MAYOR UNIVERSITY OF SAN ANDRÉS

FACULTY OF ENGINEERING
BASIC COURSE

QUANTITATIVE ANALYTICAL CHEMISTRY (QMC - 202)

INSTRUMENTAL ANALYSIS (PRQ–402)

Teacher: Mg Sc Eng. ARMENIO SILVA MANZANEDA

AUXILIARY: Univ. ZEGARRUNDO LIRA VERONICA ALEJANDRA
GROUP: A
MANAGEMENT:1/2020

PRACTICE NThe1–THIRD PARTIAL

CHROMATOGRAPHY: SEPARATION METHODS AND
NORMALIZATION OF INTERNAL AREAS AND PATTERNS
➢ Separation Methods
A solute with a retention time of 407 s has a base width of 13 s for
a column of 12.2 m in length. Calculate:
a) The equivalent height of the theoretical plate and the number of theoretical plates.
b) If a nearby peak is eluted at 424 s with a base width of 16 s, calculate the
resolution of the two compounds.
RPTA: a. 0,78 mm–15681; b. 1,17.

2. A packed column for gas chromatography had an inner diameter of

5.0 mm. The volumetric flow rate at the outlet of the column was 22.5 ml/min.
the porosity of the column was 0.45. What will be the linear flow rate in cm/s.
RPTA: 4.24 cm/s

3. Substances A and B have retention times of 16.40 and 17.63 minutes.

respectively, in a column of 30.0 cm. A species that does not retain passes through
From the column in 1.30 min. the peak widths (at the base) for A and B are 1.11 and 1.21
min. respectively. Calculate:
a) The resolution of the column.
b) The average number of dishes in column A.
c) The height of the plate.
d) The length of the column needed to achieve a resolution of 1.5.
e) The time necessary for substance B to elute in the longest column.
f) The height needed to achieve a resolution of 1.5 using the column
original of 30.0 cm and in the time given originally.
RPTA: a. 1,06; b. 3493; c. 8,7x10-3cm; d. 65,76 cm; e. 38,64 min; f. 4,3x10-3cm.

AUX. V. ALEJANDRA ZEGARRUNDO LIRA

4. An open tubular column used for gas chromatography had a diameter
interior of 0.25 mm. A volumetric flow rate of 1.0 ml/s was used. find the
linear flow velocity in cm/s at the column outlet.
RPTA: 2037 cm/s.

5. A capillary column has a length of 30 cm and an exterior diameter of 0.530 mm. The wall
The interior is coated with a stationary phase layer of 3.1 µm thickness. A solute
the non-retained solute takes 2.16 minutes to traverse it, while a certain solute has a
retention time of 17.32 min.
a) Calculate the linear velocity and the flow of the mobile phase.
b) Calculate the solute capacity factor and the time fraction it spends in the
stationary phase.
c) Calculate the distribution coefficient of the solute.
RPTA: a. 13,9 cm/min–0,03 ml/min; b. 7–0,875; c. 294.41

6. (a) A chromatographic column with a length of 10.3 cm and an inner diameter of 4.61 mm
it is packed with a stationary phase that occupies 61% of its volume. If the flow rate
It is 1.13 mL/min to find the linear flow velocity in cm/min.
a) How long will the solvent take (which is the same time that a solute takes to dissolve)?
held) in crossing the column?
b) Find the retention time of a solute that has a factor of
retention or a capacity of 10.0

RPTA: a. 17,4 cm/min; b. 0,593 min; c. 6,53 min.

7. A chromatographic procedure separates 4.0 mg of an unknown mixture in a

column of 40 cm in length and 0.856 cm in diameter.
a) What size should the column be to separate 100 mg of the same mixture?
b) If the flow rate is 0.22 mL/min in the small column, what flow rate should be used in
the big column?
c) If the mobile phase occupies 35% of the column volume, calculate the linear velocity.
of flow in the small column and in the large column?
RPTA: a. 4,28 cm; b. 5,5 mL/min; c. 1,09 cm/min.

