Continuous Flow Analysis Methods David Davey University of South Australia. 21 February
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1 Continuous Flow Analysis Methods David Davey University of South Australia 21 February
2 Flow Analysis Methods 1950s-60s Segmented Flow Methods Clinical Analysis Skeggs 1970s Flow Injection Analysis Chemical Analysis Ruzicka and Hansen, Pungor Tecator Lachat GlobalFIA FIAlab 1980s Sequential Injection Marshall, Ruzicka Lab on a valve 1990s MicroTotal Analysis Systems Lab on a chip 21 February
3 21 February
4 Continuous Flow Analysis Methods David Davey University of South Australia 21 February
5 Reagent Carrier Flow Cell Sample Pump Sample Sensors WASTE 21 February GND
6 Lab on a Chip 21 February
7 Segmented Flow Analysis Automated systems in many older laboratories 3 AIR Pump 5 Detector Mixing coil 1 2 Sample Reagent Basic Layout of SFA instrument 21 February
8 SFA Bolus= Volume of Sample and Reagents which remains largely intact in the flow system Manifold= The collection of tubing and connectors which: transports sample and reagent, creates the bolus, promotes mixing, and delivers the reaction product to a detector 21 February
9 Segmented Flow Analysis AIR 21 February
10 Some SFA Features Samples are pumped into the manifold Air separates sample boluses where reaction occurs Air (and some sample) must be removed before the detector Steady state signals are sought, and dispersion is thus small Manifold tube diameters are usually 2-3 mm. Flow is controlled and turbulent Sample throughput is 2-5 per minute. 21 February
11 Flow Injection Analysis Basic Layout of FIA instrument 1 Pump Sample valve 3 Detector 4 2 Mixing coil 1 4 Carrier or Reagent 1 Reagent 2 21 February
12 Some FIA Features Samples are injected/intercalated into the manifold Air bubbles are not wanted! Steady state signals are not required Manifold tubes are less than 1 mm in diameter Flow is controlled and mostly laminar Sample throughput can be very fast, and is often < 1 sample per minute 21 February
13 Flow Analysis Methods Segmented Flow SFA Tubing 2 mm ID Air segmented Sample 1-2 ml Flow Injection Analysis FIA Tubing < 1 mm ID Sample µL Microconduits Channels 0.5 mm 21 February
14 Flow Analysis Methods Segmented Flow SFA Tubing 2 mm ID Air segmented Sample 1-2 ml Flow Injection Analysis FIA Sequential Injection Analysis SIA Tubing < 1 mm ID Sample µL Microconduits Channels 0.5 mm 21 February
15 Flow Analysis Methods Segmented Flow SFA Flow Injection Analysis FIA Sequential Injection SIA Holding & Mixing Coil Channels < 1 mm ID Multdirectional Use of Syringe Pumps Samples10µL-100 µl 21 February
16 Sequential Injection Analysis,SIA SIA uses the same chemistry as FIA and the reaction manifold is similar. A holding and mixing coil is a key component of the design. Samples and Reagents are loaded using:- a multi-directional valve minipumps or multistep syringes under PC control 21 February
17 SIA setup Peristaltic Pump or Multi-step Piston Wash Reagent Detector Holding Coil Reaction Coil Light source Standard Sample 21 February
18 1. Flow Injection Analysis Automated systems in most laboratories small samples are introduced into a continuous liquid stream for controlled reaction and delivery to a detector FIA requires excellent time and volume control an important analytical tool in determining large numbers of samples rapidly and precisely 21 February
19 FIA Physical Needs Multichannel Pump Sample Injection Valve Flow Through Detector 21 February
20 Flow Injection Analysis Basic Layout of FIA instrument 1 Pump Sample valve 3 Detector 4 2 Mixing coil 1 4 Carrier or Reagent 1 Reagent 2 21 February
21 Flow Injection Analysis continued 1 Carrier The carrier may provide some medium control, but not form a product with the analyte. The carrier could control interfering species through ph control By complexation of an interfering compound 21 February
