PHAY 0002 Term 2

What is LogP useful for

As a measure of hydrophobicity of a drug, useful in drug design and development

Advantages of scatter plots

As a useful summary of a set of bivariate data (two variables), usually drawn before working out a linear correlation coefficient or fitting a regression line. It gives a good visual picture of the relationship between the two variables, and aids the interpretation of the correlation coefficient or regression model

Fluorescence: benzene rings

Benzene rings can rotate about centre bond. Only when the molecule is flat can the pi electrons delocalise over both rings, moderately fluorescent

Rate of a non- 1:1 reaction

Eg. Haber process: N2 + 3H2 —-> 2NH3. In this case, one mole of nitrogen reacts with three molecules of hydrogen to produce two moles of ammonia. For every mole of nitrogen consumed, three moles of hydrogen will be consumed, so the hydrogen will be consumed three times faster than the nitrogen. Similarly, for every one mole of nitrogen consumed, two moles of ammonia will be formed, so the rate for formation of ammonia will be double the rate of consumption of nitrogen

In fluorescence work deviations from linearity at ___ concentrations commonly occur

High. At high concentration the fluorescence emission becomes concentrated near the side of the sample cell to which the existing radiation enters. As the exciting radiation passes through the sample its intensity falls and hence the fluorescence produced also decreases

What does studying chemical kinetics help us to understand

How quickly a reaction is likely to proceed and which factors influence reaction rate, including concentration of the reactants (collision more likely), temperature (more energy, can overcome activation energy), pressure (more gas in confined space), catalysis (decreased activation energy). Can also give us important insight into the mechanisms of reactions

In order to perform rate calculations, it is necessary for us to consider

How the rate of reaction depends on other factors. The rate of reaction often depends on the concentration of one or more reactants. The rate of reaction is often proportional to the concentrations of the reactants raised to a certain power

When molecules absorb UV-visible radiation, the absorbed energy is gradually converted into

Kinetic energy (heat) as a result of collisions between molecules. In the case of a few excited molecules they rid themselves of the excess energy by emitting the absorbed energy as light- fluorescence

Liquid-liquid separation

Two different molecules present in one sample may be separated by simply giving them the choice between different (Immiscible) solvents

Vacuum distillation

Used to purify liquids boiling above 200 degrees (lower pressure lowers boiling point), can be applied to both simple and fractional distillation

Simple distillation

Used to purify liquids with boiling points above 200 degrees at atmospheric pressure from other compounds, boiling points need to be as different as possible

Measuring fluorescence: exciter and detector monochromator

Used to select the excitation or emission wavelengths of interest. The monochromators needs to have a high light gathering power. A filter can be used instead of a monochromator, however, filters are less versatile than monochromators

Fractional distillation

Used to separate liquids with close boiling points (less than 25 degrees difference)

if measurements are made on the mixture at λ1 and λ2 then

a pair of simultaneous equations may be set up from which the two unknown concentrations can be calculated

The more ways a particular state can be achieved

The more likely or the greater the probability of finding the system in that state. Nature proceeds towards the states that have the highest probabilities of existing

Reactions are usually described in terms of the order

The power to which the concentration of a species is raised in the rate law. The value of m is the order of reaction with respect to A and the value of n is the order of the reaction with respect to B. The overall order of the reaction is the sum of m and n

There are an infinite number of different normal curves each with their own mean (u) and standard deviation (sigma), however, all normal curves may be transformed to the

Standardised normal curve. If the population mean, u, is subtracted from the measurements ie. (X -u ) then the mean value for all normal curves wil be 0

for a mixture of two substances which have dissimilar chromophores

each substance will have different powers of light absorption at some wavelength or wavelengths in the spectrum

The chromatogram

graph showing the detector response as a function of elution time

spectroscopy: what is a

the constant a is called the absorption coefficient. It represents the absorbance of a solution of unit concentration when measured in a cell of unit path length. The units for the absorption coefficient will be dependent upon those used for concentration and path length

Beer-Lambert law

the dependence of absorption on concentration and path length, A= acl

the values of the molar absorption coefficients can be determined from

measurements made on pure solutions of a and b of known concentrations, therefore if A(λ1) and A(λ2) are measured for a mixture of a and b then values of Ca and Cb may be found by solving equations 1 and 2

if l is in cm and c is in moldm-3 then a is called the

molar absorption coefficient and given the symbol epsilon. It will have the units dm-3mol-1cm-1 or (M-1cm-1)

Spectroscopy: general requirements

molecules must contain conjugated double bonds. While all molecules do show electronic spectra, there are certain requirements if the absorption is to be within the range 200-900nm

spectroscopy: if l is in cm and c is expressed in %m/v, then the absorption coefficient is called

the specific absorbance and given the symbol A(1%, 1cm). The specific absorbance represents the absorbance of a 1%m/v solution in a cell of path length 1cm. It is widely used for analytical work. The BP gives values of A(1%, 1cm) for virtually all its UV-visible assay procedures

Normal vs reversed phase

1) NP: polar stationary phase (silica/alumina), RP: non-polar stationary phase (C4, C18) 2) NP: non-polar mobile phase, RP: polar mobile phase 3) NP: compounds elute in order of increasing polarity ie. Least polar first, RP: compounds elute in order of decreasing polarity ie. Most polar first 4) NP: most commonly used in TLC and flash column chromatography (but can run NP-HPLC mainly chiral work), RP: most commonly used in HPLC (but can run RP-TLC and flash columns)

What happens when compounds are soluble in both layers of a separation

1) Partition- the distribution of a solute between two solvents 2) compound present in both phases: according to its relative solubility in each, varies based on solvent and the solute 3) the solute will distribute itself between the two liquid in accordance with its partition coefficient

Common types of chromatography (7)

1) TLC 2) column 3) HPLC 4) GC 5) SEC 6) ion exchange chromatography 7) chiral chromatography

Use these rules to work out if it is not an aliphatic amine

1) is there a nitrogen-containing/amine functional group 2) does it have a single bond attached 3) is that single bond attached to a double bond

Analyte interactions: size exclusion

1) large molecules are excluded 2) small molecules penetrate pores of particles

Benefits of gas chromatography (8)

1) quantitative precision (but technically difficult) 2) separating power better than that of HPLC 3) easily automated 4) can be used for compounds without chromophores 5) cheap 6) no mobile parts and low wear and tear (low maintenance) 7) works with extremely low amounts of sample (potentially detecting parts per trillion) 8) no solvent waste (ecological)

Errors can be classified into three main types

1) random errors 2) systematic errors 3) gross errors

Solid phase extraction (4)

1) used in sample preparation (pharmaceuticals, blood urine) to remove matrix interferences such as proteins 2) concentrates sample of interest 3) solid phase similar to types of stationary phase used in chromatography- can be highly specialised for certain types of compounds 4) active substances can be: Unretained (matrix interferences adsorbed) Retained (matrix interferences washed through)

The first law of thermodynamics

1) the application of the conservation of energy principle to heat and thermodynamic processes 2) the energy of an isolated system is constant or 3) energy can neither be created or destroyed or 4) energy can be transformed from one form to another but not created or destroyed

spectroscopy: analysis of mixtures

1) the assay of a substance spectrophotometrically generally assumes that the measured absorbance is due purely to the species of interest 2) in this case of a mixture of substances it is quite probable that no region of the UV-visible spectrum can be found in which just one component absorbs; however it is often still possible to determine each component of the mixture by making measurements at a number of wavelengths

HPLC: how it works and method (3)

1) the basic operating principle of HPLC (high performance liquid chromatography) is to force the analyte through a column of the stationary phase by pumping a liquid (mobile phase) at high pressure through the column 2) compound within the sample are mainly separated in terms of their ability to interact with the stationary phase 3) the column is attached, at its end, to a suitable detector which measures-indirectly- the amount of compound by integrating the generated 'peak'

what does the value of T (or I) depend on (3)

1) the path length of the cell 2) concentration of the absorbing substance 3) nature of substance (ability to absorb UV or visible light)

Drugs in development must be pure

1) to ensure it is the drug that has the therapeutic effect and not impurity 2) therefore before any new compound is tested at all (in vitro or in vivo) it is purified and fully characterised. Separation is also used as part of the quality control and monitoring process

