How much blood did you use? What was done to the first sample? What different was done to the second sample? A current of 1. When a similar solution was mixed with 0. What is the concentration of the ascorbic acid in the sample? Standard Addition with Multiple solutions. The first solution on the left has no standard. Why does it have color? This approach is common in the analysis of metal ions. Now that the basics of the method of standard addition have been outlined, and the basics of the experiment explained, let's perform the technique in the laboratory.
First, prepare the ppm aluminum stock solution in water, and then use it to prepare a 1-ppm standard solution. Carefully add 5. This step enables the 8HQ to dissolve in the aqueous phase. Verify the pH with a pH indicator stick. This buffer helps neutralize the acid in the 8HQ solution when combined. Now prepare the samples, in this case by extracting the aqueous sample into the organic phase using liquid-liquid extraction.
Place six mL separatory funnels onto ring-stand rings inside the hood. Make sure all glassware is scrupulously clean, as dirty glassware will skew results. Sequentially label the funnels "blank", "0", "1", "2", "3", and "4". Using a pipette, add 25 mL of the unknown aluminum solution to each of the five separatory funnels labeled "0" through "4".
Prepare the blank by adding 25 mL of deionized water to the funnel labeled "blank". Next, add 1, 2, 3, and 4 mL of the 1-ppm standard solution to the corresponding numbered funnels.
Add no standard solution to the blank or 0 funnels. Perform a liquid-liquid extraction by adding 10 mL of chloroform to each flask. Shake the funnel vigorously, and occasionally vent the funnel to release pressure buildup. Place the funnel back into the ring, and allow the liquid layers to separate. Next, collect the chloroform phase in a clean, dry, and labeled mL beaker.
Since chloroform has a higher density than water, it is the lower layer in the funnel. Transfer the chloroform extract into a mL volumetric flask, and cap each flask to prevent evaporation. Perform a second liquid-liquid extraction on the remaining aqueous solution, by adding 10 mL of chloroform to each funnel. Shake the funnel, as before, to transfer any remaining analyte to the chloroform phase. There should be no yellow color left in the top aqueous phase.
Repeat the second extraction for each funnel, then collect the chloroform phases in corresponding labeled beakers. Pour the collected chloroform into their respective volumetric flasks, and dilute to the mark with fresh chloroform. To remove trace water, add about 1 g of anhydrous sodium sulfate to each of the six mL beakers.
Transfer the solutions back into their respective beakers, and swirl to facilitate dehydration of the sample. Set up the fluorimeter according to the manufacturers instructions and set the voltage to V. Next, open the data acquisition program on the computer. Use sample 2 to determine the best excitation and emission wavelengths.
From the fluorescence plot, determine the maximum wavelength for excitation. Set the instrument to that excitation wavelength value, in this case nm.
Next, determine the emission wavelength by performing a scan from — nm. From the resulting fluorescence plot, determine the maximum wavelength and set the emission wavelength, in this case nm. Measure each sample, including the blank at the selected excitation and emission wavelength. Record each fluorescence intensity reading. Plot the fluorescence intensity of each of the five samples versus the amount of aluminum added to the sample.
Determine the least squares value of the resulting plot, and record the slope and intercept. The plot of fluorescence intensity vs. The amount of aluminum in the sample can then be calculated using this line. Since the amount of unknown added was 25 mL, the determined value, 2. This gives a final result of 0. This is quite close to the known value of 0. Now, let's look at some other analytical techniques that can have skewed results due to matrix effects.
Atomic absorption spectroscopy is an analytical method that measures the absorbance of light by a target analyte in the gaseous phase. For most samples, a simple calibration curve relating absorption to sample concentration, can serve as a reliable method to quantify an unknown concentration.
However, this technique can lose accuracy if other components of the mixture interact with the target analyte and suppress or enhance absorption. The standard addition method can be used in this case to account for the effects of these interactions, especially in samples where the matrix cannot be removed prior to analysis. Instrument calibration plays a crucial role in the accuracy of a measurement. ICP-MS is a comparative method, meaning that the measurement of an unknown sample is based on the measurement of a chemical standard.
Thus, the uncertainty of a measurement of an unknown can't be better than the uncertainty of the calibration. The method of standard addition can therefore be used to create a calibration curve that is more accurate than the standard method, and accounts for matrix interactions in the sample. Many biological molecules are analyzed using high-performance liquid chromatography, or HPLC.
HPLC is a technique that separates and analyzes complex mixtures based on molecule properties such as polarity, charge, and size. The time at which the analyte leaves the column enables the user to identify each component in the mixture. Biological molecules can often interact in a mixture, and are greatly affected by the matrix they are suspended in. Often, the method of standard addition is used to create a calibration curve that accounts for these affects.
You've just watched JoVE's introduction to the method of standard addition. You should now understand how to perform the technique to account for matrix effects in sample analysis. A scan of the excitation wavelength from — showed the highest absorption at nm, so the excitation monochromator was set for that value. Then the emission scan was performed from — nm, and the strongest signal was found to be at nm. These are the wavelengths that are used for all of the samples.
A plot of fluorescence Figure 3 vs. Since the amount of unknown added was 25 mL, then the 2. Figure 3. Fluorescence of the samples. Figure 4. The standard addition calibration plot. The method of standard additions is often the technique utilized when accurate quantitative results are desired, used in analytical analysis such as atomic absorption, fluorescence spectroscopy, ICP-OES, and gas chromatography. Hope you don't mind: I reference this blog in the documentation and show how to use the add-in to get the same results.
The add-in grabs the inverse prediction points from a separate JMP table and copies results back into that table for convenience. It also offers a four-parameter logistic model and handles multiple by variables. Kindly, Russ. I'm glad that you liked it, Russ! By all means - please do reference it whenever it could be of use to your add-in's users! You must be a registered user to add a comment. If you've already registered, sign in. Otherwise, register and sign in.
Sign In. Turn on suggestions. Auto-suggest helps you quickly narrow down your search results by suggesting possible matches as you type. Showing results for. Show only Search instead for. Did you mean:. Choose Language Hide Translation Bar. Determining chemical concentration with standard addition: An application of linear regression in JMP. Created: Jun 4, AM. Overcoming Matrix Interferences with Standard Addition An effective and commonly used technique to overcome matrix interferences is standard addition.
Procedurally, here are the steps for preparing the samples for analysis in standard addition: 1 Obtain several samples of the solution containing the analyte in equal volumes.
After adjusting the axes and adding some captions, I get the following plot: This plot illustrates the key idea behind using this calibration curve. First, let's run the linear regression again by "Fit Model" under the "Analyze" menu. Here is the output from "Fit Model". A prediction interval takes into account two sources of variation: Variation in the estimation of the mean x-value. Variation in the sampling of a new observation. Here is the output that has been added to the bottom of this results window.
Conclusion Standard addition is a simple yet effective method for determining the concentration of an analyte in the presence of other chemicals that interfere with its analytical signal. References J. Everyone's tags 5 : analytical chemistry. Article Labels. There are no labels assigned to this post. Article Tags. Tags: analytical chemistry. Lou V wrote: Eric, Were any replicate samples taken in this study?
Eric Cai wrote: Hi Lou, If I understand what you mean by "replicate samples" correctly, you are wondering if multiple absorbances were measured at each concentration of silver. Phil Kay wrote: Hi Eric, Great post. A perfect example of how useful JMP is for analytical chemists. Lou V wrote: Hi Eric, This inverse prediction provided for a very useful tool for analyzing my mortgage data.
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