Errors and Significant figures

Systematic (determinate) -consistent error. Can be

detected & corrected
Errors

Random (indeterminate)-due to limitations of physical

measurement. Cannot be eliminated.

Systematic error

(i) operational & personal e.g. mechanical loss of

material during analysis.or inability to detect
colour changes at end-point of titration.
(ii) instrumental or reagent errors- faulty calibration
of balances, attack of reagents upon glassware.
(iii) errors of method - incorrect sampling & from
incompletenes of a rx.
(iv) additive & proportional errors.

Systematic error can be reduced by

(a) calibration of apparatus & application of correction factors
(b) running a blank determination – separate determination with a
blank under identical experimental conditions
(c) running a control - with a quantity of a standard substance with
approx the same weight of constituent as contained in sample.

(d) use of independent method of analysis

(e) running parallel determinations - serves as a check on the precision
of result-does not guarantee accuracy
(f) use of standard addiion/internal standard or
isotopic dilution.
(g) amplification methods - useful when small amount of material is to
be analysed.

CH4304 Laboratories 1
Significant figures and computations.

1. The number of significant figures in a value is the minimum number of

digits required to write the value in scientific notation. e.g. 0.00035 =

3.5 x 10-4 i.e. two significant figures. 0.000 0045 = 4.5 x 10-6, two

significant figures.

2. The first uncertain digit in a calculated result should be the last

significant figure. e.g.

0. 002364  ±0. 000003

=0 . 0946 ±0 .0002 4 
0. 02500  ±0. 00005 

2. 364 x10-3
2. 500 x10 -2
¿
righ
¿
¿
¿
¿
¿ ¿
¿
¿

3. For addition and subtraction the last significant figure is determined by

the decimal place of the least certain number 21.29965 + 12.2 = 33.5

4. For multiplication and division the number of figures is usually limited by

the factor with the least number of digits. Retain in each factor one

more significant figure than is contained in the factor having the largest

uncertainty e.g. 1.26 x 1.236 x 0.6834 x 24.8652

should be carried out as 1.26 x 1.236 x 0.688 x 24.87 and the result

expressed to three significant figures

CH4304 Laboratories 2
5. For values expressed as logs the number of figures in the mantissa should
equal the number of significant figures in the original value.

Calculating Uncertainty

1. For addition and subtraction use absolute uncertainty

e 4= e 2 2 2
1 e 2 e 3

e.g. 1.76 ( ± 0.03) + 1.89 (± 0.09) - 0.59 (± 0.02)

= 3.06 ± e4 = 3.06 ± 0.041

1. For multiplication and division use relative uncertainties


e= ∑  e 2
1 .76 ±0 . 03  x1 .89 ±0 . 02
0 . 59±0. 02 

relative uncertainty (0.03)/(1.76) x 100 = 1.70%

e=  1 . 70 2 1 . 06 2  3 . 39 2 =3 . 94
5 . 64 ±3. 94 =5 .6 ± 0. 2 

CH4304 Laboratories 3
Mean & Standard Deviation
When a quantity is measured with the greatest exactness of which the

instrument, method and observer are capable of it is found that the results

of successive determinations differ among themselves to a greater or lesser

degree the average value is accepted as the most probable – this may not

always be he true value

Difference between the measured and the true value = absolute error and

this is a measure of the accuracy of a result.

The true or absolute value of a quantity cannot always be established

experimentally so the observed value must be compared to the most

probable value
∑ xi
Sample mean x= i
n

The spread of values is measured most efficiently by the sample standard

deviation s

∑x(-)i2
s= i
n− 1

s2 = variance
R.S.D. relative standard deviation
s
=
x

Sometimes referred to as the coefficient of variation C.V.

s x 1 0 0
C= . V .
x

CH4304 Laboratories 4
Example 1

Analysis of a sample of iron ore gave the following % values for the iron

content:

7.08 7.21 7.09 7.16 7.14 7.07 7.14 7.18 7.11

x = 7.13 s = 0.047 C.V. = 0.66%
The greater the number of measurements (n) the closer the sample mean

(x) approach the population mean (µ). The standard error of the mean sx
s x
s
=
±
n

For example 1 sx = ± 0.014. If 100 measurements were made denominator

=100 1/2 so

sx = ± 0.0047 so the precision of a measurement may be improved by

increasing the number of measurements.

