Molar excess quantity

It is of importance to both experimentalists and theoreticians that the differential and the integral molar excess quantities should not be confused. 

Finally, in analogy with Eq. 6.1-12, we define a partial molar excess quantity by the relation... 

So the remaining partial molar excess quantities can all be expressed as simple functions of the activity coefficient. 

A 10% molar excess of the oxonium salt with regard to the carboxylic acid gives slightly higher yields than does an equimolar quantity. 

It was mentioned earlier (Sec. 8.6) that for iodo-de-diazoniation no catalyst is necessary because the redox potential of the iodide ion (E° = 1.3 V) is sufficient for an electron transfer to the arenediazonium ion. The reaction was actually observed by Griess (1864 c). Four iodo-de-diazoniation procedures are described in Organic Syntheses. For the syntheses of iodobenzene and 4-iodophenol (Lucas and Kennedy, 1943, and Daines and Eberly, 1943, respectively) KI is used in equimolar quantity and in 1.2 molar excess. However, for 2-bromoiodobenzene and for 1,3,4-triiodo-5-nitrobenzene (replacement of a diazonio group in the 4-position by iodine), up to... 

Add N-acetyl homocysteine thiolactone (Aldrich) to the bicarbonate reaction mixture to obtain a concentration representing a 10- to 20-fold excess over the amount of amines present. For protein thiolation, add the same molar excess of thiolactone reagent to the water reaction medium, and then slowly add an equivalent molar quantity of silver nitrate (AgNO j). Maintain the pH at 7.0-7.5 with periodic addition of NaOH. 

Add a quantity of succinic anhydride to the reaction medium to provide at least a 5-10 molar excess of reagent over the amount of amines to be modified. Even greater molar excess may be required for total blocking of all the amines of some proteins. When adding solid succinic anhydride, multiple additions may be done to maintain solubility of the reagent in the reaction solution. The anhydride also may be dissolved in dry dioxane before addition to aid in dissolution. 

Add iodoacetate to a concentration of 50mM in the reaction solution. Alternatively, add a quantity of iodoacetate representing a 10-fold molar excess relative to the number of —SH groups present. An estimation of the sulfhydryl content in the protein to be modified can be accomplished by performing an Ellman s assay (Chapter 1, Section 4.1). Readjust the pH if necessary. To aid in adding a small quantity of iodoacetic acid to the reaction, a concentrated stock solution may be made in the reaction buffer, the pH re-adjusted, and an aliquot added to the protein solution to give the desired concentration. 

Add a quantity of BMPA to the protein solution to obtain at least a 5-fold molar excess of maleimide reagent over the amount of thiol present in the protein. The final concentration of organic solvent in the protein solution should not exceed 10 percent to prevent protein precipitation. Mix thoroughly to dissolve. 

Add a quantity of Aminoethyl-8 in methanol to equal a 25-fold molar excess over the amount of sulfhydryl present (including the amount of DTT added). The solution in methanol should be made concentrated enough so only a small amount of methanol has to be added to the reaction solution (i.e., no more than 10 percent of the final volume). A second addition of modifying agent may be made after 1 hour to drive the reaction more completely toward total —SH aminoalkylation. 

Add a quantity of 2-bromoethylamine to obtain a 10-fold molar excess over the number of sulfhydryls present in the sample, including any added DTT. 

Add a quantity of glutaraldehyde equal to a 10-fold molar excess over the amount of amines to be modified. A typical concentration of glutaraldehyde in the reaction mixture is 1.25 percent. In some cases, trial experiments will have to be done to check for solubility of the resultant modified protein. Scale back the quantity of glutaraldehyde added if precipitation occurs. 

Add a quantity of adipic acid dihydrazide or carbohydrazide (Aldrich) to the protein solution to obtain at least a 10-fold molar excess over the amount of aldehyde functionality present. High molar ratios are necessary to avoid protein conjugation during the reaction process. If the concentration of aldehydes is unknown, the addition of 32mg adipic acid dihydrazide per ml of the protein solution to be modified should work well. 

