1. INTRODUCTION:

For the analysis and purification of low to moderate molecular weight, thermally labile compounds, supercritical fluid chromatography (SFC), a type of normal phase chromatography, has been employed since 1962. Additionally, chiral chemical separation can be accomplished using it. High-performance liquid chromatography (HPLC) and SFC uses similar principles, but SFC commonly uses carbon dioxide as the mobile phase, necessitating pressurisation of the entire chromatographic flow path. The term "convergence chromatography" is occasionally used to refer to supercritical fluid chromatography since the supercritical phase represents a condition in which attributes of both liquid and gas converge. With reduced viscosities and higher diffusion qualities, supercritical fluids (SF) have densities and dissolving capabilities that are comparable to those of some liquids. Accordingly, SF employed in chromatography as mobile phases should act as both material transporters, similar to how mobile phases in gas chromatography (GC) work, and solvents, similar to how solvents in liquid chromatography (HPLC), dissolve these chemicals. Supercritical Fluid Chromatography (SFC) is the name of this type of chromatography.

2. HISTORY^3,4

· Use of Super Critical Fluid Chromatography (SFC) Was First Proposed In 1958 By J. Lovelock.

· First Reported Use Was In 1962 by Klesper et.al., Who Separated Thermally Liable Porphyrins

· Use of Super Critical Fluid Chromatography (SFC) Was First Proposed In 1958 By J. Lovelock.

· vFirst Reported Use Was In 1962 by Klesper et.al., Who Separated Thermally L. Lovelock.

· Use of Super Critical Fluid Chromatography (SFC) Was First Proposed In 1958 By J. Lovelock.

Table 1. The Milestones in Development of The SFC Technique

Year	Discovery
1822	Phenomenon of the critical point reported
1869	First systematic study of critical region
1958	Use of supercritical fluids (SFs) as chromatographic mobile phases suggested
1962	Use of SFs as eluents for chromatographic separations demonstrated
1966	First chromatogram published
1966	Introduced flame-ionization detection (FID) to supercritical fluid chromatography (SFC)
1967	The name “Supercritical Fluid Chromatography” used for the first time
1967	Introduced ultraviolet-visible (UV-VIS) detection to SFC
1969	First report on use of mobile-phase modifiers (i.e., binary mobile phases)
1969	Differential refractometry detection reported for SFC
1970	Use of pressure programming reported
1970	Heat-of-adsorption detection in SFC reported
1971	Doubts about packed columns
1972	Automated fraction collection discovered
1978	Coupling mass spectrometry (MS) to SFC reported
1978	First report on use of (negative) temperature programming
1978	First report on use of simultaneous temperature-density programming
1981	Use of capillary columns (open tubular) in SFC reported
1981	Fluorometric detection in SFC reported.
1981	Use of mobile phase compositional gradient programming reported
1982	First commercial SFC introduced by Hewlett-Packard
1983	Thermionic (nitrogen-phosphorus) and Fourier transform infrared (FT-IR) detection in SFC reported
1985	Chiral separation demonstrated
1985	Multidimensional SFC reported
1985	First report on coupling supercritical fluid extraction (SFE) to SFC (on-line)
1985	The first report of the analysis of a pharmaceutical by SFC
1986	Commercial capillary SFC introduced
1986	Solute derivatization in SFC analysis reported
1986	Flame-photometric and ion-mobility detection in SFC reported
1988	First use of additives in SFC reported
1988	Journal of Supercritical Fluids – first journal devoted entirely to SFC applications
1988	Nuclear magnetic resonance (NMR), electrochemical detection (ECD), and microwave-induced plasma coupled to SFC
1989	Sulfur chemiluminescence and supersonic jet spectroscopy detection in SFC reported
1989	Solvent density and polarity deconvoluted
1992	Second-generation commercial hardware introduced for high efficiency-packed columns; independent flow control under both pressure and composition gradient conditions
1999	Emphasis shifts towards semi-preparative scale in pharmaceutical industry
2004	T. Berger awarded the Martin Gold Medal by the Chromatographic Society for work in SFC
2006	The first direct multi-gram purification of all four isomers of unnatural amino acids using SFC with stacked injection reported
2007	The establishment of a solvent cycle with a possibility for modifier control in simulated moving-bed plant for preparative SFC reported
2007	Use of preparative scale packed column SFC for drug discovery and development
2008	Ultra-fast SFC introduced

3. Definition:⁵

Supercritical fluid chromatography (SFC) is a chromatographic technique that uses sub-critical (liquid) and supercritical CO₂ as the primary solvent in the mobile phase, usually accompanied by an organic solvent.