Distribution of standard errors

If a large number of replicate observations are made (>50) of a continuous

variable the results obtained will usually be distributed about the mean in a

roughly symmetrical manner-Gaussian Error Curve.

12
y= (-exµ)σ /2
δπ2

CH4304 Laboratories 5
68% of all values fall within one S.D. of the mean, 95% will fall within 2 S.D

(1.96). and 99.7 will fall within 3 S.D of mean.

From e.g. 1 S.D. = 0.047 so 68% of values will lie ± 0.047 of the mean. 95%
will lie between ± of the mean value i.e. there is a 5% (1 in 20) os a

resultdiffering from the mean by more than ± 0.094%.

CH4304 Laboratories 6
Reliability of results

Statistical figures obtained from a set of data are of limited use by

themselves

a) reliability of results

Analysis of results

b) comparison of results with true value or

with other sets of data

Rejection of results should be considered only when a suitable statistical

test has been applied or when there is an obvious chemical or instrumental

reason

E.g. 2. The following data were obtained for the determination of Zinc in a

sample of dust:

4.3 µg g-1, 4.1µg g-1 , 4.0µg g-1 , 3.2µg g-1 . Should the value 3.2 be rejected.

Apply Q test

questionable value - nearest value 3.2 - 4.0 0.8

Q= = = = 0.727
largest value - smallest value 4.3 − 3.2 1.1
If Q (observed ).>Q (tabulated) discard the questionable point. Q for 4

points from table = 0.831 so result should be retained.

CH4304 Laboratories 7
 Correlation and regression

calibration: construct calibration curves by measuring analytical signal for

each standard & plotting against concentration

• two statistical operations should be performed on calibration curve

• Correlation coefficient {r}: to determine if graph is linear or

in the shape of a curve

• Least square error associated with “best fit” line/curve

nåxi y i -å x i åy i
r=
 [nåx 2i − åxi 2 ][ nåy 2i − åy i 2 ]

n: number of data points

• r must be close to 1 for good fit

•
Linear Regression-“ least squares”

2 assumptions

• errors in y values substantially greater than errors in x values

• uncertainties (standard deviations) in all y values are similar
eqn of line y = mx + b

point (xi, yi) vertical deviation (di) = yi - y

di = yi-y = y-(mx + b)

di2 = (yi - mx - b)2 : least squares

CH4304 Laboratories 8
 ∑  xi yi ∑ xi
∑ yi n
¿
rli
¿
¿ ÷D
¿

¿
b=
¿
2
∑  xi ∑  xi yi
∑ xi ∑ yi
¿
rli
¿
¿  ÷D
¿

¿
D=
¿
2
∑  xi  ∑ x i
∑ xi n
¿
rli
¿
¿
¿
∣¿∣¿
m=¿ ¿
¿
¿
¿

Standardised Acid and Standardised Base

CH4304 Laboratories 9
Standard HCl

Procedure

1. Use the information provided in Table 1 to calculate the volume

of
concentrat (37 wt %) HCl that should be added to 1 L of distilled
water to produce 0.1 M HCl, and prepare this solution.

2. Dry primary grade sodium carbonate for 1 hr at 110oC and cool it in a

desiccator.

3. Weigh four samples containing enough Na2CO3 to react with 25 mL

of 0.1M HCl and place each in a 125 mL flask. As you are ready to
titrate each one, dissolve it in about 25 mL of distilled water.

2HCl + Na2CO3 CO2 + 2NaCl

FW = 105.989

Add 3 drops of bromocresol green indicator to each and titrate one

rapidly (to a green colour) to find the approximate end-point.

4. Carefully titrate each sample until it just turns from blue to green.
Then boil the solution to expel CO2. The solution should return to a
blue colour.

