Module’1’Experimental’Procedures’ Experiment’1.’What ... · Module’1’Experimental’Procedures’ ’ Experiment’1.’What’sintheSpectrometer? ’ • Identify

Module 1 Experimental Procedures Experiment 1. What’s in the Spectrometer?

• Identify and ident

elements ify the pa

of th the of t

spectrometer he light.

shown in Fig. 9 in the open spectrometer unit

Experi ment 2. Lambert-‐Beer’s Law

• Prepare flasks.

0.01, 0.02, 0.03, 0.04 and 0.05 M aqueous solutions of CoCl in volumetric

calculations. hexahydride

When (CoClmaking

6Hup O) your and solutions, be sure

note that you will be using cobalt 2

2

• 0.01 0.05 M

• 2

chloride

and Your . TA will prepare a solu

to tion use witthe h unk

proper nown

molecular concentrat

mass ion, b

in etwyour een

Make sure that the absorbance spectrometer is computer via USB port—you shoul

plugged in and connected to the

• Kimwipe, and place the cuvette in the holderSpectraSuite program. Fill a cuvett

d e wbeit ableh w

toater, w hear

ipe t its f

he sides of tan running—

he cuvetand ope

spectrometer Under

te wn thiteh a

the Spectrometer picture, expand

menu menu

select [+], and

Rescan expand

Devices. Properties

Fin[+] d the absorbance

spectrometerserial number

s corresponds to the spectrometer.

to check that the

• the screen, right click the spectrometer picture and choose Show Graph.

and

ll

select Remove Spectrometer. If ther Right e is no

click graph

on disany played

other on

ClIntegration ick the Pause [

• Correction box. This box should remain checked throughout all experiments.

►

Time = ] 10 button ms, Average

and change = 10,

the and parameters Boxcar = 10.

at the Check

top the of the

Electscreen ric Dark

to

Clin ickthe Plaspectrum y [ ] to

over begin

time, the ac

clqickuis ition ont. he When

darkyou light

no longer bulb [

see ] major

reference to stor

fluctuations

spectrum. Check the Strobe/Lamp Enable box and repeat, clicking e the

on dathe rk

•

yelshould be accessible. Clow light bulb [ ] t

lick it to enter absorbance modeo store reference spectrum. Now

andthe the

Absorbance

•

Remove n click Pause

[. ] icon

and empty your

Take then fill with the solution. P

cuvette.

a spectrum by clicking lacPlay, e in

Wash holder.

with

your first sample solution, empty, and

spectrum. You can change the viewing then clicking

parameters Pause

of when the graph

you feel using

you [have ] toa autogood

-‐scaparameters

le, [ ] , toand vert[ical [

of the graph using the scrolling wheel on the mouse. ]to ly scale the

pan spectrum, the spectrum.

]

•

toggle and You to manually

can also zoom set

in the and zoom out

Clthicke File the

menSave

u. [ Under ] icon

Desired to save

Specthis

trum, spectrum.

ypselect

that the file t e is “.OOI” and the appendage to

DoProcessed

t

pressingthe

n’

Save. file

access

name Spectrum this

is “.ProcSpec”

funand ction

m through akebefore sure

window first. If the Save button isn’t activated, try clicking elsewhere in the Save

1

• Remove, the new solution,

empty, and acquire rinse the

a new cuvette

spectrum, with your

and save. next sample.

• solution.

Continue Fill

for the all cuvette samples

with of

After all spectra have been taken, click Overlay Spectral Data [ ] icon and select one of your saved spectra. Select Processed and

•

your graph.

processed

spectra. This should overlay

510

all then of clyour ick

spectra . Repeat onto the

for same all of

Cwindow lick on

at the the spectrum bottom

graph of the

area, screen

then and

type hit e

“nter.

” Tinto he a

the bsorb

Wavelength ance val

(nm) text

• respoverlaid

of

Do aect

spectra ues

ive colat 510 nm will

your

linear fitors. to t

Rhe ecord funct

tionhese

Avalappear ues in you

to r the noteb

right ook

of . the text window in their

approximate unknown sample

uncertainty and using

of the the = f(c

fit ). Read

measurement? calculate

off the its vaconcentration.lue of the absorb

Whanceat for

What do you think the biggest is th

the e

Experi

sou

m

rces

ent

of

3.

error

Arom

are?

ati

c Hydrocarbons • Prepare

and placea icuvetn hold

te ehar. lSet f to

up twothe -‐thiinstrument rds full of hexane,

•Obtain and store the dark spectrum and the reference spectrum of hexane.

using the wipe parameters

the sides in with Experiment

a Kimwipe

2.

