Pchem Exam 3 (spectroscopy and PK)

what do spectroscopic techniques provide

snapshots of a molecule as different types of energy affect a molecule differently

IR uses

provides a method to determine the presence of functional groups

how IR works? (what are atoms constantly doing and what does IR do to them)

atoms are in constant motion and these movements are called vibrations. IR radiations has just the right amount of energy to stretch or bend a bond (but not break it)

this results in the molecule absorbing energy resulting in an absorption band at that frequency (so all bonds have a frequency range where they will absorb IR energy)

type of movement for molecules in IR (6)

symmetric stretching, asymmetric stretching, wagging, twisting, rocking, and scissoring 😜

where do most organic molecules absorb energy (prob don’t need to know)

the mid infrared region (4000 cm^-1 - 6000 cm^-1)

what does an OH group look like on IR

broad band and on the left side of the graph

IR NH₂ how does it look

has two fangs

what units are IR energy represented in and which is the more common one for us

wavenumber (what we use) and wavelength

relationship between wavenumber and wavelength

higher wavenumber correlates to a lower wavelength (which makes sense because a lower wavelength means higher energy)

atomic mass trend in IR

wavenumber (frequency) increases with increasing atomic mass

put these compounds in order of highest to lowest wavenumber

N-H, C-H, O-H

O-H > N-H > C-H (atomic mass trend)

bond order trend in IR

wavenumber (frequency) increases with increasing bond order

triple bond > double bond > single bond

how does NMR work? what happens to atoms?

excites protons so that their spins align against a magnetic field

the output is a range of frequencies that correspond to the type of proton

NMR uses

determine exact structure of compound and impurities

NMR shift; what does it tell us abt a proton

tells us what type of proton we have. strongly influenced by the surrounding electron density (electronegativity)

where is downfield on NMR and what does it mean for electronegativity

to the left; higher ppm; proton next to a more electronegative atom

where is upfield on NMR and what does it mean for electronegativity

to the right; lower ppm; proton next to a less electronegative atom

NMR integral; what does it say abt the proton

how many protons in relation to other peaks (RATIO so 1:2:3 cld be CH, CH₂, CH₃ or CH₂, CH_4, CH₆, etc)

NMR splitting; what does it say abt the proton

how many protons are next to the proton we are looking at

meaning of singlet, doublet, triple, quadruplet

H is next to 0 H’s, H is next to one H, H is next to two H’s (CH_2,), H is next to 3 H’s (CH_3,)

if your looking at the CH in CH₂CHCH₃ then its two diff types of protons on each end so wld be like doublet of triplet or triplet of doublet but he wld prob not ask that like 99% sure

when are protons equivalent and how does that look on NMR

when there is a plane of symmetry and the equivalent protons will show up as a single peak

what does it mean if there is a plane of symmetry and two H’s on the same C are still showing up as two different peaks

it means there is a double bond (OLEFIN) and double bonds are not rotatable so it means the H’s wouldn’t be equivalent

olefin

c--c DOUBLE bond

what can splitting in NMR be useful to find out

location of groups on an aromatic ring/cyclohexane ring

what does it mean if you see 4 signals in the aromatic region? how many substituents?

4 H’s , 2 substituents

NMR aromatic ring region

7-8 from class (6.5-9 in acc chart)

NMR x axis

ppm

UV (UV-vis) what do the compounds do when exposed to UV light

compounds absorb UV light thru excitation of electrons

what does a compound need to be UV active

accessible electrons; compounds with pi bonds

when is UV absorption more intense

conjugation; the more double bonds there are

what is absorbance intensity proportional to

concentration (amount of light absorbing substance (solute) in a solution (aka pi bonds))

beer’s law

A = Ebc

E= extinction coefficient (like a greek weird E)

b = path length

c= concentration (amount of light absorbing substance (solute) in a solution (aka pi bonds))

UV detection method (what enzyme wld u use)

ornithine aminotransferase

UV detection method (what compound wld u use and y)

