To elaborate a bit on the previous response: Although we think of both spectrophotometers and HPLC UV detectors as measuring absorbance, this is incorrect. Both systems measure transmittance the fraction of the incident light that is transmitted by the flow cell. They calculate absorbance the negative log of transmittance. This may sound like a distinction without a difference, but it's important because the process involves measuring potentially small differences in large values.
To oversimplify a bit, photodetector noise is roughly proportional to the square root of the light intensity signal. For a given light source and flow cell path length, two things limit the amount of light available: the cross-sectional area of the cell a bigger cell can capture more light , and the slit width of the monochromator this controls the bandpass; a wider range of wavelengths will contain more light.
In HPLC, minimizing extra-column volume is critical, so an HPLC detector will be designed with a very narrow cell typically, 1 mm diameter but because spectral resolution is not very important, the slit width or bandpass values are typically large.
As DR pointed out, the very narrow flow cell requires precise alignment with the optical bench. In a spectrophotometer, the sample cell can have a very large cross-section, but spectral resolution is very important. A spectrophotometer will thus typically be designed with a large cell and a narrow bandpass. If I were less long-winded, I'd simply say that spectrophotometers are designed around spectral constraints, while HPLC detectors are designed around spatial constraints.
High Performance chromatographic techniques employed pressurizing the mobile phase to move it through the stationary phase more quickly. Eventually, the name was changed to High Performance to reflect the improved separating performance of this pressurized technique.
Rather, a basic overview will provide enough information to allow the pharmacokineticist to appreciate the science. Imagine a bottle of 10, marbles, each with a different diameter, and we want to select marbles of a specific diameter. The filters will have different size holes.
The holes would range from just larger than the smallest diameter to much larger than the largest diameter. If we place these filters in series inside of a large tube, we would then pour the bottle of mixed marbles into one end of the tube and wait for the marbles to come out the other end of the tube. The smallest marbles will easily navigate the filters because they will fit through any of the holes.
Thus, we would expect the marbles to come out of the tube starting with the smallest and ending with the largest. Please note that in true size exclusion chromatography, the larger molecules exit first and the smaller molecules exit last based on the construction of the columns.
This results in small molecules having to travel through these pores in the beads while large molecules simply travel around the beads. Despite this difference from the example, the concept is the same. Figure 2: HPLC chromatogram of standard stock solution of canagliflozin Chromatogram showed sharp peak with negligible tailing at a retention time of 4.
Figure 3: RP-HPLC overlay chromatograms of serial dilutions of canagliflozin Linear concentrations showed sharp peaks with uniform symmetry at a retention time of 4. The regression coefficient and Eqn. Furthermore, detection limit depends upon instrument sensitivity as low detection limits give high sensitivity. The results concluded that developed method was linear according to least square method of analysis.
A negligible variation in interday repeatability and intraday reproducibility studies among these developed analytical methods exhibited accurate precision for series of measurements Table 1. This was found to be accurate as percent recovery observed was high i. Previous reports also indicated that CFZ was most stable under different stress conditions like oxidation, thermal hydrolysis and photolytic exposure with negligible degradation [ 15 ].
Furthermore, no new drug peak emerged in analysis of bulk drug after storage at high temperature and humidity, which confirms the stability indicating property of the proposed method.
Thus, both UV and HPLC methods justified good agreement with the analysis of labelled claim for the tablets and were endorsed for rapid determination of CFZ in routine analysis [ 16 ].
Furthermore, the p-value for marketed product was greater than that from standard degree of freedom, implying that there is negligible difference in drug assay in both UV and HPLC methods, thus both methods were considered as statistically insignificant.
Table 7 enlists the summary of all the parameters that were analysed by both analytical methods. The cost effective, simple and low cost reagents in spectrophotometric method allow routine use in pharmaceutical research. The overall results from both spectrophotometric and HPLC methods demonstrate rapid determination of CFZ and is endorsed for routine analysis for quality control purpose.
The authors are grateful to Zydus Cadila Limited, Ahmedabad for providing gift sample of canagliflozin for this research work.
The Indian Journal of Pharmacy was started in as "a quarterly journal devoted to the Science and practice of Pharmacy in all its branches". Detection and quantitation limits Series of diluted standard solutions were prepared and analyzed by both methods. Specificity A sample solution of tablet was prepared in the test concentration range and injected into the chromatograph, to evaluate possible interfering peaks. Ruggedness Ruggedness of the proposed method was determined by analysis of sample solution prepared by proposed methods between different time intervals, days and analysts.
Figure 2. Figure 3. Table 1 Result from system suitability study. Table 2 Validation parameters of evaluated methods. Precision The precision data obtained for the evaluated methods are demonstrated in Table 2. Accuracy Accuracy was investigated by means of recovery studies using the developed methods. Table 3 Recovery study results of repaglinide for UV method.
Specificity The chromatogram obtained with the tablet sample solution with excipients shows no interfering peaks in the retention time of repaglinide. Table 5 Statistical comparison of results obtained by the proposed methods for the analysis of repaglinide in tablets. Edinburgh: Churchill Livingstone; Budavari S, editor. The Merck Index, an encyclopedia of chemicals, drugs and biologicals. Reynolds JE. London: Pharmaceutical Press; Martindale, the complete drug reference; p.
Goyal A, Singhvi I. Visible spectrophotometric methods for estimation of repaglinide in tablet formulation. Indian J Pharm Sci. Sharma S, Sharma MC. Simultaneous determination of repaglinide in pharmaceutical dosage form using indigo carmine. Amer Eurasian J Sci Res. Simultaneous specrophotometric estimation of metformin and repaglinide in a synthetic mixture.
Development of spectrofluorimetric and HPLC methods for in vitro analysis of repaglinide. Gandhimathi M, Renu TK. Anal Sci. Determination of repaglinide in pharmaceutical formulation by HPLC. J Applied Sci Res.
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