Direct Current measurements 1

INSTRUMENTS

Digital multimeter

A digital multimeter is designed to measure direct- and alternating current (DCA, ACA), direct- and alternating voltage (DCV, ACV) and resistance (OHM). The type of the quantity to be measured can be set with the button in the middle by turning it to the appropriate range of the desired quantity (Figure 1.). Use COM and V/Ω connectors when measuring voltage and COM and A when current. (COM = common, grounding)

Figure 1. The settings of the

digital multimeter.

Further scales within the fields show the maximum of the measurable value belonging to the certain position. For example in the DCV field with the setting of 200m, 2, 20, 200; maximum of 200 mV, 2 V, 20 V or 200 V can be measured. If the voltage in the electric cicrcuit is higher than the maximum of the current setting, “1” will appear on the screen indicating an error. In this case change the settings by turning the button to a higher value. Always choose the optimal setting that enables the most precise measurement. For instance, when the multimeter shows 1.3 V at setting 20 and 1.34 V at setting 2, choose the latter one.

20A A COM V/Ω

ACA ACV

DCV

OHM

DCA

Direct Current

voltage

resistance

Alternating Current

voltage

connections for

measuring

voltage/resistance

connections for

measuring current

Direct Current

current

on/off switch

Alternating Current

current

Direct Current measurements 2

MEASUREMENTS

I. Determining the resistance values

Figure 2. Circuit to prove

Ohm’s law.

1. Assemble a connection according to Figure 2. Connect multimeters switched to voltmeter (DCV) and amperemeter (DCA) settings at the points marked "V" and "A", respectively. Connect resistor R1 as it is shown on the figure.

2. Change the voltage of the direct voltage power-supply unit and measure 5-6 voltage-current value pairs. Increase the voltage in the range of 0-12 V, in approximately 2 volt steps (you do not need to set integer values of voltage). Collect the values measured in a table.

3. Change resistance R1 to R2 and repeat measurement steps 1-2 with resistance R2, as well.

4. Plot current data measured for both resistance R1 and R2 as function of the voltage in one graph on a graph paper. The graph will be similar to Figure 3. Fit lines to the plotted points.

Figure 3. Current-voltage

characteristics of ohmic

resistor.

5. Choosing a section on the line corresponding to a change of at least 5 V, read voltage difference (∆V) and current difference (∆I) along the section, then calculate the values of resistances using the following equation:

I

VR

∆

∆= (1)

II. Determining the total resistance

1. Ask the lab instructor whether to determine the total resistance of a parallel or a series connection. Write down the choice in your lab manual.

2. Assemble the connection shown in Figure 2. in a way that instead of resistor R connect resistors R1 and R2 in series or in parallel connection into the circuit. Draw the circuit diagram according to the chosen connection type.

3. Similar to the previous task, measure 5-6 voltage-current value pairs. Collect the measured values in a table.

4. Plot the current-voltage data into your previous graph and fit the characteristic line as you did in the previous experiment.

5. Calculate the total resistance using the slope of the function and equation (1).

6. Determine the expected total resistance of the connection using R1 and R2 values calculated in task I. Calculate the expected value of the total resistance using the equation of the connection and compare this with the value calculated based on the graph.

III. Characteristics of the incandescent lamp

1. Connect the incandescent lamp to the place of the resistor in the circuit according to Figure 2.

2. Measure 5-6 voltage-current value pairs up to 12 V. Collect the measured values in a table.

3. Plot the current-voltage characteristics on a graphpaper.

4. Note how the resistance of the incandescent lamp changes with the increase of voltage, and explain the phenomenon by giving the reason for the change.

IV. Characteristics of the diode

1. Set the power supply to zero volt. Connect the diode to the place of the resistor in the circuit according to Figure 2. Switch the voltage source to the 0-2 V range.

2. Determine which is the forward and backward biased direction of the diode: read the current flowing through the diode at 0.7 V, then connect the diode the other way round and read the value again. In the case of forward connection even 0.5 A current could flow through the diode while in the case of backward connection the magnitude of current flowing through is in the microampere range (µA).

3. Measure 5-6 voltage-current value pairs in both forward and reverse connections between 0.5 and 0.8 V increasing the voltage in 0.05 V steps. When connected in forward bias, the voltage cannot be increased over a given maximum, since the current flowing through the voltage source is limited to 0.5 A. At the end of the measurement turn the power supply immediately to 0 V position. Collect the measured values in a table.

4. Plot the values for the forward and backward connection on the same graph. Mark voltage values belonging to backward connection with negative sign in the negative field of the axis (left of the y axis).

5. Explain the graph.

Alternating Current measurements 1

INSTRUMENTS

Oscilloscope

Figure 1. The operating panel

of the oscilloscope. Buttons

coloured black are to be kept

pressed during

measurement.

The oscilloscope used during the measurement (Figure 1) is a two-channel oscilloscope with two independent input channels (11, 12). One can select the the input signal to be shown on the screen by pressing button YA or YB (17, 18). Only one of the inputs will be used during the measurement.