5. Carefully add HCl from the buret until the solution turns green again.
A blank titration can be performed by using 3 drops of indicator in 50
mL of 0.05 M NaCl. Subtract the volume of HCl needed for the blank
solution from that required to titrate Na2CO3.

6. Calculate the mean HCl molarity, the standard deviation and the
relative standard deviation.

CH4304 Laboratories 10
Standard NaOH

1. A 50 wt % aqueous NaOH is provided. Normally this solution is

prepared in advance and allowed to settle. Na2CO3 is insoluble in this
solution and precipitates. The solution should be stored in a tightly
sealed polyethylene bottle and handled gently to avoid stirring the
precipitate when the supernate is taken. The density is close to 1.50g
of solution per mL.

2. Primary standard-grade potassium hydrogen phthalate should be dried

for 1Hr at 110oC and stored in a dessicator.

COOK COOK
+ + H2O
NaOH
COOH COONa
F.W. = 204.22

3. Boil 1.0L of water for 1Hr to expel CO2. Pour the water into a
polyethylene bottle which should be tightly capped. Calculate the
volume of 50% aqueousNaOH needed to produce 1.0L of
approximately 0.1 M NaOH. transfer this to the water bottle, mix well
and allow to cool.

4. Weigh four samples of the solid KH(C8H4O4) (MW 204.23)and

dissolve each in approx. 25 ml of distilled water in a 125ml flask. Each
sample should contain enough solid to react with about 25ml of 0.1M
NaOH. Add 3 drops of phenolphthalein ndicator to each and titrate one
of them rapidly to find the approximate end-point. The burette should
be fitted with a loose fitting cap to minimise entry of CO2.

5. Calculate the volume of NaOH required for each of the other three
samples and titrate them carefully swirling the flask occasionally. The
end point is the firstappearance of a faint pink colour. This colour will
disappear as CO2 from the air dissolves in the solution.

6. Calculate the average molarity, the standard deviation, and relative

standard deviation.

CH4304 Laboratories 11
Name Approximat Approximat mL of Reagent
e weight e molarity needed to prepare 1
percent L of 1.0 M
solution

Acetic acid 99.8 17.4 57.5

Hydrochloric acid 37.2 12.1 82.6
Nitric acid 70.4 15.9 62.9
Perchloric acid 70.5 11.7 85.5
Phosphoric acid 85.5 14.8 67.6
Sulphuric acid 96.0 18.0 55.6
Ammonia* 28.0 14.5 69.0
Sodium Hydroxide 50.5 19.4 51.5
Potassium hydroxide 45.0 11.7 85.5

*28wt% ammonia is the same as 56.6 wt% ammonium hydroxide

CH4304 Laboratories 12
Preparation of buffers
Buffer solution can be defined as an equilibrium mixture of a conjugate acid-
base pair which is capable of resisting substantial; changes in pH upon the
addition of small amounts of acidic or basic substances. With some
exceptions, either the acid or the base of the conjugate pair is a weak
electrolyte.

The Henderson-Hasselbalch equation is a mathematical statement which

defines the pH of a solution of a conjugate acid-base pair in terms of the
dissociation constant of the weak acid-base pairs and the equilibrium
concentrations of the acid and its conjugate base. This equation is extremely
useful in calculating the pH of the buffer.

Figure 1: Preparation of a 0.02M Tris buffer at pH 8.0.

Buffer A and B are prepared in the same way except 0.5 mole of NaCl is
added to flask B before the volume is brought to 1 L for buffer B.

CH4304 Laboratories 13
Ion-exchange chromatography - spectrophotometric
determination of the composition of a sample of VOSO4

VOSO4 supplied commercially contains impurities H2SO4 and H2O. A solution

will be prepared from a known mass of reagent. The VO2+ can be assayed
spectrophotometrically and the total cation (VO2+ and H+) can be assayed by
ion-exchange. Together these measurements enable the determination of
the quantities of impurity in the sample.

Ion exchange resins are polymer matrices containing charged site which are
used to separate charged species .