Charge at physiological pH will determine

1) types of drug-target binding interactions 2) solubility (uptake and distribution) 3) potential salt forms (for formulation)

Functional groups which tend to decrease fluorescence

-COOH, -NO2, -NO, -F, -Cl, -Br, -I

If a reaction is second order overall, then a plot of ln ([A]0[B]t/[B]0[A]t) against t will give

A straight line that passes through (or close to) the origin and that has a slope of k([B]0 - [A]0), from which k can be calculated

The rate of reaction is not constant. At the beginning of the reaction

(T = 0, [A] is large), the rate of reaction is high (steep gradient) and later in the reaction (t > 0, [A] is lower), the rate of reaction is lower (shallower gradient. There is often a relationship between the concentration of the reactant and the rate of reaction

Functional groups which usually enhance fluorescence

-OH, -OCH3, -NH2, -NHR, -NR2

What is work

1) defined as movement against a force. Work (J) = opposing force (N) x distance (m) 2) one Joule is the amount of work required to displace an object one meter against a force of one Newton

HPLC vs GC

1) HPLC: compounds within the sample must be soluble in the mobile phase, GC: compounds within the sample must be volatile at the operating temperature 2) HPLC: compounds can be of a wide range of chemistry and MW, GC: compounds are of a narrow range of chemistry and MW 3) HPLC: high back pressure (very time consuming maintenance), GC: low back pressure (very low maintenance)

HPLC detection (4)

1) UV absorbance (most common) 2) fluorescence 3) refractive index 4) mass spectrometry (LC-MS)

Drawbacks of HPLC (6)

1) UV-VIS remains the most popular detectors for quantitative purposes 2) this means that only compounds with a chromophore can be analysed 3) creates large volumes of organic solvent waste 4) drugs analysed must be soluble in the mobile phase 5) it works under high pressures and wear and tear of the pump is significant 6) needs lots of maintenance

What is entropy

1) a measure of disorder or measure of the molecular randomness of a system 2) a thermodynamic function that describes the number of arrangements (positions and/or energy levels) available to a system in a given state

Single point calibration

1) a solution containing a known concentration (standard solution) of the compound to be measured in the sample is injected, find out the relation concentration (area under curve) 2) then the sample with unknown concentration of the compound is injected, the new AUC is compared with the previous to know the concentration in the sample

Experimental measurement of fluorescence: the following are required (6)

1) a source of excitation 2) a means of selecting the required excitation wavelength 3) sample cell 4) a means of selecting the required emission wavelength 5) detector to measure the intensity of the selected fluorescence 6) a means of displaying the reading/plotting the spectrum

Thin layer chromatography method

1) a thin layer of silica/alumina is spread over a glass/aluminium plate (polar stationary phase) 2) this stands in a small volume of non-polar solvent which migrates up the plate (non-polar mobile phase) 3) analytes move up the plate and are separated according to their Kx

Several modes of separation (5), what do they rely on

1) adsorption 2) partition 3) ion exchange 4) exclusion 5) chiral Rely on different chemical and physical mechanisms: ionic, dipolar, non-polar. These properties determine whether a component (analyte) prefers the mobile phase or stationary phase

High Performance Liquid Chromatography (HPLC): uses

1) analytical: identify/quantify multiple analytes in complex mixtures (small scale) 2) preparative: purification (large scale)

Component retention (4)

1) by TLC retention expressed in terms of Rf 2) by column chromatography, retention is expressed in terms of column volumes (CV) 3) Elution methods developed using TLC are generally transferable to column chromatography 4) the relationship between Rf and CV is reciprocal

two methods are commonly used for converting the measured absorbance into a concentration:

1) calibration curve 2) the Beer-Lambert law with a given value for the absorption coefficient

spectroscopy: simultaneous equations - characteristics

1) can be used for a mixture containing any number of components, however, with manual instruments it is restricted usually to mixtures of four or less absorbing components 2) many computerised UV-visible spectrophotometers perform the calculations automatically 3) with diode array computer controlled instrument as many as 12 components may be determined in a single mixture

Ion exchange: order of elution

1) charge (amount and type) 2) complementary charge retained more

SPE vs chromatography (4)

1) column much smaller than chromatography 2) specialised columns more common 3) typically analytes are strongly retained on the SPE column Then impurities are washed away Rather than both analyte and impurities passing through at different rates 4) used for sample preparation - then further chromatographic methods used after this

Chromatographic methods are primarily used for: (2)

1) compound mixture analysis (analytical) 2) purification (preparative)

Analyte interactions: partition

1) cross section of open tubular column 2) solute dissolved in liquid phase bonded to surface of column

Extraction method

1) crush tablets into fine powder with pestle and mortar 2) extract with organic solvent 3) centrifuge and take the supernatant (separated from the excipients)

Steps for hypothesis testing (6)

1) decide on a null hypothesis, Ho 2) decide on an alternative hypothesis, H1 3) calculate the test statistic 4) from tables find the critical value for the test statistic 5) compare calculated and tabulated values of the test statistic 6) state conclusion and any assumptions made

Third law of thermodynamics (4)

1) defines absolute zero on the entropy scale 2) at absolute zero temperature, a perfect crystal has zero entropy 3) in a perfect crystal at absolute zero temp there will only be one possible arrangement for minimum energy 4) hence S = 0 at T = 0K so we can calculate absolute values for S. When the temp increases above 0K, the atoms and/or molecules in a solid start vibrating, so vibrational energy modes or energy levels become available, increasing disorder then entropy starts to rise from S = 0J/molK

Gas chromatography: order of elution

1) depends on volatility or molecular weight 2) high volatility/lowMW elutes first

What is the second law of thermodynamics (4)

1) describes the relationship between entropy and the spontaneity of natural processes 2) the entropy of an isolated system increases in the course of a spontaneous change: in an isolated system, natural processes are spontaneous when they lead to an increase in disorder, or entropy 3) deltaStotal > 0 or heat does not spontaneously flow from cold body to a hot body: deltaSuniv = deltaSsys + deltaSsurr 4) is deltaS universe is positive for a process/reaction, then it is spontaneous

Recrystallisation method

1) dissolve impure crude material in minimum volume hot solvent 2) filter any insoluble material 3) allow solution to cool slowly 4) produce crystals As it cools the solution becomes saturated with desired compound and crystallises (may need to scratch bottom flask), cold solution does not become saturated with impurities as the concentration is lower (hence only 10-15% impurities tolerated)

Ways to purify a compound (7)

1) distillation (for liquids) 2) filtration (solids in liquids) 3) centrifugation (solids in liquids) 4) recrystallisation (for solids) 5) liquid-liquid extraction (for solutions) 6) chromatography 7) solid phase extraction

Entropy vs enthalpy (5)

1) entropy reflects the useless energy in a system (ie. The energ not available to do work) 2) an increase in total entropy (system and surroundings) indicates a spontaneous process 3) entropy is an inconvenient term because it requires one to consider the effect on the surroundings 4) it is better to look at free energy as a criterion for spontaneity 5) clearly a balance exists between deltaH and deltaS which relates to Gibb's free energy, G

The study of kinetics forms an important central part of many aspects of pharmacy including: (4)

1) enzyme kinetics: how the concentration of a substrate changes with time as it is converted to a product, catalysed by an enzyme 2) pharmacokinetics: how the concentration of a drug changes with time in different locations in the body (absorption, distribution, metabolism, excretion) 3) drug stability: how the concentration of an active pharmaceutical ingredient in a formulation changes with time 4) radio pharmacy: how the radioactivity of a radiopharmaceutical decreases with time

spectroscopy: calibration curve construction

1) five or more standard solutions are prepared from a sample of the pure material that is to be determined and their absorbance measured 2) the values are then used to construct a calibration curve 3) the sample absorbance is measured and its concentration read off the calibration curve

What is heat and heating (3)

1) heat may be defined as energy in transit from a high-temp object to a lower temp object 2) an object doesn't possess heat; the appropriate term for the microscopic energy in an object is internal energy 3) the internal energy may be increased by transferring energy to the object from a higher temp object- this is properly called heating

Instrumental chromatography

1) high-performance liquid chromatography (HPLC), ultra performance liquid chromatography involves more pressure 2) gas-liquid chromatography (GC) 3) combined methods - typically with mass spectrometry: get info about the molecular mass of peaks, useful for unknowns, compare to database 4) LC-MS LC-MS/MS GC-MS

