The reagents used in the various tests come in a variety of forms. These include pure solid and liquid chemicals as well as solutions made up from several chemicals. Because protein test reagents need careful care in preparation, storage, and use, there are special sections provided for each of them. Many analytical reagents need similar care, and so review of information given for the protein reagents will give information relevant to other applications as well. Definitions of some concentration units used are provided under Solution Concentration Definitions.
Whenever a solution is to be prepared, the accuracy requirements of the preparation must be known and appropriate equipment selected. For example, dilution of Reagent and Reference Dye concentrates must be done as accurately as possible using volumetric flasks. Another instance when accurate measurement must be used is in preparation of the "Working" Reference Dye Solution. Measuring with a graduated cylinder or beaker is not accurate enough for preparing these solutions.
When small volumes of a solution are to be measured accurately, a pipet is commonly used.
If accuracy requirements are not given explicitly, assume that variance within 1 digital of the smallest digit given is acceptable. For example, if 2 is specified, 1 to 3 is acceptable; if 2.0 is given, 1.9 to 2.1 is required.
When reagents are purchased as concentrates or pure chemicals, they are stable, and the shelf life is not a consideration unless otherwise noted. Once a reagent is prepared for use, its quality may deteriorate with time. For example, dye solutions used in protein tests change concentration with time due to evaporation of water. This is not important for concentrates since the water lost is replaced when the concentrate is diluted to the correct volume with water. Evaporation loss is important, however, for ready-to-use solutions.
Protein testing involves several solutions of Acid Orange 12 dye. Each test is made using Reagent Dye Solution. A measurement is made using the Digital Colorimeter which is set using Reagent Dye Solution and "Working" Reference Dye Solution. Standard Reference Dye Solution is used to verify the correct concentration of the Reagent Dye Solution and the "Working" Reference Dye Solution. The Reagent Dye Solution and Standard Reference Dye Solution are available in ready-to-use form. They are also available as concentrates to be diluted with distilled water and acetic acid before use. The "Working" Reference Dye Solution is prepared from the Reagent Dye Solution. Preparation, storage, and use of these solutions are described in the following sections. See Storage and use of Prepared Dye Solutions.
Reagent Dye Solutions Using Concentrates
Solution Concentration Definitions
X-Fold Dilution: A weighed amount of material is mixed with a weighed amount of solvent. The solvent weight to be used is X-1 times the weight of the material being diluted. For example, to obtain a 10-fold dilution (x = 10), the ratio of weight of sample to solvent is 1:9.
NOTE: Ratios are based on WEIGHT not volume. When a procedure gives a volume of solvent to be added to a specified weight of sample, the density of the solvent has been taken into consideration.
Percent (w/w) or Percent (weight to weight) means grams of material dissolved per 100 g solution.
NOTE: If w/v or w/w is not specified, assume w/w is desired. The difference between the two values depends on the density of the solution.
If the percent needed is based on anhydrous material and hydrated is to be used, multiply the percent by the formula weight of the material being used and divide by the formula weight of the material specified. Assume anhydrous material is intended unless hydrated is indicated. This substitution is permissible only when water solutions are being prepared.
Preparation of Standard "66" Reference Dye Solution -- (0.660 g/l)
Pour Reference Dye Concentrate (50 ml) into the one liter (1000 ml) Volumetric Flask. Be particularly careful not to spill any. If any solution should spill, throw away the entire preparation, rinse the volumetric flask, and start with a new bottle of concentrate. The Standard Reference Dye Solution must be prepared exactly. It is the standard to which all measurements are compared. NOTE: Prepared Standard Reference Dye Solution (PN 30-0627 or 30-0613) is available from UDY Corporation.
The Reference Dye Concentrate bottle and cap must be rinsed with distilled water at least three (3) times. Each time, the rinse water is transferred into the one liter Volumetric Flask. This should achieve complete transfer of the Reference Dye Concentrate. Fill the one liter flask just below the calibrated mark with distilled water. Add one drop octyl alcohol, if necessary, to eliminate foam. Tap the sides gently to remove any air bubbles. Use dropper bottle with distilled water to fill to the mark. If too much water is added, discard and start again. Mix after capping the flask by inverting at least ten (10) times. This is your Primary Reference Dye Solution. Store in a well sealed glass bottle, NOT the volumetric flask. Discard solution over one year old regardless of how it is stored. Temperature of the distilled water used should be 20 "3oC.