CH=CH2 CH=CH2

Styrene CH=CH2

divinylbenzene

CH-CH2 CH-CH2 CH-CH2

SO3-H+ SO 3-H+
CH-CH2 CH-CH2 CH-CH2 CH-CH2 CH-CH2

SO 3-H+ SO3-H+

• Anion exchangers contain bound positive groups e.g. –NR3+

• Cation exchangers contain bound negative groups e.g. –SO3-

CH4304 Laboratories 14
Procedure

It is initially necessary to determine the ion-exchange capacity of the resin

(moles
of Na+/gram of resin). Weigh accurately about 2.000g of Amberlite 120A
resin in the Na form. Make a slurry of the resin in distilled water and pour
into the glass chromatography column. The level of the water should always
cover the resin. Never let the resin run dry

NaCl
HCl

+
Na H
+ +
+ Na
Na + +
+ H Na
+ H
Na +
+ Na
Na + +
H Na

titrate with NaOH

NaCl HCl

VOSO4.H2SO4

+
H
VO2+
+
H VO 2+
+
H +
H

H 2SO 4 titrate with NaOH

CH4304 Laboratories 15
2. Conversion of the resin to the H+ form. Pipette 5.0mL of 1.0M acid
(H2SO4 or HCl) onto the column and allow to equilibrate. Wash off excess
acid with the distilled water until a neutral eluate is obtained (use litmus
paper).

3. Determine ion exchange capacity of resin in moles Na+/g resin.

Pipetted 2.0mL of standardised 0.500M NaCl solution onto the column
and allow to equilibrate for 5 minutes. Wash with about 25 mL of distilled
water and collect the eluate in a 150mL flask. Add three drops of
phenolphthalein indicator and titrate with standard 0.100N NaOH.

4. VOSO4 solution: weigh accurately, approximately 0.50g of VOSO4 and

make up to 100mL with distilled water. Using the molar absorptivity (ε)
of the vanadyl ion at 750nm, VO2+ = 18M-1cm-1 it is possible to calculate
the amount of pure VOSO4 in the sample.

5. Following regeneration of the resin as previously described, (2 above) the

procedure is repeated for the vanadyl sulphate solution to determine the
total cation content, i.e. VOSO4 + H2SO4 .Care should be taken not to
exceed the exchange capacity of the resin.

4. The water in the sample can be calculated from a accurate measurement

of the initial weight. i.e. original weight - (VOSO4 + H2SO4)

5. Express your answer as (VOSO4)x(H2SO4)y(H2O)z

Example

(a) In an experiment to determine the purity of Vanadyl Sulphate

2.000 g of ion-exchange resin in the H+ form were used. 20.00 ml

of 0.100M NaCl was put on the column and allowed to equilibrate.

The eluate was collected and titrated against 0.010M NaOH, the

titre was 32.0 ml . What is the ion-exchange capacity in mols/g of

resin.

CH4304 Laboratories 16
Ans

32.0 x 0.010 M = 3.2 x 10-4 mols NaOH = 1.6x10-4 mols /g resin

(b) b) Assuming the VOSO4 was absolutely pure what is the

maximum volume of solution that can be put on the column such

that the ion-exchange capacity is not exceeded given that a

solution of Vanadyl sulphate was prepared by dissolving 0.1420g in

100mls of H2O. .

Ans: VOSO4 mol wt = 163 g/mol so if 100% pure the 0.1420g of

VOSO4 = 8.7 x 10-4 mol. Resin capacity for VO2+ ion is 1.6x10-4 mols, so

the maximum capacity of the resin for VOSO4 (if 100% pure ) is 18.37

ml of solution

(c) c) A solution of Vanadyl sulphate was prepared by dissolving

0.1420g in 100mls of H2O. 10.0 ml of the solution was put on the

regenerated resin (H+ form) and allowed to equilibrate. The eluate

was collected and titrated against 0.010M NaOH. The titre was

18.0 ml. The absorbance of the solution in a 1cm cuvette was 0.126

at 750nm when ε =18cm-1M-1. .

