TAC 357 – Problem Set 1

 
 
⎛ Runk ⎞⎛ Vstd ⎞⎛ AUFSunk ⎞
External Standard: Cunk = Cstd ⎜ ⎟⎜ ⎟⎜ ⎟
⎝ Rstd ⎠⎝ Vunk ⎠⎝ AUFSstd ⎠

⎛ Runk ⎞ ⎛ Cunk ⎞
⎜ ⎟ unk .run ⎜ ⎟ unk .run
⎝ R int .std ⎠ ⎝ C int .std ⎠
Internal Standard (RATIO): =
⎛ Runk ⎞ ⎛ Cunk ⎞
⎜ ⎟ std .run ⎜ ⎟ std .run
⎝ R int .std ⎠ ⎝ C int .std ⎠

⎛ concentrationA ⎞
Internal Standard (Response Factor): RFA = ⎜ ⎟
⎝ responseA ⎠

⎛ RFA ⎞
Internal Standard (Relative Response Factor): RRFA − IS = ⎜ ⎟
⎝ RFIS ⎠

⎛ Runk ⎞
Internal Standard (Relative Reponse Factor): Cunk = ⎜ ⎟ ( RRFunk − IS )( CIS )
⎝ RIS ⎠

⎛ Cunk ⎞ ⎛ Runk ⎞
Standard Addition - Spiking (Volume Change Negligible): ⎜ ⎟ = ⎜ ⎟
⎝ VC ⎠ ⎝ VR ⎠

ALSO KNOW: Resolution Formula, Theoretical Plate Counts (Half-Height, Tangential &
5σ)

EXTERNAL STANDARD:

1/ Three standard solutions of G and H were analyzed using a fixed loop injector on an
HPLC instrument. The results are as follows:

Run Standard G Standard H

Ppm Area Ppm Area
1 25.0 4635 10.0 2875
2 50.0 9265 20.0 5755
3 75.0 13910 30.0 8620

A 24.0mg sample containing G & H that was dissolved in 200mL of mobile phase was
analyzed on the same instrument. The peak areas were G = 8422 & H = 6256.
Calculate the %w/w G & % w/w H in the sample. ( 37.9%G & 18.1% H).

2/ A HPLC is used to determine the concentration of caffeine using the External

Standard Method. The external standard was prepared by an analyst as follows:

1/ Concentrated standard: 0.5301grams of caffeine is placed in a 200mL

volumetric flask and diluted to the mark using mobile phase.
TAC 357 – Problem Set 1  
 
2/ Injection Standard: 1.5mL of the concentrated standard is placed in a 25mL
volumetric flask and diluted to the mark with mobile phase.

The analyst then injected 30 µL of the Injection Standard into a HPLC with the
absorbance detector AUFS parameter set to 1.5. The resultant area count was 123,450.

The analyst then injected 15 µL of the as-received sample #12-065 into the same HPLC
with the absorbance detector parameter adjusted to 0.75. The resultant area count was
111,225. What is the concentration of the unknown in ppm? (143 ppm)

INTERNAL STANDARD

1/ A HPLC was used to determine the concentration of a mixture containing A and B

using S as the internal standard.
A solution containing 130.0mg of A and 120mg of S was dissolved in 100mL of mobile
phase. When a sample of this solution was injected, the peak area response ratio was
as follows: A:S = 5.0 : 4.0
A second solution containing 145mg of B and 130mg of S was dissolved in 100mL of
mobile phase. When a sample of this solution was injected, the peak area response
ratio was as follows: B:S = 7.3 : 3.5
When 200.0mg of an unknown mixture containing A and B was mixed with 110mg of S
and subsequently dissolved in 100.0mL of solvent and injected under the same
chromatographic conditions, the peak area response ratio was as follows:
A:B:S = 6.0 : 5.0 : 4.5
Calculate the percent composition (%w/w) of A and B. in the unknown mixture. (63.6%
A & 32.7% B)
2/ A HPLC was used to determine the concentration of a mixture containing G and H
using I as the internal standard.

A solution containing 20.0mg of G, 15.0mg of H and 30.0mg of I was dissolved in

100mL of mobile phase. When a sample of this solution was injected, the peak area
response ratio was as follows: G : H : I = 8.4 : 6.3 : 2.4

When 145.0mg of an unknown mixture containing G and H was mixed with 20.0mg of I
and subsequently dissolved in 50.0mL of solvent and injected under the same
chromatographic conditions, the peak area response ratio was as follows: G : H : I = 6.2
: 7.8 : 4.4

Calculate the percent composition (%w/w) of G and H. in the unknown mixture. (3.70%
G & 4.66% H).
TAC 357 – Problem Set 1  
 
STANDARD ADDITION

1/ A HPLC is used to determine the concentration of W and Z in a sample submitted to

a laboratory for analysis. When a sample of the unknown solution is injected, the peak
area response ratio is A:B = 14.3 : 6.5

When a solution containing 4mL of unknown + 6mL of 8mg A/10mL is injected under the
same chromatographic conditions, the resultant peak area response ratio is

A:B = 15.5 : 2.0

When a solution containing 3mL of unknown + 3mL of 9mg B/10mL is injected under the
same chromatographic conditions, the resultant peak area response ratio is

A:B = 10.4 : 8.6

Calculate the concentration of A and B in the unknown solution in ppm.

(A = 475ppm & B = 1100ppm)

2/ A HPLC is used to determine the concentration of A and B in a sample submitted to a

laboratory for analysis.

The first injection contained 300.0mg of an unknown solid of A & B dissolved in 50.0mL
of mobile phase. The peak area response ratio is A:B = 8536 : 9217

The second injection contained 3mL of the original unknown solution + 4mL of a
solution containing 87.5mg A in 25.0mL of mobile phase. The resultant peak area
response ratio is A : B = 9178 : 3261

When a solution containing 6mL of the original unknown solution and 3mL of a solution
containing 56.5mg B in 100.0mL of mobile phase the resultant peak area response ratio
is

A:B = 5036 : 7152

Calculate the percent composition (%w/w) of A and B in the unknown mixture.

(38.32% A & 14.6% B)

STANDARD ADDITION – SPIKING

1/ A GC is used to determine to concentration of a substance by the ‘spiking’

method. When an analyst injected 15 µL of an unknown solution into the GC, a single
peak with a height of 9.3cm is observed. The analyst then prepared a solution by adding
20mg of pure standard to 100mL of the original unknown solution and injected 10 µL
TAC 357 – Problem Set 1  
 
into the same GC under the same conditions and observed the same single peak, but
with peak height of 96mm. What is the concentration of the substance in the unknown
solution in ppm? (365ppm)

NOTE: Assume there is negligible volume change when the unknown is ‘spiked’ with
the standard.

2/ A GC is used to determine to concentration of a substance by the ‘spiking’ method.

When an analyst injected 20 µL of an unknown solution into the GC, a single peak with
an area count of 915 456 is observed. The analyst then injected 20 µL of a ‘spiked’
solution (50 µL of pure standard with a density of 0.80g/mL added to 1mL of unknown)
into the same GC under the same conditions and observed the same single peak, but
with a peak area of 917 538. What is the concentration of the substance in the unknown
solution in mg/ml? (17 588 mg/ml)

NOTE: Assume there is negligible volume change when the unknown is ‘spiked’ with
the standard.