总游离亚硫酸盐检测试剂盒 Total and Free Sulphite Assay Kit 货号:K-SULPH Megazyme中文站

总游离亚硫酸盐检测试剂盒

英文名:Total and Free Sulphite Assay Kit

货号:K-SULPH

规格:40 assays (manual) / 400 assays (microplate)

A rapid, simple, reliable and accurate method for the measurement and analysis of total sulfite (sulphite) and free sulfite in wine, beverages, foodstuffs and other materials. Supplied as a “ready to use” liquid stable formulation that is suitable for manual, auto-analyser and microplate formats.

Suitable for manual, auto-analyser and microplate formats.

Colourimetric methods for the determination of Total and Free
Sulfite in wine, fruit juice, foodstuffs and other materials

Principle:
The Total Sulfite assay is based on the reaction principle
between thiol groups and Ellman’s reagent


The Free Sulfite assay is based on the reaction principle of
SO2, fuchsin and aldehyde binding

Kit size: 40 assays (manual) / 400 (microplate)
/ 400 (auto-analyser)
Method: Total sulfite: Spectrophotometric at 405 nm
Free sulfite: Spectrophotometric at 575 nm
Total assay time: Total sulfite: ~ 6 min
Free sulfite: ~ 9 min
Detection limit: Total sulfite: ~ 5 mg/L
Free sulfite: ~ 2 mg/L
Application examples:
Wine, fruit juice, seafood, food stuffs and other materials
Method recognition:
Validated for red and white wines at the Bundesamt für Weinbau, Austria.
Used widely in the wine industry

Advantages

  • ”Ready to use" liquid stable Formulation
  • Very competitive price (cost per test)
  • All reagents stable for > 18 months
  • Very rapid reaction
  • Mega-Calc™ software tool is available from our website for hassle-free raw data processing
  • Standard included
  • Suitable for manual, microplate and auto-analyser formats

 Q1. What concentration of acetaldehyde in the sample causes interference in the total sulphite (TSO2) assay?

Interference by acetaldehyde is observed when the acetaldehyde concentration is higher than approximately 250 mg/L in the 0.05 mL sample using the TSO2 Manual Assay Procedure (see Table 1). 
At acetaldehyde concentrations higher than 250 mg/L the total sulphite reaction is slower than stated in the TSO2 Manual Assay Procedure but generates the expected absorbance change when the reaction is allowed to complete.  At a concentration of 1250 mg/L acetaldehyde in the sample, the TSO2 reaction takes ~ 15 minutes to complete (at 25°C).

[Acetadehyde]

(mg/L)

[SO2]

mg/L

 

A1 

 

A2 

 

ΔAtotal SO2 

 

% error

0 

300

0.042

1.477

1.435

0.0

31 

300

0.041

1.458

1.417

1.3

63 

300

0.042

1.502

1.460

-1.7

125

300

0.042

1.498

1.456

-1.4

250

300

0.041

1.466

1.425

 0.7

625

300

0.042

1.347

1.305

 9.1

1250

300

0.045

0.279

0.234

83.7

 

 

 

 












TABLE 1. 
Acetaldehyde Interference in the TSO2 Manual Assay.  Reactions were performed using the TSO2 Manual Assay Procedure in 1 cm path-length cuvettes at 25°C.

Q2. Should the pH of the sample be adjusted even for samples in acidic media?

The pH of the assay solution after the sample is added should be the same as that of the assay buffer that is supplied with the kit.
Low sample volumes (e.g. 0.1 mL) are not likely to affect the pH of the assay solution and therefore may not require pH adjustment.
Samples above 0.1 mL are more likely to affect the pH of the assay solution and therefore the pH of these samples should be adjusted as described in the data booklet, prior to addition to the assay.

Q3. How stable are the sulphite standards and samples?

When the samples are prepared and are stored at the appropriate pH (~ pH 8 for total sulphite and ~ pH 4 for free sulphite) they will be expected to lose ~ 2% sulphite per hour.

Q4. There is an issue with the performance of the kit; the results are not as expected.

If you suspect that the Megazyme test kit is not performing as expected such that expected results are not obtained please do the following:

  1. Ensure that you have tested the standard sample that is supplied with the Megazyme test kit.
  2. Send the results of the kit standard, blank samples and the results obtained for your sample, in the relevant MegaCalc spreadsheet (if available) to Megazyme ([email protected]). Where available the relevant MegaCalc spreadsheet can be downloaded from where the product appears on the Megazyme website.
  3. State the kit lot number being used (this is found on the outside of the kit box).
  4. State which assay format was used (refer to the relevant page in the kit booklet if necessary).
  5. State exact details of any modifications to the standard procedure that is provided by Megazyme.
  6. State the sample type and describe the sample preparation steps if applicable.