Add Scale

a the drop

grof aph saturated such that

naphthacene

dropwise until peaks are you can resolve

to the peaks, hexane

and cuvette continue

and to acquiradd

e naphthacene a spectrum.

• spectrum each time, but be sure to rinse the cuvette with hexane each time. procedure for anthracene and

very naphthalene

prominent. . It Save is not

the necessar

spectrum y to take

and a repeat referen

the ce

OpeOptions”.

n your Under saved

the spectra. Right click on spectrum graph and select “Graph Layer

• At Selecti

the on wbottom

indowright . Sel

Peaks ect the Show

tab, select Pea

your k Ba

spectral ine Layer

line andfrom

corner of the graph, selselect the Peaks

pr[ethe ss OK

Spectrum .

Source

] icon. The Peak FinPeak

din Finding g toolbar

toolshoubar ld ato ppopen ear direct

the ly Peaabkove. Propert

Select ies the wCindowonfigur

. e Set[ the ] icon in the

absorbance Width until the

somewhat number

below of peaks

the

•

found peaks of interest and increase the Minimum

BaselPeak ine

you see on the graph. Click “Close” to exit

appr the w

oachindesow the . number of significant peaks

Using each of

the

•

the Previous and

in the Peak Finding tsignificant

ool peaNexks t aPeaknd

[

bar) in your notrecord

eb the

] buttons, position the green cursor on

Use the wavel→ength of the peak with the

ookwavelengths .

(from the Wavelength box

for estimate

the n=1 the

n=2 transition. Using the highestone-‐dimensional

wavelength

length, l, of each compound. Plot the length as a Particle-‐ as the

in ph-‐a-‐Box oton energy

of coordinated hydrocarbon rings. function of the number

model,

Experiment 4. Fluorescence of Quantum Dots

2

•

•

Tadescribke ab

ed sorptearl

ion ier.

spectra of three samples of quantum dots using the procedure

an Locate

electrical the fluorescence

outlet, in pulsed spectrometer

flash mode . Check

and tha

on. t the

PlacXe el iga htcuv sou ged

windows containing dots etterce wiis thpl ufoug

quantum r clinear to

• detAs

ectbefore, or shou

make ld be

sure plugged

that in SpectraSuite to the

in hola der stand

at -‐90°alone

is recognizing angl cuvete.

te holder. The source and

•

Rescanspectrometers from the menu.

Devices, checking that the serial number matches, the and right removing

device any by

other using

Cand lick

expand the spectrometer

range of the [+]. wavelengths Be sure

picture that these

and under fluorescence

the expanded wavelength

menu parameters [+], select Acquisition

obtained from your absorbance measurements. are

If in not, the

• contact your TA. Make a 1 second

sure that the acquisition

the Strobe/Lamp Enable box. You should hear an electrical whine from theintegration time, 1 scan

is paused, to average,

and then and

change a boxcar

acquisition of 10. Also,

parameters

•

check lamp.

[ √ to ]

Acquire maximum peak.

spectra Save each spectrum. for each sample, recording the color and the wavelength of the

• Use quantum

the dots. equation

1.70 to estimate

and the

effective masses For CdSe, E = eV and = 0.13 =

0.45size a

arof e ththe e

either hole effective

band do masses,

in the which conduction

gap

account and approximately

m *c me m *v me

not move freely) where m

v

e

alence bands re

is for the

s

an fact pectively el

the mass of electron. that the

(

Howelecectron

trwoelnsand

l do in

your sou

results of discrep

agree with the

rces ancy? particle in a box expression? What do you think might be

Experiment 5. Chloroform •

thStudy

a sample eir func

the tisetup ons. Be

and sure identifyto sketch

the elements of the homemade interferometer and

• FTIR spectrometer?

holder and detector be placed the interferometer

to make the interferometer and take notes.

a fully Whe

functional re should

Turn you move

on the the laser source. Can you see the interference p

interferometer would delay stage?

be varied Try

to it take out.

an During FTIR

a spectrum? scan, attern

what ? What

element will happen if

• interferogram look like with and without a sample?