4. Principle:⁶

Supercritical fluid chromatography is accepted as a column chromatography method along with gas chromatography (GC) and high-performance liquid chromatography (HPLC). Due to the properties of supercritical fluids, SFC combines each of the advantages of both GC and HPLC in one method.

5. SFC Solvents:⁶

Table 2: SFC Solvents

Solvent	Critical Temperature (°C)	Critical Pressure (bar)
Carbon dioxide (CO₂)	31.1	72
Nitrous oxide (N₂O)	36.5	70.6
Ammonia (NH₃)	132.5	109.8
Ethane (C₂H₆)	32.3	47.6
n-Butane (C₄H₁₀)	152	70.6
Diethyl ether (Et₂O)	193.6	63.8
Dichlorodifluoromethane (CCl₂F₂)	111.7	109.8
Tetrahydrofuran (THF, C₄H₈O)	267	50.5

6. Important Terminology⁷

· Critical Temperature (TC)

The maximum temperature at which a gas can be converted into a liquid by an increase in pressure.

· Critical Pressure (PC)

The minimum pressure which would suffice to liquefy a substance at its critical temperature. Above the critical pressure, increasing the temperature will not cause a fluid to vaporize to give a two-phase system.

· Critical Point:

The characteristic temperature (Tc) and pressure (pc) above which a gas cannot be liquefied at its critical point.

· Supercritical Fluid:

The defined state of a compound, mixture, or element above its critical pressure (pc) and critical temperature (Tc)

· Reduced Temperature: (TR)

The ratio of the temperature (T) in the system to the critical temperature (Tc) Tr = T/Tc

· Reduced Pressure (PR)

The ratio of the pressure in the system (p) to the critical pressure (pc). pr = p/pc

7. Properties of Supercritical Fluid⁸

Table 3: Typical Properties of Gas, Liquid, And Supercritical Fluid of Typical Organic Compounds (Order of Magnitude)

	Density (g/mL)	Diffusivity (cm2/s)	Dynamic Viscosity (g/cm s)
Gas	1 x 10^-3	1 x 10^-1	1 x 10^-2
Liquid	1.0	5 x 10^-6	1 x 10^-4
Supercritical Fluid	3 x 10^-1	1 x 10^-3	1 x 10^-2

8. Choice of Supercritical Fluids Solvent⁹

· Supercritical fluids should be inexpensive.

· They should be innocuous.

· They should be non-toxic substances.

· Insupercritical fluid, the analysts dissolved in them can be easily covered by simply allowing the solutions to equilibrate with the atmosphere at relatively low temperatures.

9. Working^3,6

Figure 2: Working of SFC

In SFC, the mobile phase is first pumped as a liquid, and it is then heated above its supercritical temperature before entering the analytical column, bringing it into the supercritical region. The sample is injected into the supercritical stream using an injection valve before being introduced to the analytical column. A pressure restrictor, which is positioned either after the detector or at the end of the column, keeps the gas supercritical as it moves through the column and into the detector. The restrictor, which comes in both fixed and variable forms, is an essential component since it maintains the mobile phase's supercritical state throughout the separation and frequently needs to be heated to avoid clogging.

10. Instrumentation Of SFC ^5-15

10.1 Mobile Phase

Supercritical fluid chromatography (SFC) uses a supercritical fluid as the mobile phase. Carbon dioxide is a commonly used supercritical fluid since its critical temperature is just 31.1 degrees C and its critical pressure is only 7.38 MPa.

It has the following advantages:

· It is nontoxic and chemically inert.

· It is non-flammable.

· It is non-polar and dissolves oils and fats as well.

· Since solvent removal is simple and carbon dioxide is easily converted to a gas at standard temperatures and pressures, component concentrations may be calculated with greater accuracy.

· High-purity carbon dioxide can be obtained at a low price, so low running costs can be realized.

· Since carbon dioxide emitted from petrochemical factories is collected, refined, and used, it does not increase carbon dioxide emissions.

