Lc nmr

LC-NMR

1. 1 Prepared By: Syed Rashed Faizan Mehdi 170717885012 M. Pharm, Pharmaceutical Analysis Dept. of Pharmaceutical Analysis DECCAN SCHOOL OF PHARMACY LC-NMR And Applications Of NMR Spectroscopy Guided by : DR. Mohammed Younus M. Pharm, Pharmaceutical Analysis. Ph D Dept. of Pharmaceutical Analysis DECCAN SCHOOL OF PHARMACY

2. 2 TABLE OF CONTENTS CONTENTS Introduction History LC-NMR Principle of LC & NMR Instrumentation of LC-NMR LC Components Interface Components NMR Components Modes of LC-NMR Technology to improve sensitivity of LC-NMR Method LC Methods NMR Method Solvent suppression method Applications Of NMR

3. 3 NMR is one of the most powerful techniques for the structural elucidation of organic compounds. Separation and isolation of the individual components of a sample mixture are the normal steps before analyzing their structures by NMR. Prior to isolation, LC/MS is used routinely to analyze the components of the mixture to evaluate the need to isolate the compound(s) of interest. In many cases, however, NMR is necessary for the identification of ambiguous structures. Even though hyphenated LC-NMR has been known since the late 1970s , the technique was not implemented widely until the last two decades . For a more complete structural analysis , LC-MS and LC- NMR have been combined (LC-NMR-MS), with its efficacy demonstrated successfully. Introduction

4. 4 NMR is one of the most powerful techniques for the structural elucidation of organic compounds. The first on-line LC–NMR experiments were performed in the late 1970s by Watanabe and Niki who demonstrated stopped-flow measurements of a mixture of known compounds. The conventional NMR probe was transformed to a flow-through probe by the introduction of a thin-walled Teflon capillary within a standard NMR tube and spectra were recorded with sample rotation . The first real sample analyzed by LC–NMR was military jet fuel using normal phase column and deuterated chloroform and Freon In theory, the physical coupling of LC with NMR could save a lot of time and was already proposed over 20 years ago. However , a successful and practical LC–NMR couplin g has been achieved only in the last decade. History of NMR

5. 5 High performance liquid chromatography (HPLC) is basically a highly improved form of column liquid chromatography . Instead of a solvent being allowed to drip through a column under gravity , it is forced through under high pressures of up to 400 atmospheres. That makes it much faster. All chromatographic separations, including HPLC operate under the same basic principle; separation of a sample into its constituent parts because of the difference in the relative affinities of different molecules for the mobile phase and the stationary phase used in the separation. Principle of LC (HPLC) Types of HPLC Normal phase HPLC Reverse phase HPLC Size-Exclusion HPLC 4. Ion-Exchange HPLC

6. 6 The principle behind NMR is that many nuclei have spin and all nuclei are electrically charged . If an external magnetic field is applied , an energy transfer is possible between the base energy to a higher energy level (generally a single energy gap). The energy transfer takes place at a wavelength that corresponds to radio frequencies and when the spin returns to its base level, energy is emitted at the same frequency. The signal that matches this transfer is measured in many ways and processed in order to yield an NMR spectrum for the nucleus concerned. Principle of NMR

7. 7 above, relates to spin-½ nuclei that include the most commonly used NMR nucleus, proton (1H or hydrogen-1) as well as many other nuclei such as 13C, 15N and 31P.

8. 8 Instrumentation of LC-NMR

9. 9 Instrumentation of LC-NMR Diagrammatic Representation of LC-NMR

10. 10 Instrumentation of LC-NMR Representation of LC-NMR

11. 11 As shown in the schematic diagram in Figure above, HPLC instrumentation includes a pump , injector , column , detector and data acquisition and display system . The heart of the system is the column where separation occurs . LC Unit

12. 12 1) Solvent Reservoir  : Mobile phase contents are contained in a glass reservoir. It is usually a mixture of polar and non-polar liquid components whose respective concentrations are varied depending on the composition of the sample. 2) Pump  : A pump aspirates the mobile phase from the solvent resorvoir and forces it through the system’s column and detector . Depending on a number of factors including column dimensions , particle size of the stationary phase , the flow rate and composition of the mobile phase , operating pressures of up to 42000 kPa (about 6000 psi) can be generated. 3) Sample Injector  : The injector can be a single injection or an automated injection system. An injector for an HPLC system should provide injection of the liquid sample within the range of 0.1-100 mL of volume with high reproducibility and under high pressure (up to 4000 psi). LC Unit

