BCH 314 TUTORIAL 1

1) To what volume must you dilute 10 cm3 of a 0.5 mol dm-3 glucose solution
in order to obtain a final glucose concentration of 0.2 mol dm -3? What is
the dilution factor?
2) (i) Complete the following table, the data in which is to be used in
constructing a standard curve for compound X. The stock solution of X
has a concentration of 1000 g ml-1 and the volume of the sample prior to
adding colour reagent is 2 ml.

g X ml Stock ml H2O Stock dilution factor (n-fold) g ml-1 X

10
20
30
40
50

(ii) What is the molar concentration of glucose (molecular mass = 180.16)

in a 2 mg ml-1 solution?
3) Sugar solution (0.5 ml) was assayed by the Nelson-Somogyi method and
yielded an A520 of 0.2 in a 1 cm light path. If the slope of the standard curve
for this sugar (i.e. the K-value) was 5 x 10-3 absorbance units g-1,
calculate its concentration in g ml-1.
4) Assume that, after reacting with alkaline copper, glucose and maltose
have the same molar absorptivity (molar extinction coefficient) at 520 nm.
Calculate the ratio of the absorbance of 100g ml -1 of glucose to that of
100 g ml-1 of maltose at 520 nm.
5) Most pure proteins are insoluble in pure distilled water but dissolve in
dilute salt concentrations. However, the addition of high concentrations of
neutral salts to an aqueous solution of protein causes it to precipitate.
What is this phenomenon referred to as? Suggest a molecular explanation
for the observation that high concentrations of added salts decrease the
solubility of proteins.
6) Calculate the ionic strengths of 1.0 M solutions of NaCl, (NH 4)2SO4, and
K3PO4. In which of these solutions would a protein be expected to be most
soluble and least soluble? Explain.
7) Separate samples of a protein-containing solution are analyzed by SDS-
PAGE and cation exchange column chromatography, giving the results
shown in the figure below:
 What conclusions can you make concerning the composition of the
original protein solution?
8) Write notes on isoelectric focusing (IEF) with particular reference to its
separation principles and applications.
9) How can isoelectric focusing be used in conjunction with SDS gel
electrophoresis? Briefly outline the advantages of Continuous Flow
Gradient Gels over Traditional Gradient Gels.
10) Give an explanation at a molecular level of how ethidium bromide interacts
with DNA.
11) Assume the isoelectric point (pI) of 6-phosphogluconate dehydrogenase
is 6. Explain why the buffer used in DEAE cellulose chromatography must
have a pH greater than 6 but less than 9 in order for the enzyme to bind to
the DEAE.
12) Given the following facts:

Protein pI Mol. wt
Chymotrypsinogen 9.4 23200
Haemoglobin 7.0 64500
Lysozyme 11.1 14100
Ovalbumin 4.9 45000

i) Draw the elution profile of a mixture of these proteins that would be

obtained by passing them through Sephadex G-100.
ii) What would be the order of elution of haemoglobin, lysozyme and
ovalbumin from an ion exchange column of DEAE cellulose eluted with a
gradient of increasing ionic strength at i) pH 8.0, ii) pH 6.0, and iii) pH 4.0?
Give a brief explanation in support of your answer in each case.
13) A mixture of the following four proteins was purified:
 protein pI Mol. wt.
A 6.0 30,000
B 7.2 30,500
C 7.2 100,000
D 8.6 30,000
Step 1 was used to separate A from B + C + D
Step 2 was used to separate C from B + D
Step 3 was used to separate B from D.

Match each step with the appropriate technique listed below and answer
the accompanying question(s)

Technique Question
a) Ion-exchange chromatography on What is the order of elution
DEAE at pH 8 from the column? Which
protein(s) is (are) expected to
interact most strongly with the
column? Why?
b) Ion-exchange chromatography on (Same as above)
phosphocellulose at pH 9
c) Molecular Which protein(s) elute(s) first
exclusion chromatography and last?
If none is the answer to any of the above steps, explain.