8.(I/2018) Substances A and B have a resolution of 1.06. In addition, the number of plates
for each substance it is 3493 and 3397 respectively. The difference between the widths
The peak at the base of B and A is equal to 0.1. Find the retention time of A.
RPTA: 16.4.

AUX. V. ALEJANDRA ZEGARRUNDO LIRA

9. (a) When a 10.0 mL solution containing 234 mg of pentanol was analyzed (PF=
88.15) and 237 mg of 2,3-dimethyl-2-butanol (PF= 102.17), the area ratio of the peaks
of pentanol: 2,3-dimethyl-2-butanol was 0.913:1.00. Assuming that pentanol is the
internal standard, find the response factor of 2,3-dimethyl-2-butanol.
Considering the chromatograms for pentanol and 2,3-dimethyl-2-butanol their
W½ are respectively 2.2 and 1.5 minutes (the peak heights are 41.4 and 76.0 mm)
respectively, calculate the areas of their peaks (taking into account that they resemble
A Gaussian peak is equal to 1.064 times the peak height times W1/2.
RPTA: a. F = 1,081; b. 121,3 mm*min.

10. In an open tubular column with coated walls, 1000 cm in length and 0.25
mm in diameter, the carrier gas (helium) flows at a speed of 37 cm/s. The time of
retention, tr, for the dean is 1.27 min, and the width at half height of the peak is
0.88s. Calculate the capacity factor for the decane, the number of effective trays of the
column and plate height.
RPTA: k´ = 1,82; N = 41 539 platos; H = 0,024 cm/plato.

11. Consider a chromatography experiment in which two components with factors of

retention k1= 4 y k2= 5, they are injected into a column with N = 1x103theoretical plates. The
The retention time for the component that is least retained is t.R(1)= 10 min.
a) Calculate tM, tR(2)and the width to half of the height (W ½) for each peak. Calculate
In addition, the width W at the base of each peak.
b) Calculate the resolution of the two peaks.
RPTA: a. 2; 12-0,74; 0,89-1,265; 1,518; b. 1,44

12. In a column of 122 cm in length and at a temperature of 160ºC, the obtained

siguientes tiempos de retención en minutos: pico del aire 0.90, heptano 1.22 y octano 1.43.
The width at the base of the peaks was 0.14 min for heptane and 0.20 min for octane.
a) Calculate the relative retention and the resolution for these peaks.
b) What length should the column have to achieve a resolution of 1.5?
RPTA: a. tr´heptano = 0,32 min; tr´octano = 0,53 min; b. 180 cm.

13. The separation by CG of four alcohols A, B, C, and D has been carried out using a
a column of 5000 theoretical plates, obtaining the following through the chromatogram
t dataM= 1.30; tRA= 4.59; tRB= 9.26; tRC= 10.02 and tRC= 18.5 min. Calculate the resolution
achieved between the first and third compound.
RPTA: 9.85

AUX. V. ALEJANDRA ZEGARRUNDO LIRA

14.(FINAL II/2019) With a gas-liquid chromatograph and a packing column of 40
The following data was obtained:

Compound Tr, min W, min

Air 1.9 -

Methylcyclohexane 10 0.76

Methylcyclohexene 10.9 0.82

Toluene 13.4 1.06

Calculate:
a) The average number of dishes.
b) The standard deviation in a)
c) The average height of the plate for the column
RPTA: a. 2718; b. 142,31; c. 0,015 cm.

15. The following data correspond to a liquid chromatography column:

LENGTH 24.7 cm
FLOW SPEED 0.313 ml/min
VM 1,370 ml
And 0.164 ml
The chromatogram of a mixture of species A, B, C, and D provides the following data:

TIME OF BEAK WIDTH

RETENCION (min) AT THE BASE (W) (min)
Not retained 3.1 -
A 5.4 0.41
B 13.3 1.07
C 14.1 1.16
D 21.6 1.72
Calculate:

a) The number of dishes for each peak.

b) The mean and the standard deviation of N.
c) The height of the plate for the column.
d) The retention factor for A, B, C, and D.
e) The distribution constant for A, B, C, and D.

