basics for qnmr - help & resources for mnova nmr and other ...€¦ · basics for qnmr peter...
TRANSCRIPT
![Page 1: Basics for qNMR - Help & Resources for Mnova NMR and other ...€¦ · Basics for qNMR Peter Lankhorst, Joep van Rijn & John Gauvin September 2017. Page 1 Quantitative NMR: The principle](https://reader033.vdocuments.us/reader033/viewer/2022050515/5f9f87347586f82b50171a4f/html5/thumbnails/1.jpg)
Basics for qNMR
Peter Lankhorst, Joep van Rijn & John Gauvin
September 2017
![Page 2: Basics for qNMR - Help & Resources for Mnova NMR and other ...€¦ · Basics for qNMR Peter Lankhorst, Joep van Rijn & John Gauvin September 2017. Page 1 Quantitative NMR: The principle](https://reader033.vdocuments.us/reader033/viewer/2022050515/5f9f87347586f82b50171a4f/html5/thumbnails/2.jpg)
Page 1
Quantitative NMR: The principle
Purity determination
P = purity
MW = molecular weight
n = number of protons
W = weight
A = NMR area
st
x
st
st
x
x
st
st
xx P
W
W
MW
MW
n
n
A
AP
Ax ~ nx
![Page 3: Basics for qNMR - Help & Resources for Mnova NMR and other ...€¦ · Basics for qNMR Peter Lankhorst, Joep van Rijn & John Gauvin September 2017. Page 1 Quantitative NMR: The principle](https://reader033.vdocuments.us/reader033/viewer/2022050515/5f9f87347586f82b50171a4f/html5/thumbnails/3.jpg)
Page 2
3 types of qNMR applications
Concentration of producs and impurities
• Down to ppm level is possible
Purity determination of standards for other techniques: HPLC.
• Highest accuracy and precision required.
Composition of complex mixtures.
• Many compound classes at the same time. E.g. metabolomics
![Page 4: Basics for qNMR - Help & Resources for Mnova NMR and other ...€¦ · Basics for qNMR Peter Lankhorst, Joep van Rijn & John Gauvin September 2017. Page 1 Quantitative NMR: The principle](https://reader033.vdocuments.us/reader033/viewer/2022050515/5f9f87347586f82b50171a4f/html5/thumbnails/4.jpg)
Page 3
NMR linearity
Range 1…..1300
R2 = 0.99999
Range 1…240000
R2 = 0.99989
![Page 5: Basics for qNMR - Help & Resources for Mnova NMR and other ...€¦ · Basics for qNMR Peter Lankhorst, Joep van Rijn & John Gauvin September 2017. Page 1 Quantitative NMR: The principle](https://reader033.vdocuments.us/reader033/viewer/2022050515/5f9f87347586f82b50171a4f/html5/thumbnails/5.jpg)
Page 4
Diacetyl impurities Quantitative NMR Dynamic range
![Page 6: Basics for qNMR - Help & Resources for Mnova NMR and other ...€¦ · Basics for qNMR Peter Lankhorst, Joep van Rijn & John Gauvin September 2017. Page 1 Quantitative NMR: The principle](https://reader033.vdocuments.us/reader033/viewer/2022050515/5f9f87347586f82b50171a4f/html5/thumbnails/6.jpg)
Page 5
Quantitative NMR Dynamic range
![Page 7: Basics for qNMR - Help & Resources for Mnova NMR and other ...€¦ · Basics for qNMR Peter Lankhorst, Joep van Rijn & John Gauvin September 2017. Page 1 Quantitative NMR: The principle](https://reader033.vdocuments.us/reader033/viewer/2022050515/5f9f87347586f82b50171a4f/html5/thumbnails/7.jpg)
Page 6
Quantitative NMR Dynamic range
![Page 8: Basics for qNMR - Help & Resources for Mnova NMR and other ...€¦ · Basics for qNMR Peter Lankhorst, Joep van Rijn & John Gauvin September 2017. Page 1 Quantitative NMR: The principle](https://reader033.vdocuments.us/reader033/viewer/2022050515/5f9f87347586f82b50171a4f/html5/thumbnails/8.jpg)
Page 7
Acetaldehyde 7 ppm
Quantitative NMR Dynamic range
![Page 9: Basics for qNMR - Help & Resources for Mnova NMR and other ...€¦ · Basics for qNMR Peter Lankhorst, Joep van Rijn & John Gauvin September 2017. Page 1 Quantitative NMR: The principle](https://reader033.vdocuments.us/reader033/viewer/2022050515/5f9f87347586f82b50171a4f/html5/thumbnails/9.jpg)
