Reference no: EM131030798

Analytical methods

NMR Demo: Sample preparation and operation in NMR spectroscopy and/or an investigation into the sensitivity of NMR in comparison with other analytical techniques.

Aims

This short practical demonstration aims to familiarise you with the process of preparing a sample and obtaining a selection of NMR spectra from it in order to be able judge the sensitivity of the technique.

Forensic Science students: your session will focus on the applicability of 1H NMR to a quantitative analysis of paracetamol

Chemistry students: your session will focus on sample preparation for 1H and 13C NMR for qualitative analysis for a typical organic substrate

Learning Outcomes:

Following this practical you should:

1. Understand how to prepare an appropriate sample of a solid or liquid or solution for routine NMR analysis.

2. Be able to describe the basic instrumentation and procedures for a routine NMR analysis and able to predict timescales and duration for routine NMR analyses.

3. Be able to evaluate and comment on the sensitivity and therefore the sample requirements and application of the technique to quantitative or qualitative analysis.

Practical:

Forensic Science Students: Following a ‘tour' of the instrument the laboratory exercise will be based on a demonstration of sample preparation (eg of a selected drug substance (paracetamol) at low concentration eg 50 mgL-1 in water) and instrument operation to small student groups. Limits of detection/quantitation will be estimated from the spectral results and this information will be linked to the summatively assessed exercise completed in A402 so take note of the sensitivity of the instrument/issues and limitations/information that can be obtained from NMR.

Chemistry students: Following a ‘tour' of the instrument the laboratory exercise will be based on a demonstration of sample preparation (eg of an organic substance such as citronellal in CDCl3) to small student groups. Amount and concentration of sample, preparative steps, solvent selection, and relative duration of 1H and 13C will be covered.

Health and Safety info

Students will not prepare or handle chemical samples in this session as these will be prepared by staff in the session (or pre-prepared by staff before the session) the session is merely a demonstration and explanation of the equipment.

The NMR machine on which the samples will be run houses a powerful magnet which will attract and affect materials made with ferro magnetic materials. People with heart pacemakers and other biostimulation devices or with implants should avoid close approach to the magnet (< 2m) and a warning to the group of this feature before entering the NMR lab is imperative. Otherwise an NMR machine is entirely safe for purely biological life forms although it is important that anyone approaching the magnet divests themselves of metal items such as keys, and watches, and magnetic storage devises including credit cards, mobile phones, memory sticks as the information on them can be wiped by close proximity to a powerful magnet.

Note: NMR magnets a commonly cryogenically cooled and there is a natural leak of gaseous nitrogen into the lab. If the air sensor alarm sounds (or at the direction of the staff member present) all personnel should leave the laboratory as if it were a fire alarm.

AIMS

1. Development of an understanding of the fundamentals of HPLC and GC.

Consideration of the underlying theory of separation processes.

2. Development of practical skills that allow the separation of the components of a mixture.

3. Consideration of the theory and application of sample preparation for solid, liquid and air samples.

4. Prediction and interpretation of 1H and 13C NMR spectra of molecules of moderate complexity and to understand the basic instrumental aspects of the techniques.

5. Prediction and interpretation of the mass spectra of molecules of moderate complexity and to understand the basic instrumental aspects of the techniques.

6. Prediction and interpretation of IR spectra of molecules of moderate complexity and to understand the basic instrumental aspects of the techniques.

7. Prediction and interpretation of UV/Vis spectra of molecules of moderate complexity and to understand the basic instrumental aspects of the techniques

LEARNING OUTCOMES

1. Demonstrate an understanding of the theory of analytical separations. (examination assessment)

2. Apply this theory to a practical end. (examination and laboratory assessment)

3. Develop chromatographic analyses using the above methods. (examination and laboratory assessment)

4. Choose an appropriate sample preparation method for solid, liquid or air samples. (examination and assessment)

5. Select an appropriate technique for use in structural elucidation of molecules of moderate complexity (examination)

6. Predict and interpret 1H and 13C NMR spectra, IR spectra, Mass-spectra and UV/Vis spectra for molecules of moderate complexity (examination and assignment)

7. Elucidate structures of unknown compounds given a selection of their spectra (examination and assignment)

8. Identify and describe the basic instrumental aspects of the main spectroscopic techniques (examination)

Assessment

There is one combined LC (summative) & NMR (formative no submission) laboratory practical to complete this semester. The laboratory assignment (across sem 1 and 2) in total is worth 20% of this 20 credit module. You will be required to prepare your samples as you deem appropriate (see following instructions). The laboratory technician will run these on your behalf and the data will be made available to you once the samples have been run (you will be notified of their availability by email). You will then be required in Excel to prepare an appropriately labelled calibration curve AND calculate the Quality Control (QC) concentration and the Relative Standard Deviation (RSD) based on your results. You should submit this as a single Excel document online via the submission portal of the module site no later than 1.00 pm on the day exactly 2 weeks after your lab session.

Feedback

The summative laboratory is based on appropriate sample preparation and quality control for samples to be analysed by LC. Final assessment marks will be awarded on the quality of your preparation skills and portrayal of analytical data with your calculations of QC/RSD information. This will be in the University guidelines of 20 working days once the final rotation of labs has been completed.

