Colorimetry Overview

The basic principle of continuous flow colorimetry is to eliminate chemical analysis by hand-mixing of reagents in individual items of glassware and to substitute a continuously flowing stream of liquid reagents circulating through a closed system of tubing. Reference and sample solutions are introduced by turns into this flowing stream of color-forming reagents.

The stream is segmented into small discrete liquid increments (slugs) by providing frequent bubbles of air or other gas that entirely fill the stream tubing bore. While moving through the system, each of these liquid increments is subjected to carefully controlled conditions specific to a particular assay, such as reagent concentration, incubation temperature, reaction time, and so on, to generate a color reaction.

This color reaction conforms to Beer’s law (the light absorbance of a solute at a particular wavelength is a function of its concentration in the solution, so that absorbance measurements can be used to measure concentration.) At the end of its flow path each reacted segment passes through a light absorbance cell where its concentration is read by a colorimeter. The recorded result for each sample and standard thus does not represent a single reacted aliquot but is the sum of the measurements for a large number of liquid-increment subsamples.

The color reactions on which the process is based are, with only minor modifications, the same as the ones that have long been accepted for manual colorimetric assays (e.g., Murphy/Riley 1962 for ortho-phosphate.) Automated methods cannot be any more accurate than the manual methods on which they are based, but they are less subject to variability since they eliminate the errors of consistent practice involved in analyzing large batches of samples by hand.

Segmented vs. laminar flow

The principal problem of continuous-flow methods is obtaining sufficient mixing of liquids within the tubing system. Aerial photographs show strikingly that when two rivers whose waters are different colors join, the waters derived from each tributary can maintain their separate identity for miles downstream with very little mixing. The following illustration shows a similar mixing problem when two reagent streams A and B are brought together at low velocity at a tubing junction:


A second mixing difficulty can be caused by laminar flow of liquid within the tubing. As shown schematically in the next illustration, the effect of wall friction and fluid viscosity on flow rate at different distances from the center of a tube is to produce a condition in which the flow rate is faster in the center and slower at the outer edge of the tube. If part of the fluid races ahead and part lags behind, this can lead to contamination from one sample to the next.


One solution is to increase fluid velocity in the system. Complete mixing can result if the two fluid streams are brought together at high velocity, giving sufficient friction at the wall of the tubing to produce turbulent flow. One may increase fluid velocity by a) increasing reagent and sample flow rate or b) decreasing tube bore diameter. The former leads to greater use of reagents and may be unusable because there is not enough sample. The latter increases the likelihood of obstruction in the tubing by precipitates or by particulate matter from the sample.

A different approach is to keep the flow rate relatively low and the tube bore diameter safely larger than any particulate obstructions that are likely to occur, and to introduce bore-filling gas bubbles into the stream. As shown below, each small aliquot of liquid between two bubbles is well mixed by turbulence due to wall friction, and laminar flow and cross-contamination between samples is prevented by complete separation between each pair of liquid slugs. The bubbles continually clean the system by wiping the walls of the tubing and driving forward any stationary liquid film that might contaminate following samples.


Standard materials and calibrants
Calibration of continuous-flow apparatus is achieved by including standard solutions of known analyte concentration in each batch of samples analysed. A sequence of four or five such standards at the beginning of a run gives absorbance data for these known concentrations from which one can calculate a regression curve to generate concentration values from sample absorbances that fall between the standard values. Within-run drift correction is achieved by including check and recalibrant standards every ten or twenty samples.

For typical analyses performed in this lab (for nitrate, ammonium, ortho-phosphate, total nitrogen and total phosphorus) 1000ppm standard stocks are made from appropriate dry reagents (KNO3, NaNO2, (NH4)2SO4, and KH2PO4.) Working standards made from these stocks are checked by analyzing Environmental Protection Agency certified Nutrient-1 quality control solutions of known analyte values. Additionally, digests for total nitrogen and total phosphorus are checked by digesting and analyzing E.P.A. Nutrient-2 QC solutions formulated to challenge digestion techniques.

Sample preparation and handling

Samples must be free of turbidity and particulate matter. Any such substances must be removed before analysis by filtering or centrifugation.

Strongly colored samples may contribute confounding absorbance at the analytical wavelength.

Water samples which cannot be analyzed immediately after collection must be preserved for shipment. The E.P.A. publication Methods for Chemical Analysis of Water and Wastes lists acceptable preservation methods and holding times for many analytes; this list is available for reference here:

* [E.P.A. sample preservation guidelines] – NEED TO ADD

Detailed sample-handling instructions specific to this laboratory are available in the document

* [Sample handling and preservation] – NEED TO ADD

Coakley, W. A. 1981.
Handbook of Automated Analysis: Continuous Flow Techniques. Marcel Dekker, Inc., New York.

Furman, W. B. 1976.
Continuous Flow Analysis: Theory and Practice. Marcel Dekker, Inc., New York.

Murphy, J. and J. Riley. 1962.
A modified single solution for the determination of phosphate in natural waters. Anal. Chim. Acta v.27 p.31-36.

U. S. Environmental Protection Agency. 1983.
Nitrogen, Nitrate-Nitrite. Method 353.2 (Colorimetric, Automated, Cadmium Reduction). pp.353-2.1 — 353-2.5. In Methods for Chemical Analysis of Water and Wastes, EPA-600/ 4-79-020. U.S.E.P.A., Cincinnati, Ohio, USA.

U. S. Environmental Protection Agency. 1983.
Nitrogen, Ammonia. Method 350.1 (Colorimetric, Automated Phenate). pp.350-1.1 — 350-1.4. In Methods for Chemical Analysis of Water and Wastes, EPA-600/ 4-79-020. U.S.E.P.A., Cincinnati, Ohio, USA.

U. S. Environmental Protection Agency. 1983.
Phosphorus, All Forms. Method 365.1 (Colorimetric, Automated, Ascorbic Acid). pp.365-1.1 — 365-1.7. In Methods for Chemical Analysis of Water and Wastes, EPA-600/ 4-79-020. U.S.E.P.A., Cincinnati, Ohio, USA.

U. S. Environmental Protection Agency. 1983.
Sample Preservation. pp. xv-xx. In Methods for Chemical Analysis of Water and Wastes, EPA-600/4-79-020. U.S.E.P.A., Cincinnati, Ohio, USA.