HPLC Instrumentation

Question 1

Which of the following are not characteristics of ideal MP in HPLC?
a. Pure
b. Chemically intert
c. Can solubilize the sample
d. Only A and B is correct
e. A, B, and C are correct

Answer 1

e. A, B, and C are correct

Question 2

In the treatment of MP in HPLC, why do we need to filter before putting it in the column?

Answer 2

To avoid particulate matter that may clog the column

Question 3

In the treatment of MP in HPLC, we are required to degas the MP. What are some ways that we can do to achieve this?

Answer 3

1. heating with stirring
2. applying vacuum
3. passing nitrogen or helium
4. ultrasonication

Question 4

Why is acetonitrile a stronger MP in RPLC than water?

Answer 4

Acetonitrile, being less polar, interacts less with the SP allowing analytes to elute faster

Question 5

In HPLC, there are 2 elution techniques. Isocratic elution and gradient elution. In isocratic elution, the MP composition remains constant throughout, which leads to band broadening. What does this imply to the chromatogram?

Answer 5

later eluting peaks are broader, while earlier ones are sharper

Question 6

Gradient elution is continuous and step-wise. Its MP composition is changed during the separation process. What are some advantages of this elution technique?

Answer 6

1. Shortening time of analysis
2. Reduces tailing, giving sharp peaks
3. increases sensitivity of analysis
4. decreases retention of later-eluting components

Question 7

HPLC contains a high pressure pump. What is its significance?

Answer 7

1. provides high pressure/constant and continuous flow of MP
2. controls flow rate

Question 8

The high pressure pump in HPLC can be divided into two types. The reciprocating pump and the syringe/displacement pump. How do they differ?

Answer 8

Syringe/displacement pump: pulse-free, small capacity, no gradient elution, limited MP capacity
Reciprocating pump: widely used, small internal volume, high pressure, gradient elution, constant flow, no need for pulse damper

Question 9

Rheodyne injectors are a specific brand of manual injectors in HPLC. It is composed of the load position and the inject position. The load position works when the sample is injected, it goes to the sample loop and any excess will go to waste. What is the purpose of the inject position?

Answer 9

Once loaded, the sample will go from the sample loop to the column

Question 10

List the Instrumentation of HPLC

Answer 10

1. reservoir for solvents (MP)
2. high pressure pump
3. sample inlet device/injection port
4. column
5. detector
6. data acquisition and control system (recorder)
7. fraction collector

Question 11

In HPLC, this is the place where the MP is in contact with the SP, forming an interface with enormous surfaces

Answer 11

column

Question 12

The column in HPLC produces _____ of the analytes in the mixture

Answer 12

separation

Question 13

Describe the HPLCs column

Answer 13

1. usually made from stainless steel
2. usually has straight shape
3. length of the column is variable

Question 14

In HPLC, it is a device for continuous registration of specific physical properties of the column effect

Answer 14

detector

Question 15

in HPLC, this is where the computer program controls all parameters of HPLC such as eluent composition, column temp, and injection sequence.

Answer 15

data acquisition and control system (recorder)

Question 16

In HPLC, this collects pure compounds once it goes out of the column

Answer 16

Fraction collector