14) Describe in detail the basic principle of gas chromatography with particular
reference to mobile and stationary phases, capillary columns and
separation conditions.
15) Write short descriptions that explain clearly the following (concerning
chromatography):
(i) Resolution
(ii) Efficiency
(iii) Selectivity
16) You wish to purify an ATP-binding enzyme from a crude extract that
contain several contaminating proteins. In order to purify the protein
rapidly and to the highest purity, you must consider some sophisticated
strategies, among them affinity chromatography. Explain how affinity
chromatography may be applied to this separation and explain the
physical basis of the separation.
17) Concanavalin A, a protein that binds carbohydrates, can be purified by
affinity chromatography on Sephadex. The column distribution (available
partition) coefficient for concanavalin A is greater than 1.0. Explain how
will this affect a molecular weight determination of concanavalin A by gel
filtration chromatography?
18) Discuss the techniques of Gas Chromatography and High Performance
Liquid Chromatography. Elaborate on the types of compounds most suited
 to each technique and give details of the advantages of a mass
spectrometer as a detector.
19) Mass spectrometry is widely used as a detection system in gas
chromatography. Describe what the mass spectrum of a molecule
indicates (i.e. what does the mass spectrum show). What is the major
advantage of obtaining a mass spectrum of a chromatographic peak?

MEMO BCH 314 Tutorial 1

1) C1V1 = C2V2 i.e 0.5 x 10 = 0.2 x V2, therefore V2 = 5/0.2 = 25 ml. Dilution
factor is 2.5
2) (i)
g X ml Stock dilution factor (n- ml H2O g ml-1 X
Stock fold)
10 0.01 200 1.99 5
20 0.02 100 1.98 10
30 0.03 66.67 1.97 15
40 0.04 50 1.96 20
50 0.05 40 1.95 25

(ii) 1 M glc = 180.16 g l-1`

2 mg ml-1 = 2 g l-1
Molar conc. of glc = (2 x 1) / 180.16 = 0.011 mol l -1
3) K-value = change A/C, i.e. 5 x 10-3 = 0.2/C, C = 0.2/5 x 10-3 (this comes
from 0.5 ml) i.e. 0.5 ml gives 0.2/5 x 10-3, therefore 1 ml = (0.2 x 1)/(5 x 10-
3
x 0.5) gml-1
4) Convert conc into Moles/ml thus: glc: 100/180 Moles/ml = (100/180) x
10-6 Mol/ml = (100/180) x 10 -6 x 103 Mol/l = 0,56 x 10 -3 Mol/l. What is
maltose? = 2 x glc, mol wt = 360. For maltose = (100/360) x 10 -6 x 103
Mol/l = 0,28 x 10-3 Mol/l
Therefore Aglc/Amalt = 0,56 / 0,28 = 2 ratio glc : malt = (2 : 1)
5) Low levels of salt can increase the solubility of proteins by permitting ionic
interactions with charged groups on the protein surface. However, as salt
concentration increases, the added ions compete with the charged protein
molecule for hydrating water molecules, become less soluble, and
precipitate, a process called salting-out. Removal of excess salt will
provide a more favourable environment for rehydration of the protein.
6) I =1/2 ci Zi2
1 M NaCl is 1 M in Na+ and Cl-; therefore I=1/2(1.0x12+1.0x12) =1.0
1 M (NH4)2SO4 has 2 M NH4++1 M SO42-; therefore I=1/2(2.0x12+1.0x22) =
3.0
1 M K3PO4 has 3 M K++1 M PO43-; therefore I=1/2(3.0x12+1.0x32) =
6.0
Proteins are salted out at high ionic strengths. Hence a protein should be
most soluble in 1M NaCl and least soluble in 1 M K 3PO4
7) The original solution contains two proteins that differ in net charge (or pIs)
but have the same subunit mol. wt.
8) The fundamental premise of IEF is that a molecule will migrate so long as
it is charged. Should it become neutral, it will stop migrating in the electric
field. IEF is run in a pH gradient where the pH is low at the anode and high
at the cathode. The pH gradient is generated with a series of zwitterionic
chemicals known as carrier (poly) ampholytes. When a voltage is
applied, the ampholyte mixture separates in the capillary. Ampholytes that
are positively charged will migrate towards the cathode whilst those
negatively charged migrate towards the anode. The pH then will decrease
at the anodic section and increase at the cathodic section. Finally, the
ampholyte migration will cease when each ampholyte reaches its
isoelectric point and is no longer charged. Initially, a solute with a negative
charge will migrate towards the anode where it encounters buffer of
decreasing pH. Finally, the solute encounters a pH where its net charge
becomes zero, the pI and migration halts. The greater the number of
ampholytes in solution, the smoother the pH gradient. The pH of the
anodic buffer must be lower than the pI of the most acidic ampholyte to
prevent migration into the analyte. Likewise, the catholyte must have a
higher pH than the most basic ampholyte. It is apparent that the EOF
(electroosmotic flow) and other convective forces must be suppressed if
IEF is to be effective. The capillary walls can be coated with
methylcellulose or polyacrylamide to suppress EOF. The coating tends to
suppress protein adsorption as well. IEF is generally used for high
resolution separations of proteins and polypeptides but could be used for
any amphoteric substance, provided a series of ampholytes that cover the
entire pI range is used. In addition to performing high resolution
separations, IEF is useful for determining the pI of a protein. IEF is
particularly useful for separating immunoglobulins, haemoglobin variants
and post-translational modifications of recombinant proteins.
Applications: clinical chemistry and forensic pathology, agriculture,
taxonomy, immunology and microbiology, food analysis, cancer
research.