How to choose method of separation

1) how much do you have? 2) how many components are there? 3) what are the physical properties of the components

As the sign of delta G indicated if a process/reaction is spontaneous and as T is always positive, then

1) if delta G < 0, (negative) the process is spontaneous 2) if delta G >0, (positive) the process is non-spontaneous 3) if delta G = 0, neither the forward nor the reverse process is favoured. So the reaction is at equilibrium

Drugs in the clinic must be pure

1) impurities could be harmful and/or cause additional effects 2) can also alter the ability to formulate a drug correctly 3) can affect stability and shelf-life

3 types of impurities can be removed by recrystallisation

1) insoluble material 2) small quantities unreacted starting materials and by products formed from side reactions 3) coloured by products

Internal standard

1) internal standards can be added to improve the accuracy and reliability 2) should be structurally similar to the analyte and elute close to it in the chromatogram: Not part of the sample being analysed Well resolved Stable Available in pure form

Predicting polarity and solubility (5)

1) is it a salt (look for permanent charges, charged molecules extremely polar) 2) polar functional groups which may ionise (CA or amine, extremely polar or polar depending on pH) 3) can it hydrogen bond (donors or acceptors, polar) 4) can it establish permanent dipole-dipole interactions (electronegative atoms, esters, ethers etc, mildly or weakly polar) 5) can it establish induced dipole attraction (van der waals/London forces only)- usually only C and H, non polar

Exclusion chromatography: uses, involves

1) makes use of a uniform, highly porous, non ionic gel: small molecules are retained in the pores of the gel, large molecules are not retained (are excluded) 2) separation involves sorting molecules by size 3) gel permeation chromatography uses polymers that swell in organic liquids eg. Polyacrylamide gels 4) gel filtration chromatography uses polymers that swell in water eg. Sephardic (polysucrose polymer)

Analyte interactions: ion-exchange

1) mobile anions held near cations that are covalently attached to the stationary phase 2) anion-exchange reoin; only anions are attracted to it

Stationary phase: reverse phase

1) modified silica 2) less polar compounds more strongly retained (hydrophobic interactions), less polar solvents increase elution

Size exclusion, gel permeation: order of elution

1) molecular size 2) low size retained more-large size elutes first

General observations (for silica-based chromatography) (4)

1) more polar compounds have lower Rf (ie. Elute slower) 2) less polar compounds have higher Rf (ie. Elute faster) 3) increasing eluent polarity can increase compound Rf 4) decreasing eluent polarity can decrease compound Rf

Solvent strength: for normal phase chromatography

1) more polar solvents (or solvent mixtures) elute analyte more rapidly ie. Increase solvent strength increases Rf, decreasing solvent strength decreases Rf, opposite is true for reversed phase chromatography 2) column chromatography frequently uses mixtures of solvents to achieved good separation

Reasons for using fluorescence (3)

1) more selective than absorption. This is because a substance will absorb radiation at one wavelength and emit at another. The chances of finding two substances which absorb and emit the same wavelengths will be less than the chances of finding two substances which simply absorb the same wavelength 2) not all substances fluorescence. This can be both an advantage and disadvantage. The advantage is that it makes fluorimetry more selective for those compounds that do fluoresce 3) this technique is much more sensitive than absorption spectrophotometry. This is the main reason for using fluorimetry

Enthalpy, H (state function) (3)

1) most chemical experiments are carried out at constant pressure, p 2) under such conditions the work done by the system as a result of the expansion is not zero 3) reactions occur that result in a change given as delta H at constant pressure. H = qp = U + P dV (work)

Why is chromatography relevant to pharmacists (4)

1) most frequently used analytical technique in pharmaceutical analysis 2) an essential part of the drug discovery and development process (synthesis/purification, characterisation, chiral separations, ADME, clinical trials) 3) most appropriate way of monitoring drug stability, breakdown products, impurities 4) rapidly entering the BP as the method of choice for assay of purity (in drugs) and content (in formulated products)

Flash column chromatography (5)

1) not effective for large amounts of material 2) preparative TLC can do up to 5-25mg 3) move to column chromatography 4) effectively just an upside down 3D version of TLC 5) uses: compound purification

Basic quantitative HPLC: internal standard

1) one solution containing a known conc of the compound to be measured in the sample AND a known conc of a (internal) standard is injected to find out the response factor 2) then the sample together with the same known conc of the (internal) standard is injected to find out the AUC of the compound to find out the response factor and this is used to calculate the concentration

Basic quantitative HPLC: single point calibration

1) one solution containing a known conc of the compound to be measured in the sample is injected to find out the area under the curve (AUC) 2) then the sample is injected to find out the AUC of the compound

Drawbacks of gas chromatography (2)

1) only good for thermally stable and volatile compounds 2) may require sample derivatisation

How column chromatography works (6)

1) pack column with silica/other stationary phase 2) Equilibrate with eluting solvent 3) load crude sample onto top of column 4) flush though the column with appropriate solvent (eluent), usually under air pressure 5) collect eluent fractions in numbered vials 6) analyse these by TLC to identity clean fractions to be combined and evaporated to yield purified material

Why might we use Hess's law?

1) perhaps a reaction that can't be isolated from others and occur at the same time eg. Formation of carbon monoxide from carbon and oxygen. When carbon burns in air, it forms both carbon dioxide and carbon monoxide. The formation of carbon monoxide can't be separated from that of carbon dioxide 2) if reactions proceed so slowly they can't effectively be measured with a calorimeter eg. Rusting of iron is a very slow process and the temp changes in the surroundings are too small to measure 3) in order to measure the heats of these reaction enthalpies the method is: delta H overall reaction = delta H step 1 + delta H step 2 etc

Chromatography: order of elution: TLC/HPLC

1) polarity 2) normal phase: polar retained more - non polar elute first 3) reverse phase: non-polar retained more - polar elutes first

Population mean, sample mean, population standard deviation and sample standard deviation symbols

1) population mean: u 2) sample mean: x bar 3) population standard deviation: sigma 4) sample standard deviation: s

Calibration curves: method (5)

1) prepare stock solution of analytical standard 2) dilute it to give series of known concentrations 3) analyse by HPLC and calculate AUCs 4) plot peak area against concentration 5) analyse unknown sample and determine concentration by interpolation on the calibration curve

Benefits of HPLC (5)

1) quantitative precision 2) established and well-developed technology 3) variety of columns and detectors available 4) easily automated 5) no destruction or degradation of sample

Gas chromatography (6)

1) sample (in solution) is applied to the end of a heated capillary column where it evaporates 2) the evaporated components of the mixture migrate at different rates through the column, carried by the gaseous mobile phase 3) the rate of elution depends mainly on the volatility of the components 4) the basic operating principle of GC is to force the analyte through a column of the stationary phase by the gas (mobile phase) at high pressure and high temp through the column 5) compounds within the sample are mainly separated in terms of their volatility (interactions with the stationary phase are minimal) 6) the column is attached to a suitable detector which measures-indirectly-the amount of compound by integrating the generated 'peak'

Reasons for sampling (4)

1) saves time 2) some measurements are destructive 3) a representative sample allows conclusions to be drawn about the population 4) statistics is all about measuring a sample and making predictions abou the population

Reasons for sampling (4)

1) saves time: only a few measurements are taken. This saves money, effort etc 2) for an infinite population it is of course impossible to measure the whole population 3) some measurements are destructive eg. Red blood count-if the population was measured the patient would have no blood left 4) a representative sample allows conclusions to be drawn about the population. This is what statistics is all about- measuring a sample and making predictions about the population. Only if the sample taken is representative of the population can we expect to make meaningful predictions. If our sample is not representative we will not make good predictions

Adsorption chromatography

1) separation based on differences between the adsorption affinities of the sample analytes for the surface of a solid stationary phase 2) based on non-covalent interactions: ionic (too strong and not ideal), H-bonding, electrostatic, dipole-dipole 3) used for organic molecules

Partition chromatography

1) separation based on differences between the solubility of the sample analytes in the mobile and stationary phases, stationary phase = immobilised liquid 2) based on non-covalent interactions: electrostatic, dipole-dipole, van der waals 3) used or polar organic molecules

