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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.


obs_exp6_1
Fig. 1: Chromatogram of machine oil


Table 1: Peaks of machine oil and their Rf value

Peak No. Rf
1 0.04
2 0.09
3 0.16
4 0.32
5 0.38 (Substance 1)
   
Peak No. Rf
6 0.46
7 0.53
8 0.57
9 0.86
10 0.91



obs_exp6_2
Fig. 2: Chromatogram of substance 1 (Rf = 0.38) of machine oil


obs_exp6_3
Fig. 3: Derivatized image of machine oil + sample


obs_exp6_4
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.

obs_exp6_5
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.