HPLC- General Principle and Procedure for Sample Preparation
Authors: Ramya, R.S1, Manimaran, B2, Ranjith, M3
1ICAR- National Bureau of Agricultural Insect Resources, Bengaluru, India
2ICAR- Indian Agricultural Research Institute, New Delhi, India
3Directorate of Plant Protection, Quarantine and Storage, Faridabad, India

High-performance liquid chromatography (sometimes referred to as high-pressure liquid chromatography), HPLC, is a chromatographic technique used to separate a mixture of compounds in analytical chemistry and biochemistry with the purpose of identifying, quantifying and purifying the individual components of the mixture. HPLC is distinguished from ordinary liquid chromatography because the pressure of HPLC is relatively high (~150 bar, ~2000 PSI), while ordinary liquid chromatography typically relies on the force of gravity to provide pressure. HPLC relies on the pressure of mechanical pumps on a liquid solvent to load a sample mixture onto a chemistry column, in which the separation occurs. In HPLC, the analyte is forced through a column of the stationary phase (usually a tube packed with small round particles with a certain surface chemistry) by pumping a liquid (mobile phase) at high pressure through the column. The sample to be analyzed is introduced in a small volume to the stream of mobile phase and is retarded by specific chemical or physical interactions with the stationary phase as it traverses the length of the column. The amount of retardation depends on the nature of the analyte, stationary phase and mobile phase composition. The time at which a specific analyte elutes (comes out of the end of the column) is called the retention time and is considered a reasonably unique identifying characteristic of a given analyte. The use of pressure increases the linear velocity (speed) giving the components less time to diffuse within the column, leading to improved resolution in the resulting chromatogram.

Sample preparation for determination of haemolymph sugars

  • Haemolymph samples are collected from individual insects in 1micro litre microcaps by clipping an antenna, puncturing an intersegmental membrane, or in some cases, puncturing the cuticle on the metathorax.
  • Before sample collection, the insects are immobilized by slow cooling, a procedure which also helps to reduce possible hypertrehalosemic or hyperglycaemic effects from handling.
  • Hemolymph samples are mixed into 5-15 micro litres of cold 70% ethanol for deproteinization and lipid removal and then centrifuged at 12,000 RPM (Fisher Microcentrifuge) for 5 min.
  • The supernatant is used directly for analysis and spotted onto plates as previously described.
  • If samples cannot be analyzed within a few hours, 5-10 micro litre of the supernatant is removed to a second vial, dried under nitrogen and frozen until the analysis can be made.
  • In most cases the major problem in holding samples is solvent loss; adjustments for this problem can be made by the addition of an internal standard such as sucrose.
Sample preparation for determination of amino acids

  • Insects collected are dried at 50 degrees Celsius and grinded until powdered and homogenized.
  • A 3g uniformly blended sample placed in a pre-weighed Whatman cartridge with 600 ml of hexane in a Soxhlet Flask during 10 hours to remove the lipid content.
  • 1mg freeze-dried insect sample (decolored and without carbohydrates) is hydrolyzed with 200 micro litre of HCl 6N during 20 hours at 110 degrees Celsius or 400 micro litre of methane sulfonic acid 4N for tryptophan during 22 hours at 110 degree Celsius.
  • Hydrolysis loss of amino acids is minimized, (providing an inert atmosphere, before sealing the sample tube) when the oxygen is excluded by a combination of nitrogen flushing and evacuation.
  • After hydrolysis, the HCl is evaporated on the Speed Vac concentrator.
  • This dried hydrolysate is kept at -4 degrees Celsius, and then it is dissolved in water, diluted at 1:10 and filtered through a 0.22 micro metre.
  • A 50 micro litre of the sample should be run as the standard chromatographic procedure.
  • The hydrolysate for tryptophan determination is kept at -4 degrees Celsius without drying since the methane sulfonic acid is non-volatile.
Sample preparation for determination of triacylglycerols

  • The fat bodies of insect are dissected after immobilization in cold.
  • Each of the dissected fat bodies is transferred individually into a glass vial with 100 micro litre of chloroform/methanol (1:1, v/v) containing BHT (25.0 mg/mL).
  • The samples are sonicated for 15 min and stored at −28 ◦C when needed.
  • The isolation of the TGs from the individual fat body tissues using semipreparative TLC.
  • Briefly, the tissue in a vial is crushed using a small glass rod, extracted several times with chloroform and the combined total extracts are separated on TLC plates with hexane/diethyl ether/formic acid (80:20:1) in the mobile phase.
  • The TG zone is scraped off the plate and extracted with freshly distilled diethyl ether.
  • The solvent is evaporated under an argon stream to dryness and the TGs are reconstituted in chloroform to a concentration of 10.0 mg/mL.
  • The TG samples are stored in sealed amber glass ampoules at −28 ◦C
Sample preparation for determination of pheromones

  • The ovipositor tips of the females containing the pheromone glands are excised and immersed in hexane for 30 min.
  • The crude extract is used for chiral HPLC analysis without purification.


References:

Adachi, Y., N. C. Do, M. Kinjo, S. Makisako, R. Yamakawa, K. Mori and T. Ando (2010). Positions and stereochemistry of methyl branches in the novel sex pheromone components produced by a lichen moth, Lyclene dharma dharma. J Chem Ecol., 36: 814-823.

Guevara, O. L. D., P. Padilla, L. Garcia, J. M. Pino and J. Ramos. (1995). Amino acid determination in some edible Mexican insects. Amino Acids., 9: 161-173.

Fell, R. D. (1990). The qualitative and quantitative analysis of insect haemolymph sugars by high performance thin layer chromatography. Comp. Biochem. Physiol., 95(4): 539-544.

Kofronova, E., J. Cvaska, V. Vrkoslav, R. Hanus, P. Jiros, J. Kindl, O. Hovorka and I. Valterova. (2009). A comparison of HPLC/APCI-MS and MALDI-MS for characterising triacylglycerols in insects: Species-specific composition of lipids in the fat bodies of bumble bee males. Journal of Chromatography., 877: 3878-3884.



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