Ion-exchange chromatography

1) separation based on differences in ion exchange affinities eg. Differences in charges 2) based on ionic forces: ionic forces, not covalent 3) used for cations, anions, proteins, peptides, amino acids and nucleic acids

Exclusion chromatography: based on

1) separation based on exclusion effects eg. Differences in size and shape 2) based on exclusion interactions: molecular size, shape 3) used for proteins and nucleic acids

Basic quantitative HPLC: multiple points calibration

1) several solutions containing different known conc of the compound to be measured in the sample are injected and a calibration curve for the conc - AUC created 2) then the sample is injected to find out the AUC of the compound

Predicting the order of elution (4)

1) should be possible for related groups of molecules 2) identify the point of difference (are the more or less polar relative to one another?) 3) more polar = stronger interaction in normal phase 4) more polar = weaker interaction in reverse phase

The organic solvent used for extraction must meet a few criteria: (5)

1) should readily dissolve substance to be extracted 2) should not react with the substance to be extracted 3) should not react with or be miscible with water (the usual second solvent) 4) should ideally have a low boiling point so it can be easily removed from the product 5) common extraction solvents are diethyl ether, dichloromethane and ethyl acetate

Stationary phase: normal phase

1) silica, alumina 2) more polar compounds more strongly retained (eg. H bonding), more polar solvents increase elution

Relative hydrophilic/hydrophobic properties of drugs crucial to influence (5)

1) solubility 2) absorption 3) distribution 4) metabolism 5) excretion Too polar: does not cross cell membranes across gut wall Too lipophilic: poorly absorbed, likely to be taken up into fat tissue and not circulated

Internal Standard Method

1) solution 1 contains a known concentration of the analyte and known concentration of an internal standard 2) solution 2 contains an unknown concentration of the analyte (g. An extract from a formulation) and the same known concentration of internal standard as solution 1 3) the two solutions are analysed by HPLC and all the peak AUCs are determined. For each solution, the AUC of the sample peak and the standard peak are compared to give a response factor

Mobile phase and solvent strength

1) solvents for normal phase chromatography: the complete range, but rarely greater than 20% of very polar solvents (MeOH, EtOH) 2) solvents for reverse phase chromatography: very polar solvents (H2O, MeOH, MeCN) 3) a strong solvent (high eo) for normal phase chromatography is a weak solvent for reverse phase and vice versa

when a beam of radiation passes through a sample eg. a solution (3)

1) some of the radiation is absorbed by the sample 2) some of the radiation is reflected/scattered 3) some radiation passes straight through

Gel electrophoresis

1) specialised example of size-exclusion chromatography = 2) uses agarose or polyacrylamide, also pass electricity through 3) separates based on size and charge 4) used for proteins and nucleic acids 5) visualised using UV and stains

Thin layer chromatography (TLC): phases

1) stationary phase (generally polar) 2) mobile phase (mixture of different solvents) 3) detection: visualisation (coloured spots), chemical reaction (staining), UV/fluorescence

What is bomb calorimetry

1) used to determine the heat of combustion of a gas, liquid or solid 2) combustion is an exothermic reaction with oxygen being consumed 3) the sample to be burnt is prepared in an oxygen-charged bomb and the reaction initiated with an electrical charge 4) the change in temp resulting from combustion is reflected in the change of water temp, combined with the mass of the sample. This is used to calculate the heat of combustion in degrees C/g

Washing

1) very common to use an organic solvent to perform the reaction in 2) therefore your compound may already be in the organic layer 3) you could extract it into another solvent but more common to wash with water 4) removes ionic and water soluble impurities from reaction mixture through extraction into water 5) acidic and basic molecules can be removed from a reaction mixture: Converting them to salts, extracting them into water, acid-base extraction

Optimising separation

1) when scouting TLC solvent systems, it is important to realise a low Rf (0.15-0.35) is preferred because a lower Rf means a greater CV 2) large CVs indicate increased compound-silica contact time, improving the chances of component resolution 3) CV is a measure of compound retention 4) change in CV is the measure of compound resolution 5) to separate adjacent compounds, a large change in CV is desirable

For a second order reaction, the integrated rate law is

1/[A]t - 1/[A]0 = kt which can also be written as: 1/[A]t = 1/[A]0 + kt . This is the equation of a straight line. This form of the integrated rate law will give a straight line if 1/[A]t is plotted against t. The intercept on the vertical axis will give the value of 1/[A]0 from which the initial conc of A can be determined, gradient gives k

Endothermic meaning

A process that absorbs energy as heat

Exothermic meaning

A process that releases energy as heat

Chemical reactions do not occur in one step but rather by a series of steps. The series of steps is termed

A reaction mechanism

The electronic transition is described as

A singlet-singlet transition

A sample refers to

A sub set of a population ie. A set of some of the measurements which comprise the population

What is Le Chatelier's principle?

A system at equilibrium, when subjected to a perturbation, responds in a way that tends to minimize its effect If deltaG < 0, the reaction proceeds: left to right If deltaG > 0, the reaction proceeds: right to left

what does isothermal mean

A system that is kept at a constant temperature by adding or subtracting heat from the surroundings

Enthalpy can be defined as

A thermodynamic variable equal to the heat absorbed at constant pressure. Exothermic = -delta H, endothermic = +delta H

Length of steps of absorption and loss of excess vibrational energy

Absorption step very rapid, approximately 10-15 s, loss of vibrational energy is longer and the overall fluorescence process is characterised by a half life of typically 10-8 to 10-7 s

Plot of fluorescence intensity vs concentration

According to the equation or fluorescence intensity, a linear dependence of intensity of concentration should be obtained. Just because the theoretical equation predicts a linear dependence it does not necessarily mean that in practice th equation holds for your particular compound or concentration range. Always check experimentally

PKa of aliphatic amines and carboxylic acids

Aliphatic amines: typically above 8 Carboxylic acids: typically below 5 Thus these functional groups are always mostly ionised at physiological pH

What is the partition coefficient

Also known as distribution coefficient, the ratio of the concentration of the solute in one liquid over the concentration in the other, dynamic equilibrium between two liquids, temperature dependent, constant at constant temperature over a limited range of concentrations

What is the rate law for a reaction

An experimentally-determined equation that expresses the rate of a reaction in terms of the molar concentrations of species involved is known as the rate law for that reaction

Since the rate of reaction, v, must have the units of concentration per time, the units of rate constant, k, must be

Appropriate

Uses of TLC-monitoring reaction progress

At various time intervals during a reaction, samples of the reaction mixture are taken and subjected to TLC analysis, monitoring purity of fractions during column chromatography

Internal standard: equation

Because the concentration of the internal standard is always the same in all injections and you know AUC is proportional to the concentration: Response factor 1 = AUC drug/AUC internal standard

Photodecomposition

Because the intensity of the exciting radiation is very intense the sample can undergo significant photodecomposition. Samples and standards should not be exposed to the radiation for longer than necessary

What is bio-luminescence

Biochemical reactions that produce light. Glow worms etc

normal deviate (z)

By dividing the deviations by the standard deviation, a, then the deviations will all be in terms of the number of standard deviations from the mean of zero and is called the normal deviate

However, while the absolute enthalpy of a substance cannot be determined, the absolute entropy of a compound or element

Can be determined, and the more positions or energy levels available to a molecule, the higher its entropy

Ion-exchange chromatography: cation and anion exchangers

Cation exchangers (stationary phase is -ve) Anion exchangers (stationary phase is +ve)

The greater the quantity of heat transferred to the system (or surroundings) the more

Chaotic motion it has. This suggests: deltaS is proportional to q. Because energy has a spontaneous tendency to flow as heat from a hot body to a cold one, then a given quantity of energy stored at a high temp has a lower entropy than the same quantity of energy stored at a lower temp. DeltaS = q/t

Skewed data

Commonly encountered, with skewed distributions the median value is often more useful than the mean as measure of the average value

The units of the rate of reaction are always

Concentration divided by time so moldm-3s-1 if the time is measured in seconds

In most cases, enthalpy (H) is heat transferred at

Constant pressure

Pure substances and mixtures

Contain only one thing (one type of atom or molecule), pure solid has fixed melting point, pure liquids have fixed boiling point. Mixtures contain more than one thing and do not have exact properties (depends on how much of each component), formed by a physical change so can easily be separated into pure substances -process known as purification

All molecular interactions are fundamentally electrostatic in nature and can be described by some variation of

