Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

Molecules to Cells - Practicals > Spectrophotometry and Chromatography > Flashcards

Spectrophotometry and Chromatography Flashcards

You may prefer our related Brainscape-certified flashcards:
Organic Chemistry 101 flashcards
1
Q

What is gel filtration chromatography ?

A

A method of separating substances according to their molecular sizes

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

What are the two other names for gel filtration chromatography ?

A
  1. Gel permeation chromatography

2. Size exclusion chromatography

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

What happens to substances that interact weakly with the stationary phase ?

A

They will pass through with minimal delay

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

What happens to substances that interact strongly with the stationary phase ?

A

They will be slowed the the greatest extent

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

What is the liquid mobile phase collected as as it leaves the column ?

A

Fractions

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

What flows through the column ?

A

The liquid mobile phase

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

Where is the stationary phase packed into ?

A

A column

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

What is the mobile phase ?

A

A liquid or gas that acts as a solvent carrying the components of the mixture through the stationary phase

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

What is a chromatographic bed ?

A

A column or a thin layer of the stationary phase material

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

What is the stationary phase ?

A

A solid, gel or an immobilised liquid

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

What is chromatography ?

A

A method for separating the individual components of a mixture on the basis of differences in physical or chemical characteristics

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

Does the Beer-Lambert law have units ?

A

No

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

What is the Beer-Lambert law ?

A

Relates the absorbance of a solution to the concentration of the solute and thickness of the solution

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

What is the absorbance of a compound in a solution directly proportional to ?

A

The concentration of the compound

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

What are the two light sources of spectrophotometers and what do they emit ?

A
  1. A tungsten filament lamp - Visible light

2. Deuterium lamp - UV light

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

What are the essential components of a single beam spectrophotometer ?

A
  1. Light source
  2. Monochromator
  3. Cuvette containing sample
  4. Detector
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

Give an example of a spectral shift in the human body ?

A

Oxygenation of haemoglobin causes a colour change from the purplish hue characteristic to the brilliant scarlet colour of arterial blood

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

What can chemical modification of a compound produce ?

A

Spectral shifts

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

Why is nucleic acids absorbance maximum at 260nm ?

A

Due to the presence of aromatic bases

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
20
Q

Why is proteins maximum absorbance at 280nm ?

A

Due to the presence of tyrosine and tryptophan

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
21
Q

What is the characteristic absorbance of DNA ?

A

260nm

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
22
Q

What is the characteristic absorbance of proteins ?

A

280nm

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
23
Q

What can be used to identify a compound ?

A

The unique absorption maxima and minima

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
24
Q

What is the ability of a compound to absorb light a function of ?

A

Its chemical structure

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
25
Q

Why do haemoglobin solutions appear red ?

A

The blue-green wavelengths are absorbed by haemoglobin and the red wavelengths are transmitted to the eye

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
26
Q

What wavelengths are human eye photoreceptors sensitive to ?

A

400 - 700nm

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
27
Q

What are two photoreceptors in the human eye ?

A
  1. Rhodopsin

2. Visual pigments

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
28
Q

Why do substances that absorb visible radiation appear coloured ?

A

The selective absorption of certain wavelengths from the complete spectrum present in ordinary white light

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
29
Q

Where do conjugated double bonds appear in the human body ?

A

Many of the compounds that appear coloured to the eye, for example chlorophyll and haemoglobin

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
30
Q

At visible wavelengths, what way are electrons whose energy levels are closest together ?

A

The ones that are excited

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
31
Q

At visible wavelengths, what strength is the energy of the radiation ?

A

Relatively weak

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
32
Q

What are conjugated double bonds ?

A

Alternating and single double bonds

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
33
Q

What is the wavelength of maximum absorption defined as ?

A

Lambda max

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
34
Q

What is the absorbance maximum of tyrosine and tryptophan centered on ?

A

A wavelength of 280nm

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
35
Q

What is the absorption spectrum of a compound shown by ?

A

As a plot of incident lights absorbed by the compound as a function of wavelength

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
36
Q

What is the absorption spectrum of a compound ?

A

The fraction of radiation absorbed by the material over a range of frequencies

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
37
Q

What are two examples of biologically important aromatic compounds ?

A

Tyrosine and tryptophan

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
38
Q

What are transitions between the pi orbitals in aromatic compounds induced by ?

A

UV radiation

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
39
Q

What do visible and UV radiation induce changes in ?

A

The energy of the molecules outer electrons

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
40
Q

What effects how radiation interacts with matter ?

A
  1. The energy of the radiation

2. The properties of the molecules involved

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
41
Q

What wavelengths are most useful to biochemists and why ?

A

200 - 800nm range

These are the UV to visible wavelengths

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
42
Q

When is their less energy contained in radiation ?

A

The longer the wavelength

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
43
Q

What happens to energy, the longer the wavelength ?

A

The less energy is contained in the radiation

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
44
Q

What is the wavelength related to ?

A

The energy of the radiation

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
45
Q

What is the symbol for wavelengths ?