13. 13 4)Columns  : Columns are usually made of polished stainless steel, are between 50 and 300 mm long and have an ID( 2 and 5 mm).They are commonly filled with a stationary phase with a particle size of 3–10 µm . Columns with internal diameters of less than 2 mm are often referred to as microbore columns . Ideally the temperature of the mobile phase and the column should be kept constant during an analysis. 5) Detector  : The HPLC detector, located at the end of the column detect the analytes as they elute from the chromatographic column. Commonly used detectors are UV-spectroscopy, fluorescence, mass-spectrometric and electrochemical detectors. 6) Data Collection Devices  : Signals from the detector may be collected on chart recorders or electronic integrators .The computer integrates the response of the detector to each component and places it into a chromatograph that is easy to read and interpret . LC Unit

14. 14 1) Di r e c t coup li n g : It i n clu d e d i r e ct f l o w o f L C e ff l u e n t in to N MR f l o w c e ll a n d co n ti nu o u s r e cor d i n g o f sp e c t ra ü p o s t - col u m n s p litter v al v e - s w itc h i n g i n ter f a c e i.e BN M I ( B r u ker N M R - Ma s s Sp e ctr om e try I n ter f ac e ) LC-NMR Interface 2) I nd i r e c t co u p li n g : ü i n ter me d ia t e s t o rage lo o p w h ich tr an sfe r o u tlet of lc to N MR flow c e ll a t s p e c i f i e d t i m e i n te r v al ü SPE un it fig: 3 6 L o op Ca s se t t e

15. 15 NMR Unit Basically NMR instrumentation involves the following units. 1. A magnet to separate the nuclear spin energy state. 2. Two RF channels , one for the field/frequency stabilization and one to supply RF irradiating energy. 3. A sample probe containing coils for coupling the sample with the RF field; it consists of Sample holder , RF oscillator , Sweep generator and RF receiver. 4. A detector to process the NMR signals. 5. A recorder to display the spectrum . B o B 1 M a gn e t Rec o r d er Fr e qu e n c y Generator Detect o r S N

16. 16

17. 17 MAGNETS It is used to supply the principal part of the field Ho, which determines the Larmor or precessional frequency of any nucleus. The stronger the magnetic field , the better the line separation of chemically shifted nuclei on the frequency scale. The relative populations of the lower energy spin level increases with the increasing field , leading to a corresponding increase in the sensitivity of the NMR experiment. 1. It should give homogeneous magnetic field i.e.; the strength and direction of the magnetic field should be constant over longer periods. 2. The strength of the field should be very high at least 20,000 gaus . TYPES OFMAGNETS 1. PERMANENT magnets 2. ELECTRO magnets and 3. SUPER CONDUCTING magnets Features

18. 18 A Big Stainless Steel Dewar • use persistent superconducting magnets to generate the B field ; • at low temperatures (less than 6 K, typically) the resistance goes to zero – that is the wire( eg.Nb alloy) is superconducting ; • To maintain the wire in its superconducting state the coil is immersed in a bath of liquid helium ( 4 K, expensive ); • “heat shield” kept at 77 K by contact with a bath of liquid nitrogen ( cheap ) to reduces the amount of liquid helium boils off; • vacuum flask so as to further reduce the heat flow. 1. Strongest Magnet; 2. Stable & homogeneous magnet field Bo; 3. Low running cost. SUPERCONDUCTING MAGNETS ( scm ): Advantages ( scm ):

19. 19 It is not easy or convenient to vary the magnetic field of large stable magnets, however this problem can be overcome by superimposing a small variable magnetic field on the main field . Using a pair of Helmholtz coils( A  Helmholtz coil  is a device for producing a region of nearly uniform magnetic field, named after the German physicist Hermann von  Helmholtz) on the pole faces of the permanent magnet does this. These coils induce a magnetic field that can be varied by varying the current flowing through them . The small magnetic field is produced in the same direction as the main field and is added to it. The sample is exposed to both fields, which appear one field to the nucleus. MAGNETIC COILS

20. 20 THE PROBE UNIT It is a sensing element of the spectrophotometer system. It is inserted between the pole faces of the magnet in X-Y plane of the magnet air gap an adjustable probe Holder. So the sample in NMR experiment experiences the combined effect of two magnetic fields ie Ho and RF (EMR). The usual NMR sample cell is generally made up of the glass , which is strong and cheap. It consist of a 5 mm outer diameter and 7.5 cm long glass tube containing 0.4 ml of liquid. The sample tube in NMR is held vertically between the poles faces of the magnet. The probe contains a sample holder, sweep source and detector coils, with the reference cell. The detector and receiver coils are orientated at 90 to each other. The sample probe rotates the sample tube at a 30-40 revolutions on the longitudinal axis. Each part of the sample tube experiences the same time average the field.