Page 8
Amoxicillin Purity
The problem:
• Production batches were rejected. Purity on HPLC was too high!!! > 100.5% means out of spec
• Purity determination with USP standards is required
Strategy:
• Check standards with quantitative NMR
![Page 10: Basics for qNMR - Help & Resources for Mnova NMR and other ...€¦ · Basics for qNMR Peter Lankhorst, Joep van Rijn & John Gauvin September 2017. Page 1 Quantitative NMR: The principle](https://reader033.vdocuments.us/reader033/viewer/2022050515/5f9f87347586f82b50171a4f/html5/thumbnails/10.jpg)
Page 9
Amoxicillin
(ppm)
1.01.52.02.53.03.54.04.55.05.56.06.57.0
7 65
4
3 21 1dmso
acetonitril
(ppm)
1.01.52.02.53.03.54.04.55.05.56.06.57.0
*
*
**
DSM
USP
![Page 11: Basics for qNMR - Help & Resources for Mnova NMR and other ...€¦ · Basics for qNMR Peter Lankhorst, Joep van Rijn & John Gauvin September 2017. Page 1 Quantitative NMR: The principle](https://reader033.vdocuments.us/reader033/viewer/2022050515/5f9f87347586f82b50171a4f/html5/thumbnails/11.jpg)
Quote CS Marcel van Tilborg
“HPLC is like you girlfriend, she will tell you what
you want to hear.
NMR is like you mother, she will tell you the truth”
Page 10
![Page 12: Basics for qNMR - Help & Resources for Mnova NMR and other ...€¦ · Basics for qNMR Peter Lankhorst, Joep van Rijn & John Gauvin September 2017. Page 1 Quantitative NMR: The principle](https://reader033.vdocuments.us/reader033/viewer/2022050515/5f9f87347586f82b50171a4f/html5/thumbnails/12.jpg)
Page 11
Complex mixtures - wine
glycerol
Tartaric
acid
Lactic acidInternal standard
butanediol
Succinic
acid
![Page 13: Basics for qNMR - Help & Resources for Mnova NMR and other ...€¦ · Basics for qNMR Peter Lankhorst, Joep van Rijn & John Gauvin September 2017. Page 1 Quantitative NMR: The principle](https://reader033.vdocuments.us/reader033/viewer/2022050515/5f9f87347586f82b50171a4f/html5/thumbnails/13.jpg)
Page 12
Complex mixtures - wine
proline
Galacturonic
acid
Vitamin C
tyrosine
![Page 14: Basics for qNMR - Help & Resources for Mnova NMR and other ...€¦ · Basics for qNMR Peter Lankhorst, Joep van Rijn & John Gauvin September 2017. Page 1 Quantitative NMR: The principle](https://reader033.vdocuments.us/reader033/viewer/2022050515/5f9f87347586f82b50171a4f/html5/thumbnails/14.jpg)
Page 13
Complex mixtures – wine
Benzoic acid
![Page 15: Basics for qNMR - Help & Resources for Mnova NMR and other ...€¦ · Basics for qNMR Peter Lankhorst, Joep van Rijn & John Gauvin September 2017. Page 1 Quantitative NMR: The principle](https://reader033.vdocuments.us/reader033/viewer/2022050515/5f9f87347586f82b50171a4f/html5/thumbnails/15.jpg)
Key aspects for qNMR
• Biological variation/sample inhomogeneitySample variation
• Changes over time?Keep track of your
standards
• Simple?Accurate weighing
• When internal standard in solution is addedAccurate pipetting
• Which pulseprogram to use?
• 30˚ or 90˚ pulse?
Relax and remember your T1 lessons
Page 14
![Page 16: Basics for qNMR - Help & Resources for Mnova NMR and other ...€¦ · Basics for qNMR Peter Lankhorst, Joep van Rijn & John Gauvin September 2017. Page 1 Quantitative NMR: The principle](https://reader033.vdocuments.us/reader033/viewer/2022050515/5f9f87347586f82b50171a4f/html5/thumbnails/16.jpg)
Page 15
Important tips for accurate weighing
1.Use tweezers
2.Clean the table
3.Clean the weighing platform of the balance before weighing.
4.Put an empty vial on the balance a few minutes before first
weighing.
5.Check if there are no magnetic objects near the balance.
6.Use vials with a narrow rim
7.Tap the vial before the weighing
8.Do not add material while the vial is on the balance!!
9.Do not attach stickers to the vial!!
