Biotin Immunoaffinity HPLC Column


The Biotin Immunoaffinity HPLC Column detects Biotin (Vitamin H, Coenzyme R, Vitamin B7) in food products, cell culture samples by Affinity Chromatography. The HPLC measuring range is linear of 5ng to 40ng Biotin per injection (R2=0.994). The limit of detection is 2ng of biotin per injection (three times of signal/noise ratio). If the given dilution steps are obeyed, the Biotin contents of 20 to 160 ng/g lie within the linear working range of the method. If the contents of used samples are higher than the cited range, extracts or the IAC column elutes should be diluted in a suitable manner. The lower limit of quantization is 10ng/g of Biotin in the sample.  Recovery rates are >85% when Biotin contents in buffer mixtures are analysed in the range of 0.1 to 5µg per IAC.

SKU: BTSA317005 Categories: ,

Biotin Immunoaffinity HPLC Column

The Biotin Immunoaffinity HPLC Column is For Research Use Only

Size: 3ml, 10 columns

Assay Principle

Many methods of biotin determination based on HPLC-UV (High Performance Liquid Chromatography Ultraviolet) detection may show either low sensitivity or low selectivity. This depends on the dilution factor of the matrices and if problematic matrices are applied.  This method of content determination of Biotin combines the high selectivity of affinity columns with its potential to concentrate elute and use high sensitivity biotin detection by post-column labeling with fluorescein- streptavidin conjugate.

Sample Preparation:
Biotin samples are to be extracted and analyzed with the method of Bachas et al. [N.G. Hentz, L.G. Bachas Methods Enzymol. 1997; 279:275-86], e.g. vitamin tablets, liquid vitamin preparations, cell culture extracts. Example: 25g vitamin containing tablets are dissolved in 100ml  PBS. The resulting extract may be filtered through a 0.45µm membrane filter.
Enrichment Step IAC:
4ml extract (containing the quantity of Biotin from a 1g sample of above-mentioned sample preparation is followed) is diluted with a total volume of 20ml PBS and then applied in a reservoir on top of the BioTeZ-Immunoaffinity Column. The optimal flow rate through the gel is between 1 to 3ml/min.

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Product Developed and Manufactured in Germany by BioTeZ Berlin-Buch GmbH

Additional Information

After the whole sample has passed through the gel, the latter is washed with 5ml of PBS. Remaining liquids in the gel are removed by applying either pressure from top of the column or pressure from the bottom.

The sample reservoir on top of the BioTeZ-Immunoaffinity Column is removed, and an appropriate vial is placed below the affinity column. The bounded biotin is eluted by using a total volume of 3ml of HPLC grade methanol.  The elution process is performed in two steps. First, an amount of 1ml methanol is applied. Once this amount has passed through the column, there should be a waiting time of 30 seconds. After that, the second portion of 2ml of methanol is eluted through the column. The flow rate should lie below 3ml/min. The remaining methanolic solutions are eluted by application of slight under- or overpressure. All methanolic fractions are unified to give the column elute.

The column elute may be injected into the HPLC directly or, if concentrations are very low, concentrated by evaporation at 50°C for 1h (e.g. using VLM evaporator), re-dissolved in HPLC solvent and finally injected into the system. For the latter case, please see the sample calculation in which the sample concentrate is re-dissolved in 0.4ml HPLC solvent.


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