AUX. V. ALEJANDRA ZEGARRUNDO LIRA

RPTA: a. 2775–2472–2364–2523; b. 2534–174,11; c. 0,0097 cm; d. 0,74–3,29–
3.55–5.97; e. 6.20–27.49–29.64–49.85.

16. Using the data from the previous problem, calculate the chemical species B and C:
a) The selectivity factor.
b) The resolution.
c) The length of the column needed to have a resolution of 1.5.
d) The time needed to separate B and C with a resolution of 1.5.

RPTA: a. 1,08; b. 0,72; c. 104 cm; d. 14,10 min.

17. Two solutes have a relative retention of 1.08 and capacity factors k'1 = 5 and k'2 =
5.4. The number of theoretical plates is practically the same for both compounds.
Calculate the number of dishes needed to achieve a resolution of 1.5 and for
And for a resolution of 3. If the theoretical plate height is 0.20 mm. Calculate the length.
from the column to achieve a resolution of 1.5.
Note: for peaks with the same number of theoretical plates, the resolution can
to express oneself as:

RPTA: (1,5) = 8649; 1,73m; (3) = 34596; 6,92 m.

18. Based on distribution studies, it is known that species M and N have coefficients
distribution between water and hexane of 6.01 and 6.20 (K = [M]H2O/[M]HEX). The two species
they have to be separated by elution with hexane over a silica gel column that
contains water on its surface. It is known that the Ve/Vm ratio for the
The packaging is 0.422. Calculate:
a) Selectivity factor.
b) How many plates will be needed to produce a resolution of 1.5.
c) What length should the column have if H = 2.2x10-3cm.
d) If a µ = 7.10 cm/min is used, how much time will be needed to elute each one?
of the species.

RPTA: a. 1,03; b. 81012; c. 178,23 cm; d. 91 min.

19. The mobile phase takes 3.0 min to pass through a column, and a solute has a time of
retención de 9,0 min. a) Calcular el factor de capacidad. b) ¿Qué fracción de tiempo pasa
the solute in the mobile phase inside the column? c) If the volume of the stationary phase

AUX. V. ALEJANDRA ZEGARRUNDO LIRA

 it is one-tenth of the volume of the mobile phase in the column (VS = 0.10 VM), calculate
the distribution coefficient of this system.

RPTA: a. 2; b. 0,33; c. 20.

A capillary column is 30 m long and has an inner diameter of 0.530 mm. The wall
The interior is coated with a stationary phase layer of 3.1μm thickness. A solute
non-retained takes 2.16 minutes to cross it, while a certain solute has a
retention time of 17.32 min. a) Calculate the velocity and the flow of the mobile phase. b)
Calculate the solute capacity factor and the fraction of time spent in the phase
stationary. c) Calculate the partition coefficient of the solute.

RPTA: a. 13,9 m/min–3ml/min; b. 7–0,875; c. 294–295.

21. The figure shows the chromatogram of a mixture of two components in a column.
packed liquid chromatography, 25 cm. The flow rate was 0.40 mL
min-1.

a) Determine the retention factors for the two components

b) Calculate the resolution of the two peaks
c) Find the average height of the plate
d) What length of the column would be needed to achieve a resolution of 1.75?

AUX. V. ALEJANDRA ZEGARRUNDO LIRA

 e) Let's assume that the length of the column is fixed at 25 cm and that the material of
packaging is fixed. What measures could you take to increase
the resolution to achieve baseline separation?
f) Is there any measure that you could use to achieve a better separation in
less time with the same column as in the previous section?

RPTA: a. 5-9; b. 1,025; c. 0,397 cm; d. 72,85 cm; e. Si la Fase Estacionaria es constante
(neither L varies, nor particle size, diameter or thickness of the liquid layer, that is N)
we can act on the mobile phase: 1. Change the eluent, use other solvents
with greater elution capacity. 2.Addition of substances to the aqueous phase, such as
tampons (pH change and nature of the tampon), ionic pairs, forming ligands
of complexes, etc. 3. Temperature variation. f) - increasing the flow of the phase
mobile - elution in GRADIENT - varying the temperature. Acting on the phase
Stationary, the resolution can be varied by acting on: - Column length
(increases retention and analysis times, therefore) -Particle size -
Diameter of the column - Thickness of the liquid layer.