10.Close the doors of the balance fully!!
11.Wait until the balance has stabilized before zeroing!!
12.Store the vials close to the balance
13.Do not use vials immediately after opening the package
![Page 17: Basics for qNMR - Help & Resources for Mnova NMR and other ...€¦ · Basics for qNMR Peter Lankhorst, Joep van Rijn & John Gauvin September 2017. Page 1 Quantitative NMR: The principle](https://reader033.vdocuments.us/reader033/viewer/2022050515/5f9f87347586f82b50171a4f/html5/thumbnails/17.jpg)
Page 16
Is weighing the most important source of errors?
Batch weighing Succinic acid
S0503 A 99.61% avg 99.57%
S0503 B 99.58% stdev 0.03%
S0503 C 99.53% max 99.61%
S0503 D 99.56% min 99.53%
S0503 E 99.58%
Idea:
• weigh more compound and standard
• use microbalance (0.001 mg read-out)
![Page 18: Basics for qNMR - Help & Resources for Mnova NMR and other ...€¦ · Basics for qNMR Peter Lankhorst, Joep van Rijn & John Gauvin September 2017. Page 1 Quantitative NMR: The principle](https://reader033.vdocuments.us/reader033/viewer/2022050515/5f9f87347586f82b50171a4f/html5/thumbnails/18.jpg)
Page 17
Important tips for accurate weighing
1.Use tweezers
2.Clean the table
3.Clean the weighing platform of the balance before weighing.
4.Put an empty vial on the balance a few minutes before first
weighing.
5.Check if there are no magnetic objects near the balance.
6.Use vials with a narrow rim
7.Tap the vial before the weighing
8.Do not add material while the vial is on the balance!!
9.Do not attach stickers to the vial!!
10.Close the doors of the balance fully!!
11.Wait until the balance has stabilized before zeroing!!
12.Store the vials close to the balance
13.Do not use vials immediately after opening the package
![Page 19: Basics for qNMR - Help & Resources for Mnova NMR and other ...€¦ · Basics for qNMR Peter Lankhorst, Joep van Rijn & John Gauvin September 2017. Page 1 Quantitative NMR: The principle](https://reader033.vdocuments.us/reader033/viewer/2022050515/5f9f87347586f82b50171a4f/html5/thumbnails/19.jpg)
Page 18
Requirements for NMR internal standards
High purity, typically > 99%
Not hygroscopic
Stable over a long period, chemically
inert
Non volatile
Simple compound, withone or two NMR
signals
Soluble
NMR signals in non-crowded
area of spectrum
![Page 20: Basics for qNMR - Help & Resources for Mnova NMR and other ...€¦ · Basics for qNMR Peter Lankhorst, Joep van Rijn & John Gauvin September 2017. Page 1 Quantitative NMR: The principle](https://reader033.vdocuments.us/reader033/viewer/2022050515/5f9f87347586f82b50171a4f/html5/thumbnails/20.jpg)
Page 19
Amongst others:
O O
OH O-
K+
O
OH
O
OH
OO
CH3
CH3
CH3N+
O-
O
Potassium biftalate Maleic acid
p-Nitrotolueen dimethoxybenzeen
NMR standard examples
![Page 21: Basics for qNMR - Help & Resources for Mnova NMR and other ...€¦ · Basics for qNMR Peter Lankhorst, Joep van Rijn & John Gauvin September 2017. Page 1 Quantitative NMR: The principle](https://reader033.vdocuments.us/reader033/viewer/2022050515/5f9f87347586f82b50171a4f/html5/thumbnails/21.jpg)
Doing the mathematicsT1 relaxation with 90˚ pulse
Page 20
results• 𝜏 = interpulse delay experiments
• When 𝜏 =T1 63% recovery to equilibrium
Example:• 99% rec
- 𝜏 /T1 = ln(1−0.99)= -4.6
𝑀𝑧(𝜏)
𝑀𝑧,𝑒𝑞= 1 − 𝑒−𝜏/𝑇1
1−𝑀𝑧(𝜏)
𝑀𝑧,𝑒𝑞= 𝑒−𝜏/𝑇1
ln(1−𝑀𝑧(𝜏)
𝑀𝑧,𝑒𝑞)= - 𝜏 /T1
• 99% recovery > d1= 4.6*T1
• 99.9% recovery > d1= 6.9*T1
• 99.99% recovery > d1= 9.2*T1
𝑀𝑧(𝜏) = 𝑀𝑧,𝑒𝑞(1 − 𝑒−𝜏/𝑇1)
![Page 22: Basics for qNMR - Help & Resources for Mnova NMR and other ...€¦ · Basics for qNMR Peter Lankhorst, Joep van Rijn & John Gauvin September 2017. Page 1 Quantitative NMR: The principle](https://reader033.vdocuments.us/reader033/viewer/2022050515/5f9f87347586f82b50171a4f/html5/thumbnails/22.jpg)
21
NMR – sensitivity & quantification
Z
Y
X
Z
Y
X
Z
Y
X
Bo
![Page 23: Basics for qNMR - Help & Resources for Mnova NMR and other ...€¦ · Basics for qNMR Peter Lankhorst, Joep van Rijn & John Gauvin September 2017. Page 1 Quantitative NMR: The principle](https://reader033.vdocuments.us/reader033/viewer/2022050515/5f9f87347586f82b50171a4f/html5/thumbnails/23.jpg)
22
Net magnetization with 30˚ or 90˚?