The oscilloscope amplifies the input signal electronically. The degree of amplification can be changed by the vertical sensitivity knobs (7, 10). The value shown on the knobs is the vertical sensitivity which shows the voltage corresponding to 1 cm on the vertical axis of the screen. To get the voltage of a signal (in volts), we multiply the signal height on the screen (in cm-s) by the vertical sensitivity (in V/T that is V/cm).

Similarly, horizontal sensitivity is indicated on the scale of the knob adjusting the horizontal running speed of the electron beam (5). Horizontal sensitivity in ms/T and µs/T that is ms/cm and µs/cm shows the time corresponding to 1 cm on the horizontal axis.

Red auxiliary buttons on the adjusting knobs allow continuous adjustment.

Values shown by the sensitivity knobs are only valid if red auxiliary buttons are turned to the far right side!

Sinusoid generator

The audio frequency signal generator produces a sinusoidally changing electric voltage. Using the amplitude and frequency buttons, the maximum voltage (Vmax) and the frequency (f) can be adjusted, respectively. At the 100× setting of the frequency switch, the value read on the disc is to be multiplied by 100 Hz, while at the kHz setting, multiplying by 1 kHz is needed.

Digital multimeter

For the usage of digital multimeter see the detailed description of the tasks of Direct current measurements. For measuring alternating current, connect the COM and V/Ώ ports of the digital multimeter to the signal generator. Turn the middle button to the ACV panel.

Synchronization

1. In order to get an immobile image on the screen, ratchet oscillation must be synchronized with the input signal. This can be done in three ways:

2. Switch on the oscilloscope and the sinusoid generator and connect the generator output to the input of the oscilloscope. The end of the cable supplied with the coaxial connector connects to the oscilloscope, while the end with banana jacks to the signal generator.

3. Internal synchronization: Press button 14, marked INT (Figure 1). Then the oscilloscope will synchronize ratchet oscillation with the input signal using an internally produced starting signal (trigger). External synchronization: Press button 15, marked EXT and connect the examined signal to the synchronous input of the oscilloscope (Figure 1, connection 16). Set the threshold voltage of start with adjustment knob 6. Automatic synchronization: Press button 13, marked AUTO on the oscilloscope. If the input signal is too low, the synchronization won’t work, the monitor stays blank.

Alternating Current measurements 2

MEASUREMENTS

I. Voltage measurement

1. Connect the oscilloscope to the signal generator and set a random amplitude and frequency value on the generator

When connecting the banana jacks to the generator always connect the thinner, earth connector to the proper output of the signal source (bottom)! The wrong connection results in short-circuit!

2. Try the synchronization methods described in the Synchronization chapter. Then, by changing the vertical and horizontal sensitivity of the oscilloscope set the image of the signal on the monitor.

3. Set a random amplitude and frequency value on the generator. Then, by changing the vertical and horizontal sensitivity of the oscilloscope (Figure 1, 7 / 10 and 5) and with the help of the image shift buttons (Figure 1, 8 / 9 and 4) set the image of the signal on the monitor. Note the vertical (V/cm) and the horisontal (ms/cm vagy µs/cm) sensitivity (Table 1,).

4. Measure the amplitude of the signal (one square unit equals one centimeter) on the screen. Multiply this value with the vertical sensitivity, read from the scale of the vertical amplification knob, to obtain maximum voltage in volts (Vmax). Calculate the effective voltage (Veff)!

5. Measure the effective voltage using the digital multimeter and compare it with the calculated value. Connect the digital multimeter to the output of the signal generator without disconnecting the oscilloscope.

6. Read the length of a period of the signal (in centimeters) on the screen. Multiply this value with the horizontal sensitivity (ms/cm or µs/cm), read from the scale of the horizontal amplification knob, to obtain the perod time (T). Calculate the frequency (f)! Compare the calculated value of the frequency with the value set on the signal generator.

7. Change the value of the amplitude and the value of the frequency on the generator and repeat the measurements. Collects the results in a table, according to Table 1!

Table1 voltage measurement

nr. of the

measurement

vertical sensitivity

(V/cm)

amplitude

(cm) Vmax (V)

Veff (V)

calculated

Veff (V)

measured

1

2

frequency measurement

nr. of the

measurement

horizontal sensitivity (ms/cm or µs/cm)

period

(cm)

period time

(s)

frequency (s-1)

calculated

frequency (s-1)

measured

1

2

II. Test of the series resonance circuit

1. Use the red field of the circuit box on the table to generate dampened oscillations (Figure 2). Continuous lines show connections that already exist behind the red panel, dotted lines show the elements you need to connect with external wires. Connect the proper elements along the dotted line then turn on the switch on the front of the box. You obtain a circuit containing a resistor (R), a coil (L) and a capacitor (C).

2. Turn the red switch to the “Auto” position, so that the circuit in the box will automatically switch on and off the voltage source, producing damped oscillations over and over again.

3. Connect the two ends of the capacitor to the oscilloscope and set the image of the damped oscillation on the screen of the oscilloscope. If the image is not stable, connect the synchronizing output of the signal to the synchronization input of the oscilloscope and switch the oscilloscope to external synchronization mode (Figure 1, 15).

4. Connect points A and B of the circuit by a cable, disconnecting (short circuiting) the resistance from the circuit

5. Measure the period time of the oscillations and calculate their frequency (resonant frequency) of the circuit. In order to make a more accurate measurement, measure the total length of several periods and divide it by the number of periods.

