Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N31/00—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods
  • G01N31/16—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods using titration
  • G01N31/166—Continuous titration of flowing liquids
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
  • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
  • G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
  • G01N21/79—Photometric titration
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
  • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
  • B01J2219/0068—Means for controlling the apparatus of the process
  • B01J2219/00702—Processes involving means for analysing and characterising the products
  • B01J2219/00707—Processes involving means for analysing and characterising the products separated from the reactor apparatus

Definitions

• the present invention relates to an improved analytical method and apparatus therefor, in particular to a method and apparatus for titration.
• Many compounds have physicochemical properties which vary according to their chemical or physical environment, which properties can be investigated by changing that environment and observing the effects on the test compound.
• properties are ionisation state, solubility, partitioning between e.g. organic and aqueous phases or into micelles or liposomes, the strength of ligand binding or metal complexing and hydrophobicity, which can vary with environmental parameters such as pH, ionic strength, or the concentrations of other species in the system.
• Analytical chemists studying the properties of chemical or biological molecules have long counted titration amongst the major tools of their trade as it allows one parameter of a system, e.g. the pH of a solution, to be varied by dropwise addition of one or more reagents whilst other parameters of the system remain essentially constant, allowing the effects of the variation to be studied effectively in isolation.
• An example of a property which can be determined by titration is the pKa (or dissociation constant) of an ionisable group of a compound, which can be defined as the pH at which the group is 50% ionised.
• the level of ionisation of a given ionisable group at any pH can be directly calculated once the pKa is accurately known.
• a given molecule may have multiple pKas if it contains more than one ionisable group. As a molecule's state of ionisation can alter other properties such as hydrophobicity and aqueous solubility, knowledge of the pKa(s) of a potential drug molecule is of great importance.
• test compound are labour intensive, with each dropwise addition of reagent followed by a delay for the mixture to equilibrate before the taking of a reading.
• the accuracy of conventional techniques is limited by the size of drops added, which can vary with the skill of the operator, and also the concentration of the test compound is altered as each dropwise addition of a reagent dilutes the test sample.
• relatively large amounts of test compound are required for standard titrimetric techniques, for example if the titrimetric analysis is to determine the pKa of a compound and is monitored by a UV spectrophotometer then 1 mg of test compound may be required. If the same analysis is monitored by a pH meter over 3mg of the compound may be required.
• the present invention provides a method of continuous titration in which at least one parameter of at least one compound in a test mixture may be monitored as the composition of the mixture is continuously varied.
• the continuous variation may be characterised by changing concentration of one or more species or components in the mixture, for example a continuous, preferably linear increase or decrease in the concentration of the species or component.
• At least two fluid streams are continuously mixed to form a test mixture stream which passes through a spectrophotometric detection zone.
• the volume to volume ratio of at least two of the component streams in the mixture entering the detection zone is continuously variable with time, by alteration of the relative proportions of the component streams forming the test mixture.
• three or more component fluid streams are continuously mixed to form the test mixture stream, the volume to volume ratio of two of these component streams being continuously varied with time by alteration of their relative proportions in the forming of the test mixture.
• the present method has the further advantage that, as solutions are not added dropwise but are continuously mixed in varying proportions, the accuracy is no longer limited by the size of drops added. Furthermore, the process can be speeded up considerably; as mixing is continuous, there is no waiting time whilst the mixture equilibrates after addition of each drop. The limiting step may then be the flow rate achievable through the pumps, mixers and tubes used.
• a further advantage of the present method is that it can better take advantage of the rate of data sampling at the detector which, in a modern instrument such as a diode array spectrophotometric detector with fixed geometry optics, can be very high e.g. 100 readings per second may be possible although in practical embodiments, 10-30 readings per second, e.g. 20 per second may be taken.
• High data sampling rates allow the option of "data smoothing" or noise reduction. For example if 20 readings per second are taken, these can be averaged over 10 readings to give an effective sampling rate of 2 per second. This averaging can provide more sensitive detection than conventional methods of spectrophotometric detection.
• the present invention provides a method of continuous titration in which a flowing fluid stream comprising a compound under test is mixed with at least one additional flowing fluid stream to form a test mixture stream and the test mixture stream is passed, preferably at a constant flow rate, through a spectrophotometric detection zone at which readings relating to at least one physical or chemical parameter of the compound under test may be taken.
• the test mixture stream is mixed from three fluid components; the first, the volume of which preferably remains constant as a percentage of the total volume of the test mixture stream, comprises the compound under test. The concentration of this compound in the mixture stream therefore remains constant.
• the % volumes of the second and third components are preferably variable in inverse proportion to one another; as the % volume of one rises, the % volume of the other falls, so as to keep the total volume of the mixture constant.