Calculate the molar ratio as

(VOSO4)1.00 (H2SO4)y(H2O)z

Ans: A = εc so c= A/ε = 0.126/18 = 7.0 x10-3M in 10 mL ⇒ 7.0

x10-5 mol VOSO4 = 0.01141 g

 18 .0  0 .010 M 
Titre 18.0 ml ⇒ mol of (VOSO4 + H2SO4)
 1000 2

= 9 x 10-5 mol ⇒ 2.0 x 10-5 mol H2SO4 = 1.96 x10-3 g H2SO4

wt of H2O = 0.01420 (in 10 mls) – (0.01141 + 1.96 x10-3)g =

8.3 x 10-4 g H2O = 4.6 x10-5 mol

(VOSO4) 7.0 x 10-5 (H2SO4) 2.0 x 10-5 (H2O) 4.6 x10-5

CH4304 Laboratories 17
 (d) = (VOSO4)1.00 (H2SO4)0.29(H2O)0.66

Gas Chromatography

Gas chromatography is an instrumental technique for the separation of

volatile molecules. The mobile phase is an inert gas generally nitrogen or
helium and the stationary phase is a nonvolatile liquid chemically bonded onto
a quartz capillary column of very narrow diameter.. The column is placed in a
temperature programmable oven and the Nitrogen passes through it. Gas
chromatography involves a sample being vapourised and injected onto the
head of the chromatographic column. Samples are injected onto the column
via a heated injection port and are transported through the column by the
flow of inert, gaseous mobile phase; and are separated on the basis of having
different affinities for the stationary phase. As each component exits (or
elutes) from the column it enters a detector and produces a signal peak. The
peak area is proportional to the concentration or mass of that component in
the sample. Qualitatively, the identity of unknown components can be
determined by comparing their retention times with known standards.
Quantitatively, the concentration of a given component can be determined
by measuring the peak area. The samples are detected as they exit the
column. The integrator gives an output of peak height versus time, called a
chromatogram from which samples may be identified and concentrations
evaluated.

injector detector integrator

N2

column

oven

CH4304 Laboratories 18
Background

Chromatographic techniques are used to perform qualitative and quantitative

analysis on complex mixtures. As the mixture of components to be separated
is carried through thecolumn, separation is affected because of the varying
attraction each component of the mixture has for the stationary phase.
Substancesthat are more strongly attracted to the stationary phase will
becarried down the column at a slower rate than those with a lesser
attraction.. The temperature of thecolumn and, to a lesser degree, the flow
rate of the mobile phase alsoaffect the degree of separation. The time
required for a particularcomponent of a mixture to pass from the injection
port, through the column, to the detector is its retention time. Under
identicalconditions, the same substance will have the same retention time
inrepeated analyses. For this reason, retention time can be usedqualitatively
to identify a substance in a mixture.
If a quantitative determination is desired, the method of internal
standards is normally employed. An internal standard is needed
when conditions such as sample size of gas flow cannot be replicated
exactly between trials. A compound is chosen to serve as an
internal standard that has physical properties similar to those of the
substance you wish to measure. A calibration curve is constructed
in which the ratio of the peak area of the compound of interest (the
analyte) to that of the internal standard is plotted on the y-axis
versus the concentration of the analyte on the x-axis. The peak area
ratios will be proportional to the analyte concentration, whereas the
peak area of the analyte alone will not be. Peak areas are calculated
by the electronic integrator.

In this experiment you are provided with an unknown alcohol in water and you
are required to

1. identify the unknown on the basis of its retention time using the

standards provided

CH4304 Laboratories 19
2. quantify the concentration of butanol in water by internal
standardisation using one of the standards provided.

3. calculate N (the number of theoretical plates required to elute the

unknown) and the resolution wrt the internal standard

EXPERIMENTAL PROCEDURE:
A: Identification of unknown in alcohol mixture
B: quantitative analysis of butanol in a butanol:water mixture.

A: identification of unknown.