A

Lambda

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
46
Q

What can wavelengths be measured in ?

A
  1. Metres
  2. Millimetre
  3. Micrometre
  4. Nanometre
  5. Picometre
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
47
Q

What are the different types of radiation that make up the electromagnetic spectrum characterized by ?

A

Their wavelengths

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
48
Q

What is the electromagnetic spectrum associated with ?

A

The transmission of energy in waveform

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
49
Q

What does the electromagnetic spectrum consist of ?

A

Diverse types of radiation

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
50
Q

At visible wavelengths, what strength is the energy of the radiation ?

A

Relatively weak

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
51
Q

In gel filtration chromatography, what is the stationary phase ?

A

A synthetic polymer with a molecular structure that contains a range of molecular sized holes

52
Q

In gel filtration chromatography, what does the term excluded mean ?

A

When the size of the molecule is greater than the largest of the molecular holes in the stationary phase. They move down a column at the greatest speed and elute from the column first.

53
Q

In gel filtration chromatography, what does the term included mean ?

A

Small molecules can penetrated the buffer inside the gel particles and therefore constantly move into and out of the the gel particles and partition between the volumes of buffer that are outside and inside the gel network

54
Q

Why do smaller molecules spend half their time in the moving interstitial liquid and what does this result in ?

A

The volumes of the buffers that are outside and inside the gel network are approximately equal.

They move at half the speed of the larger molecules

55
Q

What is gel filtration chromatography often used for ?

A

Investigating relative molecular mass of a molecule

56
Q

In the practical, what is the stationary phase used ?

A

Sephadex

57
Q

What is sephadex ?

A

1-6 linked glucose polymer (dextran)

58
Q

What determines the range of the size of molecules that can penetrate the gel (in the practical) ?

A

The degree of cross linking of the dextran and the resultant pore size in the gel beads

59
Q

We used sephadex G-25, what is it used for ?

A

Very highly cross linked and excludes molecules with a relative molecular mass greater than 5000

60
Q

What molecule has a relative molecular mass greater than 5000 and what happens to them in gel filtration (practical)?

A
  1. Proteins

2. Proteins are excluded from sephadex G-25

61
Q

What does sephaadex G-25 also do ?

A

Removes inorganic salts from protein preparations

62
Q

What is desalting ?

A

Removing inorganic salts from protein preparation

63
Q

Other than separation, what can gel filtration also be used for ?

A

Treating protein with a reagent

64
Q

How is a protein treated with a reagent in gel filtration ?

A

The protein moves through a zone of the reagents and it reacts efficiently, since at each layer it meets fresh reagent after it has partly reacted. It is then separated from the reagent by continuation of the elution.

65
Q

Why was haemoglobin chosen in this practical ?

A

It is coloured and changes in its colour can be brought about by the reaction with reagents in chromatography

66
Q

Why do colour changes occur in haemoglobin during gel filtration chromatography and what do they illustrate ?

A
  1. The changes in oxidation state of the haem moiety

2. Haemoglobin function and dysfunction

67
Q

What is a porphyrin ring system ?

A

Conjugate acids of ligands that binds metals to form complexes

68
Q

What does haem consist of ?

A

A porphyrin ring system to which is bound a single ferrous ion (fe^2+)

69
Q

How many bonds does the ferrous ion have ?

A

Six coordination bonds

70
Q

Where are the bonds located in the ferrous ion ?

A

4 to nitrogen atoms in the plane of the porphyrin ring and two perpendicular to the plane of the ring

71
Q

What helps prevent conversion of the ferrous iron to the ferric state ?

A

The coordinated nitrogen atoms which have an electron donating character

72
Q

When can we see a single broad absorption band in the visible region, which is a purple/red colour ?

A

When a histidine residue of a polypeptide chain fills one of the plane coordination positions and the sixth position is empty

73
Q

When can we see two very sharp absorption bands and a scarlet colour is visible on haemoglobin ?

A

The last valency is filled by an oxygen molecule (oxyhaemoglobin)

74
Q

When is methaemoglobin formed ?

A

Ferricyanide can be used to oxidise the the ferrous iron of haemoglobin to ferric ion

75
Q

What does ion-exchange chromatography depend on ?

A

Electrostatic attraction between species of opposite charge

76
Q

What do ion exchangers usually consist of ?

A

Modified derivatives of some support material such as cellulose

77
Q

How has DEAE-cellulose been modified ?

A

So it carries an ionizable tertiary amine group

78
Q

What is the pKa of DEAE cellulose and where will it be protonated ?

A
  1. Roughly 10

2. pH of 7

79
Q

When is an ionizable group protonated ?

A

1 pH unit below the pKa

80
Q

When is an ionizable group deprotonated ?

A

1 pH unit above the pKa

81
Q

What binds to DEAE cellulose at pH of 7

A

Anions

82
Q

How is CM-cellulose been chemically modified ?

A

So it carries a carboxymethyl group

83
Q

What is the pKa of CM-cellulose ?