• the variable components may comprise buffer solutions, solvents, test reagents, organic and aqueous phases or other fluid components which may be varied relative to one another to alter the physical or chemical environment of the compound under test.
• further fluid components may be included in the test mixture, at constant or variable volume.
• salt solutions may be employed to maintain a chosen ionic strength
• indicators may be added or the amount of water (or other solvent) may be adjusted to compensate for changes made to the volume of other fluid components.
• variable components comprise two linearising buffers - that is two buffers whose relative proportions may be altered to produce a linear pH gradient.
• These buffers will desirably be formed from components such as an acid and a basic salt of the same compound so that the overall chemical composition of the mixture remains constant during titration and no additional ionic species are introduced.
• This uniformity of chemical environment gives a measure of predictability to the behaviour of compounds introduced into the titration system, as the behaviour of some compounds can alter if the chemical environment changes significantly even, in rare cases, leading to the compound precipitating from solution as a solid salt forms.
• a test mixture stream is formed from three components: a constant volume of sample solution and two linearising buffer solutions the volumes of which vary in inverse proportion to one another.
• the absorbance is measured (at one or more wavelengths, at least one of which will be a wavelength at which there is an absorbance difference between the ionised and unionised forms of the compound) as the proportions of the buffers are varied to produce a linear pH gradient.
• the pKa of the test compound is the pH at the mid-point of the absorbance change. If the test compound has more than one ionisable group, more than one absorbance change may be observed. The mid-point of the second change then corresponds to the pKa of the second ionisable group.
• the present invention provides an analytical device comprising at least two input ports in fluid communication with a common channel, and a detection zone having an input in fluid communication with the common channel and an output, the device further comprising a spectrophotometric detector for monitoring fluid flowing through the detection zone and producing data relating to at (east one chemical or physical characteristic of a component of the fluid.
• Control means may be associated with the input ports for controlling the relative amounts of fluid introduced into the common channel through each port.
• the detector may be any suitable spectrophotometric (i.e. radiation-detecting) analytical detector e.g. an ultraviolet or visible range spectrophotometer, a fiuorimeter, a polahmeter, a colourimeter, or a light scattering, optical rotation or circular dichroism detector.
• the control means for controlling the relative amounts of fluid introduced into the common channel through each port may be e.g. a pump controller such as is commonly used with HPLC instruments.
• one or more of the input ports may have associated with it a syringe by which a fluid may be introduced through the port into the common channel, the plungers of the syringes being moved mechanically under the control of e.g. a computer.
• the skilled man will be able to envisage other means by which the input of fluids into the common channel may be controlled, such that the proportions of the fluids making up the test mixture and the rate of flow of the test mixture along the common channel through the detection zone may be controlled.
• the use of syringe pumps or pump mixers based on those employed in HPLC instruments, in combination with small-bore tubing and microanalytical detectors in-line, such as fixed geometry optics spectrophotometers means that very small volumes of test mixture may be used. Consequently, smaller quantities of test compound are needed than were required for traditional titration methods.
• Automatic syringes have, in particular, the advantages of low dead volume (avoiding the dead volume of a separate pump) and being easily programmable for automation.
• an HPLC mixer pump is connected to reservoirs of each fluid component of the test mixture.
• the mixer pump takes the fluid containing the test compound at a constant rate and mixes it with a first buffer solution pumped at an increasing rate and a second buffer solution pumped at a decreasing rate, so that the total volume and flow rate of the resulting mixture remains constant, but the relative amounts of each component of the flowing mixture change over time.
• the changing proportions of the two linearising buffer solutions in the mixture preferably result in changing the pH of the mixture as a whole and are desirably controlled to give a linear pH change over time.
• Such a system may be used to determine e.g. the pKa(s) of a test compound.
• an autosampler carousel contains reservoirs of a number of solubilised compounds to be tested, and a number of automatic syringes each contain a reservoir of one other fluid component of the test mixture, for example a first and a second buffer solution.
• the first buffer solution is then pumped at an increasing rate from a first automatic syringe to a mixing chamber and a second buffer solution is pumped to the mixing chamber at a decreasing rate, so that the total volume and flow rate of the resulting mixture remains constant, but the relative amounts of each component of the flowing mixed buffer stream change over time.
• the changing proportions of the two linearising buffer solutions in the mixture preferably result in changing the pH of the mixture as a whole and are desirably controlled to give a linear pH change over time.
• the autosampler takes a sample of fluid containing one of the test compounds and injects it, at a constant rate, into the mixed buffer stream to form the test mixture stream, which passes through the detector.
• Such a system may be used to determine e.g. the pKa(s) of a test compound.
