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Biomedical Lab > Lab 10 > Flashcards

Lab 10 Flashcards

1
Q

Amino acids have a ___ group and a ____ group

A

Carboxyl group (COO-) and an amino group (NH2)

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2
Q

what is the isoelectric point?

A

The pH at which a protein carries no net charge

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3
Q

Below the isoelectric point, proteins carry a net ___ charge

A

POSITIVE

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4
Q

It is preferable that the polar and charged amino acids lie where on the protein? Why?

A

On the surface so that the protein can form hydrogen bonds by donating or accepting a proton

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5
Q

What lies in the MIDDLE of a folded protein?

A

A hydrophobic core

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6
Q

Chromatography is a procedure used to….

A

Separate individual compounds present in a sample based on the difference of their physical and chemical properties and to determine the relative concentration of the compound in the mixture

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7
Q

What are some physical properties that that chromatography can separate

A

Size, binding affinity

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8
Q

What are some chemical properties that chromatography can separate

A

Polarity, charge

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9
Q

Before a sample is analyzed using chromatography, it must be….

A

Completely soluble in solvent

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10
Q

The technique of chromatography consists of 3 steps:

A
  1. Transfer of sample onto or into the chromatography device
  2. Movement/migration through the chromatography device
  3. Detection of the compounds
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11
Q

The 2 compounds present in all types of chromatography are the ___ phase and ___ phase

A

Stationary phase and mobile phase

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12
Q

The higher the hydrophobicity value, the _____ the hydrophobicity

A

Greater

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13
Q

Name 4 very hydrophobic amino acids

A

Leucine, isoleucine, phenylalanine

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14
Q

Detection of the compounds in chromatography is performed how?

A

Visually using colorimetric reagents or by spectrophotometric analysis

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15
Q

Give an example of a compound that forms colored products with reagents

A

Amino acids with ninhydrin

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16
Q

How are colorless compounds detected in chromatography?

A

By measuring the absorbance of the elute using a spectrophotometer

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17
Q

In some cases, a ________ is incorporated into the matrix to visualize spots under _____ uv (____nm)

A

In some cases, a FLUORESCENT COMPOUND is incorporated into the matrix to visualize spots under SHORT-WAVE uv (254nm)

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18
Q

Stationary phase is also known as a “____”

A

Matrix

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19
Q

The stationary phase can be a ____ or _______

A

The stationary phase can be a THIN LAYER ON SOLID SUPPORT (glass/plastic) or PACKED IN A COLUMN

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20
Q

How are the compounds separated in chromatography?

A

Based on their relative affinity to the stationary and mobile phase

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21
Q

If the compound has a greater affinity for the mobile phase as compared to the stationary phase, the compounds will move with the ____ phase

A

Mobile

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22
Q

Each compound in the sample mixture moves _____ through the stationary phase

A

At different speeds

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23
Q

WHY does each compound in the sample mixture move at different speeds through the stationary phase?

A

Because of the relative affinity of each compound to the mobile phase

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24
Q

Explain NORMAL PHASE chromatography

A

Normal phase chromatography has a relatively more POLAR stationary phase and is usually made of SILICA gel

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25
Q

In normal phase chromatography, will polar or nonpolar compounds bind strongly to the matrix while ____ compounds will move quickly

A

In normal phase chromatography, the stationary phase is polar. THEREFORE, polar compounds will bind strongly to the matrix while hydrophobic compounds will move quickly

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26
Q

Is silica gel acidic or basic?

A

Acidic

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27
Q

Since silica gel is acidic….

A

It offers poor separation of basic samples and causes a deterioration of acid-labile colecules (easily destroyed by an acidic environment)

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28
Q

In reversed phase chromatography, the matrix particles (silica gel) have been coated with compounds with ______ side chains

A

HYDROPHOBIC

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29
Q

In reversed phase chromatography, the matrix particles have been coated with compounds with hydrophobic side chains. What does this mean?

A

The matrix has a higher affinity for hydrophobic compounds than polar compounds. Thus, the highly polar compounds are easily eluted using a polar mobile phase (eluding solvent)

30
Q

Ion-exchange chromatography has a ____ matrix which binds to which molecules?

A

Ion-exchange chromatography has a CHARGED matrix which binds to molecules with an opposite charge

31
Q

In ion exchange chromatography, negatively charged proteins will bind to a ____ charged column

A

Positively

32
Q

The mobile phase can be a ___ or ____

A

Liquid or gas

33
Q

The mobile phase moves through the ___ phase via…..