Some other inorganic candidates that may be employed but are seldom used as principal mobile phases in a supercritical state, are xenon (Xe), nitrous oxide (N₂O),water (H₂O), sulphur hexafluoride (SF₆), ammonia (NH₃), and freons. Alkanes, such as n-butane (C₄H₁₀), propane (C₃H₈), and ethane (C₂H₆) have also been used, but they are highly flammable, making them the least preferred candidates for mobile phases.

10.2 MODIFIER:

The solvating power of a supercritical fluid is manipulated by changing its density. An increase in density increases the solvating power of the supercritical fluid. In addition, the oven temperature can be varied to allow the selectivity of a supercritical fluid. The polarity of a supercritical fluid is altered by the addition of an organic modifier such as methanol or acetonitrile.

Table 4: Frequently Used Modifiers in SFC

Modifier	Temp. (°C)	Pressure (atm)	Molecular mass	Dielectric constant at 20°C	Polarity Index
Methanol	239.4	79.9	32.04	32.70	5.1
Ethanol	243	63.0	46.07	24.30	4.3
1-Propanol	263.5	51.0	60.10	20.33	4.0
2-Propanol	235.1	47.0	60.10	18.30	3.9
1-Hexanol	336.8	40.0	102.18	13.3	3.5
2-Methoxy ethanol	302	52.2	76.10	16.93	5.5
Tetrahydrofuran	267	51.2	72.11	7.58	4.0
1,4-Dioxane	314	51.4	88.11	2.25	4.8
Acetonitrile	275	47.7	41.05	37.50	5.8
Dichloromethane	237	60.0	84.93	8.93^b	3.1
Chloroform	263.2	54.2	119.38	4.81	4.1
Propylene carbonate	352	-	102.09	69.0	6.1
N,N-dimethylacetamide	384	-	87.12	37.78^b	6.5
Dimethyl sulfoxide	465	-	78.13	46.68	7.2
Formic acid	307	-	46.02	58.5^c	-
Water	374.1	217.6	18.01	80.1	10.2
Carbon disulfide	279	78.0	76.13	2.64^c	-

10.3 Stationary Phase:

A neutral substance known as the stationary phase serves as a source of "friction" for some sample molecules as they glide through the column. Polar molecules are drawn to silica, which causes them to tightly bind and hold until enough mobile phase has passed through to draw them away. The resolution and speed of the experiment are influenced by the interactions between the stationary phase and mobile phase features.

10.4 PUMP:

Supercritical fluid chromatography (SFC) system uses a pump as a pressure source for precision pumping of a compressible fluid.

Three Types of Pumps are Used in SFC:

10.4.1 Pneumatic Amplifier Pump:

This pump is composed of two cylinders that are different in piston cross-sectional area. The piston cross- sectional area ratio between the two cylinders equals the pressure amplification factor from the low-pressure cylinder to the high-pressure cylinder and equals the flow rate attenuation factor from the high-flow-rate cylinder to the low-low-rate (high-pressure) cylinder. In practice, an area ratio of 5: 10 is recommended for reasons such as safety, reliability of ultrahigh-pressure seals and connectors, fluid com- pressibility, and high-pressure cylinder volume.

10.4.2 Reciprocating Piston Pump:

The reciprocating piston pump is a continuous-flow pump similar to an HPLC pump. Three major differences of a reciprocating pump from a liquid chromatographic (LC) pump are the addition of a pump cooling system, the requirement of a pulse dampener, and the greater minimization of post pump interface volume. Commercially reciprocating pumps are available which have feed. back control to compensate for fluid compressibility, minimizing pressure ripple and, thus, producing reproducible results.

10.4.3 Syringe Pump:

This pump is widely employed for both open-tubular- column and packed-column SFC applications. Microprocessor control allows reproducible SFC fluid pressure or density programming.

10.5 Injection System:

Injectors act as the main site for the insertion of samples. Injectors come in a variety of forms and depend on a wide range of variables. The sample size for packed columns must be minimal, and it will vary depending on the column's diameter. The usage of larger volumes is possible for open tubular columns. Depending on how the sample must be inserted into the instrument, different injectors are utilised in both situations.

A loop injector is primarily utilised for initial testing. The supercritical fluid is passed via a chamber that has been filled with the sample before being pushed down the column. Before continuing with the full elution at greater pressures, it uses a low-pressure pump. The sample volume can be easily controlled with an inline injector. The (exactly measured) sample is injected into a stream of eluent by a high-pressure pump, where it is then carried through the column. This approach offers more flexibility and specific dilutions.