22. A solute with a retention time of 407 s has a base width of 13 s for
a column of 12.2 m in length. Calculate the equivalent height of the theoretical plate and the
number of theoretical plates.

RPTA: N=1.57x104H=0.78 mm

A peak with a retention time of 407 s has a base width of 13 s, a peak

The next one eludes at 424 s with a base width of 16 s. Calculate the resolution of the
two compounds.

RPTA: R=1,17

24. A chromatogram with ideal Gaussian bands has tR= 9 min and W1/2= 2 min.
determine:
a) How many theoretical plates are present.
b) If the column is 10 cm long, what will the A.E.P.T. (equivalent height of
theoretical dishes.
c) If the A.E.P.T. of the column found is greater by 0.5 mm than another column X in the
same conditions, which column is more efficient.

RPTA: a. 1,1x102; b. 0,89 mm.

AUX. V. ALEJANDRA ZEGARRUNDO LIRA

 ➢ NORMALIZATION OF AREAS AND INTERNAL PATTERNS
25. The area normalization method was applied for the determination of a mixture.
of alcohols: n-butyl, iso-butyl, sec-butyl, and tert-butyl. With the purpose of
to obtain the relative response factor, a standard solution of the alcohols was prepared
and was injected into the gas chromatograph, the results are as follows:

Alcohol Weight taken (g) Peak area, A (cm2)

n-butyl 0.1731 3,023
isobutyl 0.1964 3,074
sec-butyl 0.1514 3,112
tert-butylic 0.1826 3,004
For a sample containing only the four alcohols, the following data was obtained:

Alcohol Peak area, A (cm)2)

n-butyl 1,731
isobutyl 3,753
sec-butyl 2,845
tert-butylic 1,117
Calculate the weight percentage of each alcohol in the mentioned sample.

RPTA: a. 18,2; 44,1; 25,36; 12,4 %

26. The area normalization method is applied for the determination of n-alcohols.
butyl (nB), iso-butyl (iB), sec-butyl (sB), and tert-butyl (tB). The results for a
pattern sample were:

Complete the table above. For a sample that contains only the four alcohols,
they obtained the following data:

RPTA: 18,18; 43,99; 25,36; 12,46

Auxiliary V. Alejandra Zegarrundo Lira

27. In the internal normalization method, the calculation of the composition is performed in a way
similar to that of the percentage area, but considering the response factors. It is desired to
determine by the method of internal normalization the mass composition of a
sample consisting of 4 esters of butanoic acid. A reference solution of
these esters lead to the following values of the relative response factors of
methyl butanoate (BM), ethyl (BE) and propyl (BP) with respect to butyl butanoate
(BB): FBB= 1; FBM= 0.919; FBE= 0.913; FBP= 1.06. Calculate the composition of the sample to
starting from the following data, which have been obtained from a chromatogram recorded with
the same chromatographic conditions as the reference solution.

RPTA: (16,61%; 16,63%; 33,38%; 33,37%)

28. To determine the serotonin content S (5-OH tryptamine) in a sample, the following is performed:
the corresponding analysis is done as follows: 1 mL of the sample is taken and
add 1 mL of a standard solution containing 30 ng of N-methyl serotonin.
The obtained solution is treated to eliminate interferences; the analyte is extracted and the
internal standard by solid phase extraction and 20μL is injected into a chromatograph
liquid. a) Why is an internal standard added in the stage prior to extraction? b)
Knowing that injecting pure patterns (5ng) of serotonin and N-methylserotonin results in
They obtained areas of 30885982 and 30956727, what will be the concentration?
of serotonin in the sample knowing that the areas corresponding to the peaks of
serotonin and N-methyl serotonin in the sample chromatogram were 2573832 and
1719818, respectively.

RPTA: 45 ng/mL.

29. The relative areas of the peaks obtained by gas chromatography for a mixture of
methyl acetate, methyl propionate, and methyl butyrate were 17.6; 44.7; and 31.1
respectively.
Calculate the percentage of each compound if the relative detection responses were
respectively 0.65; 0.83 and 0.92.

