8304462 173 Food Tests and Chromatography

8/8/2019 8304462 173 Food Tests and Chromatography

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Bfu FactsbmHowto identify oods:FoodTests ndChromatography

Food tests and chromatography are techn iques used fo r therecognition of biologically important chemical compounds (examquestions on these techniques are common). Th e use of thesetechniques is not limited only to the food industry. Detectives canuse chromatography to analyze fibres found at a crime scene andwhen used in conjunction with sniffer dogs, gas chromatographyis a common way to identify explosives in airports, it has also beenused in schools to perform drug screening.

Why do we need two methods for identifying foods?Food tests are less precise. they basically identify foods into thecategory of proteins, lipids, starch, cellulose, reducing sugars ornon-reducing sugars, whereas paper chromatography can identifyspecific molecules, fo r example, finding ou t which amino acids ormonosaccharides are contained in a mixture.

Food TestsIt is \rorth earninghe ood estsas t canmeaneasymarks n an exam, t is pure ecall. In an examquestion lwaysquote he reagent sed,method, tartingolourandendcolour or a positiveesult.Table 1. Food tests

BiologicalMolecule Reagent/s usedto test Method Starting Colour EndColour(positive result)Reducing Sugar(for exampleglucose, fructose,maltose.

Benedict 's solution( I lVo CuSO,) Add 2cm3 Benedict's solution an equalamount of the test sample, heat over awater bath.

Blue Orange/Red recipitate.

Non-reducingSugar (sucrose) Benedict 's solut ion( l lVo CuSOu), Cl,

NaHCO,l. Boil test sample with HCI (t o break

th e glycosid ic bonds).2. Neutralise with NaHCO.,/alkali(otherwise the HCI would react with

the Benedict 's solution).3. Add Benedict's solution to the test

sample, heat over a water bath.

Blue Orange/Red precipitate

Starch Iodine solution (KI) Add a few drops of Iodine solution tothe test sample.

Yellow/brown Blue-blackLipids The Emulsion Test

ethanol (C.H'OH)and water

l. Shake some of the test sample withabout 4cm3 of ethanol.

2. Decant the liquid into a test tube ofwater, leaving any undissolvedsubstances behind.

Clear Formation of a cloudywhite emulsion.

Proteins Biuret reagent(alkalineCUSO,)Add a few drops of Biuret reagent tothe test sample

Blue Lilac/purple precipitateCellulose Schultze's solution Add a few drops of Schultze's solution

to the test sampleYellow Purple

Three tips on food tests with sugarsl. To distinguish betwecn reducing ard non-reducing sugars, fiIst test a sample for reducing suga.rs,o see f there are any present, f nopositiveresult s achieved est for non-reducingsugars.2. wren testing for a reducing sugar it is not possible to distinguish between glucose and fructose, pa!,er chromatographywould needto be used.3. When testing for a non-reducingsugar the solution will be neutralised when it stops fizzing,


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173.How to dentify oods:Food estsandchromatography Bio Factsheettt,wyv.urri cu um-D ess. o.ukChromatography

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There are many different types of chromatography:l. Paper chromatography is the simplest, but does not alwaysgive very clean separation. It can be used for separatingaminoacids, anions, RN A fingerprinting, separating an d testinghistamines and ant ib iot ics.Thin layer chromatography (TLC) usesa thin layer of celluloseor si l ica coated onto a plast ic or glass sheet. This is moreexpensive, but gives much better and more reliable separationthis can be used fo r detecting pesticide or insecticideresiduesin food. It can also be used in forensics to analyze the dvecomposition of fibres.Column chromatography uses a glass column filled withcellulose slurry. Large samples can be pumped through thecolumn and the separated ractions can be collected for furtherexperiments, so this is preparative chromatography as opposedto analytical chromatography.

High performance liquid chromatography (HPLC) is animproved form of column chromatography that deliversexcellent separationvery quicklv. It can be to used test watersamples o look for pollution by analysing metal ions an dorganic compounds in solutions. It uses l iquids which ma yincorporate hydrophilic, insoluble molecules.Gas chromatography is used in airpor-ts o detect bombs andis used s forensics in many different ways. It is used to analyzefibres on a person's body and also analyze blood found at acrime scene. In gas chromatography helium is used to move agaseousmixture through a column of absorbent material.Electrophoresis uses an electric current to separatemoleculeson the basis of charge. It can also be used to separateon thebasisof molecular size,and as such s used n DNA sequencing.

4.

5.

