Flow Injection Analysis and ApplicationsFlow Injection Analysis and Applications

Western Kentucky UniversityWestern Kentucky UniversityChemistry DepartmentChemistry DepartmentFall Graduate SeminarFall Graduate Seminar

Presented by: Dheyaa AlkarawiPresented by: Dheyaa Alkarawi

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OutlineOutline

• IntroductionIntroduction• Theory and application Theory and application • ConclusionConclusion• ReferencesReferences• AcknowledgeAcknowledge• QuestionsQuestions

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IntroductionIntroduction

• Flow injection analysis (FIA): a high throughput sampling technique used in combination with other instruments.

• In FIA, analytes are detected after sequential insertion of a discrete sample solution into an unsegmented continuously flowing stream. Even this definition, however, is frequently made obsolete by new developments.

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The main advantage of Flow Injection is the ability to automate a wide range of wet chemical assays expediently.

applicationsapplications

• Flow injection analysis designed as tool of for automation of laboratory assays, also FIA is a tool of research in a variety of fields:

Industrial, clinical, environmental, agricultural, metallurgical, geological, food mixture, pharmaceutical, and biotechnological applications.

FIA methods of analysis could be very useful for water analysis FIA was exploited to analyze the pollutants in water samples from a stream, river,

lake.

Due to its flexibility, FIA can be combined and integrated with other instruments (HPLC, AA, GC, CE…) to provide a robust research tool.

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Theory and application Theory and application

Flow Injection Analysis (FIA) is group of a flow-based techniques;

Presently FIA techniques fall into four categories:

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1.Flow Injection Analysis (FIA)  

2. Sequential Injection analysis (SI)

3. Bead Injection (BI)

4. Sequential Injection Chromatography (SIC)

Abs

orba

nce

Time (s)

0

2

5

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Sample injected into a continuously moving streamReagent merged with sample, generating a colored productColor intensity measured in detector

Flow Injection Analysis (FIA)Flow Injection Analysis (FIA)

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Flow injection analysis to determine chloride IonFlow injection analysis to determine chloride Ion

Sampling rate was approximately 120 sample/h can be analyzed Each sample was injected 4 times. At 480nm

J. Ruzicka & E.H. Hansen, “Flow Injection Analysis” 2nd ed. J. Wiley, N.Y. 19887

480nm

Sequential Injection Analysis (SIA)Sequential Injection Analysis (SIA)

Sample (A red)  and reagent (B blue) are injected sequentially, by means of a multiposition valve (MPV), into a carrier stream, driven by a single syringe pump, placed upstream of the valve.

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As the  reaction product (C yellow) starts to form at the interface of stacked zones the flow stopped to allow product to form The flow is then reversed (D) to further promote mixing and to transport the reaction mixture into the detector for monitoring.

ADV:

Robustness and reliability.Stability of flow.Low reagent consumption even lower than FlA teq.Low waste generationSpeed of response: the readout is available within 30 seconds after sample injection

Features:

Both sample and reagent injected as finite segments

Discontinuous operation the flow can be stopped, slowed, accelerated, reversed at will

Sequential Injection Analysis (FIA)Sequential Injection Analysis (FIA)

Multiposition valve (MPV),

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Configuration of SI-LOV system for the determination of ethanol in beverages was successfully analyzed, including red and white wine, beers and various  spirits.

Fig. 2.2 ADH, alcohol dehydrogenase; NAD+ Nicotinamide adenine dinucleotide; Buffer, phosphate buffer pH 9.5; W, waste; SP, syringe pump HC, holding coil; FC, flow cell; P, peristaltic pump; Detector.

Fig 2.3Variation of the absorbance with the increase of the concentration of ethanol by (A) initial rate measurements and (B) “A linear dynamic application “ peak height measurement. at 340 nm.

Susana S.M.P. Vidigal, Ildiko V. T ´ oth, Ant ´ onio O.S.S. Rangel ´2011

Variant of ethanol Conc In the range of 0.00–0.040% (v/v) was monitored.

The objective of this work was to study the potential of the sequential injection-lab-on-valve (SI-LOV) format for the miniaturization of enzymatic assays, by using different measurement modes (peak height and initial-rate-based measurement).

Table 1. Comparison of the results obtained for the analysis of different beverages accordingto the reference and the developed procedures

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BEAD INJECTIONBEAD INJECTION

Bead Injection (BI): volume of suspension of microbeads are injected into a carrier stream, where the beads with a suitable bioligand ion exchange group or C-18 group are trapped within  a selected location (flow cell).

Step 1

Step 2

Step 3

Step 4On-column Off-column

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The sample is injected and transported downstream and when the sample reaches the bead layer, its components react with functional groups on the bead surfaces.

The main  advantage of BI is accumulation of target analyte on bead surfaces, while the non retained matrix is being removed.

A) carrier solution and bead suspension were aspirated respectively.

12Y.-L. Yu; Jiang, Y; R.-H. He. Development of a miniature analytical system in a lab-on-valve for determination of trace copper by bead injection spectroscopy, Talanta, 88 (2012) 352– 357

LOV-BIS system incorporating a multipurpose flow cell for copper measurement by bead LOV-BIS system incorporating a multipurpose flow cell for copper measurement by bead injection spectroscopyinjection spectroscopy

B) variants of copper conc. were analyzed. The absorbance in the flow cell was real-time monitored and recorded by spectrophotometer. Samples Certified (micg g−1) Found (micg g−1)

rice 4.9 ± 0.3 4.5 ± 0.5

Human hair 23 ± 1.4 22 ± 1

Water 51 ± 2 ng g−1 49 ± 2 ng g−1

Table 1.Determination of copper contents in various samples by LOV-BIS platformLOV-BIS platform

Sequential Injection Chromatography (SIC)Sequential Injection Chromatography (SIC)

Sequential Injection Chromatography (SIC) is a combination of two technologies:

A)Liquid Chromatography (LC).