6. Measure the height of two consecutive positive peaks and calculate their ratio. This ratio characterises the degree of damping.

7. By disconnecting the wire between points A and B of the circuit connect the resistance into the resonance circuit.

8. Measure again the self – frequency (resonant frequency) of the resonance circuit and compare it with the values without resistance.

9. Measure again the value of damping and compare it with the value measured without resistance.

10. Make a drawing of the two curves of damped oscillations in your notebook on a single graph and write down you experiences during measurement.

Figure 3. The picture of damping

oscillation on the oscilloscope’s

monitor.

III. Test of the diode

1. Connect the two cables of the panel testing the diode to the signal generator (black banana jack to the bottom connector), then connect the two ends of the resistor to the input of the oscilloscope (Figure 3).

Figure 3. Connection to test the

diode.

2. Connect a cable between points A and B (Figure 3) to make a short circiut. The image of the signal will appear on the screen of the oscilloscope. Adjust the signal if necessary.

3. Remove the short cable and connect the diode between points A and B. Notice how the image of the voltage signal changes.

4. Change the direction of the diode. Notice again, how the image of the voltage signal changes.

5. Draw all the three signals on a graph paper and explain the how and why the signals changed.

y1

y2

y1

y2

R

signal generator

diode

to oscilloscopeA B

Figure 2. Schematic

drawing of the

circuit box with the

series resonance

circuit.

RLRLR

L

CA

B

to oscilloscope

manual

control

mode

selector

manual automatic

in

to synchronization

(red field)

Electrical conductance

INSTRUMENTS

Conductometer

Turning on, rinsing

After turning on, let the instrument warm up for approx. 5 min.

Rinse the electrode thoroughly before the first measurement with distilled water by moving up and down the flask on the electrode several times. When finished, gently wipe off the excess water on the outside with a piece of tissue paper. Repeat the rinsing with distilled water after each measurement.

Exchanging the solution

The simplest way to exchange the solutions is to pull out the wooden cube from below the flask, pull the flask off the electrode, put up the flask with the new solution, and push back the cube (Figure 1.).

Upon each exchange rinse the electrode with the new solution the same way as you did with distilled water.

When measuring conductance for a series of solutions, always go in increasing order.

Figure 1. Measurement

setup, exchanging the

solution (a). The scale of

the conductometer (b).

a.

b.

Choosing the measuring range

The numbers on the measuring range switch of the instrument (5 mS, 15 mS, 50 mS, 150 mS etc.) represent the maximum measurable value of a given setting. Always choose the range which provides the greatest accuracy, namely the one at which the pointer stands in the middle or upper-third region of the scale. If the pointer swings to the extreme right, the range is too low: in such a case quickly switch back to the previous (larger) range.

Calibration

The instrument must be calibrated at each change of the measuring range. This is done by holding down the CALIBRATION button, and setting the pointer to the red triangle found on the right-hand side of the scale with the calibration knob. When ready, release the button.

Reading the scale

The reading of the scale is done depending on the measuring range. For measuring ranges beginning with the digit 5 (0,5; 5; 50; 500 mS) the upper scale must be used, while for those beginning with the digit 1 (0,15; 1,5; 15; 150 mS), use the lower scale. The maximum value of the scale always corresponds to the measuring range.

To instrument

Wooden block

MEASUREMENTS

I. Determining the cell constant

1. Measure the conductance (G) of the 10 mM concentration KCl solution.

Convert the mS values to S before doing the calculation.

2. Calculate the cell constant (K) using equation (1) below.

1− = K mG

σ

(1)

3. Read the specific conductance (σ) of the 10 mM solution from the following table, corresponding the the temperature in the lab.

Table 1. Specific conductance

of the 10 mM concentration

KCl solution.

Temperature(°C) Specific conductance (S·m-1)

19 0.1251

20 0.1278

21 0.1305

22 0.1332

23 0.1359

24 0.1386

25 0.1413

30 0.1552

II. Determining specific conductance and molar conductance

1. Measure the conductance of the 100 mM and 1 M concentration KCl solutions. After each measurement rinse the electrode several times with distilled water!

2. Using equation (1) and the value of the cell constant determined in the previous part, calculate the specific conductance of the 100 mM and 1 M KCl solutions.

3. Calculate the molar conductivity (Λ) of all three solutions by using equation (2).

2Sm

c mol

σ Λ =

(2)

Note that for concentrations: 1 M = 1 mol/dm3 = 103 mol/m3

4. Summarize the values of conductivity and molar conductivity in a single table for all the three solutions.

Table 2. Parameters of the KCl solution conductance

C ( ) G ( ) σ ( ) Λ ( )

III. Determining an unknown concentration

1. Measure the conductance of each of the KCl solutions in the second series, having different percentile concentrations.

2. Plot the conductance values versus concentration on a graph paper. Fit a line to the data points.

3. Measure the conductance of the solution of uknown concentration and read the concentration corresponding to the measured conductance from the graph.

Refractometry 1

INSTRUMENTS

Abbe-type refractometer

Figure 1. Abbe-type

refractometer.

