12.01 Determination of the isoelectric point of an amino acid (glycine)

Transcription

1 Biochemistry LEB 12 Determination of the isoelectric point of an amino acid (glycine) What you can learn about Isoelectric point Acidic anions Basic cations Zwitterions Equivalence (inflection) points pks value Titration Motor piston burette What you need: Experiment P with Cobra3 Basic-Unit Experiment P with Cobra3 Chem-Unit Principle and tasks: Amino acid molecules carry both acid and amino groups. They can therefore form both acidic anions and basic cations. The ph at which these two types of ions are both present in the same concentration is called the isoelectric point. This point is to be determined by recording the titration curve for the amino acid glycine. Cobra3 Basic-Unit, USB Cobra3 Chem-Unit Power supply, 12 V/2 A Data cable, plug/socket, 9 pole Software Cobra3 ph/potential Measuring module ph/potential ph electrode, polythene sheathed Cobra3, sensor C Cable, Cobra3 Basic-Unit Software Cobra3 Chem-Unit ph electrode, plastic body, gel-filled, BNC Immersion probe NiCr-Ni, 50/1000 C Cable, Cobra3 Chem-Unit Motor. piston burette, univer. 50 ml Magnetic stirrer Mini/MST Magnetic stirring bar 30 mm, cylindrical Volumetric pipette, 50 ml Rubber stopper, d =18/14 mm, 1 hole Pipettor Wash bottle, plastic, 500 ml Glass beaker DURAN, tall, 250 ml Glass beaker DURAN, tall, 50 ml Volumetric flask 500 ml, IGJ 19/ Funnel, glass, m d = 50 mm Spoon with spatula end, l = Balance MXX-212R, 210 g/0.01 g Buffer solution, ph 4.01, 1000 ml Buffer solution, ph 10.01, 1000 ml Caustic soda solution, 1.0 M, 1000 ml Hydrochloric acid, 1.0 mol/l, 1000 ml Glycocoll, glycine, 100 g Water, distilled, 5 l PC, Windows XP or higher Titration curve for hydrochloric acid glycine solution against 1 mol/l NaOH Experimental set-up using the Basic-Unit (P ) Determination of the isoelectric point of an amino acid (glycine) P /40 74 Laboratory Experiments, Biology PHYWE Systeme GmbH & Co. KG D Göttingen

2 Determination of the isoelectric point of an amino acid (glycine) LEB Related topics Isoelectric point, acidic anions, basic cations, zwitterions, equivalence (inflection) points, pk s value, titration, motor piston burette Principle and task Amino acid molecules carry both acid and amino groups. They can therefore form both acidic anions and basic cations. The ph at which these two types of ions are both present in the same concentration is called the isoelectric point. This point is to be determined by recording the titration curve for the amino acid glycine. Equipment (for the experimental procedure using the Chem Unit) Cobra3 Chem Unit * 1 Cobra3 power supply Data cable RS * 1 Software Cobra3 Chem Unit * 1 ph electrode, gel-filled, BNC * 1 Immersion probe NiCr-Ni, Teflon * 1 Cable, Chem Unit / motor piston burette * 1 Motor piston burette, universal type, 50 ml Magnetic stirrer mini, plastic (ABS) Magnetic stirring rod, cylindrical, l = 30 mm Volumetric pipette, 50 ml Rubber stopper, 14/18, 1 hole, d = 7 mm Safety pipettor Flip Wash bottle, 500 ml Beaker, DURAN, tall form, 250 ml Beaker, DURAN, tall form, 50 ml Volumetric flask, 500 ml Funnel, glass, d = 55 mm Spoon with spatula end, l = 150 mm, steel, wide Balance MXX-212R, 210 g/0.01 g, RS232, 230 V Buffer solution ph 4.01, 460 ml Buffer solution ph 10.01, 1000 ml Sodium hydroxide standard solution, 1 mol/l, 1000 ml Hydrochloric acid standard solution, 1 mol/l, 1000 ml Glycine, 100 g Distilled water, 5 l PC, Windows 95 or higher Hazards and safety measures According to its concentration, sodium hydroxide solutions cause severe burns or act as irritant to skin, eyes and mucous membranes. Spray from them irritates the respiratory organs. The corrosion destroys tissue and causes severe pain. Must be kept out of the reach of children. According to its concentration, hydrochloric acid causes severe burns or acts as irritant. Vapours irritate respiratory organs, whereby the mucous membranes of the upper respiratory tract are particularly affected. Concentrated acids destroy skin and textiles. Fig. 1: Experimental set-up using the Chem Unit Phywe Series of publication Laboratory Experiments Biology PHYWE SYSTEME GMBH & Co. KG D Göttingen P /40 163

