Video Densitometry

Video densitometry has been developed in the last few years and is now being deployed throughout industry and research. Such instruments use an imag

Figure 4 (See Colour Plate 27). Separation of pesticides in tap water on an HPTLC silica gel plate by AMD. Multi-wavelength (six wavelengths) evaluation permits resolution by optical means of fractions insufficiently separated. Reprinted from Camag literature, CAMAG, Muttenz, Switzerland.

Wavelength: 366 nm mm

Figure 5 Fluorescence scan of dansylcadaverine derivatized fatty acids separated on an HPTLC silica RP18 plate.

Wavelength: 366 nm mm

Figure 5 Fluorescence scan of dansylcadaverine derivatized fatty acids separated on an HPTLC silica RP18 plate.

ing system consisting of a high resolution CCD camera with a zoom attachment to focus and enlarge the chromatogram, if required and a suitable illumination system. The camera is linked to a computer (usually a PC) and a video printer. The software controls the camera, as well as all parameters such as brightness, contrast, colour balance and intensity. These can be saved for future use or kept as a record of the results. The chromatogram can be presented as an image on the video printer and can be quantified to obtain the concentration of analytes using the mathematical procedures used in scanning densitometry. As in spectrodensitometric scanning, the software does all the necessary calculations to determine the concentration of analytes. For weakly Suorescing analytes, a small camera aperture (F : 22) can be used with long time integration. This enables the imagining of Suorescing compounds which are often invisible to the human eye. The images can be annotated and that annotation stored separately for

Correlation of spectra without RF value

Correlation of spectra with RF value

Correlation of spectra without RF value

Substance Correlation

Ethylmorphine 0.9924

Codeine 0.9905

Nalorphine 0.9848

Morphine 0.9839

Correlation of spectra with RF value

Substance Correlation

Morphine 0.9839

Atenolol 0.9223

Salbutamol 0.9174

Sotalol 0.8939

Figure 6 (See Colour Plate 28). UV spectra of codeine, ethylmorphine and unknown (morphine) overlaid. Spectra of codeine and ethylmorphine taken from spectral library. Spectrum of morphine taken from chromatogram. Reprinted from Camag literature, CAMAG, Muttenz, Switzerland.

Fatty acid analysis

Stdmix Std mix Stdmix Fresh oil Old oil

Figure 7 Video scan of separation of corticosteroids on an HPTLC silica gel plate. Detection reagent: blue tetrazolium solution. Spot application with automatic equipment.

Figure 9 Video scan of separation of pre-derivatized saturated fatty acids on an HPTLC RP18 plate. The plate was scanned at 366 nm to produce fluorescent zones. Band application with automated equipment.

Track 3 Sample a

Peak Start

Maximum

Area

Subst

Peak Start

Maximum

Area

Subst

Rf

H

Rf

H

[%]

Rf

H

A

%%]

1

0.016

0.0

0.065

606.4

10.76

0.097

50.9

5076.4

9.53

8

2

0.097

50.9

0.126

488.3

8.67

0.146

87.9

3432.6

6.44

7

3

0.146

87.9

0.174

780.2

13.85

0.206

12.1

5353.3

10.05

6

4

0.227

0.0

0.271

1094.6

19.42

0.316

13.8

9440.8

17.72

5

5

0.324

0.0

0.368

844.7

14.99

0.397

240.7

7487.0

14.05

4

6

0.397

240.7

0.417

555.2

9.85

0.462

6.1

4914.6

9.22

3

7

0.543

26.1

0.587

509.7

9.05

0.640

10.6

6065.0

11.38

2

8

0.794

70.3

0.854

755.8

13.41

0.915

46.6

11519.2

21.62

1

Total height, 5634.96; total area, 53288.8.

Figure 8 Video scan of separation of corticosteroids on an HPTLC silica gel plate.

Figure 10 Separation of dye mixture developed on an HPTLC spectrodensitometric scan with video scanning.

readiness in annotating further images. Such images can be stored in a variety of files which can then be used in a number of well-known office programs, such as Word, and PowerPoint.

The illumination system needs a number of features in order to get the best results from the CCD camera unit. Illumination from above is necessary, both visible light and UV light at 254 and 366 nm (depending on the chromatogram). However, it is essential that the light fittings do not interfere with the camera's field of view. Lighting from below the plate can in some cases also prove advantageous in giving a bright image.

Figure 7 illustrates a video print of a separation of corticosteroids developed on an HPTLC silica gel plate. The steroids were detected with blue tetra-zolium reagent. Figure 8 shows the scan taken using the software option available. RF data is recorded in the table below. Figure 9 illustrates a further video print, this time of fluorescent chromatographic zones, photographed under UV light (366 nm). This is a

silica gel plate with toluene as mobile phase. Comparison of

separation of derivatized saturated fatty acids from C6 to C24 (conditions as in Figure 5).

Although it is possible to quantify results from the video scan, they are not as accurate as those obtained from a spectrodensitometer. Figure 10 and Table 1 show a comparison of the CV for a six-component dye test mixture separated on an HPTLC layer. Whereas the CVs for spectrodensitometric scan are below 2%, those for the video imaging system are typically from 2 to 4%. As most USP (United States Pharmacopoeia) and EP (European Pharmacopoeia) monographs accept CVs of + 6% in most, if not all, cases, the use of video densitometry is acceptable. However, it should be remembered that for fluorescence quenching and absorption measurements below 254 nm, video densitometry will not show any detection. This is one of the present limitations of the technique. Some substances do require shorter wavelength UV light for their detection. In these instances spectrodensitometry is presently the only solution.

Table 1 Comparison of coefficient of variance (CV) with video scanning and spectro-densitometric scanning. Separation of dyes on an HPTLC silica gel plate using toluene as mobile phase

Dye

RF

Video scan with white light

Spectrodensitometric scan at 592 nm

Meanvalue (%) CV(%)

Mean value (%) CV(%)

Black

0.04

Grey

0.10

99.4 3.50

101.5 0.83

Red

0.17

102.8 3.10

97.8 0.31

Blue

0.23

103.0 3.52

101.2 1.90

Pink

0.36

99.7 3.46

98.3 0.96

Yellow

0.51

98.6 1.30

98.8 0.56

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Solar Panel Basics

Global warming is a huge problem which will significantly affect every country in the world. Many people all over the world are trying to do whatever they can to help combat the effects of global warming. One of the ways that people can fight global warming is to reduce their dependence on non-renewable energy sources like oil and petroleum based products.

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