PHAR2811 Drug Design and Discovery UVSpectrophotometry 2020 PDF

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Summary

UV SPECTROPHOTOMETRYObjectives: To revise and build on the principles introduced in BIOL To teach you to use a spectrophotometer in the UV range to: o identify and quantify a biochemical o measure the concentration of the active ingredient of a pharmaceutical preparation How the Objectives will be a...

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PHAR2811_Drug Design and Discovery UV Spectrophotometry

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UV SPECTROPHOTOMETRY Objectives: • •

To revise and build on the principles introduced in BIOL1007 To teach you to use a spectrophotometer in the UV range to: o identify and quantify a biochemical o measure the concentration of the active ingredient of a pharmaceutical preparation

How the Objectives will be achieved: • • • •

You must read the relevant section in this manual You will identify an unknown DNA base by UV Spectrophotometry. You will determine the concentration of acetylsalicylate (aspirin) in commonly available pharmaceutical tablets You will be given example problems to give you experience at a range of applications for Spectrophotometry.

Why do All This? Spectrophotometry using wavelengths in the UV range of the electromagnetic spectrum increases the versatility and the range of applications of this very common technique. Principles and techniques covered in this session will be used in many of the practicals this semester and possibly next semester and next year

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PHAR2811_Drug Design and Discovery UV Spectrophotometry

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SAFETY Additional Current risk

controls required

Eye/Skin contact

Use of PPE (gloves, labcoat, safety glasses, filled-in shoes), handle in fume hood

1 (low)

no

dissolved in 50% methanol

Eye/Skin contact

Use of PPE (gloves, labcoat, safety glasses, filled-in shoes), handle in fume hood

1 (low)

no

acetyl salicylate

Use of PPE (gloves, Overdose/bleeding labcoat, safety glasses, with large filled-in shoes), handle in quantities ingested fume hood

1 (low)

demonstrator to hold stock

Hazard

aspirin measurement

dissolved in 0.05 M HCl

aspirin measurement

aspirin measurement

Associated

Existing risk control

Task

harm

2

Residual risk

PHAR2811_Drug Design and Discovery UV Spectrophotometry

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1. INTRODUCTION How do we detect colourless compounds?

Proteins and nucleic acids absorb UV light.

What determines the λ a molecule will absorb at?

The amount of energy absorbed is inversely proportional to the λ.

In BIOL1007 we used Spectrophotometry to: • Identify DNA by its absorption spectrum It would be nice if the compounds we wanted to measure were coloured but unfortunately, most of the compounds of biochemical and pharmaceutical interest are not coloured. Of the four major groups of biopolymers; carbohydrates, lipids, nucleic acids and proteins, two (carbohydrates and lipids) do not absorb visible or UV light and therefore cannot be quantified directly by spectrophotometry. To measure the concentration of these compounds we first must form a coloured derivative in a colorimetric assay. This type of reaction is covered in your second practical when the concentration of glucose is estimated. Some molecules, while usually appearing colourless to the naked eye, do absorb UV light. Proteins and nucleic acids both exhibit this property. Some proteins, for example, haemoglobin, myoglobin and cytochrome c are coloured but this is due to a prosthetic iron-containing group (heme) attached to the protein polymer. The molecular structure of a compound dictates the wavelength of light (λ) it will absorb. Essentially molecules can exist at different energy levels, “allowed” by the structure. When certain molecules are irradiated some electrons are promoted to higher energy levels. The amount of energy absorbed (∆E) to achieve this transition will determine the λ of light absorbed. This ∆E is inversely proportional to the wavelength of light absorbed ie. ∆E = hc/λ, where h is Planck’s constant and c is the velocity of light.

Compounds with aromatic rings will absorb strongly in the UV region.

Valence or outer electron transitions require more energy than molecular rotations and vibrations, hence we observe UV and visible absorption when electronic transitions occur and infra-red (IR) absorption when molecular vibrations occur. IR spectroscopy can provide information on the conformation of a peptide backbone. Absorption of radio waves will result in spin orientation in a magnetic field. This is the basis of NMR.

The side chains of Phe, Tyr and Trp give proteins their absorption at ~280 nm.

Now let’s look a little more closely at proteins and nucleic acids, to see why these two biopolymers absorb UV light. One common chemical structure, which is associated with UV absorption, is the aromatic ring. Structures with conjugated double bonds (alternating double bonds), particularly ring systems, provides a cloud of delocalised π electrons above and below the plane of the ring. When irradiated, the π electrons in aromatic rings undergo electronic transitions hence absorb light very strongly in the UV region. 3

PHAR2811_Drug Design and Discovery UV Spectrophotometry

Electronic transitions in the peptide backbone give proteins their absorption ~200 nm.

Nucleic acids absorb light at ~260 nm because of the bases; A, G, C, T and U.