RPTA: 14,8%; 8,1%; 37,1%.

AUX. V. ALEJANDRA ZEGARRUNDO LIRA

30. When 1.06 mmol of 1-pentanol is dissolved and prepared by gas chromatography
1.53 mmol of 1-hexanol yielded peaks with relative areas of 992 and 1570 units.
respectively.
a) Calculate the response factor for hexanol with respect to pentanol as
internal pattern.
b) When 0.57 mmol of pentanol is added to a problem that contains hexanol,
the relative areas of the chromatographic peaks are 834:816 (pentanol: hexanol).
How much hexanol does the problem contain.

RPTA: a. 0,848; b. 0,47.

31. Cinnamaldehyde is the compound that determines the flavor of cinnamon. It is also
a powerful antimicrobial, which is part of certain essential oils. The response
from gas chromatography (GC) with an artificial sample that contains six
components of essential oils and methyl benzoate as an internal standard is shown in
the following figure:

Aux. V. Alejandra Zegarrundo Lira

The following figure is an enlargement of the region near the peak of cinnamic aldehyde.

Calculate:

a) The number of theoretical plates for the compound under study.

b) The fused silica column has a diameter of 0.25 mm x 30 cm and a length of
column respectively, and with a particle size of 0.25μm. Determine the
theoretical plate height.
c) Quantitative data is obtained using methyl benzoate as an internal standard for
the compounds cinnamaldehyde and thymol as shown in the following table.
Knowing that the relative area values for each compound are calculated
dividing the area of the component tip/internal pattern area, calculate: the area
relative to the two previously mentioned compounds, determine the
calibration curve equations for each component and based on the data
From the previous section, determine which component has the highest sensitivity.

RPTA: a. 23104; b. 0,13 cm; c. 1,110–1,991–3,910–7,104.

Aux. V. Alejandra Zegarrundo Lira

32.(II/2019) The relative peak areas for the five peaks obtained by chromatography
gases in the separation of five steroids are indicated below. Also, it
they provide the relative responses of the detector for the five compounds. Calculate the
percentage of each compound in the mixture.

Relative response of
Compound Relative area of the peak
detector

Dehydroepiandrosterone 27.6 0.7

Estradiol 32.4 0.72

Estrone 47,1 0.75

Testosterone 40.6 0.73

Estriol 27.3 0.78

RPTA: 14.99–18.10–27.40–22.99–16.52–100.00% respectively.

33. It is desired to determine the concentration of ethanol in blood using a method.

chromatographic using internal standard. For this, the samples are processed in the same way.
series of aqueous solutions of ethanol pattern and a serum sample: an aliquot of
0.01 ml of each solution is diluted with 0.1 ml of a standard solution of 2-propanol 0.1
mg/ml. Inject 0.1 µL of each solution into a CG equipped with an FID detector.
(flame ionization), an integrator and a PEG-400 (polyethylene glycol) column at 80OC. The
the results obtained were as follows:

Ethanol patterns Integrator measurement

(mg/ml) Ethanol peak Propyl alcohol peak
0.5 5518 12754
0.75 7563 11893
1.0 10350 12084
1.25 13935 12870
1.5 15628 12314
Blood sample 9862 12604
Build the corresponding calibration graph and from this graph determine the
concentration of ethanol in the blood sample.

RPTA:

AUX. V. ALEJANDRA ZEGARRUNDO LIRA

34. A chromatographic analysis using the internal standard method yielded the following
resultados:
Sample:
Compound Area Concentration (g/L)
1 2001
2 2732
3 1759
E. Internal 2582 0.3621
4 1417

Calibration solution:
Compound Area Concentration (g/L)
1 1434 0.2101
2 2467 0.3762
3 1396 0,2559
E. Internal 2421 0.3621
4 1942 0.2776

Calculate:

a) The response factors of the compounds.

b) The concentrations of compounds 1, 2, 3, and 4.

RPTA: a. 0,39–1,21–0,47–0,70; b. 0,11 g/L–0,12 g/L–0,14 g/L.

AUX. V. ALEJANDRA ZEGARRUNDO LIRA