3 LEB Determination of the isoelectric point of an amino acid (glycine) Do not inhale vapours or spray. Avoid skin contact. When working with these chemicals wear appropriate protective apparel, protective gloves and safety glasses. First aid: Thoroughly rinse the affected skin or eyes (opened wide) with plenty of water. When eyes are affected, seek immediate medical attention. After accidents or on feeling unwell, fetch medical attention at once. When inhaled: Fresh air, keep respiratory tract free. On respiratory distress: Transport to medical attention in a half-sitting position. Waste disposal: Dilute the solutions with water, neutralize them (ph 6-8) and wash them down the drain. Preparatory work Prepare the solution required for the experiment as follows: 0.1 molar hydrochloric acid glycine solution: Weigh 3.75 g of glycine into a 50 ml beaker. Transfer this weight quantitatively into a 500 ml volumetric flask (rinse the beaker several times with distilled water). Use the volumetric pipette to add 50 ml of 1 mol/l hydrochloric acid to this flask and make up to the mark with distilled water. Set-up and procedure (using the Chem Unit) Set the apparatus up as shown in Fig. 1 Connect the control input of the motor-driven piston burette to the TTL output of the Chem Unit with the cable specifically for this Connect the ph electrode to the ph input and the immersion probe to the temperature input T1 of the Chem Unit Call up the Cobra3 Measure programme in Windows and assign the Chem Unit as measuring instrument Set the measurement parameters as shown in Fig. 2. In the <Displays...> menu, set both Digital display 1 and Diagram 1a (range 0-30 ml) to ph (range 1 to 13) Set the temperature compensation in the <Preferences / ph> menu to temperature probe T1: ph(comp.t1) Calibrate the ph electrode by pouring the two buffer solutions into separate 50 ml beakers and calling up <Calibrate / ph>. Should the electrode which is to be used have already been recently calibrated, however, then a new calibration is not necessary (automatic saving of calibration data) Follow the operating instructions for the motor-driven piston burette to fill it with sodium hydroxide solution Set the motor-driven piston burette to <Dispense>, <Dispensing volume = 0.2 ml> and <Rate = 100 ml/min> Place the 200 ml beaker, containing a magnetic stirring rod, on the magnetic stirrer Use the 50 ml volumetric pipette twice to pipette 100 ml of the prepared hydrochloric acid glycine solution into the beaker Fix the discharge tube of the motor-driven piston burette, the ph electrode and the immersion probe to the electrode holder of the motor-driven piston burette, using the bored rubber stopper for the immersion probe. Adjust the electrodes and the tip of the discharge tube so that they are completely immersed in the solution Adjust the stirrer to a medium stirring speed (Take care: Do not allow the magnetic stirring rod to hit against the ph electrode) Start the titration with <Continue> and <Start measurement>. The first measured value (V = 0 ml, for the starting point) is immediately acquired by the programme and the motor-driven piston burette dispenses the first portion of sodium hydroxide solution The titration is now automatically carried out, in accordance with the set parameters, until a total of 30 ml of sodium hydroxide solution has been added. The programme then automatically stops the measurement Fig. 2: Measurement parameters (Chem Unit) Fig. 3: Titration curve for hydrochloric acid glycine solution against 1 mol/l NaOH 164 P /40 Phywe Series of publication Laboratory Experiments Biology PHYWE SYSTEME GMBH & Co. KG D Göttingen