With mixing, add a quantity of the mono(lactosylamido) mono(succinimidyl)suberate in dry DMF to the protein solution to result in a 10-20 fold molar excess of reagent over the amount of protein present. Depending on the desired application for the lactosyl-modified protein, several different molar ratios of reactant-to-protein may have to be tried to optimize the resulting modification level. 

Add at least a 10-fold molar excess of NEM over the amount of sulfhydryls present in the reaction. Alternatively, add an equal mass of NEM to the amount of macromolecule present. To facilitate the addition of a small quantity of reagent, a more concentrated stock solution may be prepared in buffer and an aliquot added to the reaction medium. Make the stock solution up fresh, and use it immediately to prevent loss of activity due to maleimide group breakdown. 

Add EDC (Thermo Fisher) to the above solution to obtain at least a 10-fold molar excess of EDC to the protein. Alternatively, a 0.5-0.1 M EDC concentration in the reaction mixture usually works well. To make it easier to add the correct quantity of EDC, a higher concentration stock solution may be prepared if it is dissolved and used immediately. To prepare the peptide-protein conjugate, add the solution from step 3 to 10 mg of EDC in a test tube. Mix to dissolve. If this ratio of EDC to peptide or protein results in precipitation, scale back the amount of carbodiimide addition until a soluble conjugate is obtained. For some proteins, as little as 0.1 times this amount of EDC may have to be used to maintain solubility. 

Weigh out 2 mg of sulfo-SMCC and add it to the above solution. Mix gently to dissolve. To aid in measuring the exact quantity of crosslinker, a concentrated stock solution may be made in water (or DMSO) and an aliquot equal to 2 mg transferred to the reaction solution. If a stock solution is made, it should be dissolved rapidly and used immediately to prevent extensive hydrolysis of the active ester. As a general guideline of addition for a particular protein activation, the use of a 40- to 80-fold molar excess of crosslinker over the amount of protein present usually results in good activation levels. 

Since the active ester end of the molecule is subject to hydrolysis (half-life of about 20 minutes in phosphate buffer at room temperature conditions), it should be coupled to an amine-containing protein or other molecule before the photolysis reaction is done. During the initial coupling procedure, the solutions should be protected from light to avoid decomposition of the phenyl azide group. The degree of derivatization should be limited to no more than a 5- to 20-fold molar excess of sulfo-SBED over the quantity of protein present to prevent possible precipitation of the modified molecules. For a particular protein, studies may have to be done to determine the optimal level of modification. 

Add 50 pi of the NHS-PEGg-maleimide solution to the 1ml dendrimer solution and mix thoroughly to dissolve. This represents approximately a 14-fold molar excess of crosslinker over the quantity of dendrimer present, if a G-3 PAMAM dendrimer is used with an ethylenediamine core. The optimum molar ratio of crosslinker-to-dendrimer should be determined experimentally for best performance of the resultant conjugate in its intended application. If enough material is available, doing a series of experiments at different mole ratios of crosslinker-to-dendrimer will help to optimize the resultant conjugate. 

Add a quantity of triethylamine to the solution so that a 25 percent molar excess over the amount of anhydride will result (1.25 ml for the G-5 dendrimer). 

Add 200 mg of sodium carbonate per ml of the dendrimer solution prepared in step 1 and a quantity of epibromohydrin equal to a 285-fold molar excess over the amount of... 

Add a 3-fold molar excess of biotinylation reagent over the molar quantity of dendrimer present. For the use of sulfo-NHS-LC-biotin (MW 556), this represents the addition of 2.1pmol or 1.16mg. This reaction ratio will result in a modification level of about 2.5 biotin groups per dendrimer. Other molar ratios also may be used, depending on the desired level of modification and the intended use for the conjugate. 