A

Stationary phase via capillary action or with the use of pressure

34
Q

How many solvents can be mixed together in varying proportions to obtain the best polarity (or pH) for separation?

A

2 or more

35
Q

What are some common mobile phases used in liquid chromatography?

A

Water (polar solvent), ethanol and methanol (less polar solvents)

36
Q

For more sophisticated chromatographic separations, what kind of solvents are used?

A

Other organic solvents such as aniline and octane

37
Q

Name 3 types of chromatography

A

Size exclusion chromatography, thin layer chromatography, high performance (or pressure) liquid chromatography SEC, TLC, HPLC

38
Q

What kind of chromatography was used to separate DNA and proteins based on their size or molecular weight in previous labs

A

Size exclusion chromatography (SEC)

39
Q

Which chromatography is a simple and rapid technique?

A

Thin layer chromatography

40
Q

What chromatography is used in this last lab?

A

Thin layer chromatography

41
Q

Explain the process of thin layer chromatography

A

The sample is spotted onto a stationary phase fixed onto the surface of a glass plate. The bottom edge of the plate is placed in a mobile phase. The mobile phase moves through the stationary phase by capillary action. A compound with GREATER AFFINITY TO THE MOBILE PHASE MOVES UP THE GLASS PLATE ALONG WITH THE MOBILE PHASE

42
Q

After the separation is complete, the TLC plate is called a _____

A

Chromatogram

43
Q

Most compounds in TLC are _____ and visualized how?

A

Colorless, visualized using UV light or by using a reagent which reacts with the compound to give a colored spot on the plate

44
Q

What does RF value stand for?

A

Retardation factor

45
Q

Rf values are used to….

A

Compare the movement of the individual compounds in the sample mixture

46
Q

Rf value =

A

Distance traveled by the compound in the mixture/ distance traveled by the mobile phase

47
Q

The vale for Rf is always between __ and ___

A

0-1

48
Q

How can compounds with the same Rf values be compared?

A

They can be compared with a known standard to assist in identification of the compound

49
Q

Amino acids are _____

A

Colorless and cannot be visualized by our eyes

50
Q

How can amino acids be visualized?

A

By using a color reagent (ninhydrin)

51
Q

Ninhydrin will react with most amino acids to give what color?

A

Purple

52
Q

What test is commonly used to visualize TLC spots?

A

The Ninhydrin test

53
Q

HPLC stands for….

A

High performance (or pressure) liquid chromatography

54
Q

HPLC uses a ___ stationary phase packed in a ____

A

Solid stationary phase packed in a column

55
Q

The mobile phase is also known as the ____

A

Eluent

56
Q

In HPLC, what happens to the mobile phase?

A

It is pushed through the packed column under constant pressure using pressure pumps

57
Q

How many pumps are often used in HPLC? WHY?

A

2
So that the polarity of the mobile phase can be automatically changed during the run

58
Q

Reverse phase HPLC is most common in chromatographic analysis of what?

A

Drugs and body fluids

59
Q

Many drugs are hydrophilic or hydrophobic?

A

Hydrophobic

60
Q

Since many drugs are hydrophobic, ____ matrices must be used because…..

A

Hydrophobic matrices must be used so that they could retain hydrophobic compounds (such as drugs) and remove polar contaminants

61
Q

In HPLC, a spectrophotometer continuously measures the absorbance of the _____

A

Eluent as it leaves the column

62
Q

A CHROMATOGRAPH is a……

A

Recording of absorbance changes

63
Q

When a compound is eluted, there is a ___ in absorbance

A

Increase

64
Q

When a compound is eluted, there is an increase in absorbance, recorded as a ____ on the chromatograph

A

Peak

65
Q

The chromatograph not only shows a peak, but also….

A

The time of the elution, and the area of the peak

66
Q

The area of the peak is used to calculate……

A

The concentration of the compound (by measuring the area under the curve)

67
Q

What is retention time? (Rt)

A

The time required for eluting the compound through the column

68
Q

A compound attracted more strongly by the mobile phase will have a ____ retention time

A

Shorter, as it will move rapidly through the column

69
Q

The identity and concentration of the saluted compounds is determined by comparing…

A

The retention times and the chromatographs with known standards

70
Q

Salicylic acid has a small retention time, meaning it is relatively ____

A

Polar

71
Q

Between lysine, glycine, and phenylalanine, which is the most hydrophilic (_____est Rf value)

A

Lysine is the most hydrophilic (lowest Rf value)

72
Q

A higher retention time = the ____ spinning in the column

A

Longer

Biomedical Lab (10 decks)

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