Coulombs law. Eg. Why do cyclohexane and water spontaneously separate? This question can be answered using entropy/enthalpy based answer: non-polar substances are not soluble in water. As an empirical description water and oil do not mix

Recrystallisation: limitations

Crude material containing only up to 10-15% impurities can be purified by this method, anything greater use another method

We can employ Hess's law using tabulated bond energies to calculate heats of reaction:

Delta H = sum (energy of bonds broken) - sum (energy of bonds made)

Can use a constant volume bomb calorimeter to calculate

Delta H. Usually delta U is used but in our analysis we will not take into account the change in pressure in the cylinder although it can have a small effect on the final reaction equilibrium and value of delta U

What is the standard enthalpy of formation

Delta Hf, the symbol for a standard reaction enthalpy for the formation of a compound from its elements in their reference states. The reference state of an element is its most stable state at the specified temperature and 1 bar

What is the enthalpy change of vaporisation

DeltaHvap. The enthalpy change when 1 mole of the liquid converts to gas at its boiling point with a pressure of 1 bar (100kPa). For water, the enthalpy change of vaporisation +41 kJmol-1, meaning that it takes 41 kJ to change 1 mole of water into steam. If 1 mole of steam condenses into water, the enthalpy change would be -41 kJ. Changing from liquid to gas needs heat; changing gas back to liquid releases exactly the same amount of heat

What is the rearrangement of the intergrated (first) rate law to its logarithmic form useful for

Determining the time required for the conc of A to reach a certain value. This form of the integrated rate law will give a straight line if ln[A]t is plotted against t. A straight line has the form y = a + bx where a is the intercept on the y axis and b is the gradient. The intercept on the vertical axis will give [A]0 from which the initial conc of A can be easily determined, and the gradient of the line will give the value of -k, easily allowing determination of the rate concentration

Retention factor (Rf)

Distance between the middle of the eluting spot and the baseline, Rf = distance spot ran/distance solvent ran

Inferential statistics (what should we do next)

Distinguish true differences from random variation, allows hypothesis testing

How can the energy of a system change

Due to a difference in temp between it and its surroundings. Energy has been transferred as heat

Open system meaning

Energy and matter can transfer

Closed system meaning

Energy transfers only

Quantities calculated from a sample are only

Estimates of the true value and will be subject to uncertainty

What are the uses of integrated rate laws

Evaluating the rate constant of a reaction, predicting the concentration of a species at a particular time and predicting the time required for the concentration of a species to reach a certain value

What are surroundings

Everything else besides the system

the relationship between I and path length

Experimentally it is found that I decreases exponentially with an increase in path length, l. This was first observed by Bouger and independently by Lambert.

If ECl is small (<0.01) ie. At low concentrations then, what does the equation indicate

F = k' x quantum efficiency x Io x ECl. The equation indicates: 1) the intensity of the emitted radiation is directly proportional to concentration- this makes fluorescence useful analytically 2) fluorescence is directly proportional to the intensity of the excitation source. The brighter the lamp the greater the fluorescence 3) fluorescence depends upon the molar absorption coefficient- the stronger the absorption the greater the fluorescence

Not every molecule which absorbs radiation will lose the energy as

Fluorescence. Due to collisions between molecules will lose all the energy as kinetic energy. The ratio of the number of molecules which fluoresce to the number which actually absorb energy is termed the quantum efficiency

What types of compounds exhibit fluorescence

Fluorescent compounds generally have a rigid structure, molecules which have a large number of delocalised electrons are often fluorescent providing the structure is sufficiently rigid.

Washing: add acid to the aqueous layer

Low pH, base will be protonated, forms salt, goes into water + HCl layer (polar, acidic)

the relationship between concentration and I

I decreases exponentially with increasing concentration. Light must pass through more solution (increased path length), interacts with more molecules and T decreases

percentage transmittance =

I/Io x 100

Descriptive statistics (summarise data)

Identify patterns in data (plots), leads to hypothesis generating

If you are given a set of data that show how the concentration of a substance varies with time and you plot 1/[A]t versus t

If the points fall along a straight line then that demonstrates the reaction is second order with respect to that sustance. This is the easiest way to demonstrate that a reaction follows second order kinetics

Miscible meaning

If they are similar they mix in every proportion, only one layer or phase

Immiscible meaning

If they are too different = solvents don't mix, form separate layers or phases, exploited in liquid-liquid separations

What is purification

In a chemical context is the physical separation of a chemical substance of interest from foreign or contaminating substances

Chemi-luminescence definition and example

In some chemical reactions the product molecules are left in an excited state. The light emitted when these molecules return to the ground state is referred to as chemi-luminescence. Eg. Reaction of luminal with an oxidising agent and peroxide

Steps of fluorescence (3)

In this process the emitted radiation is of lower energy than the absorbed radiation. 1) molecules in the ground state absorb light and are excited to any one of the vibrational levels in the 1st excited electronic state 2) the excess vibrational energy is then lost by collisions with other molecules until the molecules are in the v = 0 vibrational level of the first excited state 3) finally the molecules get rid of the remaining energy by emitting as radiation and dropping down to any one of the vibrational levels in the ground electronic state

For a second order overall reaction, the integrated rate law is

In which [A]0 and [B]0 are the initial concentration and [A]t and [B]t are the concentrations at time (t) of A and B respectively

Increasing the conc of reactant generally results in

Increased reaction rates as there is a greater probability of molecules colliding (more molecules within the same space)

What is the simplest way to determine the order of a reaction

Integrate the rate law and see if a given set of experimental data fit the integrated rate law for a first-order process or second-order etc.

Internal potential energy meaning

Intermolecular interactions

What is the energy of a system

Its capacity to do work

What is a state function

Its value only depends on the current state and not how it got there eg. Internal energy

Energy, work, quantity of heat units

Joules = N m = m2 kg/s2

The distribution coefficient, Kx

Kx = C(stat)/C(mob), where: C (stat) = conc of compound X in the stationary phase C (mob) = conc of compound X in the mobile phase Each compound will have a different Kx Chromatographic separations can be altered by changing the nature of the stationary and/or mobile phases

Units of k: third order

L2mol-2S-1

Units of k: second order

Lmol-1S-1

A useful rearrangement of the integrated rate law to its logarithmic form (first order) is

Ln[A]t = ln[A]0 - kt

The experimental determination of the rate law of a reaction can often be quite challenging. In principle, we could

Measure the concentrations of all the reactants at a variety of time points during the reaction and try to determine how the rate of reaction depends upon these concentrations, but it will then be difficult to know which reactant is affecting the reaction rate in which way

The value of A and Ea in the Arrhenius equation can be determined experimentally by

Measuring the rate constant of a reaction at a series of different temps. By using the logarithmic version of the Arrhenius equation: ln k = lnA - Ea/RT, we can plot ln k against 1/T and this should give a straight line. The intercept on the y axis will give ln A from which the value of A can be easily calculated. The gradient of the line will give -Ea/R from which Ea can be easily obtained by multiplication by -R. Once the valves of A and Ea are known, the rate of reaction can be predicted for any temp

Kinetic energy meaning

Motion of its parts- molecules, atoms and electrons

Measuring fluorescence: detector

Must be as sensitive as possible. Generally a photomultiplier tube. The sensitivity varies with wavelength

We can see that a spontaneous process takes place

Naturally, without outside intervention or stimulus. An explosion is fast and spontaneous, while the process of rustng is slow and also spontaneous. Whether spontaneous or not this does not tell us the rate of reaction. We need kinetics to aid in the determination of the rate of reaction

If a reaction decreases the number of gaseous molecules, deltaS is

Negative

Force units

Newtons. N = m kg/S2

If the excitation wavelength set to a value such as A and the analyser scanned

No fluorescence would be observed as no molecules are absorbing energy. At a wavelength such as B, some radiation is being absorbed and hence some molecules are being excited. On scanning the analyser a small fluorescence peak (dotted spectrum) would be obtained. The wavelength of maximum emission will, however, correspond to the optimum wavelength of emission for the compound. With the analyser set to this value it is now a simple matter to scan the excitation wavelength to find the optimum wavelength for excitation

isolated system meaning

No transfers of energy or matter

Fluorescent impurities

Often the limiting factor is the fluorescence from impurities in the reagents used and the cell itself. The four sides of a curette often have slightly different background fluorescence intensities and consequently the cell should always be placed in the holder the same way round

Recrystallisation: suitable solvent

One in which the compound to be purified is insoluble when the solvent is cold and soluble when solvent is hot (can use mixture of solvents), based on theory of saturated solutions

Conc-time graph: 1: 1 mol reaction

One mole of A reacts to produce one mole of B. This means that for every mole of A consumed, a mole of B is formed

What is the isolation method

One or more of the reactants is used in very large excess. Eg. V = k[A]m[B]n. If we use a very large excess of B, then the concentration [B] will hardly change during the reaction. The rate of reaction will then only depend upon the concentration of [A], making it much easier to analyse. The rate law effectively becomes v = k'[A]m. The initial rate of reaction (Vo) can then be measured for a variety of different initial concentrations of A, allowing determination of the value of m, the order of reaction with respect to A. We could then repeat the investigation using a very large excess of A, examine how the rate depends upon the conc of B and hence determine the value of n, the order of reaction with respect to B.

