Lab Sessions

Question 1

TLC
sp
mp
to separate what
How to identify analytes
How to achieve better separation

Answer 1

sp silica is polar

mp different ratios of petroleum ether and acetone is non polar and adjustments made to how non polar it is

can separate spinach pigments

Retardation factor: used to compare analytes to known values

Achieve separation by adjusting polarity of mp

Question 2

Formula for retardation factor

Answer 2

Distance traveled by pigment /
Distance traveled by solvent (solvent is the range of mp)

Question 3

How to achieve better separation in TLC

Answer 3

Use a range of mp

eg petroleum ether 60:40 acetone produces the best separation due to the chlorophyll being a relatively polar compound

The range of mp are all relatively less polar than silica

Question 4

Mode of separation in TLC

Answer 4

Adsorption

Question 5

Polarity of silica

Answer 5

Polar

Question 6

GC
mp
sp
mode of separation
sample
Oven
Internal standard

Answer 6

mp gas eg helium nitrogen inert gases
sp column packed or capillary
mode = partition
Oven: temperature programming or isothermally
Sample: must be volatile
Calibration curve produced by preparing standards of varying concentrations of ethanol and adding a constant amount of propanol to each

Peak area of ethanol divided by propanol in the chromatogram was graphed against concentration

Question 7

Explain partition

Answer 7

Partitioning between mp and sp

Question 8

Which has a shorter retention time ethanol or propanol

Answer 8

ethanol has less carbons than propanol therefore has a shorter retention time

Question 9

What is a use for GC

Answer 9

Can be used to determine the % alcohol in a product using an internal standard method

Question 10

HPLC
mp
sp
pump and injector
If sample is like the mp
Detector

Answer 10

mp liquid is polar eg methanol water ratio
sp liquid coated in a column eg C18 is non polar because it is a long chain carbon
Injecting 10ul
Depending on polarity of the sample it will have more of an affinity with the mp or sp
Sample will elute faster and will have a shorter retention time
Photo diode array

Question 11

mp is polar and sp in non polar

Answer 11

Reverse phase chromatography

Question 12

why is C18 is non polar

Answer 12

because it is a long chain carbon

Question 13

How to avoid long retention times in HPLC

Answer 13

Make the mp more like the sample

Question 14

Area under a chromatogram?
If there’s two peaks

Answer 14

Peak area relates to concentration
Two peaks there is two components in the sample

Question 15

Photo diode array detector
How to identify caffeine

Answer 15

Light of all wavelengths are passed through the sample and only some will be absorbed depending on what chloroform’s are present in the sample

Caffeine absorbs at 273nm :

If the sample absorbs at 273 there will be a peak at this wavelength

Question 16

Alternative detectors

Answer 16

Photo diode array
Mass spec detector (no debate as to what the sample is, gives unique fingerprints)

Question 17

If a substance absorbs UV light

Answer 17

If it has certain chloroforms ( functional groups) present cause the substance to absorb UV light at a wavelength

Question 18

Keeping the mobile phase constant in HPLC is called

Answer 18

Isocratic elution.

Question 19

Changing the mobile phase to achieve better separation in HPLC

Answer 19

Gradient elution

Question 20

Sharpness of peak
How well separated the peaks are

Answer 20

Sharpness of peak = efficiently n, the number of plates
Separation = resolution

Question 21

Adequately resolved peaks

Answer 21

Can see where one peak end and one peak brings
Resolution value greater than 1.5

Question 22

How to improve poor resolution

Answer 22

Adjust parameters

Parameters

Flow rate
Type of column
Type of mobile phase

Question 23

T = 10%

Answer 23

T% = .1

Question 24

Concentration must be in

Answer 24

moles per litre

Question 25

Energy of an element

Answer 25

E = hf AN

Question 26

1ppm in grams per litre

Answer 26

1 x 10 to the -3

Question 27

Units of epsilon

Answer 27

Litres moles per centimetre

Question 28

T% = 40%

Answer 28

T = 0.4

Question 29

A1 / A2

Answer 29

C1 / C2

Question 30

High frequency waves have high or low energy

Answer 30

High frequency
Short wavelength
High energy

Question 31

Is blue or red light higher frequency?

Answer 31

Blue light is 400nm
Red light is 700nm
Blue is shorter therefore high frequency and higher energy