Clinical Chemistry (CHE221) Professor Hicks Lecture 4 Chloride Ion and Determination of its Concentration titration method titrate mercuric ion into the sample Hg 2+ (aq) + 2Cl - (aq) HgCl 2 (aq) stock Hg 2+ chloride ion is consumed eventually all used up, then Hg 2+ + dephenylcarbazone (blue) excess mercury reacts with dephenylcarbazone turning solution blue + 2 drops indicator (diphenylcarbazone) 2000 µl protein-free serum sample (contains Cl - ) all the stock solutions have been prepared for you 1
Do we really have to create a Folin-Wu protein-free filtrate? NO! Today you will work with a pseudo-serum that contains no protein Why doe proteins have to be removed? proteins have numerous sites that could bind Hg 2+ - amine and carboxylic acid functional groups at the ends of proteins (aka N terminus and C terminus) - amino acid side chains with O, S, N calculations for titration method stock Hg 2+ + 2 drops indicator (diphenylcarbazone) 2000 µl protein-free serum sample (contains Cl - ) 2
spectrophotometric method Hg(SCN) 2 (aq) + 2Cl - (aq) HgCl 2 (aq) + 2SCN - (aq) added in excess reacts completely SCN - + Fe +2 (aq) FeSCN 2+ (aq) (intense red color) Hg(SCN) 2 and HgCl 2 are covalent compounds do not dissociate into ions when dissolved amount formed = amount Cl - in sample Amount SCN - released is equal to amount HgCl 2 formed HgCl 2 is soluble -Hg 2 Cl 2 mercury(i) chloride is insoluble Protocol is wrong red product formed is FeSCN 2+ not Fe(SCN) 3 protocol says reaction does not follow Beer s Law fact: Beer s Law is not a property of a reaction if a reaction gives a non-linear calibration curve some possible reasons are 1) absorbance is not in a measurable range (0.10 1.0) 2) the instrument is out of whack 3) your model is out of whack 3
(except when it is not directly proportional?)??? protocol says intensity of the color (we can only assume they mean absorbance) is proportional to chloride concentration protocol also says reaction does not follow Beer s Law fact 2 : Beer s law states that concentration is proportional to A - the absorbance of the red product cannot be proportional to chloride and not obey Beers Law at the same time so what part, of GenChem, was this Pointe Scientific scientist sleeping through? Complex Ions! Fe 3+ + SCN 1- FeSCN 2+ (red) FeSCN 2+ + SCN 1- Fe(SCN) 1+ 2 etc. LeChateliers Principle says: adding modest amounts of SCN - will form mostly FeSCN 2+ at higher concentrations SCN - the FeSCN 2+ will react further to form Fe(SCN) 1+ 2 etc. reducing the absorbance due to FeSCN 2+ 4
Fact: Pointe Scientific s equation is based on Beer s Law Pointe scientific Pointe Scientific protocol calls for construction of a calibration curve to determine the linear range so that the unknown concentration can be calculated in this range as absorbance of unknown conc. of calibrator = conc. of unknown absorbance of calibrator which is derived from Beer s Law A (unknown) = ε b c(unknown) A (calibrator) = ε b c(calibrator) conc. of calibrator = conc. of unknown ε cancels because it is the same substance causing absorbance in calibrator and unknown b cancels because the same cell is same size cell are used for in calibrator and unknown c (calibrator) and conc. of calibator cancel because they are the same exact thing written in different notation 5
BUT, Pointe scientific s method of calculation will not work on any linear range but only ones that extrapolate back to zero we cashed your check already! 0.70 looks nice and linear here lets use Beers Law in this range 0.20 0.40! Pointe scientific CEO 0.60 0.50 0.40 0.30 0.20 0.10 concentration more than doubles 20-42 mmol/l but absorbance changes only 0.60 0.62! 0 10 20 30 40 50 Describes a line with A on y axis, C on x axis, slope εb and y-intercept of zero Beer s Law A = εb * c + 0 y = m * x + b Everything you ever wanted to know about Beers Law Calibration Curves data can deviate from Beers Law for reasons other than absorbances that are outside the measurable range a calibration curve relates concentration to absorbance with no assumption of any theory 0.70 0.60 0.50 0.40 0.30 0.20 0.10 hard to tell where it hit when the graph is almost horizontal graph is curved but concentration can be determined from A??? the limitation on the range of A that can be related to concentrations is in reading the calibration curve not the range that Beers Law is obeyed or the range your assumed reaction occurs in direct proportion to the amount of reagents added uncertainty in concentration gets big! 0 10 20 30 40 50 6
