Results And Discusson Bismuth Fluorescence Intensity Scans Biology Essay

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To ascertain that we are at the correct fluorescent wavelength for Bi, fluorescent intensity scans were taken from 452 nm to 492 nm. Three different fluorescent intensity scans, one for each of blank, 0.5 ppb Bi, and 1 ppb Bi were taken. The scans not only show that we are at the right fluorescent wavelength i.e., 473.0 nm but also show that the blank has no Bismuth contamination in it. The fluorescent signal intensities for 0.5 ppb Bi and 1 ppb Bi were good and the figure also shows that the fluorescent signal intensity is proportional to the concentration of Bi.

Influence of hydrochloric acid concentration on fluorescence intensity of Bi:

To determine the optimal acidity conditions for the generation of Bi hydride, the influence of different concentrations of HCl on fluorescence intensity of Bi was investigated. Fluorescence intensity for 1 ng/mL Bi was measured at 5% (v/v), 10% (v/v), 15% (v/v), 20% (v/v), and 25% (v/v) HCl concentrations. The fluorescence signal increased very slightly and reached a maximum at 10% (v/v) HCl, but the signal was observed to decrease slightly after 10% (v/v) HCl. Figure shows that 10% (v/v) HCl gave the best signal even though the signal was almost equal for all the concentrations. A concentration of 10% (v/v) hydrochloric acid was used during subsequent Bi fluorescence intensity measurements.

Influence of sodium tetrahydroborate concentration on fluorescence intensity of Bi:

To optimize the amount of reducing agent, the effect of sodium tetrahydroborate concentration on fluorescence signal intensity of Bi was investigated. To improve stability, Sodium tetrahydroborate solutions were prepared in 0.4% (w/v) sodium hydroxide. In this study, it was found that different concentrations of NaBH4, 0.5, 1.0, 1.25, 1.5, 1.75 and 2.0% (w/v), affected the fluorescence signal of 1 ng/mL Bi. Results from figure indicated that the fluorescence signal increased upon increasing NaBH4 concentration. From these results, it was determined that 2.0% NaBH4 in 0.4% (w/v) NaOH and 10% (v/v) HCl provided the best results for HG-LIF Bi measurements.

Influence of Peristaltic pump flow rates on Fluorescence Intensity of Bi:

The effect of peristaltic pump flow rate on the magnitude of 1 ng/mL Bi fluorescence signal was studied. As seen from figure, the signal of Bi increased gradually when the peristaltic pump flow rate was increased between 10 and 30 (a.u.). Flow rate of 15 (a.u.) was selected as the optimum flow rate for the rest of the study.

In this study, argon was used as carrier gas to carry the volatile hydride to the flame atomizer and hydrogen flame was used for the atomization of the hydrides. The flow rates of argon and hydrogen were also optimized.

Table: Peristaltic pump flow rates of Acid/Sample and NaBH4

Table shows the corresponding volumetric flow rates for the solutions.

Pump Flow rate

(a.u.)

Acid/Sample

(mL/min)

NaBH4

(mL/min)

10

3.1

1.2

15

4.4

1.8

20

5.7

2.3

25

7.1

2.8

30

8.4

3.3

Influence of monochromator slit width on Fluorescence Intensity of Bi:

Figure shows the influence of monochromator slit width on magnitude of fluorescence signal of blank and 1 ng/mL Bi. The results indicated that the blank signal increased slightly with increase of slit width. As seen from figure, the signal of 1 ng/mL Bi increased linearly when the slit width was increased from 1000 to 2000 µm. Increase in the magnitude of signal after 2000 µm was not linear. So, 2000 µm slit width was selected in our work in order to obtain highly stable fluorescence measurements.

Influence of Masking Reagent Concentration on Fluorescence Intensity of Bi:

Masking agent helps to nullify the effect of contaminant elements on hydride generation during real sample analysis. A masking reagent of thiourea-ascorbic acid was chosen and the optimal concentration of masking reagent was investigated. Figure shows that the addition of masking reagent has no influence on the fluorescence signal of Bi and 0.3% (w/v) thiourea-ascorbic acid was prepared for real sample analysis. In this study, while analyzing real samples, all the solutions including calibration standards and real samples were made in 0.3% (w/v) thiourea-ascorbic acid.

Fluorescence of Bi as a function of Laser Energy:

The effect of laser energy on fluorescence signal intensity of 1 ng/mL Bi was investigated. Figure shows the power dependence of the fluorescence signal. The fluorescence signal increased with laser energy in a linear way, when the laser energy was increased between 60 - 70 mJ. But, after 70 mJ there was a slight decrease in the signal. The results indicated that saturation occurred at 70 mJ which is important because LIF measurements conducted with laser energies above the saturation depend weakly on the laser power. Hence 70 mJ laser energy was used for the rest of the study.