1. Before the measurement open the pair of prisms (Figure 4, 1) by loosening the closing screw and tilting the refractometer backward.

2. Wash the prisms thoroughly with distilled water and wipe their surfaces with a soft wiper or filter paper. This must be done before every measurement.

Do not touch the surface of the prisms by hand or by hard objects (e.g. the dropper).

3. Put 1-2 drops of solution to be measured on the surface of the lower prism and close the prisms by tilting the refractometer forward and holding the lower prism horizontally.

Use separate droppers to each solution. The refractometer can only be used with enough solution between the prisms, otherwise the viewfield is dark.

4. Point the lamp to the mirror of the refractometer (Figure 4, 2), and look into the right eyepiece (5), tilt the mirror until the field of vision becomes the brightest.

5. Find the borderline of the dark and bright fields by turning the large knob on the left (3). If the borderline is blurred and multicoloured, use the compensator (a knob on the right side of the refractometer, 4) to make it sharp.

6. Using the large knob on the left side, set the borderline exactly to the point of intersection of the crosshair in the right eyepiece (5).

7. Look into the left eyepiece (6) and read the refractive index of the solution on the left scale. The right scale shows the percentile concentration for a glucose solution. Please keep in mind that it does not work for other solution.

Figure 2. How to read the scale of the

refractometer. The position of the

horizontal line indicates the refractive

index on the left side (1,352). In case

of glucose solution the concentration

can direcly measured by using the

scale on the right side (14%).

MEASUREMENTS

I. Checking the calibration of instrument by distilled water

1. Measure the refractive index of distilled water.

2. Write down the measured value and compare it to the theoretical value of 1.333 (at room temperature).

II. Studying glucose solutions

1. Determine the refractive indices and percentile concentrations of given glucose solutions by averaging 2-2 measured values for each.

Between two measurements open the prism pair and clean it thoroughly.

III. Determination of blood serum protein content

1. Mix the serum with the dropper before use.

2. Determine the refractive index of blood serum by averaging the results of two measurements. Drop new serum samples onto the prisms each time.

3. Determine the protein content of the serum according to Table 2. The light grey rows show the normal range, while the dark grey row marks the normal average. Note which range your data fall in.

IV. Determination of the refractive index of glycerol solutions

1. Measure the refractive indices of the glycerol solutions with known concentrations. Start with the lower concentration and finish with the highest.

2. Measure the refractive index of the glycerol solutions with unknown concentration.

3. Plot the refractive index against the concentration for the solutions with known concentration values including the value for distilled water. Fit a line to the data points.

4. Read the concentration of the glycerol solution with unknown concentration (Y) from the diagram (calibration curve) using the refractive index measured.

Spectroscopy and spectrophotometry

INSTRUMENTS

The structure of the Kirchoff-Bunsen-type prism spectroscope

Figure 1. Kirchhoff-Bunsen-type

prism spectroscope.

Spectrophotometer

Figure 2. The front panel of

the spectrophotometer used

on the practical.

Calibration of the spectrophotometer

1. Close the shutter by turning it to position 0. This creates total darkness for the detector, when light cannot reach the detector in this case, therefore the measured absorption should be maximal.

2. Turn the Zero setting knob until you can reach the maximal value (∞) on the upper (extinction) scale. Do not turn this knob from now on.

Steps of the measurement

1. Switch on the lamp.

2. Open the shutter by turning it to position 1.

3. Set the required wavelength with wavelength setting knob.

4. Put one cuvette filled with distilled water into one of the cuvette holders, then push the cuvette inside.

5. Turn the Gain knob (bottom) to set the pointer back to 0 absorption. This step is important to substract the absorption of the water from that of the solution (in our measurement we consider the absorption of water to be zero).

6. Put the other cuvette filled with the solution into the other cuvette holder and move the holder and cuvette to the line of detection.

7. Read the absorbance (extinction) value from the upper scale (E). The reading is accurate when the pointer overlaps its mirror image.

8. To make a measurement at a different wavelength, repeat steps 2-6. To measure a new solution at the same wavelength, repeat steps 5-6.

prism

scale

focal plane

light

source

rés

observer

collimatoreyepiece

slit

1

0

nm

shutter

1

0

nm

cuvettedetector

wavelengthsetting

zero knob

gain knob

MEASUREMENTS

I. Study of an emission spectrum

1. Switch on the Na-lamp and wait for 5 minutes, until it warms up.

2. Push the entrance slit of the collimator tube of the spectroscope close to the lamp. First close the slit then open it slowly until a set of lines appears.

3. If the image is blurred, you can make it sharp with the focus knob.

4. Examine the emission spectrum of sodium using the spectroscope. Take notes and make drawings of your experiences on the lines you see (colour, position, intensity).

II. Absorption spectrum of oxy-haemoglobin of blood using direct vision spectroscope

1. Fill the plastic container with distilled water and add 5-6 drops of blood and mix it until it becomes light pink, but it is still very transparent. (You can find blood on the table of the Centrifugation practice.)

2. Put the container in front of a light source (use sunlight through the window or the lamp of the Refractometer) and look through the container using a direct vision spectroscope.

