biochemical tests

Question 1

what is meant by ‘making serial dilutions’ ?

Answer 1

make up a set of dilutions diluted by a particular factor each time

Question 2

what is meant by ‘making a series of dilutions’?

Answer 2

choose a set of concentrations to make up, usually with even intervals

Question 3

what is the equation for making a dilution series?

Answer 3

C1 x V1 = C2 x V2

c1= conc of stock solution
v1= volume of stock solution used to make new conc
c2= conc of solution you’re making
v2= volume of new solution you’re making

Question 4

what are the two methods for judging the concentration of sugar present in an unknown sample?

Answer 4

-comparing to known standards
-colorimetry

Question 5

describe how comparing to known standards would help identify the sugar concentration of an unknown sugar solution?

Answer 5

-make up a range of known concentrations of sugar (known standards)
-then carry out a valid Benedict’s test on each of the known standards and the unknown solution

Question 6

give examples of things to ensure when carrying out a valid benedict’s test?

Answer 6

-give each solution the same amount of time before colour is recorded
-same conc of benedicts
-volume of samples same
-same temp for heating

Question 7

what is the problem with comparing to known standards?

Answer 7

it is subjective- based on people’s own judgement

Question 8

what is the basic idea behind colorimetry?

Answer 8

when light is shone on a sample of liquid, not all the light will be transmitted through it- some will be absorbed

Question 9

why are different coloured filters placed in front of the light in the colorimeter?

Answer 9

to select the wavelength of light that the solution will absorb the most, and so makes the colorimeter more accurate

Question 10

what do we have to do before we use a colorimeter to determine the concentration of an unknown sugar solution?

Answer 10

-set up the colorimeter with a blue filter
-calibrate the colorimeter with water
-make up a series of known concentrations of sugar solution
-carry ot a valid benedicts test on all the known and unknown concentrations
-place the tubes in a cold bath to quickly cool

Question 11

describe how we we would use the colorimeter to determine concentration of sugar solutions?

Answer 11

-place each known concentration in a cuvette and read the % absorbance using the colorimeter
-plot a calibration graph to show the absorbance at different known concs

x axis on the graph will be conc of sugar solution, y will be % absorbance

-use the colorimeter to read the % absorbance for the unknown sugar conc
-use graph to read off conc of sugar for this absorbance

Question 12

what is chromatography?

Answer 12

a technique that is used to separate different substances so that they can be identified

Question 13

what factor is used to separate different substances in chromatography?

Answer 13

solubility in the solvent

Question 14

if a substance is completely soluble in the solvent, where will it be found on the chromatography paper?

Answer 14

on the origin line

Question 15

if a substance has the highest solubility in the solvent, where will it be found?

Answer 15

on the solvent front

Question 16

describe a method for chromatography?

Answer 16

-draw a line at the bottom of the chromatography paper with pencil; this is the origin line

-place a small amount of the sample onto the paper at the origin, let it dry before adding more drops to build up the concentration

-place the paper in the solvent up to the origin line and allow the solvent to run

-before the solvent reaches the top of the paper remove the paper, let it dry and draw the line where the solvent reached; the solvent front

Question 17

what are the two ways of identifying the substances that have been separated during chromatography?

Answer 17

-comparing to known standards
-calculation of Rf values and comparison to Rf values in tables for the same solvent

Question 18

explain how comparing to known standards in chromatography works?

Answer 18

unknown substance and a series of known standards spaced out along the origin line. They all move up the paper and whatever is in the unknown substance can be identified if it moves the same distance as one of the known standards

Question 19

what is the Rf value?

Answer 19

the position the substance has travelled to compared to the position of the solvent front

Question 20

what is the equation to work out Rf value?

Answer 20

distance travelled by compound from the origin // distance travelled by the solvent from the origin

Question 21

what is the maximum Rf value?

Answer 21

1 (would be completely soluble)

Question 22

describe the test for reducing sugars?

Answer 22

add equal volume of benedict’s reagent to the liquid sample
heat the mixture in a gently boiling water bath for five minutes
if reducing sugar is present the solution turns from blue to brick red

Question 23

describe the test for non reducing sugars? (sucrose)

Answer 23

first carry out a valid benedicts test and if the solution remains blue then….

-add equal volume (eg 2cm3) of dilute hydrochloric acid to liquid sample
-and place the test tube in a gently boiling water bath for 5 minutes
-slowly add some sodium hydrogen carbonate solution to the test tube in order to neutralise the HCl (benedict’s won’t work in acidic conditions)
-use pH paper to check the solution is alkaline
-retest the solution by carrying out a benedicts test and observing from a colour change of blue to orange-brown (this is because of the reducing sugars which are present from the hydrolysis of the non-reducing sugars)

Question 24

describe the test for lipids?

Answer 24

add ethanol, shake, add water, shake
a milky-white emulsion indicates lipids

Question 25

describe the test for proteins?

Answer 25

add biurets reagent to solution
colour change from blue to purple if protein is present