so what should we do? 1) follow the Pointe Scientific protocol and calculate as they indicate 2) when plotting the calibration curve plot concentrations 0-120 mmol/l (not just 80-120 as Pointe indicates) - a 120 mmol/l stock will be provided. Prepare samples 20, 40 60 80, and 100 mmol/l by diluting with water 1) using the calibration curve determine the unknown concentrations graphically 2) let me know how it turns out by comparing the three sets of results in your discussion - make a table of chloride concentration found and method used chloride ion major extracellular anion = chloride (major intracellular anions = organic phosphates) counter ion to maintain charge balance most dietary chloride is absorbed - no energy must be put in, diffuses in with Na + Reference Ranges serum 98-107 mmol/l urine 110-250 mmol/l (during day) 7
passive transport movement of substances to regions of lower concentration (often across cell membrane) results eventually in all concentrations being equal happens because molecules are always in random motion requires no input of energy two types passive transport simple diffusion by direct migration of molecules moving randomly (small molecules CO 2, O 2, N 2 etc.) facilitated diffusion involving a facilitator such as a carrier protein (larger molecules like sugars, other proteins) substances that are constantly consumed can be delivered by passive transport because their concentration is constantly being lowered active transport transport that requires energy ATP ADP decomposition most common source of energy pumps substances to places where they need to be at higher concentration for cell function Example higher concentration in cells Na + lower concentration outside cells 8
waste of energy Free Energy (G) ATP if no attempt is made to harness it both release heat heat ADP heat active transport some of the energy can be used to drive active transport Free Energy (G) ATP less heat ADP active transport driven by ATP ADP hydrolysis like bowling ball being used as a counterweight to lift lighter object 9
diagnostic value of chloride ion cystic fibrosis high sodium and chloride levels in sweat indicate CF diagnose acidosis/alkalosis detect instrument problems by anion gap Acidosis respiratory acidosis result of retention of CO 2 CO 2 + H 2 O H + + HCO 3 - chronic metabolic acidosis result of production of organic acids at faster rate than they can be destroyed Example - Ketoacidosis diabetics cannot get glucose into cells b/c requires insulin use fat as energy source producing ketoacids acetone acetoacetic acid beta-hydroxybutyric acid 10
Acidosis and hypochloremia / hyperchloremia chloride levels usually follow sodium levels exception: metabolic acidosis and alkalosis metabolic acidosis - hyperchloremia (high Cl - ) without hypernatrinia (high Na + ) metabolic alkalosis hypochloremia (low Cl - ) without hyponatrinia (low Na + ) Anion Gap reference range = 10-20 mmol/l total positive charge = total negative charge Na + + + Ca 2+ + Mg 2+ = Cl - -HCO 3- + HPO 2-4 + HSO 4- + Org. Acid + protein not all cations (positive ion) are measured not all anions (negative ions) are measured ion gap = Na + + -Cl - -HCO - 3 ion gap increases if HPO 4 2- or HSO 4 1- increases uremia or renal failure ion gap decreases if Ca 2+ increases hypercalcemia (rare) = HPO 2- Na + + -Cl - -HCO- 4 + HSO 4- + Org. Acid + protein - Ca 2+ -Mg 2+ 3 more than one analysis with an ion gap outside reference range probably an instrumental problem ion gap increases if Org. Acids go up ketoacidosis ion gap decreases if albumin (a protein) hypoalbuminemia (rare) 11