Optimal conditions for HG-LIF Bi:

Parameters

Bi

Acid Concentration

10% (v/v)

Tetra-hydro borate Concentration

2.0% (m/v) in 0.4% (w/v) NaOH

Peristaltic pump flow rate

15 a.u.

Monochromator slit width

2000 µm

Masking Reagent Concentration

(Thiourea-Ascorbic Acid)

0.3% (m/v)

Calibration and Analytical Figures of merit

In order to determine the sensitivity and limit of detection of the HG-LIF method, calibration curves were performed and typical calibration curves for Bi are shown in Figures. Fluorescence intensity of Bi was found to be linearly proportional to the concentration of Bi. All the calibration curves showed good linearity. After optimizing the HG-LIF response as a function of HG reagents, Limit Of Detection (LOD) were calculated by dividing three times the standard deviation of the fluorescence signal of 16 blanks by the slope of the corresponding calibration lines.

LOD = 3σ/m

Where, σ is standard deviation (noise) and m denotes slope of calibration line (sensitivity).

The limit of detection for Bi fluorescence at 473.000 nm was determined to be 0.03 ng/mL.

At high concentration of Bi sometimes the fluorescence emission has exceeded the capacity of photomultiplier tube. Hence, Neutral Density (ND) filters were used to limit the amount of light reaching the detector. When using these filters, the actual signal intensity was determined by multiplying the observed intensity with the attenuation factor of the corresponding filter. In this study, ND 0.3, ND 0.6, ND 1, ND 2 filters were used.

Table Attenuation factors of the ND filters at different wavelengths

Wavelength (nm)

ND 0.3 filter

ND 0.6 filter

ND 1 filter

ND 2 filter

Table Analytical figures of merit for HG-LIF Bi

Analytical Figures

Bi

Linear Range (ng/mL)

Limit of Detection (ng/mL)

Limit of Quantification (ng/mL)

Relative Standard Deviation (%)

Analysis of a Multielement Standard and a Standard Reference material

Laser Induced Fluorescence has high spectral selectivity resulting from both laser excitation source and the monochromator. The high selectivity of the HG-LIF method is expected to result in high accuracy for Bi, even in complex sample matrices. To check the accuracy of the method a standard reference material (NIST 1643e Trace elements in water) that contains low concentrations of several elements, was analyzed. The certified value of Bi in the standard reference material was 13.0 ± () ng/mL. The result of HG-LIF determination was 12.79 ± 0.17 ng/mL. The close agreement of these two values indicates the accuracy of the HG-LIF approach.

A Multielement Standard Solution V for ICP was analyzed by the HG-LIF method, and recovery experiments were carried out after the spike of 10 ng/mL Bi. Recovery studies were carried out with and without the addition of 0.3% Thiourea-Ascorbic acid (Masking Reagent). The recoveries with and without masking were 97.52% ± (), and 55.45% ± () respectively. These results emphasize the need of masking reagent while analyzing real samples.

Analysis of Real samples

Once the optimal conditions for the generation and collection of Bismuth hydride gas had been established, the proposed HG-LIF method was applied to determine trace levels of Bi in real samples.

To further demonstrate the accuracy of optimized HG-LIF method, a Pepto-Bismol () sample digestion solution was analyzed. The certified value of Bi in the Pepto-Bismol was 10.08 ng/mL; therefore the Bi concentration in the digested solution was 10.08 ng/mL. The result of HG-LIF determination was 10.44 ± 0.15 ng/mL (n=3). The results indicated that the developed HG-LIF method was very accurate and precise.

Determination of Bi in different kinds of Tea leaves

In order to demonstrate the application of HG-LIF technique to complex samples, different kinds of tea leaves were analyzed for Bi content. The results of the analyses are summarized in table. As could be seen, the contents of Bi in decaffeinated tea samples were low, especially for decaffeinated breakfast tea. The means of Bi contents were 37.28 ng/g and 0.31 µg/g in decaffeinated breakfast tea and green tea, respectively. In caffeinated earl-grey tea and breakfast tea, the means of Bi contents were 0.88 and 1.39 µg/g, respectively. The results revealed that the amount of Bi in caffeinated tea samples was significantly greater than that in decaffeinated tea samples.

Table Bismuth contents of different kinds of tea leaves

Sample

Number of sample

Mean

Standard Deviation

Decaffeinated Breakfast tea

37.28 ng/g

Decaffeinated Green tea

0.31 µg/g

Caffeinated Earl-grey tea

0.88 µg/g

Caffeinated Breakfast tea

1.39 µg/g

GERMANIUM

Effect of Acidity on Fluorescence signals of Ge

In this study of Germanium determination by Hydride Generation method, the fluorescence signals were affected by type of acid medium. Previously different optimal acidity conditions were suggested for the generation of Germanium hydride [Xuo, Xuo]. In this study we have investigated the effect of O-Phosphoric acid and Hydrochloric acid on Fluorescence signal intensities of Germanium.