What does LogP measure

Only unionised form of the drug between octanol and water

What is the standard enthalpy of formation

Or standard heat of formation of a compound. The change of enthalpy from the formation of one mole of the compound from its constituent elements, with all substances in their standard states at 1 atmosphere

Properties of most drugs

Organic molecules with acid/base properties, non-polar weak acids or bases eg. Aspirin or codeine, behaviour as solutes will depend on their ionisation state (depend on pH). Changing the pH will change the distribution of charged to uncharged species and thus affect the solubility of the compound. These two factors: polarity of solvent and pH behaviour are widely used methods of extraction

P equation

P = conc of drug in octanol / conc of drug in water. Hydrophobic molecules prefer octanol layer - high P, hydrophilic prefer water - low P. Typically represented as LogP, can be estimated using computational software CLogP

pressure, stress units

Pa = N/m2 = kg/m s2

Chromatography definition

Physical method of separation, in which the components to be separated are distributed between two phases, one of which is stationary whilst the other moves in a definite direction

Polar stationary phases

Polar compounds are retained more strongly than lipophilic compounds

Potential energy meaning

Position or configuration of its parts

If a reaction increases the number of gaseous molecules, deltaS is

Positive

The intensity of fluorescence will be directly proportional to

Quantum efficiency. A non-fluorescent compound will have a quantum efficiency of zero. F=k x quantum efficiency x (Io - I) where k is the constant of proportionality. The fluorescence emission occurs in all directions and in practice only part of the emitted radiation is measured, k can therefore be considered as an instrumental constant

Delta G = delta G +

RT ln Q where temp in K Q = reaction quotient indicating the state of a system at a given instant

The second type of second order reaction is one in which the rate of reaction depends on conc of both

Reactant species each raised to the first power. This is the most common type of second-order reaction and examples include most SN2, E2 and nucleophilic addition-elimination reactions. VA = -d[A]/dt = vb = -d[B]/dt = k[A][B[

Le Chatelier's Principle can be applied to

Reactions in a state of dynamic equilibrium. It's applicable to how changes to the position of eqm occur if you change concentration, pressure or temperature. However, remember that catalysts have no effect on the position of eqm.

When chemical change occurs bonds are

Rearranged. The bonds in the reactants are broke and new bonds are formed to produce the products eg. The bonds in the propane and the oxygen need to break and new bonds in the carbon dioxide and oxygen have to form

What does LogD represent

Relative distribution of all species (ionised and unionised)

Non-polar stationary phases

Reversed phase has bonded lipophilic groups, lipophilic compounds are retained more strongly than polar compounds

What are Lipinskis rules

Rule of 5 suggests drug like molecules have a log P of less than 5, derived from an analysis of drugs from the World drug database, guide for design of orally bioavailable drugs. Molecular weight less than 500 Da Fewer than 5 H bond donors Fewer than 10 H bond acceptors LogP less than 5

Entropy can be defined as this equation

S = k ln W K= Boltzmann's constant (a proportionality constant equal to the ideal gas constant divided by Avogadro's number) W = represent the different states available Ln = natural log

We might imagine S is proportional to W however

S is an extensive property- double N and S will double. While W is a measure of the possible number of states, leading to S = K ln W and a mathematical theory that relates the entropy S, or degree of disorder of a system, to the thermo-dynamic probability W, where W is the number of microscopically distinct states of a system

Units of k: first order

S-1

Central limit theorem

Sampling distribution of the sample mean will have its own population mean and population standard deviation

It is not always necessary to record the complete spectrum

Simply measuring the fluorescence intensity at the optimum wavelength for emission is often adequate. Recording a spectrum, does however, give an extra check that the measured signal is due to the compound of interest and is not interfered by any other signals. Unlike absorption spectrophotometry it is necessary to have a standard solution for calibration. It is not possible to quote the equivalent of a A(1%, 1cm) value

Analyte interactions: adsorption

Solute adsorbed on surface of stationary phase

Partially miscible meaning

Solvents not too similar but not too different, may mix at determinate proportions, depends on the composition

Mobile phase definition

The phase that moves in a definite direction. Consists of the sample being separated/analysed and the solvent that moves the sample through the column

Delta G only applies to

Standard state conditions, where temp is 25 degrees C and all gas pressures are at 1atm. Thus delta G only allows us to predict if a reaction is spontaneous at these conditions. However, most reactions don't occur at these conditions. We need a more general equation that allows us to determine the free energy change (delta G) and to predict if a reaction is spontaneous for all other conditions

The chromatographic process: diagram

Strongly retained analyte = larger Kx Weakly retained analyte = smaller Kx

What is solubility dependant on

Structure and functional groups in the molecule: 1) ionic compounds are the most polar ones 2) ionisable functional groups may become ionic under certain conditions (pH) 3) functional groups which can form hydrogen bonds are also polar

Standardised normal curve

Subtract population mean (u) from measurements ie. (X - u) then all normal curves will have a mean of zero

We will see on many occasions when we are studying kinetics that often it is essential to

Take the data that are presented and to transform them into data that can be plotted graphically to give a straight line. Useful info can then be obtained from the gradient and intercept of that straight line.

to describe/quantify the amount of radiation which passes through the sample, a quantity called the transmittance, T is defined:

T = I/Io. Transmittance is a ratio and therefore has no units. It takes values between 0 and 1, though it is often as a percentage value (0 to 100%)

When t = t1/2, the value of [A] will be half of [A]0 so

T1/2 = ln2/k units are: time-1

With an endothermic reaction, the system absorbs energy from the surroundings because

The products have higher potential energy (requie more energy to form) compared to the reactants. The difference in energy must be absorbed from the surroundings for the reaction to occur

If you are given a set of data that show how the conc of a substance varies with time and you plot [A]t versus t, if the points fall along a straight line then

That demonstrates that the reaction is first order with respect to that substance. This is the easiest way to show that a reaction follows first order kinetics.

The ranges of values used for an Arrhenius plot often mean

That it is difficult to extrapolate the line of best fit back to the vertical axis. After determination of the gradient, b, of any straight line (y = a +bx) the intercept on the y axis, a, is easily calculated by taking any point on the line of best ft and substituting in to the rearranged equation a = y -bx

A characteristic of the fluorescence process is

That the spin quantum number of the electron does not change during the electronic transition

The reaction between the rate of reaction and temp is described by

The Arrhenius equation: k = Ae-Ea/RT k = rate constant A = preexponential factor/frequency factor (same units as k), represents the total frequency of collisions Ea = activation energy E-Ea/RT = fraction of collisions that are sufficiently energetic to initiate reaction R= gas constant T= temp in Kelvin

The relationship between Io and I is given by

The Beer-Lambert law: I = Io 10 -ECl, where E is the molar absorption coefficient, c is conc and l is path length

Washing: add base to the aqueous layer

The acid will be deprotonated, forms a salt which is not soluble in the non-polar layer, but moves into aqueous layer

What is the molar heat capacity

The amount of at energy required to raise the temperature of one mole of a material by 1 degree celsius

As the first step in the process is absorption, the intensity of fluorescence emission depends on

The amount of energy absorbed. The more energy absorbed the more energy that can possible be emitted

What is the specific heat capacity

The amount of energy required to heat 1 kg of a substance by 1 degrees celsius

What is the heat capacity

The amount of heat energy required to raise the temperature of a certain amount of material by 1 degree (or 1 kelvin). C = q/delta T

