### G. Campbell - Biophysical Measurements and Instrumentation

#### Chapter 4

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Measuring Water Potential

In soil, and within living organisms, the moisture measurement which is relevant to water transport and growth is the water potential. The water potential is the potential energy per unit mass of water in a system. The potential energy of the water in an organism or soil is determined by the position (with respect to some reference level) of the water in a gravity field, the adsorptive forces binding water to a matrix, the concentration of dissolved substance in the water, and the hydrostatic or pneumatic pressure on the water. The total water potential is the sum of all these components, and is written as:

(1)

where the subscripts are for gravitational, matric, osmotic, and pressure potentials, respectively. In a given situation, some of the components of the water potential may not be important. For example, we normally do not consider the gravitational potential in calculation of water uptake by plants (except for tall trees) or the osmotic potential in water transport in soil. Careful attention needs to be given to the potentials acting under a given set of circumstances in order to decide which measurement is appropriate for describing the driving force for water flow.

Numerous methods have been devised for measuring water potential in soil and plant systems. We will discuss several of the most useful. The methods we will discuss are based on one of two principles. One class of instruments balances liquid water at a known water potential against water at an unknown potential across a semipermeable membrane. At equilibrium, the potential of the water in the measuring system is equal to the unknown water potential. Instruments which use this principle are the tensiometer for measuring matric potential of soils and other colloidal systems and the pressure bomb which is used to measure water potential of plant leaves.

The second class of instruments measures the relative humidity in equilibrium with a sample at unknown water potential. A relation derived from the first law of thermodynamics and the perfect gas law relates water activity, aw, to water potential:

(2)

where R is the gas constant (8.31 J mole-1 K-1), T is the Kelvin temperature, and Mw is the molecular weight of water. When the gas phase is in equilibrium with the liquid phase, then the relative humidity, hr, in the gas phase is equal to aw.

In principle, this measurement is simple, but in practice it requires measurement of humidity in the range of 0.95 to 1.0 with a precision of around 7 parts in 105. A special psychrometer is used for the measurement, and special precautions are required to obtain the needed precision. Instruments using this principle are called thermocouple psychrometers or thermocouple hygrometers depending on the way they are read.

Figure 1. Diagram of Pressure Bomb apparatus.

The pressure bomb is an instrument used for measuring the water potential of leaves. Figure l shows a typical pressure bomb arrangement. A leaf is severed from the plant and placed in the bomb with its petiole or stem protruding through the seal. Pressure is applied to the tissue until water just appears at the cut end of the petiole or stem. When water appears, the pressure applied to the tissue is equal to the negative of the leaf water potential. In terms of the principles mentioned earlier, consider the xylem of the leaf as being a continuous porous system, impermeable to air, but permeable to solutes and water. Since the xylem is vented to the atmosphere through the seal of the bomb, the potential of the water in the xylem, when water just appears at the cut surface, is equal to that of free water, or zero. The water potential of the cells is in equilibrium with the xylem potential so at the end point, the water potential of the cells is also zero. The water potential of the cells is the sum of the applied pneumatic pressure and the potential of the cell: P + = 0, so = -P.

It is worth noting that the tissue water potential is = p + o, where p is the turgor pressure and o is the osmotic potential of the cell sap. When the leaf wilts is zero, and the water potential of the leaf is equal to the osmotic potential of the cell sap. The pressure bomb can therefore be used for direct measurements of osmotic potential of wilted leaves. If several measurements are made at different leaf water contents, a pressure - volume curve can be constructed, from which osmotic potential at any leaf water content can be determined. More detail on this method is given in Campbell (1985).

Several precautions are necessary to assure accurate measurements with the pressure bomb. It is important to prevent water loss from the leaf during the time the measurement is being made. The entire range of water potentials from full to zero turgor for most leaves is covered with a change in water content of about l0%. Thus a loss of only a small fraction of the leaf water during the measurement can result in errors of several hundred joules per kilogram.

Another precaution is related to the rate of pressure increase in the bomb. The rate of pressure increase must be slow enough to give the water in the leaf time for equilibration. If the pressure is increased too rapidly the pressure reading will be too high. One method of determining whether the rate of pressure increase is too rapid is to take a measurement, then release the pressure until the water just disappears from the cut surface, then increase the pressure again to obtain a second reading. The two readings so obtained should be the same. If the second reading is lower, the rate of pressure increase was probably too high.

Another source of error results from the fact that air usually passes through some of the xylem vessels as the pressure is being increased, and fluids other than water in the phloem or xylem can coat the cut surface and cause bubbling which is often difficult to distinguish from the real end point. If one wipes the cut surface during the early stages of pressurization, it is usually possible to detect the proper end point. With twigs, it also helps to strip away the bark down to the cambium.
Some tissues have very porous xylem which fills with water as the measurement is being made. The storage of water in this tissue causes the pressure readings to be too high. Other leaves, such as sunflower or sugar beet have large fleshy petioles which are easily crushed and difficult to seal in the pressure bomb. With all of these, it is often best to cut a section from the leaf, leaving a length of mid-rib or other conducting tissue attached. Pass the conducting tissue through the seal to indicate the end point. Any xylem which is attached to the mesophyll should provide the necessary connection for detecting the end point.