An in-column injector is helpful for samples that don't need to be diluted or interact right away with the eluent. This enables the mobile phase to pass through the packed column without first passing through the sample, and vice versa.

Agilent Technologies has unveiled a different injection technique for their SFC equipment, the so-called feed injector. This feed injector adds the sample volume to the mobile phase flow, raising the overall flow rate for a brief period of time. Normally, a loop is switched in-line with the mobile phase flow channel to inject the sample.

Types of Injectors are as below:

10.5.1 Loop Injection

· Low pressure feed pump Needed to fill the loop

· Mostly for preliminary tests of column performance and elution parameters

10.5.2 In-Line Injection:

· Flexibility for changing injected volume

· High-pressure pump required to inject feed solution

· Injected stream dissolved in eluent flow

10.5.3 In-Column Injection

· Permits injection of the feed solution directly onto the column

· No dilution required

10.6 Oven

The mobile phase is heated to the necessary temperature in the oven, as previously mentioned. The critical temperature of the supercritical fluid is always the desired temperature in SFC. These ovens have exact controls and are the same for SFC, HPLC, and GC. The oven is typically made so that there are no heat gradients greater than 0.1°C between any two positions in the area of the oven where a column is installed.

10.7 COLUMNS:

Both packed columns and open tubular columns are used in SFC. Packed columns can provide more theoretical plates and handle larger sample volumes than open tubular columns. Because of the low viscosity of supercritical media, columns can be much longer than those used in LC and column lengths of 10 to 20 m and inside diameters of 50 or 1010um are common with open tubular columns. For difficult separations columns 60 m or longer have been used. Open tubular columns are similar to the fused-silica wall-coated (FSWC). Packed columns are usually made of stainless steel. 10-25 cm long. More than 100,000 plates have been achieved with packed columns.

Many of the column coatings used in LC have been applied to SFC as well. Typically, these are polysiloxanes chemically bonded to the surface of silica particles or to the inner silica wall of capillary tubing Film thicknesses are 0.05 to 0.4 um.

Some of the common stationary phases available as capillary columns are as follows:

1. SB-methyl-100: This is a 100% methylpolysiloxane stationary phase and is considered the least polar phase. Flexible siloxane bonds contribute to excellent diffusion characteristics and high efficiencies. This phase is often used as the first choice in analysis of unknown samples in SFC because of the high resolution it delivers and the volatility-based elution order of solutes.

2. SB-phenyl-5: This stationary phase, which contains 5% phenyl substitution, has a slight amount of polarizability with increased capacity for polar solutes.

3. SB-phenyl-50: This stationary phase has 50% polarizable phenyl groups present on the silica surface and exhibits apparent dipoles of varying intensity depending on the local environment. This phase is moderately polar in a general sense and exhibits good selectivity for isomers containing polar functional groups, due to slight differences in the isomers' abilities to introduce dipoles within the stationary phase.

4. SB-biphenyl-30: This stationary phase has 30% biphenyl substitution which provides enhanced polarizability when compared to phenyl- substituted stationary phases. It is in the middle polarity range among the SFC stationary phases. 5. SB-cyanopropyl-25: This phase has 25% cyanopropyl substitution and often gives longer retention for polar solutes.

5. SB-cyanopropyl-50: This is the most polar stationary phase currently available in capillary SFC. It has 50% cyanopropyl substitution and is useful for polar analytes such as pharmaceuticals.

6. SB-smectic: This phase is a liquid polysiloxane. Its selectivity is based on solute size and shape. Solutes are separated based on molecular geometry, with the length-to-breadth ratio determining elution or der within an isomeric series.

Column Storage:

Prior to long-term storage, columns utilised in SFC conditions should be flushed with 10 column volumes of methanol or ethanol. Methanol or ethanol might be used to store the column.

10.8 Detector:

One of the biggest advantages of SFC over HPLC is the range of detectors. It is possible to use the flame ionisation detector (FID), which is typically part of a GC system, with SFC. Given that FID is a very sensitive detector, such a detector can improve the quality of SFC analyses. More simply than with an HPLC, SFC can also be connected with a mass spectrometer, a UV-visible spectrometer, or an IR spectrometer. SFC can be connected to additional detectors used with HPLC, such as thermionic or fluorescence emission spectrometers.