In a 10mL volumetric flask make up 3 standards containing 0.20mL each of

butanol, ethanol, and propanol in distilled water. Inject each standard onto
the GC and record the retention times. Inject the unknown provided.
Comparison of the Rt of each of standards against the components of the
unknown should allow direct identification of the unknown(s)

The Quantitation of Butanol

Prepare a series of standards ranging from 2.0% to 8.0% of butanol as
indicated below using pipettes to deliver each liquid. Use the 50ml flasks
and make up to the mark with water

After the four standards have been prepared, add 2.0mL of propanol to
each mixture via pipette. Propanol will serve as the internal standard in
this analysis.

standard volume of butanol volume of propanol Total volume

CH3CH2CH2CH2OH CH3CH2CH2OH

1.0mL 2.0 mL 50.0

2.0mL 2.0 mL 50.0
3.0 mL 2.0 mL 50.0
4.0 mL 2.0 mL 50.0

Swirl each mixture thoroughly to ensure complete mixing, then cover

each solution to avoid evaporation.

CH4304 Laboratories 20
Obtain 50.0 mL of an unknown butanol:Water mixture from your
instructor. Using a 1.0 mL pipet, add exactly 2.0 mL propanol to the
unknown. Swirl the mixture thoroughly and cover to retard evaporation.
.

SETTING UP THE Gas Chromatograph

You will use the HP5890gas chromatograph in conjunction with the
integrator

The integrator output will list the peak number

Peak # Retention time Area counts % Area

(mins)
1 x.xx xxxxxx xx.x
2
3

Experiment should be run isothermally. Your instructor will select the temperature

CALCULATIONS:
1. Calculate the ratio of peak area values of butanol to propanol.
2. Using EXCE L prepare a calibration curve by plotting the
ratios of concentration of butanol/propanol on the x-axis versus the average
of the ratios of the peak area of butanol to peak area of propanol.
3. Use EXCELto perform a regression analysis on the calibration
curve .
4. Calculate the concentration of butanol in the unknown sample from
the regression analysis basing your calculation on the straight line
equation form, y = m x + b.
5. Turn in the graph of the calibration curve, the regression analysis,
the calculation of the unknown and the Report Sheet.

A major source of precision error in GC is from poor injection technique.

Both manual and automatic injection methods are available. If automatic
equipment is available use it.

CH4304 Laboratories 21
 If not several approaches can be used to try and reduce error.
The first step is to ensure that the syringe is properly filled containing no
air bubbles. The instructors will demonstrate how air bubbles can be
eliminated .The sample should be injected rapidly as a “plug” to avoid band
broadening and poor resolution.
a) draw the sample into the syringe barrel
b) draw 2-3µl of air into the barrel
c) insert needle into injection port and allow to heat for a few
seconds
d) Inject quickly but without excessive force as the syringes are
fragile
e) When the syringe has been removed check for any un-injected
sample and subtract from nominal injection volume
f) Clean the syringe using diluent (water in this case) six times
before the next injection.
g)

CH4304 Laboratories 22
 High Performance Liquid Chromatography

HPLC is a technique suitable for separation of most organic compounds and is

the most often encountered instrumental quality control method. Separation
occurs between a liquid mobile phase consisting of degassed very high purity
organic/aqueous solvents which may or may not be buffered and a stationary
phase which is chemically bonded to a support material. The most frequently
used stationary phase is Octadecylsilane ODS which consists of a long
hydrocarbon chain, it is a non-polar material.

uv-vis detector
pump injection loop

column

mobile
phase integrator

.
The purpose of this experiment is to determine the concentration of
caffeine in common beverages using a reversed-phase column,
Equipment and Materials

1. HPLC System with variable wavelength detector.

2. Reversed-phase column (C-18); Ultrasphere ODS, 4.6 x 150 mm, 5 micron
particles coated with octadecylsilane groups.
3. 25-µL flat nose syringe.
B. Solutions the mobile phase is is 40:60 CH3OH:H2O

CH4304 Laboratories 23
Part A:
.
Identify caffeine in beverages on the basis of its retention time (tr)and
determine its concentration by standard curve.
Stabilize the system with the methanol/water solvent at a flow rate of
about 1.0 mL/min. Flush the injection port with at least 3-4 syringe volumes of
mobile phase.