• phase partition fluids which may be employed include oil-in-water emulsions or emulsions of other organic solvents in aqueous solvents (e.g. octanol in water), surfactant micelles (e.g. sodium dodecyl sulphate (SDS) micelles) and phospholipid, e.g.
• DMPC liposomes but the skilled man will be able to select an appropriate mixture to suit the test compound, from his own knowledge.
• a linear pH gradient test mixture stream may be formed as discussed above and brought into contact with a flowing organic phase (e.g. octanol) stream, for example using a microscale chemical processing device being developed by CRL (Central Research Laboratories Ltd., associated with EMI Group pic) and BNFL (British Nuclear Fuels Limited). This device is specifically designed to allow aqueous and organic phases to flow in contact with each other and then be clearly separated. Details may be found in "Eureka, Transfers Technology" October 1997, page 42. From the difference in the pKa of the test compound with and without contact with the organic phase, the partition coefficient may be calculated.
• the fluids whose proportions are to be varied may include one or both of the binding reagents themselves, and/or salt solutions or buffers for controlling the ionic strength and/or pH of the mixture.
• test solute may be introduced in the manner discussed above, as a constant proportion of the test mixture.
• pH being varied as a function of time by the mixing of e.g. two buffers
• the ligand of interest is titrated against water or a solvent of relevance in the presence of solute, thus giving a continuous, preferably linear, gradient of ligand concentration.
• An example of such a system is nickel(ll):Ethylenediamine.
• Possible interactions which could be studied using the techniques and apparatus of the present invention include those between enzymes and their substrates or cofactors, chelators and metal ions, receptors and their agonists or antagonists, antibodies and their antigens, or the strength of interaction in any form of complex or specific binding pair.
• the data produced could be analysed using traditional techniques. This approach could be advantageous over other approaches as no dilution factor need be corrected for.
• the automatic syringes, or reservoirs and mixer pump, discussed above may be replaced by other pumping systems which can handle very small volumes with high precision and accuracy.
• Other suitable pumping systems include peristaltic pumps (although these may lead to pulsing of the pumped mixture) and digital on-off valve pumps in microtubing.
• the apparatus may comprise two or more different sorts of pump. Where a mixer pump is not employed, desirably some other means of efficiently mixing the components of the test mixture stream will be used, for example a mixing coil, a mixer T-piece or a spin-mixer.
• the mid-point (inflexion point) of this curve can be determined by curve-fitting, or by taking the 1st derivative of the absorbance readings against pH, which gives a peak corresponding to the point of inflexion.
• Use of the first derivative plot allows pKas which lie close to the ends of the pH gradient to be determined, as the gradient need only run a short way past the inflexion point for the first derivative plot to peak and begin its down-turn.
• the inflexion point of the curve- fitted absorbance trace can only easily be determined if the lowest and highest absorbance levels can be seen on the trace, which requires a longer span of the pH gradient, as can be seen from figure 11.
• TFA target factor analysis
• PCA - refs D. Perez-Bend ito, Analyst, Vol. 115, 689-698 (1990) and E.R. Malinowski, Factor Analysis in Chemistry, 2nd Ed. 1991 , pub. Wiley, New York
• PCA - refs D. Perez-Bend ito, Analyst, Vol. 115, 689-698 (1990) and E.R. Malinowski, Factor Analysis in Chemistry, 2nd Ed. 1991 , pub. Wiley, New York
• the method of continuous titration permits the creation of a fast linear pH gradient over a wide pH range, with the use of appropriate buffers as described below. This in turn allows the speedy determination of pKa values.
• the apparatus will generally be fitted with a pH meter in addition to a spectrophotometric detector, so that absorbance can be determined over time or against pH.
• the pH electrode may not be able to respond quickly enough, giving erroneous readings. In this situation, the gradient can instead be calibrated using compounds of known pKa.
• the present invention is particularly advantageous in the analysis of poorly soluble compounds, as only very small concentrations and volumes of solutions are required i.e. 100s down to 10s of micrograms per millilitre, and 100s of microlitres, rather than 100s of millilitres for traditional methods. Also, the use of highly sensitive detectors combined with high data sampling rates allows the introduction of noise-reduction techniques and means that much less concentrated test solutions may be used. Furthermore, it is not necessary to know the concentration of the test compound, because the output can be presented graphically and the changes in e.g. absorbance are plotted rather than absolute values, graphical shape changes showing the changing ionisation state, phase or other changes in the test compound. It is sufficient that the concentration is such that the chromophore is detectable by the spectrophotometer.
• the present invention provides methods and apparatus which will assist in overcoming these problems.
• problems in relation to sample purity when compounds are synthesised as part of a library and the level of automation achievable by the use of continuous titration methodology as described herein may provide a means for overcoming those problems also.
• Fig. 1 is a diagrammatic representation of apparatus according to a first embodiment of the invention
• Fig. 2 is a diagrammatic representation of apparatus according to a second embodiment of the invention.
• Fig. 3 is a diagrammatic representation of the plumbing connections of the apparatus of Fig 2;
• Fig. 4 is a diagrammatic representation of the electrical connections of the apparatus of Fig 2;
• Fig. 5 is a diagrammatic representation of the electrical trigger events controlling the apparatus of Fig 2;
• Fig. 6 is a detailed diagram showing the connections within the terminal block of the apparatus of Fig 2;
• Fig. 7 is a diagrammatic representation of apparatus according to a third embodiment of the invention.
• Fig. 8 is a diagrammatic representation of apparatus according to a fourth embodiment of the invention
• Fig. 9 is a diagrammatic representation of the detector connections of the apparatus of Fig 8;
• Fig. 10 is a flow-chart depicting the control sequence for the autosampler in the apparatus of Fig. 8;
• Fig. 11 is a diagrammatic representation of the relationship between pKa, absorbance and 1st derivative of absorbance for a species having a single ionisable group in which the ionised and unionised forms have different absorbance profiles;
• Fig. 12 shows a standards (calibration) curve derived from titration data obtained in accordance with the invention for compounds of known pKa;
• Fig. 13 is a plot of pH against time for the linear gradient.
• Fig. 14 is an absorbance curve for 4-CN phenol run on the gradient of Fig. 13;
• Fig. 15 is a plot of the first derivative of the absorbance readings plotted in Fig.
• Fig. 16 is an absorbance curve for an endpoint titration (Example 4);
• Fig. 17 is a calibration curve for an endpoint titration (Example 4);
• Fig. 18 is a plot of pH against % acid for the linear gradient on which the standards (calibration) curve of fig 12 was produced;
• Fig. 19 is a calibration curve for an endpoint titration (Example 5);
• Fig. 20 is a calibration curve for a complexometric titration (Example 6);
• Figs. 21 to 24 show the use of curve fitting as a data processing method
• Fig. 25 is a plot of pKa as determined using the apparatus of Fig. 8 vs. Literature pKa values for 10 compounds;
• Fig. 26 is a plot of pKa as determined using the apparatus of Fig. 8 vs. Literature pKa values for a further 10 compounds;
• Fig. 27 is a plot of pKa as determined using the apparatus of Fig. 8 vs. Literature pKa values for 35 compounds. Examples
• the apparatus was assembled from equipment already available in the laboratory, and consisted of the following units:
• DAD diode array detector
• the system initially developed used four solutions mixed into a linear gradient
• Fig. 1 The sample at constant % volume was titrated with acid (as in Fig. 1) or base and the % volume of salt solution was decreased as the acid or base increased, to maintain ionic strength within acceptable limits.
• the system was buffered by a constant % volume of buffer solution. Refinement of the buffering system has allowed this to be reduced to three components; a sample solution, the amount of which is not varied over the time that the gradient is run, and two linearising buffer solutions, one acidic and one basic, which are varied linearly over time in inverse proportion to one another. See Fig. 2.
• the sample containing the test compound is drawn at a constant rate from the autosampler into channel A of the HP1050 pump.
• varying amounts of the other components are drawn into the pump.
• Universal buffer component B (basic component, see further below) is drawn into channel B from a reservoir.
• universal buffer compound A (acidic component) is drawn into channel C.
• One of the buffer components rises from zero or a low % volume of the test mixture at the start of the gradient to e.g. 80% or more of the mixture at the end.
• the other buffer falls from e.g. 80% or more of the mixture to zero or a low final concentration.
• test compound solution channel A
• other components e.g. water, surfactant micelles, reactant(s)
• the mixed components pass from the outlet of the HPLC pump to the Spectrophotometer (Kontron 440DAD) and then to waste, optionally via a pH meter which may be used to monitor the correct operation of the system, e.g. to check the linearity of the pH gradient formed.
• Kontron 440DAD Spectrophotometer
• pH meter which may be used to monitor the correct operation of the system, e.g. to check the linearity of the pH gradient formed.
• Tubing may suitably be 1/16" OD PEEK or stainless steel tubing.
• the apparatus can be set to run through a repeating cycle during which there may be four distinct phases: 1 ) The buffers and any other components are pumped through the HPLC pump at constant rates in fixed ratios to give a stable starting point for the gradient. 2) The gradient is run by varying the ratios of the buffer components. 3) The final conditions of the gradient may be maintained for a short period before 4) the system recycles (which may include flushing with water of other suitable solvent at the end of the cycle), in preparation for the drawing up of the next sample.
• analogue data from the spectrophotometer and pH meter are fed to the terminal block and thence to the PC for capture and analysis by Dasylab software. Any other software capable of capturing and manipulating analogue data would be suitable.
• This embodiment is limited to four analogue outputs from the spectrophotometer, the four data channels from the spectrophotometer are connected to shielded inputs 1 to 4 on the terminal block and the analogue signal from the pH meter is connected to terminal 5. Shielding of the cables reduces interference from high frequency instrumental noise.
• the connections within the terminal block are shown in more detail in figure 6.
• the autosampler is connected to the pump, spectrophotometer and terminal block. These contacts are digital signals which specify the start and finish of the experimental cycle, these contact closure events are driven by the autosampler.
• the signals to the pump and spectrophotometer are contact closures, the signal to the terminal block is a contact opening. This is shown in more detail in figure 5.
• Figure 7 shows a similar apparatus arrangement to that of figure 2, but the buffer components are introduced into the mixer from automatic syringes rather than being drawn up by the mixer pump from a reservoir. Any extra components such as micelle suspension for a partitioning experiment may also be introduced by syringe as may the test samples, if desired, although if multiple samples are to be tested the use of an autosampler instead of a pump or a syringe provides a convenient means of automation.
• Figures 8 and 9 show another apparatus embodiment which uses automatic syringes for delivery of the gradient-forming components.
• This apparatus uses the following units:
• Pulsed lamp power supply (Cathodeon - C720)
• the two flow streams from the syringe dispensers are mixed using a mixing T which has a total volume of 4 ⁇ l.
• the flow stream then passes through a coil to aid mixing and then on to the injection port, located on the autosampler.
• the autosampler can inject samples in to the flow stream at this point. From the injection site, the stream flows on to the spectrophotometer's remote flow cell and then out to waste.
• FIG 9 can be seen the electrical and fiberoptic connections associated with the detector used with the apparatus of Fig. 8.
• the deuterium lamp is controlled via a transistor-transistor logic (TTL) signal which in-turn controls the power circuitry in the power supply.
• TTL transistor-transistor logic
• the deuterium lamp should be warmed up before commencing experiments. This is typically for about half an hour.
• the TTL signal is controlled via the computer, allowing the lamp to be turned off and on automatically.
• a transmission fibre optic runs from the lamp to transmit the light from the lamp to the cell holder.
• the cell holder is used to position the flow cell in-line with the light path.
• the position of the receiving fibre can be adjusted within the cell holder, and then fixed in place using a locking screw.
• This fibre then connects to the MMS spectrophotometer.
• the MMS 12-bit adapter electronics perform the data capture from the spectrophotometer, under the control of signals from the CIO-DAS 16jr computer board.
• the main control program has been programmed as a LabView Virtual Instrument. It is used to control the main peripherals :
• the lamp for the spectrophotometer i.e. turn on/off.
• the gradient module (sends trigger signal, and configures)
• the autosampler (sends the correct control strings)
• the spectrophotometer (provides clock pulses and receives triggers)
• the main control program initially configures all the external peripherals and brings them into a ready state.
• the user can select a filename for where they want all the data to be saved from the run about to commence. Once this is completed, the instrument enters a holding state where the user can either run experiments individually totally under their control, or they can set a programmed number of samples to run continuously until completion.
• the Sirius Gradient System includes an 80C552 microprocessor based control board, two Sirius syringe dispensers, a Datavision LCD and a keypad.
• the LCD and keypad provides a simple user interface that allows a user to programme gradient control variables for the flow stream.
• the gradient system can also be controlled via an RS-232 interface.
• the gradient module has an embedded software program that allows the user to set up experimental parameters for generating the gradient.
• the set up parameters for the gradient control are :
• the gradient reset time in seconds (e.g. if the gradient goes from low to high, the gradient needs to be reset back to low before the next sample is injected).
• the post gradient time in seconds (this pushes the end of the gradient through to the flow cell).
• the gradient control protocol can be started using an external trigger signal, supplied automatically from the main control computer. When this signal is detected the gradient module begins its operational run, at the end of which it automatically reloads, then waits for the next trigger signal for the start of the next experiment.
• FIG. 10 shows a flow chart of the autosampler control.
• the arm then moves to the injection point.
• a trigger is sent to the gradient module to start the gradient flow and also to the deuterium lamp.
• data collection begins at 0.5 second intervals.
• the sample is injected into the gradient stream.
• the instrument actually captures six scans from the spectrophotometer, averages the last five scans and uses this average as the stored scan. This is done by calling a CIN (Code interface node) which liaises with the CIO-DAS 16/Jr board, collects the 256-absorbance spectra and returns this data array to the VI which saves it on disk in the specified file.
• CIN Code interface node
• the data capture routine has been simplified by implementing hardware to control and time the data acquisition of the signal from the diode array.
• the hardware is encompassed in the Zeiss electronics, along with the signal conditioning electronics.
• the data capture routine is required to send a trigger signal to indicate that a scan is required.
• the Zeiss electronics controls the diode array and the data capture board.
• the Zeiss electronics also conditions the signal from 0V to +2.5V, making full use of the resolution of the Analogue-to- digital converter (ADC).
• ADC Analogue-to- digital converter
• a dark scan (lamp off) is made, and this result is subtracted from all other scans (lamp on).
• the signal is sampled six times, the first scan is discarded and the remaining five scans are then averaged. This averaged scan is then saved to disk. This takes about
• the resultant data file contains 256 wavelengths of data for each sample.
• Diode array data is in terms of "energy counts" which need to be converted to Absorbance data using the equation:
• l(sample) is the intensity or energy count of the gradient with sample for a particular channel or wavelength
• l(dark) is the dark current
• l(ref) is the initial reference count (buffer B plus sample).
• the convert program allows the user to specify which wavelengths need to be extracted for use in the data processing algorithm, and formats the resultant data file.
• the 1st derivative program must be run first to calculate the pH gradient.
• the pH gradient is calculated by using data from compounds that have well defined pKas, and have thus been termed 'standards'. This then provides the pH scale required for the TFA algorithm.
• a blank sample (just water or appropriate solvent) and calibration standards must be run.
• the blank sample provides a blank profile, providing absorption information due to the gradient and the water. This must be subtracted from all the standard/sample runs. This then provides an absorption profile purely due to the standard/sample.
• the data processing algorithm uses a linear fit algorithm to smooth the data and then performs the derivative upon the slope of the linear fit. The user is able to specify the number of points over which the fit is applied (it must be an odd number of points). The data processing algorithm is applied to each point in the data file. Once this has been completed, peaks need to be found. This is done by dividing the data into cells (user specified size), and in each cell, searching for peaks that fit the criteria for a minimum or maximum peak.
• Figure 2 shows a diagrammatic representation of apparatus used to form a buffered linear pH gradient. The following pKa determination experiment was performed on this apparatus.
• a linear pH gradient was created by mixing a sample solution, the amount of which is not varied over the time that the gradient is run, and two buffer solutions, one acidic and one basic, which are varied linearly over time in inverse proportion to one another.
• the two buffers have a common component to which an acidic component is added to form buffer A and a basic component is added to form buffer B.
• the buffers were made up as follows: Solution C: Common Component (1 litre) Into 1 litre of water:
• Butylamine (FLUKA 19480) 29.256g (39.696 cm 3 ) 0.4M
• Figure 18 shows that the pH gradient is essentially linear from pH 3 to 11.
• Compounds of known pKa were run in a continuous (rather than stepped) gradient on the apparatus of Fig. 2, in which the amount of buffer A ran from 80% to 0% and of buffer B from 0% to 80% of the test mixture over 4 minutes.
• the sample solution was kept constant at 20%.
• the HP1050 pump was used again with buffer B introduced via channel B and buffer A via channel C.
• the flow rate of the test mixture stream from the mixer to the detector was 1 cm 3 mm
• Time to peak maximum is from the start of the instrument cycle (when the autosampler first goes into a new sample container).
• Figs. 13,14 and 15 show the calibration curve (Fig. 13) with the absorbance curve at 290nm (Fig. 14) and the 1st derivative plot (Fig. 15) for 4-CN phenol run on the above gradient.
• the pKa value corresponding to the time of the peak maximum (361 seconds) is read from the calibration curve for this pH gradient, the pKa derived is 7.64.
• the expected result is 7.7 (derived from traditional stepwise titration using the Sirius PCA101 instrument).
• the method could be further enhanced by incorporation of the calibration curve into the data handling routines, for example the computer which stores the absorbance readings generated by the detector may be programmed to find the first derivatives of these readings, determine the time of the peak reading and, for example using a look-up table derived from the calibration curve absorbance readings, produce an output reading giving the pKa of the sample.
• the pKa reading would then be the only output - no calculations would be required on the part of the operator.
• the buffer recipes have been further optimised to improve gradient linearity while maintaining physiological ionic strength without significantly reducing the buffer capacity.
• the Components were also chosen with minimal UV/visible absorption characterisfics. Recipes are shown below and compared to the recipe of Example 2A above:
• Component A (acidic buffer):
• Component B (basic buffer):
• buffer component B is dispensed at a flow-rate of 1 ml/min and sample injected downstream (from the CAVRO autosampler) at a flow-rate of 0.25ml/min to produce a total flow of 1.25ml/min. Before each experiment a dark spectrum (lamp off) is recorded. After the flow has reached the Hellma flow- cell (model 178.713, path length 10mm, volume 8 ⁇ L) the deuterium lamp (Cathodeon) is switched on from the Cathodeon C720 deuterium pulsed lamp power supply and a reference energy spectrum recorded with sample and buffer B present.
• the deuterium lamp Cathodeon
• the gradient is started and run over a time period of 240 seconds during which the buffer components are varied linearly over time in inverse proportion to one another, starting with component B (basic buffer) and changing to component A (acidic buffer) at the end of the 240s time period.
• the total gradient flow-rate is maintained at 1 ml/min.
• buffer A and sample are allowed to run through for a short period of time to push the end of the gradient through the flow-cell before switching back to buffer B, to restore the initial conditions ready for the next sample.
• spectra are recorded at 0.5 second intervals for the duration of the gradient using a Zeiss 256 wavelength photodiode array and 12-bit data capture electronics. Each spectrum consists of the average of five scans using an integration time of 50 milliseconds and records the energy count per diode channel minus the dark current.
• Sample Preparation Typically, a 1-1 Omg sample is weighed into a vial to which 1 ml methanol is dispensed, to aid sample dissolution, followed by 10ml de-ionised water (>10 14 M ⁇ ). The solutions are drawn into 5ml disposable syringes and dispensed directly into test tubes through disposable nylon filters to remove undissolved solid. The test-tubes are transferred directly to the Cavro autosampler unit for sample analysis. The sample flow makes up 20% of the total flow so that typical sample concentrations at the flow-cell detector are 10 "3 - 10 "5 M.
• the first stage is to establish the pH scale using the peak maximum time in the first derivative of the absorbance curve.
• Several wavelengths are used (benzoic acid - 235nm; KHP - 278nm; p-nitrophenol - 321 nm; phenol - 235nm) and the energy spectra converted to absorbance:
• A Iog10( (I (ref) - 1 (dark)) / (I (sample) - 1 (dark)) ) where: l(sample) is the intensity or energy count of the gradient with sample for a particular channel or wavelength; l(dark) is the dark current; and l(ref) is the initial reference count (buffer B plus sample).
• Blanks are processed first and subtracted from all sample and standard spectra. Peak times of the standards are plotted against the known pKa values and the calibration history saved to file. For a given set of experimental conditions the calibration regression equafion has been shown to be remarkably consistent for periods of weeks reducing the necessity of more than daily calibration.
• sample data can be processed. Typically, up to twenty evenly spaced wavelengths are selected for sample analysis ranging from 210 - 350nm and energy counts converted to absorbance as above. Internal referencing can also be applied by selecting a non-absorbing region of the spectrum (usually 420-440nm) and establishing a baseline from a Blank to correct for any drift.
• Target Factor Analysis is applied to determine the pKa values of samples.
• the first derivative method can also be applied to samples with non-overlapping pKa values.
• the continuous titration method has proven an accurate method for determination of pKas across a wide pH range and for compounds with multiple pKas which have been difficult to determine by traditional methods.
• This behaviour can be studied using the continuous titration method and apparatus described above by including a fourth component in the gradient mixture.
• This component comprises micelles formed from surfactants such as sodium dodecyl sulphate (SDS).
• SDS sodium dodecyl sulphate
• concentration of surfactant in the fourth component must be high enough that in the final test stream, mixed from the four components, the surfactant is present in excess of its critical micelle concentration (CMC) and micelles are formed.
• CMC critical micelle concentration
• the partitioning coefficient can be determined by the following equation:
• Vo can be calculated from the CMC, micelle radius and the aggregation number of the surfactant (number of molecules required for each micelle), factors which would be readily available to or calculable by the skilled man.
• the continuous titration apparatus was set up with the following sample queue:
• Vo is taken as the volume of SDS micelles.
• ionic strength the ionic strength of the buffer stream
• KHP potassium hydrogen phthalate
• test solute KHP
• the test solute KHP was introduced in the sample stream of the apparatus of Fig. 2 at a constant 20% of the final mixture, as in Example 2 above.
• the two components of the gradient (from 80% to zero and zero to 80% respectively) are 0.05M KOH and water.
• the end-point indicator was introduced via the sample stream (2 drops phenolphthalein solution in 20cm 3 of sample).
• test solute KHP was titrated by the KOH stream. As soon as all compound has been titrated there is a large increase in pH and rapid change of the ionisation state of the indicator and hence a rapid colour change.
• the system was calibrated by the use of KHP standards of known concentration.
• Example traces obtained are shown in Figure 16.
• Phenolphthalein indicator 0.33g phenolphthalein dissolved in 10cm MeOH and 10cm 3 H 2 O. Excess phenolphthalein precipitates out with time.
• the diode away detector was set up to detect at four wavelengths: 540nm, 550nm, 560nm and 570nm.
• Solvent A H 2 O Solvent B: 0.05N KOH
• the solvents were dispensed from 5cm 3 syringes.
• the flow rate through the flow cell was 0.8cm 3 min "1 .
• Gradient time was 240s with a pre-gradient flow of 75s and post-gradient flow of 90s followed by a post-gradient restoration period of 45s, giving a 7.5 minute cycle time overall.
• the autosampler was set up with a dispensing rate of 0.2cm 3 min "1 and a 7.5 minute cycle time. Blanks, standards and samples were run in the following order:
• the data was captured using Dasylab and analysed using the first derivative method following 3 data point smoothing.
• the data was analysed at 540nm.
• the standard curve was plotted (see Fig. 19) and can be represented by the equafion
• Zinc ions form a complex with Xylenol orange to give an intense red colour absorbing at 580nm.
• EDTA ethylenediaminetetracetic acid
• the Zn ++ preferentially forms a complex with the EDTA. Once all the Zn ++ has formed the EDTA-Zn ++ complex, then the Xylenol orange is once again in the free form, which is yellow in appearance and so a corresponding decrease in absorbance at 580nm is observed.
• Zinc standards Zinc nitrate was chosen as the salt for formation of the standards.
• the concentration of the zinc in the test mixture stream must be less than the maximum concentration of the EDTA, to ensure that all zinc is complexed by the EDTA, leaving the indicator in the free, uncomplexed form.
• the Zn ++ concentrafion in the measurement stream must be less than 14.976mM.
• 1.03g of Zn(NO 3 ) 2 .6H 2 O was weighed out and dissolved in 50cm 0.1 N acetate buffer (pH 4.9) to give a zinc stock solufion of concentrafion 69.25mM. From this, standards were prepared:
• the autosampler was set up with the 7.5 minute cycle time and a 0.2mM min "1 dispensing rate and the standards were run against the EDTA gradient to establish a calibration curve (see Fig. 20).
• the peak times in the first derivative of the absorbance data at 570nm were as follows:
• the first derivative curve could not be fitted to the absorbance data involving high concentration standards. This is thought to be due to insufficient buffering of the standard solutions, which therefore did not give a clear colour change in the xylenol orange indicator.
• a linear calibration curve was obtained (Fig. 20). This experiment shows clearly that the continuous titration method can be used for complexometric titrations. In the example shown here buffering capacity was insufficient, but this could be improved by using buffer for making up the EDTA solution.
• the third method of analysis which can be applied to the data generated in the continuous titration spectroscopic methods of the present invenfion is "curve fitting".
• This method of analysis can be used with much smaller spectral changes, is fairly insensitive to non-linearity in the pH gradient, requires data from only a single wavelength, does not require data smoothing and is not very computationally intensive.
• absorbance data from the titration of a sample compound S obtained using a linear pH gradient formed as in Example 2 was normalised as described below. Data from four wavelengths was used. The minimum and maximum absorbance for each wavelength was determined and the data scaled between zero and one, using the equafion:
• Abs new Abs o s - Abs min (Abs ⁇ - Abs ⁇ )
• the spectral change can be defined as a logistic function:
• A, B and D are constants and x is the dependant variable.
• A, B, and D are found by trial and error fitting, and x is time. This function was fitted to the spectral data using the "solver" function in Microsoft ExcelTM, by minimising the residual sums of squares and fitting A, B and D.
• the first derivative of the logistic is defined as:
• the partition coefficient (log P) can be found from the following equafion:
• log P is approximately equivalent to the difference between pKa and pKa'.
• the confinuous titration apparatus may be used for determining log P values by carrying out two titrations, the first a standard continuous titration as in Example 1 and the second with the addition of an octanol or other organic phase stream in contact with the aqueous sample stream. Given an adequate contact area between the aqueous and organic streams, partitioning will occur between the two phases. The aqueous and organic phases are then separated and the aqueous stream passed through the detector.
• microscale chemical processing device being developed by CRL and BNFL. This device is specifically designed to allow aqueous and organic phases to flow in contact with each other and then be clearly separated. Details may be found in "Eureka, Transfers Technology" October 1997, page 42. If equal flow rates are used, this will be equivalent to equal volume ratios in a traditional partitioning experiment.
• the spectral data which would be expected from such an experiment can be modelled as follows:
• pKa pKa of molecule.
• a 0 absorbance of the unionised species.

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