Q5. Can K-SULPH be used to measure sulphite in food samples?

Yes.  K-SULPH can be used to measure total sulphite and free sulphite in food samples using the standard sample preparation procedures given below.

Sample preparation for Total Sulphite (TSO2)
Homogenise approx. 5 g of sample with 60 mL of distilled water using a mortar and pestle or standard homogeniser for approximately 2 min.  Adjust to approximately pH 8.0 using 1 M NaOH or 1 M HCl.  Quantitatively transfer the mixture to a 100 mL volumetric flask, fill up to mark with distilled water, mix and filter through Whatman No. 1 filter paper or centrifuge at 13000 x g.  If required, dilute the sample using 20 mm sodium phosphate buffer (pH 8.0).

Sample preparation for Free Sulphite (FSO2)
Homogenise approx. 5 g of sample with 60 mL of distilled water using a mortar and pestle or standard homogeniser for approx. 2 min.  Adjust to approximately pH 4.0 using 1 M NaOH or 1 M HCl.  Quantitatively transfer the mixture to a 100 mL volumetric flask, fill up to mark with distilled water, mix and filter through Whatman No. 1 filter paper or centrifuge at 13000 x g.  If required, dilute the sample using 1% (w/v) citric acid.

Notes:
1.It is highly recommended that samples are tested immediately after sample preparation.
2.The amount of sulphite obtained in the final sample must be within the detectable range of the test.  Since the amount of sulphite present will vary between samples the amount of original sample used in the preparation method may have to be experimentally determined.

Q6. Can you explain, step by step, how to follow the method and perform the kit assay?

For users who are not familiar with how to use the Megazyme tests kits then it is recommended that they follow this example, e.g. D-Fructose/D-Glucose Assay kit K-FRUGL (http://secure.megazyme.com/D-Fructose-D-Glucose-Assay-Kit):

1. The kit components are listed on pages 2-3 of the kit booklet.
2. Prepare the kit reagents as described on page 3.
3. For separate measurements of glucose and fructose follow procedure A on page 4.
4. Pipette the volumes listed for water, sample, solution 1 and solution 2 into 3 mL, 1 cm pathlength cuvettes. Duplicate sample assays and duplicate blanks are recommended. Mix the contents of each cuvette by inversion (seal the cuvette using parafilm or a plastic cuvette cap – do not use a finger) then after ~3 min record the first absorbance reading of each cuvette at 340 nm (this is reading A1).
5. Then add suspension 3 and mix the contents of each cuvette by inversion. Incubate for 5 minutes then record the absorbance reading of each cuvette at 340 nm (this is reading A2). NB. It is essential that the reaction is compete. To assess this, record the absorbances at ~ 2 minute intervals and until the absorbance plateaus. A stable absorbance indicates that the reaction is complete. If the absorbance continues to increase then continue to record absorbances until it plateaus and only then record absorbance reading A2.
6. Then add suspension 4 and mix the contents of each cuvette by inversion. Incubate for 5 minutes then take absorbance reading of each cuvette at 340 nm (this is reading A3). NB. As above, assess that the reaction has completed by take subsequent readings at ~2 min intervals.
7. For simple, automated results analysis, input the absorbance readings (A1, A2, A3) for samples and blanks into the 
K-FRUGL MegaCalc.

To ensure that the assay is working, and being performed correctly it is recommend that the test is performed using the standard sample that is provided with the kit and to obtain the expected values before proceeding to test real samples.
It is recommend that new users also watch 
this video which highlights how to perform the assays.
Many of the other Megazyme test kits follow a similar format.

Q7. The pH of my sample is low (pH ~ 3.0), do I need to adjust this before I use the sample in the kit assay?

The final pH of the kit assay after the sample is added should not change from what it should be (as stated in the kit for the assay buffer). If it does change then the sample will require pH adjustment. In most cases the sample volume being used is low relative to the final assay volume and in this case the pH of the kit assay is unlikely to be affected.

Q8. How can I work out how much sample to extract and what dilution of my sample should be used in the kit assay?

Where the amount of analyte in a liquid sample is unknown, it is recommended that a range of sample dilutions are prepared with the aim of obtaining an absorbance change in the assay that is within the linear range.
Where solid samples are analysed, the weight of sample per volume of water used for sample extraction/preparation can be altered to suit, as can the dilution of the extracted sample prior to the addition of the assay, as per liquid samples.

Q9. I have some doubts about the appearance/quality of a kit component what should be done?

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