LAB 2

Question 1

what is spectroscopy

Answer 1

a technique that measures the amount of visible or ultraviolet light absorbed by molecules in a solution.

Question 2

how can we use spectrometry to identify molecules present in a solution

Answer 2

different molecules absorb light at different wavelengths

Question 3

what can spectrometry be used for

Answer 3

to determine the concentration of a particular molecule in a solution or to identify unknown molecules in a solution based on their absorbance characteristics

Question 4

what is the visible spectrum

Answer 4

refers to light wavelengths that are visible to the human eye and covers the wavelengths in the 400 to 700 nm range
it sits between the infrared (longer) and the ultraviolet (shorter) bands of the electromagnetic spectrum

Question 5

what does the wavelengths that get reflected or absorbed depend on

Answer 5

the chemical groups making up a molecules

Question 6

what is the absorbance maxima

Answer 6

each chemical will have a particular wavelength that is absorbed more than any other wavelength

Question 7

how do some molecules appear colorless molecules in a solution

Answer 7

they reflect wavelengths in the electromagnetic spectrum that are outside the light spectrum our eyes can see

Question 8

how can we measure colorless molecules in a solution

Answer 8

by using instruments capable of detecting other wavelengths on the electromagnetic spectrum

Question 9

how does a spectrophotometer work

Answer 9

light of a single wavelength is sent through a solution and the amount of light that is passed through the solution can be measured using a detector
it measures the amount of light that passes through the solution by comparing the amount of light going into the solution (incident light) with the amount of light coming out of the solution detector (transmitted light)

Question 10

what should you set the wavelength to when using a spectrophotometer

Answer 10

the absorbance maxima of the chemical substance you are measuring

Question 11

what does the spectrophotometer calculate

Answer 11

the log ratio of incident and transmitted light and displays this as an absorbance reading

Question 12

why does the absorbance reading have no units

Answer 12

its a ratio

Question 13

what is the relation of concentration to absorbance

Answer 13

the more molecules there are in a solution (higher concentration) the more light will be absorbed and the higher the absorbance reading will be

Question 14

what are the 2 variables that affect an absorbance reading at a specific wavelength

Answer 14

concentration and path length

Question 15

what is Beer’s law

Answer 15

states that the concentration of a solution is linearly proportional to the absorbance of that solution

Question 16

what is Lambert’s law

Answer 16

states that the intensity of the transmitted light is linearly proportional to the path length

Question 17

what is beer-lambert’s law

Answer 17

absorbance = molar absorption coefficient * concentration * path length

Question 18

what is the molar absorption coefficient

Answer 18

a measure of how strongly a chemical absorbs light at a particular wavelength and is an intrinsic property of the molecule

is a constant value at a particular wavelength for a particular molecules

a measure of 1 mol/L solution at a specified wavelength and a standard light path of 1 cm

Question 19

what is a reference sample/blank and what is it used for

Answer 19

a reference sample contains everything in an assay, except the compound that is being measured and is used to xero the spectrophotometer before measurements are taken

Question 20

what is a standard curve/calibration curve

Answer 20

a type of graph that relates the concentration of a molecule to its absorbance at a particular wavelength

Question 21

what is the standard curve used for

Answer 21

can be used to determine the concentration range over which the beer-lambert law applies for a particular molecule

Question 22

when does the beer-lambert relationship apply when using the standard curve

Answer 22

only applies when there is a linear relationship between concentration and absorbance
- at higher concentrations it becomes non-linear due to multiple factors

Question 23

how is a standard curve made

Answer 23

by measuring the absorbance of a series of standards - a solution containing a known concentration of a chemical substance
the absorbance values can be plotted against the known concentrations of these standards, forming the standard curve graph

Question 24

what are enzymes

Answer 24

proteins that catalyse biological reactions

Question 25

how do enzymes catalyse reactions

Answer 25

they increase the rate of a reaction (conversion of substrates into product) by lowering the activation energy needed to start the reaction

Question 26

what does an enzyme assay investigate

Answer 26

how well an enzyme catalyses a reaction and is essential to the study of enzyme kinetics and inhibition

Question 27

what can influence the rate of an enzyme catalyses reactions

Answer 27

- temp - pH - substrate concentration - enzyme concentration - the presence of an enzyme inhibitor

Question 28

how do we avoid the influence of factors that affect the rate in the lab

Answer 28

by running enzyme assays using standard lab conditions or standard biological conditions

Question 29

what is alcohol dehydrogenase

Answer 29

an enzyme produced in the liver that breaks down primary alcohols into aldehydes

Question 30

what are dehydrogenase enzymes usually involved inn

Answer 30

in redox reactions meaning they need a co enzyme to reduce/oxidize for the reaction to proceed

Question 31

what is the co factor in the case of human alcohol dehydrogenase

Answer 31

NAD+ is reduced to NADH + H when ethanol is oxidized to acetaldehyde

Question 32

how can enzyme assays track a reaction

Answer 32

by measuring the appearance product or disappearance of substrate

Question 33

what are the 2 substrates in alcohol dehydrogenase catalysed reactions

Answer 33

ethanol and NAD+

Question 34

what are the 2 products in alcohol dehydrogenase catalysed reactions

Answer 34

acetaldehyde and NADH/H+

Question 35

why do we measure the absorbance of NADH rather than acetaldehyde or ethanol

Answer 35

ethanol doesn't absorb well at any of the wavelengths acetaldehyde's absorbance maxima does not overlap with any of the others maxima NADH has a second peak thats in our range that we can measure

Question 36

why do we measure the initial rate/initial velocity

Answer 36

the rate of an enzyme reaction may vary considerably during the course of an enzyme assay

Question 37

what does the slope of the tangent of the progressive curve indicate

Answer 37

V0 (initial velocity/rate)

Question 38

what happens when substrate concentration is increased

Answer 38

as substrate concentration increases the initial rate of reaction will get closer and closer to Vmax resulting in a hyperbolic curve on the V vs [S] plot

Question 39

how does an inhibitor affect enzyme activity

Answer 39

the presence of inhibitors can change the rate of an enzyme-catalysed reaction by interfering with their ability to catalyse the reaction

Question 40

how do we determine which inhibitor is present

Answer 40

the kinetic parameters for an enzyme assay in the presence or absence of an inhibitor can be compared to determine what sort of inhibitor the compound is and how effectively the compound inhibits the reaction