The Tensiometer

The tensiometer consists of a water-filled porous ceramic cup attached to a vacuum gauge. Figure 2 shows a typical unit. Water is withdrawn from the cup by the matric forces in the soil until the pressure potential inside the tensiometer is equal to matric potential of the soil water. The cup is permeable to salts but not to air and soil colloids, so the osmotic potential of the soil solution has no effect on the reading. The useful range of the tensiometer is about 0 to -80 J/kg. Very accurate measurements are possible in this range, and this is the range in which most moisture flow occurs in soil. In theory, the tensiometer could be used to measure matric potentials much lower than -80 J/kg, since the adhesive and cohesive forces of water are sufficient to withstand tensions of many hundreds of joules per kilogram (c.f. in the xylem of plants), but impurities in the water and on the surfaces inside of the tensiometer, and dissolved gases in the water cause cavitation when the absolute water pressure approaches zero. Once a gas phase in present in the tensiometer, negative pressures are impossible.

Figure 2. A form of tensiometer for measuring soil water potential.

The Thermocouple Psychrometer

The thermocouple psychrometer measures the humidity of air in equilibrium with a sample of plant, soil, or solution. Equation 1 can then be used to infer the water potential of the sample from the humidity measurement. The humidity measurement is made by enclosing a small thermocouple in a sealed chamber with the sample, allowing time for vapor equilibrium, and measuring the wet bulb temperature depression. Two methods have been used to measure wet bulb depression. In one, the thermocouple has a small ceramic bead surrounding the measuring junction. This bead is dipped in water before the thermocouple is placed over the sample. When the wet junction is placed over the sample, it cools by evaporation, and the temperature of the wet ceramic (which is measured by the thermocouple) is read as the wet bulb temperature.

In the other method, the thermocouple is cooled using the Peltier effect. A current is passed through the junction for a specified time until water has condensed on it. The wet bulb reading is the temperature of the wet junction as the water evaporates.

The psychrometer equation could be used to find h, but it is usually easier and more accurate to relate cooling to the water potential by calibrating the psychrometer with salt solutions of known water potential. The water osmotic potentials of the salt solutions can be calculated using

(3)

where c is the concentration (moles/kg), R is the gas constant, T is kelvin temperature, n is the number of osmotically active particles per molecule of solute (2 for KCl and NaCl), and f is the osmotic coefficient. Values for f as a function of c are listed in Table 1.

Table 1. Osmotic coefficients (f ) of NaCl and KCl solutions

 c NaCl KCl c NaCl KCl 0.1 0.932 0.927 1.6 0.962 0.904 0.2 0.925 0.913 2.0 0.983 0.912 0.3 0.922 0.906 3.0 1.045 0.937 0.4 0.920 0.902 4.0 1.116 0.965 0.5 0.921 0.899 5.0 1.192 0.7 0.926 0.897 6.0 1.271 1.0 0.936 0.897

The basic idea of the thermocouple psychrometer can be incorporated into a number of devices for measuring water potential or component potentials. A commercial sample chamber psychrometer is shown in Fig. 3.

Figure 3. C-52 Sample Chamber Psychrometer from Wescor, Inc., Logan, UT.

It is useful for measuring osmotic potential when solutions of unknown concentration are absorbed in filter paper disks and placed in the chamber. Osmotic potentials of soil solution, body fluids, and sap expressed from plant tissue can be determined in the manner. Various sizes of chamber can be used with the sample chamber psychrometer, so soil samples and leaf disks can be used. It should be pointed out, however, that equilibrium time with these samples is much longer than with osmotic samples. This causes no serious problems with soil samples, but excision and equilibration of leaves in the psychrometer chamber can cause errors as large as several hundred joules per kilogram due to changes which take place in the tissue during equilibration and adsorption of water vapor by the leaf surface (Campbell, 1985).

Figure 4. SC-10A Thermocouple Psychrometer Sample Changer from Decagon Devices, Inc., Pullman WA.

A sample changer which is better suited to soil samples is shown in Fig. 4. This instrument can equilibrate 9 samples at once, and uses the ceramic bead psychrometer, so that readings over dry samples can be obtained. It has also been successfully used to measure water activity of food and fiber samples.

The thermocouple psychrometer can also be placed in a porous ceramic cup and buried in the soil. Figure 5 shows a cutaway view of the Wescor soil psychrometer. This instrument measures the water potential of the soil in situ, and must therefore be calibrated at a number of temperatures to determine the appropriate calibration factor to use at a given soil temperature. These units have also been used to measure the water potential in the xylem of trees and in potato tubers.

A fourth version of the thermocouple psychrometer is designed into a special clamp which can be used on leaves. The leaf psychrometer is shown in Fig. 6. This instrument measures the water potential of the leaf in situ, and, when properly used, is the most accurate method of assessing leaf water potential. The chamber is sealed to the leaf, and an aluminum clamp assures that the system stays isothermal. A thin layer of Styrofoam insulation covered with aluminized mylar surrounding the unit helps keep the temperature stable.