What would of

the the

Move to the Nic

S

oledow

• computer interface. Under the Win

Exp et

t 380 FT-‐IR and open the OMNIC software program on the

ClIR ickscan on. tUnder he

the Cicon ollect

to header, open a dia

set logue top menu, click on New Window.

window that allows you to configure the

to Interferogram. Click OK to exit. the number of scans to 16 and “Final format”

3

mariusztwardowski

Text Box

• Put perform

an empty a background

thin cuvette scanin

scan of your sample. What . the

holder and flush with nitrogen for 60 seconds, and

• What do you expect to see in the spectrum?

can Fill

be the

concluded cuvette with

from chloroform the observed

and then interferogram?

perform a

Set the spectrometer

• concland making

usions. the appropriate

to the transmission change, and

mode repe

via at clicking the proc

the edurExep toSet v eicon rify

aygaourin

several Where do

peaks you

from expect

the the CHpeaks Cl3 dat

to a aappear nd calcu

if lathe te wsample here the

is deuterated deuterated

(CpeaDCks l3)?shou Pickld

beWh.

about μ•

at Remember do

the

? e s K represen

relation t? Would its va

from lues b

our e differen

discussion t for

of CHClharmonic

3 and CDoscillators. Cl3? What

Repeat the experiment with deuterated chloroform and compare your results to

Experi

prediction. What are some possible sources of any

ment 6. Essential Oils

discrepancies?

•

•

Put with nitrogen for 60 seconds to remove water and C

an IR card in the card holder in the FTIR instrument, O2 vapors

close . the cover and flush

•

Recoran IR card, repeat the procedure and take the spectrum of the sample

d the background spectrum. Put several drops of limonene on . the window of

• Repeat the procedure with the carvone sample Try or internet

to assign sources. as many

functional groups in the spectrum as possible using literature

• relatively low? Recal

Why l the ex

are pressionsome vibrational

for the vibfrequencies rational reson

relatively ance frequ

high encyand some .

What differentiate between S and R enantiomers using FTIR ab

are the differences between the spectra of the two compounds?t

Caωn0

sorp ion spectroscop

y? you

Experiment 7. Protein Secondary Structure Determination

o• Before starting, set the temperature on the heating bath to 50 C and turn on the peristaltic pump. While you are taking room temperature spectra, the bath will heat up.

• Using D2O as the solvent, make three 100 μL solutions with 20 mg/mL concentrations of the three proteins you'll be studying: one for Lysozyme, one for Myoglobin, and one of Ribonuclease A.

• Purge the spectrometer with nitrogen for 4 minutes and collect a background spectrum of the empty spectrometer with 2 cm-1 resolution and 16 scans. Save the spectrum. In experimental setup, set this background as the default background.

• Clean the CaF2 windows and Teflon spacer with water. Dry windows with lens paper. Dry Teflon spacer with a kimwipe.

• Load the CaF2 cell with 30 μL of D2O. Do not use the heating coil. The cell should be assembled in this order: tapered metal ring, tapered metal ring (opposite orientation), thick flat metal ring, lead gasket, CaF2 window, 50 μm Teflon spacer, CaF2 window, lead gasket, thin flat metal ring, threaded sealing cap. To load the cell, carefully slide in cell

4

components up to and including the Teflon spacer. Drop 30 μL of sample into center of window. Gently lower remaining stacked components onto sample drop and screw the threaded sealing cap down. Tighten firmly by hand until you encounter resistance. Verify that there are no bubbles in your clear aperture.

• Place the entire cell in the Nicolet spectrometer and purge with nitrogen for 4 minutes. • Collect a D2O spectrum with 2 cm-1 resolution and 16 scans. Save spectrum. Collect a

D2O background spectrum, save, and set it as the default background. • Remove cell, dissemble, clean windows with water, and load Lysozyme.

-• Collect a 16 scan, 2cm 1 resolution spectrum of Lysozyme. Save the spectrum. • Remove cell, dissemble, clean windows with water, and load Myoglobin. • Collect a spectrum after purging. • Repeat for RNase A. • After collecting a room temperature RNase A FTIR spectrum, unscrew sealing cap and

slide on heating coil. Retighten. Make sure the heating coil orientation is correct such that it will stick out of the top window of the spectrometer when the cell is loaded.

• After purging, set the temperature of the bath to 100oC. • Collect spectra of RNase A as the temperature rises at ~7oC increments. Save the spectra

and record the temperature. The maximum temperature should be ~81 oC. • Turn off heater and allow to cool. When the cell is cool enough to handle, disassemble

and clean. • Compare Lysozyme, Myoglobin, and RNase A room temperature spectra in the 1600-

1700 and 1900-1300 cm-1 ranges. Can you distinguish different secondary structures from the Amide I spectra?

• Compare the RNase A temperature-dependent spectra over the same ranges. To observe changes, subtract the room temperature spectrum from the others (control, right click selects both and then select the subtract button). What changes? What is happening to the protein as you heat it?

• Experiment 8. 15-‐MHz NMR

•

•

Appendix this experiment.

