Standard and Dilution Making Protocols

 

EQUIPMENT


·         4 or 5-place weighing balance

·         30-mL plastic beakers

·         100-mL plastic bottles w/lids

·         100-mL volumetric flasks

·         ICP (inductively coupled plasma spectrophotometer)

·         Automatic pipette w/macro-tips, w/micro-tips

·         colored marking tape and pen

·         triple-distilled water (TDW) system

·         double-distilled water (DW) system

·         disk, computer and spreadsheet software


 

REAGENTS

            Triple-distilled water (TDW) or double-distilled water (DW) – Each laboratory has water purification systems that produce TDW and standard tap for DW. Check with lab supervisors on correct TDW system operation. Use water directly from systems. See comments concerning silicon standards.   

            Stock solutions, any element (1000-mg/L) - Use “Dilut-it” standards and/or buy standards already prepared by VWR or Fisher. Follow instructions for the standards purchased.  See comments below on storage of standards. This standard solution is referred to as “stock solution”.  Keep these stored in plastic bottles. Standards made in water should be stored in the refrigerator.

            Working standards, any element (100-mg/L) - Prepare a “working” standard of each element by placing 10-mL “stock” solution (1000-mg/L) into a 100-mL volumetric flask and make to volume with TDW. Calibrate the automatic pipette using the balance for precise measurement of the “stock solutions” to make the working standards (see procedure A, #3 for automatic pipette calibration instructions).  Store these in plastic bottles, if possible. Use these “working” standards to prepare individual standards for the ICP.   

            Individual standards, any element - Make the following individual standards by pipetting the proper amount of “working” standard (100-mg/L) into a 100-mL volumetric flask, adding diluted or undiluted matrix extraction solution depending on sample preparation, and making to volume for the ICP. Place all the element(s) desired to be run on the ICP in the same volumetric flask. For example:  transfer the proper amount of Al, Fe, Si, and P to the same 100-mL volumetric with a calibrated automatic pipette. Calibrate the automatic pipette (see procedure A, #3 for automatic pipette calibration instructions) using the balance for precise measurement of the “working” standard solutions to make individual standards. Keep individual standards in plastic bottles, if possible. Some matrix solutions are only stable for a couple weeks or months. 

 

                        Standard                                              mL “working”

                          mg/L                                               standard solution

                              0                                                              0

                              1                                                              1

                              5                                                              5

                            10                                                            10

 

COMMENTS

            Wear eye protection and rubber gloves when making standards or working around concentrated hazardous elemental standards (e.g. Cd, As, Pb, Hg). Check with laboratory supervisor on the proper operation of the automatic pipette and changing from macro to micro tips. Calibrate settings of automatic pipette each time standards are made. Calibrate the 4-place weighing balance each day. Keep equipment covered when not in use. Heavy items should not be placed on the weighing table. Keep working area very clean.

Standards should be at room temperature before extracting standard solution with the automatic pipette. TDW should also be at room temperature, i.e. winter water may be colder from the attic double-distilled apparatus. Never pipette directly from standard containers and never pour used standard back into standard container. Discard all standards into appropriate waste containers. Use plastic containers as much as possible as they cause less contamination with certain elements. 

When purchasing known standards, buy 100-mL stock as the shelf-life may be less than one year. If using large quantities of standards often, then larger quantities may be appropriate. Fisher and VWR (other possible sources) have certified stock standards that can be used to make ICP working standards. Be careful to read the exact concentration of the stock solution - some may be 1,000-mg/L and others may be 10,000-mg/L.  Refrigerate individual standards to reduce microbial growth.

Special care must be taken to prevent silicon contamination from glass. Plastic lab ware must be used in all procedures and sample tubes for the ICP. Water from purification systems that are silica-based should not be used.  Use DW to avoid silicon contamination. If possible, check the TDW and DW on the ICP for levels of silicon.

 

PROCEDURE

A.      Standard Making

1.         Determine element concentrations needed for ICP standards, if possible. It is recommended that a 3-point standard curve be used for the ICP.  If unknown element concentrations vary or it is not possible to evaluate the element concentration needed, then use a high standard (50-mg/L), a low standard (1-mg/L) and a quality control (QC) standard (5-mg/L).  If the concentrations are in a narrow range and low or high, use a 3-5 point standard curve.  The ICP operator may be able to evaluate large sets of samples before ICP run is made so appropriate standards can be prepared.  These can be changed depending on the concentration of samples. The different elements can be varied as long as the ICP protocol is set to recognize the amounts used for each element.  See curve example in table below.

           

Element

Blank 3% HNO3

1st std.
ppm

2nd std.
ppm

3rd std.
ppm

QC std.
ppm

Aluminum

0.00

1.0

10

50

5

Iron

0.00

1.0

10

50

5

Manganese

0.00

0.5

1

5

2

Phosphorus

0.00

0.5

1

5

2

Silicon

0.00

1.0

10

50

5

 

2.         Place approximate amount of “stock” element standard or “working” element standard needed into a 30-mL plastic beaker and allow it to come to room temperature. Pour only the approximate amount needed because unused standard should never be put back into original container. Discard unused standard in appropriate waste container.

