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Chemical detection of Aflatoxins in dairy products

High performance liquid chromatography

High performance liquid chromatography (HPLC) is a widely used analytical technique. It allows samples to be separated and their components determined.

The basic principle of HPLC is passing a sample through a column packed with tiny particles (usually less than 10um diameter) and applying high pressure to increase the resolution and speed of the technique.

There is solvent constantly flowing through the system. This solvent is called mobile phase. To ensure the solvent is constantly moving through the system a pump is employed. This gives the required pressure to allow mobile phase and sample to move through the column with ease and speed. The sample is injected into the mobile phase so it can proceed directly to the column. The sample comes out from the column (elutes) and proceeds to the detector. This monitors the refractive index difference between the pure mobile phase entering the column and the column elute.

The signal from the detector is sent to a data recorder. This measures the time of each component (retention time) and the size of the signal (peak area).
By injecting a known standard a retention time and peak area are achieved. If an unknown sample is then injected and peak obtained at the same retention time.
The peak area can be used to calculate the amount of standard material in the unknown by proportion.

HPLC is especially important for evaluation of non-volatile compounds, providing rapid determination of additives, contaminants and natural components of food products.

Mycotoxins present in food products

Mycotoxins are substances created by fungal secondary metabolic processes which are in connection with specific disorders in animals and humans. The action of toxicity in animals is as harmful as the fungal species which produce these compounds.

Aflatoxins in milk products

A notable example of mycotoxins is aflatoxin that usually brings about liver damage and cancer, reducing the milk production and inhibiting immune system.

Aflatoxin M1, is a toxin found in milk extracted from ingested aflatoxin. Identification of M1 is important since it is likely to be found in milk which is an essential source of nutrition for human.

There are significant regulations (throughout the world) concerning the presence of aflatoxin M1 in dairy products.
Afla M1 HPLC is a rapid aflatoxin test from which accurate numerical results can be achieved. Using monoclonal affinity chromatography, Afla M1 can isolate aflatoxin M1 from dairy products.

About the Author

Read more: Aflatoxins. Other subjects: Dayspring | Lynmar

What is HPTLC (High Pressure Thin Layer Chromatography)?

What is High Pressure Thin Layer Chromatography? How does it differ from Thin Layer Chromatography? Is there a link between HPLC and HPTLC?

Features of HPTLC
1. Simultaneous processing of sample and standard - better analytical precision and accuracy less need for Internal Standard
2. Several analysts work simultaneously
3. Lower analysis time and less cost per analysis
4. Low maintenance cost
5. Simple sample preparation - handle samples of divergent nature
6. No prior treatment for solvents like filtration and degassing
7. Low mobile phase consumption per sample
8. No interference from previous analysis - fresh stationary and mobile phases for each analysis - no contamination
9. Visual detection possible - open system
10. Non UV absorbing compounds detected by post-chromatographic derivatization

Steps involved in HPTLC

1. Selection of chromatographic layer
2. Sample and standard preparation
3. Layer pre-washing
4. Layer pre-conditioning
5. Application of sample and standard
6. Chromatographic development
7. Detection of spots
8. Scanning
9. Documentation of chromatic plate

Selection of chromatographic layer

· Precoated plates - different support materials - different Sorbents available
· 80% of analysis - silica gel GF · Basic substances, alkaloids and steroids - Aluminum oxide
· Amino acids, dipeptides, sugars and alkaloids - cellulose
· Non-polar substances, fatty acids, carotenoids, cholesterol - RP2, RP8 and RP18
· Preservatives, barbiturates, analgesic and phenothiazines- Hybrid plates-RPWF254s

Sample and Standard Preparation

To avoid interference from impurities and water vapours
Low signal to noise ratio - Straight base line- Improvement of LOD
Solvents used are Methanol, Chloroform: Methanol (1:1), Ethyl acetate: Methanol (1:1), Chloroform: Methanol: Ammonia (90:!0:1), Methylene chloride : Methanol (1:1), 1% Ammonia or 1% Acetic acid
Dry the plates and store in dust free atmosphere

Activation of pre-coated plates

Freshly open box of plates do not require activation
Plates exposed to high humidity or kept o­n hand for long time to be activated
By placing in an oven at 110-120ºc for 30’ prior to spotting
Aluminum sheets should be kept in between two glass plates and placing in oven at 110-120ºc for 15 minutes.

Application of sample and standard

· Usual concentration range is 0.1-1µg / µl
· Above this causes poor separation
· Linomat IV (automatic applicator) - nitrogen gas sprays sample and standard from syringe o­n TLC plates as bands
· Band wise application - better separation - high response to densitometer

Selection of mobile phase

- Trial and error
- one’s own experience and Literature
- Normal phase
- Stationary phase is polar
- Mobile phase is non polar
- Non-polar compounds eluted first because of lower affinity with stationary phase
- Polar compounds retained because of higher affinity with the stationary phase
· Reversed phase
- Stationary phase is non polar
- Mobile phase is polar
- Polar compounds eluted first because of lower affinity with stationary phase
- Non-Polar compounds retained because of higher affinity with the stationary phase
- 3 - 4 component mobile phase should be avoided
- Multi component mobile phase o­nce used not recommended for further use and solvent composition is expressed by volumes (v/v) and sum of volumes is usually 100
- Twin trough chambers are used o­nly 10 -15 ml of mobile phase is required
-· Components of mobile phase should be mixed introduced into the twin - trough chamber

Pre- conditioning (Chamber saturation)

· Un- saturated chamber causes high Rf values
· Saturated chamber by lining with filter paper for 30 minutes prior to development - uniform distribution of solvent vapours - less solvent for the sample to travel - lower Rf values.

Chromatographic development and drying

· After development, remove the plate and mobile phase is removed from the plate - to avoid contamination of lab atmosphere
· Dry in vacuum desiccator - avoid hair drier - essential oil components may evaporate

Detection and visualization

· Detection under UV light is first choice - non destructive
· Spots of fluorescent compounds can be seen at 254 nm (short wave length) or at 366 nm (long wave length)
· Spots of non fluorescent compounds can be seen - fluorescent stationary phase is used - silica gel GF
· Non UV absorbing compounds like ethambutol, dicylomine etc - dipping the plates in 0.1% iodine solution
· When individual component does not respond to UV - derivatisation required for detection

Quantification

· Sample and standard should be chromatographed o­n same plate - after development chromatogram is scanned
· Camag TLC scanner III scan the chromatogram in reflectance or in transmittance mode by absorbance or by fluorescent mode - scanning speed is selectable up to 100 mm/s - spectra recording is fast - 36 tracks with up to 100 peak windows can be evaluated
· Calibration of single and multiple levels with linear or non-linear regressions are

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Supelco, a division of Sigma-Aldrich® (Nasdaq: SIAL ), announced today the launch of Ascentis Express Peptide ES-C18, a high-speed, high-performance liquid chromatography (HPLC) column based on a new 160 angstrom Fused-Core™ particle design.

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