Components of both proteins and nucleic acids contain these conjugated ring structures. The amino acids phenylalanine, tyrosine and tryptophan all have side chains with aromatic structures and all absorb strongly around the 270 to 290 nm region. This property is exploited when measuring proteins in the UV region; we typically measure the absorbance of a protein solution at 280 nm. Proteins absorb strongly at a second region around 200 nm. This is the result of electronic transitions in the peptide backbone. The problem with using absorption spectroscopy in this region is that many, many compounds absorb here, including contaminants in the solvent. Your samples and solutions must be very pure before you can use these wavelengths. We will restrict ourselves to absorbance around 280 nm in classes this semester. Nucleic acids (DNA and RNA) contain bases: adenine (A), guanine (G), cytosine (C), uracil (U) and thymine (T). These compounds are heterocyclic ring structures ie have conjugated double bonds in a ring which contains both carbon and nitrogen atoms. They also have delocalised π electrons and as such exhibit strong absorption in the UV region. They tend to absorb light around 260 nm. Their absorption properties are also dependent on conformation eg. double-stranded DNA does not absorb as strongly as single stranded DNA. The reason for this so called hyperchromic effect will be covered in your lecture course.

2. EXPERIMENTAL 2.1 Experimental Overview Obtain the UV spectrum of an unknown DNA base and, based on the λmax and extinction coefficient, identify it.

Determine the concentration of acetyl salicylic acid in an aspirin tablet

2.2 Experimental Considerations 2.2.1 Identifying an unknown DNA base •

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Each base solution has a concentration of 60 µM.

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PHAR2811_Drug Design and Discovery UV Spectrophotometry

•

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Because you are working in the UV range you will need to use quartz cuvettes. Plastic and glass absorb strongly at wavelengths 90% of the initial incident light, IO and the detector will have trouble discriminating accurately between the 2 intensities. At the other end of the range absorbances of >1.0 represent less than 10% of the light transmitted (very concentrated solutions). The problem here is the log scale. A measurement of 2% transmitted light gives an absorbance of 1.6 while 1% transmitted light gives 2.0 This is a very big difference in the absorbance value for a small difference in light intensity and the detector has to be very good to accurately measure the difference. This is particularly a problem if the blank solution also has considerable absorbance. We will use the spectrophotometer to measure the concentration of acetyl salicylate in a solution containing a dissolved aspirin tablet. We will need to dilute this solution so that it is within the working range of the spectrophotometer. If this range is between 0.1 and 1.0 then it is best to aim for the middle of the range, ~0.5. This will give us maximum flexibility in the measurement on either side. You will use the extinction coefficient for acetyl salicylate and the equation A = εcl to work out this “ideal” concentration. Once this is established, work out the concentration of acetyl salicylate in your solution using the amount on the packaging as a guide. This concentration will have to be in milliMolar (mM) the same as the ideal concentration. Then work out how much you need to dilute your solution to get it to the “ideal” concentration. The step-wise guide is below.

2.3.3 Determining acetylsalicylic acid levels in aspirin tablets Various brands of commercial tablets containing aspirin will be analysed for its composition. In this experiment, you will determine the concentration of aspirin in the commercial tablet provided. 



Weigh one tablet and dissolve it in 100 mL of 0.1 M HCl:Methanol (1:1). You will need to filter the solution once you have added the solvent to remove particulate matter from the tablet. This must all be done in the fumehood. Make a record of the mass of aspirin and total mass of the tablet you will analyse in your lab notebook similar to table 3 8

PHAR2811_Drug Design and Discovery UV Spectrophotometry











 

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From the amount of acetyl salicylate quoted by the manufacturer of the tablet calculate an appropriate dilution. See below for the strategy. Make up your dilution with water (once the acetyl salicylate is dissolved in the acidified methanol it shouldn’t come out of solution if diluted in water), with a 3 mL final volume. Baseline-correct the spectrophotometer between 340 and 240 nm with water in a quartz cuvette using spectrum mode. Discard this water solution and fill the same cuvette with the diluted tablet suspension. Obtain the spectrum and, using the peak function, the absorbance at the peak wavelength (~275 nm). Calculate the true amount of acetyl salicylate in your tablet (mg). Calculate the % mass of aspirin in the tablet. Compare your results with the manufacturer claims? How do they stack up? What are the possible errors in determining the correct composition?

Strategy for calculating the dilution of your tablet suspension. Step 1: What is the amount of active ingredient in the tablet as quoted by the manufacturer? Example: Suppose you have a tablet containing 500 mg acetyl salicylate. Step 2: How many mmoles is this? Using the example 500 mg/180.15 (molecular weight acetyl salicylate) = 2.77 mmoles. Step 3: What is the [acetyl salicylate] of your tablet suspension? In this example it would be 2.77 mmol/100 mL = 27.7 mM (mmol/L) = 27.2 µmol/mL = 27.7 nmol/µL Step 4: What concentration do you need to give an absorbance of ~0.5 at 275 nm? Following the example using A = εcl  0.5/1.0 (extinction coefficient) = 0.5 mM Step 5: How many mmoles do you need? Here is where the 3 mL quartz cuvette is important. You need 0.5 mmoles per litre = 0.5 µmol/mL = 1.5 µmol/3 mL (volume required for quartz cuvette) OR 1,500 nmol in 3 mL final volume. Step 6: What volume of the tablet suspension will deliver 1,500 nmol? If the tablet suspension in the example has ~25 nmol/µL (27.7 to be exact but 25 will do for this calculation) then you will need 1500/25 = 60 µL in a final volume of 3 mL Dilution factor 3000/60= 50

