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# 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 100µg ml -1 of glucose to that of
100 µg 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:

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. 13) A mixture of the following four proteins was purified: . 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.0. 12) Given the following facts: Protein pI Mol.4 23200 Haemoglobin 7.0 64500 Lysozyme 11. and iii) pH 4.0? Give a brief explanation in support of your answer in each case.9 45000 i) Draw the elution profile of a mixture of these proteins that would be obtained by passing them through Sephadex G-100. 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. lysozyme and ovalbumin from an ion exchange column of DEAE cellulose eluted with a gradient of increasing ionic strength at i) pH 8. 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. ii) pH 6.1 14100 Ovalbumin 4. wt Chymotrypsinogen 9.0. ii) What would be the order of elution of haemoglobin.

0 30.2 100. Elaborate on the types of compounds most suited .2 30. you must consider some sophisticated strategies.6 30.000 B 7. 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. A 6. among them affinity chromatography. wt. can be purified by affinity chromatography on Sephadex. 17) Concanavalin A.000 D 8. capillary columns and separation conditions. protein pI Mol.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. a protein that binds carbohydrates. 14) Describe in detail the basic principle of gas chromatography with particular reference to mobile and stationary phases. explain. In order to purify the protein rapidly and to the highest purity. 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.0. Explain how affinity chromatography may be applied to this separation and explain the physical basis of the separation.500 C 7. The column distribution (available partition) coefficient for concanavalin A is greater than 1. 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.

0x12+1.011 mol l -1 3) K-value = change A/C. What is the major advantage of obtaining a mass spectrum of a chromatographic peak? MEMO BCH 314 Tutorial 1 1) C1V1 = C2V2 i. what does the mass spectrum show).e. 6) I =1/2 ∑ ci Zi2 1 M NaCl is 1 M in Na+ and Cl-.98 10 30 0.5 ml gives 0.0 1 M (NH4)2SO4 has 2 M NH4++1 M SO42-.0 Proteins are salted out at high ionic strengths. and precipitate. 19) Mass spectrometry is widely used as a detection system in gas chromatography. of glc = (2 x 1) / 180. therefore 1 ml = (0.05 40 1. Hence a protein should be most soluble in 1M NaCl and least soluble in 1 M K 3PO4 . Dilution factor is 2. ml H2O g ml-1 X Stock fold) 10 0. mol wt = 360. the added ions compete with the charged protein molecule for hydrating water molecules. therefore V2 = 5/0.97 15 40 0.2/5 x 10-3 (this comes from 0. For maltose = (100/360) x 10 -6 x 103 Mol/l = 0.e 0.16 g l-1` 2 mg ml-1 = 2 g l-1 Molar conc.99 5 20 0. therefore I=1/2(1.03 66.0x12+1.01 200 1. C = 0. a process called salting-out.5 2) (i) g X ml Stock dilution factor (n.02 100 1.2 x 1)/(5 x 10- 3 x 0. Describe what the mass spectrum of a molecule indicates (i.56 x 10 -3 Mol/l. 5 x 10-3 = 0.16 = 0.67 1.e. What is maltose? = 2 x glc.96 20 50 0.2 = 25 ml.0x22) = 3.5 x 10 = 0. therefore I=1/2(2. therefore I=1/2(3.2/C.95 25 (ii) 1 M glc = 180.0x12+1. However. 0.56 / 0.0x32) = 6.28 x 10-3 Mol/l Therefore Aglc/Amalt = 0. become less soluble.5) gml-1 4) Convert conc into µMoles/ml thus: glc: 100/180 µMoles/ml = (100/180) x 10-6 Mol/ml = (100/180) x 10 -6 x 103 Mol/l = 0. i.5 ml) i. to each technique and give details of the advantages of a mass spectrometer as a detector.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. Removal of excess salt will provide a more favourable environment for rehydration of the protein.04 50 1.2 x V2.2/5 x 10-3. as salt concentration increases.0 1 M K3PO4 has 3 M K++1 M PO43-.e.0x12) =1.

wts. Should it become neutral. taxonomy. provided a series of ampholytes that cover the entire pI range is used. food analysis. 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. the smoother the pH gradient. IEF is run in a pH gradient where the pH is low at the anode and high at the cathode. The capillary walls can be coated with methylcellulose or polyacrylamide to suppress EOF. The pH then will decrease at the anodic section and increase at the cathodic section. the ampholyte migration will cease when each ampholyte reaches its isoelectric point and is no longer charged. The pH of the anodic buffer must be lower than the pI of the most acidic ampholyte to prevent migration into the analyte. 3rd=chymotrypsinogen. agriculture. it will stop migrating in the electric field. the pI and migration halts. the catholyte must have a higher pH than the most basic ampholyte. haemoglobin variants and post-translational modifications of recombinant proteins. IEF is generally used for high resolution separations of proteins and polypeptides but could be used for any amphoteric substance. Ampholytes that are positively charged will migrate towards the cathode whilst those negatively charged migrate towards the anode. wt on the basis of differing pI’s. 9) Isoelectric focusing can separate proteins of the same mol. In addition to performing high resolution separations. Initially. 8) The fundamental premise of IEF is that a molecule will migrate so long as it is charged. a solute with a negative charge will migrate towards the anode where it encounters buffer of decreasing pH. Likewise. Applications: clinical chemistry and forensic pathology. Finally. It is apparent that the EOF (electroosmotic flow) and other convective forces must be suppressed if IEF is to be effective. IEF is particularly useful for separating immunoglobulins. IEF is useful for determining the pI of a protein. 12) i) 1st=Hb. cancer research. 4th=Lysozyme . The coating tends to suppress protein adsorption as well. 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. immunology and microbiology. Together a great resolution of large numbers of proteins can be achieved. wt. SDS gel electrophoresis can then separate proteins with the same pI’s on the basis of differing mol. the ampholyte mixture separates in the capillary.7) The original solution contains two proteins that differ in net charge (or pI’s) but have the same subunit mol. the solute encounters a pH where its net charge becomes zero. 10) Ethidium bromide contains a planar (flat) group that easily slides (intercalates) between the stacked bases of the DNA molecule. 2nd =ovalbumin. The greater the number of ampholytes in solution. Finally. When a voltage is applied. The pH gradient is generated with a series of zwitterionic chemicals known as carrier (poly) ampholytes.

Lys and HB (+ve) and elute without appreciable exchange. B & C are weakly negatively charged and come out second. Helium and Argon are the three most commonly used carrier gases. Order: i) lys’ ii) Hb’ iii) Ovalbumin At pH 6: Ovalbumin (-ve) and exchanges on DEAE. through which the mobile phase) is passed. step 3 = a). B is negatively charged and sticks to resin.000. depending upon the analysis. Commonly used stationary phases include the polyethylene glycols. either selective. C = 100.500. The selectivity of a packed GC column is largely determined by the chemical properties of the liquid stationary phase. and these are coated onto the support to give from 1% to 25% loading.000 . ii) ovalbumin pH4: All 3 proteins (+ve) and elute directly at the same time from column without exchanging.and methyl vinyl silicone gums (so-called OV phases). More often the phases used are high boiling point organic compounds.g. Too high a temperature results in excessive column bleed owing to the sample being volatilized off. 13) DEAE is an anion exchanger step 1 = a). . Hb. or non- selective. contaminating the detector and giving an unstable recorder baseline. The basic requirements for any GLC stationary phase are that it must be involatile and thermally stable at the temperature used for analysis. A is strongly negatively charged at this pH and sticks most tightly to the column. D is positively charged at this pH and elutes step 2 = c). succinic and phthalic acids. At pH 8: ovalbumin (most –ve. The choice of phase for analysis depends on the compound under investigation and is best chosen after reference to the literature. Lysozyme (+ve) and passes through without exchange. D comes out first (positively charged at pH 8.. The most negatively charged protein will bind more tightly to DEAE cellulose. B (MW) = 30.80 cm 3 min-1. where separation occurs by utilization of different chemical characteristics of components. At pH 8. where separation is achieved on the basis of differences in boiling points of the sample components. silicone grease) is supported on an inert granular solid. The operating temperature for the analysis must be compatible with the phase chosen for use. No separation. Such phases are of two types. methyl phenyl. This material is packed into a narrow coiled glass or steel column 1-3 m long and 2-4 mm internal diameter. D = 30. followed by HB. A stationary phase of a high boiling point liquid material (e. Order: i) lys. They are passed through the column at a flow rate of 40 . ii) Protein binding to ion exchange resins is a function of their net charge. Apiezon L and esters adipic. C comes out first & D last 14) Nitrogen.