The intensity of fluorescence will be proportional to

The amount of light absorbed (difference between intensity of Io and I), thus: F is proportional to (Io - I)

Gibb's approach allows a researcher to calculate

The change in free energy in the process, such as in a chemical reaction, and how fast it will happen. Since virtually all chemical processes and many physical ones involve such changes, his work has significantly impacted both the theoretical and experiential aspects of these sciences. G = H -TS and deltaG = deltaH -TdeltaS

It is very convenient to use bond energies to calculate heats of reaction, however

The bond energies are only average values and can result in significant differences between experimental measurement using heats of reactionsand theoretical calculations. It is also important to remember that you can only use bond enthalpies directly if everything you are working with is in the gas state

What do integrated rate laws relate

The concentration of a reactant at any time during the reaction to the initial concentration of that reactant and the time elapsed

With second order reactions, the rate of reaction depends upon

The concentration of one species raised to the second power (ie. The conc squared). Eg. If conc of reactant doubles, rate x 4, half life increases

Quantitative HPLC: what is the response of the detector to a compound proportional to

The concentration of the compound in the sample, within a certain range of concentrations only, low and high limits of detection

The rate concentration is independent of

The concentrations of the species involved in the reaction but is influenced by other factors such as temperature

Entropy, like enthalpy is a state function and its value only depends on

The current state and not on how it got there. Delta S = S final - S initial

The heat of the reaction is

The difference between the energy needed to break the bonds in the reactants and the energy released in the formation of the bonds in the products. These bond energies termed average bond energies (of dissociation) are determined by examining the heats of various reactions in which each type of bond is broken

Delta G determines

The direction and extent of chemical change. A decrease in Gibbs free energy (ie. Delta G < 0) is the condition required for a reactionto go. Remember that deltaG is meaningful only for changes in which the temperature and pressure remain constant. These are the conditions under which most reactions are carried out in the laboratory. The system is usually open to the atmosphere (constant pressure) and the process is started and ended at room temp (after any heat that has been added or which was liberated by the reaction has dissipated)

What is fluorescence

The emission of light by molecules

What is internal energy

The energy associated with the random, disordered motion of molecules eg. A room temp glass of water sitting on table has no apparent energy, either potential or kinetic. But on the microscopic scale it is a mass of high speed moleculs travelling at hundreds of metres per second

What is the heat of combustion

The energy released as heat when one mole of a compound undergoes complete combustion with oxygen

What does Hess's law say

The enthalpy change accompanying a chemical change is independent of the route by whcih the chemical change occurs. The overall enthalpy change will be exactly the same whether you do it in one step or many steps.

Liquid-liquid separation relies on

The fact that the different components of a mixture have different solubility properties, uses two Immiscible solvents to extract the desired compound from one solvent to another eg. Organic compounds are extracted from aqueous solution to organic solvent, separating funnel used, organic product will be soluble in organic solvent (organic layer)

Quenching

The fluorescence of a substance is generally more affected by its environment than is the absorption of a sample. When the fluorescence is decreased by the presence of another substance the phenomenon is termed quenching

The instantaneous rate of reaction at any time t is given by

The gradient of the curve at that time. If we wanted to manually determine the rate, we could draw a tangent to the curve and calculate the gradient by dividing the change of conc of A by the change in time

A useful measure of a first order reaction is

The half life of the reactant. This is the time taken for the concentration of the reactant to fall to half of its original value. The half life is a constant and is independent of concentration for a first order reaction

The change in internal energy of a system is equal to

The heat added to the system minus the work done by the system. Delta U = Q - W

The greater the space or volume available to particles

The higher the positional entropy. So solids will have the smallest volume and relatively few positions available for particles, while gases have a much greater volume with many positions available for gas particles to adopt

Fluorescence: lower limit of detection

The lower limit of detection is the lowest concentration of the substance that can be determined. The main reason for using fluorescence is because of its very low detection limits. A knowledge of the factors that affect the detection limit are important

Fluorescence: methyl groups

The methyl groups stop the two benzene rings from being in the same plane and hence pi electrons can not delocalise over both rings, non-fluorescent

The number of molecular interactions between the functional groups is enormous. Huge number of weak molecular interactions within a folded protein are balanced by

The molecular interactions of the unfolded protein with surrounding water molecules. The protein is held in a delicate balance between powerful countervailing forces contributed primarly by molecular interactions. It is the small difference between these large effects that determines direction of the folding reaction. The eqm constant is generally small. A small change in pH or temp can tip the balance

Temp is an important factor in determining the significance of deltaS surrounding and

The overall sign for deltaS universe ie. Whether or not a process or reaction is spontaneous. We can express deltaS surroundings in terms of the enthalpy change for the system, deltaH, as follows: deltaS = -(deltaH/t) where deltaH is in joules and absolute temp in K

What is a system

The portion of the universe that we are considering

A histogram shows

The range of the data, but also gives a very clear picture of how the data is distributed

The coefficient k in the rate law is called

The rate constant and is characteristic of the reaction being examined

The rate of reaction for a particular species is defined as

The rate of change of concentration of that species with respect to time

The rate of reaction for A —> B can be defined as

The rate of consumption of A or as the rate of formation of B as both are equal

For a second order overall reaction that is first order with respect to each reactant, the rate of consumption of A is equal to

The rate of consumption of B- that is, for every mole of A that reacts, one mole of B will also react. The integrated law is appropriate if the initial concentrations of A and B, [A]0 and [B]0 are different. In the case where the initial concs are the same, the concs of A and B will be identical at all times during the reaction as they are decreasing at the same rate. In the case [B]0 - [A]0 = 0, and we cannot use the integrated law above

A reaction is first order if

The rate of reaction depends upon the concentration of only one species raised to the first power. If conc of reactant doubled, rate doubled. Has a constant half life eg. SN1, E1, radioactive decay

Reactions can be described as zero order if

The rate of reaction does not depend on the conc of any of the reactants. In these rare cases, the reaction rate remains constant, irrespective of concentration

What is chemical kinetics the study of

The rates of chemical reactions

Studying chemical kinetics allows us to examine

The rates of eg. SN1 and SN2 reactions mathematically and enables us to determine what conc of a given species is present at a certain time, or how long it will take for the reaction to reach a certain percentage completion.

The rate of a chemical reaction is dependent on

The reaction mechanism. An SN1 reaction is unimolecular; its rate is dependent only on the conc of one species (substrate). An SN2 reaction is bimolecular; its rate depends on conc of substrate and nucleophile

Increasing the temp at which a reaction takes place also generally increases

The reaction rate. This increase occurs by two mechanisms: firstly, the probability of collisions taking place is increased (molecules move faster) and secondly, there is greater probability that the colliding molecules will have sufficient energy for the collision to result in a successful reaction (molecules have more kinetic energy)

Hydrophobic substances are structurally incapable of forming hydrogen bonds. So why do they spontaneously separate?

The reason is that water molecules adjacent a hydrocarbon loose rotational and translational freedom to maintain molecular interactions.Water adjacent to a hydrocarbon becomes ordered loweringn its entropy to maintain good molecular interactions. The entropic contribution leads to unfavourable free energy of mixing oil and water

The change in positional entropy is dominated by

The relative number of gaseous molecules in a chemical reaction. If increases, positive or if negative, decreases

Chromatography: order of elution, depends on

The relative order of elution of the different molecules depends primarily only in the interactions they have with the stationary phase

Layout of measuring fluorescence diagram

The right angled layout is used to prevent the high intensity light beam Io impinging directly on the entrance slit of the analyser monochromator

Unlike reaction rates that depend on the pathway of a reaction (ie. Whether or not a catalyst is used), state functions like entropy are

The same regardless of pathway

To make predictions/draw conclusions it is generally necessary to know

The shape of the distribution

Solubility

The solubility of a compound will depend on the nature of the forces acting between solute and solvent Ionic compounds: more soluble in polar solvents than non-polar solvents Covalent compounds: more soluble in non-polar solvents Like dissolves like

The equation for fluorescence intensity doesn't hold under high concentrations ie.

