Sometimes I get negative OD readings (at 540 nm) when determining reducing sugars using glucose as standard (Miller, 1959). When I use Microsoft Excel to plot the glucose standard curve, the line that gives the highest R-square value (0.99) doesn't always pass through the origin, and when I try to make it pass through the origin, R-square value decreases (around 0.96 or 0.95). If I use the equation of the line that gives the highest R value, I will be getting higher values for glucose when the OD reading is negative. I am sure that negative OD means no sugar. Should I just enter sugar concentration for samples with negative OD540 as zero? What is the best way to handle this?
I agree with everyone who said you shouldn't be getting negative values and that the machine should be recalibrated of this continues. Your reference sample should contain no analyte and its reading should be identical to background. It is possible that the device you're using is broken and "forgets" the reference value. The easiest way to check it is to blank the machine and then read the reference sample (not blank again!) every few samples to check if it still is at 0.00. If it's not, none of your readings is reliable.
Regarding your analysis, Excel is really stubborn when it comes to linear regression and in general is not great for this purpose. Your standard curve should always cut the Y axis at the value that represents 0 analyte. If you blanked the spec at the start this will always be 0;0. The problem is the assumptions behind plotting of the trend line in excel are different and this is the line of the best fit, not always biochemically relevant and you have to force it through 0;0 or your linear regression won't be accurate. It is Aldo not essential to always have R2=0.99. 0.95 is perfectly fine too. To improve that you could, or even should, remove the standard values that are outside the linear range. However, you !UST keep in mind that if any of your samples falls out of the linear range, it has to be diluted, concentrated or discarded. In the first two examples you correct the reading value to the real value manually later.
The negative value is direct imprint of error brought by improper handing of instrument may be calibration or reference sample. Such value must removed by re-calibrating instrument and choosing proper reference.
1. See that temperature of sample and reference must be same.
2. Machine must be calibrated
3. Use chemicals and other reagent from same source.
4. Avoid handling error.
Hii...
Mushafau Adebayo Oke..
Even i also faced such problems few months back..Following points may help to sort out the problem.
1. Check the machine properly calibrated or not.
2. Check your reagents (all the reagents should be prepared in same batch)
3. Are you getting straight line graph for the glucose standards with R2 value >0.98, if yes then there is problem with your samples (may be samples are too diluted/ or have negligible activity).
4. Always use positive control, e.g. Commercial enzyme (Cellulase from Aspergillus, Sigma) for each reaction.
Best wishes..
It indicates that your detector has some moisture or there is an overheating or filter is unclear. Or there is a background contamination in your reagents and samples.
Hi,
I suggest:
1- Check if the instrument is calibrated.
2-Check physical properties of DNS.
3-Repeat the experiment in more replicates.
Best,
Check the sensitivity of your method and instrument expanding the range of standards..All spectrophotometric methods work well within their detection limit..
I agree with everyone who said you shouldn't be getting negative values and that the machine should be recalibrated of this continues. Your reference sample should contain no analyte and its reading should be identical to background. It is possible that the device you're using is broken and "forgets" the reference value. The easiest way to check it is to blank the machine and then read the reference sample (not blank again!) every few samples to check if it still is at 0.00. If it's not, none of your readings is reliable.
Regarding your analysis, Excel is really stubborn when it comes to linear regression and in general is not great for this purpose. Your standard curve should always cut the Y axis at the value that represents 0 analyte. If you blanked the spec at the start this will always be 0;0. The problem is the assumptions behind plotting of the trend line in excel are different and this is the line of the best fit, not always biochemically relevant and you have to force it through 0;0 or your linear regression won't be accurate. It is Aldo not essential to always have R2=0.99. 0.95 is perfectly fine too. To improve that you could, or even should, remove the standard values that are outside the linear range. However, you !UST keep in mind that if any of your samples falls out of the linear range, it has to be diluted, concentrated or discarded. In the first two examples you correct the reading value to the real value manually later.
I also concur that negative value may be due to machine problem .
Because I have also experience this problem, I have found that the problem may also due to disparity in the volume of the DNS reagent used for both the SUBSTRATE STANDARD (spectro zero) and that of the test enzyme (analytes).
Do ensure equal volumes and reconfirm. I am sure it will work
Volume disparity often occur when you compare large samples and you want to ensure equal time of measurement for enzyme incubation (hydrolysis). Usually, one will hastily add DNS reagent leading to probable disparity in the amount of added DNS reagent.
Hi Mushafau Adebayo Oke,
Check the age of DNS reagent,
Prepare freshly every time before going to use
Check the concentration of NaOH/ volume? how much amount you are going to add in to the DNS reagent. It may cause loss of sugar during longer incubation period.
Minimise the handling errors
As every one said above calibrate/check the detector of the instrument.
All the Best
Once the blank is 0, reference samples should not be less than zero or even negative. It should be above zero. Adam Ostrowski, can you clarify your reference being zero as you stated above after blanking?
Dear Mushafau Adebayo Oke,
I certainly agree that there may be problem with instrument & if so require recalibration. However, one might often face such a problem if you have not prepared blank properly. For instance, either you might have not added specific volume of the solvent (such as buffer, 80% ethanol or 0.1/0.5/5% TCA) in which you have/prepared sugar [irrespective of if you have extracted from any sample or obtained through enzyme (cellulase, amylase) activity] or added distilled water in place of the actual solvent. Can you please let me know, how exactly you prepared the blank and what are the samples in which you are estimating sugars?
Can you also let me know, what exactly is the composition of DNS reagent you used and how exactly you performed the assay? Did you add Rochelle's salt along with DNS or you added it after heating sample with DNS (without Rochelle's salt) reagent?
You can prepare every time a solution with a known concentration of glucose to be sure if is a problem of a calibration curve or of a reagent that you use for the assay.
As suggested by many, I will recommend you to prepare a standard curve of know glucose concentration with OD. The same can be used to calculate the concentration of Glucose or any other reducing sugars in the sample.
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