22 Flow Injection Analysis continued 1 4 Reagent(s) FIA relies on carefully optimised conditions and selective chemistry and/or a selective detector to pick out a key compound in a sample. Colorimetric or Fluorimetric Analysis are dominant methods employed. UV-visible detector measures the absorbance of the product of reaction with the analyte, or Fluorescing product gives out light to a detector Chemiluminescent product gives out light to a detector 21 February
23 Methods characterised by Dispersion FIA & SIA Dispersion is the process which is utilised in FIA (& SIA) to create unique analysis methods Dispersion Factor, Df defines the amoiunt of mixing employed 21 February
24 Dispersion is measured relative to a Steady State result D f =1 A Steady State in FIA is a signal which is the maximum achievable for that manifold. The observed response could be decided by sample volume kinetics mixing characteristics 21 February
25 Low dispersion Flow Injection Analysis sample delivery to electrodes, optodes, atomic spectrometers etc with minimal treatment Medium dispersion sample reaction in multipath manifolds with processes in-stream such as preconcentration, sample-reagent mixing, heating and time delays to achieve a particular sensitivity before a product reaches the detector High dispersion methods using enhanced concentration gradients eg in-stream titrations 21 February
26 Dispersion Coefficient D f = C o /C = H o /H Flow Injection Analysis D f range Low dispersion 1 3 Medium dispersion >3 6 (Ruzicka -10) High dispersion > February
27 Low dispersion Flow Injection Analysis Samples are delivered to a detector with minimal change Applications include:- Electrochemical Methods Atomic absorption Some adjustment may be made to the carrier stream TISAB, buffer Low concentration of the analyte 21 February
28 Examples ph, pf, pca ph 4.6 Pump Sample valve Mixing coil Water TISAB February
29 Medium dispersion Urea in serum ml/min Flow Injection Analysis Sample 30 µl Urease 0.5 Hypochlorite nm Phenol 1.6 Standards 4-25 mm Samples 21 February
30 Other Examples Phosphate in water or soil via vanadomolybdate polyphosphate complexation Sulfur dioxide in beverages forms a dyestuff with para-rosaniline at 550 nm. Ammonia or Chloramine in water via indophenol reaction Nitrite in food via azo-dye formation 21 February
31 High Dispersion Peak Width Measurements FIA provides extraordinary control of mixing processes. Thus in-stream reactions have found peak width as the parameter useful In-stream titration Concentration zones EDTA reagent Ca electrode 21 February
32 Information in a peak The response curve is a repository of information peak height or area = k [analyte] peak width = k log [analyte] 21 February
33 Flow Injection Analysis High dispersion - In-stream titrations EDTA reagent Ca electrode Ca electrode EDTA reagent Analyte Ca February
34 Flow Injection Analysis High dispersion - In-stream titrations t equivalence = k log[ca ++ ] t equivalence 21 February
35 Some Questions to try 21 February
36 Examine the literature to find details for: Phosphate in water or soil via vanadomolybdate polyphosphate complexation under acid conditions Ammonia or Chloramine in water via indophenol reaction Nitrite in food via azo-dye formation with napthylamine 21 February
37 Ques1. Design 2 flow manifolds for N speciation Nitrogen speciation is a key measure in determining water quality. Measure ammonia using the NH 3 electrode Nitrate is first reduced on line to form nitrite which then reacts with napthylamine reaction to form an azo dye before a colorimetric end-point 21 February
38 Design a manifold for SO2 in Wine Ques 2. Sulfur dioxide is a key preservative in beverages. It also has regulated levels under export rules. SO 2 is determined after p-rosaniline reaction under acid conditions, and the response read colorimetrically at 550 nm Total sulfur dioxide requires bound material, e.g. the bisulfite addition complex with aldehyde, be broken up first. 21 February
39 Examine the literature to find details for: Phosphate in water or soil via vanadomolybdate polyphosphate complexation under acid conditions Ammonia or Chloramine in water via indophenol reaction Nitrite in food via azo-dye formation with napthylamine 21 February