Different types of detectors are used in conjunction with SFC. Some of the commonly used detectors are as follows:

(a) Flame ionization

(b) Ultraviolet

(d) Chemiluminescence

(e) Fourier transform-infrared

(f) Mass spectrometer

(g) Electrochemical

(h) Electron capture

(i) Thermionic ionization

(j) Light scattering

(k) Nitrogen-phosphorus

11. NEW ADVANCES IN SFC^{16,17,18,19,20}

11.1 Ultra-High Performance Supercritical Fluid Chromatography–Mass Spectrometry (UHPSFC-MS):

UHPSFC, which offers a number of advantages, is currently a very intriguing adjunct approach to HPLC and GC. The capacity to separate chiral and other isomers, rapid analysis, high separation efficiency, variety of applications and technique development, smaller pressure drops, orthogonality to RP-HPLC separations, lower operating costs, and environmental friendliness are a few of these. Due to significant advancements in the ESI and APCI atmospheric pressure ionisation procedures, coupling of UHPSFC with MS has been made easier. UHPSFC-MS is preferred for the investigation of complex matrices such biological materials, foods, and natural products because to its improved sensitivity and selectivity.

11.2 Ultra-High-Performance Supercritical Fluid Chromatography Coupled with Tandem Mass Spectrometry for Screening of Doping Agents I: Investigation of Mobile Phase and MS Conditions:

In both ESI+ and ESI- modes, a typical mixture of 31 acidic and basic doping agents was examined. The mobile phase consists of 2% water and 10 mM ammonium formate in the CO2/MeOH. To interface UHPSFC with MS, ethanol is introduced as a makeup solvent. It offered a satisfactory MS response for all doping agents and was able to offset the detrimental effects of the 2% water addition in ESI- mode. Only one chemical (bumetanide) showed a significantly greater MS response (4-fold) under UHPLC-MS/MS settings, but sensitivity was improved by 5-100-fold in UHPSFC-MS/MS compared to UHPLC-MS/MS for 56% of compounds.

11.3 UHP SFC Coupled with Tandem MS for Screening of Doping Agents II: Analysis of Biological Samples:

For the first time, UHPSFC-MS/MS's potential and applicability for detecting doping in urine samples were put to the test. Two separation techniques—UHPLC-MS/MS and UHPSFC-MS/MS in both ESI+ and ESI- modes—were used to evaluate a group of 110 doping agents with various physicochemical features for this aim. In terms of selectivity, sensitivity, linearity, and matrix effects, the two methods were contrasted. As anticipated, extremely varied retentions and selectivities were attained in UHPLC and UHPSFC, demonstrating a favourable complementary relationship between these analytic approaches. These two approaches can be used for screening because they both produced appropriate peak shapes and MS detection capabilities in less than 7 minutes of analysis time. For 46% of the tested compounds, method sensitivity was found to be comparable, whereas higher sensitivity was seen for 21% of the tested compounds in UHPLC-MS/MS and for 32% in UHPSFC-MS/MS.

11.4 Opportunities and Challenges of Liquid Chromatography Coupled to Supercritical Fluid Chromatography:

For the separation of ionizable and neutral solutes, two-dimensional separation using a mix of liquid chromatography and supercritical fluid chromatography appears to present new options. For the separation of neutral molecules, noteworthy orthogonality is demonstrated by two-dimensional LC SFC or SFC LC combinations.

11.5 A Compound Post-Column Re-Focusing Approach On Supercritical Fluid Chromatography:

To achieve both actual concentration enhancement of the analyte and signal enhancement in UV-Vis detection, a post-column refocusing technique for SFC analysis is used. Refocusing of the collected analytes is possible by heart-cutting and moving a chosen fraction from the SFC flow onto a trapping column with a flushing solvent after the depressurized CO2 has been removed. Signal-enhancement ratios between 2.2 and 6.4 were achieved for four sample compounds eluting with very variable amounts of SFC modifier (methanol) by carefully choosing the trapping stationary phase and the two solvents. Because the UV sensitivity of the analytes was found to differ significantly under SFC and LC conditions, the actual concentration enhancement was lower (ratios between 1.7 and 2.9).