B contains some useful information and photographs to help you through

Fill from

a the sample

magnet and insert the sample. top of

vial the about vial, t

¾ hen full ploface etaha plnolast. ic Plcaap ce on an the O-‐ring top,

about lift the

half top

a panecentimeter

l on the

5

• Turn on the 15 MHz spectrometer and the oscilloscope.

on the spectrometer are connected as shown in the figure below. Verify that the blue cables

•

is Step off. 1: These View pulse.

switches Make

let us sure that the A pulse switch is on and the B pulse switch

disconnect connect A-‐B

the OUT

blue to C

cable hanne

from send electronic signals (pulses) to the magnet. Next,

loose. Remember this configuration; l 1 on

the theA-‐B oscil

IN loscope, port on the spectrometer and use it to

pulse generator’s input into the magnet) every you

time lyou etting

want the

to MIXER view

OUthe T cable hang

will connect the cables in this pulse (the

until Change

you the areparameters able to view

on ththe e pulsoscilloscope

e. Meas(vertical and horizontal zoom and

fashion. pan)

Whthe

ypuls wo

euld us weing want tothe curs

usorse a son qthuare oes pulscillos

ecour

fopee and record the length, or duration, of

r th. Whis pur

at pois thsee? purpose of the RF pulse?

6

• Step return

2: inView g the

FIcaDbl.es Set to up the the origoscilloinal set

scopupe (Ato view

OUT to the oscilloscope). This is the-‐B IN and

the Afree -‐B OUT

indu ctreion conne

decactey d(FI, MIXER D) by

receivinexperiment

g the to pobserve ulse. Adjust

the FIthe D, the

oscilloscope time-‐domain configuration

response you will

of usethe thr

sample oughout

after the

oscilsignifica

lating nce signaof this

l; this valu

is e? t he HowFI Dis . tMehe asFIurD e the

so fthat que

you’re

signal rperodu

ncced?y o Wf thable e F

to observe an

signal? have to mix hy

ID

What information does it give us? Why do we is the it . an Whoscilat ilsa tthing e

•

signal

With thefrequ

from the sample with a reference signal? Step

the

coming

frequency ency 3: Tun

adjue

and stto setreson

watch to coa

an

the rse, t frequen

oscilloscope. turn th

cy e tuni

an

ng d optimize knob on the

sample pulse

oscillations in the FID should change as you The

adjust intensity of the FID

generaheight.

and tor number to adjus

of t

oscilyou’ve

lations found

shoulthe resonant

d decrea se frequency, as you get

you closer to the

the reso

tuning. nant fr

The eque

number ncy. Whe

of n

intoscilensitlations.y of

the NowFID aadjust s you

the do. Not

height e the

of peathe shoulsample d see

k that changes in an the exponential

the magnet and

decay

most dramatically watch without

the

remember it—later, when you need to “reach and

• Consample is at the optimum height and FID”,

cept

this

check:

is the peak you’ll be monitoring. you the see t

Adjua maximum st

in the intensity of the

Before proceeding, check that yohe lu undargestthe ru

FID.bber

o-‐ring so that the

that cause the FID signal to decay. What are their relative ertime stand

frames? the thre

What e proces

does sesit

mean to have a 90 degree or a 180 degree (π/2

• Step other angles that would achieve the same result? rotated?

4: Create

Why does

a π/2

finding

pulse.

the 90 degree pulse maximize or π) puls

our e? What

signal? exactly Are there

is being any

Watch the scope as you do this. T

Wure n kthe now

A -‐tWihatd thlengt kno

h b of tothe adpuljusse t this e π/2 puls

we

reach hen length

we .

a maximum

move are several

the A-‐possible

in the intensity of the FID (the peak you noticed

Width knomaxima—you b more than

should make sure that the one in you Step

use 3). doesn’t There

• Step observe

5: Create your 90

a degree π pulse.

pu Doulse. M

bleae thsuere halfway. and record

Next, change instrument

width of pulstehe tol a engtπ h of the pulse.

setup to

thmaximum, e correspo

and ndi

how ng FID

the lootime k like

delay ? Con

of siderthe

response whether y

should ou expe

pulsct e th(1e8 s0i)gnal . What should

Step 4. Reconnect AB OUT to AB IN and MIXER OUT to Chn 1 compare on the scope

to the to be

FID at of a

changing without

The FID shoulany

d other appea

settings.

to have r to be fl

Observe at. If your

the FID FID is notand

entcompare irely fla

wit, wthha yot urwoul pred dcaicuse tions

it.