3. Compare the spectrum you see with or without the container.

4. Describe the spectrum and the changes in your notebook. Explain how it differs from the spectrum of the white light.

III. Absorption spectrum of oxy-haemoglobin of blood using spectrophotometer.

The guide for using the spectrophotometer can be found in the Instruments section.

After the measurement rinse the cuvettes with distilled water. Hold the cuvettes by their matte sides, do not touch the transparent side of the cuvettes!

1. Switch on the spectrophotometer.

2. Fill a cuvette with distilled water and the other with the diluted blood from the previous excercise. Place both cuvettes in the cuvette holders of the spectrophotometer. Light should go through the clear sides of each cuvette.

3. Check the settings of the spectrophotometer.

4. Measure the absorbance at different wavelengths from 500 nm to 600 nm in steps of 5 nm. Read the value on the upper scale (E). Don’t forget to set the absorbance of distilled water set to zero with the gain knob after changing the wavelength!

5. Plot the absorbance values (E) as a function of wavelength on a graph paper.

6. Make notes of your measurements. Write down the wavelengths at which the absorption was the highest. Compare this spectrum to the spectrum of oxy-haemoglobin observed with the direct vision spectroscope.

IV. Determining the concentration

1. Check the settings of the spectrophotometer.

2. Measure the absorbance of the provided potassium dichromate (K2Cr2O7) solutions of known concentrations at 495 nm.

3. Plot the absorbance values (E) for the known concentrations against the concentration (c) on graph paper. Fit a calibration line to the data points.

4. Measure the absorbance of the solution of unknown concentration.

5. Find the unknown concentration using the calibration line and the absorbance value measured for the unknown solution.

Polarimetry

INSTRUMENTS

Polarimeter

1. Set to 0 degrees range of the polarimeter.

2. Switch on the Na-lamp and wait for 5 min to reach its full intensity.

3. Take the tube out of the polarimeter, unscrew the upper cap with the rubber ring and remove the glass cover plate.

4. Fill the tube with the solution to be measured or the pure solvent (distilled water) and place the glass cover plate back on top of the tube. Avoid bubble formation.

5. Close the tube with the cap, wipe the glass cover plates and put the tube back into the polarimeter.

6. Use the metal ring on the eyepiece to bring the image in the viewfield into focus. Then rotate the analyzer slowly until then you observe a dark grey homogeneous viewfield.

7. Read the rotation angle on the vernier scale.

The vernier scale

The vernier scale is an auxiliary scale that enables a more accurate reading of lengths or angles. The principle of its use is that 9 divisions of the main scale correspond to 10 divisions of the vernier scale. Therefore, one division of the vernier scale equals 0.9 divisions of the main scale.

The main and the vernier scale can slide past one another. The integer value can be read on the main scale as the closest value on the left side of the zero of the vernier scale. The first decimal can be read on the vernier scale as the number of the division where the lines of the scales have an exact match (Figure 5).

The vernier scale of the polarimeter has 20 divisions, that allows a reading of the angles with the precision of 0.05 °.

Figure 5. Determination of

the decimal using the vernier

scale.

MEASUREMENTS

I. Determining the specific rotation of glucose

1. Fill the polarimeter tube with distilled water.

2. Place the tube into the polarimeter and read the rotation angle.

3. Fill the polarimeter tube with the 10% glucose solution.

4. Place the tube into the polarimeter and read the rotation angle.

5. Subtract the rotation angle of the distilled water from the measured value.

6. Determine the specific rotation of glucose ([ ]20Dα ) using this corrected value and the

equation below (the length of the polarimeter tube is 1.9 dm).

[ ]lcD

αα 10020 = (1)

II. Determining unknown glucose concentrations

1. Fill the polarimeter tube with the glucose solution of unknown concentration (X%).

2. Place the tube into the polarimeter and measure the rotation angle.

3. Subtract the rotation angle of distilled water from the measured value (see Ist task).

4. Determine the concentration (c) of the glucose solution by using equation (1) (use the corrected rotation angle and the previously determined specific rotation).

5. Repeat the measurement with the XX% glucose solution.

III. Determine the specific rotation of saccharose using a calibration curve

1. Measure the rotation angle of known concentration saccharose solutions in an order of elevating concentrations.

2. Subtract the rotation angle of the distilled water from the measured values (see Ist task).

3. Plot the corrected rotation angles as a function of concentration on graph paper and fit a line to points.

4. Use the slope c

α∆ ∆

of the line to determine the specific rotation of saccharose [ ]( )20

Dα

using the following equation: [ ]100

20 l

c Dαα =∆∆

(2)

IV. Determining saccharose concentration of unknown concentration using a calibration curve

1. Measure the rotation angle of a saccharose solution of unknown concentration (Y%).

2. Read the concentration of the solution from the calibration curve plotted in the previous task.

Viscosity of fluids

MEASUREMENTS

Perform three measurements in each experiment and use the average for calculations.

Measuring the viscosity of glycerol solutions

Use exactly the same volume of solution for every measurement! Measure the solutions in elevating concentration order!

1. Measure 20 cm3 of distilled water with the measuring cylinder and pour it into the higher sphere of the Ostwald’s viscosimeter. Suck the solution up slowly to the right side of the U-shaped tube. When the meniscus reached the sign above the upper sphere, close the tap.