A

Roughly 4

84
Q

What happens to CM-cellulose at a pH of 7 ?

A

The carboxyl group is fully ionized and therefore negatively charged

85
Q

What is CM-cellulose called and what does it bind ?

A
  1. Cation exchanger

2. Positively charged speices

86
Q

What is an anion exchanger ?

A

Binds negatively charged species

87
Q

What are some ionizable molecules ?

A
  1. Several side chain groups of the amino acid residues on proteins, for example lysine and glutamic acid
  2. The N-terminal amino and the C-terminal carboxyl groups
88
Q

What does the overall charge a protein carries at a particular pH depend on ?

A

The pKa values of its constituent charged amino acids

89
Q

What is the isoelectric point of a protein?

A

Is the point at which the protein carries no net charge

90
Q

When is a protein negatively charged ?

A

If pH is greater than pI

91
Q

When is a protein positively charged ?

A

If pH is less than pI

92
Q

What is the pI of acidic proteins ?

A

pI less than 7

93
Q

What is the pI of basic proteins ?

A

pI greater than 7

94
Q

What happens when a mixture of proteins is passed through a column of DEAE cellulose ?

A

Positively charged proteins pass straight through whereas negatively charged proteins stick to the column

95
Q

How could you elute the bound proteins from the column ?

A
  1. Lowering the pH will neutralize the negative charges on the protein molecules and cause them to fall off
  2. Raising the concentration of anions in the mobile phase will elute protein from the column since there will be more negative ions in the medium to compete with the protein for the positive charges of DEAE cellulose
96
Q

What are three applications of gel filtration chromatography ?

A
  1. Determination of relative molecular size of proteins
  2. Separation of molecules on the basis of size
  3. Removal of inorganic ions from preparations of protein
97
Q

What are three applications of ion exchange chromatography ?

A
  1. Separation of molecules on the basis of charge
  2. Water softening, removal of Ca ^2+
  3. Analytical chemistry- high performance liquid chromatography
98
Q

What are protons present as in solutions ?

A

There hydrated forms, as hydronium ions

99
Q

Do weak acids dissociate completely in water ?

A

no

100
Q

What are present in a solution at equilibrium ?

A

Both acid and conjugate base

101
Q

What is defined by the dissociation constant Ka ?

A

The strength of the acid HA

102
Q

What do square brackets indicate ?

A

Concentration in moles per litre

103
Q

The stronger the acid…….

A

The larger the value of the dissociation constant

104
Q

How can the pKa of any weak acid be determined ?

A

Titration with a strong base

105
Q

What is a buffer region ?

A

The flat region of a curve which extends 1 pH unit either side of the pH of the acid, there is resistance to a change in pH

106
Q

Explain Le Chatelier’s Principle.

A

If a dynamic equilibrium is disturbed by changing the conditions, the position of equilibrium shift to counteract the change to re-establish an equilibrium

107
Q

What is the pH of the equimolar solution equal to ?

A

The pKa of acid

108
Q

What is a buffer solution ?

A

A mixture of a weak acid and its conjugate base or a weak base and its conjugate acid

109
Q

Where does the innate resistance of biological fluids to changes in pH come from ?

A

The presence of cellular constituents that acts as buffers eg. proteins

110
Q

What is the shape of a titration curve of any weak acid described as ?

A

Henderson-Hasselbalch equation

111
Q

What is the pKa of amino acid carboxyl groups ?

A

1.8-2.4

112
Q

What is the pKa of amino acid amino group ?

A

8-11

113
Q

What is the pKa of the aspartate group ?

A

3.7

114
Q

What is the pKa of the lysine group ?

A

10.5

115
Q

What is nicotinamide adenine dinucleotide (NAD+) ?

A

A coenzyme present in all living cells

116
Q

What is nicotinamide adenine dinucleotide involved in ?

A

Involved in redox reactions, transferring electrons from one molecule to another

117
Q

What are the two forms nicotinamide adenine dinucleotide found in ?

A
  1. The oxidised NAD+ accepts electrons from other molecules

2. Reduced to NADH which can be used as a reducing agent

118
Q

What is the absorption maximum of NAD+ ?

A

260nm

119
Q

What is the second absorption maximum of NAD+ ?

A

340nm

120
Q

What is the measure of enzyme activity ?

A

The change in absorbance per minute

121
Q

What is the initial rate ?

A

The initial linear portion of the reaction

122
Q

What is the function of glucose oxidase in ion exchange chromatography ?

A

Catalyzes the oxidation of glucose to glucono-1,5-lactone, which hydrolyzes to gluconic acid

123
Q

What enzyme is required to catalyze the oxidation of glucose to glucono-1,5-lactone which then hydrolyzes to gluconic acid ?

A

Cofactor flavin adenine dinucleotide (FAD)

124
Q

What colour is FAD ?

A

Yellow

125
Q

What colour is FADH2 ?

A

Colourless

126
Q

What does FAD work as ?

A

Initial electron acceptor

Molecules to Cells - Practicals (5 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions