In this reaction, we are going to be monitoring the bleaching of New Fuchsin dye in a series of sodium hydroxide solutions. Therefore, we will be monitoring the loss of the reactant as the reaction proceeds. The reaction will be taking place under pseudo-first order reaction conditions, where one of the reagents (NaOH) needs to be in large excess compared to the other reagent. As we want to monitor the loss of dye by UV-Vis absorption, this predetermines the dye concentration range suitable for the limits of detection of the UV-Vis spectrometers, and it is usually easiest to make this the limiting reagent, with the non-observed species being in excess.

Once we have identified what we intend to measure, we will need to find a suitable wavelength to use to record the change in concentration during the reaction. We will also need to determine a suitable starting concentration. Ideally, this would use as much of the working range of the instruments as possible. I.e. we want to start with a concentration of New Fuchsin which is close to the upper limit of detection for the UV-Vis spectrometer. If we were monitoring the formation of the product, we would determine a suitable concentration to reach when the reaction had gone to completion.

Determining these parameters will require some trial and error, then iterating to find a suitable concentration. Initially, aim to record a scanning spectrum of a solution of the dye where the absorbance for the λmax is close to the upper limit of detection, i.e. an absorbance between 1.0 and 1.5. We are monitoring a coloured species, with a visible absorbance, consider what would be appropriate scanning ranges for this spectrum.

A starting point is to record the supplied dye at its provided concentration. Whilst this will be off-scale, the initial scanning spectrum will allow you to start to see where the λmax will be, and will help to direct the level of dilution required.

Once you have a good scanning spectrum for the dye at its maximum concentration, you should perform a series of dilutions within the linear range of the instrument (0.1 to 1.5 abs) and determine the ε value.

With a sensible initial concentration for New Fuchsin determined, we need to decide what quantities of the two reactants will be combined. This could be equal quantities, or different quantities of one versus the other. The cuvettes being used can take a volume between 2.5 mL and 4 mL. Mixing is also a consideration, as the reaction starts as soon as any of the starting reagents are combined. Consider the equipment provided and how this can be achieved. E.g. using a micropipette to add the first reagent into the cuvette in two portions is not a problem, as the reaction will not have begun. However, if you have to add the second reagent in two portions (and potentially different volumes), this may be problematic as this will delay being able to record the reaction in the photometer.

With both the initial concentration, and the reagent volumes having been determined, it is now possible to prepare a stock solution of the dye which can be used in all of the kinetic measurements. This stock solution will have a higher concentration than the initial starting concentration which had been determined, as the reagent mixing in the cuvette results in a dilution.

Once you have prepared this stock dye, it is sensible to take the intended dye volume in a cuvette and make up to the total reaction volume with water (ie add the same volume of water as there would be for sodium hydroxide in a rection run) and re-record the UV-Vis spectrum to check the initial starting dye concentration is as intended.

It is necessary to determine a suitable run-time (timebase) over which each kinetic measurement can be recorded, along with a suitable sampling rate. These factors depend in part on the choice of technique (UV-Vis) and instrumentation used. The run-time also needs to be realistic to achieve good data recording, considering the time taken to mix the reagents and get the cuvette into the photometer. However, we also don’t want the reaction time to be so long that the measurements take a huge amount of instrument time. The chosen timebase then affects the available range of concentrations of the reagent in excess - the shorter this timebase becomes, the more limiting range of suitable values.

Kinetic measurements need to include sufficient datapoints, i.e. the instrument has a sampling rate which is fast enough for the duration of the measurement. The photometers being used here have a maximum sampling rate of 1 Hz. A suitable sampling rate needs to be chosen in conjunction with the timebase to give a sensible number of datapoints over the course of the reaction. For instance, a 30 second timebase with a 1 Hz sampling rate would only give 30 datapoints, which is rather insufficient for analysis. The photometers here aren’t limited by the maximum number of datapoints they can record, but this can be an issue with some instruments. E.g. stopped flow apparatus often has a fixed number of datapoints that can be stored in the buffer, so if the timebase is lengthened, the sampling rate would need to be reduced. In our case, there is no limit to the number of datapoints, so even for a longer timebase it is fine to run with a maximum sampling rate.

With New Fuchsin being the limiting reagent, for pseudo-first order reaction conditions, we know that the sodium hydroxide solutions will need to be present in large excess (at least several orders of magnitude). This reagent needs to be recorded using a variety of concentrations, so that we can later determine the second-order k value from the measured kobs values. Ideally, a set of regularly spaced concentrations is desirable, and there is benefit from this concentration range being as wide as possible, but without causing an excessively long timebase to be used. Selecting concentrations will involve some trial and error, then choosing whether the next run should be a higher or lower concentration. The plots below give an idea of reaction profiles based on a 200 second timebase.

Following the concentration determination step above, a full kinetic series can be recorded. You will need to have sufficient independent datapoints (kobs vs hydroxide concentration) to enable an accurate determination of the second-order rate constant, k. As an absolute minimum, your graph should consist of 6 different concentrations, with at least 10 datapoints. Datapoints should be fully independent to be included in your analysis; see the note below on replicate runs.

Replicate runs

In order to record an independent replicate set of data, it is necessary to ensure that any random errors are truly random in the measurement. To record a true replica datapoint at the same concentration as another datapoint, this would necessitate the preparation of a new hydroxide solution. The use of a solution which had been used for the same measurement would only be a replica measurement in respect to cuvette volume measurements, instrumentation issues etc, but would not help with any errors in the preparation of the solution. This is not to say that recording multiple runs from the same solution isn’t useful, as they can help with some errors, particularly in data collection, but they are not true replicas.

Running repeats from the same solution is entirely sensible if you think a data collection might have been affected by other factors, such as delays in starting the run, volume measurement issues, poor mixing, air bubbles etc.

It is worth noting that if you are thinking about running true repeats, it may be more desirable to make up another different concentration, rather than preparing a true replica of an existing measurement.

Systematic errors

It is worth noting that there are some systematic errors potentially present in this kinetics practical. The most obvious potential error sources are that all the solutions are determined from the same stock solutions, these being the stock dye solution and the stock sodium hydroxide. In the case of the dye solution, this will actually be changing in the course of the reaction, and it is this concentration we are determining using UV-Vis in the course of the reaction. The kinetics of the reaction do not depend on this solution being of a known concentration and can be eliminated as a source of error. The sodium hydroxide solution is important, and the stock solution provided is obtained commercially with a batch certificate indicating the solution has been tested (via titration) and falls within the specified concentration range.

Absorbance values with compact photometers

You may notice during kinetic runs that the absorbance values from the compact photometers differ from those obtained for the solutions when recording the scanning spectrum. There is a number of reasons why this can occur, but they are unimportant for the kinetic data we are recording, as we are measuring the change in values using the photometers, not the absolute value for a given solution.