Bo Z
Y
X
Z
Y
X
Z
Y
X
![Page 24: Basics for qNMR - Help & Resources for Mnova NMR and other ...€¦ · Basics for qNMR Peter Lankhorst, Joep van Rijn & John Gauvin September 2017. Page 1 Quantitative NMR: The principle](https://reader033.vdocuments.us/reader033/viewer/2022050515/5f9f87347586f82b50171a4f/html5/thumbnails/24.jpg)
23
How long do you need to wait?
0
20
40
60
80
100
120
0 5 10 15 20 25
NET
MZ
(%)
INTERPULSE DELAY (S)
T1 relaxation 2.5s
@ 30˚ @ 90˚
![Page 25: Basics for qNMR - Help & Resources for Mnova NMR and other ...€¦ · Basics for qNMR Peter Lankhorst, Joep van Rijn & John Gauvin September 2017. Page 1 Quantitative NMR: The principle](https://reader033.vdocuments.us/reader033/viewer/2022050515/5f9f87347586f82b50171a4f/html5/thumbnails/25.jpg)
0
20
40
60
80
100
120
0 10 20 30 40 50 60
NET
MZ
(%)
INTERPULSE DELAY (S)
T1 relaxation 10s
24
How long do you need to wait?
@ 30˚ @ 90˚
![Page 26: Basics for qNMR - Help & Resources for Mnova NMR and other ...€¦ · Basics for qNMR Peter Lankhorst, Joep van Rijn & John Gauvin September 2017. Page 1 Quantitative NMR: The principle](https://reader033.vdocuments.us/reader033/viewer/2022050515/5f9f87347586f82b50171a4f/html5/thumbnails/26.jpg)
25
Relax and go 90’s
0,0
2,0
4,0
6,0
8,0
10,0
12,0
14,0
16,0
18,0
0 100 200 300 400 500 600 700 800 900 1000 1100
SiN
ore
lati
ve
Number of scans
SiNo vs number of scans
Sensitivity improves
With 2 with a double
amount of scans.
~4x as much scans are
needed to achieve same
SiNo.
@ 90˚
@ 30˚
![Page 27: Basics for qNMR - Help & Resources for Mnova NMR and other ...€¦ · Basics for qNMR Peter Lankhorst, Joep van Rijn & John Gauvin September 2017. Page 1 Quantitative NMR: The principle](https://reader033.vdocuments.us/reader033/viewer/2022050515/5f9f87347586f82b50171a4f/html5/thumbnails/27.jpg)
-
20,00
40,00
60,00
80,00
100,00
120,00
140,00
160,00
180,00
200,00
0 20 40 60 80 100 120 140 160 180 200
SIG
NA
L IN
CR
EA
SE (
REL)
TIME SPENT (S)
Signal increase per unit time
30dg, 2.5s
45dg, 2.5s
90dg, 2.5s
30dg, 10s
45dg, 10s
90dg, 10s
26
Relax and go 90’s
30˚ pulse needs ~2x more
measurement time for the same SiNo
@90˚ @30˚@45˚
@30˚
@45˚
@90˚
![Page 28: Basics for qNMR - Help & Resources for Mnova NMR and other ...€¦ · Basics for qNMR Peter Lankhorst, Joep van Rijn & John Gauvin September 2017. Page 1 Quantitative NMR: The principle](https://reader033.vdocuments.us/reader033/viewer/2022050515/5f9f87347586f82b50171a4f/html5/thumbnails/28.jpg)
Thank you!
Acknowledgments
DSM
• Peter Lankhorst
• Joep van Rijn
Page 27