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Chem 241L Final

Terms in this set (99)

Experiment 1
Extraction of Ethanol from Beer - Gas-Liquid Chrom.
solvent-solvent extraction
it is necessary to isolate the compound of interest, the analyte, from the rest of the compounds in the sample matrix

Transfer the analyte from its original solvent, to a second solvent which only the analyte is soluble
- the best solvent to use is one in which the analyte has a higher solubility than it does in the orginal solvent
Partition Coefficient
the concentration of solute in each layer

K = [S] in organic / [S] in aqueous
q
the fraction of solvent left behind

1-q = what was extracted
the stationary phase is the
packed column
the partitioning process
the volatile organic molecules moving between the mobile gas phase and the liquid stationary phase

the gas molecules will dissolve into the liquid phase and diffuse back out of the liquid into the gas phase

The longer a molecules stays dissolved in the stationary phase, the more time it will take before it elutes from the column

THIS IS ALL G-L CHROM
in experiment 1
- a thermal conductivity detector
-packed stationary phase (carbowax 20M can interact with polar molecules, such as alcohols)
if you had a mix of non-polar compounds
then elution ordered would be based on vapor pressures or boiling points

if there was a mixture of non polar and poalr then nonpolar would elute first becuase the polar compounds would interact strongest with the Carbowax
experiment 1
this is the experiment where we used the syringe and went to the thermal conductivity room - a two week experiment
week 1
standard calibration - have to determine partition coef.
weed 2
standard addition

- does not require that all ethanol be extracted
- does not require you to determine K or the fraction of ethanol left behind
k
ethanol extracted/ethanol known - ethanol extracted
experiment 2
isoelectric point of BSA
using slab electrophoesis
carried out on a a thin flat layer or slab of a porous semisolid gel or membrane in a buffer solution
strips are made out of
celllulose polyacetate
the electric field
(E) is determined by dividing the applied voltage by the distance between the two electrodes

E = V/d
positively charged molecules will migrate towards the cathode
negatively charged molecules towards the anode
the rate at which the molecule vibrates
u(ep)
u(e)
= q/f x E

q is the charge and f is the frictional force
smaller and more charged particles move the furthest
a smaller +1 charge will move further than a larger +1 molecule
for molecues like aspartic acid with 3 ionizable protons
pI = pka1 + pka2 / 2

you cant do this with BSA however
isoelectric point
only affected by those equilibrium reactions that involve chemical species with a zero net charge
voltage was set to
300

24 AMP
Ponceau Stain
contains trichloroacetic acid, which is corrosive
the second tray was filled with
5% acetic acid - the stain binds to the acid and removes the excess stain revealing the pink band, which is the BSA sample
experiment 3
pKa of Acid-Base indicator Bromothymol blue
A = Log (Io/I)
Io - the irradiance of the light measured in the absense of absorbing solution, which would be water

I - the irradiance of the light measured after it passes through a solution
plot of absorbance vs. wavelength
spectrum
% T = I/Io x 100
pay close attention because the i's are reversed from the one above
A = log (Io/I)
or -log(T)
molar absorbtivity
info on ow much light a given molecule acn absorb at a particular wavelength
Ka
acid dissociation constant
the larger the Ka
the further the equilibrium lies to the right, and thus the stronger acid
Ka
concentration of products/reactants
pKa
-log(Ka)
the acidic form of BTB
yellow (has an OH in the structure) the resonance gives it its acidity

the basic form is blue (has a ketone in the structure)
the structural changes
gives it the color change
the first part of the experiment
use a spec to determine the wavelength of maximum absorbance
for both acidic and basic forms
ph = 10 for basic (pH > pka)
ph = 5 for acidic (pH < pKa)
set the absorbance from
240-800 nm

- took measurments in 10 nm increments
extraction of BTB
solvent solvent extraction

the solvent used is ethyl acetate
the second part of the exeperiment
set the spec to the basic form of BTB (615 nm which was calculated from the first part of the experiment)

then 8 different buffer solutions with different pH

these were graphed (absorbance vs the pH) - the inflection point of this curve is where the concentration of the acidic form a is equal to the basic form!
Then you use beers law to find the
concentration
the two extraction samples from the first part of the experiment
recorded at the acidic wavelength
Experiment 4
The extraction and recrystallization of benzoic acid
testing the purity of recrystallized benzoic acid
by measuring its melting point
carboxylic acids
are not very soluble in water, but their conjugate bases are
benzoic acid is going to be more soluble in organic solvents
benzoate (the charged conjugate base) is going to be more soluble in aq phase
the majority of benozic acid in the form of the benzoate anion will be present in the
aqueous layer
the goal is to get the impurities into the organic layer
and the benzoate anion stays in the aq phase which is what we want
once the organic phase is removed
dilute acid is added until the aq layer ph is less than the pka of the compound -- this reforms the benzoic acid (reacidfies it)
the purer the substance
the higher the melting point and the narrower its melting point range
recrystalization took place in the
buchner funnel
acid base extraction is where
we used the separatory funnel