(i) Using the pure caffeine provided make up a stock solution of approx 0.1%
(ii) Prepare a series of standards by serial dilution of 0.01%, 0.005%,
0.001%, 0.0005% caffeine in water.
(iii) Load 50 µL of each onto the column, When the injection loop (20 µL)
is properly flushed and filled, injected onto column.. Begin recording the
chromatograms at an absorbance range (AUFS) such that the entire peak
appears on the chromatogram.
(iv) note the retention time and the area associated with each peak.
Construct a calibration graph, and find the best fit regression line
(v) Take a sample of beverage. Filter to remove any undissolved material and
inject onto the chromatograph. The caffeine can be identified by its
retention time and the concentration calculated from its peak area.
..

Part B

You are required to calculate certain performance characteristics of your

separation.

(vi) Prepare solutions of 0.1% NaNO3 , 0.1% benzoic acid and 0.1%
caffeine as well a mixture of 0.1% caffeine and 0.1% phenol. NaNO3 will
be used to estimate tm as it will not be retained by the stationary phase
as it is ionic and will elute with the mobile phase.
(vii) Inject 50 µL of each onto the column, note the retention time. The
chromatograms for caffeine, phenol and the caffeine/phenol mixture
should be measured at 254 nm and the retention times correspond to tr.
for each compound. The chromatogram for NaNO3 should be recorded
at 210 nm. The retention time measured is tm.

CH4304 Laboratories 24
(viii) calculate t’r(the adjusted retention time), k’ (the capacity factor), α
(the relative retention), N (the number of theoretical plate), H (the
height equivalent to a theoretical plate) and R the resolution using the
information supplied in the table.

Quantity equation Parameters

C
Partition coefficient K=
s
Cm Cs = conc of solute in stationary phase

CH4304 Laboratories 25
 Cm= conc of solute in mobile phase
Adjusted retention t 'r =t r −t m tr= retention time of solute of interest
time tm = retention time of unretained solute
Vs = volume of stationary phase
Capacity factor k'=t r ' /t m=KV s /V m Vm = volume of mobile phase
ts ts = time solute spends in stationary
k'=
tm phase
tm = time solute spends in mobile phase

subscripts 1 and 2 refer to two solutes

Relative retention t' k' K w = width at base
α= r2 = 2 = 2
t' r1 k'1 K 1 w1/2 = width at half height
δ = std deviation of band
Number of Plates 16t 2r 5 . 55t2r x = distance travelled by centre of band
N= 2 = 2
w w 1/2 L = length of column
N = number of plates on column
Plate height δ L 2 ∆tr = difference in retention times
H= =
x N ∆Vr = difference in retention volumes
Resolution Δt ΔV r wav = average width at baseline
R= r =
wav w av N = number of plates
α = relative retention
  
k '2
R= 
N α −1
4 α 1k 'av k 2' = capacity factor for second peak

k 'av = average capacity factor

DETERMINATION OF NITROGEN BY KJELDAHL METHOD

The method
The Kjeldahl digestion converts nitrogen compounds (proteins, amines,
organic compounds) into ammonia compounds. Free ammonia is released by
the addition of caustics, which is then expulsed by distillation and
subsequently titrated.
Kjeldahl tablets or copper sulphate/selenium salts accelerate the reaction.

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The tablets consist of sulfate and metal salts. The sulfate salt raises the
boiling point of sulfuric acid, while the selenium, coppers, titanium, or
mercury salts shorten the digestion time.

Digestion

catalyst
CHNO + H2SO4 CO2 + SO2 +H2O +(NH4)2SO4
sample

Weigh out accurately part of the organic sample (1.5 - 2.0g). Add 10g of
potassium sulphate, 0.2g selenium or 0.5g anhydrous copper sulphate and

pour 25ml of concentrated sulphuric acid into the flask in such a way as to

wash down any solid adhering to the neck. Prepare three sample flasks and

one blank flask. Allow to digest under vacuum for 2 to 4 hours. The addition

of some glass beads to the flasks before boiling speeds up the digestion

process.

When cool carefully add 100ml of water to each flask and transfer to the

distillation apparatus.

