Instrumental Analysis

Lab. 3211

Sequential Injection Analysis

Determination of Phosphate Ion

TA: Timo Kikas

Spring 2000, Section III

Sequential Injection Analysis - Introduction

Sequential injection analysis is a variation of the technique of flow injection analysis.
These are flow-based technologies that bring speed, automation of solution handling,
miniaturization, reagent economy, minimized waste generate, and low cost to the
analytical laboratory. For a detailed description of the fundamentals of FIA, refer to the
book, Flow Injection Analysis, 2nd edition, by J. Ruzicka and E. H. Hansen, Chemical
Analysis Series, Vol. 62, Wiley-Interscience, New York, 1988.

Figure 1. Main steps of Sequential Injection Analysis

In conventional flow injection analysis, the sample zone is injected into a flowing carrier
stream, and auxiliary reagents are merged with it on the way to the detector, but
sequential injection is based on a different approach (Figure 1). Using a multiport
selection (rather than injection) valve, a wash solution, sample zone and reagent zone(s)
are sequentially injected (drawn) into a holding coil connected to the common port (CP).
In this way, a stack of well-defined zones is obtained (Figure 2), which is then propelled
through a reactor into a detector. This flow reversal creates a composite zone in which
sample and reagent zones penetrate each other, due to combined axial and radial
dispersion. For a kinetic based assay, a selected section of the interdispersed reagent and
sample zone may be arrested in the observation field of the detector by stopping the flow.
The reaction rate during the stopped flow period can be monitored, and the changing

signal reflects the rate of the chemical reaction. The benefit of the stopped flow
technique is that it extends the reaction time, saves reagents, and eliminates interference
from background signals and lag phases.

Figure 2. Experimental setup

The measuring cycle of the SIA technique consists of the following operations: (1)
pumping of carrier/wash solution with the selector valve in position 8, to fill the holding
coil and reactor coil lines and detector; (2) aspiration of a few microliters of sample
solution with the selector valve in position 2, while the pump makes a small reverse step;
(3) aspiration of reagent solution with the valve in position 3, using a small pump reverse
step; (4) a forward pump move with the valve in position 8, with the pumping time
adjusted so that a preselected section of the interdispersed sample/reagent zone is injected
through the reactor into the detector; (5) a stopped flow period for reaction rate
monitoring (Figure 3.); and (6) a forward pump step which expels all sample and reagent
as well as the majority of the carrier/wash solutions from the valve and from the flow
channel. When the chemical reaction is fast and when peak height is measured rather
than reaction rates, step 5 is omitted. If two reagents are used, the optional port 4 and an
appropriate flow programming are used to introduce the second reagent. The total
volume of the dispersing step(s) should be at least 4 times the volume of the reactor coil,

detector and aspirated sample and reagent volumes (aspirated + RC + D) to assure

complete washing of the system at the end.

Figure 3. Stopped flow timing in one reagent experiment.

In order to avoid carryover when changing from one sample solution to another, the
auxiliary waste line (position 1) is used to discard the sample solution trapped in the
sampling line by aspirating into the holding coil and then pumping to the auxiliary waste.
The sampling line should be kept short, with an internal diameter of 0.5 mm.
The precise and reproducible choreography of zone sampling, sequencing, merging, and
data collection can be performed only under computer control, using Alitea Instruments
USA custom designed FIAlab for Windows software, which is compatible with both SIA
and FIA modes. For details of operation and computer configuration, consult the FIAlab
for Windows Software Users Guide, provide on the CD that accompanies the instrument.
The SIA technique is a versatile and powerful flow method that allows precise
measurements using only a single line system. Since the flow is not continuous (it only
occurs during the sequencing and measurement steps), reagent consumption and waste
disposal are minimized. In addition to single- and two-reagent chemistries, it is possible
to carry out multireagent chemistries, chemical separations, preconcentration, matrix
removal and multidetector-based assays with SIA.