21. 21 THE RADIOFREQUENCY GENERATOR Using an RF oscillator creates the radio frequency radiation, required to induce transition in the nuclei of the sample from the ground state to excited states. The source is highly stable crystal controlled oscillator . It is mounted at the right angles to the path of the field of wound around the sample tube perpendicular to the magnetic field to get maximum interaction with the sample. The oscillator irradiates the sample with RF radiation. Radio frequencies are generated by the electronic multiplication of natural frequency of a quartz crystal contained in a thermo stated block. To achieve the maximum interaction of the RF radiation with the sample , the coil of oscillator is wound around the sample container . The RFO coil is installed perpendicular (90 ºC ) to the applied magnetic field and transmits radio waves of fixed frequency such as 60,100,200 or 300 MHz to a small coil that energies the sample in the probe . This is done so that the applied RF field should not change the effective magnetic field in the process of irradiation

22. 22 • High resolution NMR requires linewidths of 1 Hz or less • Magnetic field across the sample must be homogeneous so that the corresponding variation in the Larmor frequency is small. • Surround the sample with a set of shim coils , each of which produces a tiny magnetic field with a particular spatial profile to canceling out the small residual inhomogeneities in the main magnetic field. • Modern spectrometers might have up to 40 different shim coils labeled according to the field profiles they generate, such as x , y, z , z 2 , z 3 , z 4 , z 5 , xy , xz , yz , x 2 -y 2 , etc … • Shimming, the process to optimize the shims, requires skill and experience because various shims will interact with each other. Shim Coils

23. 23 Synthesizer: RF source which produces a stable frequency which can be set precisely. RF amplifier: boost this small signal to a power of 100 W or more to provide enough energy to excite the NMR active nuclei in the sample. Attenuator: altering the RF power level in units of decibels (dB) All under computer control The Transmitter Channel

24. 24  1. Continuous Flow(on flow):- Eluent sampled in “real- time” as flowing through NMR Detection Coil 2. Stopped Flow:- Pump is stopped at desired location and data acquired 3. Time Slices:- Regions, or “time-slices” of interest are analyzed 4. Peak Parking:- Peaks of interest are “parked” in off-line sample loops 5. Peak Trapping:- Solid Phase Extraction cartridges are used to “re-concentrate” samples Modes of LC-NMR

25. 25 1) On flow mode:  The outlet of the LC-detector is connected directly to the NMR probe . While the peaks are eluting , NMR spectra are continuously acquired . The chromatographic system is used to move the samples/peaks through the NMR cell  Equipment: - Any HPLC system, which delivers a stable pulse free flow. - LC-NMR Probe LC-NMR interface not required With any of the LC-NMR interface this working mode is also possible, however they are not required . Modes of LC-NMR

26. 26 2) Stopped Flow method The outlet of the to the LC-detector is connected directly NMR probe. A LC-detector ( normally UV ) is used to detect peaks eluting from the column. When a peak is detected , the flow continues until the peak arrives in the NMR cell. At this time, the chromatography ( pump, data acquisition, gradient ) stops and the NMR experiments are performed . Once the NMR experiments are completed , the chromatography resumes until the next peak is found. This process can be repeated several times within one chromatogram Equipment :- HPLC system  - LC-NMR Probe - Controlling station Modes of LC-NMR

27. 27   3) Time slice method  It include to stop the flow at short interval over the chromatography peak to time slice different part of chromatography run. It is useful if there is poor chromatography separation or if compound under study have poor or no UV chromophore or if the exact chromatography retention time is unknown . The data from such a time slice experiment referred as a total NMR chromatogram ( tNMRc ) Modes of LC-NMR

28. 28 4) Peak Parking method The outlet of the LC-detector is connected to the sample loops of the BPSU-36 or BPSU-12. A LC-detector ( normally UV ) is used to detect peaks eluting from the column. A detected peak is moved into one of the sample loops without interrupting the chromatography. When the chromatography is completed , the HPLC pump is used to transfer the peaks from the loops into the NMR probe Equipment :- Any HPLC system - Pump under control for transfer - LC-NMR Probe -BPSU-12 (Bruker Peak Sampling Unit – 12) - Controlling station Modes of LC-NMR

