Exam I Flashcards
1
Q
What is the range of a P10 micropipette?
A
.2 microliters to 10 microliters
2
Q
What is the range of a P-20 micropipette?
A
2 microliters to 20 microliters
3
Q
What is the range of a P200 micropippette?
A
20 microliters to 200 microliters
4
Q
What is the range of a P1000 micropippete?
A
200 microliters to 1000 microliters
5
Q
What are important considerations for micropipettes?
A
Never
- over-rotate
- use without a tip in place
- lay the pipetteman down when it contains liquid
- let the plunger snap back
- immerse the barrel of the pipetman
Always use a disposable tip
6
Q
How can you express concentration?
A
Mass % = mass of solute/volume of solvent
Parts per million = 1g/1000000 mL
Molarity (M) - moles of solute/L of solvent (used most often in this class)
Morality (m) - moles of solute/ kg of solvent
7
Q
What are the three variations of expression consentration?
A
Weight per volume %
Volume per Volume %
Weight per Weight %
8
Q
How is molecular weight expressed?
A
G/mol
9
Q
How is the molecular weight of a protein expressed?
A
Daltons = g/mol
10
Q
What is the equation to calculate the amount of solid to weigh out to make a given volume of solution at a particular concentration?
A
G/mol * mol/L * L = g
MW of solid * required molarity * required volume = amount to be weighed
11
Q
How do you convert from % solution to molarity?
A
Molarity = (% solution * 10)/MW
12
Q
What does percent mean?
A
Parts per hundred grams or hundred ml
13
Q
What is the general formula to convert dilutions?
A
C1V1 = C2V2
14
Q
What involves techniques to monitor the interaction of light with matter?
A
Spectrophotometer
15
Q
What is a form of electromagnetic radiation that has two components: electric and magnetic, which oscillate perpendicular to each other?
A
Light
16
Q
What equation gives the energy of electromagnetic radiation (EM)?
A
E = hv
V is frequency, Hz, 1/sec
H is planck’s constant = 6.62410^-34 Jsec
17
Q
What equation gives frequency?
A
V=c/lambda
C = speed of light (2.998*10^8 m/s)
Lambda is the wavelength
18
Q
What is the range of wavelengths for visible light?
A
340 to 800 no
19
Q
What is the range of wavelengths for UV light?
A
200 to 340 no
20
Q
In what state do the electrons spin opposed to each other?
A
Singlet
21
Q
In what state do electrons spin parallel to each other?
A
Triplet
22
Q
Which state has the lower energy?
A
Triplet
23
Q
Singlet to triplet transition has a very low what?
A
Probability
24
Q
What transition of state to state is more probable?
A
Singlet to singlet
25
When energy goes from ground state to a singlet we say that the light was what?
Absorped
26
When energy goes from a singlet to ground state we call it what?
Fluorescence
27
When energy goes from a triplet to ground state we call it what?
Phosphorescence
28
When wavelengths are absorbed the transmitted wavelengths will be seen as what?
Complementary color
29
What is a plot of the amount of light absorbed by a sample as a function of wavelength?
Absorption spectrum
30
What molecules go from sigma to anti bonding sigma?
Aliases
31
What molecules go from pi bonds to anti bonding pi bonds?
Alkenes
Carbonyl compn
Sulfur
Halogen compounds
32
What molecules go from non bonding to anti sigma bonding?
Oxygen, nitrogen, sulfur, and halogen compounds
33
What molecules go from non bonding to anti pi bonds?
Carbonyl compounds
34
What is a weak transition in the range of 200 to 800 nm?
Non bonding to anti pi bonds
35
What is an intense transition in the range of 200 to 800 nm?
Pi to anti pi bonds
36
Increasing the number of conjugate does what?
Increase the wavelength absorbed
37
What is a covalently unsaturated group responsible for electronic absorption?
Chromosphore
38
What is a saturated group with non bonded electron which when attached to a chromophore alters both the wavelength and intensity of the absorption?
Auxochrome
39
What is an increase in absorption intensity?
Hyperchromic shift
40
What is a decrease in absorption intensity?
Hypochromic shift
41
What is a shift in absorption to a longer wavelength due to a substitution or solvent effect
Red shift (Bathochromic shift)
42
What is a shift in absorption to a shorter wavelength due to substitution or solvent effects?
Hypsochromic shift (blue shift)
43
What equation gives absorbance?
A = 2 - log %T
44
What is the Beer-Lamber law?
A = molar absorptivity (extinction coefficient) * concentration (M) * path length (cm)
45
Does Absorbance have units?
NO
46
What equation gives %T using the Beer-Lambert’s Law?
%T = 10^(2 - epsilon * c * l)
47
What is lost at high concentration?
Linearity
48
What is constant for a particular substance at a given wavelength?
Molar absorptivity or extinction coefficient
49
What equation can be used if the sample is the same but measured at the same wavelength? ie to find a concentration.