Experiment 3: SIA Determination of Phosphate Using Two Reagent Solutions

Principle:
The analytical procedure is based upon the following reactions:
7H3PO4 + 12(NH4)6Mo7O24 + 51H+ 7(NH4)4PO4 12MoO3 + 51NH4+ + 36H2O
Mo(VI) - YELLOW COMPLEX + ascorbic acid Mo(V) - BLUE COMPLEX
The carrier is distilled water. Reagent 1 is a 0.0025 molar solution of heptamolybdate.
Reagent 2 is 5% ascorbic acid (w/v) in a 10% aqueous solution of glycerin. The glycerin
aids in preventing the colored complex from adhering to the walls of the flow-through
cells upon injection of the sample into the carrier stream. The sample is sandwiched
between the two reagent solutions and merges with the reagents on the way to the
detector. The phosphate in the sample combines with the heptamolybdate, forming a
yellow colored complex. This yellow complex then reacts with the ascorbic acid, which
reduces the molybdenum from the +6 state to the +5 state, there by forming a blue
colored complex, which has an extremely high absorptivity and is measured
spectrophotometrically at 660 nm. The height of the recorded peak is proportion al to the
concentration of phosphate. The calibration curve linearity and slope depend upon the
extent of reaction, i.e., how much of the blue complex has been formed. This is a
function of the kinetic nature of the SIA procedure, in that the reaction may not reach its
steady state value, but rather some fraction of the steady state value, since it is a slow
reaction. The extent of reaction is dependent on the particular reaction system. Addition
of antimony(III) catalyzes the reduction by ascorbic acid.
Solutions and Chemicals Required:
Reagent 1. In a 100 mL volumetric flask, place approximately 50 mL of distilled water
and carefully add 1.3 mL of concentrated nitric acid. To this add 0.309 g of ammonium
heptamolybdate, (NH4)6Mo7O24H2O, and dilute to the mark with distilled water. This
results in a solution, which is 0.2 M in nitric acid and 0.0025 M in heptamolybdate.
Reagent 2. In a 100 mL volumetric flask, place 5 g of ascorbic acid, approximately 50
mL of distilled water, and 10 mL of glycerin. Mix thoroughly prior to diluting to the
mark with distilled water. This results in a solution, which is 5% (w/v) in ascorbic acid.
Standard solutions. Standard solutions of phosphate (as P) in the range of 5-50 ppm P
are made by suitable dilution of a stock solution containing 1000 ppm P (0.440 g of
anhydrous KH2PO4 per liter).
Assembly and Initialization of the System
Turn on the detector and allow it to warm up for several minutes to stabilize. Set the
monochromator at 660 nm.
The aspiration lines are 0.5 mm i.d. tubing (2 L/cm). The end of the pump tube is
immersed in distilled water. The holding coil is connected to the common port of the
valve, the reaction line/detector to port 8, reagent 1. to port 2, reagent 2. to port 3, sample

line to port 4 and auxiliary waste line to port 1 (home position). The sample line should
be as short as possible to minimize the volume of sample required to flush it.
To initialize the system fill all lines with the appropriate reagents. The holding coil and
reactor coil lines are first filled with carrier, followed by filling the individual aspiration
lines at ports 2, 3, and 4, and then flushing the excess solutions from the holding coil to
the auxiliary waste. Use MachineInit.fia program.
In programming the system for measurements, it is necessary to first flush the sample
aspiration line with fresh sample by aspirating sample equal to at least four volumes of
the aspiration line into the holding coil. You can estimate the aspiration line volume from
its dimensions (2 L/cm). Then carrier is pumped to discharge the excess sample in the
holding coil to waste. The system is then ready to sample and measure.
Calibration
Make triplicate measurements on each standard solution by inserting the sample
aspiration line in the appropriate standard solution and running the program
OnePeakPO4.fia". From the measurements, determine the appropriate calibration range.
Prepare a calibration curve from the average of each reading.
Soil Sample Handling
Firstly five grams of the soil sample is weighed. The sample is then put into the oven at
100C and dried for one hour. Sample is cooled down and weighed again. The water
content of the soil is calculated in milligrams per a kilogram of dry soil. Three milliliters
of distilled water is added to dry soil sample and mixed thoroughly. Resulting solution is
then decanted and filtered, drawn into the plastic syringe and microfiltered into the vial.
This sample is then used as unknown solution and measured as calibration solutions.

Questions
1. What steps would you have to go through in phosphate measurement if you were
operating manually?

2. What is the sequence of the sample and reagents drawn in the holding coil? What
should be the sequence if you wanted to do triplicate measurement at once?

3. What would be another way to handle the soil sample?

THINGS REQUIRED IN THE LAB REPORT.

1.
v
v
v

Introduction (20 points)

Theoretical principle of instrument
Apparatus
Theoretical description of experiment

2. Experimental part (20 points)

v Description of the work done
v Results and calculations (Water content of the soil, phosphate content of the soil
sample, standard deviations, accuracy for quality control sample)

3.
v
v
v
v

Discussion (40 points)

Analysis of results (Discuss precision and accuracy)
Error analysis (include possible error sources)
Conclusions
Summary

4. References (5 points)

5. Answers to the questions (15 points)