hence. giving rise to baseline variation. to ensure that the compounds to be separated are kept in the vapour state and that analysis time is reasonable. Consequently some instruments have two identical columns and detectors. Consequently. The column is maintained in an oven at an elevated temperature. In WCOT columns the stationary phase is coated directly onto the walls of the capillary tubing. 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 are two types of capillary column systems known as wall-coated open tubular (WCOT) and support-coated open tubular (SCOT). 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. As there is only a small amount of stationary phase present. In GLC. however. one set of which is used as a reference. In SCOT columns a support material is bound onto the walls of the capillary column. 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. and the stationary phase is coated onto the support. assuming equal bleed from both columns. In the separation of compounds of widely differing polarity or Mr it may be advantageous to increase the temperature gradually. This. which can be operated in either of the two modes: In isothermal analysis a constant temperature is employed. The currents from the two detectors are opposed. 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. partition coefficients are particularly sensitive to temperature so that analysis times may be regulated by adjustment of the column oven. also known as porous layer open tubular (PLOT) columns for adsorption work. SCOT systems are therefore considerably simpler to use for quantitative analyses than are WCOT systems. only very small amounts of samples may be chromatographed. 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. though. This is referred to as temperature programming. Their efficiency. 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 . the resulting current gives a steady baseline as the column temperature is raised. often results in excessive bleed of the stationary phase as the temperature is raised. The remainder of the sample is vented to waste. is less than that of WCOT systems but considerably better than that of conventional GLC columns.

However. As an alternative approach. efficiency of the column is the degree to which it keeps zones from spreading).. The contaminating proteins are washed from the column and the bound protein eluted with ATP. 16) Affinity chrom is an elegant. 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). where separation is achieved purely on the basis of differences in boiling points of the sample components). may hydrolyse ATP and destroy the affinity probe. To identify compounds. The mol. authentic samples of the test compounds can be used for calibration or a mass spectrometer can be used. or empirically determined combination of these. or another protein in the crude extract. wt. The technique is based on the specific. strong binding of a substrate. Selectivity: the degree to which the column thermodynamically distinguishes between the 2 components. (i.g.or methylene analog). 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.e. 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. (remember larger proteins are eluted first. substrate analog. imido. determination will be low. inhibitor or antibody to a protein. (where separation occurs by utilization of different chemical characteristics of components or non- selective. since concanavalin will appear as a smaller protein which occupies more of the space inside the gel matrix. The sample is dissolved in organic solvent (hexane. The GC technique is applied extensively in: . pentane etc) and then injected using a microsyringe through a septum in the injection port. therefore making peaks narrower (by changing selectivity of column stationary phase). construct the affinity probe using a nonhydrolysable analog of ATP (e. increased ionic strength. they pass through a detector that is connected via an amplifier to a chart recorder. the protein of interest. which in turn. Polar samples (non- volatile) are derivatised (e.g. An ATP-binding protein(s) would be predicted to bind to affinity probe. by methylation). pH alteration. 17) Concanavalin A will have a larger Ve than expected since it binds to the carbohydrate gel matrix. As compounds emerge from the column.) The sample is soluble in the carrier gas. The spacer allows the protein to bind the affinity probe with minimal steric or chemical interference from the soluble support. small proteins last). rather specific method to separate a protein or an enzyme from a mixture. records a peak as each analyte passes through the detector.

when seen as part of the mass spectrum. bromine. representing a unique fingerprint of a molecule that can be used in identification. is referred to as the MOLECULAR ION. In the detector. 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. The relative percentages of these cluster ions provide more clues useful in unraveling the identity of a parent molecule from its molecular fingerprint. The masses of these fragments are used to deduce the structure of the parent compound. analysis of biological materials (detection of pesticides). 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). which is in the vapour phase. chlorine. the ion fragments generate an electrical signal that is proportional to the number of ions. due to the presence of functional groups in the molecule and their interconnection. and a few other elements. water and air contamination. prevention of pollution by detecting gas leaks. The sample. The data system records these electrical signals and then converts them into a mass spectrum. Occasionally the molecule is so totally fragmented by the ionizing process that little or no molecular ion is seen. 19) Mass spectrum is a recording of the masses of each of the ionized fragments. These clusters represent “naturally occurring impurities” or isotopes that are present for carbon. The mass spectra of certain compounds exhibit clusters of mass peaks. 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. The ionized parent molecule. mineral-oil and gas- exploration industries (finding gas and oil fields by ultra trace analysis of methane/ethane). Certain fragments are more prone to form from the parent molecule than others. 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. wine and food industry (for identification of flavour compounds). nitrogen. sulfur.