Figure 5. Ceramic cup soil psychrometer from Wescor, Inc., Logan, UT

Figure 6. Wescor L-51 Thermocouple psychrometer and leaf clamp for measuring leaf water potential in situ.

Several precautions are necessary for accurate measurements with the psychrometer method for measuring water potential. The measurement is very sensitive to differences in temperature between the measuring junction and the sample. A 1C temperature difference between the measuring junction and the sample will result in an error of 12000 J/kg. We would like measurement errors to be smaller than about 10 J/kg, so temperature differences must be kept smaller than about 0.001C. The measurement is also very sensitive to contamination in the sample chamber. Contaminants take up water and slow or prevent equilibrium. At humidities near 1.0, many clean surfaces and almost any contamination on a surface will take up water. The importance of cleanliness cannot be over emphasized in the use of the psychrometer.

The theoretical range of the Peltier-cooled thermocouple psychrometer (the Wescor units) is 0 to around -5000 J/kg. They are limited on the dry end by the ability of the Peltier effect to cool the junction sufficiently for water to condense. The ceramic bead psychrometers (Decagon units) are not limited on the dry end because water is placed on the wet junction. On the wet end, the practical range without special precautions and techniques is around -50 J/kg for both types. Samples wetter than -50 J/kg are difficult to equilibrate.

The Hydraulic Press

The equipment discussed so far is quite slow and cumbersome. For many studies it would be desirable to have same portable and fast method for estimating water potential. A device constructed from a hydraulic jack is available from Decagon which presses leaf tissue between a rubber membrane and a Plexiglas plate. If the tissue is free to deform under pressure, then the pressure applied to the tissue will be transmitted to the water in the tissue, and water will be expressed from the tissue. The pressure required to express water from the tissue should be related to the water potential. Because of the irregular shape (on a micro-scale) of tissue, a small part of the tissue is always subjected to greater pressure than the average for the tissue, when pressure is first applied. A small amount of water is therefore expressed at low pressure, and the pressure required to do this is apparently uncorrelated with water potential. If one continues to increase the pressure until the intercellular spaces infiltrate with water and water comes out around the edge of the tissue, then the pressure is uniform on all parts of the tissue, and this can be used as the end point of the reading. The pressure at which the tissue starts to darken (cell wall air space fill with water) correlates well with the zero turgor water potential (measured with the pressure bomb). Additional study is necessary to finally establish a theoretical basis for the hydraulic press, but this correlation with daytime pressure bomb measurements allows one to use the press for quick (but relatively imprecise) estimates of osmotic potential or daytime water potential of plants. These measurements could be useful in screening breeding lines or assessing water potentials of tree seedlings before or after transplanting.

Summary
The various methods for measuring water potential, along with their precautions, ranges of operation, and principles of operation are summarized in Table 2.

EXPERIMENT

Use the tensiometer to measure soil water potential, the pressure bomb to measure leaf water potential, and the sample chamber psychrometer to measure osmotic potential. From the leaf water potential and osmotic potential measurements, calculate the turgor pressure of the leaf.

Questions:

1. Briefly describe the measurement principle for each measurement you made, and tell the precautions needed for accurate measurement.

2. Why is it necessary to freeze the leaf before expressing sap for the osmotic measurement?

References
Campbell, G. S. 1985. Instruments for measuring plant water potential and its components. in Marshall, B, and Woodward, F. I., Instrumentation for Environmental Physiology. Cambridge Univ. Press, Cambridge, p.193-209.

Campbell, G. S. 1988. Soil water potential measurement: an overview. Irrig. Sci. 9:265-273.

Table 2. Comparison of methods for measuring water potential

 Method Measures Principle Range Precautions Tensiometer matric potential balances internal suction against external matric potential through a porous cup 0 to -80 J/kg difficult to keep system gas free Pressure bomb water potential of plant tissue (leaves); can also measure osmotic potential balances external pneumatic potential against cell water potential to produce zero potential in the xylem 0 to -6 kJ/kg sometimes difficult to see endpoint; use slow pressure increase (2 psi/s); prevent water loss from leaf; Hydraulic press (jack) probably measures osmotic potential; measures matric potential of frozen leaves principle not worked out. Calibration based on correlation with other methods. 0 to -6 kJ/kg Many endpoints visible. Care needed to choose the right one. Sample chamber psychrometer matric plus osmotic potential of soils, leaves, and solutions Measures hr of vapor equilibrated with sample. Uses eq. 2 to get potential 0 to -6 kJ/kg (some methods go to -100 kJ/kg) very sensitive to dirt and temperature fluctuations; has problems in the 0 to -100 J/kg range; requires frequent calibration in situ soil psychrometer matric plus osmotic potential in soil same as sample changer 0 to -5 kJ/kg same as sample changer in situ leaf psychrometer water potential of leaves same as sample changer 0 to -5 kJ/kg same as sample changer; should be shaded from direct sun; must have good seal to leaf