2. Open the tap and use the stopwatch to measure the time (t) elapsed until the liquid pours down from upper sign to under sign.

3. Use the method described above to measure the 10, 20, 40, 50 and 60% glycerol solutions in elevating concentration order. After each measurement pour the glycerol solution back to the glass bottle.

4. Calculate the absolute (ηx) and relative (ηrel) viscosity of glycerol solutions using the equations (1) below. Collect the results in a table. You may find the viscosity of distilled water (ηw) in Table 1. Read the temperature of the room from the lab thermometer. Density of various glycerol solutions (ρx) can be found in Table 2.

The density of the distilled water (ρw) is 997 kg/m3 at room temperature.

= = =x x x x xrel x w

w w w w w

η ρ t ρ tη η η

η ρ t ρ t (1)

5. Plot the absolute viscosities versus concentration on a graph paper! Connect the points considering the viscosity of distilled water (0% glycerol)! Explain the graph!

At the end of the practice wash the viscosimeter several times with distilled water!

Table 1. Viscosity of distilled

water (ηw) at different

temperatures.

Temperature (°C) Viscosity (mPa·s)

10 1.307

20 1.002

24 0.911

28 0.833

30 0.798

Table 2. Density of glycerol

solutions (ρx).

Glycerol concentration (%) Density (kg/m3)

10 1026

20 1052

40 1104

50 1130

60 1156

Surface tension

MEASUREMENTS

Measure each solution three times in each experiment and use the average value for further calculations.

I. Measuring surface tension of distilled water

Wash the stalagmometer at least twice with distilled water before the measurements, and then rinse it with the liquid to be measured. Keep the stalagmometer and the capillary tubes clean!

1. Loosen the clamp on the rubber tube attached to the stalagmometer.

2. Suck the liquid above the upper sphere using the syringe.

3. Tighten the clamp and open the stopcock (or remove the syringe) to make the liquid drop out of the stalagmometer in a countable way.

4. Count the drops (nw) falling from the end of the capillary while the meniscus lowers from the upper sign to the lower sign.

5. Calculate the surface tension (αw) using the following equation (absolute measurement method):

ww

w

Vg

2r n

ρα =π⋅

(1)

The volume of liquid between the two signs is V = 2.3 cm3.

The radius of the capillary is r = 0.19 cm.

The density of distilled water at room temperature is: ρw = 997 kg/m3.

g = 9.81 m/s2

Do not forget to convert the units if it is necessary before the calculations!

II. Measuring surface tension of drug solutions with relative measuring method

1. As in the previous measurement, fill up the stalagmometer with the alcoholic and aqueous drug solution and count the drops (nx) corresponding to the volume between the two signs.

2. Calculate the surface tension (αx) using the following equation (relative measurement method):

=x w x

w x w

n

n

α ρα ρ

(2)

The density of the aqueous drug solution is: ρaqu = 1059 kg/m3.

The density of the alcoholic drug solution is: ρalc = 898 kg/m3.

The concentration is 10 % (10 g in 100 ml) for both solutions.

Do not forget to convert the units if it is necessary before the calculations!

III. Calculation of the effective dosis of drug solution

Calculate how many drops contain a given amount of active substance (ask the instructor for exact numbers) in case of the aqueous and alcoholic drug solutions from Experiment II.

IV. Measuring concentration of alcohol solution using the capillary method

1. Pour the lowest known concentration alcohol solutions into the wider branch of the U-shaped tube (Figure 1.). Place the rubber plug to the mouth of the tube and gently push it down untill the solution fills up the capillary (thinner branch of the U-shaped tube), and pushes the air bubbles out of it. Wipe off any effluent solution from the capillary top with a piece of paper towel. Remove the plug and wait until the fall of the meniscus stops.

Figure 1. Measuring the capillary

elevation.

2. Measure the capillary elevation (Figure 1., h).

3. Repeat the measurement with the other alcohol solutions, in an order of increasing concentrations. Finally perform the measurement with the unknown concentration alcohol solution.

4. Using the measured values of the capillary elevation (h) calculate the surface tension of all the alcohol solutions (α) using the following equation:

2

ghrρα = (3)

The density of alcohol solutions of different concentrations can be read from Table 1.

If the actual concentration is not present in the table, determine it by interpolation.

The radius of the capillary is r = 0.25 mm.

The density of the unknown alcohol solution at room temperature: ρ = 945 kg/m3.

g = 9.81 m/s2

Do not forget to convert the units if it is necessary before the calculations!

5. Plot the calculated surface tensions (vertical axis) against the concentration (horizontal axis) and determine the concentration of the unknown alcohol solution.

Table 1. Density of alcohol

solutions (ρ).

Alcohol concentration (%) Density (kg/m3)

0 998.2

10 981.9

20 968.7

30 953.9

40 935.2

50 922.7

60 913.9

70 891.1

80 867.6

90 843.6

100 818.0

h

Adsorption and swelling

INSTRUMENTS

Torsion balance

Calibration

1. With empty tray switch the balance to open position (Frei).

2. Set the pointer of the balance to zero.

3. Using the rear calibration screw, bring the zeroing pointer to 0 position. Do not touch this screw during further measurements.