Background:

Correct use of Gilson pipettes (remind yourself of how to use these correctly before the lab session!)

Appropriate calculations to perform

Mobile phases (preparation, isocratic, gradient)

Reservoirs

Pumps

Detector (although these will be discussed in further detail next week)

Column (selection, phases, guard column)

Injector type (Rheodyne, volume)

Method (setting up, appropriate settings)

Troubleshooting (peak shape, leaking, piston seals, back pressure, erratic pressure, flat/wavering baselines, retention time drift)

Choosing the most appropriate wavelength (UV spec, Beer Lamberts Law)

Setting the most appropriate wavelength

Influence of wavelength on detector response

DAD & information can be obtained

MS Ionisation principles (Soft)

Fragmentation

Voltage optimisation

Sensitivity

If you are not familiar with this terminology or content then read, for example:

Bayne & Carlin, Forensic Applications of High Performance Liquid Chromatography, CRC Press Taylor & Francis Group, 2010

Chromatography Lab

Part A Calibration Curve preparation (prior calculations required!)

Instructions

For this lab session you ONLY have to prepare a calibration curve based on the following information, but you will have to do your calculations PRIOR to attending this session. Your samples will be racked and stacked in the trays provided and these will be run for you. It is essential that you have performed appropriate initial reading on the drug in question (to determine appropriate concentrations) and are careful with the preparation of your curve (pipetting skills, quality etc) as the appropriateness of the curve and its linearity will form the basis of this assessment.

You have been asked by a senior analytical chemist to provide proof that your scientific analytical method for paracetamol is reliable and robust before they instruct you to perform an analysis on their behalf. To this end they have asked you to create a calibration curve covering the expected blood therapeutic to fatal levels of this drug, with an associated quality control and RSD samples as well.

You will have to find out some more information about this drug: find out the therapeutic and fatal blood drug concentrations of paracetamol, usually expressed in mg/L (or possibly μg/ml, which is the same) - note where a mass of drug is indicated this refers to a dose and not a blood drug concentration - do not confuse the two!.

Possible Sources:

1) Library - check out the drug monographs from Baselt: ‘Toxic Disposition of Drugs and Chemicals in Man' (various versions) - take care to read the whole monograph!

2) Internet: (WITH CAUTION!) but note that paracetamol has different names in different countries (eg acetaminophen in USA)

You will need to construct a calibration curve covering this concentration range using a pure drug stock solution that has been prepared for you at the following concentration - paracetamol (2000mg/L) and will be available in the lab on arrival. To do so you will need to do your calculations PRIOR to attending this lab (time is limited and you will not have enough time to calculate and prepare your curve, so don't forget to bring your calculations with you!)

You will have to prepare your standards from this stock solution. It is likely that you will need to consider an intermediary stock solution in order to obtain an accurate calibration curve.

You should prepare FIVE standards you want to use for your calibration curve using GILSON pipettes only (Note you will NOT be using 10 ml volumetric flasks) - and don't forget about your blank!

Calculate what you intend the final concentration of each standard is to be and what volumes you need to take from the stock solution and water to dilute to achieve this concentration but bear in mind that you will prepare your calibration solutions directly into an autosampler vial (minimum volume 0.5ml, maximum capacity volume 1.8 ml) so you will have to think carefully about your volumes that you need to pipette and ensure they will fit in the vial!

Use this table to provide the following information:

Calibrant number	Concentration (mg/L)	Volume of stock (ml)
Blank	0	0
1
2
3
4
5

You will also need to include your full calculation of at least 1 concentration to illustrate that you understand the principles behind this calculation.

1) Using your notes, prepare your set of calibration standards that cover the appropriate concentration range using the apparatus provided - make sure you have labelled your glassware appropriately.

2) Make sure the vials have your name and your concentrations written on them. Rack these samples into the tray provided as well as documenting the appropriate details on the logsheet provided next to the tray.

3) Leave a copy of your calculations and table of concentrations with your name clearly printed on it.

PART B QC & RSD Preparation

QC: Prepare 1 quality control (QC) sample in duplicate at a concentration of 50mg/L. You will have to calculate how to prepare this sample from the stock solution of 2000mg/L.

RSD: Prepare 5 samples at a concentration of 100mg/L. You will have to calculate how to prepare these samples from the stock solution of 2000mg/L.

Rack these samples into the tray provided as well as documenting the appropriate details on the logsheet provided next to the tray.

Accuracy: Take note, this QC has already been analysed and your results will be compared against this analysis. If your calculated analytical result deviates <3% from the previously analysed QC, then you will score 100% for this part of the assessment ie 10 marks out of 10. If however your calculated result indicates a 10% deviation then you will score 6 marks out of 10 for this part of the assignment and so on according to the accuracy of your data.

Precision: The preparation of your 5 samples will be used to calculate the RSD. This calculation will measure how precise you are in repetition of a single sample concentration preparation. It also gives an indication of the performance of the HPLC system (which is known to be 0.5%). As with the accuracy, the RSD data will be compiled and assessed as indicated by the tables below:

I NEED A DOCUMENT LOOKED AT FOR TOMORROW ITS CHROMATOGRAPHY LAB AND ITS ASSESSED I NEED IT FOR EARLY TOMORROW MORNING