Paper ChromatographyOn what principledoespaperchromatographywork?The pr inc ip le i s based on rhe so lub i l i t y o f chemica ls (ca l led'solutes') i.e. how fast they dissolve in a solution. A solvent canthen separatethe solutes for example, if a solute is very soluble itwill dissolve quickly in the solvent and as the solvent travels up themedium (paper) by capillary action it will separateout first on thepaper before the solvent dissolves out the next solute. The resultof this is seeing spots on the paper which representseparatesolutes.What types of biochemical molecules can be tested for?1. coloured molecules for example, ink, chlorophyll and fruit juice,

these do not have to be stained as they are already coloured.2 . Co lou r less m o lecu les fo r exam p le , an am ino ac id o rmonosaccharidemixture. These have to be stained for example.ninhydrin is used to stain amino acids.MethodA Chromatography tank should be set up as below:

chromatographytank

chromatographypaperonglnso l ven t

Pour some solvent into a chromatography tank and seal it, thisis so that the atmosphere is saturated with solvent vapour.Different solvents are suitable for different tasks, but they areusually mixtures of water with organic liquids such as ethanolor propanone.Place a drop of the mixture to be separated onto a sheet ofchromatography paper near one end, this is the origin of thechromatogram. The spot should be small but concentrated, thisis achieved by spotting and drying, spotting and drying and soon , drying the spot stops it spreading. Repeat fo r any othermixtures. Label the spots with pencil, as ink may dissolve.

3. Place the chromatography sheet into the tank so that the originis just above the level of solvent, and leave for several hours.The solvent will rise up rhe paper by capillary action carryingthe contentsof the mixture with it . An y solutesdissolved n thesolvent wi l l be partit ioned between th e organic solvent (themoving phase) and the water, which is held by the paper (thestationary phase). The more soluble a solute is in the solventthe further up the paper it will move.4. when the solvent has nearly reached the top of the paper, thepaper is removed and the position of the solvent front marked.The chromatogram may need to be developed to make the spotsvisible for example,someamino acidsstainpurple with ninhydrin.What to do if the separation of molecules is poorSometimes chromatography with a single solvent is not enough toseparateall the constituentsof a mixture, the spots may appear as asmear. I f this happens separation ca n be improved by two-waychromatography.The chromatography paper is tumed through 90" and run a secondtime in a different second solvent. Solutes that didn't separate none solvent will separate n another because they have differentsolubilities. The diagram below demonstrated this:

rotateth rough90"

rid

original spotSecond run

original


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173.How to dentify oods:FoodTests ndChromatography Bio Factsheetlrytyr.g11vviul um-o ress.c o. uk

Analysis of the ChromatogramMethods for analysing a chromatogram that can be used are:l. Run known substance/salongside unknown and s pot positions

compared to the known substances.2. Work out how far the substance has moved compared with the

solvent, his is called R, value (hint: it is always ess han I ). Theformula for R. value is:Distancemoved by the spot

Distancemoved by the solvent rontThese methods are possible as a particular substancewill travel thesame distance up the paper when a known solvent is used understandard condit ions.Howeve r , t hese two m e thods can on ly be used to iden t i f ysubstances. Another method must be adopted to quantify th esubstances. .e. how to work out how much is there of each aminoacid or monosaccharide in the sample.A colorimeter is used to detect the shadeof the colour - the darkerthe colour, the more the amino acid or sugar here s i.e. the colorimetermeasures concent ra t ion o f samples by measur ing the co lourconcentration within them.

4. Elute the spot i.e. cut it out and dissolve into a set amount ofsolvent, place it in a cuvette.

original spot First run5. Put th is in to the co lo r imeter and read of f the percentage

transmission for example if the percentage transmission was60Va this value can now be read off the calibration curve todetermine the concentration.

Typical Exam QuestionThe amino acids in a sample of fruit juice were separatedby paperchromatography. The pattern obtained is shown below:

so lven tfront

origin

Mix Glycine Alanine Leucinel. Which component is the most soluble?2. How many amino acids are found in the mixture?3. What amino acids are not found in the mixture?4. If the mixture contained 4 amino acids but only 3 were seen on

the chromatogram, what does it mean?5. What is the R, value of Leucine?6. If the mixture was a protein, how would you separate t out into

its amino acids?7. How would you work out the amount of glycine in the mixture(n o details needed)?Answers1. The one furthest away/Alanine.2. Three.3. Leucine.4. One amino acid is insoluble in that solvent.5 . 6 t8 -0 .75 .6. Hydrolyse with protease or acid and then run chromatography.7. Use a colorimeter.A.,Je" LdEan*:Ilu FtLt.shtLtwu rctatn h.l dlp \rnto h\ Johil Cre.hdLghCwbtltanPrcx tunk House, )05 lintg sece$dnrtgb,t, Shmphire,TFl IIA-.

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concentrations of for example. amino

on a graph

| | r lUtJ[J.3v 0.41,t 05uPlace each concentration into a cuvette (a flat sided test tube),then place in the colorimeter and read off the percentagetransmission.

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8304462 173 Food Tests and Chromatography

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