Analytic Techniques

Question 1

Extraction in protein purification

Answer 1

have to lyse cell membranes to remove proteins

Question 2

What do you treat protein mixtures with during extraction?

Answer 2

protease inhibitors

Question 3

Where are proteins in centrifugation?

Answer 3

lighter proteins stay suspended in the supernatant

heavier cell debris and such moves to the bottom

Question 4

How can you use salt to separate proteins?

Answer 4

initially the salt will increase solubility of the protein by breaking apart side chain reactions

then, when enough salt is added it will compete with the protein for solvent to be broken down and the protein will precipitate out

use the optimal amount of salt that the protein precipitates out

Question 5

Thin layer chromatography

Answer 5

use polar silica as stationary phase

separate compounds on polarity

use Rf ratio

Question 6

What type of molecules move the furthest in thin layer chromatography?

Answer 6

nonpolar substances

interact poorly with the polar stationary phase

Question 7

Rf

Answer 7

ratio of how far a substance traveled in TLC

has to be between 0-1

Question 8

Iodine in TLC

Answer 8

helps us visualize the compounds

Question 9

High performance liquid chromatography

Answer 9

uses a polar stationary phase

polar materials have a longer retention time

Question 10

Reverse phase HPLC

Answer 10

uses a nonpolar stationary phase

Question 11

Gas chromatography

Answer 11

mobile phase is gas and stationary phase is liquid

compounds with low boiling points evaporate first

Question 12

Size exclusion chromatography

Answer 12

captures small molecules in gel

Question 13

Anion-exchange chromatography

Answer 13

has positive beads to attract anions

Question 14

Cation-exchange chromatography

Answer 14

has negative beads to attract cations

Question 15

Salt buffer in ion-exchange chromatography

Answer 15

releases attracted molecules

Question 16

Affinity chromatography

Answer 16

ligand binds to a specific receptor in the column

Question 17

Electrophoresis anode and cathode

Answer 17

anode is positive

cathode is negative

Question 18

SDS limits

Answer 18

does not break down disulfide bridges

need a reducing agent to break down disulfide bridges

Question 19

What travels the furthest in SDS page?

Answer 19

smaller molecules

Question 20

Isoelectric focusing

Answer 20

a type of electrophoresis where molecules will end up at pH that equals there pI

Question 21

Immunoassays

Answer 21

rely on antibodies for specificity/identification

Question 22

Example of an immunoassays

Answer 22

Western blotting

Question 23

ELISA

Answer 23

antigens marked with fluorescent labels bind the molecule of interest can use spectroscopy to see how many molecules binded

Question 24

Beer-Lambert Law

Answer 24

A = ecl

Question 25

At what wavelength do proteins have max absorbance? Why?

Answer 25

in the UV spectrum as aromatic/conjugated pi bonds

Question 26

Extraction

Answer 26

separate liquid mixtures into organic and aqueous phases

Question 27

Organic phase in extraction

Answer 27

non-polar

Question 28

Aqueous phase in extraction

Answer 28

polar

Question 29

Where are uncharged acid and bases found in extraction?

Answer 29

in the non-polar organic phase

Question 30

Distillation

Answer 30

separates liquids by their boiling point

Question 31

Fractional distillation

Answer 31

can separate liquids with similar boiling points

Question 32

Vacuum distillation

Answer 32

lowers atmospheric pressure to allow high boiling points to evaporate

Question 33

Recrystallization

Answer 33

recrystallize a solid precipitate to filter out impurities that may have been in the original precipitate

Question 34

Nucleation

Answer 34

initiates the recrystallization process

Question 35

Spectroscopy

Answer 35

analyzes molecules based on interaction with electromagnetic radiation

Question 36

How does IR radiation work?

Answer 36

makes polar bonds bend and stretch

Question 37

Where is O-H found in IR?

Answer 37

a broad peak at 3100-3500

Question 38

Where is C-H found in IR?

Answer 38

a stretch at 3000

Question 39

Where is C=O found in IR?

Answer 39

a sharp peak at 1650-1780

Question 40

UV-Vis Spectroscopy

Answer 40

looks at movement of electrons good for determining conjugated pi systems

Question 41

How can you find the color of a compound from UV-VIs spectroscopy?

Answer 41

colors that you don't absorb give the color of the conjugated system

Question 42

What is UV-Vis spectroscopy good at measuring?

Answer 42

conjugated pi systems / aromatic compounds

Question 43

Where are less shielded protons found in NMR?

Answer 43

downfield to the right

Question 44

What makes a proton less shielded in NMR?

Answer 44

the presence of electronegative species that remove ED

Question 45

How many number of signals are found in NMR?

Answer 45

number of sets of equivalent hydrogens / carbons

Question 46

Where is the position of signals in NMR?

Answer 46

look at shielding to shift upstream or downstream

Question 47

What does the size of the signal in NMR tell us?

Answer 47

more protons means more area/larger size

Question 48

How is splitting in NMR determined?

Answer 48

n+1, where n is the number of adjacent hydrogens

Question 49

Is there splitting in 13C NMR?

Answer 49

no

Question 50

Mass spectroscopy steps

Answer 50

1) Ionize by shooting electrons at molecule 2) Accelerate in E-field 3) Curve in B-field to tell us the mass

Question 51

Parent ion in mass spectroscopy

Answer 51

Generated by the loss of only on electron Is the peak the furthest to the right on graph

Question 52

What can the parent ion tell us in mass spectroscopy?

Answer 52

the molecular weight of the molecule

Question 53

Parent ion abbreviation

Answer 53

M+