29. 29   5) Peak Trapping method  The outlet of the LC-detector is connected to the SPE unit . A LC-detector ( normally UV ) is used to detect peaks eluting from the column. A detected peak is moved trapped on a SPE cartridge with out interrupting the chromatography . When the chromatography is completed, the chromatography solvents are removed and the peak is transfer with fully deuterated solvents into the NMR probe  Equipment :- Any HPLC system  - pump under control for transfer - LC-NMR Probe - SPE system - Controlling station Modes of LC-NMR

30. 30 1) LC method  a) On line SPE method b) On line column trapping method c) Use of semi micro column 2) NMR method high strength magnetic field high sensitivity probe 3) Solvent suppression method a) presaturation b) soft pulse multiple irradiation c) WET method Technology to improve sensitivity of LC-NMR method

31. 31  a) On line SPE method It is important to eliminate unnecessary fractions by efficient pretreatment , introducing only the targeted component to the column and controlling overloading. The SPE cartridge absorbs the desired peak. After the sample is dried with N2 gas and the contents are finally eluted from the cartridge into the NMR flow probe . SPE principle   Solid phase extraction involves the separation of components of samples in solution through their selective interaction with and retention by a solid, particulate sorbent . The specific hydrophobic organic functional moieties are chemically bonded intimately to a solid surface , such as powdered chromatographic grade silica. 1) LC method

32. 32 These groups will interact with hydrophobic organic compounds by Vander Waals forces and extract them from an aqueous sample in contact with the solid surface. SPE uses the affinity of solutes dissolved or suspended in a liquid (known as the mobile phase) for a solid through which the sample is passed (known as the stationary phase ) to separate a mixture into desired and undesired components. The result is that either the desired analytes of interest or undesired impurities in the sample are retained on the stationary phase. The portion that passes through the stationary phase is collected or discarded , depending on whether it contains the desired analytes or undesired impurities.   Contd …

33. 33 If the portion retained on the stationary phase includes the desired analytes, they can then be removed from the stationary phase for collection in an additional step, in which the stationary phase is rinsed with an appropriate eluent .   SPE DEVICES Several SPE configurations are used which are as following: o Cartridge o Disk o Micropipette tip o 96-well plate o Coated fiber   Contd …

34. 34 CARTRIDGE The most popular SPE configuration is the cartridge. It is carried out using a small packed bed of sorbent with a nominal particle size of 50-60 µm contained in a cartridge made from a polypropylene syringe barrel , fitted with luer tip , so that a needle can be affixed to direct the effluent to a small container or vial. The sorbent being retained in position by use of fits . Frits are made of polytetrafluoroethylene (PTFE), polypropylene or stainless steel with a porosity of 10 to 20 µm and thus offer little flow resistance. The sorbent generally occupies only the lower half of the cartridge, leaving space above to accommodate several milliliters of the sample solution or washing and solvents. Contd …

35. 35 Advantages of catridges (SPE) 1. Highly economical as nondeuterated solvents and HPLC buffers are used 2. The final transfer volume of 200–500 μl is deuterated 3. SPE uses less solvent than liquid-liquid extraction (LLE) 4. SPE is faster (at least 5 times) 5. High capacity 6. Total SPE costs are considerably less than LLE 7. High selectivity : broad choice of bonded phases and solvents 8 . Automation much easier Contd …

36. 36 b) On line column trapping   In this method, after separation using a conventional column , concentration is first done in a trap column , and the sample is separated again using a semi-micro column then introduced to NMR. Concentration by this technique is highly effective . Once sufficient sample has been collected on the trap, the flow reversed and the solute is transported to the NMR for further analysis c) Use of semi micro column The highest sensitivity is provided when all of the components separated by HPLC are introduced to the flow-cell of NMR. However, the peak volume separated by HPLC is greater than the flow-cell capacity (normally about 30 μL to 120 μL ) therefore, only part of the component is actually the target of measurements 1) LC method

37. 37 The method of using columns with an internal diameter of around 2 mm , known as semi-micro columns , is a peak concentration method suited to LC-NMR. The volume of a semi-micro column is around 1/5 of a conventional column , and since the required amount of solvent is reduced in proportion to the elution, highly concentrated sample solutions can be introduced to LC-NMR On line column trapping.

