Observation
The chromatographic profile of the sample was simple, showing different peaks of Machine oils (Fig. 1) but we select the sharp and major peak (Fig. 2). Machine oil is not UV active, so the plates were derivatized using the 5 % sulphuric acid in methanol (Fig. 3). Peak of machine oil was identified using the solvent system [n-Hexane: Diethyl ether: MeOH:: 7:2:1.5 (v/v)] with the Rf value of 0.38 ± 0.02 at 450 nm (Fig. 4). Table 1 shows the appearance of different peaks in machine oil and their Rf values.
Fig. 1: Chromatogram of machine oil
Table 1: Peaks of machine oil and their Rf value
|
|
Fig. 2: Chromatogram of substance 1 (Rf = 0.38) of machine oil
Fig. 3: Derivatized image of machine oil + sample
Fig. 4: 3D display of substance 1 of machine oil peaks
The linearity of the proposed method was confirmed in the range of 2-16 µg of substance 1 for machine oil. A linear regression of the data points for substance 1 is resulted in a calibration curve with the equation Y=1569.021 + 53.047x [regression coefficient (r2) = 0.96790, standard deviation (S.D.) = 2.52%] (Fig. 5). Substance 1 content in milk samples were calculated and depicted in Table 2.
Fig. 5: Calibration curve of substance 1 in machine oil
Table 1: Cane sugars and lactose content in milk
Milk Samples (%) | Substance1 (%) | SD (%) |
---|---|---|
Pure Milk | 0 (naturally occurring) | |
Milk sample 1 | 0.32 | ± 0.05 |
Milk sample 2 | 0.40 | ± 0.07 |
Milk sample 3 | 0.49 | ± 0.02 |
The linearity, accuracy in terms of recovery % and precision was considered for the method. Validation of the method at three concentration levels was carried out by the standard recovery formula returned a mean of 85%. Precision (repeatability) was determined by running a minimum of four analyses and the coefficient of variability was found to be 0.365 %. The limit of detection (LOD) and quantification (LOQ) was found to be 0.156 and 0.475 µg respectively.