B) Sequential Injection Analysis (SIA).

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Sequential Injection Chromatography (SIC)Sequential Injection Chromatography (SIC)

A) Firstly, the system is filled with an eluent solution, A precise volume of sample (red) is aspirated via multi-position valve (MPV) by flow reversal.

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B) The MPV is then switched from sample port to the column port, and the sample passes through the column while analytes are retained.

C) eluent is aspirated  via MPV by flow reversal.

D) Flow is reversed and a gradient of eluent is passed via MPV into the column and the separated analytes are measured as they flow through the detector.

15Basic combinations of on-line coupling of flow-processing devices and different separation techniques

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Determination of polycyclic aromatic hydrocarbons using lab on valve dispersive liquid–liquid microextraction coupled to high performance chromatography

The extraction procedure was done within a few seconds. And included:

4 mL of an aqueous solution containing the 15 PAHs + 900 µL acetonitrile + 100 µL trichloroethylene were aspirated into extraction chamber.

A cloudy solution was formed.. Then an aliquot of 20 µL of the separated phase was aspirated into an injection loop and then to the HPLC column for separation and detection.

Compound Abbreviation

Peak Order

Naphthalene Nap 1Acenaphthene Acp 2

fluorene Flu 3phenanthrene PA 4

anthracene Ant 5fluoranthene FL 6

pyrene Pyr 7benz[a]anthrace

neBaA 8

chrysene Chr 9benzo[b]fluorant

heneBbFl 10

benzo[a]pyrene BkFl 11benzo[a]pyrene BaP 12

indeno[1,2,3-cd]pyrene

IP 13

dibenzo[a,h]anthracene

DBA 14

benzo[g,h,i]perylene

BghiP 15

Table 4: The elution order and retention time of 15 PAHs

On-line DLLME–HPLC chromatograms of water spiked with 0.02 mg/L of each PAH. PAHs were preconcentrated from 4 ml of sample

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No PAHs were found in tap water.

In rain or stream water samples only PAHs of 2 or 3 rings were detected.

Those PAHs consist of five or six rings only found in highly contaminated sites.

Application to real Samples:

ConclusionConclusion Four FIA techniques were described. However, the goal of any approach to automation of a

reagent based assay is to design a method that will:

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Finally, The automated MSFIA–DLLME proposed procedure offers significant saving of reagents and time compared to other techniques.

1. Provide reproducible and accurate data. 2. Achieve a high sampling frequency

3. Minimize reagent consumption and waste generation.

4. Simplify the assay protocol, while making the system transparent to the user.

5- Be versatile, and can be combined to various instruments to provide a power and solid instrumentation can be used in different ways for qualitative purposes.

In addition, An analytical methodology for the determination of PAHs in aqueous sample was developed. The results showed good performance of the analytical protocol.

AcknowledgeAcknowledge

My research adviser: Dr. Eric Conte The department chair and research adviser: Dr . Stuart Burris Dr. Kevin Williams Evaluation committee The audience

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ReferencesReferences • Clavijo, S.; Avivar, J.;Suarez, R.; Cerda, V. Analytical strategies for coupling separation and flow-injection

techniques, Trends in Analytical Chemistry. 67 (2015) 26–33 

• Zacharis, C.-K.; Theodoridis, G.-A.; Voulgaropoulos, A.-N. Coupling of sequential injection with liquid chromatography for the automated derivatization and on-line determination of amino acids, Talanta. 69 (2006) 841–847 

• Fernández, M.; Clavijo, S.; Forteza, R.; Cerdà, V. Determination of polycyclic aromatic hydrocarbons using lab on valve dispersive liquid–liquid microextraction coupled to high performance chromatography, Talanta. 138 (2015) 190–195 

• Ayyildiz, H.- F.; Kara, H. A Highly Efficient Automated Flow Injection Method for Rapid Determination of Free Fatty Acid Content in Corn Oils, J Am Oil Chem Soc. (2014) 91:549–558 

• Ružicka, J., & Hansen, E. H. (1988). Flow injection analysis (Vol. 62). John Wiley & Sons. 

• Clavijo, S.; Fern´andez, M.; Forteza, R.; Brunetto, M.-R.; Cerdà, V. Online coupling lab on valve-dispersive liquid– liquid microextraction multisyringe flow injection with gas chromatography-mass spectrometry for the determination of sixteen priority PAHs in water, Anal. Methods. 2014, 6, 3335

 • Boonjob, W.; Sklenářová, H.; Barron, L.; Solich, P.; Smith, N. Renewable sorbent material for solid phase

extraction with direct coupling of sequential injection analysis-bead injection to liquid chromatography-electrospray ionization tandem mass spectrometry, Anal Bioanal Chem. (2015) 407:5719–5728

 • Danchana, K.; Maya, F’; Wilairat, P.; Uraisin, K..; Cerdà, V. Spectrophotometric determination of bromide in water

using the multisyringe flow injection analysis technique coupled to a gas-diffusion unit, Anal. Methods. 2015,7, 4202-4208

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