Table 2 Brand and active ingredient quoted by manufacturer Tablet weight (g) Weight aspirin in tablet (mg) Absorbance (275 nm) [Acetyl salicylate] (mM) ε = 1.0mM -1cm -1 @275 nm Dilution factor [acetyl salicylate] in the 100 mL tablet suspension (mM) Amount of acetyl salicylate (mg) recovered mol. Wt. = 180.15 % recovery of aspirin based on the manufacturer’s reported content

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PHAR2811_Drug Design and Discovery UV Spectrophotometry

Table 3, using the example above Brand and active ingredient quoted by manufacturer

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Fantastic aspirin, 500 mg

weight (g) from balance

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Absorbance (275 nm) from spectrophotometer

0.51

[Acetyl salicylate] (mM) (use ε = 1.0mM -1cm -1 @275 nm)

0.51

Dilution factor (3000 final volume in µ L/60 volume stock in µL) [acetyl salicylate] in the 100 mL tablet suspension (mM) =0.51*50 Amount of acetyl salicylate (mg) in the tablet = 25.5 (mmol/L)/10  #mmol in 100 mL * 180.15 (mol. Wt.) % Recovery of aspirin= 459.4*100/500

50 25.5 459.4 91.9

2.3.4 Paracetamol Below are the details for paracetamol, another commonly used analgesic which has strong UV absorbing properties which are commonly used when determining its concentration and purity. For next week… Using the information in the table below, construct a protocol to analyse the concentration of paracetamol in a commercially available paracetamol tablet. H N

CH 3 C O

HO

Acetaminophen or paracetamol, the active ingredient in many pain killers absorbs in the UV range with a λmax at 250 nm in neutral solutions (methanol usually). Its extinction coefficient is 14 mM1 cm-1. Each commercially available tablet (Tylenol, Panadol or Herron) contains 500 mg paracetamol. Its molecular weight is 151.1 UV spectrum of Paracetamol reproduced from technical notes

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PHAR2811_Drug Design and Discovery UV Spectrophotometry

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4. ASSESSMENT There will be a page in your PHAR2811 Lab Archives ELN for this experiment. Make sure you include the following in the space provided. This will be due by the beginning of your lab in week 4. The mark for this lab forms 5% of your final PHAR2811 mark. IDENTIFYING YOUR DNA BASE (5 marks) MSDS

Attach the MSDS for methanol and answer the safety question

1.0

Image of the absorption spectrum of your unknown base

1.0

λ max and calculation of ε with units Table 1 filled in

Identification of the base with comparison to literature values of λ max and ε.

3.0

DETERMINING the AMOUNT of acetyl salicylate in aspirin tablets (10 marks) Table 3 filled in containing: Protocol

Your aspirin tablet values, with calculations explained

6.0

Protocol to analyse a commercial paracetamol tablet

4.0

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PHAR2811_Drug Design and Discovery UV Spectrophotometry

Using the Shimadzu Spectrophotometer Choosing the MODE Select 2….Spectrum MODE for the absorption spectrum Select 1…..Photometric MODE for absorbance measurements at one wavelength (standard curves and unknowns). If you are not on the MODE MENU screen hit the return button….that usually gets you there.☺

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PHAR2811_Drug Design and Discovery UV Spectrophotometry

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Spectrum MODE settings: For Obtaining an Absorption Spectrum Once in Spectrum mode make sure you have the following settings. If you want to change one of the parameters hit the appropriate button (1 toggles between Absorbance and %transmittance; 2 changes the wavelength; 3 the full scale etc.). Hit the enter button after any change.

F1

F2

F3

F4

1. Put your cuvette, filled with water, into the spectrophotometer and hit the F1 button below the BaseCorr…this will correct the baseline between 650 nm and 350 nm. This will take a few minutes

2. Empty the cuvette and then fill with the coloured dye solution. Hit START and allow the instrument to produce the spectrum on the screen. Take a photo or sketch the spectrum.

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PHAR2811_Drug Design and Discovery UV Spectrophotometry

Once the spectrum is complete….

F1

F2

F3

F4

Hit the button below the PEAK and the λmax and the absorbance at that wavelength(s) will be displayed. Record these in your lab notebooks.

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PHAR2811_Drug Design and Discovery UV Spectrophotometry

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Photometric MODE settings: For absorbance measurements at one wavelength (standard curves and unknowns).

Once in Photometric MODE select your wavelength by hitting the GOTO WL button, typing in the wavelength you want (in nm) then hitting enter.

Put your cuvette, filled with water or blank solution, into the spectrophotometer and hit the auto zero button…it will ping when it is ready. Make sure the display is reading 0.00 before you continue.

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Empty the cuvette and then fill with the MOST dilute solution first. Take the absorbance reading then move onto the next most dilute solution. Tip the solutions back into the test-tubes after each measurement and drain the cuvette quickly on tissue between readings. You don’t need to rinse the cuvette thoroughly if you go from most dilute to most concentrated....