- used sodium carbonate and ethyl acetate
- also used sulfuric acid to reacidify it
Experiment 5
Determining Myoglobin's weight by Gel-filtration
SEC
a form of LC that utilizes a gel stationary phase to separate molecules
in this experiment
an aqueous mobile phase is used --> gel filtration chrom. (This is what we did)

when a organic solvent is used as the mobile phase --> GPC (fyi)
gel stationary phase
the gel particles are porous and cross-linked polymer

we used sephadex g50 i this experiment - has a fractionation range for globular proteins of 1500-30,000 daltons
GFC is going to separate molecules
based on their size
smaller molecules will enter many more
pores. this makes them travel slower, they travel through the sephadex

sephadex is cross linked dexatran
Vr = Vo + KVi
Vr = retention volume
Vo = void volume
K = distribution coefficient for given solute
Vi = internal pore volume
the retention volume
the volume of the mobile phase needed to elute a given solute from the column
the void volume
the volume of solvent between the gel particles in the column
more clearly
K = Vr-Vo/Vm-Vo
Vm = Vo + Vi
...
for solute molecules, such as dexatran blue
totally excluded form the pores of the gel stationary phase, Vr = 0 and K = 0

Solute molecules that are smaller than the pores can completely penetrate the gel therefore Vr = Vm and K = 1
molecules that have molecular weights that fall within the pore size range of the gel stationary phase
will be eluted between K values of 0 and 1
a graph was constructed for the standards
log of MW vs retention volume
the top layer in the column is
G-25
the phosphate buffer was at ph
7
Hemoglobin (68000)
Ovalbumin (45,000)
Cytochrom C (12,300)
wavelength was set at 280 nm
only hemoglobin and cyt c asorb light in the visible region

Ovalbumin - doesn't absorb in the visible region but most amino acids absorb in the UV region and ovalbumin absorbs highest at 280
the greatest absorbance of ovalbumin
is the most intense fraction - therefore the 16th fraction was most intense so that was the retention volume (Vr)
hemoglobin had a brown color
cyto had a pinkish brown color
Experiment 6
Extraction from Spinach using LC
used reversed phase chromatography
also used solvent solvent extraction to get out the pigments
plant pigments
porphyrins - chlorophylls a and b
carotenoids - xanthophylls (yellow) and carotenes (orange yellow)
flavenoids - flavonol
for photosynthesis to proceed
the plant has to produce chlorophyll at a rate equal to or great than the rate at which it breaks down
there are 70% xanthophylls in plants
and 30% carotenes
the final color of the leaf is a function of
pH of the sap and amount of anthocyanin production
the pigments are broken down with
acetone - breaks the pigments apart

and mixed with NaCl - this results in two phase, aq and organic - NaCl is added to help drive the pigments into the orgnic layer
- cant use water because this wouldn't drive them into the organic phase
salting out effect
this is what NaCl is considered
- typically results in the solubility of nonelectrolytes generally being smaller in the aq electrolyte solutions (NaCl) than in water
the aqueous phase (relatively polar) - these are xanthophylls
the organic phase (nonpolar) - these are carotenes
relatively polarity
carotenes - chlorophylls - xanthophylls
in this experiment we are using
reversed phase LC becaue the stationary phase in nonpolar and the mobile phase is polar

also using a step-gradient elution - the composition of the mobile phase changes with time
begin with a more polar mobile phase in order to remove the
polar compounds
two parameters used to describe the efficiency of separation are
retention (alpha)
resolution (R)
alpha = tr2 - tr1 / tr1 - tm
R = 2(tr2 - tr1) // w2 + w1
the peak width
a function of the overall chromatographic efficiency of the column
also used beers law in this experiment
the absorption spectra of isolated plant pigments are important becuase they indicate which wavelength will give the maximum signal per mole of pigment
column is packed with
C18 - stationary phase
mobile phase 1
most polar phase

the third mobile phase has the most ethyl acetate

water >> acetonitrile >> ethyl acetate (least polar)
xanthophylls (most polar)
carotenes (least polar)
spect set to
450 nm
experiment 7
glucose in sports drinks
a selective chemical receptor
is associated with a trandsducer which translates the recognition event into a physically measurable value
the bio assay
B-D-glucose + o2 gets converted to D-glucolacetone by glucose oxidase

H2O2 plus ferrOcyanide gets converted into ferrIcyanide by horseradish peroxidase (which is a yellow solution)
measured at
420 nm
there were how many standards
7 standards (we knew the concentration of these standards)
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