4 Determination of the isoelectric point of an amino acid (glycine) LEB At the end of the measurement, save the data in menu <File> <Save measurement as...> Results and evaluation Fig. 3 shows the titration curve, as it is displayed by the programme after ending the measurements. The prepared hydrochloric acid glycine solution contains glycine and hydrochloric acid in a ratio of 1:1. The amino groups of the glycine are converted by the acid to the protonized cation form: H 2 -COOH + H 3 O + + Cl - > + H 3 -COOH + H 2 O + Cl - On the addition of sodium hydroxide, the ph of the solution continually increases and the concentration of the cations continually decreases: + H 3 -COOH + Cl - + OH - + Na + > + H 3 -COO - + H 2 O + Cl - + Na + At the first equivalence point of the titration, the addition of an equimolar quantity of base has quantitatively split the proton from the carboxyl group. The glycine is present here in the form of zwitterions (externally neutral). The ph at this point is called the isoelectric point (pi), as the number of amino acid cations is here equal to the number of amino acid anions. As the titration is continued, the increase in ph continues, the concentration of zwitterions decreases and that of the amino acid anions increases, until, at the second equivalence point, only amino acid anions are present in the solution: + H 3 -COO - + OH - + Na + > H 2 -COO - + H 2 O + Na + In principle, therefore, the glycine cation (see above) represents a two-proton acid, and the titration curve so exhibits two inflection points (equivalence points). The software enables the equivalence point of the titration to be automatically determined. Should your software not enable this to be done, then you can have it determined by using differentiation. For this, call up the menu <Analysis> / <Channel modification> and select there the options <Differentiate>, <Modify left axis> and <Add new channel>. Select the function <Calculate> for the differentiated measurement curves to be displayed (Fig. 3). To have the two curves displayed underneath each other, reset the displayed ph range under <Measurement>, <Display options> and <Left or right y-axis>. Use the <Survey> function to have a vertical line fitted through the peak of the differentiated curve. The intersection of this line with the titration curve gives the equivalence point (point of inflection). The coordinates of this equivalence point are displayed numerically in a yellow field. Unlike the first equivalence point in this titration, the ph increase at the second equivalence point is too low. To determine this second equivalence point, therefore, first enlarge the display of the second portion of the titration curve with the zoom function (Fig. 4) and then proceed as described above.the pk S1 value can be read from the curve as 5 ml (half of the consumption of alkali up to the first equivalence point, see Fig. 5). The pk S2 value can be read as 15 ml (half of the difference in the consumption of alkali up to the first and the second equivalence point). Fig. 4: Magnified display of the right portion of the titration curve Fig. 5: Reading of the pk S value Phywe Series of publication Laboratory Experiments Biology PHYWE SYSTEME GMBH & Co. KG D Göttingen P /40 165

5 LEB Determination of the isoelectric point of an amino acid (glycine) The respective buffering regions of the aqueous glycine solution lie in the regions of ph around the pk S values. In these regions, the ph changes only slightly despite the addition of base or acid. At the equivalence points, however, a large change in the ph is caused by only small additions of base or acid. In aqueous solution, and in dependence on the ph, glycine forms not only cations, but also anions and neutral zwitterions. At the isoelectric point, the number of cations is the same as the number of anions, the concentration of zwitterions is highest here. In the titration described here, this point is identical with the first equivalence point (pi = ph = 6.12). In the example shown, the equivalence points are at V = ml and V = ml, and at ph values of 6.12 and The respective pk S values were calculated to be 2.34 and Theoretically, the equivalence points of the amount of hydrochloric acid glycine used should lie at V = ml and V = ml, with ph values of ph = 6.01 (ph = 0.5 pk S pk S2 ) and ph = (ph = pk b log c b ) Experimental procedure using the Basic Unit The experiment using the Chem Unit described above can be analogously carried out using the Basic Unit. For this, the entries in the list of materials which are marked with an asterisk must be replaced by the materials listed below. The set-up and procedure are then also slightly different (see below, in particular Fig. 6 and Fig. 7). Fig. 7: Measurement parameters (Basic Unit) The values for glycine given in the literature are: pk S value of the carboxyl group: 2.34 pk S value of the ammonium group: 9.71 (pk b value: 4.29) Isoelectric point: 6.03 The values found experimentally therefore comply quite well with the theoretical values. Fig. 6: Experimental set-up using the Basic Unit 166 P /40 Phywe Series of publication Laboratory Experiments Biology PHYWE SYSTEME GMBH & Co. KG D Göttingen

6 Determination of the isoelectric point of an amino acid (glycine) LEB Changes in the equipment required for use of the Basic Unit: Cobra3 Basic Unit, USB Software Cobra3 ph/potential Measuring module ph/potential ph Electrode, plastic, gel-filled Temperature sensor, semiconductor type Cable, Basic Unit / motor piston burette Set-up and procedure using the Basic Unit Prepare the set-up as in Fig. 6 Connect the control input of the motor-driven piston burette to output S1 of the Basic Unit with the cable specifically for this Connect the ph electrode to the ph measuring module and the temperature sensor to input S2 of the Basic Unit Call up the Cobra3 Measure programme in Windows and assign Cobra3 ph/potential as measuring instrument Set the measurement parameters as shown in Fig. 7 Set the temperature compensation to <Automatic> (temperature probe S2) Carry on from here as with the Chem Unit Note The measurement can also be carried out without a temperature probe, but then the temperature of the solution must be entered by hand in the menu <Preferences / ph>, or, when using the Basic Unit, in the field <Temperature compensation>. Phywe Series of publication Laboratory Experiments Biology PHYWE SYSTEME GMBH & Co. KG D Göttingen P /40 167

7 LEB Determination of the isoelectric point of an amino acid (glycine) Room for notes 168 P /40 Phywe Series of publication Laboratory Experiments Biology PHYWE SYSTEME GMBH & Co. KG D Göttingen