A1 c2 = A2 C1
A1/c1 = A2/c2
50
What are some applications of UV-Vis spectrophotometry?
Determine chromophore composition
Cell density
Determination of concentration
Measure structural changes/reaction - example - enzyme assays
51
What are two other ways the extinction coefficient can be expressed?
E% = (Emolar *10)/MW
Emg/ml = Emolar/MW
52
What can happen to a molecule at different conditions ie pH change?
Structural changes that effect absorbance and wavelength.
53
Shifts in absorbance and wavelength can be used to monitor what?
Reactions
54
When measuring turbidity or cell density, what is measured?
The scattering of light
55
Proteins can absorb at what two wavelengths?
220 nm peptide bonds
| 280 nm aromatic side chains
56
Why do we usually only use the 280 nm wavelength?
Too much background absorbance at 220 nm
57
Spectrophotometer is preferred to analyze proteins because of what?
The sample is recoverable
58
What is the part of a molecule that absorbs light from the UV-vis part of the spectrum called?
Chromophore
59
N->pi* is weak or intense?
Weak
60
Pi->pi* is weak or intense?
Intense
61
What is the Ligand-Field Theory?
D-block elements/transition state metals also absorb light in the visible range
62
Absorption shifts where the wavelength increases are called what?
Red shifts or bathochromic shifts
63
Absorption shifts that decrease the wavelength are called what?
Blue shifts or hypsochromic shift
64
Increases in absorbance are called what?
Hyperchromic shift
65
Decreases in absorbance are called what?
Hypochromic shifts
66
With increasing aromatic rings the length of the wavelength does what?
Increase
67
Which Amino acid residues have the ability to absorb at 280 nm?
Tryptophan, Tyrosine, Cystine (a dimer of cysteine)
68
Is cytosol reducing or oxidizing?
Reducing
69
Is air reducing or oxidizing?
Oxidizing
70
What measurement is used to determine the optical density of cells?
600 nm
71
Why does DNA have a higher UV absorption compared to proteins?
The presence of aromatic rings
72
Why does DNA absorption increase as it melts?
Non bonded electrons interact with pi bonding
73
List some limitations of the Beer-Lambert law
Deviations in epsilon at high concentration due to electrostatic interactions between molecules, changes in refractive index, and shifts in the chemical equilibria
Scattering of light due to particulates in the sample
Fluorescence or phosphorescence of the sample
Non monochromatic radiation
Stray light
74
What is used to indirectly detect protein concentrations?
Either copper chelation chemistry
Or
Binding to dye
75
What are the three methods that use copper chelation chemistry
Biuret method
Lowry Assay (Folin-Cicalteu reagent)
BCA method
76
What is the name of the method to determine protein concentration through binding to dye?
Bradford Assay (coomassie Blue)
77
How does the Biuret Method work?
Cu2+ binds to protein and gets reduced to Cu+ which forms a Biuret complex that absorbs light at 550 nm (blue-purple)emitted
78
What are the advantages of the Biuret Method?
Applicable to any protein irrespective of the presence of aromatic amino acids
79
What are the disadvantages of the Biuret Method?
Not very sensitive (requires a large amount of protein)
80
How is the Lowry Assay different from the Biuret Method?
A phosphomolybdic-phosphotungstinic acid solution is used. This allows tyrosine, tryptophan, cysteine, histidine, and asparagine to enhance the amount of color used (the contribute additional reduction fo the phosphomolybdic/phosphotungstinic acid complex)
81
What are the advantages of the Lowry Assay?
100 fold more sensitive than Biuret
82
What are the disadvantages of the Lowry Assay?
Absorbance depends on the composition of the protein which makes a protein stand hard to find
Many substances can interfere (Tris buffer, ammonium sulfate, EDTA, reducing agents,
83
What wavelength is absorption found at for the Lowry Assay?
750 nm
84
How is BSA different than the Biuret method?
Bichinochonic acid is added to the solution
85
At what wavelength does absorption occur when using the BCA method?
562 nm
86
What are the advantages of the BCA method?
Colored complex is stable (100 times more sensitive than biuret method)
87
What are the disadvantages of the BCA Method?
Bicinchonic acid is expensive
Composition is dependent on cysteine/cystine, tyrosine and tryptophan (increase the color)
88
What is the process of the Bradford Assay?
An acidic solution of Coomassie Brilliant Blue G-250 is used
89
At what wavelength does unstable (cationic) CBB absorb?
465 nm
90
At what wavelength does stable (anionic) CBB absorb at?
595 nm
91
What are the advantages of the Bradford Assay?
Very sensitive, fast, inexpensive, compatible with a wide range of substances, dye-protein complex is stable, but requires a standard bovine albumin serum (BSA)
92
What do you plot to generate a standard curve when using the Bradford Assay?
Absorbance vs concentration
93
What is the minimum Rsquared value that is acceptable when generating a standard curve?