9) Isoelectric focusing can separate proteins of the same mol. wt on the

basis of differing pIs. SDS gel electrophoresis can then separate proteins
with the same pIs on the basis of differing mol. wts. Together a great
resolution of large numbers of proteins can be achieved.
10) Ethidium bromide contains a planar (flat) group that easily slides
(intercalates) between the stacked bases of the DNA molecule. The fixed
position of this group and its close proximity to the bases cause the dye
bound to the DNA to display an increased fluorescent yield under UV
light.
11) The buffer pH must be above 6 but below 9 to assure that the protein is
negatively charged and the DEAE groups are positively charged.
12) i) 1st=Hb, 2nd =ovalbumin, 3rd=chymotrypsinogen, 4th=Lysozyme
 ii) Protein binding to ion exchange resins is a function of their net charge.
The most negatively charged protein will bind more tightly to DEAE
cellulose. At pH 8: ovalbumin (most ve, followed by HB. Lysozyme (+ve)
and passes through without exchange. Order: i) lys ii) Hb iii) Ovalbumin
At pH 6: Ovalbumin (-ve) and exchanges on DEAE. Lys and HB (+ve) and
elute without appreciable exchange. Order: i) lys, Hb, ii) ovalbumin
pH4: All 3 proteins (+ve) and elute directly at the same time from column
without exchanging. No separation.
13) DEAE is an anion exchanger
step 1 = a). D comes out first (positively charged at pH 8; B & C are
weakly negatively charged and come out second; A is strongly negatively
charged at this pH and sticks most tightly to the column.
step 3 = a). At pH 8, B is negatively charged and sticks to resin; D is
positively charged at this pH and elutes
step 2 = c). B (MW) = 30,500; C = 100,000; D = 30,000 ; C comes out first
& D last