Measurement

1. Switch the balance to closed position (Fest).

2. Use a forceps to place a gelatine piece onto the tray.

3. Switch the balance to open position (Frei).

4. Rotate the main pointer of the balance until the zeroing pointer is set exactly to zero.

5. Read the mass of the gelatin on the inner scale.

6. Set the switch to closed position (Fest) and take out the gelatine piece.

MEASUREMENTS

I. Determine the specific surface of “Carbo medicinalis” (activated charcoal)

1. Take three test tubes; put one charcoal pill in each.

2. Pump 10 cm3, 15 cm3, 20 cm3 methylene blue solution into the test tubes respectively. Use the big bottle which pumps 5 cm3 methylene blue per each press.

3. After shaking/vortexing the tubes thoroughly, when the pills completely fell apart, put one drop from each test tube on filter paper. Check wheteher the blue colour of the solution appear on the edge of the black circle of the pill, which means, that the charcoal pill got saturated and cannot bind more methylene blue.

4. To determine the exact amount of dye, which saturates one charcoal pill, choose the last unsaturated solution (with no blue colour) and add another 1 cm3 methylene blue to it, using an automatic pipette. Check its discoloration again and repeat the process until you get a pale blue colour on the filter paper. If you recorded how much methylene blue solution was pipetted into the tube (all but the last cm3), you can calculate the total amount of dye adsorbed by the charcoal.

5. Use this volume and the concentration of the stock dye to calculate the adsorbed mass of methylene blue.

6. Using this value, calculate the actual free surface of a charcoal pill.

7. With the determination of the volume of one pill, calculate the specific surface for unit volume of a charcoal pill.

8. Calculate the specific surface for unit mass of the carbo medicinalis (actual free surface of one kilogram active charcoal).

Use the following information for the calculation:

A surface of 1 m2 of charcoal is able to adsorb 1 mg methylene blue.

The mass density of the charcoal: ρ = 2.0 g/cm3

The carbon content of a pill: m = 0.125 g.

The concentration of the methylene blue solution: c = 0.2 % (given in g/100 ml).

II. Determine the time dependence of swelling in gelatine

1. Weigh separately 6 gelatine pieces and record their dry mass (md) using a table like table 1.

2. Pour water into 6 Petri-dishes.

3. Dip the first gelatine piece into a Petri-dish. Start a stopwatch at dipping and take the gelatine out after 30 seconds. Dry them up quickly and gently between two filter papers then weigh them again and record their swollen mass (swollen mass, ms).

4. Repeat the previous step with the second and third pieces of gelatine after 1 and 2 minutes of soaking respectively.

5. The other three pieces can be put into the dishes simultaneously, you will be able to measure their masses one by one when the given time is elapsed (3, 6 and 10 minutes). Dry them up quickly and gently between two filter papers then weigh them again and record their swollen mass (swollen mass, ms).

6. Determine the water content (mw=ms-md) and the relative water content (i=mw/md) from the data.

7. Plot the relative water content against time. The plot should be similar to a saturation curve.

8. Determine the maximum relative water content (imax) using the curve. This value shows the mass of the water intake compared to the dry mass in the case of maximum swelling.

9. Determine the speed constant of the swelling (k) with the help of the time required for the half-maximal saturation:

1/2

ln2k

t=

(1)

Table 1

Swelling of gelatine flakes

gelatine flake time elapsed

[t], (minutes)

dry mass

(md)

swollen mass

(ms)

water content

(mw=ms–md)

relative water

content

(i=mw/md)

1 0.5

2 1

3 2

4 3

5 6

6 10

imax =

Centrifugation

MEASUREMENTS

I. Determination of the volume of erythrocytes

1. Prepare hypertonic (2.0% NaCl), isotonic (0.9% NaCl), and hypotonic (0.5% NaCl) blood suspensions by mixing equal volumes (0,5 – 0,5 ml) of shaked anticoagulated (or defibrinated) blood and different NaCl solution. Mix gently and keep for 30 minutes (incubation) at room temperature. Do not pipette the blood up and down to avoid the haemolysis.

During incubation you can perform tasks II. and III.

2. After 30 minutes shake them again gently and fill 1-1 hematocrit capillaries with each of the suspensions: tilt the small dish and immerse one end of the capillary at an angle. Capillary forces slowly suck up the fluid the capillary tube. If necessary, move up and down the capillary or gently tap the lower end of the capillary to the bottom of the vessel.

3. Seal one end of the capillaries by pushing them into the plasticine, then twist and pull them out of the plasticine. Form at least 5 mm long plug.

4. Place the capillaries into the hematocrit centrifuge, with the sealed ends outwards. Put the lid onto the rotor and rotate the screw on top to tighten it.

5. Centrifuge the samples for 2 minutes at 6000 rpm. Press the set button and use the +/− buttons to set time and rpm values. The centrifuge stops automatically and after the sound you can open it with the lid button. (Pressing the start/stop button again will restart the centrifuge, so don’t do it!)

