Module’1’Experimental’Procedures’ Experiment’1.’What ... ·...
Transcript of Module’1’Experimental’Procedures’ Experiment’1.’What ... ·...
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
• 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
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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5.35 / 5.35U Introduction to Experimental ChemistryFall 2012
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