NADH bc its UV active

how does UV detection work if you wanna see how active an enzyme is but its not UV active

ex) ornithine aminotransferase (O) (not UV active)

want to see how fast O is working but not UV active so u cant see under light

nadh is showed under UV so u use it

nadh reacts w O and the light keeps getting dimmer as NADH gets used and turns to NAD+ which means the O enzyme is working (active) to convert this

by measuring how much light nadh has over time you can see how active the enzyme (O) is

this method is used to see if the enzyme is working and at what rate it’s working

fast drop (steep) of nadh means enzyme is working rly fast rate and slow drop (not steep slope) shows enzyme working slow

UV-vis units you measure and graph x/y axis

Absorbance (y axis) and wavelength (x axis/ nm)

fluorescence; how is it diff than UV-vis

certain molecules can be excited by UV/visible light BUT will emit radiation at a longer wavelength

what is happening at A, B, C

A = excitation

B = relaxation

C = emission

between excitation, relaxation, and emission which are radioactive transitions

excitation and emission

relaxation is a non radioactive transition

how does excitation happen and what does it mean when a molecule is at a higher energy level

when UV or visible light shines on a molecule electrons get excited

if the molecule is at a higher excited energy level it got more/absorbed more light

what is relaxation

as its on a higher excited energy level for a while the molecule starts to loose energy and starts relaxing (slanted line)

no light is emitted and this is why molecules emit light at a higher wavelength (lower energy) bc some is lost during relaxation

what is emission

when the electrons fall back down to ground state

as they fall they release energy as light known as fluorescence

relationship between energy of the particles and wavelength

higher E means lower wavelength

If a molecule is excited at a wavelength of 340 will the emitted fluorescence be greater or less than 340

greater bc the emission will be a lower E bc some got lost in relaxation so the wavelength wld be like 500 nm

uses of fluorescence and when is a molecule fluorescent

to measure binding of a drug to a receptor/target, used for detecting fluorescent impurities

drugs are fluorescent when they have extended pi systems

is fluorescence predictable?

no

is quinine naturally florescent

yes bc it has an extended pi system

what molecules need to be cleaved to be fluorescent

sugars and peptides don’t allow a normally fluorescent molecule to be fluorescent so u cut them off with enzymes

what enzymes cleave the sugar and peptide bond

glycosidase and protease (cleave amide bonds between n and carbonyl c)

how can u measure an enzymes activity with cleaving in fluorescence

as you cleave the sugar or peptide bond that compound will start be be fluorescent

so you can measure the brightness of the compound and that will tell you the enzyme activity

more fluorescent means. more enzyme being used

why are we covering PK (MS, HPLC, like how all the stuff we did is not overlapping)

to identify to compound and find its concentration

radiometric assays (whaat do they do)

they take a small molecule and tag it w a radiolabel

to do this they replace for example a ¹²C with a ¹⁴C and then can follow the drug

common radio label substrates

³H, ¹⁴C, ³²P

what is the radio labels detected by

scintillation counter or MS

they will detect the radio active molecule —> send signal —> results in peak

what do you usually need to do for radiometric assays

purification

methods of purification (2)

1) HPLC - as it comes off can detect if molecule is radioactive bc installed radio label on substrate (usually MS is done right after; HPLC-MS)

2) onbead purification

drawbacks to radiometric assays

special precautions needed for radioactive material

synthesis/ purchase of radiolabeled materials (expensive)

benefit of radiometric assay

selective (can detect between isotopes C12 vs C14)

traceable

what was radiometric assays replaced by

MS

PK graph x and y axis

x axis: time

y axis: amount of drug in blood

PK graph first half vs second half

first half: absorption

second half: elimination

PK graph what is the peak and what does it mean

Cmax, highest concentration of drug in blood

PK graph where is t_^1/2

in the middle (half the conc of Cmax then from there find the time)

PK graph what is the bottom point and what does it mean

Cmin: lowest concentration of drug in blood

what can PK graph show

look how much drug in blood, plasma, target, receptor