Major NMR instrument makers include Oxford Instruments, Bruker, Spinlock SRL, General Electric, JEOL, Kimble Chase, Philips, Siemens AG, Varian, Inc. and Agilent Technologies, Inc..
chemical shift is the resonant frequency of a nucleus relative to a standard.
Principle The fundamental principle of separation involved is Adsorption. The other principles involved are Ion-exchange: It is used to separate the mixture containing similar charged ions. Ion-pair: It is the alternate to ion-exchange, used for the components which are difficult to separate using covalent bonded ion-exchangers. Gel permeation: It is separated on the basses of difference in the size & shape of the solute molecules. In HPLC ,based on polarity there are two phase Normal phase Reversed phase Normal phase: Stationary phase Polar Mobile phase Non-polar Reversed phase : Stationary phase Non-polar Mobile phase Polar
based on Elution there are types Isocratic separation Gradient separation Isocratic separation: Mobile phase & the strength of elution is maintained constant. Gradient separation: Mobile phase & the strength of elution is varied.Instrumentation Mobile phase reservoir columns Pumps Sample injection system Detectors
Mobile phase reservoir It can be an aqueous-organic mixture or buffer solution or a mixture of organic solvents. The mobile phase is pumped under pressure from one or several reservoirs and flows through the column at a constant rate. Eluting power of the Mobile phase is determined by its overall polarity, the polarity of the stationary phase and the nature of sample component.
Solvent degassing system The constituents of the mobile phase should be degassed and filtered before use. Methods are employed to remove the dissolved gases in the mobile phase are include: Heating and stirring, Vacuum degassing with an aspirator and with an air- soluble membrane, Filtration through 0.45 filters, Ultra sonication or combination of these methods. HPLC system is also provided an online degassing system, which continuously removes the dissolved gases from the mobile phase.
Characteristics of columns and column packingsThe column is the heart of HPLC separation processes.Most column packing used for HPLC separations make use of silica particles (SiO2 x H2O).It consist of a network of siloxane linkages (Si-O-Si) in a rigid three - dimensional structure containing inter connecting pores.Thus wide ranges of commercial products are available with surface areas ranging from 100 and 800 m/g and particle sizes from 3 to 50 µm.
The useful pH range for column is 2 to 8, since siloxane linkages are altered below pH 2 while at pH values above 8 silica may dissolve. Category Particle (µm ) Bonding L1 1.5 to10 C18 L2 30 to 50 C18 L7 1.5 to10 C8 L8 3 to 10 NH2 L11 1.5 to10 Phenyl L17 7 to 11 Cation Exchange
Pumpso The most important component of HPLC in solvent delivery system is the pump, because its performance directly effects the retention time, reproducibility and detector sensitivity.o It should generate high pressure upto 6000psio Pumps are 2 types: o Constant Displacement Pumps: Constant flow of mobile phase Eg: Syringe type pumps, Reciprocating pumps o Constant Pressure Pumps: Non-pulsating solvent flow but has very small capacity. Eg Pneumatic pumps o The reciprocating pump with twin or triple pistons is widely used, as this system gives less baseline noise, good flow rate, reproducibility etc
Sample injection system Two means for injection on the column are Injection into a flowing stream and Injection into a stop flow injection. These techniques use a syringe or an injection valve. Automatic injector is a microprocessor-controlled version of a manual universal injector. Usually, up to 100 samples can be loaded into the auto injector tray. The system parameters such as flow rates, gradient, run time, volume to be injected, etc., are chosen, stored in memory and sequentially executed on consecutive injections.
DetectorsThe purpose of detector is to monitor the eluent coming out of the column. Generally two types of detectors are used in HPLC.a. Bulk property detectors: These detectors are based on differential measurement of a property. Eg: refractive index, conductivity and dielectric constant detectors.b. Solute property detectors: Solute property detectors respond to a physical property of the solute, which is not exhibited by the pure mobile phase. Eg: UV detector, fluorescence detectors, electro-chemical and radioactivity detectors, electron capture detector are suitable for gradient elution.
The mobile phase is pumped at high pressur into the column. The sample is introduced at the top of the column by using suitable injector system. As the mobile phase flows down the separation of components occur. The column effluent is made to pass through a detector which produces electrical signals proportional to the characteristics of the solute molecules which are amplified & recorded using a potentiometric recorder as chromatographs.