• the FID signal decays. What is the net magnetization of the sample at this time?

having Step

instrument.

6:

nonzero oscillations in intensity? Explain

both Create

A Set and two

the B π/2

delay on will

pulses

time allow

to

usin

10 us g

ms to differensend on the

more t chan

the state

pulse complicated n

generator.

els.

of the system right after

Turn pulse on the B Channel—

the units on the pulse programmer display and especially the Be

white carsignals eful to

to nothe

dot that te

represents pulse using

a the decimal

same point. technique

Switch as before.

to FID view What

and do y

adjust ou expe

B-‐WIDct th

TeH c otorr feisndpo a ndπing /2

7

•

FIchannelStep

D to

7:

look and one on tCreate

like?

a

Why

π/2he B channel–π

is the

sequen

FID not

ce.?

zero after sending two π/2 pulses, one on the A

echoforming

ex pera 18

i0ment. degree pulse with a delay

As itime n Step 5, double the width of your B pulse,

Transform, and adjust Wh

the at dfrequency o you obs

of erthe ve?

spectrometer of Set 10 up msthe . Thscope until

is is the

you to view

setup

observe the for Fa

a ourpeak.

spinier

Record maximum,

the location

the FID agaiFWHn. Me

M, asas of best this

you peak and the peak width (estimate the full width at half

would expect to see urmultiple e the fr

can) eque

and

peaks nc

point 11). for y note o

the units. Now set up the scope to view

after Expt Why don’t we ethanol f the FID

(if and you’re

compare not sure

this why, to the

revisiFT. t We

have this

using experiment?

this instrument Calculate

between the energ

2 y peaks differen

with ce a (inthese Hz)

multiple that we

peaks would

in expa ect15

2 ppm difference from each tother. o MHsee z

• CStep ompare this to

8: Dephasinthe line

T

g and wiLifetime dth you o

inbs glycerol.erve in th

Here e FT.

w e will study the dephasing and

lifetime 2

of glycerol in water. We

T

will focus on the rates due to dynamical dephasing ( ) due to evolving interactions (e.g. “collisions”) within or among molecules, the lifetime of our nuclear spins ( 1), and inhomogeneous dephasing (Δ) due to static variation in the environments around different glycerol protons. Because solutions with different glycerol concentrations have different environments around the protons, the

To

extent

save

of inhomogeneity and the rates of dynamical dephasing processes may vary from sample to sample. time, each

group will conduct this experiment

yielconcent

just one

d concentration of glycerol, and the data from different groups will

using be combined to

YoΔ: ur FFind the

ration-‐dependent resul

ID will dpulsewidth ecay approximately

of pulse A ts. which

exponentially. creates a π/2 pulse in your sample.

results In gener

in al a thdrop e decay

in your rate

FID includes

intensity contributions

to 1/e (37%) Measure

its maximum, the time

from of initial

delay

inhomogeneity, dephasing, value. that

Tand lifetime, i.e. Δ, T:

2, and T12 Find the pulsewidth

.

You are now set up to perform of a pulse spin echo

B which experiment.

creates a Useπ pulse a delay

in time your

of sample. 2.5

accurate initially.

way Record

to do ththis e int

is ensitto measure

y of the the echodistance using hor

from izontal the highest

cursors. The most ms

points rather than trying to the lowest

this the baseline. The echo wil

to l alwaysmeasure

appear the distance

at twice between the delay

the time—highest

whypoint ? Repeat

and

no point.

echo procedure,

can be increasing measured,

the recording delay time for each measurement by

n’t decayed by 50 ms, increase the step size to 10 ms. the delay time and the intensity

2.5

If the echo hasfor ms until

each

Tpuls1: e ("inversion Set up your pulsewidths

recovery" measurement). such that pulse

To A is

begin, a π pulse

measure and pulse

the maximum B is a π/2

(initial)

pulse pulse A

B. and amplitude

B of the FID following pulse B A Next, turn on

As and measure the

without pulse on.

before, start at a maximum time delay

(initial) of 2.5

amplitude ms, and

of continue the FID following

to make

8

measurements in 2.5 ms steps. If the echo hasn’t decayed by 50 ms, increase

What step size to 10 ms.

the

would processes y

and

delay time ou for expethe ct spin to measurements ch

inveglydo Δ

echo ange

and with

rcseiorn o, l Tconcent2, and T correspond to and which

values of Δ, T2, and T1 were found in your rexperiments? ecovery

ratat

1ioneach? Pcoloncet intentrnsatiiotyn. v eWhrsusat

Experim

your ex

ent 9. 3

pect

0

at

0-‐

ions?

