Flow Injection Analysis: An Overview

By Vasco Medeiros, BScReviewed by Emily Henderson, B.Sc.

Developed in the early 70s, flow injection analysis (FIA) automates many processes associated with wet chemistry. The instrumentation garners all reagents and mixes them while plotting the reaction rate over time. It does this by recording the change in the hue of the solution, which is proportional to the varying concentrations of reagents in said solution. Common analytes that are assayed in this methodology are chloride, nitrate, phosphate, and more. This technique measures the concentration of a given sample or rates of reaction through spectroscopic means.  

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How the Apparatus Works

The significant components of any FIA hull are the reagent manifolds, the peristaltic pump, the sample loop, the mixing coil/chamber, the detector, and a waste collection box. Some auxiliary features may be present in some FIA machinery, including a filter, a heater for color development, etc.

Firstly, the sample is drawn from the autosampler to the sample loop. In tandem, the peristaltic pump draws in the carrier and reagent solutions from their respective manifolds. As this is being accomplished, the software is running to acquire a real-time data view. The lab-on-valve- (LOV) will inject the sample solution from the loop into the carrier system, and the resulting sample & reagent mixture will travel to the mixing coils. Once the carrier and first reagent become homogenous, the mixture will travel back into the LOV, mixing with reagent two and flowing through a secondary mixing coil and flow cell. Only here will the color difference become appreciable, and the sample intensity can finally be measured. This is traditionally done using a spectrophotometer coupled with a photomultiplier tube. After this analysis, the resultant liquid will flow through to waste collection. 

It is important to denote that for any given reaction/analyte that one wishes to observe, appropriate standards must also be injected so that a calibration/standard curve can be plotted. These standard solutions are also loaded within the autosampler. Only after we compare these light-dependent signals with our standards can we fully grasp the integrity of the data. The software is put in place to help measure the signal of light bounding of the sample within the flow cell.

Using FIA To Determine the Amount of Phosphate in Soil Samples

A standard example of FIA taught in many labs and universities is the correlation between a molybdate coloring reagent and the concentration of an analyte such as phosphate. For this specific methodology, a reducing solution of ascorbic acid is needed, as is the target sample, the coloring agent, a carrier sample, and a dilutant such as Bray No. 1 solution. Bray solutions are designed to extract adsorbed forms of phosphate and are used to assay soils with a more basic pH.

Once the standards have been prepared, a curve can be plotted to establish the correlation between color and phosphate concentration. These standards are variable, though most laboratories have the highest data point at around 20ppm of phosphorus, while the lowest data point should hover around 5ppm of phosphorus. A blank should also be employed, consisting solely of the matrix solution.

When fashioning the matrix solution, it is important to use deionized water rather than distilled water so that no contaminants or free ions (phosphorus, for example) can compromise the results. These standards are placed into the autosampler and compared to the sample.

Applications In Enzyme kinetics

FIA practices determine the activity of a given reaction based on the spectroscopic properties of said reaction. One practical application of this can be found when assaying the kinetic property of certain enzymes, such as malate dehydrogenase (MDH). By introducing the catalytic moieties present in this enzyme to oxaloacetate and α-ketoglutarate, the absorbance (substrate change per second) can be determined, equating to the turnover rate of the enzyme. This process is accomplished using the FIA method previously described, incorporating a potassium phosphate buffer and varying concentrations of OAA and α-ketoglutarate from their respective manifolds.

In this case (Medeiros V et al. (2021)) induced an R81H neutral substitution mutation on the Rossmann fold of human MDH2 to examine the difference in catalytic turnover (k_cat) and substrate affinity when compared to the wild type. They found that introducing the imidazole group of histidine in the R81H mutation shows an appreciable difference in turnover (kcat =0.01 ± 2 *10-3 s-1) compared to the wild-type turnover (k_cat= 20 ± 39 s-1). The turnover rate of OAA at physiological levels is 48000 s^-1; therefore, this mutant would not function at normal metabolic levels.

Based on findings in (Babker A et al. (2019)), the appreciable increase in α-ketoglutarate binding specificity compared to OAA equates to a lesser affinity for malate. By downregulating this mechanism, less NAD and lactate will propagate the proliferation of glioblastoma cells, which will, in turn, suppress tumorigenesis and metastasis in certain forms of cancer. Suppose one were to generate a successful mutant and monitor an appropriate enzymatic turnover rate using FIA. In that case, this mutation could be introduced into plasmids such as pET28a and issued as a therapeutic.

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