A Gallery of Citric Acid Photomicrographs
a variety of illumination techniques)
Citric acid is a weak organic acid found
in citrus fruits and some vegetables. Lemons contain the highest
percentage of the chemical – about 8% by mass. Its naturally sour
taste results in its being added to many soft-drinks as a
flavouring. In addition, it acts as a natural preservative in
many food items.
Citric acid was first isolated from lemon juice in 1784 by the Swedish chemist Carl Wilhelm Scheele. In 1893, C. Wehmer found that the penicillium mold could be used to produce citric acid from sugar. Since fruit was readily available, this type of biological or microbial synthesis seemed unnecessary. However, when the first world war disrupted Italian citrus imports, microbial synthesis of the compound became industrially important.
2 – Hydroxy – 1, 2, 3 – propanetricarboxylic acid, or citric acid, whichever you prefer, contains three carboxylic acid groups (-COOH), as can be seen below. (Both illustrations were produced using HyperChem Pro software.) Each of these groups can lose a proton in solution, resulting in a citrate ion. These citrate ions are used to make buffers that control the acidity (pH) of pharmaceutical and food products.
For this article, a specimen was prepared by placing a small quantity of the pure white crystalline solid on a microscope slide, covering with a cover-glass, and heating gently with an alcohol lamp until the crystals melt. (Melting temperature is 100 degrees Celsius.) Melt specimens usually re-crystallize almost immediately after cooling. This is not the case with citric acid. The images in the article were taken after about 90% of the melt had solidified, five weeks, after the slides had been removed from the heat of the flame! Patience is a virtue when dealing with this compound! (Special care must be taken not to overheat the sample while melting. If this occurs, decomposition of the citric acid produces unsightly bubbles in the finished slide.)
Note: The MSDS safety document for the compound states the following:
WARNING! CAUSES SEVERE EYE IRRITATION. CAUSES IRRITATION TO SKIN AND RESPIRATORY TRACT.
The following three images show typical fields between crossed polars. The black circles are gas-filled bubbles that formed as decomposition occurred during the melting process. Magnification has increased with each photomicrograph.
By using elliptically polarized light, (obtained by the use of two lambda/4 compensators), the background is coloured off-white instead of the normal black.
Three images of the same field, with increasing magnification, again using elliptically polarized light, are shown below. The irregularly shaped gaps are voids formed by overheating during the melting process. Note that the first image in the article is identical to the first image below. Photoshop CS was used to “invert” the colours of the earlier image.
Several finger-shaped crystal structures on the slide are shown below. (Elliptically polarized light)
It is difficult to maintain a constant distance between slide and cover-glass while making a melt specimen. Very different crystal thickness’s sometimes occur. The following image shows a particularly thick area of crystal growth on the slide. (Plane polarized light)
By replacing the polarizing condenser with a dark-ground condenser, it is possible to display the circular decomposition voids more vividly.
If a phase-contrast condenser is used with a normal, (non-phase) objective, a different sort of dark-ground image is formed which is highlighted by coloured interference patterns. Four examples are shown below.
melt specimens displayed the strange crystal structures that can be
seen below. (Plane-polarized light.)
By using a phase-contrast condenser with non-phase objective, the structures are revealed in a different light.
Similar structures, taken with elliptically polarized light, and transformed within Photoshop using the “Invert (colour)” command, can be seen below.
Proper phase-contrast illumination, (phase-contrast condenser and phase objective), gives another view of the structures.
Again, normal phase-contrast illumination was used to show the following interesting fields.
patterns occurred regularly near the edge of the cover-glass.
They were likely formed by the Permount, and had nothing to do with the
citric acid structures. (Phase-contrast)
unlikely that we could go a single day without eating or drinking
something containing citric acid or citrate ions. I hope that
this article has given you a different perspective on this common
The images in the article were photographed using a Nikon Coolpix 4500 camera attached to a Leitz SM-Pol polarizing microscope. Images were produced using several illumination techniques: dark-ground, phase contrast and polarized light. Crossed polars were used in all polarized light images. Compensators, ( lambda and lambda/4 plates ), were utilized to alter the appearance in some cases. A 2.5x, 6.3x, 16x or 25x flat-field objective formed the original image and a 10x Periplan eyepiece projected the image to the camera lens.
Published in the July
2007 edition of Micscape.
Please report any Web problems or offer general comments to the Micscape Editor.
Micscape is the on-line monthly magazine of the Microscopy UK web
site at Microscopy-UK
© Onview.net Ltd, Microscopy-UK, and all contributors 1995 onwards. All rights reserved. Main site is at www.microscopy-uk.org.uk with full mirror at www.microscopy-uk.net .