Appendix G

Operation manual for the Hach DR/2000 spectrophotemeter

The Hach DR/2000 spectrophotemeter is a portable programable single beem spectrophotometer. We will be using its programable features for the determination of total protein using the Bradford Method. In all other proceedures we will be using the instrument as an ordinary spectro-photometer and determinin concentrations from readings on standards and your knowledge of Beer-Lamberts law.

Figure 1 shows the spectrophotometer controls, indicators and connections, and their functional descriptions are given in Table 1

Testing with programmed methods

The DR/2000 Spectrophotometer Procedures Manual provides, step-by-step procedures for performing all the factory-entered methods. The intent of the material presented in this instrument manual is to provide supplemental information on how the instrument operates to perform the necessary functions and how to use the various special operating features offered. Once you become familiar with the instrument, the instructions in the procedure manual will be sufficient to analyze your samples.

The following graphic presentation is typical of most programmed test methods and is run in the Momentary mode. A more detailed discussion follows.

Colorimetric testing with preprogrammed calibrations can be divided into four general phases: spectrophotometer setup, sample preparation, zeroing the instrument and measuring the prepared sample. In the following paragraphs, the scope of each phase is described in detail.

Instrument Setup

There are a number of instrument operating features that are available as configuration menu options that have not been included in this annotated instruction manual. You may wish to consider at some fuyture ime. Included in these options are Constant On/Momentary status, data transmittal interval, etc. Refer to configure programming. An operating feature that is not selected through the configuration menu but is described under Configure Programming is the beeper selection.

Spectrophotometer setup in this test situation is limited to selecting the method or program number assigned to the desired calibration and selecting the wavelength. Prompting messages will appear in the display at the appropriate times to guide the operator through the procedure. When the instrument is turned on, an initial display of self-test with a momentary appearance of the software version number will appear. The software version number is replaced by a countdown sequence beginning with 15. At the end of the countdown, the first operational prompt to appear will be the method prompt:

45.7 nm

METHOD #?

If you wish to select the same program that was last used, you can save keystrokes by answering the method prompt by pressing the METH key. The last used method will be recalled even if the instrument was turned off. Since the wavelength setting has not changed, the prompt to adjust the wavelength will not appear in the display and the instrument will go to the zero prompt (for example mg/l CaC03 Mg). If a different method is desired, proceed as follows to set up the instrument.

The method or program number can be entered with the numeric keys or it can be scrolled to, using the Down or Up Arrow key. Holding the arrow key down allows rapid scrolling. A list of the tests with their number is affixed to the underside of the sample compartment cover (light shield). To enter the number with the numeric keys, key in the number and press the READ/ENTER key. If the number is not valid, an error signal will sound (if beeper is on) and the display will momentarily read:

45.7 nm

INCORRECT #

After approximately one second, the display will return to the prompt for the method number. Re-enter the method number properly or use the Down Arrow key to scroll forward or the Up Arrow to scroll backward through the method menu and select from the displayed methods. During scrolling, the method number is displayed in the large digits and the unit of measure and symbol for the test subject are displayed in the text area; for example,

P225 457 nm mg/1 CaCO3 Mg

Once the proper method appears in the display, accept it by pressing the READ/ENTER key. Unless the instrument is already adjusted to the correct wavelength, the display will next prompt you to set the wavelength value; for example,

P225 457nm DIAL nm TO 522

Adjust the wavelength control (see Figure 1) until the nanometer readout matches the prompted value. Always approach the desired wave-length from the high side for best accuracy and repeatability. When the values are equal, press the READ/ENTER key to proceed.

Note

If the wavelength was not set properly according to the programmed wavelength (within +- nm), the wavelength number in the display will flash. You may, however, perform the test with an alternate wavelength by pressing the READ-ENTER key when the alternate value is in the display. The nanometer display will flash continually, indicating the recommended wavelength is not being used.

Sample Preparation

The next task in the colorimetric test is the preparation of the test sample. If the zero solution (or blank) to be used requires some special treatment, it too is prepared at this time. Generally, sample preparation consists of adding the contents of a premeasured reagent powder pillow to 25 mL of the sample and allowing time for a color reaction to take place.

Note

It is important to observe the waiting period specified in the particular test procedure to be certain that the color (due to the reaction of the reagent with the substance being measured) develops fully. Many procedures also give a maximum time limit after which the color may begin to fade.

The DR/2000 Spectrophotometer has color development times programmed into the method software, and the operator is notified with a series of short beeps when the time has elapsed. The time must be initiated, however, by pressing SHIFT TIMER at the time the countdown should begin.

Zeroing the Instrumenmt

The instrument must be zeroed for each test or series of tests. This establishes a zero reference for the measurement and is done by placing a solution recognized as the zero concentration in the cell holder and pressing the ZERO key. The prompt display indicating readiness for this function appears as:

522 nm mg/1 CaC03 Mg

Place the zero solution (blank) in the instrument (with the 25-mL mark to the left or right) and press the ZERO key. See Figure 9. While the instrument is zero calibrating (which may take up to 8 seconds), the display will read:

522nm WAIT

When the zero calibration is completed, the display will show a zero result:

0.00 522 nm mg/1 CaC03 Mg

You are now ready to measure unknown samples.

Note

Once the zero reference point has been established, often several samples can be measured merely by placing them into the cell holder and closing the light shield. If in the MOMENTARY mode, you must press the READ/ENTER key for each measurement. The instrument can be re-zeroed at any time by placing the zero solution (blank) into the instrument and pressing the ZERO key.

Measuring the prepared sample

In the CONSTANT ON mode, just place the prepared sample into the cell compartment (with the 25 mL mark to the left or right as shown in Figure 10) and close the light shield. The test result will be displayed immediately and will be updated every 0.4 of a second. In the MOMENTARY mode, however, you must press the READ/ENTER key when ready to take the reading. The display will show:

522 nm WAIT

In about 6 seconds, the test result will appear; for example:

1.00 522 nm mg/1 CaC03 Mg

The corresponding absorbance or percent transmittance values can be displayed simply by pressing SHIFT, ABS or SHIFT, %%, respectively. SHIFT, CONC will restore the concentration display.

Note

The absorbance or percent transmittance measurements can be made without selecting a method. When the method prompt is displayed, press SHIFT, ABS or SHIFT, %%. After establishing the zero reference point, absorbance or percent transmittance measurements will be displayed.

Using the 1-CM Cell Adapter

Standard 1-cm square cuvettes can be used with the DR/2000 Spectro-photometer when Hach's 1-cm adapter (optional) is installed in the cell holder. One-centimeter cuvettes are not supplied with the instrument but are available as optional accessories either individually or in optically matched pairs.

The adapter is placed in the instrument cell compartment with the handling tab to the rear. This position will orient the adapter correctly in the light path. When using glass cuvettes, place them in the adapter with the clear sides in the left-to-right optical path. The cell compartment cover must be closed while taking readings. If operating the instrument in direct sunlight, use the light shield cap supplied with the accessories to cover the cell holder.

Walter I. Hatch
wihatch@smcm.edu

August 12, 2012