40 Selective determination of orthophosphate and total inorganic phosphates in detergents by flow injection photometric method Liu Jing-fu and Jiang Gui-bin, Talanta Vol 52, June 2000, S C HC CC DB 30µL RC D R 1.2 ml/min Pump 620 nm 21 February
41 Questions on orthophosphate and total inorganic phosphate Manifold Assume HC is 100 cm of 1 mm tube. What is its volume (π R 2 L assume π =3)? How long will fresh sample take to travel through the coil? If RC has the same volume as HC, what is the approximate time taken by a new sample to reach the detector D? What do HC, CC and RC represent? Let s allow C and R to flush the detector cell (volume 0.2 ml) twice between introducing sample for reaction. For one sample, how many repeat analyses per minute might be possible? 21 February
42 Questions & Answers on orthophosphate and total inorganic phosphate Manifold Assume HC is 100 cm of 1 mm Internal Diameter tube. What is its volume (π R 2 L assume π =3)? How long will fresh sample take to travel through the coil? Volume = π x(0.05^2)x100 ml = approx 0.8 ml 60 sec x 0.8/1.2 = 40 s If RC has the same volume as HC, what is the approximate time taken by a new sample to reach the detector D? HC 40 s ; RC 20 s (flow rate 2.4) gives Total 60 s 21 February
43 Questions on orthophosphate and total inorganic phosphate Manifold cont d What do HC, CC and RC and DB represent? HC heated coil; CC cooling coil; RC analysis reaction coil; DB debubbler. Detergents are poly phosphates, and reaction is needed to break them down to PO 3-4. Frothing and thus bubblers need to be removed at DB. Let s allow C and R to flush the detector cell (volume 0.2 ml) twice between introducing sample for reaction. For one sample, how many repeat analyses per minute might be possible? The question is best seen as having A sample loaded to the injection valve. C and R flush the detector twice, volume 2 x 0.2 ml. Time 60 x 0.4/2.4 s=10 s Note that the injection valve has easily been rinsed and filled in this time. 60 x 0.03/1.2=1.5 s. So Repeat analyses (peak responses) would be possible 6 times per minute, 360 per hour. In 21 February reality 2012 rates such as per hour are achieved. 43
44 Ammonia in Water NH3 + HOCl NH2 Cl + PhenylOH NH2 Cl + H2O NH2 Phenyl OH 0.3 S A B µL RC1 RC2 RC3 D C 1.0 Pump 660 nm ml/min 21 February
45 Ammonia Manifold Questions Assume 1 mm tubes throughout. RC1 is 10 cm long. RC2 and 3 are 50 cm long. If these are the major volume elements, how long would a sample take to reach the detector? What would you suggest A, B and C are? Let s allow reagents to flush the whole manifold between introducing fresh sample for reaction. For one sample type, how many repeat analyses per hour might be possible? This is called the through put. Sample carryover implies reaction of one sample bolus with another. Suggest a maximum repeat sample injection for this system. 21 February
46 Ammonia Manifold Questions Assume 1 mm tubes throughout. RC1 is 10 cm long. RC2 and 3 are 50 cm long. If these are the major volume elements, how long would a sample take to reach the detector? From previous question, volumes are = 0.88 ml Each manifold component has its own flow rate so times are 4.8, 12 and 8 s total time = 25 s What would you suggest A, B and C are? A good answer would be: A ph control; B HOCl; C phenol Let s allow reagents to flush the whole manifold between introducing fresh sample for reaction. For one sample type, how many repeat analyses per hour might be possible? This is called the through put. Since 25 seconds are needed per sample, throughput= 60 x 60/25 =144 h February
47 Ammonia Manifold Questions Sample carryover implies reaction of one sample bolus with another. Suggest a maximum repeat sample injection for this system. I interpret this to mean how many 100 µl samples could squeeze into the manifold. It is roughly 1 in RC1; 4 in both RC2 and RC3. 4 in RC3 at 3 ml/min would imply a sample every 8/4 s or 1800 h -1! However if the volume D were 0.2 ml, 2 samples would end up in the detector cell at once! A mixed signal (carryover would result). The detector volume (and response time) is the ultimate limit on the analysis speed. 21 February
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