14) Nitrogen, Helium and Argon are the three most commonly used carrier
gases. They are passed through the column at a flow rate of 40 - 80 cm 3
min-1. A stationary phase of a high boiling point liquid material (e.g.,
silicone grease) is supported on an inert granular solid. This material is
packed into a narrow coiled glass or steel column 1-3 m long and 2-4 mm
internal diameter, through which the mobile phase) is passed. The
selectivity of a packed GC column is largely determined by the chemical
properties of the liquid stationary phase. The basic requirements for any
GLC stationary phase are that it must be involatile and thermally stable
at the temperature used for analysis. More often the phases used are
high boiling point organic compounds, and these are coated onto the
support to give from 1% to 25% loading, depending upon the analysis.
Such phases are of two types, either selective, where separation occurs
by utilization of different chemical characteristics of components, or non-
selective, where separation is achieved on the basis of differences in
boiling points of the sample components. The operating temperature for
the analysis must be compatible with the phase chosen for use. Too high
a temperature results in excessive column bleed owing to the sample
being volatilized off, contaminating the detector and giving an unstable
recorder baseline. The choice of phase for analysis depends on the
compound under investigation and is best chosen after reference to the
literature. Commonly used stationary phases include the polyethylene
glycols, methyl phenyl- and methyl vinyl silicone gums (so-called OV
phases), Apiezon L and esters adipic, succinic and phthalic acids.
 The are two types of capillary column systems known as wall-coated open
tubular (WCOT) and support-coated open tubular (SCOT), also known as
porous layer open tubular (PLOT) columns for adsorption work. In WCOT
columns the stationary phase is coated directly onto the walls of the
capillary tubing. As there is only a small amount of stationary phase
present, only very small amounts of samples may be chromatographed.
Consequently, a splitter system has to be used at the sample injection port
so that only a small fraction of the injected sample reaches the column.
The remainder of the sample is vented to waste. The design of the splitter
is critical in quantitative analyses in order to ensure that the ratio of
sample chromatographed to sample vented is always the same. In SCOT
columns a support material is bound onto the walls of the capillary
column, and the stationary phase is coated onto the support. The
capacity of SCOT columns is considerably higher than that of the WCOT
columns and consequently small samples can be injected directly onto
such columns without the need for a splitter system. SCOT systems are
therefore considerably simpler to use for quantitative analyses than are
WCOT systems. Their efficiency, though, is less than that of WCOT
systems but considerably better than that of conventional GLC columns.

The column is maintained in an oven at an elevated temperature, to

ensure that the compounds to be separated are kept in the vapour state
and that analysis time is reasonable. The column temperature must be
within the working range of the particular stationary phase and is chosen
to give a balance between peak retention time and resolution. In GLC,
partition coefficients are particularly sensitive to temperature so that
analysis times may be regulated by adjustment of the column oven, which
can be operated in either of the two modes: In isothermal analysis a
constant temperature is employed. In the separation of compounds of
widely differing polarity or Mr it may be advantageous to increase the
temperature gradually. This is referred to as temperature programming.
This, however, often results in excessive bleed of the stationary phase as
the temperature is raised, giving rise to baseline variation. Consequently
some instruments have two identical columns and detectors, one set of
which is used as a reference. The currents from the two detectors are
opposed, hence, assuming equal bleed from both columns, the resulting
current gives a steady baseline as the column temperature is raised.