Influence of O-phosphoric acid concentration on Fluorescence intensity of Ge

To determine the optimal acidity conditions for the generation of Ge hydride, the influence of different concentrations of O-phosphoric acid on fluorescence intensity of Ge was studied. Fluorescence intensity for 100 ng/mL Ge was measured at 0.5M, 1M, 2M, and 3M Phosphoric acid concentrations. The results obtained from different concentrations of Phosphoric acid are shown in figure. The fluorescence signal increased very slightly and reached a maximum at 2M Phosphoric acid, but the signal was observed to decrease slightly after 2M Phosphoric acid. The results revealed that 2M Phosphoric acid gave us best signal and hence 2M Phosphoric acid was retained for the subsequent Ge measurements.

Influence of hydrochloric acid concentration on fluorescence intensity of Ge

To optimize the acidity conditions for the generation of Ge hydride, experiments were carried out using different concentrations of Hydrochloric acid. In this study, it was found that different concentrations of Hydrochloric acid, 0.5% (v/v), 1% (v/v), 2% (v/v), 3% (v/v), and 4% (v/v), affected fluorescence signals of Germanium. Results obtained for different hydrochloric acid concentrations were shown in figure. The maximum intensity was found at 2% (v/v) Hydrochloric acid and the intensity dropped rapidly after 2% (v/v) Hydrochloric acid.

Influence of sodium tetrahydroborate concentration on fluorescence intensity of Ge

To optimize the amount of reducing agent, the effect of sodium tetrahydroborate on fluorescence signals of Ge was studied by using 2M Phosphoric acid and 1% HCl. The results obtained for different concentrations of sodium tetrahydroborate were shown in figure. The results indicated that the fluorescence signal of Germanium reached a maximum when 0.5% Sodium tetrahydroborate was used in combination with 2M Phosphoric acid. When 1% (v/v) HCl was used as acid medium, the signal reached a maximum at 1% (w/v) Sodium tetrahydroborate concentration. From the results, it was determined that 0.5% (w/v) NaBH4 in 0.1% NaOH as stabilizer and 2M H3PO4 provided the best results for HG-LIF Ge.

Influence of Peristaltic pump flow rate on Fluorescence intensity of Ge

The effect of peristaltic pump flow rate on the magnitude of 100 ng/mL Ge fluorescence signal was studied. As seen from figure, the signal of Ge increased gradually when the peristaltic pump flow rate was increased between 15 and 25 (a.u.). Flow rate of 20 (a.u.) was selected as the optimum flow rate for the rest of the study.

In this study, argon was used as carrier gas to carry the volatile hydride to the flame atomizer and hydrogen flame was used for the atomization of the hydrides. The flow rates of argon and hydrogen were also optimized.

Influence of Masking reagent Concentration on Fluorescence intensity of Ge

Masking agent helps to nullify the effect of contaminant elements on hydride generation during real sample analysis. A masking reagent of thiourea-ascorbic acid was chosen and the optimal concentration of masking reagent was investigated. Figure shows that the addition of masking reagent has no influence on the fluorescence signal of Ge and 0.3% (w/v) thiourea-ascorbic acid was prepared for real sample analysis. In this study, while analyzing real samples, all the solutions including calibration standards and real samples were made in 0.3% (w/v) thiourea-ascorbic acid.

Table Optimal conditions for HG-LIF Ge

Parameters

Ge

Acid Concentration

Sodium tetrahydroborate Concentration

Peristaltic pump flow rate

Masking Reagent Concentration

Calibration and analytical figures of merit

In order to determine the sensitivity and limit of detection of the HG-LIF method, calibration curves were performed and typical calibration curves for Ge are shown in Figures. Fluorescence intensity of Ge was found to be linearly proportional to the concentration of Ge. All the calibration curves showed good linearity. After optimizing the HG-LIF response as a function of HG reagents, Limit Of Detection (LOD) were calculated by dividing three times the standard deviation of the fluorescence signal of 16 blanks by the slope of the corresponding calibration lines.

LOD = 3σ/m

Where, σ is standard deviation (noise) and m denotes slope of calibration line (sensitivity).

Under optimal conditions the limit of detection obtained for Ge at 303.900 nm was 0.1 ng/mL.

Calibration curve plot HG-LIF Ge 09/20/12 and 09/18/12

Table Analytical figures of merit for HG-LIF Ge

Analytical Figures

Ge

Linear Range (ng/mL)

Limit of Detection (ng/mL)

Limit of Quantification (ng/mL)

Relative Standard Deviation (%)

Analysis a certified reference material and multielement standards

ICP SRM 51844 Recovery study

ICP SRM 51740 Recovery study

1643e NIST SRM recovery study

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