 3.        Calibrate the automatic pipette using the balance. Make sure the macro or micro tip is securely fitted to the pipette. Place weighing dish on 4-place balance, tare, and pipette desired amount needed to make standard into dish. For example: if pipetting 5 mL, it should weigh 5 grams. If not, the automatic pipette should be adjusted lower or higher until the weight reads exactly 5.000 g on the balance.  Use a new dry pipette for each pipetting/weighing.

4.         When pipetting different elements and/or amounts, use a different pipette tip each time.  In the Pedology Laboratory the tips are rinsed, placed in acid bath for at least one hour, TDW rinsed at least twice, and air-dried.

5.         Place standards in plastic bottles and label each with elements, concentration, matrix, date, and your initials.

6.         ICP operator will set the protocol for the element(s) prepared. The instrument will test the protocol and wave lines available that work best for the matrix and concentrations.  If all lines are low (UV) wavelength (<260) or high (VIS) wavelength (>260), the integration time can be shortened on the wave line not being used.

7.         Many matrix extracting solutions need to be diluted, i.e. selective dissolution procedures (sodium pyrophosphate, ammonium oxalate (AOD), and sodium citrate, sodium bicarbonate and sodium dithionite (CBD), ammonium acetate (NH4OAC) or potassium chloride (KCl)).  Sample dilution should be 1:10 or less if the concentrations are low.  Concentrations too low may not give good numbers.  See procedure B, dilution preparation below.  High salt contents may cause the ICP jet to clog.    

8.         Place prepared element standards in the batch run with unknown samples on the ICP auto-sampler for internal standard checks. 

9.         Prepare a spreadsheet to calculate your unknown samples and the actual sample concentrations.

 

B.   Dilution Preparation

1.                 Determine the dilution needed for analysis on the ICP.  Usually a 1:10 (1 to 10) dilution is necessary for most extractants such as AOD, pyrophosphate, and CBD.  Potassium chloride needs a 1:3 or less, but salts up the ICP jet.  Clarification: pipette 1-mL of unknown sample and add 9-mL of appropriate diluents. Total volume will be 10-mL and dilution factor equals 10. Another example: prepare a 1:3 dilution; pipette 1-mL of unknown sample and add 2-mL TDW. Total volume will be 3-mL and dilution factor equals 3.

2.                 Program the automatic pipette to dilute mode.  Press “F” (function key), then press “C” (clear), press “F” (function key), then “D” (dilute key). If the laboratory does not have a programmable automatic pipette that has a dilution mode, then a standard automatic pipette is satisfactory. Check the calibration amounts the same as with the programmable automatic pipette using the balance. The standard automatic pipette will require two steps – picking up the unknown amount, dispensing, picking up the matrix solution amount, and dispensing; whereas, the programmable automatic pipette will pick up the unknown amount, then matrix amount and dispense together.  Remember to mix all dilutions and standards. 

3.                 Make the dilution desired. Rinse tips several times before pipetting the next sample or standard. Use a different tip after 5 samples. Do not run the automatic pipette again to “clean out” the tip; just allow it to drain freely along the side of the plastic ware.   

4.                 Prepare standards for ICP in same matrix as extractant. If a 1:10 dilution is used for unknown samples then the standards are made in a 1:10 matrix solution. Prepare the matrix dilution the same as for unknown samples except in larger amount. For example:  take 100-mL AOD extractant and add to 900-mL of DW to make a 1:10 matrix solution.

 

REFERENCES

Klesta, Eugene K. and Joan K. Bartz.  1996.  Quality Assurance and quality control. p. 19-48.  In  D. L. Sparks (Editor), Methods of Soil Analysis - Chemical Methods, Part 3.  Soil Sci. Soc. Am., Monograph No. 5, Madison, WI. 

 

Shugar, Gershon J., Ronald A. Shugar, Lawrence Bauman, and Rose Shugar Bauman.  1981.  Chemicals and preparation of solutions.  p. 431-451.  In The Chemical Technicians’ Ready Reference Handbook, 2nd edition.  McGraw-Hill Book Company.  New York. 

 

http://www.lachatinstruments.com/tools/tools.asp

 

http://www.jtbaker.com/conversion/conversions.htm?#particle

 

CALCULATIONS

           

            Example standard preparation:

To prepare 100-mL of 5 mg/L Al solution using a 100 mg/L Al working standard, use the following equation:

 

c1v1 = c2v2       where:    c1 = concentration of solution 1 (your working standard)

                                                v1 = volume of solution 1

                                                c2 = concentration of solution 2 (your 5 mg/L Al solution)

                                                v2 = volume of solution 2

 

Then,   c1 = 100 mg/L

            v1 =  ?

            c2 = 5 mg/L

            v2 = 100 mL

 

By solving the equation, v1 = 5-mL. This means you have to pipette 5-mL of the 100 mg/L working standard into a 100-mL volumetric flask and bring to volume with matrix dilution, mix well and transfer to labeled container. This will make a 5 mg/L Al solution to use on the ICP.

 

Miscellaneous:

            ppm   =  mg/mL   =   mg/1000 mL  =  mg/L     

%  =  ppm  X  10-4