MHz

E

N

xplai

MR

n any

discrepancies. Do your results match

The detailed operating procedures are presented in Appendix C. It is essential that you read them before conducting experiments, for the safety of the NMR machine and for the success of your measurements. Make sure that you have

thoroughly read all the NMR material in this manual and the Appendix before beginning.

Samples D is a mixture.

A, SpecB, and trum I

Take C contain spectra

nterpretationacetone, of all four

anethanol, samples

d Chemical Determin

• and methanol in

three samples and the identity and mole fractions to determine

of the the

ationsome identity

order.

of the Sample

first

• Rationalize the line positions, integrals, and splitting patterns that you obs

mixture in Sample erve.

D.

For oscillation

the sample frequency

containing determined

methanol in the

calculate

ion,

•

this previous

the chemical shift in ppm from the

value to the spectrum in the frequency domain experimental obtained in thi

section s sect

and compare

in w

protons on adjacent atoms in ethanol using your spectrum.

the units are ppm. For

hich

the sample containing ethanol, calculate the J-‐coupling parameter values for

• the Observe

FID the is visible.

FID: Enter For

the Time Domain

solution ‘acqi’ w

‘A’ indowyou

Measuremen, should hit ‘FID’,

see andts

two adoscillation just the ver

frequencies. tical scale unti

By l

•

moving oscilExamine

lation frequencies in Hertthe

the

cursor

linewidth

between the peaks of the two oscillations; calculate the two

: Bring tz.he cursor close to a peak. Enter nl dres (nearest line

•

display experiment. Integrate

resolution).

your spectrum

Compare this value to the linewidth from the TeachSpin

button to clear

to all define resets

those (zeroes)

: Clalready ick

regions

[partial in memory.

integral] Click to display th

and e inte

use gral the lineleft . Tmouse ype cz

using used to

the undo middle

mistakes. mouse

The button parameter you wish to

setintegrate. s the inte

[resets] The grals

right scale

mouse and can

button be ad

can juste

be d

contwill

rolno. w Plbeace

to gai

qt

while is in integral mode. Type re n cursor

• regPrint the spectrum

ions) to get uehe rcursor ied for

over an inte

the grdesired al value

region . Type

and ds click et ] dpir

the (displ

ds [S

a iy ntpeabutto

pl pscale pap page

k intn. egraYou

the integral values displayed underneath the spectrum. l

: Type into the command line.

9

10

Appenhtditp://lx A:

asp.colorado.edu/cassini/education/ Electromagnetic Spectrum

Please see: The Electromagnetic Spectrum

http://lasp.colorado.edu/cassini/education/

http://lasp.colorado.edu/cassini/education/Electromagnetic Spectrum.htm

Appendix B: Helpful Hints for the 15 MHz

The picture above shows the three main components of the instrument setup. The pulse generator is where you will program the pulses that get sent to the magnet: the duration of each pulse, the frequency, the delay time between pulses, and the pulse patters patterns. The magnet consists of coils of wire which create a magnetic field and a place to put a sample. The oscilloscope is where you will view both the input of the pulse generator into the magnet and the output of the sample after receiving pulses. Its controls are shown below:

1

2

3 4

5

11

The five most important knobs are outlined in solid red. In display mode, 1, 2, 4, and 5 control the panning (POSITION) and zooming (VOLTS/DIV or SEC/DIV) of the display. In cursor mode, 1 and 3 move the cursors. To change between modes, use the buttons above the vertical and horizontal controls. The five buttons in a column left of the vertical and horizontal controls allow you to change the options displayed on the right-hand side of the screen. They allow you to change between vertical and horizontal cursors when you’re in cursor mode, and when you’re in MATH MENU (in the center of the picture), they will allow you to do to cycle through operation options (add, subtract, etc.) until you find “FT”. The pictures below show a pulse, which is what the pulse programmer sends to the magnet. When you first turn on the oscilloscope, it may take some adjusting to get the view on the left, but just keep fiddling with the zoom and panning until you see a single solid line. Zooming in even further (center it before zooming to avoid losing it off screen) will give you the image on the left, which is your pulse. The pulse duration is the width of this square pulse (time is x-axis).

When you switch the cables around to view the FID, it will look something like this:

12

Photograph by Keith Nelson, Timothy Swager, andPhotograph by Keith Nelson, Timothy Swager, andMariusz Twardowski. Used with permission.Mariusz Twardowski. Used with permission.