The squared and higher terms in the exponential series have become important (ECl no longer small). In some cases at high concentrations the fluorescent signal actually decreases. This is caused by the fluorescence emission from one molecule being absorbed by another

Kinetics deals with

The study of the rates of change of various processes. We typically encounter kinetics in pharmacy when considering how the concentration of a given substance changes with time

Analyte definition

The substance being separated/analysed

Stationary phase definition

The substance fixed in place for the chromatography procedure Fluid/solvent entering the column is called the eluent Fluid/solvent leaving the column is called the eluate Process of passing liquid through a chromatography column is called elution

In an exothermic reaction such as methane burning, the heat flows from

The system to the surroundings because the products have lower potential energy (have stronger bonds) compared to the reactants

The Gibb's free energy relates

The tendency of a physical or chemical system to simultaneously lower its energy and increase its disorder, or entropy, in a spontaneous natural process

When isolated hydrocarbon molecules aggregate in aqueous solution

The total volume of interfacial water decreases. Thus the driving force for aggregation of hydrophobic substances arises from an increase in entropy of the water. The driving force for aggregation does not arise from intrinsic attraction between hydrophobic solute molecules

A population refers to

The whole set of measurements that can be made, can be finite or infinite. When measurements can be repeated only a finite number of times it is a finite population, when measurements can be repeated an infinite number of times the population is infinite

What is luminescence

There are numerous different ways in which a molecule can give rise to the emission of electromagnetic energy and the general name for such processes is luminescence. Fluorescence is just one such process

Measuring fluorescence: source

This should be as intense as possible at the wavelength of maximum absorption of the compound. The source should ideally be continuous so that any desired wavelength can be selected. The wavelength of interest is 200-500nm. The source is often a xenon arc lamp

Provided with a compound which fluoresces, how are the optimum wavelengths for excitation and emission found?

To some extent, trial and error

Internal kinetic energy meaning

Translational, vibrational, rotational

X bar and s are estimates of

U and sigma respectively

Quantities calculated from populations have no

Uncertainty to them, they are the true values (no more values to measure)

Quantities calculates from samples are subject to

Uncertainty, they are estimates of the true values

Sometimes its simpler to use an isolation method to deal with a second order overall reaction. This saves

Us having to measure the concs of both A and B throughout the reaction. By having one reactant in considerable excess, its concentration remains essentially constant as it hardly changes at all throughout the reaction. If we have a very large excess of B, then: vA = -d[A]/dt = k[A][B]. Simplifies to: VA = -d[A]/dt = k'[A] effectively becoming first order overall. We call such a reaction pseudo first order and the rate constant, k', is called the pseufo-first-order rate constant

Centrifugation

Uses centrifugal force, spins samples fast, separates even fine solid matter from a liquid, differential centrifugation - different sized materials

Measuring fluorescence: sample cell

Usually made from silica. Generally of square cross section, 1 x 1cm, with all four sides optically worked

Solubility for separation

Utilised different properties of the molecules, solubility varies with solvent and temperature, techniques that utilise these properties: filtration, centrifugation, recrystallisation, liquid-liquid separation

For the reaction A+B —-> products, the rate may be found to be directly proportional to the concentrations of the reactants A and B so:

V is proportional to [A]m[B]n or v = k[A]m[B]n

VA equation for first order reactions

VA = -d[A]/dt = k[A]

At any time, t, the rate of consumption of A (vA) is given by

VA = -d[A]/dt. This is a differential equation. Minus sign. The rate of reaction is always a positive quantity (time and conc aren't negative). Since [A] decreases with time, the valu of d[A]/dt is negative (gradient goes downhill) and the minus sign is required to make sure the rate of reaction with respect to A is positive

VA equation for second order reaction

VA= -d[A]/dt = k[A]2

The rate of formation of B, VB is given by

VB = d[B]/dt. Since [B] increases with time, the value of d[B]/dt is positive (gradient goes uphill) and the rate of reaction with respect to B is positive. In a simple 1:1 reaction, the rate of formation of B is equal to the rate of consumption of A as one mole of B is formed for every mole of A consumed

Fluorescence: rigid with both rings in same plane

Very fluorescent, pi electrons can delocalise over both rings

Why calculate the sample mean

We feel that the mean value is more reliable than any single measurement - we hope that errors in the individual measurements (some positive, some negative) cancel out

When is liquid-liquid separation used

When isolating or purifying a product, can also be used to extract a natural product, normally performed during the work up stage after a reaction to make a compound ie, when you have decided your reaction is finished and all of the starting material used up. Organic chemistry routinely used liquid-liquid extraction, washing and acid-base extractions

The normal distribution

When measurements are subject to many small random variations, the form of the distribution often conforms to a particular mathematical form - the normal curve

If you have one or more liquids present, you need an extra energy term to work out the enthalpy change

When you convert from liquid to gas, or vice versa, the enthalpy change of vapourisation

What is van's Hoff's equation

Where a plot of lnK vs 1/T should give a straight lie. With a slope = -deltaH/R and the y intercept = deltaS/R. This is a convenient way of measuring delta H and delta S (assume delta H and delta S independent of temperature - not really true, but OK for small temp changes). If delta S is positive and delta H is negative then this reaction is spontaneous under all conditions

Some functional groups can be charged, depends on

Whether a molecule is an acid or a base, pH

The normal curve

With a continuous variable (such as concentration) when the measurements are subject to many small random variations, the form if the distributions often conforms to a particular mathematica form-the normal curve. In situations where many small random variations are influencing each measurement, the total variation in the measurement will have a normal distribution, bell shaped. Large deviations occur much less often than small ones. The curve is defined by just 2 parameters: the mean (u) and standard deviation (sigma)

Fluorescence: constructing a calibration curve

With the exciter set at its optimum wavelength the emission spectra for a set of standard solutions and the unknown(s) are recorded. The fluorescence intensity values for the standard solutions can be used to construct a calibration curve from which the concentration of the unknown(s) can be determined

The standard enthalpies of formation of elements in their reference states are

Zero at all temperatures

By definition, the standard enthalpy of formation of the most stable form of any element is

Zero because there is no formation reaction needed when the element is already in its standard state

For a first order reaction, the integrated rate law is

[A]t = [A]oe-kt, where [A]t is he conc of A at time, t, [A]0 is the initial conc of A. The concentration of A decreases exponentially with time. This form of the integrated rate law allows us to predict the conc of A at any time after the start of the reaction

spectroscopy: eliminating/minimising effects of reflection and scattering

for analytical purposes we are interested in measuring the amount of radiation absorbed by the sample and therefore need to eliminate/minimise the effects of reflection and scattering. This is done by taking Io as equal to the intensity passing through the cell when filled with a blank solution (everything except the substance being measured), and I as the intensity passing through the cell when filled with sample solution

advantage of calibration curves

it can be used even if the Beer-Lambert law does not hold (ie. absorbance vs. concentration plot is curved). A pure sample of the analyte must be available

Energy as heat and work all measured in the same units:

joules where 1J = 1kgm2S-2

the wavelength selected for an assay should be chosen so that

only the substance of interest (analyte) absorbs, with no absorption from any impurities. In the diagram, λ1 would be chosen in preference to λ2 at which the impurity also absorbs. The wavelength should also correspond to an absorption maximum and not on the sloping portion of the curve, otherwise a small error in setting the wavelength will have a large effect on the absorbance value

using Beer-Lambert law and absorption coefficient

the absorbance for the sample solution is measured and the concentration calculated using the Beer-Lambert law and a given value for the specific absorbance or molar absorption coefficient. Most of the UV-visible assay procedures in the BP are of this type.

what does the molar absorption coefficient represent

the absorption of a 1moldm-3 solution in a cell of path length 1cm. It is useful for comparing the light absorbing abilities of different substances on a molecule for molecule basis. Typical values are 10, 000 - 30,000, though occasionally values as high as 100,000 dm3mol-1cm-1 are observed

the longer the chromophore,

the longer the wavelength of absorption

what is absorbance directly proportional to

the quantity log10(Io/I) is called the absorbance. The absorbance is directly proportional to the concentration of the absorbing substance and the path length. This is an extremely important relationship and is the basis of virtually all quantitative spectophotometry

if we assume that the components of the mixture do not react with one another, then the absorbance A (λ) at some wavelength λ will be given by

the sum of the absorbances. A(λ) = sum of (molar absorption coefficient of the i th component at wavelength + conc of the i th component + path length)