15) Resolution: is the measure of the ability of a column to separate 2 or

more peaks (through a combination of column efficiency and selectivity)
which takes into account zone width as well as the distance between zone
centres.
Efficiency: is the measure of the narrowness of a peak relative to the time
it takes to come out (Peaks can be resolved by greater efficiency and
 therefore making peaks narrower (by changing selectivity of column
stationary phase). (i.e. efficiency of the column is the degree to which it
keeps zones from spreading).
Selectivity: the degree to which the column thermodynamically
distinguishes between the 2 components. (where separation occurs by
utilization of different chemical characteristics of components or non-
selective, where separation is achieved purely on the basis of differences
in boiling points of the sample components).
16) Affinity chrom is an elegant, rather specific method to separate a protein
or an enzyme from a mixture. The technique is based on the specific,
strong binding of a substrate, substrate analog, inhibitor or antibody to a
protein. An initial strategy to isolate the ATP-binding protein involves
constructing an affinity resin by linking ATP to an insoluble support through
a spacer side chain of appropriate length. The spacer allows the protein to
bind the affinity probe with minimal steric or chemical interference from the
soluble support. However, the protein of interest, or another protein in the
crude extract, may hydrolyse ATP and destroy the affinity probe. As an
alternative approach, construct the affinity probe using a nonhydrolysable
analog of ATP (e.g. imido- or methylene analog). An ATP-binding
protein(s) would be predicted to bind to affinity probe. The contaminating
proteins are washed from the column and the bound protein eluted with
ATP, increased ionic strength, pH alteration, or empirically determined
combination of these.
17) Concanavalin A will have a larger Ve than expected since it binds to the
carbohydrate gel matrix.. The mol. wt. determination will be low, since
concanavalin will appear as a smaller protein which occupies more of the
space inside the gel matrix. (remember larger proteins are eluted first,
small proteins last).
18) GC: It is effective in separating molecules of low polarities (volatile
compounds) because its separation principle is based on the differences
in volatilities of compounds (The basis of separation is the difference in
partition coefficients (volatilities or boiling points) of the volatilised
compounds between liquid and gas phases as compounds are carried
through the column by carrier gas). The column is maintained at elevated
temperatures to ensure compounds to be separated are kept in a vapour
state (The injection region is maintained at a slightly higher temperature
than the column itself to ensure rapid and complete volatilisation of the
sample.) The sample is soluble in the carrier gas. Polar samples (non-
volatile) are derivatised (e.g. by methylation). The sample is dissolved in
organic solvent (hexane, pentane etc) and then injected using a
microsyringe through a septum in the injection port. As compounds
emerge from the column, they pass through a detector that is connected
via an amplifier to a chart recorder, which in turn, records a peak as each
analyte passes through the detector. To identify compounds, authentic
samples of the test compounds can be used for calibration or a mass
spectrometer can be used. The GC technique is applied extensively in:
 wine and food industry (for identification of flavour compounds), analysis
of biological materials (detection of pesticides), mineral-oil and gas-
exploration industries (finding gas and oil fields by ultra trace analysis of
methane/ethane), prevention of pollution by detecting gas leaks, water
and air contamination.
19) Mass spectrum is a recording of the masses of each of the ionized
fragments, representing a unique fingerprint of a molecule that can be
used in identification. The sample, which is in the vapour phase, is
introduced into the vacuum chamber via the interface between the GC and
Mass Selective Detector (MSD) and is ionized in the ion source. The
ionized molecule breaks into reproducible fragments that are filtered by
the mass analyzer according to their mass-to charge (m/z) ratios and are
collected by the detector. In the detector, the ion fragments generate an
electrical signal that is proportional to the number of ions. The data system
records these electrical signals and then converts them into a mass
spectrum.
Molecular fragments appear in a mass spectrum and provide clues to the
identity and molecular structure of the parent molecule (similar to the way
pieces of a jigsaw puzzle provide clues to the structure of an intact
puzzle). A mass spectra is a plot showing the mass/charge ratio (in
daltons or atomic mass units) versus abundance data for ions from the
sample molecule and its fragments. Most ions formed by GC/MS have a
charge of +1. The mass/ charge ratio of a fragment is therefore normally
equal to the mass for that fragment. The largest peak in the spectrum is
called the BASE PEAK. Certain fragments are more prone to form from
the parent molecule than others, due to the presence of functional groups
in the molecule and their interconnection. The masses of these fragments
are used to deduce the structure of the parent compound.

The ionized parent molecule, when seen as part of the mass spectrum, is
referred to as the MOLECULAR ION. Occasionally the molecule is so
totally fragmented by the ionizing process that little or no molecular ion is
seen. The mass spectra of certain compounds exhibit clusters of mass
peaks. These clusters represent naturally occurring impurities or
isotopes that are present for carbon, nitrogen, sulfur, chlorine, bromine,
and a few other elements. The relative percentages of these cluster ions
provide more clues useful in unraveling the identity of a parent molecule
from its molecular fingerprint.