The flat part to the far left is the baseline before the pulse was applied. The first large spike is mostly noise—it contains the pulse you applied, and it overwhelms your signal response until a short time later, when you see the second smaller peak next to it (see arrow). This is the signal you will monitor when trying to maximize the FID. The long oscillation afterwards is the difference between your reference pulse and the natural frequency of the proton precessions, and is what you will minimize by adjusting the frequency on the pulse generator. Afterwards your signal should look something like this:

Note that the oscillations after the signal are almost completely gone. Another helpful visual is a spin echo:

At this point in the experiment you will have set up two pulses, which are the first two FIDs you see here. The last signal, which lacks the spike characteristic of a pulse sent to the instrument, is your echo! The echo can be much less pronounced that what you see here, but you will know to always look for it at the same distance from the second pulse that the second pulse is from the first. If that doesn’t seem obvious, review the process that causes the echo. Finally, this is what a FT of your signal should look like:

13

Note how broad this peak is (and how poor the signal-to-noise is) in comparison to the nice sharp peaks you see in the 300 MHz experiment!

14

Appendix C:

How to Obtai 1

Before ejecting the

Step

previous

1: Ge

sample

t a lock

that

sig

has

nal

been

and shim

n a H NMR

•

checking the

there should always be a sample in the

Acquisition Status

left into the instrument (

wind

probeow.

) make sure that the spinner is off by

Me

• To

nu buttoeject the

nssample windo

and w and

to recover click on

the eject

spinner .

(turbine), click the

Acqi button in the

• Remove

into the the spinner

sample and that clean

is currently the outside

in the of the

probe. NMR

Carefully tube with

insert a KimWipe.

your

sample Position

tube

thallow e tube

the isample nto the

the magnetic homogeneity of the sample. to spinner be rotated

with at the a frequency depth gauge.

of 20 The Hz inside

purpose the of magnet, the spinner

increasing is to

15

• Csplimb inner

the on ladder the air

to cuin

reach

sertshion

.

the

thatop t flof owthe s throu

magnet gh t

and

carefully position the

• computer and click

20 Hz Lo

he

ck

top NMR probe. Return tubte o atnd he

•

Set adjusting the bottom parameter.

the spinner rate to by clicking the button in the Acqi window and

•

In shouthe ld command

From here, set the

you shimline -‐coilat the

have two s to r

top

choices. easof onablythe main ype

good

window, values to

t

Option 1 will automatically star

‘rts’

go

t wi

straight

thand. enter ‘

lock,

best’. This

shim,

Option 2 allows for manual shimming and locking. perform the selected experiment, which means you can to

mid-‐Step and 3.

Option 1: Automatic • Cl

of the monitor. If you do not see the Walkup button, expand theick on Walkup tab in the Tcl/dg window, which is present on the bottom left area

window.

16

• Select type of

the experiment appropriate

you solvent would

from like to

the run dropdown (in our case,

Solvent 1H).

menu,

not start automatically, click the Start ACQ button. If the

then experiment

click on does the

Option 2: Manual • Click on the Acqi Tab and the Acquisition window will appear.

•

Cl ick on Lock

•

button. Check that the LOCK is

Initially

off.

quantthe lockpower

ity, the whilspinnere

1the

rightis off

. increases). Set

lockgain

the spin The to 20 Hz (left-‐clicking a button decreases the

to 20 and the to 2spinner 8.

should now be on. Also increase

1 The lock power and lock gain levels depends on the concentration of the deuterated solvent, the number of deuterium atoms in the solvent and the relaxation time of the deuterium in that particular solvent.

17

•

Adjust the magnetic field frequency Z0 shim (units

deuThe

tegreater rium resonance field.

the number of sine waves, the poorer are H

the z), until

match a sine

of wave Z0 wi

is thseen. the

18

• If the lock is near resonance, the wave looks like:

•

PoIf the

showwavweer

amplitude 2

is too low, increase the , and, if necessary the

len ngthuntil isthe seen,

signal and is tbetween hen

50% andLock ain

, 100%G. Continue to adjust until

Loone ck

below should appear. click the h ck ste

Z0Lock on button. T e lo p function

• Ifthat phas

the e islock not

signal set proper

is maximized. ly, adjust the

decrease the Lock Gain. If lockphase

the signal appears in steps of

to –be 1 or satu+1. raYtoued w(>= ill not

100),ice

2 A too high Lock Power (>40) is saturating the lock signal. Saturation is signaled by the fact that the lock level is very erratic. Large values for Lock Gain give a noisy lock signal. Reduce Lock Power and adjust Lock Gain to maximize the lock level.