0.98
94
What is the shift in wavelengths called when comparing the absorbance spectrum with the fluorescence emission spectrum called?
Stokes shift
95
What is the Forster Resonance Energy Transfer (FRET)?
When one molecule absorbs at a shorter wavelength and emits at a longer wavelength which is then absorbed by a second molecule which may or may not emit at an even longer wavelength.
96
According to Svante Arrhenius, what is an acid and a base?
An acid is a material that can release a proton and a base is a material that can donate a hydroxide ion.
97
According to Lowry Bronsted what is an acid and a base?
Base accepts a proton, acid donates a proton when dissolved in water.
98
What is the part of the acid called after donating a proton called?
Conjugate base
99
What is the base called after accepting a proton called?
Conjugate acid
100
Strong acid and bases completely what?
Ionize in water
101
What are some strong acids?
Nitric, hydrochloric, sulfuric, perchlorate, hydrobromic, hydroiodic
102
What are poly protein/basic?
Acids and bases with two or more ionizable hydrogen/hydroxyl groups
103
What only ionizes partially?
Weak acids and bases
104
How do we define the strength of a weak acid?
The degree of dissociation or acid dissociation constant Ka.
Ka = ([H+][A-])/[HA]
105
Ka is a property of an acid or base at a given what?
Temperature
106
What is a buffer?
An aqueous solution that resists changes in pH upon the addition of an acid or base
107
What does a buffer consist of?
A pair of a weak acid and its conjugate base or a pair of weak base with its conjugate acid.
108
What is a substance that can act as an acid or a base?
Amphoteric
109
Significance of polyprotic substances?
Blood has a pH of 7.4 and is maintained by equilibrium between carbonic acid/bicarbonate/carbonate ions
Cells have a pH of 7.2 maintained by equilibrium between phosphoric acid in cells (dihydrogen/monohydrogen phosphate system
110
Is what amphoteric?
Yes
111
What is pKa?
When 50% of the molecule is dissociated
112
What is the optimum buffering capacity for a buffer?
About 1 pH unit from its pKa
113
What tend to be on the inside away from aqueous environments?
Hydrophobic amino acids like Phenylanine, Tyrosine, Tryptophan pKa ~11
114
What are some negatively charged residues of proteins?
Aspartame D and Glutamate D pKa ~5
115
What are some positively charge residues?
Lysine pKa~11 Arginine pKa~12.5 and Histidine pKa~6
116
You can estimate the charge of an amino acid if the pH is how many units away from the pKa?
2
117
What is the isoelectric point?
The pH at which all ionizable groups balance and the net charge is zero, for a protein
118
The analyses interacting the most what with the stationary phase will take longer to pass through the system?
Strongly
119
What is the strength of the analyte to interact with the stationary phase described by?
The parameter Kav or partition coefficient/distribution coefficient
120
Kav can be from what two values?
0 and 1
121
What is the distance distance moved by the substance divided by the distance moved by a solvent front called?
Retention time/retention factor
122
What are the classification of chromatography based on attractive forces?
Ion exchange anion/cation
Partition-non polar/polar
Size exclusion
Affinity
123
What are two types of resins?
Polystyrene and cellulose/dextran
124
What are the parameters of a chromatogram?
Retention time (depends on size of the column and flow rate)
125
In ion exchange chromatography (IEC), what is the stationary phase?
A matrix of beads functionalists with a charge
126
In IEC what is the mobile phase
A solvent with a salt/pH gradient buffer
127
In IEC useful buffers will be 1-1.5 units above or below the what of the protein
PI
128
Gel filtration separates based on what?
Size and shape of the compounds
129
For size affinity what is Kav?
Kav = (Ve-Vo)/(Vt-Vo)
130
If Kav is greater than 1 then the molecules is what?
Bound non-specifically to the column
131
If Kav is less than 0 then what?
There is a crack in the column
132
Ve/Vo is independent of column size and protein concentration but may be dependent on what?
Temperature for some proteins
133
Ve actually depends on the shape of the molecule there the dependence on MW is only what?
Approximate
134
What are advantages of gel filtration chromatography?
Doesn’t depend on pH, ionic strength and buffer composition.
135
What are limitation of gel filtration chromatography?
Sensitive to temperature and packing conditions
136
In affinity purification only molecules that have what will bind to the resin?
A complementary binding site
137
What are advantages of gene fusion or native
For tagged proteins
Easy purification >90% purity
Tagged protein can b used as a method of detection
Solubility and stability can be improved
Untagged proteins
Tag removal is not needed
Closer to native conditions
138
What are dissadvantages of tagged or native gene fusion?
For tagged proteins
Take my interfere with protein activity, structure, and function
Cleavage of rage may not reach 100% if cleavage site is buried
After cleavage the protein of interest may lose its solubility or remain permanently attached to the Tate due to protein-protein interactions
Untagged proteins
Purification and detection is not simple
139
What are applications of gel chromatography?
High resolution
| MW estimation