38. 38 a) High strength magnetic field NMR detection sensitivity is proportional to the magnetic field strength to the 3/2 power , and the stronger the external magnetic field is, the higher the sensitivity. Currently, the magnetic field strength has reached 1000 MHz. Magnetic fields being generated by modern instruments employing cryomagnets , field homogeneity is high and as a consequence the sample need not be rotated. 2) NMR methods b) high-sensitivity probe It is also known as a cryogenic probe that reduces the heat noise arising during NMR signal detection by cooling the coil using superconductor materials. This will eliminates the thermal electronic noise associated with the initial stages of signal detection and increases the coil quality factor. This leads to an improvement in the S/ Nratio by a factor of 3-4.

39. 39 Contd … C o n ve n t io n a l NM R pr o b e C o n t i n uo us flo w NM R p r o be

40. 40 Flow Cells – Active Volume   3mm - 60µL 4mm - 120µL 5mm - 240 µL Contd …

41. 41 Presaturation The most widespread  solvent  suppression in use is the so-called presaturation technique It depend on the phenomenon that nuclei which are unable to relax because their population in ground state and exited state is same, do not contribute to free induction decay after pulse irradiation.   Before the data acquisition, a highly selective low power pulse irradiates the desired solvent signal for 0.5 to 2 s. This leading to saturation of solvent signal frequency . During data acquisition, no irradiation should occur. This method is used for stopped flow mode . 3) Solvent supression methods  

42. 42 b) soft pulse multiple irradiation Here, presaturation is performed with the use of shaped pulse which has a broader excitation profile. This method is better suitable for suppression of multiplets  Advantage:  1. Easy to implement 2. Multiple presaturation can be possible  Disadvantage:  1. Spin with resonance close to solvent frequency will also be saturated and 2D cross peak will be absent   3) Solvent supression methods  

43. 43 c) WET method This technique contains NMR difference probe. This difference probe consists of a dual coil probe that contains two sample coils in a resonant circuit that switches between parallel excitation and serial acquisition to cancel common signals, such as solvent and solvent impurities. Essentially, this technique is based on a dual beam background subtraction, where the reference signal and sample signal that are collected simultaneously are subtracted from each other automatically. No software manipulation , pulse sequence modification, or spectrometer alteration is necessary . Hence the technique does not lengthen the pulse sequence but it reduces experimental time . It takes 50-100 ms , So it is used for on flow method. This method is used for on flow mode. 3) Solvent supression methods  

44. 44 v Other possible steps for solvent supression   Using eluents that have as few 1H NMR resonances as possible, e.g. H2O, ACN, or MeOH. Using at least one deuterated solvent , e.g., D2O (approx. $290/L), ACN-d3 (approx. $1600/L), or MeOD (approx. $3000/L). Using buffers that have as few 1H NMR resonances as possible, e.g., TFA or ammonium acetate. Using ion pair reagents that have as few 1H NMR resonances as possible , e.g., ion pairs with t -butyl groups create an additional resonance Contd …

45. 45 The information between the two (three) techniques is so orthogonal; HPLC methods resolve “ complexity of a mixture ” by separation, whereas NMR resolves virtually any structure question (especially with different experiments). The NMR can determine if the LC peak impure . LC-NMR/MS is “THE” ultimate instrument . NMR data can be taken without complete separation of mixture . It is nondestructive technique . Sample can be stored for analysis by another method. Advantages of LC-NMR

46. 46 high costs. Capital equipment costs; long experiment times ; partial use of 2H solvents. operator training requirements. Doing LC-NMR/MS requires a unique set of skills . Difficulty in solvent selection . Disadvantages of LC-NMR  

47. 47 Degradation Products. Impurities. Trace Analysis. Analysis of Mixtures. Tautomer Kinetics. Unstable Products. Natural Products. Proteins/Peptides. Application of LC-NMR  

48. 48 References: 1) L C -N M R : A p o w er f ul t oo l f o r an a ly z i n g a nd c h a r a c t e r iz i ng c o m plex c h e mi c a l mixtur e s w i t h o ut t he n e e d o f c hemi c a l s e p a r a t i o n , Bhum i ka. D. S ak hr e l i y a * , Sw a ti K a n sa r a D epar t m ent of Q ua lit y A s sura n c e , A-O ne Ph a r m a cy C o ll ege, A nas a n A h m edabad, G ujara t , Ind i a. 2) LC-NMR-Expanding-the-Limits-of-Structure-Elucidation, Nina C. Gonnella. 3) [Applications of NMR Spectroscopy Volume 2] Choudhary, M. Iqbal_ Rahman, Atta- ur - Applications of NMR Spectroscopy. Volume 2 (2015, Bentham Science Publishers Ltd) 4) Klaus Albert - On-line LC-NMR and Related Techniques (2002, Wiley)