View
5
Download
0
Category
Preview:
Citation preview
Basics for qNMR
Peter Lankhorst, Joep van Rijn & John Gauvin
September 2017
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 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 3
NMR linearity
Range 1…..1300
R2 = 0.99999
Range 1…240000
R2 = 0.99989
Page 4
Diacetyl impurities Quantitative NMR Dynamic range
Page 5
Quantitative NMR Dynamic range
Page 6
Quantitative NMR Dynamic range
Page 7
Acetaldehyde 7 ppm
Quantitative NMR Dynamic range
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 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
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 11
Complex mixtures - wine
glycerol
Tartaric
acid
Lactic acidInternal standard
butanediol
Succinic
acid
Page 12
Complex mixtures - wine
proline
Galacturonic
acid
Vitamin C
tyrosine
Page 13
Complex mixtures – wine
Benzoic acid
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 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 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 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 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 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
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)
21
NMR – sensitivity & quantification
Z
Y
X
Z
Y
X
Z
Y
X
Bo
22
Net magnetization with 30˚ or 90˚?
Bo Z
Y
X
Z
Y
X
Z
Y
X
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˚
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˚
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˚
-
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˚
Thank you!
Acknowledgments
DSM
• Peter Lankhorst
• Joep van Rijn
Page 27
Recommended