Application Used in inorganic chemistry for separation of anions & cations Used in forensic science for the separation of phenylalkyl amines from blood plasma,& for detection of poisons. Used in environmental studies. Used in bioassays of compounds. Used in controlling microbiological processes. Used in cosmetic industry for the assay & QC. Used in agrichemical industry for separation of herbicides.
NMR Spectrometry Bruker 600 MHz NMR Spectrometer.
Principle: It is based upon the spin of nuclei in an external magnetic field. In absence of magnetic field, the nuclear spins are oriented randomly. Once a strong magnetic field is applied they reorient their spins i.e aligned with the field or against the field. When nuclei are irradiated with RF radiation the lower energy nuclei flip to high state and nuclei said to be in resonance.NMR are 2types based on parameters that are measured 1. Single coil spectrometers (Measures absorption) 2. Double coil spectrometers( Measures resonant radiation)
It is further divided into Absorption type: It uses a bridge circuit for detecting radio-frequency energy absorbed through the coil surrounding the sample. Inductive type: It uses two coils perpendicular to each other & absorption of energy is from the transmitter coil resulting in the orientation of nuclei, thereby inducing voltage in receiver coils.Instrumentation Magnets Sample holder Sweep coil Transmitter Receiver Detector
MagnetsPermanent or electromagnet are used to obtain a homogeneous magnetic field. Permanent – field variation is impossible. Electromagnet – field strength is varied.As the field strength is proportional to chemical shift it must not be less than 20,000guass.Sample holder It is about 5mm in diameter & 15-20 cm in length. It must be transparent to radio frequency radiation & chemically inert. Glass tubes are used. 1-30mg of 2- 19% sample is used.
Sweep coil It is used to production of NMR spectra. It is achieved by passing direct current either through the coli that are wound around the magnetic pole or through a pair of Helmhotz coil located on either sides of the sample. Rate of sweep is important parameter because slow rate results in saturation effect where as fast rate results in ringing.Transmitter It generates of few MHz which irradiates the sample molecules. If the energy difference between the relevant spin state matches the radio frequency wave then the nuclei moves to the higher spin state. The output obtained is multiplied with the desired frequency.
Receiver Radio frequency bridge is employed under single coil instrument. In double coil, the transmitter & receiver coil are arranged perpendicular to each other & to direction of the magnetic field.Detector The NMR spectra obtained from high resolution instrument are recorded directly via computer. To direct the radio frequency signal exhibited due to resonating nuclei, the receiver coil is used.
Transmitter generates radio waves of definite frequency which passes through a receiver coil surrounding the sample. The receiver coil receives the signals & send to detector. The sweep is used to bring variations in the externally applied magnetic field Bo. The frequency of Bo is varied until it is in resonance with that of the radio wave frequency, thereby leading to absorption of energy which is recorded in the form of signals. The peak obtained in the signals represent the value of Bo at which the frequency of the sample nuclei is in resonance with that of the applied magnetic field Bo. To obtain NMR spectrum, the signals are platted again applied magnetic field Bo
Applications Used in qualitative & quantitative analysis. Used in identification testing Used in determining Hydrogen bonging present in metal chelates & organic compounds. Determination of double bond character. Determination of structure. Identification of structural isomers. Distance between Cis & Trans Isomers. Determination of optical purity.
NMR detection coupled with LC offers great promise in combining the ability to separate complex mixtures into individual components with one of the most structurally rich detection schemes available. In 1978, Watanabe reported the coupling of LC effluent to NMR using a stopped flow approach, and within 1 year, an on-line system had been reported. The major advantages of on-line as opposed to off- line NMR detection of LC are improved chromatographic resolution, consistent response, on-line data analysis, and rapid data acquisition.
Over the past 15 years, numerous groups have reported improved LC-NMR hyphenation methods, improved NMR plus sequences and increased chromatographic resolution. The drawbacks of continuous flow NMR include poorer sensitivity due to the limited time available to measure each analyze and the flow rate dependence of the NMR line width. Recently radio frequency (RF) microcoils are used for NMR spectroscopy to create 5 nL to 1- mL volume detection cells.
The sample can be transferred during the chromatographic separation and the NMR spectra are then acquired either in on-flow mode (continuously, while the chromatography is running) or in stop-flow mode (while a selected peak is parked in the NMR probe and the chromatography is paused). The Bruker LC-NMR systems are ideal for sensitive and instable compounds, fully automated analysis, or simply to provide a very convenient and quick path from LC separation to NMR results.