19

• Now, coils shim.

that Shimming surround

adjusts the

the current flow

•

Here, only the through

“spinning” a series of differently

will be adsample. (e.g. the sample-‐

val

shaped spinning) shims In

to

the Z1 – Z7

ACQUISITIONjusted.

ues of Z1C, Z2C, Z1 wi

andnd

SHZ2 ow in order to maximize the lock signal. click on IM. Start making small changes to the

•

• (or, ifWhen

necessarthe lock

y, thesignal

Logock Poes abo

wvere

su, in t100

he% , Loreck duc

up wie

In the command line Acqitype which sets the

ndtheo w)lock

Lo.ck Gain in the same window

disconnects from the window.

and shim parameters and

20

Select ,

St

, and

ep

the

3: Acquire spectra

• Main

for nt=

p8rot toon set

Menu appropriate and . Now, type in

NMtheR. nu

Then mber Setup

typof e transients (acquisitions)

Nucleusto eight, which

Solvenis t

ga to acquire and automatically process your spectrum. a good choice

•

UsualAfter

ly, the

symmetric the transients are completed, the spectrum

around spectrum

the base is not

of a prpeak. oper

ly Auto-‐phase phased;

ffav.

ththe ewill

be

spectrbaseline

displayed

um iby sn’t typing flat on and n’t

the command window or by using the macro aph

screen. iints

If one doesn’t work, try the other.o

• dscale •

If

Reference the x-‐axis disappears at any point, type the command to retrieve it.

that is used the comes spectrum

from either CHCl

to 3, your which

solvent is in

or small to TMS.

amounts In this

in example, the CDC

the l3 sol

proton vent.

21

Expand the NMR scale around 7-‐ 8 ppm: click on the Boxbutton after the arrow is close to 7 ppm. Click with the left button

button. C around 8.2 ppm. lick on the

right

• Expand

nl

Clchloroform ick on the

peak, then button. type the

With command the mouse,

(stbring

rl(7.

display

27p).

digital resolution, which for a well shimmed ands

the for red nea

cursor rest line),

very

be or close

This will place the probe should less

nlthan drto the

1 esH toz.

• Click the full If button to display the full spectrum, or type you decid

red e to uscursor

e the on TMStop signaof the

l, tynearest pe (rl=0.

line. 0p

f). Type the command

and press enter.

• Integrate the spectrum

contresets

: Click . The integr line will be displayed

clickinuousl

y. al

after the signal. Re. L To eft

cutclick the on intthe egralpart

integr abinove al

tegral

slightly each peak

befor, te ype the czsignal (clears

and allthen reset

slightly s) and

peat for each peak.

22

•

•

To display the values of

necessary make save you

vp=r file

1

•Finally, w

andthe typeintegrals ds dpir

underneath again.

the spectrum type ds dpir. If

To page

get (meaning: a hard copy

plot, wit

2

h ith your the svf

1H (‘NMR, filenam

plot e’) command.

plot the ppm scale, the plot spectrum

the integral typing

•

resets, pl pscale

plot pir pap

ex

acquisition parameters) To

some

pand see coupling

buttonspatterns, you can expand

th

regions

threshold parameter

dpf

. In by ordclicking er to ge

on t the

. pe With ak fre

of spectrum

the queleft ncymouse, , first set

with th

the and

yellow line such as to include all the peaks with significance (avoset

ithe e peheightak box pi of ckitng he

• ppfhYosignau alsz

ls). o Typcan

e print

toth dei seplayxpand

thee ped rak f

d the noise level

provides peak position in heretzgioren qby uenc

typiiesng .

constant values. , which is very

pl usppfhz eful to

pscale extract

page the cou

. Macrpling

o

EjectClick , ,

Wh, make en you have finished

• acqi lock off

sample

spin off•

= 0, make

= 0.

turbine. Wipe the sample with KimWipe, and

• Substitute your with a sealed

lockpower

sample. Insert

lock gain

insert. the sealed sample in the

•

• ‘autolock’ to no and ‘autoshim’ to no. Disable auto-‐locking and auto

Tr-‐shimming oubleshoot

features: ing

Hit ‘flags and conditions,’ toggle

If prompt the software

by right-‐is clnot icking responding:

on the desk Close

top and back

open the program. Open a command

th‘terminal’. en ente

Enter ‘su acqproc’ into the command ground, line to kill

paging the

tcurrent o ‘tools’,

acquisition, and then

respthen tur

ond,n i tru ‘srnu acoff qthe proc

spectrometer ’ to re-‐initialize

(white the spectrometer. still

t bacswitch around

If back), the software

wait a few does seconds,

not

k on before re-‐opening the software.

23

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