Neutralisation

H2SO4 + 2 NaOH Na2SO4 + 2 H2O

(NH4)2SO4 + 2 NaOH Na2SO4 + 2 NH3 + 2 H2O

Add excess of 50% sodium hydroxide solution. Distill off the ammonia into

excess standard acid or a saturated solution of boric acid.

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 +
Collection of NH3 NH3 + H3BO3 NH4 + H2BO-3

{H[B(OH)4] + NH3 NH4[B(OH)4]}

The borate formed is determined by titration with standard 0.1N HCl; one mole of HCl
being required for each mole of NH3

-
+
Titration H2BO3 + H H3BO3
{2 NH4[B(OH)4] + H2SO4 (NH4)2SO4 + 2 H[B(OH)4]}

The solution at the equivalence point contains H3BO3 and NH4CL, so an

indicator in the pH range 5-6 is necessary. Bromocresol green or

bromophenol blue.

Calculate the % Nitrogen in the sample.

What methods would you use to reduce or eliminate determinate error from

this experiment.

Karl Fischer Titration of H2O

Water can affect the chemical and physical properties of substances and
influence the quality, price and the shelf-life of a product.

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Using the Karl Fischer titration, the water content of gases, liquids and
solids can be determined easily and with a high degree of accuracy.
Determination of water content according to Karl Fischer is rapid, accurate
and reliable; it is often the method of choice in quality and in-process
control, production, research and development. It is a very widely used
procedure for measuring water in food and pharmaceutical samples.
The Karl Fischer Reagent is comprised of iodine sulphur dioxide, a base and
an alcohol.

What is a Karl Fischer chemical reaction?

I2 + 2H20 + SO2 2Hl + H2SO4

This reaction occurs in the presence of a base and a solvent (a typical

solvent could be methanol or diethylene glycol, and a base imidazole)

A chemical reaction takes place between iodine (I) and water (W) with the
reactants being in a 1:1 ratio between (I) and (W).

1x (I) + 1x(W) --> Reaction

The end-point is usually detected coulometrically using an auto-titrator.

The anode solution contains an alcohol, a base, SO2, I-, and possibly another
organic. Pyridine was the primary ingredient in Karl Fischer solutions but
has mostly been replaced. The alcohol is diethylene glycol and the base
imidazole. The anode generates I2 by oxidation of I- in the presence of H2O,
A stoichiometric reaction occurs between the alcohol, (ROH) base (B), SO2,
and I2.

B.I2 + BSO2 + B + H2O BH+I- + +B-SO3-

+
B-SO3- + ROH BH+ROSO3-
The net rx is oxidation of SO2 by I2 with the formation of alkyl sulphate.
One mole of I2 is consumed for each mole of H2O.

Once the iodine in the Karl Fischer reagent is determined, the unknown
concentration of water in the sample can be determined. ie, as 1 molI2 reacts

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with 1 mol H2O, the amount of (I2) used during the titration must be equal to
the unknown amount of water present in the sample.

Coulometric Karl Fischer: - With a coulometric Karl Fischer titration, the

amount of water present is determined by measuring the amount of current
generated during the titration (coulombs) Coulombs are a measurement of
current (amps) multiplied by the titration time in seconds. There is a
relationship between the iodine (I2) used in the titration, the samples water
content (W) and the current. ie,
Current (A) x time (secs) = Coulombs (C) ---> I + W ---> KF reaction
According to Faradays Law :- 2 x 96,485 Coulombs are needed to generate 1
mole of iodine and this iodine subsequently reacts 1 : 1 with the water in the
KF reaction.
How does a coulometric titration work?

Coulometric KF titrations are preferably carried out within the pH range 4-

7
Stage 1 Iodine generation Instead of dispensing KF reagent as in Volumetric
KF titration, the Metrohm KF instrumentation actually generates the
reagent inside the reaction cell. A current flows through the reagent
generating iodine at the anode electrode.
Stage 2 The instrument detects the end of the titration (end-point)
Stage 3 T he instrument subsequently calculates the moisture content.

Determine the moisture content of the sample provide quotingthe

average and standard deviation.

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