Add a quantity of the DyLight 649 dye to the dendrimer solution to provide at least a 1.25-fold molar excess of dye over the amount of dendrimer present (for nonaqueous reactions) or a 6-15-fold molar excess for aqueous reactions. Mix well to dissolve. The optimal amount of dye added should be determined experimentally by preparing a series of conjugates using different molar ratios of dye-to-dendrimer. 

In subdued lighting conditions, add 25-50 pi of the fluorescein solution to each ml of protein solution while mixing. Alternatively, determine the exact molar quantity of protein present and add a 25-fold molar excess of fluorescein-5-maleimide solution. 

With mixing, add a quantity of the dye solution to the protein solution to provide the desired molar excess of dye over protein. For instance, for an antibody dissolved in buffer at lOmg/ml, the addition of 66pi of dye solution will give a 10-fold molar excess of dye over protein. 

Add a quantity of the protein solution to the QD solution with mixing to obtain the desired molar excess of protein over the concentration of nanoparticles. Using a 1- to 20-fold molar excess typically works well, but optimization should be done to determine the best ratio for a particular application. 

With mixing, add a quantity of the sulfo-NHS-biotin solution to the protein solution to obtain a 12- to 20-fold molar excess of biotinylation reagent over the quantity of protein present. For instance, for an immunoglobulin (MW 150,000) at a concentration of 10 mg/ml, 20 pi of a sulfo-NHS-biotin solution (containing 8 X 10-4 mmol) should be added per ml of antibody solution to obtain a 12-fold molar excess. For more dilute protein solutions (i.e., 1-2 mg/ml), increased amounts of biotinylation reagent may be required (i.e., 20-fold molar excess or more) to obtain similar incorporation yields as when using more concentrated protein solutions. 

Add a quantity of photobiotin solution to the protein solution to give at least a 5-fold molar excess of biotinylation reagent. 

Add a quantity of the appropriate isobaric tag solution to each sample to provide a final concentration of 10-20 mM. This quantity of reagent will assure a large molar excess of reagent over the concentration of peptides present in order to modify completely all peptides at their N-terminus. Note that the e-amino groups of lysine residues also will be modified by this procedure. React for 1 hour at room temperature. 

Add a quantity of SANH or SFB solution to the oligo to provide a 40-fold molar excess of crosslinker over the amount of amine-oligo present. Mix well. 

Add a quantity of the crosslinker solution of choice (SANH or SFB) to the antibody solution to obtain the desired molar excess of reagent over the antibody. Typically, antibody modification procedures are done with 10- to 20-fold molar excess, but for dilute antibody concentrations, this may have to be doubled, depending on how many hydrazine or aldehyde groups are desired to be introduced on the modified antibody. 

Add a quantity of PDBA-NHS to the protein solution to provide the desired molar excess of crosslinker over the protein. A suggested starting point is to use a 10- to 15-fold molar excess of reagent, but the optimal amount to be added should be determined by experimentation to provide a final conjugate having the best possible properties for the intended application. The PDBA-NHS may be first dissolved in DMF as a concentrated stock solution and then an aliquot added to the reaction mixture. Mix well to dissolve. 

Add a quantity of NHS-salicylic acid methyl ester reagent dissolved in coupling buffer to the slide surface to provide at least a 2-fold molar excess of crosslinker over the quantity of amines present on the surface. The surface of the slide may be coated with a minimum solution volume of the crosslinker by layering the solution over the surface. Slide masks or gaskets may be used to isolate only certain regions for modification. Alternatively, the slide may be immersed in the crosslinker solution. The NHS-salicylic acid methyl ester may be first dissolved in DMF as a concentrated stock solution and then an aliquot... 

Add a quantity of the crosslinker solution to the protein solution to provide a 1- to 10-fold molar excess of reagent over the concentration of protein. The use of lower molar ratios will limit the potential for oligomerization of proteins in solution. A series of reactions using different concentrations of crosslinker may have to be done to determine the optimal level to use for a particular application. 

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