6. Calculate the ratio of the pellet height on the percentage of the total volume (the height of the entire suspension) to use with the supplied reader. Multiply by two because of the dilution. This will be the Packed Cell Volume (PCV, hematocrit).

7. Calculate the volume of a single erythrocyte (MCV = Mean Corpuscular Volume) in each of the three solutions. The standard number of erythrocytes is 5×106 cells/mm3.

36 /mm105

hematocrit

⋅=MCV (1)

II. Study the degree of sedimentation against time

1. Shake the Falcon-tube containing Sephadex G-75 bead suspension to disperse all of the sediment. Balance the other tube with distilled water. Place the tubes into the inner positions of the rotor. (The rotor should be balanced in four directions, do not remove the other two preprepared balancing tube from the rotor.)

2. To set time and rpm values press preset while turning the button. To spin shorter time than 1 minute you should press quick until the desired time.

Check the settings with the instructor prior to starting the centrifugation!

3. Centrifuge at 2000 rpm until 0.5 – 0.5 – 1 – 1 – 2 – 5 – 5 minutes. Measure the height of the pellet after every stops (the height of the Sephadex microbeads fraction). DO NOT shake the tube between the measurements!

4. Calculate the relative pellet height in percentage: pellet height / total solution height (pellet + supernatant) (Figure 2.).

5. Plot the relative pellet height as a function of time. The first point of the plot is 100% at zero time.

Figure 1. Change of the

sediment height vs. the time

of centrifugation.

III. The relative and absolute acceleration of the centrifuged sample

1. Measure the minimum and maximum radius of the rotation using a dividing ruler. These correspond to the distances between the rotation axis and the top and bottom point of the centrifuge tube, respectively.

2. Calculate the relative centrifugal force (RCF) for both radius at low, medium and high rotation speeds (rpm values) given by your lab instructor.

3. Plot the relative centrifugal force as a function of the number of revolutions (N). What type of function describes this dependence?

4. Check the RCF values using the provided nomogram.

supernatant

pellet

100Pellet height (%)

20

Time (min)

5 1510

Electrophoresis

HOW TO USE THE ELECTROPHORETIC DEVICE

The left display on the power supply shows the voltage in V, the right one represents the electric current flowing through the membranes, given in mA.

Figure 1. The scheme of the

electrophoretic device used on the lab.

buffer

membran

filter-

paper

lid

Power supply

running directionplace of sample

application

Volt Ampere

MEASUREMENTS

I. Electrophoresis of blood serum

1. Pour some running buffer solution in a Petri-dish and sink the membranes in the solution for 15 min.

Do not touch the membranes with your fingers, always use forceps. Make sure that the membranes do not float on top of the buffer, and do not stick to the bottom of the dish or to each other.

2. After 15 minutes take the membranes out of the buffer, blot the excess liquid from their surface with filter paper and place them into the electrophoretic device, laying them like a bridge across the platforms covered by filter-paper (Figure 1).

3. Apply a low amount of serum in a thin line perpendicular to the length of the membrane. Use the metal frame to create a very thin line (approx. 1 mm × 10 mm), close to the negative terminal, in front of the plattform. Leave a margin (approx. 5 mm) to the longitudinal strip edge on both sides.

4. Switch on the instrument and set the current to 1.5 mA per each membrane. Run the samples for 30 minutes. Check the current several times during the run, and set it back to the desired value if needed.

5. At start, after 15 minutes and at the end of running, record the voltage that is important for the calculation of electrophoretic mobility. Use the average of the 3 measured values.

Do not touch the inner parts of the instrument after switching it on! It is recommended to perform the measurement II. during the 30 minute run.

6. Switch off the instrument.

7. Take the membranes out of the chamber and put them into the amido black staining solution for 5 minutes. Amidoblack binds irreversibly to the protein components.

8. Pure destaining solution in 3 Petri-dishes, and put the membranes in the first dish after staining, for 3 minutes. After 3 minutes take the membranes out of the dish and put them into the second dish, and after another 3 minutes into the third destainer solution. The amidoblack is released from the membrane, except from the places where the protein components have bound it.

9. Finally, put the membranes on a filter paper and let them dry. Afterwards, stick them into your lab report.

10. Identify the proteins corresponding to the bands. If the applied serum-stripe was thin enough, then α- and even β-globuline fractions split up into two different components, but usually only the albumin and the three main globuline components can be distinguished.

11. Measure the distances covered by the given protein components from the place of application. Calculate the electrophoretic mobility knowing the distance between the electrodes (5.5 cm), the voltage applied and the time elapsed. Summarize the distances and the mobilities of the proteins in a table.

II. Measure the densitogram of a pre-made electropherogram

1. Switch on the densitometer with the main switch at the back. By turning the knob on the top of the instrument you may select which sample you would like to study, meaning one strip from the several one. The position of the strip is signed by the red light. If you push the flat gray button the sample steps forward below the detector, this way you can scan step-by-step the strip.

2. Choose two of the stripes on the membrane in the densitometer. Record the light absorption values in percentage, going along both of the stripes.

3. Plot the recorded absorbance values on mm-paper, and identify the protein fractions corresponding to the absorption peaks. Remember, the absorbance is proportional to the concentration (density) of the protein. Write down the differences of the two measured curves!