
3. Uses gel stationary phase to separate molecules according to their molecular
size in solution
a. Varies on protein’s ability to enter gel pores
b. Smaller molecules readily enter the pores as they travel through the
column; will be retained for a longer time than the larger molecules,
which cannot readily enter the pores
4. Can also be used to estimate molecular weight of substances
5. Must fall within fractionation range
6. Retention volume
a. 𝑉y = 𝑉> + 𝐾𝑉|
b. 𝑉> is void volume
c. 𝑉| 𝑖𝑠 𝑖𝑛𝑡𝑒𝑟𝑛𝑎𝑙 𝑝𝑜𝑟𝑒 𝑣𝑜𝑙𝑢𝑚𝑒
7. 𝑀𝑜𝑏𝑖𝑙𝑒 𝑝ℎ𝑎𝑠𝑒 𝑣𝑜𝑙𝑢𝑚𝑒 = 𝑉> + 𝐾𝑉|
8. 𝑑𝑖𝑠𝑡𝑟𝑖𝑏𝑢𝑡𝑖𝑜𝑛 𝑐𝑜𝑒𝑓𝑓𝑖𝑐𝑒𝑛𝑡
e *e e *e
a. K= T S = T S
e& e‘ *eS
9. For large molecules are totally excluded from the pores, 𝑉y = 𝑉>
10. For molecules that are smaller than the pores can completely penetrate the
gel, 𝑉y = 𝑉{ and K=1
















Experiment 6: the extraction of plant pigments from spinach and the subsequent
separation and analysis of the extracted pigments using liquid chromatography
1. Normal phase liquid chromatography: stationary phase is polar and mobile
phase is nonpolar
2. Reverse phase liquid chromatography: stationary phase is nonpolar and
mobile phase is polar
3. Isocratic elution: composition of the mobile phase remains constant
throughout the entire separation
4. Gradient elution: composition of the mobile phase changes with time (in
discrete steps or changed continuously)
5. Porphyrins
a. Chlorophylls
i. Chlorophyll a (green)
ii. Chlorophyll b (yellow-green)
iii. These two compounds always exist together; produced in
response to sunlight
iv. Plant breaks down chlorophyll molecules into smaller
molecules
v. For photosynthesis to proceed, plant has to produce
chlorophyll at a rate equal to or greater than the rate at which
it is broken down
b. Carotenoids
i. Xanthophylls (yellow)
ii. Carotenes
1. Alpha, beta, gamma (orange-yellow)
2. Lycopene (red)
iii. Found in fruits and vegetables
iv. Participate in photosynthesis also; energy absorbed by them is
transferred to chlorophylls
v. Do not break down rapidly and are present in plant leaf during
their entire lifetime
c. Flavonoids
i. Flavonol (yellow)
ii. Flavone (yellow)
iii. Anthocyanin (red, blue, purple)
iv. Flavonoids all have similar structures but differ in hydroxyl or
carboxyl group in various locations
6. Relative retention
6 *6
a. 𝛼 = T, ‘
6T“ *6‘
7. Resolution
M(6 *6 )
a. 𝑅 = T, T“
?, d?“
8. Band broadening or increase in peak width is a function of the overall
chromatographic efficiency of the column
9. Accurate measurement is only possible under isocratic elution
 10. Well separate peaks make for a good resolution
11. Narrow peaks indicate high efficiency
12. Broad peaks indicate high selectivity
13. Absorption spectra of isolated plant pigments are important because they
indicate which wavelength will give the maximum signal per mole of pigment









































Experiment 7: spectrophotometric determination of glucose in sports drinks
1. Sample dilution equations
a. Multiply original concentration by (1 mL/50 mL) to get new
concentration (to go backwards use the reciprocal)
 earlier dilution
uses C1V1 = C2V2
2. Carbohydrates
a. Glucose and fructose are monosaccharides
b. Sucrose is composed of glucose and fructose
c. In order for sucrose or any disaccharide to be digested, it must be
broken down enzymatically
3. Biosensors: Portable analytical devise that combine the selectivity of
biomolecules to some type of signal transducer
a. Biomedical: glucose sensor, sensors for the detection of genetic
defects in DNA
b. Environmental: control air pollution, detect low level toxic and
hazardous gases
c. Quantitation: pH electrodes, ion-selective electrodes
4. Enzymes are very selective and only interact with those biomolecules that
have receptor sites in which they can “fit” into and thus bind
a. Catalyze only specific chemical reactions