12. The Plus-Side of Supercritical Fluidchromatography²¹

We are already aware of SFC's quickness. But because CO2 has a lower viscosity than common reverse phase, normal phase, or HILIC solvents, it is possible to increase the flow rate and complete adequate chromatography in a much shorter amount of time. Because supercritical fluid chromatography significantly reduces the amount of hazardous waste, which frequently requires specialised disposal methods, it is also excellent for the environment. Another advantage is that hardly any dry down time is needed. Additionally, it is an LC orthogonal approach, making it more appealing to chemists who need a confirmation analysis as part of their workflow.

13. THE DOWN-SIDE OF SUPERCRITICAL FLUID CHROMATOGRAPHY²¹

Low UV sensitivity and a lack of reproducibility are two traditional drawbacks; however, this is changing as newer systems enter the market. Additionally, SFC is limited for highly polar chemicals and proteins and does not function for substances that are solely water soluble. For this analysis, big, heavy CO2 tanks are necessary. This becomes more of a problem when using preparative SFC. Larger petrol tanks may be difficult to store because to a lack of lab space and the weight limit of older buildings' floors.

14. APPLICATIONS ^18,22-34

· Supercritical fluid chromatography is used for the analysis of antihypertensive Drugs

· Supercritical fluid chromatography is used for the analysis of herbal medicines

· Supercritical fluid chromatography is used for the analysis of natural products such as

I. Alkaloids

II. Anthraquinones

III. Cannabinoids and other drugs

IV. Carotenoids

V. Coumarins

VI. Flavonoids and Phenols

VII. Lignans.

· Chiral pesticides are analysed using supercritical fluid chromatography. These include the chromatographic analysis of chiral herbicides using green mobile and chiral stationary phases.

· Supercritical fluids can be used to extract the given compounds

I. Extraction of sunflower oil

II. Extraction of Jatropha seeds

III. Extraction of sesame seed oil

IV. SFE of guava seeds and leave

V. Alcohol recovery using SFE

VI. Extraction of flavour and fragrance

VII. Spice extraction

VIII. Herbal extraction

IX. SFE of neem seeds

X. Extraction of caffeine

XI. Petroleum extraction

· SFC has met with great favour in targeted and untargeted lipid profiling due to its high efficiency, low organic solvent consumption, and the specialty for unambiguous identification of the isomeric species of some lipids

· Chromatography with supercritical CO₂ has found application in the determination, separation, and quantitative analyses of both fat- and water-soluble vitamins.

· SFC has widespread applications in pharmaceutical analysis and enantioseparation

· UHPSFC-MS/MS's potential and usability for detecting doping in urine samples. Two separation techniques, UHPLC-MS/MS and UHPSFC-MS/MS in both ESI+ and ESI modes, were used to evaluate a group of 110 doping agents with various physicochemical features for this aim.

· Supercritical fluid chromatography is used for the Rapid analysis of nine lignans in Schisandra chinensis using diode array and mass spectrometric detection.

15. CONCLUSION:

The above report shows the recent advancement in SFC. It also highlights the recent applications of SFC techniques in various analytical fields, such as pesticide analysis, vitamin analysis, enantioseparation, lipid analysis, natural products, antihypertensive drugs. SFC has proven to be an alternative technique to chiral normal-phase HPLC, offering speed, safety, versatility, and significantly reduced solvent costs. SFC can elute non-volatile and thermolabile additives that are unsuitable for analysis by GC, offering better resolution and faster separations compared to HPLC.

16. ABBREVIATIONS:

APCI - Atmospheric Pressure Chemical Ionization

ECD - Electrochemical Detection

FID - Flame-Ionization Detection

GC - Gas Chromatography

HILIC - Hydrophilic interaction liquid chromatography

HPLC - High Pressure/ Performance Liquid Chromatography

IR - Infrared Radiation

LC - Liquid chromatography

MS - Mass Spectrometry

NMR - Nuclear Magnetic Resonance

SF - Supercritical Fluid

SFC - Supercritical Fluid Chromatography

SFE - Supercritical Fluid Extraction

UHPLC - Ultra High-Pressure Liquid Chromatography

UHPSFC - Ultra High-Pressure Supercritical Fluid Chromatography.

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Received on 12.07.2023 Modified on 13.11.2023

Research J. Science and Tech. 2024; 16(1):87-96.

DOI: 10.52711/2349-2988.2024.00014