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